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TW202434621A - Polynucleotide construct and related viral vectors and methods - Google Patents

  • ️Sun Sep 01 2024

TW202434621A - Polynucleotide construct and related viral vectors and methods - Google Patents

Polynucleotide construct and related viral vectors and methods Download PDF

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Publication number
TW202434621A
TW202434621A TW112142462A TW112142462A TW202434621A TW 202434621 A TW202434621 A TW 202434621A TW 112142462 A TW112142462 A TW 112142462A TW 112142462 A TW112142462 A TW 112142462A TW 202434621 A TW202434621 A TW 202434621A Authority
TW
Taiwan
Prior art keywords
seq
construct
amino acid
nucleotide sequence
acid sequence
Prior art date
2022-11-04
Application number
TW112142462A
Other languages
Chinese (zh)
Inventor
克里斯汀 米特爾斯特德
Original Assignee
美商優莫佳生物製藥有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
2022-11-04
Filing date
2023-11-03
Publication date
2024-09-01
2023-11-03 Application filed by 美商優莫佳生物製藥有限公司 filed Critical 美商優莫佳生物製藥有限公司
2024-09-01 Publication of TW202434621A publication Critical patent/TW202434621A/en

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Abstract

Provided herein are polycistronic constructs for coexpression of a synthetic cytokine receptor complex and a chimeric antigen receptor system, as well as vectors, such as viral vectors, comprising same, cells comprising same, and methods of using same.

Description

多核苷酸構築體及相關病毒載體及方法Polynucleotide constructs and related viral vectors and methods

本發明提供用於共表現合成細胞介素受體複合物及嵌合抗原受體系統之多順反子構築體;以及包含其之載體,諸如病毒載體;包含其之細胞;及其使用方法。The present invention provides a polycistronic construct for co-expressing a synthetic interleukin receptor complex and a chimeric antigen receptor system; a vector comprising the same, such as a viral vector; a cell comprising the same; and a method of using the same.

嵌合抗原受體(CAR) T細胞療法已證明針對實體腫瘤之功效有限,部分地係由於克服實體腫瘤異質性及與免疫抑制性腫瘤微環境(TME)相關之CAR T細胞耗竭的挑戰。除此挑戰以外,在以可在治療各種疾病(包括癌症)中提供CAR之治療功效的可控制持久性之方式,將表現CAR之細胞遞送至個體的方法方面亦存在挑戰。本文提供解決此類需求之實施例。Chimeric antigen receptor (CAR) T cell therapy has demonstrated limited efficacy against solid tumors, in part due to challenges in overcoming solid tumor heterogeneity and CAR T cell exhaustion associated with the immunosuppressive tumor microenvironment (TME). In addition to this challenge, there are challenges in methods of delivering CAR-expressing cells to individuals in a manner that can provide controllable durability of the therapeutic efficacy of CAR in the treatment of various diseases, including cancer. Embodiments that address these needs are provided herein.

本文提供一種多順反子構築體,其以5'至3'順序含有:(a)第一表現卡匣,其包括編碼FRB之核苷酸序列;(b)第二表現卡匣,其包括編碼合成細胞介素γ鏈多肽之核苷酸序列;(c)第三表現卡匣,其包括編碼合成細胞介素β鏈多肽之核苷酸序列;及(d)第四表現卡匣,其包括編碼嵌合抗原受體(CAR)之核苷酸序列,其中該等表現卡匣各藉由裂解位點序列分隔開。Provided herein is a polycistronic construct comprising, in 5' to 3' order: (a) a first expression cassette comprising a nucleotide sequence encoding FRB; (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic interleukin γ chain polypeptide; (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic interleukin β chain polypeptide; and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each of the expression cassettes is separated by a cleavage site sequence.

在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在任何實施例中之一些中,編碼FRB之核苷酸序列包括SEQ ID NO: 3、13或50之核苷酸序列。在任何實施例中之一些中,FRB包括與SEQ ID NO: 4、14或51之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,FRB包括SEQ ID NO: 4、14或51之胺基酸序列。In some embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 3, 13 or 50. In some of any embodiments, the nucleotide sequence encoding FRB includes the nucleotide sequence of SEQ ID NO: 3, 13 or 50. In some of any embodiments, FRB includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4, 14 or 51. In some of any embodiments, FRB includes the amino acid sequence of SEQ ID NO: 4, 14 or 51.

在任何實施例中之一些中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在任何實施例中之一些中,編碼合成細胞介素γ鏈多肽之核苷酸包括SEQ ID NO: 15之核苷酸序列。In some of any embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 15. In some of any embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide include the nucleotide sequence of SEQ ID NO: 15.

在任何實施例中之一些中,合成細胞介素γ鏈多肽包括介白素2受體次單元γ (IL2RG)。在一些實施例中,IL2RG包括與SEQ ID NO: 16之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,IL2RG包括SEQ ID NO: 16之胺基酸序列。In some of any of the embodiments, the synthetic interleukin gamma chain polypeptide comprises interleukin 2 receptor subunit gamma (IL2RG). In some embodiments, IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16. In some of any of the embodiments, IL2RG comprises the amino acid sequence of SEQ ID NO: 16.

在任何實施例中之一些中,第二表現卡匣進一步包括編碼FRB之核苷酸序列。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在任何實施例中之一些中,編碼FRB之核苷酸序列包括SEQ ID NO: 13之核苷酸序列。在任何實施例中之一些中,FRB包括與SEQ ID NO: 14之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,FRB包括SEQ ID NO: 14之胺基酸序列。In some of any embodiments, the second expression cassette further comprises a nucleotide sequence encoding FRB. In some embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 13. In some of any embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 13. In some of any embodiments, FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. In some of any embodiments, FRB comprises the amino acid sequence of SEQ ID NO: 14.

在任何實施例中之一些中,第二表現卡匣經密碼子最佳化。In some of any of the embodiments, the second representation cassette is codon optimized.

在任何實施例中之一些中,第二表現卡匣包括與SEQ ID NO: 11之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,第二表現卡匣包括SEQ ID NO: 11之核苷酸序列。在任何實施例中之一些中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,第二表現卡匣編碼包括SEQ ID NO: 12之序列的胺基酸序列。In some of any embodiments, the second expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 11. In some of any embodiments, the second expression cassette comprises a nucleotide sequence of SEQ ID NO: 11. In some of any embodiments, the second expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12. In some of any embodiments, the second expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO: 12.

在任何實施例中之一些中,第二表現卡匣進一步包括編碼FKBP12之核苷酸序列。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在任何實施例中之一些中,編碼FKBP12之核苷酸序列包括SEQ ID NO: 21或55之核苷酸序列。在任何實施例中之一些中,FKBP12包括與SEQ ID NO: 22之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,FKBP12包括SEQ ID NO: 22之胺基酸序列。In some of any embodiments, the second expression cassette further comprises a nucleotide sequence encoding FKBP12. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some of any embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21 or 55. In some of any embodiments, FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some of any embodiments, FKBP12 comprises the amino acid sequence of SEQ ID NO: 22.

在任何實施例中之一些中,第二表現卡匣包括與SEQ ID NO: 53或56之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,第二表現卡匣包括SEQ ID NO: 53或56之核苷酸序列。在任何實施例中之一些中,第二表現卡匣編碼與SEQ ID NO: 54、57或128之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,第二表現卡匣編碼包括SEQ ID NO: 54、57或128之序列的胺基酸序列。In some of any embodiments, the second expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 53 or 56. In some of any embodiments, the second expression cassette comprises a nucleotide sequence of SEQ ID NO: 53 or 56. In some of any embodiments, the second expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 54, 57 or 128. In some of any embodiments, the second expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO: 54, 57 or 128.

在任何實施例中之一些中,合成細胞介素β鏈多肽包括介白素2受體次單元β (IL2RB)。在任何實施例中之一些中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在任何實施例中之一些中,編碼合成細胞介素β鏈多肽之核苷酸包括SEQ ID NO: 23或61之核苷酸序列。在一些實施例中,IL2RB包括與SEQ ID NO: 24或62之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,IL2RB包括SEQ ID NO: 24或62之胺基酸序列。In some of any embodiments, the synthetic interleukin beta chain polypeptide includes interleukin 2 receptor subunit beta (IL2RB). In some of any embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some of any embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide include the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, IL2RB includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some of any embodiments, IL2RB includes the amino acid sequence of SEQ ID NO: 24 or 62.

在任何實施例中之一些中,第三表現卡匣進一步包括編碼FKBP12之核苷酸序列。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在任何實施例中之一些中,編碼FKBP12之核苷酸序列包括SEQ ID NO: 21之核苷酸序列。在任何實施例中之一些中,FKBP12包括與SEQ ID NO: 22之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,FKBP12包括SEQ ID NO: 22之胺基酸序列。In some of any embodiments, the third expression cassette further comprises a nucleotide sequence encoding FKBP12. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 21. In some of any embodiments, the nucleotide sequence encoding FKBP12 includes the nucleotide sequence of SEQ ID NO: 21. In some of any embodiments, FKBP12 includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some of any embodiments, FKBP12 includes the amino acid sequence of SEQ ID NO: 22.

在任何實施例中之一些中,第三表現卡匣經密碼子最佳化。In some of any of the embodiments, the third representation cassette is codon optimized.

在任何實施例中之一些中,第三表現卡匣進一步含有編碼FRB之核苷酸序列。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在任何實施例中之一些中,編碼FRB之核苷酸序列包括SEQ ID NO: 13之核苷酸序列。在任何實施例中之一些中,FRB包括與SEQ ID NO: 14之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,FRB包括SEQ ID NO: 14之胺基酸序列。In some of any embodiments, the third expression cassette further contains a nucleotide sequence encoding FRB. In some embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 13. In some of any embodiments, the nucleotide sequence encoding FRB includes the nucleotide sequence of SEQ ID NO: 13. In some of any embodiments, FRB includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. In some of any embodiments, FRB includes the amino acid sequence of SEQ ID NO: 14.

在任何實施例中之一些中,第三表現卡匣包括與SEQ ID NO: 19之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,第三表現卡匣包括SEQ ID NO: 19之核苷酸序列。在任何實施例中之一些中,第三表現卡匣編碼與SEQ ID NO: 20之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,第三表現卡匣編碼包括SEQ ID NO: 20之序列的胺基酸序列。In some of any embodiments, the third expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 19. In some of any embodiments, the third expression cassette comprises a nucleotide sequence of SEQ ID NO: 19. In some of any embodiments, the third expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20. In some of any embodiments, the third expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO: 20.

在任何實施例中之一些中,第三表現卡匣包括與SEQ ID NO: 59之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,第三表現卡匣包括SEQ ID NO: 59之核苷酸序列。在任何實施例中之一些中,第三表現卡匣編碼與SEQ ID NO: 60或129之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,第三表現卡匣編碼包括SEQ ID NO: 60或129之序列的胺基酸序列。In some of any embodiments, the third expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 59. In some of any embodiments, the third expression cassette comprises a nucleotide sequence of SEQ ID NO: 59. In some of any embodiments, the third expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some of any embodiments, the third expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO: 60 or 129.

在任何實施例中之一些中,CAR包括胞外抗原結合域、跨膜域及含有共刺激傳訊域與主活化傳訊域(諸如CD3ζ傳訊域)的胞內域。在一些實施例中,胞外抗原結合域及跨膜域藉由諸如含有鉸鏈域之間隔子序列分隔開。在一些實施例中,胞外抗原結合域包含scFv。In some of any of the embodiments, the CAR includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain containing a co-stimulatory signaling domain and a primary activation signaling domain (such as a CD3ζ signaling domain). In some embodiments, the extracellular antigen binding domain and the transmembrane domain are separated by a spacer sequence such as a hinge domain. In some embodiments, the extracellular antigen binding domain comprises a scFv.

在任何實施例中之一些中,CAR包括scFv域。在一些實施例中,scFv域包括抗螢光異硫氰酸鹽(FITC) E2。In some of any embodiments, the CAR includes a scFv domain. In some embodiments, the scFv domain includes anti-fluorescent isothiocyanate (FITC) E2.

在任何實施例中之一些中,scFv域包括輕鏈可變域(VL)、連接子及重鏈可變域(VH)。在一些實施例中,scFv VL包括與SEQ ID NO: 30或65之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,scFv VL包括SEQ ID NO: 30或65之核苷酸序列。在任何實施例中之一些中,scFv VL包括與SEQ ID NO: 31之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,scFv VL包括SEQ ID NO: 31之胺基酸序列。In some of any embodiments, the scFv domain includes a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH). In some embodiments, the scFv VL includes a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% consistent with the nucleotide sequence of SEQ ID NO: 30 or 65. In some of any embodiments, the scFv VL includes a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% consistent with the amino acid sequence of SEQ ID NO: 31. In some of any embodiments, the scFv VL includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% consistent with the amino acid sequence of SEQ ID NO: 31.

在任何實施例中之一些中,scFv VH包括與SEQ ID NO: 34或67之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,scFv VH包括SEQ ID NO: 34或67之核苷酸序列。在任何實施例中之一些中,scFv VH包括與SEQ ID NO: 35之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,scFv VH包括SEQ ID NO: 35之胺基酸序列。In some of any embodiments, the scFv VH comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some of any embodiments, the scFv VH comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35. In some of any embodiments, the scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35.

在任何實施例中之一些中,scFv連接子包括與SEQ ID NO: 32或66之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,scFv連接子包括SEQ ID NO: 32或66之核苷酸序列。在任何實施例中之一些中,scFv連接子包括與SEQ ID NO: 33之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,scFv連接子包括SEQ ID NO: 33之胺基酸序列。In some of any embodiments, the scFv linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some of any embodiments, the scFv linker comprises a nucleotide sequence of SEQ ID NO: 32 or 66. In some of any embodiments, the scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 33. In some of any embodiments, the scFv linker comprises an amino acid sequence of SEQ ID NO: 33.

在任何實施例中之一些中,scFv包括與SEQ ID NO: 28或64之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,scFv包括SEQ ID NO: 28或64之核苷酸序列。在一些實施例中,scFv包括與SEQ ID NO: 29之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,scFv包括SEQ ID NO: 29之胺基酸序列。In some of any of the embodiments, the scFv comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some of any of the embodiments, the scFv comprises a nucleotide sequence of SEQ ID NO: 28 or 64. In some of the embodiments, the scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some of any of the embodiments, the scFv comprises an amino acid sequence of SEQ ID NO: 29.

在任何實施例中之一些中,CAR包括鉸鏈域。在一些實施例中,鉸鏈域包括短鉸鏈或中等鉸鏈域。In some of any embodiments, the CAR includes a hinge domain. In some embodiments, the hinge domain includes a short hinge or a medium hinge domain.

在任何實施例中之一些中,鉸鏈域包括CD8或IgG。在一些實施例中,CD8鉸鏈包括CD8α鉸鏈。In some of any of the embodiments, the hinge domain comprises CD8 or IgG. In some embodiments, the CD8 hinge comprises a CD8α hinge.

在一些實施例中,CD8α鉸鏈包括與SEQ ID NO: 38或114之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,CD8α鉸鏈包括SEQ ID NO: 38之核苷酸序列。在任何實施例中之一些中,CD8α鉸鏈包括與SEQ ID NO: 39或115之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,CD8α鉸鏈包括SEQ ID NO: 39或115之胺基酸序列。In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some of any embodiments, the CD8 alpha hinge comprises the nucleotide sequence of SEQ ID NO: 38. In some of any embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some of any embodiments, the CD8 alpha hinge comprises the amino acid sequence of SEQ ID NO: 39 or 115.

在任何實施例中之一些中,CAR含有跨膜域。在一些實施例中,跨膜域包括CD8或CD28。在一些實施例中,CD8跨膜域包括CD8α跨膜域。In some of any embodiments, the CAR contains a transmembrane domain. In some embodiments, the transmembrane domain includes CD8 or CD28. In some embodiments, the CD8 transmembrane domain includes a CD8 alpha transmembrane domain.

在任何實施例中之一些中,跨膜域包括與SEQ ID NO: 40之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,跨膜域包括SEQ ID NO: 40之核苷酸序列。在任何實施例中之一些中,跨膜域包括與SEQ ID NO: 41之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,跨膜域包括SEQ ID NO: 41之胺基酸序列。In some of any embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 40. In some of any embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41. In some of any embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41.

在任何實施例中之一些中,CAR包括胞內域。在一些實施例中,胞內域包括共刺激分子傳訊域。In some of any embodiments, the CAR includes an intracellular domain. In some embodiments, the intracellular domain includes a co-stimulatory molecule signaling domain.

在任何實施例中之一些中,胞內域包括4-1BB、CD3ζ及/或CD28之傳訊域。In some of any of the embodiments, the intracellular domain includes the signaling domains of 4-1BB, CD3ζ and/or CD28.

在一些實施例中,4-1BB胞內域包括與SEQ ID NO: 42或69之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,4-1BB胞內域包括SEQ ID NO: 42或69之核苷酸序列。在任何實施例中之一些中,4-1BB胞內域包括與SEQ ID NO: 43之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,4-1BB胞內域包括SEQ ID NO: 43之胺基酸序列。In some embodiments, the 4-1BB intracellular domain includes a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% consistent with the nucleotide sequence of SEQ ID NO: 42 or 69. In some of any embodiments, the 4-1BB intracellular domain includes a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% consistent with the amino acid sequence of SEQ ID NO: 43. In some of any embodiments, the 4-1BB intracellular domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% consistent with the amino acid sequence of SEQ ID NO: 43.

在任何實施例中之一些中,CD3ζ胞內域包括與SEQ ID NO: 46、70、100或118之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,CD3ζ胞內域包括SEQ ID NO: 46、70、100或118之核苷酸序列。在任何實施例中之一些中,CD3ζ胞內域包括與SEQ ID NO: 47之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,CD3ζ胞內域包括SEQ ID NO: 47之胺基酸序列。In some of any embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some of any embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47. In some of any embodiments, the CD3 zeta intracellular domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47.

在任何實施例中之一些中,第四表現卡匣包括與SEQ ID NO: 26、63、71或82之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,第四表現卡匣包括SEQ ID NO: 26、63、71或82之核苷酸序列。在任何實施例中之一些中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在任何實施例中之一些中,第四表現卡匣編碼SEQ ID NO: 27、72或127之胺基酸序列。In some of any embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82. In some of any embodiments, the fourth expression cassette comprises a nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82. In some of any embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27, 72 or 127. In some of any embodiments, the fourth expression cassette encodes an amino acid sequence of SEQ ID NO: 27, 72 or 127.

在任何實施例中之一些中,裂解位點序列各包含2A可裂解連接子序列。In some of any of the embodiments, the cleavage site sequences each comprise a 2A cleavable linker sequence.

在任何實施例中之一些中,編碼2A可裂解連接子序列之各核苷酸不同。In some of any of the embodiments, each nucleotide encoding the 2A cleavable linker sequence is different.

在任何實施例中之一些中,2A可裂解連接子獨立地為T2A、P2A、E2A或F2A裂解位點。In some of any of the embodiments, the 2A cleavable linker is independently a T2A, P2A, E2A, or F2A cleavage site.

在任何實施例中之一些中,2A可裂解連接子獨立地為P2A或T2A。In some of any of the embodiments, the 2A cleavable linker is independently P2A or T2A.

在任何實施例中之一些中,至少一個2A可裂解連接子為P2A,且編碼P2A可裂解連接子之核苷酸序列包含與SEQ ID NO: 17、25、52或58至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。In some of any of the embodiments, at least one 2A cleavable linker is P2A, and the nucleotide sequence encoding the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17, 25, 52 or 58.

在任何實施例中之一些中,編碼P2A可裂解連接子之核苷酸序列述於SEQ ID NO: 17、25、52或58中。In some of any of the embodiments, the nucleotide sequence encoding the P2A cleavable linker is described in SEQ ID NO: 17, 25, 52, or 58.

在任何實施例中之一些中,P2A可裂解連接子包含與SEQ ID NO: 18至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。In some of any of the embodiments, the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.

在任何實施例中之一些中,P2A可裂解連接子包含SEQ ID NO: 18中所述之序列。In some of any of the embodiments, the P2A cleavable linker comprises the sequence set forth in SEQ ID NO: 18.

在任何實施例中之一些中,至少一個2A可裂解連接子為T2A,且編碼T2A可裂解連接子之核苷酸序列包含與SEQ ID NO: 9至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。In some of any of the embodiments, at least one 2A cleavable linker is T2A, and the nucleotide sequence encoding the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9.

在任何實施例中之一些中,編碼T2A可裂解連接子之核苷酸序列述於SEQ ID NO: 9中。In some of any of the embodiments, the nucleotide sequence encoding the T2A cleavable linker is set forth in SEQ ID NO: 9.

在任何實施例中之一些中,T2A可裂解連接子包含與SEQ ID NO: 10至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。In some of any of the embodiments, the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.

在任何實施例中之一些中,T2A可裂解連接子包含SEQ ID NO: 10中所述之序列。In some of any of the embodiments, the T2A cleavable linker comprises the sequence set forth in SEQ ID NO: 10.

在任何實施例中之一些中,裂解位點序列中之至少一者包含弗林蛋白酶(furin)裂解位點序列。In some of any of the embodiments, at least one of the cleavage site sequences comprises a furin cleavage site sequence.

在任何實施例中之一些中,弗林蛋白酶裂解位點序列位於第一表現卡匣與第二表現卡匣之間。In some of any of the embodiments, the furin cleavage site sequence is located between the first expression cassette and the second expression cassette.

在任何實施例中之一些中,編碼弗林蛋白酶裂解位點序列之核苷酸序列包含與SEQ ID NO: 7至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。In some of any of the embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7.

在任何實施例中之一些中,編碼弗林蛋白酶裂解位點序列之核苷酸序列包含SEQ ID NO: 7中所述之序列。在任何實施例中之一些中,弗林蛋白酶裂解位點序列包含與SEQ ID NO: 8之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。In some of any of the embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises the sequence set forth in SEQ ID NO: 7. In some of any of the embodiments, the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

在任何實施例中之一些中,弗林蛋白酶裂解位點序列包含SEQ ID NO: 8之胺基酸序列。In some of any of the embodiments, the furin cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 8.

在任何實施例中之一些中,裂解位點序列包含弗林蛋白酶裂解位點序列及T2A裂解序列(furinT2A)。In some of any of the embodiments, the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furinT2A).

在任何實施例中之一些中,編碼裂解位點序列之核苷酸序列與SEQ ID NO: 5之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。In some of any of the embodiments, the nucleotide sequence encoding the cleavage site sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:5.

在任何實施例中之一些中,編碼裂解位點序列之核苷酸序列包含SEQ ID NO:5之核苷酸序列。In some of any of the embodiments, the nucleotide sequence encoding the cleavage site sequence comprises the nucleotide sequence of SEQ ID NO:5.

在任何實施例中之一些中,裂解位點序列包含與SEQ ID NO: 6之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。In some of any of the embodiments, the cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6.

在任何實施例中之一些中,裂解位點序列包含SEQ ID NO: 6之胺基酸序列。In some of any of the embodiments, the cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 6.

在任何實施例中之一些中,第一表現卡匣及第二表現卡匣藉由furinT2A分隔開,第二表現卡匣及第三表現卡匣藉由P2A分隔開,且第三表現卡匣及第四表現卡匣藉由P2A分隔開。In some of any of the embodiments, the first and second presentation cassettes are separated by furin T2A, the second and third presentation cassettes are separated by P2A, and the third and fourth presentation cassettes are separated by P2A.

在任何實施例中之一些中,構築體包括與SEQ ID NO: 1之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,構築體包括SEQ ID NO: 1之核苷酸序列。In some of any embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some of any embodiments, the construct comprises a nucleotide sequence of SEQ ID NO: 1.

在任何實施例中之一些中,構築體編碼包括與SEQ ID NO: 2之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致之胺基酸序列的多肽。在任何實施例中之一些中,構築體編碼包括SEQ ID NO: 2之胺基酸序列的多肽。In some of any of the embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some of any of the embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

在任何實施例中之一些中,構築體包括與SEQ ID NO: 48之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在任何實施例中之一些中,構築體包括SEQ ID NO: 48之核苷酸序列。In some of any of the embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 48. In some of any of the embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 48.

在任何實施例中之一些中,構築體編碼包括與SEQ ID NO: 49之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致之胺基酸序列的多肽。在任何實施例中之一些中,構築體編碼包括SEQ ID NO: 49之胺基酸序列的多肽。In some of any of the embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49. In some of any of the embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 49.

在一些態樣中,本文提供一種病毒載體,其含有任何實施例中之一些的多順反子構築體。在一些實施例中,病毒載體為慢病毒載體。In some aspects, provided herein is a viral vector comprising a polycistronic construct of any of the embodiments. In some embodiments, the viral vector is a lentiviral vector.

在任何實施例中之一些中,病毒載體進一步含有一或多種表面T細胞活化劑。在一些實施例中,該一或多種表面T細胞活化劑包括CD58、抗-CD3,或CD80。In some of any embodiments, the viral vector further contains one or more surface T cell activators. In some embodiments, the one or more surface T cell activators include CD58, anti-CD3, or CD80.

在一些態樣中,本文提供一種細胞,其含有任何實施例中之一些的病毒載體。在一些實施例中,細胞包括幹細胞或前驅細胞。在一些實施例中,幹細胞包括誘導性富潛能幹細胞(induced pluripotent stem cell;iPSC)。In some aspects, provided herein is a cell containing any of the viral vectors of any of the embodiments. In some embodiments, the cell comprises a stem cell or a progenitor cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC).

在一些實施例中,前驅細胞包括周邊血液單核細胞(peripheral blood mononuclear cell;PBMC)。在一些實施例中,細胞包括T細胞。在一些實施例中,細胞包括細胞毒性先天性淋巴球(cytotoxic innate lymphocyte;CIL)細胞。在一些實施例中,細胞包括自然殺手(natural killer;NK)細胞。In some embodiments, the progenitor cells include peripheral blood mononuclear cells (PBMC). In some embodiments, the cells include T cells. In some embodiments, the cells include cytotoxic innate lymphocytes (CIL). In some embodiments, the cells include natural killer (NK) cells.

在一些態樣中,本文提供一種轉導細胞之方法,其包括使目標細胞與任何實施例中之一些的多順反子構築體中之任一者接觸。在一些實施例中,目標細胞包括幹細胞。在一些實施例中,幹細胞包括誘導性富潛能幹細胞(iPSC)。In some aspects, provided herein is a method of transducing a cell, comprising contacting a target cell with any of the polycistronic constructs of any of the embodiments. In some embodiments, the target cell comprises a stem cell. In some embodiments, the stem cell comprises an induced enriched potential stem cell (iPSC).

在一些實施例中,目標細胞包括前驅細胞。在一些實施例中,前驅細胞包括周邊血液單核細胞(PBMC)。In some embodiments, the target cells include progenitor cells. In some embodiments, the progenitor cells include peripheral blood mononuclear cells (PBMC).

在一些實施例中,目標細胞包括T細胞。在一些實施例中,T細胞包括CD4+或CD8+ T細胞。In some embodiments, the target cell comprises a T cell. In some embodiments, the T cell comprises a CD4+ or CD8+ T cell.

在一些態樣中,任何實施例中之一些的方法進一步包括使目標細胞與以下接觸:(i)靶向內源基因中之目標位點的引導RNA(gRNA),及(ii) RNA引導核酸內切酶,從而將核苷酸序列插入內源基因中。In some aspects, the methods of any of the embodiments further comprise contacting the target cell with: (i) a guide RNA (gRNA) that targets a target site in an endogenous gene, and (ii) an RNA-guided endonuclease to insert the nucleotide sequence into the endogenous gene.

在一些態樣中,本文提供一種在目標細胞中表現嵌合抗原受體及/或合成細胞介素受體之方法。在一些實施例中,目標細胞包括幹細胞。在一些實施例中,幹細胞包括誘導性富潛能幹細胞(iPSC)。In some aspects, provided herein is a method for expressing a chimeric antigen receptor and/or a synthetic interleukin receptor in a target cell. In some embodiments, the target cell comprises a stem cell. In some embodiments, the stem cell comprises an induced enriched-potential stem cell (iPSC).

在一些實施例中,目標細胞包括前驅細胞。在一些實施例中,前驅細胞包括周邊血液單核細胞(PBMC)。In some embodiments, the target cells include progenitor cells. In some embodiments, the progenitor cells include peripheral blood mononuclear cells (PBMC).

在一些實施例中,目標細胞包括T細胞。在一些實施例中,T細胞包括CD4+或CD8+ T細胞。In some embodiments, the target cell comprises a T cell. In some embodiments, the T cell comprises a CD4+ or CD8+ T cell.

在任何實施例中之一些中,該方法係離體或在活體外進行。In some of any of the embodiments, the method is performed ex vivo or in vitro.

在任何實施例中之一些中,該方法係在活體內進行。In some of any of the embodiments, the method is performed in vivo.

在一些態樣中,本文提供一種細胞,其係藉由任何所提供實施例中之一些的方法產生。In some aspects, provided herein is a cell produced by the methods of any of the provided embodiments.

在一些態樣中,本文提供一種向個體投與一些實施例之細胞之方法。在一些實施例中,嵌合抗原受體能夠靶向與個體之疾病或病狀相關的抗原,且該個體患有能夠藉由嵌合抗原受體治療之疾病或病狀。在一些實施例中,疾病或病狀為癌症。In some aspects, provided herein is a method of administering the cells of some embodiments to an individual. In some embodiments, the chimeric antigen receptor is capable of targeting an antigen associated with a disease or condition in the individual, and the individual suffers from a disease or condition that can be treated by the chimeric antigen receptor. In some embodiments, the disease or condition is cancer.

在一些態樣中,本文提供一種向個體投與任何所提供實施例中之一些的病毒載體之方法。在一些實施例中,嵌合抗原受體能夠靶向與個體之疾病或病狀相關的抗原,且該個體患有能夠藉由嵌合抗原受體治療之疾病或病狀。在一些實施例中,疾病或病狀為癌症。In some aspects, provided herein is a method of administering a viral vector of any of the provided embodiments to an individual. In some embodiments, the chimeric antigen receptor is capable of targeting an antigen associated with a disease or condition in the individual, and the individual has a disease or condition that can be treated by the chimeric antigen receptor. In some embodiments, the disease or condition is cancer.

在一些實施例中,CAR為靶向能夠與細胞表面上與疾病或病狀相關之抗原結合之配體的CAR。在一些實施例中,所提供實施例中之任一者的CAR為針對FITC之抗FITC CAR,且該配體為由FITC及能夠與目標細胞上之表面分子或受體結合之結合分子構成的雙官能配體。在一些實施例中,該方法進一步包括投與雙官能配體以標記個體中之癌細胞,其中雙官能配體特異性結合腫瘤上表現之分子。在一些實施例中,雙官能配體為FITC-葉酸。在一些實施例中,癌症為骨肉瘤。In some embodiments, the CAR is a CAR that targets a ligand that can bind to an antigen on the surface of a cell that is associated with a disease or condition. In some embodiments, the CAR of any one of the provided embodiments is an anti-FITC CAR for FITC, and the ligand is a bifunctional ligand composed of FITC and a binding molecule that can bind to a surface molecule or receptor on a target cell. In some embodiments, the method further includes administering a bifunctional ligand to mark cancer cells in an individual, wherein the bifunctional ligand specifically binds to a molecule expressed on a tumor. In some embodiments, the bifunctional ligand is FITC-folate. In some embodiments, the cancer is osteosarcoma.

在任何實施例中之一些中,該方法進一步包括向個體投與非生理學配體。在一些實施例中,非生理學配體能夠與合成細胞介素受體結合且誘導細胞中之γ細胞介素傳訊。在一些實施例中,非生理學配體包括雷帕黴素(rapamycin)或雷帕黴素類似物。In some of any of the embodiments, the method further comprises administering a non-physiological ligand to the individual. In some embodiments, the non-physiological ligand is capable of binding to a synthetic interleukin receptor and inducing interleukin-γ signaling in the cell. In some embodiments, the non-physiological ligand comprises rapamycin or a rapamycin analog.

相關申請案之交互參考Cross-references to related applications

本申請案主張2022年11月4日申請之美國臨時申請案第63/422,920號、2023年3月1日申請之美國臨時申請案第63/449,289號及2023年5月15日申請之美國臨時申請案第63/466,714號之優先權,該等臨時申請案之名稱全部均為「POLYNUCLEOTIDE CONSTRUCT AND RELATED VIRAL VECTORS AND METHODS」,其內容以全文引用之方式併入。 電子序列表之參考 This application claims priority to U.S. Provisional Application No. 63/422,920 filed on November 4, 2022, U.S. Provisional Application No. 63/449,289 filed on March 1, 2023, and U.S. Provisional Application No. 63/466,714 filed on May 15, 2023, all of which are entitled "POLYNUCLEOTIDE CONSTRUCT AND RELATED VIRAL VECTORS AND METHODS", the contents of which are incorporated by reference in their entirety. Reference to Electronic Sequence Listing

電子序列表(260132000940SEQLIST.xml;大小246,342位元組;及創建日期:2023年10月31日)之內容以全文引用之方式併入本文中。The contents of the electronic sequence listing (260132000940SEQLIST.xml; size 246,342 bytes; and creation date: October 31, 2023) are incorporated herein by reference in their entirety.

本發明大體上係關於一種多核苷酸構築體,其包含編碼至少兩種合成受體之連續多核苷酸序列;及其使用方法。在一些實施例中,多核苷酸構築體為多順反子構築體,其編碼合成細胞介素受體、合成嵌合抗原受體(CAR)及可自由擴散的FRB,其中細胞介素受體對雷帕黴素結合有反應。有利的是,FRB降低雷帕黴素對於經工程改造以表現本文提供之多順反子構築體之細胞中之mTOR的抑制性作用。可自由擴散的FRB之表現可促進經工程改造細胞之恆定活化及增殖。The present invention generally relates to a polynucleotide construct comprising a contiguous polynucleotide sequence encoding at least two synthetic receptors; and methods of use thereof. In some embodiments, the polynucleotide construct is a polycistronic construct encoding a synthetic interleukin receptor, a synthetic chimeric antigen receptor (CAR), and a freely diffusible FRB, wherein the interleukin receptor is responsive to rapamycin binding. Advantageously, the FRB reduces the inhibitory effect of rapamycin on mTOR in cells engineered to express the polycistronic constructs provided herein. Expression of the freely diffusible FRB can promote homeostatic activation and proliferation of engineered cells.

本發明亦提供,多順反子構築體之5'至3'順序對於由構築體編碼之多肽之表現的非常重要。在一些實施例中,多核苷酸構築體之5'至3'順序改良所編碼之多肽的表現。在一些實施例中,本文提供之多順反子構築體包含編碼FRB之核苷酸在5'端,其改良FRB表現且與針對雷帕黴素介導之免疫抑制之保護增加相關。此外,意外地發現,CAR表現(即使當位於構築體之3'端時)亦足夠高以介導被其中表現多核苷酸構築體之細胞的抗原定向殺死(antigen-directed killing)。The present invention also provides that the 5' to 3' sequence of the polycistronic construct is very important for the expression of the polypeptide encoded by the construct. In some embodiments, the 5' to 3' sequence of the polynucleotide construct improves the expression of the encoded polypeptide. In some embodiments, the polycistronic construct provided herein comprises nucleotides encoding FRB at the 5' end, which improves FRB expression and is associated with increased protection against rapamycin-mediated immunosuppression. In addition, it was unexpectedly found that CAR expression (even when located at the 3' end of the construct) is high enough to mediate antigen-directed killing of cells in which the polynucleotide construct is expressed.

在一些態樣中,本文提供一種多順反子構築體,其包含四個藉由裂解位點序列分隔開之表現卡匣。在一些實施例中,四個表現卡匣包含以5'至3'順序:第一表現卡匣,其包含編碼FRB之核苷酸序列;第二表現卡匣,其包含編碼合成細胞介素γ鏈多肽之核苷酸序列;第三表現卡匣,其包含編碼合成細胞介素β鏈多肽之核苷酸序列;及第四表現卡匣,其包含編碼CAR之核苷酸序列。In some aspects, provided herein is a polycistronic construct comprising four expression cassettes separated by cleavage site sequences. In some embodiments, the four expression cassettes comprise, in 5' to 3' order: a first expression cassette comprising a nucleotide sequence encoding FRB; a second expression cassette comprising a nucleotide sequence encoding a synthetic interleukin γ chain polypeptide; a third expression cassette comprising a nucleotide sequence encoding a synthetic interleukin β chain polypeptide; and a fourth expression cassette comprising a nucleotide sequence encoding CAR.

在一些態樣中,本文提供一種病毒載體,其包含本文所揭示之多順反子構築體中之任一者。在一些態樣中,本文提供一種細胞,其包含本文所揭示之病毒載體中之任一者。In some aspects, provided herein is a viral vector comprising any one of the polycistronic constructs disclosed herein. In some aspects, provided herein is a cell comprising any one of the viral vectors disclosed herein.

在一些態樣中,本文提供一種轉導細胞之方法,其包含使目標細胞與本文所揭示之多順反子構築體中之任一者接觸。In some aspects, provided herein is a method of transducing a cell, comprising contacting the target cell with any of the multicistronic constructs disclosed herein.

在一些態樣中,本文提供一種在目標細胞中表現嵌合抗原受體及/或合成細胞介素受體之方法。在一些態樣中,本文提供一種細胞,其係藉由本文所揭示之方法中之任一者產生。In some aspects, provided herein is a method of expressing a chimeric antigen receptor and/or synthesizing an interleukin receptor in a target cell. In some aspects, provided herein is a cell produced by any of the methods disclosed herein.

在一些態樣中,本文提供一種向個體投與本文所揭示之細胞中之任一者之方法。在一些態樣中,本文提供一種向個體投與本文所揭示之病毒載體中之任一者之方法。In some aspects, provided herein is a method of administering any of the cells disclosed herein to an individual. In some aspects, provided herein is a method of administering any of the viral vectors disclosed herein to an individual.

本申請案提及的所有公開案,包括專利文獻、科學論文及資料庫,均出於所有目的以全文引用之方式併入本文中,其引用的程度如同各個別公開案以引用的方式個別地併入一般。若本文中所述之定義與以引用之方式併入本文中之專利、申請案、公開申請案及其他公開案中所述之定義相反或不一致,則本文中所述之定義優先於以引用之方式併入本文中之定義。All publications, including patent documents, scientific papers, and databases, cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall prevail over the definition incorporated herein by reference.

本文所用之章節標題僅出於組織目的且不應解釋為限制所描述之主題。 I.多順反子構築體 The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. Polycistronic Constructs

本文提供編碼一或多種個別蛋白質之多順反子構築體。在一些實施例中,多順反子構築體包含一個、兩個、三個或四個各自編碼個別蛋白質之表現卡匣。在一些實施例中,多順反子構築體包含四個各自編碼個別蛋白質之表現卡匣。在一些實施例中,表現卡匣藉由可裂解連接子分隔開。Provided herein are polycistronic constructs encoding one or more individual proteins. In some embodiments, the polycistronic construct comprises one, two, three, or four expression cassettes, each encoding an individual protein. In some embodiments, the polycistronic construct comprises four expression cassettes, each encoding an individual protein. In some embodiments, the expression cassettes are separated by cleavable linkers.

在一些實施例中,本文提供之多順反子構築體包含編碼FRB之核苷酸序列。在一些實施例中,本文提供之多順反子構築體包含編碼嵌合抗原受體(CAR)之核苷酸序列。在一些實施例中,本文提供之多順反子構築體包含編碼合成細胞介素多肽之核苷酸序列。在一些實施例中,合成細胞介素多肽包含合成細胞介素γ鏈多肽及合成細胞介素β鏈多肽。在一些實施例中,合成細胞介素γ鏈包含介白素2受體次單元γ (IL2RG)。在一些實施例中,合成細胞介素γ鏈進一步包含FRB。在一些實施例中,合成細胞介素β鏈包含介白素2受體次單元β (IL2RB)。在一些實施例中,合成細胞介素γ鏈進一步包含FKBP12。在其他實施例中,合成細胞介素γ鏈包含介白素2受體次單元γ (IL2RG)。在一些實施例中,合成細胞介素γ鏈進一步包含FKBP12。在一些實施例中,合成細胞介素β鏈包含介白素2受體次單元β (IL2RB)。在一些實施例中,合成細胞介素β鏈進一步包含FRB。In some embodiments, the polycistronic constructs provided herein include a nucleotide sequence encoding FRB. In some embodiments, the polycistronic constructs provided herein include a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the polycistronic constructs provided herein include a nucleotide sequence encoding a synthetic interleukin polypeptide. In some embodiments, the synthetic interleukin polypeptide includes a synthetic interleukin γ chain polypeptide and a synthetic interleukin β chain polypeptide. In some embodiments, the synthetic interleukin γ chain includes interleukin 2 receptor subunit γ (IL2RG). In some embodiments, the synthetic interleukin γ chain further includes FRB. In some embodiments, the synthetic interleukin β chain includes interleukin 2 receptor subunit β (IL2RB). In some embodiments, the synthetic interleukin gamma chain further comprises FKBP12. In other embodiments, the synthetic interleukin gamma chain comprises interleukin 2 receptor subunit gamma (IL2RG). In some embodiments, the synthetic interleukin gamma chain further comprises FKBP12. In some embodiments, the synthetic interleukin beta chain comprises interleukin 2 receptor subunit beta (IL2RB). In some embodiments, the synthetic interleukin beta chain further comprises FRB.

在一些實施例中,本文提供之多順反子構築體包含編碼FRB、合成細胞介素多肽及CAR之核苷酸序列。In some embodiments, the polycistronic constructs provided herein comprise nucleotide sequences encoding FRB, a synthetic interleukin polypeptide, and a CAR.

在一些實施例中,多順反子構築體包含以5'至3'順序編碼FRB之核苷酸序列、編碼合成細胞介素多肽之核苷酸序列及編碼CAR之核苷酸序列。在一些實施例中,編碼合成細胞介素多肽之核苷酸序列包含以5'至3'順序編碼FRB:IL2RG的第一核苷酸序列及編碼FKBP12:IL2RB的第二核苷酸序列。在一些實施例中,編碼合成細胞介素多肽之核苷酸序列包含以5'至3'順序編碼FKBP12:IL2RG的第一核苷酸序列及編碼sFRB:IL2RB的第二核苷酸序列。In some embodiments, the polycistronic construct comprises a nucleotide sequence encoding FRB in 5' to 3' order, a nucleotide sequence encoding a synthetic interleukin polypeptide, and a nucleotide sequence encoding CAR. In some embodiments, the nucleotide sequence encoding the synthetic interleukin polypeptide comprises a first nucleotide sequence encoding FRB:IL2RG in 5' to 3' order and a second nucleotide sequence encoding FKBP12:IL2RB. In some embodiments, the nucleotide sequence encoding the synthetic interleukin polypeptide comprises a first nucleotide sequence encoding FKBP12:IL2RG in 5' to 3' order and a second nucleotide sequence encoding sFRB:IL2RB.

在一個態樣中,本文提供一種多順反子構築體,其包含以5'至3'順序:(a)第一表現卡匣,其包含編碼FRB之核苷酸序列;(b)第二表現卡匣,其包含編碼合成細胞介素γ鏈多肽之核苷酸序列;(c)第三表現卡匣,其包含編碼合成細胞介素β鏈多肽之核苷酸序列;及(d)第四表現卡匣,其包含編碼嵌合抗原受體(CAR)之核苷酸序列,其中該等表現卡匣各藉由編碼裂解位點序列之核苷酸序列分隔開。 A.胞質FRB In one embodiment, the present invention provides a polycistronic construct comprising, in 5' to 3' order: (a) a first expression cassette comprising a nucleotide sequence encoding FRB; (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic interleukin γ chain polypeptide; (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic interleukin β chain polypeptide; and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence. A. Cytoplasmic FRB

在一些實施例中,多順反子構築體之表現卡匣編碼FRB域。FRB域為來源於mTOR蛋白激酶之大約270個鹼基對(bp)域。其可在胞質液中作為可自由擴散的可溶性蛋白質表現。In some embodiments, the expression cassette of the polycistronic construct encodes the FRB domain. The FRB domain is an approximately 270 base pair (bp) domain derived from the mTOR protein kinase. It can be expressed in the cytosol as a freely diffusible soluble protein.

在一些實施例中,多順反子構築體中之第一表現卡匣包含編碼FRB之核苷酸序列。在一些實施例中,當表現FRB時,其為可自由擴散的可溶性蛋白質。In some embodiments, the first expression cassette in the polycistronic construct comprises a nucleotide sequence encoding FRB. In some embodiments, when FRB is expressed, it is a freely diffusible soluble protein.

在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少80%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少85%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少90%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少95%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少96%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少97%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少98%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少99%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少100%一致。在一些實施例中,編碼FRB之核苷酸序列包含SEQ ID NO: 3、13或50之核苷酸序列。在一些實施例中,編碼FRB之核苷酸序列由SEQ ID NO: 3、13或50之核苷酸序列組成。In some embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 3, 13 or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 80% identical to the nucleotide sequence of SEQ ID NO: 3, 13 or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 85% identical to the nucleotide sequence of SEQ ID NO: 3, 13 or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 90% identical to the nucleotide sequence of SEQ ID NO: 3, 13 or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 95% identical to the nucleotide sequence of SEQ ID NO: 3, 13 or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 96% identical to the nucleotide sequence of SEQ ID NO: 3, 13 or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 97% identical to the nucleotide sequence of SEQ ID NO: 3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 98% identical to the nucleotide sequence of SEQ ID NO: 3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 99% identical to the nucleotide sequence of SEQ ID NO: 3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO: 3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB consists of the nucleotide sequence of SEQ ID NO: 3, 13, or 50.

在一些實施例中,FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,FRB包含SEQ ID NO: 4、14或51之胺基酸序列。在一些實施例中,FRB由SEQ ID NO: 4、14或51之胺基酸序列組成。In some embodiments, the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4, 14 or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 4, 14 or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 4, 14 or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4, 14 or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4, 14 or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence of SEQ ID NO: 4, 14, or 51. In some embodiments, FRB consists of the amino acid sequence of SEQ ID NO: 4, 14 or 51.

在一些實施例中,合成細胞介素受體複合物包含與配體或包含配體之複合物結合的胞質多肽。In some embodiments, the synthetic interleukin receptor complex comprises a cytoplasmic polypeptide bound to a ligand or a complex comprising a ligand.

有利的是,胞質FRB賦予對非生理學配體(例如雷帕黴素或雷帕黴素類似物)之免疫抑制作用的抗性。 B.合成細胞介素受體 Advantageously, cytoplasmic FRBs confer resistance to the immunosuppressive effects of non-physiological ligands such as rapamycin or rapamycin analogs. B. Synthetic interleukin receptors

在一些實施例中,多順反子構築體之表現卡匣編碼合成細胞介素受體。本發明之合成細胞介素受體包含合成γ鏈及合成β鏈,其各自包含二聚域。二聚域在非生理學配體存在下進行可控制二聚,從而活化傳訊合成細胞介素受體。In some embodiments, the expression cassette of the polycistronic construct encodes a synthetic interleukin receptor. The synthetic interleukin receptor of the present invention comprises a synthetic gamma chain and a synthetic beta chain, each of which comprises a dimerization domain. The dimerization domain can controllably dimerize in the presence of a non-physiological ligand, thereby activating the signaling synthetic interleukin receptor.

合成γ鏈多肽包含第一二聚域、第一跨膜域及介白素-2受體次單元γ (IL-2RG)細胞內域。二聚域可為細胞外(N端至跨膜域)或細胞內(C端至跨膜域以及N端或C端至IL-2G細胞內域)。The synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG). The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N-terminal or C-terminal to the IL-2G intracellular domain).

合成β鏈多肽包含第二二聚域、第二跨膜域及細胞內域,該細胞內域選自介白素-2受體次單元β (IL-2RB)細胞內域、介白素-7受體次單元β (IL-7RB)細胞內域或介白素-21受體次單元β (IL-21RB)細胞內域。二聚域可為細胞外域(N端至跨膜域)或細胞內域(C端至跨膜域以及N端或C端至IL-2RB或IL-7RB細胞內域)。The synthetic β-chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain, wherein the intracellular domain is selected from the intracellular domain of interleukin-2 receptor subunit β (IL-2RB), the intracellular domain of interleukin-7 receptor subunit β (IL-7RB) or the intracellular domain of interleukin-21 receptor subunit β (IL-21RB). The dimerization domain can be an extracellular domain (N-terminal to the transmembrane domain) or an intracellular domain (C-terminal to the transmembrane domain and N-terminal or C-terminal to the intracellular domain of IL-2RB or IL-7RB).

在一些實施例中,本文提供之多順反子構築體包含一或多個編碼合成細胞介素受體之核苷酸序列。在一些實施例中,該一或多個核苷酸序列對應於一或多個表現卡匣。在一些實施例中,本文提供之多核苷酸構築體包含一個編碼IL2RG之表現卡匣及編碼IL2RB之第二表現卡匣。In some embodiments, the polycistronic constructs provided herein comprise one or more nucleotide sequences encoding a synthetic interleukin receptor. In some embodiments, the one or more nucleotide sequences correspond to one or more expression cassettes. In some embodiments, the polynucleotide constructs provided herein comprise one expression cassette encoding IL2RG and a second expression cassette encoding IL2RB.

在一些實施例中,合成γ鏈多肽由編碼訊號肽之核酸序列編碼。在一些實施例中,合成β鏈多肽由編碼訊號肽之核酸序列編碼。熟習此項技術者容易熟悉可提供訊號以運輸細胞中之新生蛋白質的訊號肽。可採用各種訊號肽中之任一者。In some embodiments, the synthetic gamma chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. In some embodiments, the synthetic beta chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. Those skilled in the art are readily familiar with signal peptides that can provide signals to transport nascent proteins in cells. Any of a variety of signal peptides can be used.

在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少80%一致。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少85%一致。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少90%一致。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少95%一致。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少96%一致。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少97%一致。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少98%一致。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少99%一致。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少100%一致。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸包含SEQ ID NO: 15之核苷酸序列。在一些實施例中,編碼合成細胞介素γ鏈多肽之核苷酸由SEQ ID NO: 15之核苷酸序列組成。In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 80% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 85% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 90% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 95% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 96% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 97% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 98% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 99% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide are at least 100% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic interleukin gamma chain polypeptide comprise the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the synthetic interleukin gamma chain polypeptide consists of the nucleotide sequence of SEQ ID NO: 15.

在一些實施例中,合成細胞介素γ鏈多肽包含介白素2受體次單元γ (IL2RG)。在一些實施例中,IL2RG包含與SEQ ID NO: 16之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,IL2RG包含與SEQ ID NO: 16之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,IL2RG包含與SEQ ID NO: 16之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,IL2RG包含與SEQ ID NO: 16之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,IL2RG包含與SEQ ID NO: 16之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,IL2RG包含與SEQ ID NO: 16之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,IL2RG包含與SEQ ID NO: 16之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,IL2RG包含與SEQ ID NO: 16之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,IL2RG包含與SEQ ID NO: 16之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,IL2RG包含與SEQ ID NO: 16之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,IL2RG包含SEQ ID NO: 16之胺基酸序列。在一些實施例中,IL2RG由SEQ ID NO: 16之胺基酸序列組成。In some embodiments, the synthetic interleukin gamma chain polypeptide comprises interleukin 2 receptor subunit gamma (IL2RG). In some embodiments, IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, IL2RG consists of the amino acid sequence of SEQ ID NO: 16.

在一些實施例中,第二表現卡匣進一步包含編碼FRB之核苷酸序列。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少80%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少85%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少90%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少95%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少96%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少97%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少98%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少99%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少100%一致。In some embodiments, the second expression cassette further comprises a nucleotide sequence encoding FRB. In some embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 80% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 85% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 90% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 95% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 96% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 97% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 98% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 99% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO: 13.

在一些實施例中,編碼FRB之核苷酸序列包含SEQ ID NO: 13之核苷酸序列。在一些實施例中,編碼FRB之核苷酸序列由SEQ ID NO: 13之核苷酸序列組成。In some embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB consists of the nucleotide sequence of SEQ ID NO: 13.

在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,FRB包含SEQ ID NO: 14之胺基酸序列。在一些實施例中,FRB由SEQ ID NO: 14之胺基酸序列組成。In some embodiments, the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB consists of the amino acid sequence of SEQ ID NO: 14.

在一些實施例中,第二表現卡匣經密碼子最佳化。In some embodiments, the second representation cassette is codon optimized.

在一些實施例中,第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少100%一致的核苷酸序列。In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 11.

在一些實施例中,第二表現卡匣包含SEQ ID NO: 11之核苷酸序列。在一些實施例中,第二表現卡匣由SEQ ID NO: 11之核苷酸序列組成。In some embodiments, the second expression cassette comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette consists of the nucleotide sequence of SEQ ID NO: 11.

在一些實施例中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,第二表現卡匣編碼包含SEQ ID NO: 12之序列的胺基酸序列。在一些實施例中,第二表現卡匣編碼由SEQ ID NO: 12之序列組成的胺基酸序列。In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 12. In some embodiments, the second expression cassette encodes an amino acid sequence consisting of SEQ ID NO: 12.

在一些實施例中,第二表現卡匣進一步包含編碼FKBP12之核苷酸序列。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少80%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少85%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少90%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少95%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少96%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少97%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少98%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少99%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少100%一致。在一些實施例中,編碼FKBP12之核苷酸序列包含SEQ ID NO: 21或55之核苷酸序列。在一些實施例中,編碼FKBP12之核苷酸序列由SEQ ID NO: 21或55之核苷酸序列組成。In some embodiments, the second expression cassette further comprises a nucleotide sequence encoding FKBP12. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 85% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 90% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 95% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 96% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 97% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 98% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 99% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 consists of the nucleotide sequence of SEQ ID NO: 21 or 55.

在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,FKBP12包含SEQ ID NO: 22之胺基酸序列。在一些實施例中,FKBP12由SEQ ID NO: 22之胺基酸序列組成。In some embodiments, FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 consists of an amino acid sequence of SEQ ID NO: 22.

在一些實施例中,第三表現卡匣包含與SEQ ID NO: 53或56之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 53或56之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 53或56之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 53或56之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 53或56之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 53或56之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 53或56之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 53或56之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 53或56之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含含有SEQ ID NO: 53或56之核苷酸序列的核苷酸序列。在一些實施例中,第三表現卡匣由包含SEQ ID NO: 53或56之核苷酸序列的核苷酸序列組成。In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the third expression cassette comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the third expression cassette consists of a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 53 or 56.

在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 54、57或128之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 54、57或128之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 54、57或128之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 54、57或128之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 54、57或128之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 54、57或128之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 54、57或128之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 54、57或128之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 54、57或128之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼包含SEQ ID NO: 54、57或128之胺基酸序列的胺基酸序列。在一些實施例中,第三表現卡匣編碼由SEQ ID NO: 54、57或128之胺基酸序列組成的胺基酸序列。In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 54, 57, or 128. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 54, 57, or 128. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 54, 57, or 128. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 54, 57, or 128. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 54, 57, or 128. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 54, 57, or 128. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 54, 57, or 128. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 54, 57, or 128. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 54, 57, or 128. In some embodiments, the third expression cassette encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 54, 57, or 128. In some embodiments, the third expression cassette encodes an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 54, 57 or 128.

在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少80%一致。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少85%一致。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少90%一致。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少95%一致。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少96%一致。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少97%一致。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少98%一致。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少99%一致。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少100%一致。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸包含SEQ ID NO: 23或61之核苷酸序列。在一些實施例中,編碼合成細胞介素β鏈多肽之核苷酸由SEQ ID NO: 23或61之核苷酸序列組成。In some embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 80% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 85% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 90% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 95% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 96% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 97% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 98% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 99% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic interleukin beta chain polypeptide are at least 100% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotide encoding the synthetic interleukin β chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotide encoding the synthetic interleukin β chain polypeptide consists of the nucleotide sequence of SEQ ID NO: 23 or 61.

在一些實施例中,合成細胞介素β鏈多肽包含介白素2受體次單元β (IL2RB)。In some embodiments, the synthetic interleukin β chain polypeptide comprises interleukin 2 receptor subunit β (IL2RB).

在一些實施例中,IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,IL2RB包含SEQ ID NO: 24或62之胺基酸序列。在一些實施例中,IL2RB由SEQ ID NO: 24或62之胺基酸序列組成。In some embodiments, IL2RB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB comprises an amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, IL2RB consists of an amino acid sequence of SEQ ID NO: 24 or 62.

在一些實施例中,第三表現卡匣進一步包含編碼FKBP12之核苷酸序列。In some embodiments, the third expression cassette further comprises a nucleotide sequence encoding FKBP12.

在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少80%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少85%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少90%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少95%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少96%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少97%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少98%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少99%一致。在一些實施例中,編碼FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少100%一致。在一些實施例中,編碼FKBP12之核苷酸序列包含SEQ ID NO: 21之核苷酸序列。在一些實施例中,編碼FKBP12之核苷酸序列由SEQ ID NO: 21之核苷酸序列組成。In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 85% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 90% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 95% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 96% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 97% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 98% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 99% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding FKBP12 consists of the nucleotide sequence of SEQ ID NO: 21.

在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,FKBP12包含與SEQ ID NO: 22之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,FKBP12包含SEQ ID NO: 22之胺基酸序列。在一些實施例中,FKBP12由SEQ ID NO: 22之胺基酸序列組成。In some embodiments, FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 comprises an amino acid sequence of SEQ ID NO: 22. In some embodiments, FKBP12 consists of an amino acid sequence of SEQ ID NO: 22.

在一些實施例中,第三表現卡匣經密碼子最佳化。In some embodiments, the third representation cassette is codon optimized.

在一些實施例中,第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含SEQ ID NO: 19之核苷酸序列。在一些實施例中,第三表現卡匣由SEQ ID NO: 19之核苷酸序列組成。In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette comprises the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the third expression cassette consists of the nucleotide sequence of SEQ ID NO: 19.

在一些實施例中,第三表現卡匣包含與SEQ ID NO: 59之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 59之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 59之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 59之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 59之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 59之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 59之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 59之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含與SEQ ID NO: 59之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,第三表現卡匣包含SEQ ID NO: 59之核苷酸序列。在一些實施例中,第三表現卡匣由SEQ ID NO: 59之核苷酸序列組成。In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the third expression cassette comprises the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the third expression cassette consists of the nucleotide sequence of SEQ ID NO: 59.

在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60或129之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60或129之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60或129之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60或129之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60或129之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60或129之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60或129之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60或129之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60或129之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼包含SEQ ID NO: 60或129之序列的胺基酸序列。在一些實施例中,第三表現卡匣編碼由SEQ ID NO: 60或129之序列組成的胺基酸序列。In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO: 60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence consisting of the sequence of SEQ ID NO: 60 or 129.

在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼與SEQ ID NO: 60之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,第三表現卡匣編碼包含SEQ ID NO: 60之序列的胺基酸序列。在一些實施例中,第三表現卡匣編碼由SEQ ID NO: 60之序列組成的胺基酸序列。In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 60. In some embodiments, the third expression cassette encodes an amino acid sequence consisting of SEQ ID NO: 60.

在一些實施例中,第三表現卡匣進一步包含編碼FRB之核苷酸序列。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少80%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少85%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少90%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少95%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少96%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少97%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少98%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少99%一致。在一些實施例中,編碼FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少100%一致。在一些實施例中,編碼FRB之核苷酸序列包含SEQ ID NO: 13之核苷酸序列。在一些實施例中,編碼FRB之核苷酸序列由SEQ ID NO: 13之核苷酸序列組成。In some embodiments, the third expression cassette further comprises a nucleotide sequence encoding FRB. In some embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 80% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 85% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 90% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 95% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 96% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 97% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 98% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 99% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB consists of the nucleotide sequence of SEQ ID NO: 13.

在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,FRB包含與SEQ ID NO: 14之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,FRB包含SEQ ID NO: 14之胺基酸序列。在一些實施例中,FRB由SEQ ID NO: 14之胺基酸序列組成。 1.細胞內域 In some embodiments, the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the FRB comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, FRB comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, FRB comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, FRB comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, FRB comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, FRB comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, FRB consists of an amino acid sequence of SEQ ID NO: 14. 1. Intracellular domain

在一些實施例中,第一跨膜受體蛋白之細胞內傳訊域包含介白素-2受體次單元γ (IL2Rg)域。In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain.

在一些實施例中,合成細胞介素受體包含:第一跨膜受體蛋白,其包含IL-2RG細胞內域、第一二聚域;第二跨膜受體蛋白,其包含IL-2RB細胞內域及第二二聚域。In some embodiments, the synthetic interleukin receptor comprises: a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain; and a second transmembrane receptor protein comprising an IL-2RB intracellular domain and a second dimerization domain.

在一些實施例中,合成β鏈包含介白素-2受體次單元β (IL2RB)細胞內域。IL2RB亦稱為IL15RB或CD122。因此,當在本文中提及時,IL2RB亦可意謂IL15RB。即,該等術語在本發明中可互換使用。In some embodiments, the synthetic beta chain comprises the intracellular domain of interleukin-2 receptor subunit beta (IL2RB). IL2RB is also known as IL15RB or CD122. Therefore, when referred to herein, IL2RB may also mean IL15RB. That is, these terms can be used interchangeably in the present invention.

在一些實施例中,合成細胞介素受體包含:第一跨膜受體蛋白,其包含IL-2RG細胞內域、第一二聚域;第二跨膜受體蛋白,其包含IL-7RB細胞內域及第二二聚域。In some embodiments, the synthetic interleukin receptor comprises: a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain; and a second transmembrane receptor protein comprising an IL-7RB intracellular domain and a second dimerization domain.

在一些實施例中,合成β鏈包含介白素-7受體次單元β (IL7RB)細胞內域。In some embodiments, the synthetic beta chain comprises the interleukin-7 receptor subunit beta (IL7RB) intracellular domain.

在一些實施例中,合成細胞介素受體包含:第一跨膜受體蛋白,其包含IL-2RG細胞內域、第一二聚域;第二跨膜受體蛋白,其包含IL-21RB細胞內域及第二二聚域。In some embodiments, the synthetic interleukin receptor comprises: a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain; and a second transmembrane receptor protein comprising an IL-21RB intracellular domain and a second dimerization domain.

在一些實施例中,合成β鏈包含介白素-21受體次單元β (IL21RB)細胞內域。 2.二聚域 In some embodiments, the synthetic beta chain comprises the intracellular domain of interleukin-21 receptor subunit beta (IL21RB). 2. Dimerization domain

二聚域可為異二聚域,包括但不限於大小為12 kD之FK506結合蛋白(FKBP)以及FKBP12-雷帕黴素結合(FRB)域,此項技術中已知其在雷帕黴素或雷帕黴素類似物存在下進行二聚。The dimerization domain may be a heterodimerization domain, including but not limited to the 12 kD FK506 binding protein (FKBP) and FKBP12-rapamycin binding (FRB) domain, which are known in the art to dimerize in the presence of rapamycin or a rapamycin analog.

或者,第一二聚域及第二二聚域可為大小為12 kD之FK506結合蛋白(FKBP)以及鈣調神經磷酸酶域,此項技術中已知其在FK506或其類似物存在下進行二聚。Alternatively, the first dimerization domain and the second dimerization domain may be the 12 kD FK506 binding protein (FKBP) and the calcineurin phosphatase domain, which are known in the art to dimerize in the presence of FK506 or an analog thereof.

在一些實施例中,二聚域為選自以下之同二聚域: i)大小為12 kD之FK506結合蛋白(FKBP); ii)親環蛋白A (cyclophiliA) (CypA);或 iii)迴旋酶B (CyrB); 其分別具有對應非生理學配體: i) FK1012、AP1510、AP1903或AP20187; ii)環孢素-A (CsA);或 iii)庫馬黴素(coumermycin)或其類似物。 In some embodiments, the dimerization domain is a homodimerization domain selected from: i) FK506 binding protein (FKBP) of size 12 kD; ii) cyclophilin A (cyclophiliA) (CypA); or iii) gyrase B (CyrB); which respectively have corresponding non-physiological ligands: i) FK1012, AP1510, AP1903 or AP20187; ii) cyclosporine-A (CsA); or iii) coumermycin or its analogs.

在一些實施例中,跨膜受體蛋白之第一及第二二聚域為FKBP域及親環蛋白域。In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a cyclophilin domain.

在一些實施例中,跨膜受體蛋白之第一及第二二聚域為FKBP域及細菌性二氫葉酸還原酶(DHFR)域。In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

在一些實施例中,跨膜受體蛋白之第一及第二二聚域為鈣調神經磷酸酶域及親環蛋白域。In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

在一些實施例中,跨膜受體蛋白之第一及第二二聚域為PYR1樣1 (PYL1)及脫落酸不敏感物1 (ABI1)。 3.跨膜域 In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1). 3. Transmembrane domain

跨膜域為跨越膜之合成細胞介素受體之序列。跨膜域可包含疏水性α螺旋。在一些實施例中,跨膜域來源於人類蛋白。The transmembrane domain is a sequence of a synthetic interleukin receptor that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

在一些實施例中,TM域及細胞內傳訊域係來自相同細胞介素受體。在一些實施例中,合成γ鏈多肽含有IL-2RG TM域及IL-2RG細胞內域。在一些實施例中,合成β鏈多肽含有IL-2RB TM域及IL-2RB細胞內域。在一些實施例中,合成β鏈多肽含有IL-7RB TM域及IL-7RB細胞內域。在一些實施例中,合成β鏈多肽含有IL-21RB TM域及IL-21RB細胞內域。In some embodiments, the TM domain and the intracellular signaling domain are from the same interleukin receptor. In some embodiments, the synthetic gamma chain polypeptide contains the IL-2RG TM domain and the IL-2RG intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains the IL-2RB TM domain and the IL-2RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains the IL-7RB TM domain and the IL-7RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains the IL-21RB TM domain and the IL-21RB intracellular domain.

在一些實施例中,亦可包括與細胞介素受體之TM域直接相鄰之胞外域的一或多個額外連續胺基酸作為合成細胞介素受體鏈之多肽序列的一部分。在一些實施例中,包括與細胞介素受體之TM域相鄰之胞外域的1-20個連續胺基酸作為合成細胞介素受體鏈之多肽序列的一部分。該胞外域部分可為與TM序列直接相鄰(例如其N端)之1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20個胺基酸的連續序列。In some embodiments, one or more additional consecutive amino acids of the extracellular domain directly adjacent to the TM domain of the interleukin receptor may also be included as part of the polypeptide sequence for synthesizing the interleukin receptor chain. In some embodiments, 1-20 consecutive amino acids of the extracellular domain adjacent to the TM domain of the interleukin receptor are included as part of the polypeptide sequence for synthesizing the interleukin receptor chain. The extracellular domain portion may be a continuous sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids directly adjacent to the TM sequence (e.g., its N-terminus).

在一些實施例中,合成細胞介素受體能夠被非生理學配體雷帕黴素或雷帕黴素類似物結合。在一些實施例中,合成細胞介素受體對非生理學配體雷帕黴素或雷帕黴素類似物有反應,其中非生理學配體與合成細胞介素受體之二聚域的結合誘導細胞中細胞介素受體介導之傳訊,諸如經由JAK/STAT路徑。 C.嵌合抗原受體 In some embodiments, the synthetic interleukin receptor is capable of being bound by a non-physiological ligand, rapamycin or a rapamycin analog. In some embodiments, the synthetic interleukin receptor is responsive to a non-physiological ligand, rapamycin or a rapamycin analog, wherein binding of the non-physiological ligand to the dimerization domain of the synthetic interleukin receptor induces interleukin receptor-mediated signaling in the cell, such as via the JAK/STAT pathway. C. Chimeric Antigen Receptors

在一些實施例中,多順反子構築體之表現卡匣編碼嵌合抗原受體。 1. CAR構築體及編碼核苷酸 In some embodiments, the expression cassette of the polycistronic construct encodes a chimeric antigen receptor. 1. CAR construct and encoding nucleotides

在一些實施例中,CAR構築體含有細胞外結合部分、跨膜域及細胞內傳訊域。在一些實施例中,細胞內傳訊域含有共刺激傳訊域及/或活化傳訊域。在一些實施例中,CAR構築體含有細胞外結合部分、跨膜域及包含共刺激傳訊域的細胞內傳訊域。在一些實施例中,CAR構築體含有細胞外結合部分、跨膜結構域及包含活化傳訊域的細胞內傳訊域。在一些實施例中,CAR構築體含有細胞外結合部分、跨膜域及包含共刺激傳訊域與活化傳訊域的細胞內傳訊域。In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain contains a co-stimulatory signaling domain and/or an activation signaling domain. In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain comprising a co-stimulatory signaling domain. In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain comprising an activation signaling domain. In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain comprising a co-stimulatory signaling domain and an activation signaling domain.

在本文所描述之任何實施例中,CAR之結合部分可為例如抗體之單鏈可變片段區(scFv)、Fab、Fv、Fc或(Fab')2片段,及其類似者。In any of the embodiments described herein, the binding portion of the CAR can be, for example, a single-chain variable fragment region (scFv), Fab, Fv, Fc or (Fab')2 fragment of an antibody, and the like.

在一些實施例中,共刺激傳訊域用以增強在CAR與所靶向部分結合後淋巴球之增殖及存活。共刺激傳訊域之屬性(identity)僅限於在所靶向部分被CAR結合後其具有增強細胞增殖及存活活化之能力。合適共刺激傳訊域包括但不限於:CD28 (參見例如Alvarez-Vallina, L.等人, Eur J Immunol. 1996. 26(10):2304-9);CD137 (4-1BB),腫瘤壞死因子(TNF)受體家族之成員(參見例如Imai, C.等人, Leukemia. 2004. 18:676-84);及CD134 (OX40),受體TNFR超家族之成員(參見例如Latza, U.等人, Eur. J. Immunol. 1994. 24:677)。熟習此項技術者應理解,可使用此等共刺激傳訊域之序列變體,其中該等變體與其所模型化之域具有相同或類似活性。在各種實施例中,此類變體與其所源自之域之胺基酸序列具有至少約80%、至少約90%、至少約95%、至少約97%、至少約98%、至少約99%或至少約99.5%序列一致性。 In some embodiments, the co-stimulatory signaling domain is used to enhance the proliferation and survival of lymphocytes after the CAR binds to the targeted moiety. The identity of the co-stimulatory signaling domain is limited to its ability to enhance cell proliferation and survival activation after the targeted moiety is bound by the CAR. Suitable costimulatory signaling domains include, but are not limited to: CD28 (see, e.g., Alvarez-Vallina, L. et al., Eur J Immunol . 1996. 26(10):2304-9); CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor family (see, e.g., Imai, C. et al., Leukemia . 2004. 18:676-84); and CD134 (OX40), a member of the TNFR superfamily of receptors (see, e.g., Latza, U. et al., Eur. J. Immunol . 1994. 24:677). Those skilled in the art will appreciate that sequence variants of these costimulatory signaling domains can be used, wherein the variants have the same or similar activity as the domains they are modeled for. In various embodiments, such variants have at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they are derived.

在本發明之一些實施例中,CAR構築體包含兩個共刺激傳訊域。儘管特定組合包括四個所提及域之所有可能變體,但特定實例包括:1) CD28+CD137 (4-1BB)及2) CD28+CD134 (OX40)。In some embodiments of the present invention, the CAR construct comprises two co-stimulatory signaling domains. Although specific combinations include all possible variants of the four mentioned domains, specific examples include: 1) CD28+CD137 (4-1BB) and 2) CD28+CD134 (OX40).

在一些實施例中,活化傳訊域用以在CAR與所靶向部分結合後活化細胞。活化傳訊域之屬性僅受限制於在所靶向部分被CAR結合後其具有誘導選定細胞之活化的能力。合適活化傳訊域包括CD3ζ鏈及Fc受體γ。在一些實施例中,傳訊域為NKG2C或NKp44之傳訊域。熟習此項技術者應理解,可在無不利地影響本發明之情況下使用此等所提及活化傳訊域之序列變體,其中該等變體與其所模型化之域具有相同或類似活性。此類變體與其所源自之域之胺基酸序列可具有至少約80%、至少約90%、至少約95%、至少約97%、至少約98%、至少約99%或至少約99.5%序列一致性。In some embodiments, the activation signaling domain is used to activate cells after CAR binds to the targeted portion. The properties of the activation signaling domain are limited to its ability to induce activation of selected cells after the targeted portion is bound by CAR. Suitable activation signaling domains include CD3ζ chain and Fc receptor γ. In some embodiments, the signaling domain is the signaling domain of NKG2C or NKp44. Those familiar with this technology should understand that sequence variants of these mentioned activation signaling domains can be used without adversely affecting the present invention, wherein the variants have the same or similar activity as the domains they are modeled. Such variants may have at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or at least about 99.5% sequence identity with the amino acid sequence of the domain from which they are derived.

在一些實施例中,CARs可包括額外元件,諸如用於確保融合蛋白適當輸出至細胞表面的訊號肽、用於確保融合蛋白維持作為整合膜蛋白的跨膜域、及賦予識別區可撓性且允許與所靶向部分強結合的鉸鏈域。In some embodiments, CARs may include additional elements, such as signal peptides to ensure proper export of the fusion protein to the cell surface, transmembrane domains to ensure that the fusion protein remains an integral membrane protein, and hinge domains that confer flexibility to the recognition region and allow strong binding to the targeted moiety.

在一些實施例中,編碼CAR之核苷酸序列包含細胞外域、視情況存在之鉸鏈域、跨膜域及細胞內傳訊域。在一些實施例中,細胞內傳訊域包含共刺激域及活化傳訊域。在一些實施例中,共刺激及活化傳訊域為單個域,例如向細胞提供共刺激及活化訊號之單個細胞內域。在其他實施例中,細胞內傳訊域包含共刺激域或活化傳訊域。在一些實施例中,CAR包含細胞外域、CD8a鉸鏈、CD8a跨膜域、4-1BB共刺激域及CD3ζ傳訊域。在一些實施例中,核苷酸序列編碼細胞外域、CD28鉸鏈域、CD28跨膜域、CD28共刺激域及CD3ζ傳訊域。在一些實施例中,核苷酸序列編碼細胞外域、IgG4鉸鏈域、CD28跨膜域、4-1BB共刺激域及CD3ζ傳訊域。在一些實施例中,核苷酸序列編碼CAR,該CAR包含細胞外域、CD8a鉸鏈、CD28跨膜域、4-1BB共刺激域及CD3ζ傳訊域。In some embodiments, the nucleotide sequence encoding CAR includes an extracellular domain, an optionally present hinge domain, a transmembrane domain, and an intracellular communication domain. In some embodiments, the intracellular communication domain includes a co-stimulatory domain and an activation communication domain. In some embodiments, the co-stimulatory and activation communication domains are a single domain, such as a single intracellular domain that provides co-stimulatory and activation signals to cells. In other embodiments, the intracellular communication domain includes a co-stimulatory domain or an activation communication domain. In some embodiments, CAR includes an extracellular domain, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ communication domain. In some embodiments, the nucleotide sequence encodes an extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ communication domain. In some embodiments, the nucleotide sequence encodes an extracellular domain, an IgG4 hinge domain, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, the nucleotide sequence encodes a CAR comprising an extracellular domain, a CD8a hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain.

適合於所提供多順反子構築體之說明性CAR構築體提供於下: (1) scFv-CD8 TM-4-1BB IC-CD3ζ (參見例如Liu E, Tong Y, Dotti G等人, Leukemia. 2018; 32: 520-531); (2) scFv-CD28 TM+IC-CD3ζ (參見例如Han J, Chu J, Keung CW等人, Sci Rep. 2015; 5: 11483;Kruschinski A, Moosmann A, Poschke I等人, Proc Natl Acad Sci U S A.2008; 105: 17481-17486;及Chu J, Deng Y, Benson DM等人, Leukemia. 2014; 28: 917-927); (3) scFv-DAP12 TM+IC(參見例如Muller N, Michen S, Tietze S等人, J Immunother.2015; 38: 197-210); (4) scFv-CD8 TM-2B4 IC-CD3ζ (參見例如Xu Y, Liu Q, Zhong M等人, J Hematol Oncol.2019; 12: 49); (5) scFv-2B4 TM+IC-CD3ζ (參見例如Altvater B, Landmeier S, Pscherer S等人, Clin Cancer Res. 2009; 15: 4857-4866); (6) scFv-CD28 TM+IC-4-1BB IC-CD3ζ (參見例如Kloss S, Oberschmidt O, Morgan M等人, Hum Gene Ther. 2017; 28: 897-913); (7) scFv-CD16 TM-2B4 IC-CD3ζ (參見例如Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (8) scFv-NKp44 TM-DAP10 IC-CD3ζ (參見例如Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (9) scFv-NKp46 TM-2B4 IC-CD3ζ (參見例如Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (10) scFv-NKG2D TM-2B4 IC-CD3ζ (參見例如Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (11) scFv-NKG2D TM-4-1BB IC-CD3ζ (參見例如Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (12) scFv-NKG2D TM-2B4 IC-DAP12 IC-CD3ζ (參見例如Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (13) scFv-NKG2D TM-2B4 IC-DAP10 IC-CD3ζ (參見例如Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (14) scFv-NKG2D TM-4-1BB IC-2B4 IC-CD3ζ (參見例如Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);及 (15) scFv-NKG2D TM-CD3ζ (參見例如Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192)。 a. CAR細胞外域 Illustrative CAR constructs suitable for the provided multicistronic constructs are provided below: (1) scFv-CD8 TM -4-1BB IC -CD3ζ (see, e.g., Liu E, Tong Y, Dotti G et al., Leukemia . 2018; 32: 520-531); (2) scFv-CD28 TM+IC -CD3ζ (see, e.g., Han J, Chu J, Keung CW et al., Sci Rep . 2015; 5: 11483; Kruschinski A, Moosmann A, Poschke I et al., Proc Natl Acad Sci USA. 2008; 105: 17481-17486; and Chu J, Deng Y, Benson DM et al., Leukemia . 2014; 28: 917-927); (3) scFv-DAP12 TM+IC (see, e.g., Muller N, Michen S, Tietze S et al., J Immunother. 2015; 38: 197-210); (4) scFv-CD8 TM -2B4 IC -CD3ζ (see, e.g., Xu Y, Liu Q, Zhong M et al., J Hematol Oncol. 2019; 12: 49); (5) scFv-2B4 TM+IC -CD3ζ (see, e.g., Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009; 15: 4857-4866); (6) scFv-CD28 TM+IC -4-1BB IC -CD3ζ (see, e.g., Kloss S, Oberschmidt O, Morgan M et al., Hum Gene Ther . 2017; 28: 897-913); (7) scFv-CD16 TM -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell . 2018; 23: 181-192); (8) scFv-NKp44 TM -DAP10 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell . 2018; 23: 181-192); (9) scFv-NKp46 TM -2B4 IC -CD3ζ (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell . 2018; 23: 181-192); (10) scFv-NKG2D TM -2B4 IC -CD3ζ (see e.g. Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell . 2018; 23: 181-192); (11) scFv- NKG2DTM -4-1BB IC -CD3ζ (see e.g. Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell . 2018; 23: 181-192); (12) scFv-NKG2DTM - 2B4IC - DAP12IC - CD3ζ (see e.g. Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell . 2018; 23: 181-192); (13) scFv-NKG2DTM - 2B4IC - DAP10IC -CD3ζ (See, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell . 2018; 23: 181-192); (14) scFv-NKG2D TM -4-1BB IC -2B4 IC -CD3ζ (See, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell . 2018; 23: 181-192); and (15) scFv-NKG2D TM -CD3ζ (See, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell . 2018; 23: 181-192). a. CAR extracellular domain

在一些實施例中,CAR之結合部分可針對諸如由於其在細胞上過度表現或與疾病或病狀(如癌症)相關而需要靶向之任何抗原。In some embodiments, the binding portion of the CAR can be directed against any antigen that is desirable to be targeted, such as because it is overexpressed on a cell or is associated with a disease or condition, such as cancer.

在一些實施例中,CAR之結合部分對腫瘤抗原具有特異性。抗原結合域之選擇將視待治療之癌症之特定類型而定。腫瘤抗原為此項技術中熟知且包括例如:神經膠質瘤相關抗原、癌胚抗原(CEA)、EGFRvIII、IL-11Ra、IL-13Ra、EGFR、FAP、B7H3、Kit、CA LX、CS-1、MUC1、BCMA、bcr-abl、HER2、β-人類絨毛膜促性腺素、α胎蛋白(AFP)、ALK、CD19、CD123、週期蛋白B1、凝集素反應性AFP、Fos-相關抗原1、ADRB3、甲狀球蛋白、EphA2、RAGE-1、RU1、RU2、SSX2、AKAP-4、LCK、OY-TES1、PAXS、SART3、CLL-1、岩藻糖基GM1、GloboH、MN-CA IX、EPCAM、EVT6-AML、TGS5、人類端粒酶逆轉錄酶、多唾液酸(plysialic acid)、PLAC1、RU1、RU2 (AS)、腸羧基酯酶、路易斯Y (lewisY)、sLe、LY6K、mut hsp70-2、M-CSF、MYCN、RhoC、TRP-2、CYPIBI、BORIS、前列腺酶、前列腺特異性抗原(PSA)、PAX3、PAP、NY-ESO-1、LAGE-la、LMP2、NCAM、p53、p53突變體、Ras突變體、gplOO、前列腺蛋白(prostein)、OR51E2、PANX3、PSMA、PSCA、Her2/neu、hTERT、HMWMAA、HAVCR1、VEGFR2、PDGFR-β、存活素及端粒酶、豆莢蛋白、HPV E6、E7、精子蛋白17、SSEA-4、酪胺酸酶、TARP、WT1、前列腺癌瘤腫瘤抗原-1 (PCTA-1)、ML-IAP、MAGE、MAGE-A1、MAD-CT-1、MAD-CT-2、MelanA/MART 1、XAGE1、ELF2M、ERG (TMPRSS2 ETS融合基因)、NA17、嗜中性球彈性蛋白酶、肉瘤易位斷裂點、NY-BR-1、ephnnB2、CD20、CD22、CD24、CD30、CD33、CD38、CD44v6、CD97、CD171、CD179a、雄性激素受體、FAP、胰島素生長因子(IGF)-I、IGFII、IGF-I受體、GD2、o-乙醯基-GD2、GD3、GM3、GPRCSD、GPR20、CXORF61、葉酸受體(FRa)、葉酸受體β、ROR1、Flt3、TAG72、TN Ag、Tie 2、TEM1、TEM7R、CLDN6、TSHR、UPK2及間皮素。腫瘤抗原之非限制性實例包括以下:分化抗原,諸如酪胺酸酶、TRP-1、TRP-2及腫瘤特異性多譜系抗原,諸如MAGE-1、MAGE-3、BAGE、GAGE-1、GAGE-2、pi 5;過度表現胚抗原,諸如CEA;過度表現致癌基因及突變腫瘤抑制基因,諸如p53、Ras、HER-2/neu;由染色體易位產生之獨特腫瘤抗原;諸如BCR-ABL、E2A-PRL、H4-RET、IGH-IGK、MYL-RAR;及病毒抗原,諸如埃-巴二氏病毒抗原(Epstein Barr virus antigen) EBVA及人類乳突狀瘤病毒(HPV)抗原E6及E7。其他較大的基於蛋白質之抗原包括TSP-180、MAGE-4、MAGE-5、MAGE-6、RAGE、NY-ESO、p185erbB2、p180erbB-3、c-met、nm-23H1、PSA、IL13Ra2、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、β-鏈蛋白、CDK4、Mum-1、p 15、p 16、43-9F、5T4、791Tgp72、α-胎兒蛋白、β--HCG、BCA225、BTAA、CA 125、CA 15-3\CA 27.29\BCAA、CA 195、CA 242、CA-50、CAM43、CD68\P1、CO-029、FGF-5、G250、Ga733\EpCAM、HTgp-175、M344、MA-50、MG7-Ag、MOV18、NB/70K、NY-CO-1、RCAS1、SDCCAG1 6、TA-90\Mac-2結合蛋白\親環蛋白C相關蛋白、TAAL6、TAG72、TLP、TPS、GPC3、MUC16、LMP1、EBMA-1、BARF-1、CS1、CD319、HER1、B7H6、L1CAM、IL6及MET。在一些實施例中,CAR包含靶向兩種或更多種呈任何組合的本文所揭示之抗原的結合域。舉例而言:CD19及CD3、BCMA及CD3、GPRC5D及CD3、FCRL5及CD3、CD38及CD3、CD19及CD20、CD19及CD22、BCMA及GPRC5D、或CD20及CD22。在一些實施例中,CAR包含靶向相同目標蛋白上之兩種或更多種抗原,例如BCMA中之兩種抗原決定基的結合域。In some embodiments, the binding portion of the CAR is specific for a tumor antigen. The choice of antigen binding domain will depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, neuroglioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha fetoprotein (AFP), ALK, CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAXS, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxylesterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostate enzyme, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-1a, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2/neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-β, survivin and telomerase, podocyte protein, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA/MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastic protease, sarcoma translocation breakpoint, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRCSD, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. Non-limiting examples of tumor antigens include the following: differentiation antigens, such as tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutant tumor suppressor genes, such as p53, Ras, HER-2/neu; unique tumor antigens generated by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein Barr virus antigen EBVA and human papilloma virus (HPV) antigens E6 and E7. Other larger protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, IL13Ra2, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG1 6, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6 and MET. In some embodiments, the CAR comprises a binding domain targeting two or more antigens disclosed herein in any combination. For example: CD19 and CD3, BCMA and CD3, GPRC5D and CD3, FCRL5 and CD3, CD38 and CD3, CD19 and CD20, CD19 and CD22, BCMA and GPRC5D, or CD20 and CD22. In some embodiments, the CAR comprises binding domains that target two or more antigens on the same target protein, such as two antigenic determinants in BCMA.

熟習此項技術者容易熟悉針對多種多樣的腫瘤抗原之CAR。可採用此類CAR中之任一者作為CAR。多種CAR已併入經FDA批准之產品中且包括但不限於抗-CD19及抗BCMA CAR T細胞,諸如替沙津魯(tisagenlecleucel) (威爾瑞(Kymriah))、西卡思羅(axicabtagene ciloleucel) (伊斯卡他(Yescarta))、布萊奧妥(brexucabtagene autoleucel) (特卡圖斯(Tecartus))、力索嗎魯(lisocabtagene maraleucel) (布雷楊齊(Breyanzi))、艾德維賽(idecabtagene vicleucel) (阿貝克瑪(Abecma))或西達奧妥(ciltacabtagene autoleucel) (察爾維克蒂(Carvykti))。產生用於特異性靶向所需腫瘤抗原之類似構築體在熟習此項技術者之能力範圍內。Those familiar with the art will be familiar with CARs targeting a variety of tumor antigens. Any of these CARs can be used as CAR. A variety of CARs have been incorporated into FDA-approved products and include, but are not limited to, anti-CD19 and anti-BCMA CAR T cells, such as tisagenlecleucel (Kymriah), axicabtagene ciloleucel (Yescarta), brexucabtagene autoleucel (Tecartus), lisocabtagene maraleucel (Breyanzi), idecabtagene vicleucel (Abecma), or ciltacabtagene autoleucel (Carvykti). It is within the capabilities of those skilled in the art to generate similar constructs for specific targeting of desired tumor antigens.

在一些實施例中,CAR之結合部分可針對通用抗原以靶向廣泛多種腫瘤而不需要製備個別CAR構築體。被CAR識別之所靶向部分亦可保持恆定。在一些實施例中,可向個體投與配體以允許與目標細胞相互作用及與CAR之結合部分相互作用。僅小結合物分子之配體部分需要改變以允許系統靶向具有不同屬性之癌細胞。例示性CAR系統描述於以下章節中。In some embodiments, the binding portion of the CAR can be directed against a universal antigen to target a wide variety of tumors without the need to prepare individual CAR constructs. The targeted portion recognized by the CAR can also remain constant. In some embodiments, a ligand can be administered to an individual to allow interaction with the target cell and interaction with the binding portion of the CAR. Only the ligand portion of the small binding molecule needs to be changed to allow the system to target cancer cells with different properties. Exemplary CAR systems are described in the following sections.

在一些實施例中,CAR為抗CD19 CAR,且CD19 CAR之細胞外結合域對CD19 (例如人類CD19)具有特異性。在一些實施例中,CD19 CAR之細胞外域包含來源於FMC63單株抗體(FMC63)之scFv,其包含由連接子連接之FMC63的重鏈可變區(VH)及輕鏈可變區(VL)。FMC63及所衍生之scFv已描述於Nicholson等人, Mol. Immun. 34(16-17):1157-1165 (1997)及PCT申請公開案第WO2018/213337號中,其各自之全部內容以引用之方式併入本文中。例示性抗CD19 CAR與其不同部分(包括細胞外域)顯示於 1中。 In some embodiments, the CAR is an anti-CD19 CAR, and the extracellular binding domain of the CD19 CAR is specific for CD19 (e.g., human CD19). In some embodiments, the extracellular domain of the CD19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises a heavy chain variable region (VH) and a light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16-17):1157-1165 (1997) and PCT Application Publication No. WO2018/213337, the entire contents of each of which are incorporated herein by reference. Exemplary anti-CD19 CARs and their various portions (including the extracellular domain) are shown in Table 1 .

在一些實施例中,CAR為抗CD20 CAR,且CD20 CAR之細胞外結合域對CD20 (例如人類CD20)具有特異性。在一些實施例中,CD20 CAR之細胞外結合域係來源於對CD20具有特異性之抗體,包括例如Leu16、IF5、1.5.3、利妥昔單抗(rituximab)、阿托珠單抗(obinutuzumab)、替伊莫單抗(ibritumomab)、奧伐木單抗(ofatumumab)、托斯圖單抗(tositumumab)、奧尼妥單抗(odronextamab)、維妥組單抗(veltuzumab)、烏妥昔單抗(ublituximab)及奧瑞組單抗(ocrelizumab)。在此等實施例中之任一者中,CD20 CAR之細胞外結合域可包含以下或由以下組成:抗體中之任一者的VH、VL及/或一或多個CDR。例示性抗CD20 CAR與其不同部分(包括其細胞外域)顯示於 2 3中。 1)例示性CAR In some embodiments, the CAR is an anti-CD20 CAR, and the extracellular binding domain of the CD20 CAR is specific for CD20 (e.g., human CD20). In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific for CD20, including, for example, Leu16, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In any of these embodiments, the extracellular binding domain of the CD20 CAR may comprise or consist of: VH, VL and/or one or more CDRs of any of the antibodies. Exemplary anti-CD20 CARs and their different portions (including their extracellular domains) are shown in Tables 2 and 3. 1 ) Exemplary CARs

習知地,CAR藉由以下來產生:將編碼VL、VH或scFv之多核苷酸與編碼跨膜及細胞內域之多核苷酸的5'端融合,及用該多核苷酸以及用對應VH或VL (若需要)轉導細胞。關於CAR的多種變體為此項技術中熟知,且本發明考慮使用已知變體中之任一者。另外,無數的半抗原之VL/VH對及scFv為此項技術中已知的,或可藉由習知方法常規地產生。因此,本發明考慮使用任何已知的半抗原結合域。As is known, CAR is produced by fusing a polynucleotide encoding VL, VH or scFv to the 5' end of a polynucleotide encoding a transmembrane and intracellular domain, and transducing cells with the polynucleotide and with the corresponding VH or VL (if necessary). A variety of variants of CAR are well known in the art, and the present invention contemplates the use of any of the known variants. In addition, countless VL/VH pairs and scFvs of haptens are known in the art or can be routinely produced by known methods. Therefore, the present invention contemplates the use of any known hapten binding domain.

在一些實施例中,CAR為抗FITC CAR,且配體由與結合所需目標細胞(諸如癌細胞)之藥劑結合之螢光素或螢光異硫氰酸鹽(FITC)部分構成。例示性配體在下文描述。在一些實施例中,配體為FITC-葉酸。In some embodiments, the CAR is an anti-FITC CAR and the ligand consists of a fluorescein or fluorescein isothiocyanate (FITC) moiety conjugated to an agent that binds to a desired target cell, such as a cancer cell. Exemplary ligands are described below. In some embodiments, the ligand is FITC-folate.

例示性抗FITC CAR及其不同部分顯示於 4中。 Exemplary anti-FITC CARs and their different parts are shown in Table 4 .

在一些實施例中,CAR包含scFv域。在一些實施例中,scFv域包含抗螢光異硫氰酸鹽(FITC) E2。在一些實施例中,scFv域包含輕鏈可變域(VL)、連接子及重鏈可變域(VH)。In some embodiments, the CAR comprises a scFv domain. In some embodiments, the scFv domain comprises anti-fluorescent isothiocyanate (FITC) E2. In some embodiments, the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

在一些實施例中,scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,scFv VL包含SEQ ID NO: 30或65之核苷酸序列。在一些實施例中,scFv VL由SEQ ID NO: 30或65之核苷酸序列組成。In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence of SEQ ID NO: 30 or 65. In some embodiments, the scFv VL consists of a nucleotide sequence of SEQ ID NO: 30 or 65.

在一些實施例中,scFv VL包含與SEQ ID NO: 31之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 31之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 31之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 31之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 31之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 31之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 31之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 31之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 31之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,scFv VL包含與SEQ ID NO: 31之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,scFv VL包含SEQ ID NO: 31之胺基酸序列。在一些實施例中,scFv VL由SEQ ID NO: 31之胺基酸序列組成。In some embodiments, the scFv VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL comprises an amino acid sequence of SEQ ID NO: 31. In some embodiments, the scFv VL consists of the amino acid sequence of SEQ ID NO: 31.

在一些實施例中,scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,scFv VH包含SEQ ID NO: 34或67之核苷酸序列。在一些實施例中,scFv VH由SEQ ID NO: 34或67之核苷酸序列組成。In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence of SEQ ID NO: 34 or 67. In some embodiments, the scFv VH consists of a nucleotide sequence of SEQ ID NO: 34 or 67.

在一些實施例中,scFv VH包含與SEQ ID NO: 35之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 35之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 35之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 35之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 35之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 35之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 35之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 35之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 35之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,scFv VH包含與SEQ ID NO: 35之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,scFv VH包含SEQ ID NO: 35之胺基酸序列。在一些實施例中,scFv VH由SEQ ID NO: 35之胺基酸序列組成。In some embodiments, the scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv VH consists of the amino acid sequence of SEQ ID NO: 35.

在一些實施例中,scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,scFv連接子包含SEQ ID NO: 32或66之核苷酸序列。在一些實施例中,scFv連接子由SEQ ID NO: 32或66之核苷酸序列組成。In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence of SEQ ID NO: 32 or 66. In some embodiments, the scFv linker consists of a nucleotide sequence of SEQ ID NO: 32 or 66.

在一些實施例中,scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,scFv連接子包含SEQ ID NO: 33之胺基酸序列。在一些實施例中,scFv連接子由SEQ ID NO: 33之胺基酸序列組成。In some embodiments, the scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the scFv linker consists of the amino acid sequence of SEQ ID NO: 33.

在一些實施例中,scFv包含與SEQ ID NO: 28或64之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,scFv包含與SEQ ID NO: 28或64之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,scFv包含與SEQ ID NO: 28或64之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,scFv包含與SEQ ID NO: 28或64之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,scFv包含與SEQ ID NO: 28或64之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,scFv包含與SEQ ID NO: 28或64之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,scFv包含與SEQ ID NO: 28或64之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,scFv包含與SEQ ID NO: 28或64之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,scFv包含與SEQ ID NO: 28或64之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,scFv包含與SEQ ID NO: 28或64之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,scFv包含SEQ ID NO: 28或64之核苷酸序列。在一些實施例中,scFv由SEQ ID NO: 28或64之核苷酸序列組成。In some embodiments, the scFv comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv comprises a nucleotide sequence of SEQ ID NO: 28 or 64. In some embodiments, the scFv consists of a nucleotide sequence of SEQ ID NO: 28 or 64.

在一些實施例中,scFv包含與SEQ ID NO: 29之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,scFv包含與SEQ ID NO: 29之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,scFv包含與SEQ ID NO: 29之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,scFv包含與SEQ ID NO: 29之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,scFv包含與SEQ ID NO: 29之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,scFv包含與SEQ ID NO: 29之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,scFv包含與SEQ ID NO: 29之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,scFv包含與SEQ ID NO: 29之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,scFv包含與SEQ ID NO: 29之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,scFv包含與SEQ ID NO: 29之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,scFv包含SEQ ID NO: 29之胺基酸序列。在一些實施例中,scFv由SEQ ID NO: 29之胺基酸序列組成。In some embodiments, the scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv consists of the amino acid sequence of SEQ ID NO: 29.

靶向CAR及表現CAR之細胞的各種方法在此項技術中已描述,包括例如在US 2020/0123224 (其揭示內容以引用之方式併入本文中)中描述。舉例而言,螢光素或螢光異硫氰酸鹽(FITC)部分可與結合所需目標細胞(諸如癌細胞)之藥劑結合,且因此表現抗螢光素/FITC嵌合抗原受體之CAR可選擇性地靶向被結合物標記的目標細胞。在變化形式中,可使用被CAR識別之其他半抗原替代螢光素/FITC。CAR可使用此項技術中已知之各種scFv序列來產生,或scFv序列可藉由習知及常規方法來產生。螢光素/FITC及其他半抗原之其他說明性scFv序列例如在WO 2021/076788 (其揭示內容以引用之方式併入本文中)中提供。Various methods of targeting CARs and cells expressing CARs have been described in this art, including, for example, in US 2020/0123224 (the disclosure of which is incorporated herein by reference). For example, a fluorescein or fluorescein isothiocyanate (FITC) moiety can be conjugated to an agent that binds to a desired target cell (such as a cancer cell), and thus a CAR expressing an anti-fluorescein/FITC chimeric antigen receptor can selectively target target cells labeled with the conjugate. In variations, other haptens recognized by the CAR can be used in place of fluorescein/FITC. CARs can be produced using various scFv sequences known in this art, or the scFv sequences can be produced by known and conventional methods. Other illustrative scFv sequences for fluorescein/FITC and other haptens are provided, for example, in WO 2021/076788 (the disclosure of which is incorporated herein by reference).

在一個實施例中,本發明提供對此結合物分子/CAR系統之說明。In one embodiment, the present invention provides a description of this conjugate molecule/CAR system.

在一些實施例中,本發明之CAR系統利用結合物分子作為表現CAR之細胞與所靶向癌細胞之間的橋連。結合物分子為包含半抗原及細胞靶向部分(諸如任何合適的腫瘤細胞特異性配體)之結合物。可被CAR識別且結合之說明性半抗原包括:小分子量有機分子,諸如DNP (2,4-二硝基苯酚)、TNP (2,4,6-三硝基苯酚)、生物素及地高辛(digoxigenin);以及螢光素及其衍生物,包括FITC (螢光異硫氰酸鹽)、NHS-螢光素、及五氟苯基酯(PFP)與四氟苯基酯(TFP)衍生物;打結素(knottin);生替林(centyrin);及DARPin。自身可用作CAR之半抗原的合適細胞靶向部分包括打結素(參見Kolmar H.等人, The FEBS Journal. 2008. 275(11):26684-90)、生替林及DARPin (參見Reichert, J.M. MAbs2009. 1(3):190-209)。 In some embodiments, the CAR system of the present invention utilizes a conjugate molecule as a bridge between cells expressing CAR and targeted cancer cells. The conjugate molecule is a conjugate comprising a hapten and a cell targeting moiety (such as any suitable tumor cell-specific ligand). Illustrative haptens that can be recognized and bound by CAR include: small molecular weight organic molecules such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin and digoxigenin; and fluorescein and its derivatives, including FITC (fluorescein isothiocyanate), NHS-fluorescein, and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives; knottin; centyrin; and DARPin. Suitable cell targeting moieties that can themselves be used as haptens for CARs include knottin (see Kolmar H. et al., The FEBS Journal . 2008. 275(11):26684-90), triptin and DARPin (see Reichert, JM MAbs 2009. 1(3):190-209).

在一些實施例中,細胞靶向部分為以奈莫耳濃度親和力被PSMA陽性人類前列腺癌細胞結合之配體DUPA (DUPA-(99m) Tc) (K D= 14 nM;參見Kularatne, S.A.等人, Mol Pharm. 2009. 6(3):780-9)。在一個實施例中,DUPA衍生物可為與靶向部分連接之小分子配體的配體,且DUPA衍生物描述於WO 2015/057852 (其以引用之方式併入本文中)中。 In some embodiments, the cell targeting moiety is a ligand DUPA (DUPA-(99m) Tc) that is bound by PSMA-positive human prostate cancer cells with nanomolar affinity ( KD = 14 nM; see Kularatne, SA et al., Mol Pharm . 2009. 6(3):780-9). In one embodiment, a DUPA derivative can be a ligand of a small molecule ligand linked to a targeting moiety, and DUPA derivatives are described in WO 2015/057852 (which is incorporated herein by reference).

在一些實施例中,細胞靶向部分為被CCK2R陽性癌細胞(例如甲狀腺、肺、胰腺、卵巢、腦、胃、胃腸道基質及結腸之癌症;參見Wayua. C.等人, Molecular Pharmaceutics. 2013. ePublication)結合的配體CCK2R配體。 In some embodiments, the cell targeting moiety is a ligand CCK2R ligand that is bound by CCK2R-positive cancer cells (e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stromal, and colon; see Wayua. C. et al., Molecular Pharmaceutics . 2013. ePublication).

在一些實施例中,細胞靶向部分為葉酸(folate/folic acid)或其類似物,一種被癌症細胞上之葉酸受體結合的配體,該等癌症包括卵巢癌、子宮頸癌、子宮內膜癌、肺癌、腎癌、腦癌、乳癌、結腸癌及頭頸癌;參見Sega, E.I.等人, Cancer Metastasis Rev. 2008. 27(4):655-64)。 In some embodiments, the cell targeting moiety is folate (folic acid) or an analog thereof, a ligand bound by a folate receptor on cancer cells, including ovarian cancer, cervical cancer, endometrial cancer, lung cancer, kidney cancer, brain cancer, breast cancer, colon cancer, and head and neck cancer; see Sega, EI et al., Cancer Metastasis Rev. 2008. 27(4):655-64).

在一些實施例中,細胞靶向部分為NK-1R配體。例如在結腸癌及胰臟癌上發現NK-1R配體之受體。在一些實施例中,NK-1R配體可根據國際專利申請第PCT/US2015/044229 (其以引用之方式併入本文中)號中所揭示之方法合成。In some embodiments, the cell targeting moiety is a NK-1R ligand. For example, receptors for NK-1R ligands are found on colon cancer and pancreatic cancer. In some embodiments, the NK-1R ligand can be synthesized according to the method disclosed in International Patent Application No. PCT/US2015/044229 (which is incorporated herein by reference).

在一些實施例中,細胞靶向部分可為肽配體,例如配體可為呈NK1受體之內源性配體的肽配體。在一些實施例中,小結合物分子配體可為屬於靶向速激肽(tachykinin)受體之速激肽家族的調節肽。此類調節肽包括P物質(Substance P;SP)、神經激肽A (K物質)及神經激肽B (神經介肽K) (參見Hennig等人, International Journal of Cancer:61, 786-792)。 In some embodiments, the cell targeting moiety may be a peptide ligand, for example, the ligand may be a peptide ligand that is an endogenous ligand of the NK1 receptor. In some embodiments, the small binder molecule ligand may be a regulatory peptide belonging to the tachykinin family that targets the tachykinin receptor. Such regulatory peptides include substance P (SP), neurokinin A (substance K), and neurokinin B (neuropeptide K) (see Hennig et al., International Journal of Cancer: 61, 786-792).

在一些實施例中,細胞靶向部分為CAIX配體。例如在腎癌、卵巢癌、外陰癌及乳癌上發現CAIX配體之受體。CAIX配體在本文中亦稱為CA9。In some embodiments, the cell targeting moiety is a CAIX ligand. For example, receptors for the CAIX ligand are found on kidney cancer, ovarian cancer, vulvar cancer, and breast cancer. The CAIX ligand is also referred to herein as CA9.

在一些實施例中,細胞靶向部分為γ麩胺醯基轉肽酶之配體。轉肽酶例如在卵巢癌、結腸癌、肝癌、星形細胞神經膠質瘤、黑色素瘤及白血病中過度表現。In some embodiments, the cell targeting moiety is a ligand for gamma glutamyl transpeptidase, which is overexpressed in, for example, ovarian cancer, colon cancer, liver cancer, astrocytic neuroglioma, melanoma, and leukemia.

在一些實施例中,細胞靶向部分為CCK2R配體。CCK2R配體之受體尤其發現於甲狀腺癌、肺癌、胰臟癌、卵巢癌、腦癌、胃癌、胃腸基質癌及結腸癌上。In some embodiments, the cell targeting moiety is a CCK2R ligand. Receptors for CCK2R ligands are found in particular on thyroid cancer, lung cancer, pancreatic cancer, ovarian cancer, brain cancer, gastric cancer, gastrointestinal stromal cancer, and colon cancer.

在一些實施例中,細胞靶向部分為PSMA配體。In some embodiments, the cell targeting moiety is a PSMA ligand.

在一些實施例中,細胞靶向部分為FAP配體。In some embodiments, the cell targeting moiety is a FAP ligand.

在一個實施例中,細胞靶向部分之質量可為小於約10,000道爾頓、小於約9000道爾頓、小於約8,000道爾頓、小於約7000道爾頓、小於約6000道爾頓、小於約5000道爾頓、小於約4500道爾頓、小於約4000道爾頓、小於約3500道爾頓、小於約3000道爾頓、小於約2500道爾頓、小於約2000道爾頓、小於約1500道爾頓、小於約1000道爾頓、或小於約500道爾頓。在另一實施例中,小分子配體之質量可為約1至約10,000道爾頓、約1至約9000道爾頓、約1至約8,000道爾頓、約1至約7000道爾頓、約1至約6000道爾頓、約1至約5000道爾頓、約1至約4500道爾頓、約1至約4000道爾頓、約1至約3500道爾頓、約1至約3000道爾頓、約1至約2500道爾頓、約1至約2000道爾頓、約1至約1500道爾頓、約1至約1000道爾頓、或約1至約500道爾頓。In one embodiment, the mass of the cell targeting moiety can be less than about 10,000 daltons, less than about 9000 daltons, less than about 8,000 daltons, less than about 7000 daltons, less than about 6000 daltons, less than about 5000 daltons, less than about 4500 daltons, less than about 4000 daltons, less than about 3500 daltons, less than about 3000 daltons, less than about 2500 daltons, less than about 2000 daltons, less than about 1500 daltons, less than about 1000 daltons, or less than about 500 daltons. In another embodiment, the mass of the small molecule ligand can be about 1 to about 10,000 Daltons, about 1 to about 9000 Daltons, about 1 to about 8,000 Daltons, about 1 to about 7000 Daltons, about 1 to about 6000 Daltons, about 1 to about 5000 Daltons, about 1 to about 4500 Daltons, about 1 to about 4000 Daltons, about 1 to about 3500 Daltons, about 1 to about 3000 Daltons, about 1 to about 2500 Daltons, about 1 to about 2000 Daltons, about 1 to about 1500 Daltons, about 1 to about 1000 Daltons, or about 1 to about 500 Daltons.

在一個說明性實施例中,本文所描述之結合物中之鍵聯可為直接鍵聯(例如FITC之異硫氰酸酯基團與小分子配體之自由胺基團之間的反應),或鍵聯可經由中間連接子。在一個實施例中,若存在,則中間連接子可為此項技術中已知之任何生物相容性連接子,諸如二價連接子。在一個說明性實施例中,二價連接子可包含約1至約30個碳原子。在另一說明性實施例中,二價連接子可包含約2至約20個碳原子。在其他實施例中,採用低分子量二價連接子(亦即,近似分子量為約30至約300 Da之彼等連接子)。在另一實施例中,合適的連接子長度包括但不限於具有2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39或40個或更多原子之連接子。In one illustrative embodiment, the linkage in the conjugates described herein may be direct linkage (e.g., reaction between the isothiocyanate group of FITC and the free amine group of the small molecule ligand), or the linkage may be through an intermediate linker. In one embodiment, if present, the intermediate linker may be any biocompatible linker known in the art, such as a divalent linker. In one illustrative embodiment, the divalent linker may comprise about 1 to about 30 carbon atoms. In another illustrative embodiment, the divalent linker may comprise about 2 to about 20 carbon atoms. In other embodiments, low molecular weight divalent linkers (i.e., those linkers having an approximate molecular weight of about 30 to about 300 Da) are employed. In another embodiment, suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more atoms.

在一些實施例中,半抗原及細胞靶向部分可經由諸如FITC之異硫氰酸酯基團與小配體(例如葉酸、DUPA及CCK2R配體)之自由胺基團之間的反應的手段而直接結合。然而,用於連接兩個分子之連接域的使用可為有幫助的,因為其可提供可撓性及穩定性。合適的連接域之實例包括:1)聚乙二醇(PEG);2)聚脯胺酸;3)親水性胺基酸;4)糖;5)非天然肽聚糖;6)聚乙烯吡咯啶酮;7)普朗尼克(Pluronic) F-127。合適的連接子長度包括但不限於具有2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39或40個或更多原子之連接子。In some embodiments, haptens and cell targeting moieties can be directly conjugated by means of reactions between isothiocyanate groups such as FITC and free amine groups of small ligands (e.g., folate, DUPA, and CCK2R ligands). However, the use of a linker domain to connect the two molecules can be helpful as it can provide flexibility and stability. Examples of suitable linker domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) sugars; 5) non-natural peptidoglycans; 6) polyvinylpyrrolidone; 7) Pluronic F-127. Suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more atoms.

在一些實施例中,連接子可為可包括一或多個間隔子之二價連接子。In some embodiments, the linker may be a bivalent linker that may include one or more spacers.

本發明之說明性結合物為FITC-葉酸 。 本發明之說明性結合物為FITC-CA9 An illustrative conjugate of the present invention is FITC-folic acid The illustrative conjugate of the present invention is FITC-CA9 .

本發明之說明性結合物包括以下分子:FITC-(PEG) 12-葉酸、FITC-(PEG) 20-葉酸、FITC-(PEG) 108-葉酸、FITC-DUPA、FITC-(PEG) 12-DUPA、FITC-CCK2R配體、FITC-(PEG) 12-CCK2R配體、FITC-(PEG) 11-NK1R配體及FITC-(PEG) 2-CA9。 Illustrative conjugates of the invention include the following molecules: FITC-(PEG) 12 -folate, FITC-(PEG) 20 -folate, FITC-(PEG) 108 -folate, FITC-DUPA, FITC-(PEG) 12 -DUPA, FITC-CCK2R ligand, FITC-(PEG) 12 -CCK2R ligand, FITC-(PEG) 11 -NK1R ligand, and FITC-(PEG) 2 -CA9.

儘管配體及癌細胞受體結合之親和力可變化,且在一些情況下低親和力結合可為較佳的(諸如約1 µM),但配體及癌細胞受體之結合親和力一般將為至少約100 µM、1 nM、10 nM或100 nM,較佳地為至少約1 pM或10 pM,甚至更佳地為至少約100 pM。Although the affinity of binding of a ligand to a cancer cell receptor can vary, and in some cases low affinity binding may be preferred (e.g., about 1 μM), the binding affinity of a ligand to a cancer cell receptor will generally be at least about 100 μM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, and even more preferably at least about 100 pM.

結合物之實例及製備其之方法在美國專利申請US 2017/0290900、US 2019/0091308及US 2020/0023009中提供,其全部均以引用之方式併入本文中。 b.間隔子(例如鉸鏈域) Examples of conjugates and methods for preparing the same are provided in U.S. Patent Applications US 2017/0290900, US 2019/0091308, and US 2020/0023009, all of which are incorporated herein by reference. b. Spacers (e.g., hinge domains)

在一些實施例中,CAR包含鉸鏈域。在一些實施例中,鉸鏈域包含短鉸鏈或中等鉸鏈域。在一些實施例中,鉸鏈域包含CD8或IgG。在一些實施例中,CD8鉸鏈包含CD8α鉸鏈。在一些實施例中,IgG鉸鏈包含IgG4鉸鏈。在一些實施例中,IgG4鉸鏈經修飾。在一些實施例中,IgG鉸鏈包含IgG1鉸鏈。在一些實施例中,鉸鏈域包含PD1鉸鏈。在一些實施例中,鉸鏈域包含CD28鉸鏈。In some embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises a short hinge or a medium hinge domain. In some embodiments, the hinge domain comprises CD8 or IgG. In some embodiments, the CD8 hinge comprises a CD8α hinge. In some embodiments, the IgG hinge comprises an IgG4 hinge. In some embodiments, the IgG4 hinge is modified. In some embodiments, the IgG hinge comprises an IgG1 hinge. In some embodiments, the hinge domain comprises a PD1 hinge. In some embodiments, the hinge domain comprises a CD28 hinge.

在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,CD8α鉸鏈包含SEQ ID NO: 38或114之核苷酸序列。在一些實施例中,CD8α鉸鏈由SEQ ID NO: 38或114之核苷酸序列組成。In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8α hinge consists of the nucleotide sequence of SEQ ID NO: 38 or 114.

在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,CD8α鉸鏈包含SEQ ID NO: 39或115之胺基酸序列。在一些實施例中,CD8α鉸鏈由SEQ ID NO: 39或115之胺基酸序列組成。In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8α hinge comprises the amino acid sequence of SEQ ID NO: 39 or 115. In some embodiments, the CD8α hinge consists of the amino acid sequence of SEQ ID NO: 39 or 115.

在一些實施例中,CD8鉸鏈包含與SEQ ID NO: 123之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,CD8鉸鏈包含與SEQ ID NO: 123之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,CD8鉸鏈包含與SEQ ID NO: 123之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,CD8鉸鏈包含與SEQ ID NO: 123之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,CD8鉸鏈包含與SEQ ID NO: 123之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,CD8鉸鏈包含與SEQ ID NO: 123之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,CD8鉸鏈包含與SEQ ID NO: 123之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,CD8鉸鏈包含與SEQ ID NO: 123之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,CD8鉸鏈包含與SEQ ID NO: 123之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,CD8鉸鏈包含與SEQ ID NO: 123之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,CD8鉸鏈包含SEQ ID NO: 123之核苷酸序列。在一些實施例中,CD8鉸鏈由SEQ ID NO: 123之核苷酸序列組成。In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge comprises the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the CD8 hinge consists of the nucleotide sequence of SEQ ID NO: 123.

在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 119之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 119之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 119之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 119之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 119之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 119之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 119之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 119之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 119之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 119之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含SEQ ID NO: 119之胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈由SEQ ID NO: 119之胺基酸序列組成。In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the modified IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 119.

在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 120之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 120之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 120之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 120之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 120之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 120之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 120之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 120之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 120之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含與SEQ ID NO: 120之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈包含SEQ ID NO: 120之胺基酸序列。在一些實施例中,經修飾之IgG4鉸鏈由SEQ ID NO: 120之胺基酸序列組成。In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the modified IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 120.

在一些實施例中,IgG1鉸鏈包含與SEQ ID NO: 122之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,IgG1鉸鏈包含與SEQ ID NO: 122之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,IgG1鉸鏈包含與SEQ ID NO: 122之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,IgG1鉸鏈包含與SEQ ID NO: 122之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,IgG1鉸鏈包含與SEQ ID NO: 122之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,IgG1鉸鏈包含與SEQ ID NO: 122之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,IgG1鉸鏈包含與SEQ ID NO: 122之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,IgG1鉸鏈包含與SEQ ID NO: 122之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,IgG1鉸鏈包含與SEQ ID NO: 122之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,IgG1鉸鏈包含與SEQ ID NO: 122之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,IgG1鉸鏈包含SEQ ID NO: 122之胺基酸序列。在一些實施例中,IgG1鉸鏈由SEQ ID NO: 122之胺基酸序列組成。In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge comprises an amino acid sequence of SEQ ID NO: 122. In some embodiments, the IgG1 hinge consists of the amino acid sequence of SEQ ID NO: 122.

在一些實施例中,PD1鉸鏈包含與SEQ ID NO: 121之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,PD1鉸鏈包含與SEQ ID NO: 121之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,PD1鉸鏈包含與SEQ ID NO: 121之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,PD1鉸鏈包含與SEQ ID NO: 121之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,PD1鉸鏈包含與SEQ ID NO: 121之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,PD1鉸鏈包含與SEQ ID NO: 121之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,PD1鉸鏈包含與SEQ ID NO: 121之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,PD1鉸鏈包含與SEQ ID NO: 121之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,PD1鉸鏈包含與SEQ ID NO: 121之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,PD1鉸鏈包含與SEQ ID NO: 121之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,PD1鉸鏈包含SEQ ID NO: 121之胺基酸序列。在一些實施例中,PD1鉸鏈由SEQ ID NO: 121之胺基酸序列組成。In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge comprises an amino acid sequence of SEQ ID NO: 121. In some embodiments, the PD1 hinge consists of the amino acid sequence of SEQ ID NO: 121.

在一些實施例中,CD28鉸鏈包含與SEQ ID NO: 124之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,CD28鉸鏈包含與SEQ ID NO: 124之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,CD28鉸鏈包含與SEQ ID NO: 124之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,CD28鉸鏈包含與SEQ ID NO: 124之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,CD28鉸鏈包含與SEQ ID NO: 124之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,CD28鉸鏈包含與SEQ ID NO: 124之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,CD28鉸鏈包含與SEQ ID NO: 124之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,CD28鉸鏈包含與SEQ ID NO: 124之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,CD28鉸鏈包含與SEQ ID NO: 124之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,CD28鉸鏈包含與SEQ ID NO: 124之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,CD28鉸鏈包含SEQ ID NO: 124之胺基酸序列。在一些實施例中,CD28鉸鏈由SEQ ID NO: 124之胺基酸序列組成。 c.跨膜域 In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge comprises an amino acid sequence of SEQ ID NO: 124. In some embodiments, the CD28 hinge consists of the amino acid sequence of SEQ ID NO: 124. c. Transmembrane domain

在一些實施例中,CAR包含跨膜域。在一些實施例中,跨膜域包含CD8或CD28。在一些實施例中,跨膜域包含CD8域。在一些實施例中,跨膜域包含CD28域。在一些實施例中,CD8跨膜域包含CD8α跨膜域。In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises CD8 or CD28. In some embodiments, the transmembrane domain comprises a CD8 domain. In some embodiments, the transmembrane domain comprises a CD28 domain. In some embodiments, the CD8 transmembrane domain comprises a CD8α transmembrane domain.

在一些實施例中,跨膜域包含與SEQ ID NO: 40之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 40之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 40之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 40之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 40之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 40之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 40之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 40之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 40之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 40之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,跨膜域包含SEQ ID NO: 40之核苷酸序列。在一些實施例中,跨膜域由SEQ ID NO: 40之核苷酸序列組成。In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the transmembrane domain consists of the nucleotide sequence of SEQ ID NO: 40.

在一些實施例中,跨膜域包含與SEQ ID NO: 41之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 41之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 41之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 41之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 41之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 41之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 41之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 41之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 41之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,跨膜域包含與SEQ ID NO: 41之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,跨膜域包含SEQ ID NO: 41之胺基酸序列。在一些實施例中,跨膜域由SEQ ID NO: 41之胺基酸序列組成。 d.細胞內域(亦即胞內域) In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain consists of the amino acid sequence of SEQ ID NO: 41. d. Intracellular domain (i.e., intracellular domain)

在一些實施例中,CAR包含胞內域。在一些實施例中,胞內域包含共刺激分子。在一些實施例中,胞內域包含4-1BB、CD3ζ及/或CD28。在一些實施例中,胞內域包含4-1BB。在一些實施例中,胞內域包含CD3ζ。在一些實施例中,胞內域包含CD28。In some embodiments, CAR comprises an intracellular domain. In some embodiments, the intracellular domain comprises a co-stimulatory molecule. In some embodiments, the intracellular domain comprises 4-1BB, CD3ζ and/or CD28. In some embodiments, the intracellular domain comprises 4-1BB. In some embodiments, the intracellular domain comprises CD3ζ. In some embodiments, the intracellular domain comprises CD28.

在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,4-1BB胞內域包含SEQ ID NO: 42或69之核苷酸序列。在一些實施例中,4-1BB胞內域由SEQ ID NO: 42或69之核苷酸序列組成。In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain consists of the nucleotide sequence of SEQ ID NO: 42 or 69.

在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,4-1BB胞內域包含SEQ ID NO: 43之胺基酸序列。在一些實施例中,4-1BB胞內域由SEQ ID NO: 43之胺基酸序列組成。In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 80% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 85% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 90% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 95% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 96% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 97% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 98% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 99% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 100% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 100% consistent with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain consists of the amino acid sequence of SEQ ID NO: 43.

在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,CD3ζ胞內域包含SEQ ID NO: 46、70、100或118之核苷酸序列。在一些實施例中,CD3ζ胞內域由SEQ ID NO: 46、70、100或118之核苷酸序列組成。In some embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100, or 118. In some embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100, or 118. In some embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100, or 118. In some embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100, or 118. In some embodiments, the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100, or 118. In some embodiments, the CD3 zeta intracellular domain comprises the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some embodiments, the CD3 zeta intracellular domain consists of the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118.

在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少80%一致的胺基酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少85%一致的胺基酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少90%一致的胺基酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少95%一致的胺基酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少96%一致的胺基酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少97%一致的胺基酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少98%一致的胺基酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少99%一致的胺基酸序列。在一些實施例中,CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少100%一致的胺基酸序列。在一些實施例中,CD3ζ胞內域包含SEQ ID NO: 47之胺基酸序列。在一些實施例中,CD3ζ胞內域由SEQ ID NO: 47之胺基酸序列組成。 2.例示性CAR多核苷酸 In some embodiments, the CD3 ζ intracellular domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3 ζ intracellular domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3 ζ intracellular domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3 ζ intracellular domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3 ζ intracellular domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3ζ intracellular domain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3ζ intracellular domain comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3ζ intracellular domain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3ζ intracellular domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3ζ intracellular domain comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3ζ intracellular domain comprises an amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3ζ intracellular domain consists of the amino acid sequence of SEQ ID NO: 47. 2. Exemplary CAR polynucleotides

在一些實施例中,CAR為抗CD19 CAR,且在此等實施例中,多順反子載體包含含有編碼CD19 CAR之核苷酸序列的第四表現卡匣。在一些實施例中,CD19 CAR可包含訊號肽、特異性結合CD19之細胞外結合域、鉸鏈域、跨膜域、細胞內共刺激域及細胞內活化傳訊域。在一些實施例中,第四表現卡匣編碼具有 1中所述之特徵的抗CD19 CAR。 1.抗CD19 CAR及組分之例示性序列 組分 核苷酸 (SEQ ID NO) 胺基酸 (SEQ ID NO) 完全抗 CD19 CAR 104 105 細胞外結合域 CD19 scFv 106 107 CD19 scFv VL 108 109 連接子 110 111 CD19 scFv VH 112 113 鉸鏈及跨膜域 CD8鉸鏈 114 115 CD28跨膜域 116 117 共刺激傳訊域 4-1BB 99 43 活化傳訊域 CD3ζ 118 47 In some embodiments, the CAR is an anti-CD19 CAR, and in these embodiments, the multicistronic vector comprises a fourth expression cassette comprising a nucleotide sequence encoding a CD19 CAR. In some embodiments, the CD19 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds to CD19, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular activation signaling domain. In some embodiments, the fourth expression cassette encodes an anti-CD19 CAR having the characteristics described in Table 1 . Table 1. Exemplary sequences of anti-CD19 CAR and components Components Nucleotide (SEQ ID NO) Amino Acid (SEQ ID NO) Complete anti- CD19 CAR 104 105 Extracellular binding domain CD19 scFv 106 107 CD19 scFv VL 108 109 Connector 110 111 CD19 scFv VH 112 113 Hinge and transmembrane domain CD8 Hinge 114 115 CD28 transmembrane domain 116 117 co-stimulatory signaling domain 4-1BB 99 43 Activate the communication domain CD3ζ 118 47

在一些實施例中,CAR為抗CD20 CAR,且在此等實施例中,多順反子載體包含含有編碼CD20 CAR之核苷酸序列的第四表現卡匣。在一些實施例中,CD20 CAR可包含訊號肽、特異性結合CD20之細胞外結合域、鉸鏈域、跨膜域、細胞內共刺激域及細胞內活化傳訊域。在一些實施例中,第四表現卡匣編碼具有 2(具有flag之抗CD20 CAR)或 3(無flag之抗CD20 CAR)中所述之特徵的抗CD20 CAR。 2.抗CD20 CAR及組分(具有flag)之例示性序列 組分 核苷酸 (SEQ ID NO) 胺基酸 (SEQ ID NO) 完全抗 CD20 CAR 83 84 細胞外結合域 CD20 scFv 87 88 CD20 scFv VL 89 90 218連接子 91 92 CD20 scFv VH 93 94 Flag域 Flag 95 96 鉸鏈及跨膜域 CD8鉸鏈及跨膜域 97 98 共刺激傳訊域 4-1BB 99 43 活化傳訊域 CD3ζ 100 47 3.抗CD20 CAR及組分(無flag)之例示性序列 組分 核苷酸 (SEQ ID NO) 胺基酸 (SEQ ID NO) 完全抗 CD20 CAR 85 86 細胞外結合域 CD20 scFv 87 88 CD20 scFv VL 89 90 218連接子 91 92 CD20 scFv VH 93 94 鉸鏈及跨膜域 CD8鉸鏈及跨膜域 97 98 共刺激傳訊域 4-1BB 99 43 活化傳訊域 CD3ζ 100 47 In some embodiments, the CAR is an anti-CD20 CAR, and in these embodiments, the multicistronic vector comprises a fourth expression cassette comprising a nucleotide sequence encoding a CD20 CAR. In some embodiments, the CD20 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds to CD20, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular activation signaling domain. In some embodiments, the fourth expression cassette encodes an anti-CD20 CAR having the characteristics described in Table 2 (anti-CD20 CAR with flag) or Table 3 (anti-CD20 CAR without flag). Table 2. Exemplary sequences of anti-CD20 CAR and components (with flag) Components Nucleotide (SEQ ID NO) Amino Acid (SEQ ID NO) Complete anti- CD20 CAR 83 84 Extracellular binding domain CD20 scFv 87 88 CD20 scFv VL 89 90 218 connector 91 92 CD20 scFv VH 93 94 Flag field Flag 95 96 Hinge and transmembrane domain CD8 hinge and transmembrane domain 97 98 co-stimulatory signaling domain 4-1BB 99 43 Activate the communication domain CD3ζ 100 47 Table 3. Exemplary sequences of anti-CD20 CAR and components (without flag) Components Nucleotide (SEQ ID NO) Amino Acid (SEQ ID NO) Complete anti- CD20 CAR 85 86 Extracellular binding domain CD20 scFv 87 88 CD20 scFv VL 89 90 218 connector 91 92 CD20 scFv VH 93 94 Hinge and transmembrane domain CD8 hinge and transmembrane domain 97 98 co-stimulatory signaling domain 4-1BB 99 43 Activate the communication domain CD3ζ 100 47

在一些實施例中,第四表現卡匣編碼具有 4中所述之特徵的CAR。在一些實施例中,CAR為抗FITC CAR。 4.抗FITC CAR及組分之例示性序列 組分 核苷酸 (SEQ ID NO) 胺基酸 (SEQ ID NO) 完全抗 FITC Tag CAR 26、63、82 27、127 細胞外結合域 E2 scFv 28、64 29 E2 scFv VL 30、65 31 連接子 32、66 33 E2 scFv VH 34、67 35 鉸鏈及跨膜域 CD8鉸鏈及跨膜域 36 37 CD8鉸鏈 38 39 CD8跨膜域 40 41 共刺激傳訊域 4-1BB 42 43 活化傳訊域 CD3ζ 46 47 In some embodiments, the fourth expression cassette encodes a CAR having the characteristics described in Table 4. In some embodiments, the CAR is an anti-FITC CAR. Table 4. Exemplary sequences of anti-FITC CAR and components Components Nucleotide (SEQ ID NO) Amino Acid (SEQ ID NO) Complete anti- FITC Tag CAR 26, 63, 82 27, 127 Extracellular binding domain E2 scFv 28, 64 29 E2 scFv VL 30, 65 31 Connector 32, 66 33 E2 scFv VH 34, 67 35 Hinge and transmembrane domain CD8 hinge and transmembrane domain 36 37 CD8 Hinge 38 39 CD8 transmembrane domain 40 41 co-stimulatory signaling domain 4-1BB 42 43 Activate the communication domain CD3ζ 46 47

在一些實施例中,第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,第四表現卡匣包含SEQ ID NO: 26、63、71或82之核苷酸序列。在一些實施例中,第四表現卡匣由SEQ ID NO: 26、63、71或82之核苷酸序列組成。In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence of SEQ ID NO: 26, 63, 71, or 82. In some embodiments, the fourth expression cassette consists of a nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82.

在一些實施例中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少80%一致的胺基酸序列。在一些實施例中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少85%一致的胺基酸序列。在一些實施例中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少90%一致的胺基酸序列。在一些實施例中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少95%一致的胺基酸序列。在一些實施例中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少96%一致的胺基酸序列。在一些實施例中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少97%一致的胺基酸序列。在一些實施例中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少98%一致的胺基酸序列。在一些實施例中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少99%一致的胺基酸序列。在一些實施例中,第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少100%一致的胺基酸序列。在一些實施例中,第四表現卡匣編碼包含SEQ ID NO: 27、72或127之序列的胺基酸序列。在一些實施例中,第四表現卡匣編碼由SEQ ID NO: 27、72或127之序列組成的胺基酸序列。In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27, 72 or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 27, 72 or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 27, 72 or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 27, 72 or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 27, 72 or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence consisting of the sequence of SEQ ID NO: 27, 72, or 127.

在另外的實施例中,編碼CAR之說明性核苷酸序列可包含SEQ ID NO: 71,且說明性CAR胺基酸序列可包含SEQ ID NO: 72。In further embodiments, an illustrative nucleotide sequence encoding CAR may comprise SEQ ID NO: 71, and an illustrative CAR amino acid sequence may comprise SEQ ID NO: 72.

說明性核苷酸插入可包含SEQ ID NO: 73。Illustrative nucleotide insertions may include SEQ ID NO: 73.

在一些實施例中,CAR可由編碼訊號肽以傳訊在細胞中運輸CAR之核酸序列編碼。應理解,通常將自蛋白質移除訊號肽。In some embodiments, the CAR may be encoded by a nucleic acid sequence that encodes a signal peptide to signal transport of the CAR in the cell. It should be understood that the signal peptide will generally be removed from the protein.

無訊號肽之說明性CAR胺基酸序列可包含SEQ ID NO: 74。An illustrative CAR amino acid sequence without a signal peptide may include SEQ ID NO: 74.

具有訊號肽之說明性CAR胺基酸序列可包含SEQ ID NO: 75。在各種實施例中,提供表現CAR之細胞,其包含SEQ ID NO: 71或73之核酸。在一些實施例中,涵蓋嵌合抗原受體多肽,其包含SEQ ID NO: 72。在一些實施例中,涵蓋嵌合抗原受體多肽,其包含SEQ ID NO: 74。在一些實施例中,涵蓋載體,其包含SEQ ID NO: 71或73。在一些實施例中,涵蓋慢病毒載體,其包含SEQ ID NO: 71或73。在一些實施例中,SEQ ID NO: 72可包含人類或人源化胺基酸序列或由其組成。在一些實施例中,SEQ ID NO: 74可包含人類或人源化胺基酸序列或由其組成。An illustrative CAR amino acid sequence with a signal peptide may comprise SEQ ID NO: 75. In various embodiments, a cell expressing a CAR is provided, comprising a nucleic acid of SEQ ID NO: 71 or 73. In some embodiments, a chimeric antigen receptor polypeptide is contemplated, comprising SEQ ID NO: 72. In some embodiments, a chimeric antigen receptor polypeptide is contemplated, comprising SEQ ID NO: 74. In some embodiments, a vector is contemplated, comprising SEQ ID NO: 71 or 73. In some embodiments, a lentiviral vector is contemplated, comprising SEQ ID NO: 71 or 73. In some embodiments, SEQ ID NO: 72 may comprise or consist of a human or humanized amino acid sequence. In some embodiments, SEQ ID NO: 74 may comprise or consist of a human or humanized amino acid sequence.

在一些實施例中,涵蓋與SEQ ID NO: 71、SEQ ID NO: 72、SEQ ID NO: 73或SEQ ID NO: 74具有至少約80%、至少約90%、至少約95%、至少約97%、至少約98%、至少約99%或至少約99.5%序列一致性的變異核酸序列或胺基酸序列。In some embodiments, variant nucleic acid sequences or amino acid sequences having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74 are encompassed.

儘管由淋巴球表現之CAR與所靶向部分結合之親和力可變化,且在一些情況下低親和力結合可為較佳的(諸如約50 nM),但CAR與所靶向配體之結合親和力一般將為至少約100 nM、1 pM或10 pM,較佳地為至少約100 pM、1 fM或10 fM,甚至更佳地為至少約100 fM。 D.可裂解連接子 Although the affinity with which a CAR expressed by a lymphocyte binds to a targeted moiety can vary, and in some cases low affinity binding may be preferred (e.g., about 50 nM), the binding affinity of a CAR to a targeted ligand will generally be at least about 100 nM, 1 pM, or 10 pM, preferably at least about 100 pM, 1 fM, or 10 fM, and even more preferably at least about 100 fM. D. Cleavable Linker

如本文所提供,多順反子構築體之表現卡匣可藉由連接子分隔開。在一些實施例中,連接子包含用於裂解之位點,使得其為可裂解連接子。As provided herein, the expression cassettes of a polycistronic construct can be separated by a linker. In some embodiments, the linker comprises a site for cleavage, making it a cleavable linker.

裂解位點可用於多順反子構築體之設計中以達成多個基因之共表現。在一些實施例中,裂解位點包含自裂解位點。在一些實施例中,自裂解位點包含2A位點。2A肽為一類18-22個胺基酸長的肽,可誘導轉譯期間的核糖體跳躍,從而導致丟失甘胺酸與脯胺酸殘基之間的肽鍵,此允許蛋白質水解酶識別2A位點。分子生物學中最常用的2A肽包括T2A、P2A、E2A及F2A。Cleavage sites can be used in the design of polycistronic constructs to achieve co-expression of multiple genes. In some embodiments, the cleavage site comprises a self-cleavage site. In some embodiments, the self-cleavage site comprises a 2A site. 2A peptides are a class of 18-22 amino acid long peptides that can induce ribosome skipping during translation, resulting in the loss of the peptide bond between the glycine and proline residues, which allows protein hydrolases to recognize the 2A site. The most commonly used 2A peptides in molecular biology include T2A, P2A, E2A and F2A.

在一些實施例中,本文提供之多順反子構築體包含一或多個可裂解連接子。在一些實施例中,將表現卡匣分隔開之一或多個可裂解連接子相同。在一些實施例中,將表現卡匣分隔開之可裂解連接子不同。在一些實施例中,將表現卡匣分隔開之一或多個可裂解連接子包含一或多個裂解位點。在一些實施例中,該一或多個裂解位點相同。在一些實施例中,該一或多個裂解位點不同。In some embodiments, the polycistronic constructs provided herein comprise one or more cleavable linkers. In some embodiments, the one or more cleavable linkers separating the expression cassettes are identical. In some embodiments, the cleavable linkers separating the expression cassettes are different. In some embodiments, the one or more cleavable linkers separating the expression cassettes comprise one or more cleavage sites. In some embodiments, the one or more cleavage sites are identical. In some embodiments, the one or more cleavage sites are different.

在一些實施例中,除2A位點以外,可裂解連接子亦可包含另一裂解位點。在一些實施例中,額外裂解位點包含弗林蛋白酶位點。存在三種已知弗林蛋白酶位點,包括FC1、FC2及FC3。In some embodiments, in addition to the 2A site, the cleavable linker may also include another cleavage site. In some embodiments, the additional cleavage site includes a furin site. There are three known furin sites, including FC1, FC2 and FC3.

在一些實施例中,本文提供之多順反子構築體包含T2A、P2A、E2A或F2A裂解位點在可裂解連接子中。在一些實施例中,多順反子構築體包含T2A裂解位點在可裂解連接子中。在一些實施例中,多順反子構築體包含P2A裂解位點在可裂解連接子中。在一些實施例中,多順反子構築體包含弗林蛋白酶裂解位點在可裂解連接子中。在一些實施例中,多順反子構築體包含T2A裂解位點及弗林蛋白酶裂解位點在可裂解連接子中。In some embodiments, the polycistronic constructs provided herein comprise a T2A, P2A, E2A, or F2A cleavage site in a cleavable linker. In some embodiments, the polycistronic constructs comprise a T2A cleavage site in a cleavable linker. In some embodiments, the polycistronic constructs comprise a P2A cleavage site in a cleavable linker. In some embodiments, the polycistronic constructs comprise a furin cleavage site in a cleavable linker. In some embodiments, the polycistronic constructs comprise a T2A cleavage site and a furin cleavage site in a cleavable linker.

在一些實施例中,本文提供之多順反子構築體包含至少一個、至少兩個或至少三個2A可裂解連接子序列。在一些實施例中,本文中之多順反子構築體包含T2A裂解位點以及P2A、E2A或F2A裂解位點。在一些實施例中,本文中之多順反子構築體包含P2A裂解位點以及T2A、E2A或F2A裂解位點。在一些實施例中,本文中之多順反子構築體包含E2A裂解位點以及P2A、T2A或F2A裂解位點。在一些實施例中,本文中之多順反子構築體包含F2A裂解位點以及P2A、E2A或T2A裂解位點。In some embodiments, the polycistronic constructs provided herein comprise at least one, at least two, or at least three 2A cleavable linker sequences. In some embodiments, the polycistronic constructs herein comprise a T2A cleavage site and a P2A, E2A, or F2A cleavage site. In some embodiments, the polycistronic constructs herein comprise a P2A cleavage site and a T2A, E2A, or F2A cleavage site. In some embodiments, the polycistronic constructs herein comprise an E2A cleavage site and a P2A, T2A, or F2A cleavage site. In some embodiments, the polycistronic constructs herein comprise an F2A cleavage site and a P2A, E2A, or T2A cleavage site.

在一些實施例中,本文提供之多順反子構築體包含2A可裂解連接子序列。在一些實施例中,編碼2A可裂解連接子序列之各核苷酸序列不同。在一些實施例中,2A可裂解連接子獨立地為T2A、P2A、E2A或F2A裂解位點。在一些實施例中,2A可裂解連接子獨立地為P2A或T2A。In some embodiments, the polycistronic constructs provided herein comprise a 2A cleavable linker sequence. In some embodiments, the nucleotide sequences encoding the 2A cleavable linker sequence are different. In some embodiments, the 2A cleavable linker is independently a T2A, P2A, E2A, or F2A cleavage site. In some embodiments, the 2A cleavable linker is independently P2A or T2A.

在一些實施例中,2A可裂解連接子為P2A,且編碼P2A可裂解連接子之核苷酸序列包含與SEQ ID NO: 17、25、52或58至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。在一些實施例中,編碼P2A可裂解連接子之核苷酸序列述於SEQ ID NO: 17、25、52或58中。在一些實施例中,P2A可裂解連接子包含與SEQ ID NO: 18至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。在一些實施例中,P2A可裂解連接子包含SEQ ID NO: 18中所述之序列。In some embodiments, the 2A cleavable linker is P2A, and the nucleotide sequence encoding the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17, 25, 52 or 58. In some embodiments, the nucleotide sequence encoding the P2A cleavable linker is described in SEQ ID NO: 17, 25, 52 or 58. In some embodiments, the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18. In some embodiments, the P2A cleavable linker comprises the sequence described in SEQ ID NO: 18.

在一些實施例中,至少一個2A可裂解連接子為T2A,且編碼T2A可裂解連接子之核苷酸序列包含與SEQ ID NO: 9至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。在一些實施例中,編碼T2A可裂解連接子之核苷酸序列述於SEQ ID NO: 9中。在一些實施例中,T2A可裂解連接子包含與SEQ ID NO: 10至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。在一些實施例中,T2A可裂解連接子包含SEQ ID NO: 10中所述之序列。In some embodiments, at least one 2A cleavable linker is T2A, and the nucleotide sequence encoding the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the T2A cleavable linker is set forth in SEQ ID NO: 9. In some embodiments, the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. In some embodiments, the T2A cleavable linker comprises the sequence set forth in SEQ ID NO: 10.

在一些實施例中,裂解位點序列中之至少一者包含弗林蛋白酶裂解位點序列。在一些實施例中,弗林蛋白酶裂解位點序列位於第一表現卡匣與第二表現卡匣之間。在一些實施例中,編碼弗林蛋白酶裂解位點序列之核苷酸序列包含與SEQ ID NO: 7至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。在一些實施例中,編碼弗林蛋白酶裂解位點序列之核苷酸序列包含SEQ ID NO: 7中所述之序列。在一些實施例中,弗林蛋白酶裂解位點序列包含與SEQ ID NO: 8之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,弗林蛋白酶裂解位點序列包含SEQ ID NO: 8之胺基酸序列。In some embodiments, at least one of the cleavage site sequences comprises a furin cleavage site sequence. In some embodiments, the furin cleavage site sequence is between the first expression cassette and the second expression cassette. In some embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises a sequence described in SEQ ID NO: 7. In some embodiments, the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

在一些實施例中,裂解位點序列包含弗林蛋白酶裂解位點序列及T2A裂解序列(furinT2A)。在一些實施例中,編碼裂解位點序列之核苷酸序列與SEQ ID NO: 5之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。在一些實施例中,編碼裂解位點序列之核苷酸序列包含SEQ ID NO: 5之核苷酸序列。In some embodiments, the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furinT2A). In some embodiments, the nucleotide sequence encoding the cleavage site sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the cleavage site sequence comprises the nucleotide sequence of SEQ ID NO: 5.

在一些實施例中,裂解位點序列包含與SEQ ID NO: 6之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。在一些實施例中,裂解位點序列包含SEQ ID NO: 6之胺基酸序列。In some embodiments, the cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 6.

在一些實施例中,第一表現卡匣及第二表現卡匣藉由furinT2A分隔開,第二表現卡匣及第三表現卡匣藉由P2A分隔開,且第三表現卡匣及第四表現卡匣藉由P2A分隔開。 E.例示性多順反子構築體 In some embodiments, the first expression cassette and the second expression cassette are separated by furinT2A, the second expression cassette and the third expression cassette are separated by P2A, and the third expression cassette and the fourth expression cassette are separated by P2A. E. Exemplary polycistronic constructs

在一些實施例中,本文提供之多順反子構築體包含 5 6 7中所述之特徵。 表5. RACR構築體之例示性序列 組分 核苷酸(SEQ ID NO) 胺基酸(SEQ ID NO) 構築體C2.U 完全 載體 1 構築體C2.U 125 2 FRB 3 4 RACR FRB-IL2Rγ 11 12 FRB 13 14 IL2Rγ 15 16 FKBP12-IL2Rβ 19 20 FKBP12 21 22 IL2Rβ 23 24 抗FITC CAR 26、63、82 27、127 In some embodiments, the polycistronic constructs provided herein comprise the features described in Table 5 , Table 6 , or Table 7 . Table 5. Exemplary sequences of RACR constructs Components Nucleotide (SEQ ID NO) Amino Acid (SEQ ID NO) Structure C2.U complete carrier 1 Structure C2.U 125 2 FRB 3 4 RACR FRB-IL2Rγ 11 12 FRB 13 14 IL2Rγ 15 16 FKBP12-IL2Rβ 19 20 FKBP12 twenty one twenty two IL2Rβ twenty three twenty four Anti-FITC CAR 26, 63, 82 27, 127

在一些實施例中,構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少80%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少85%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少90%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少95%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少96%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少97%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少98%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少99%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少100%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含SEQ ID NO: 2之胺基酸序列的多肽。在一些實施例中,構築體編碼由SEQ ID NO: 2之胺基酸序列組成的多肽。In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the construct encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

在一些實施例中,構築體包含與SEQ ID NO: 125之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 125之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 125之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 125之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 125之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 125之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 125之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 125之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 125之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 125之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,構築體包含SEQ ID NO: 125之核苷酸序列。在一些實施例中,構築體由SEQ ID NO: 125之核苷酸序列組成。In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO: 125.

在一些實施例中,構築體包含與SEQ ID NO: 1之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 1之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 1之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 1之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 1之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 1之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 1之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 1之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 1之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 1之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,構築體包含SEQ ID NO: 1之核苷酸序列。在一些實施例中,構築體由SEQ ID NO: 1之核苷酸序列組成。 表6. RACR構築體之例示性序列 組分 核苷酸(SEQ ID NO) 胺基酸(SEQ ID NO) 構築體C2 完全載體 48 構築體C2 126 49 FRB 50 51 RACR FKBP12-IL2Rγ 53 54 FKBP12 55 22 IL2Rγ 56 57 FRB-IL2Rβ 59 60 FRB 13 14 IL2Rβ 61 62 抗FITC CAR 26、63、82 27、127 In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO: 1. Table 6. Exemplary sequences of RACR constructs Components Nucleotide (SEQ ID NO) Amino Acid (SEQ ID NO) Structure C2 complete carrier 48 Structure C2 126 49 FRB 50 51 RACR FKBP12-IL2Rγ 53 54 FKBP12 55 twenty two IL2Rγ 56 57 FRB-IL2Rβ 59 60 FRB 13 14 IL2Rβ 61 62 Anti-FITC CAR 26, 63, 82 27, 127

在一些實施例中,構築體編碼包含與SEQ ID NO: 48之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 48之胺基酸序列至少80%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 48之胺基酸序列至少85%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 48之胺基酸序列至少90%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 48之胺基酸序列至少95%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 48之胺基酸序列至少96%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 48之胺基酸序列至少97%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 48之胺基酸序列至少98%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 48之胺基酸序列至少99%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 48之胺基酸序列至少100%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含SEQ ID NO: 48之胺基酸序列的多肽。在一些實施例中,構築體編碼由SEQ ID NO: 48之胺基酸序列組成的多肽。In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the construct encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 48.

在一些實施例中,構築體包含與SEQ ID NO: 126之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 126之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 126之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 126之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 126之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 126之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 126之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 126之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 126之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 126之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,構築體包含SEQ ID NO: 126之核苷酸序列。在一些實施例中,構築體由SEQ ID NO: 126之核苷酸序列組成。In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO: 126.

在一些實施例中,構築體包含與SEQ ID NO: 48之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 48之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 48之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 48之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 48之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 48之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 48之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 48之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 48之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 48之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,構築體包含SEQ ID NO: 48之核苷酸序列。在一些實施例中,構築體由SEQ ID NO: 48之核苷酸序列組成。 表7. RACR構築體之例示性序列 組分 核苷酸(SEQ ID NO) 胺基酸(SEQ ID NO) 構築體C3.U 完全載體 101 構築體C3.U 102 103 FRB 3 4 RACR FRB-IL2Rγ 11 12 FRB 13 14 IL2Rγ 15 16 FKBP12-IL2Rβ 19 20 FKBP12 21 22 IL2Rβ 23 24 抗CD19 CAR 104 105 In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO: 48. Table 7. Exemplary sequences of RACR constructs Components Nucleotide (SEQ ID NO) Amino Acid (SEQ ID NO) Structure C3.U complete carrier 101 Structure C3.U 102 103 FRB 3 4 RACR FRB-IL2Rγ 11 12 FRB 13 14 IL2Rγ 15 16 FKBP12-IL2Rβ 19 20 FKBP12 twenty one twenty two IL2Rβ twenty three twenty four Anti-CD19 CAR 104 105

在一些實施例中,構築體編碼包含與SEQ ID NO: 103之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 103之胺基酸序列至少80%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 103之胺基酸序列至少85%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 103之胺基酸序列至少90%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 103之胺基酸序列至少95%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 103之胺基酸序列至少96%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 103之胺基酸序列至少97%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 103之胺基酸序列至少98%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 103之胺基酸序列至少99%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含與SEQ ID NO: 103之胺基酸序列至少100%一致之胺基酸序列的多肽。在一些實施例中,構築體編碼包含SEQ ID NO: 103之胺基酸序列的多肽。在一些實施例中,構築體編碼由SEQ ID NO: 103之胺基酸序列組成的多肽。In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 103. In some embodiments, the construct encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 103.

在一些實施例中,構築體包含與SEQ ID NO: 102之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 102之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 102之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 102之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 102之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 102之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 102之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 102之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 102之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 102之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,構築體包含SEQ ID NO: 102之核苷酸序列。在一些實施例中,構築體由SEQ ID NO: 102之核苷酸序列組成。In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 102. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO: 102.

在一些實施例中,構築體包含與SEQ ID NO: 101之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 101之核苷酸序列至少80%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 101之核苷酸序列至少85%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 101之核苷酸序列至少90%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 101之核苷酸序列至少95%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 101之核苷酸序列至少96%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 101之核苷酸序列至少97%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 101之核苷酸序列至少98%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 101之核苷酸序列至少99%一致的核苷酸序列。在一些實施例中,構築體包含與SEQ ID NO: 101之核苷酸序列至少100%一致的核苷酸序列。在一些實施例中,構築體包含SEQ ID NO: 101之核苷酸序列。在一些實施例中,構築體由SEQ ID NO: 101之核苷酸序列組成。 II.核酸載體 In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO: 101. II. Nucleic acid vectors

在一些實施例中,可多順反子構築體插入核酸載體中。如本文所用,術語「核酸載體」意欲意謂用於攜帶、含有或表現所關注核酸之任何核酸。核酸載體可具有特定功能,諸如表現、封裝、假模式化、轉導或定序。核酸載體亦可具有例如操縱功能,諸如選殖或穿梭載體。載體之結構可包括可進行製造且特定用途所需之任何所需形式。此類形式包括例如環狀形式(諸如質體及噬菌粒)以及線性或分支形式。核酸載體可由例如DNA或RNA構成,以及部分或完全含有核苷酸衍生物、類似物及模擬物。此類核酸載體可獲自天然來源,以重組方式製造或以化學方式合成。In some embodiments, a polycistronic construct can be inserted into a nucleic acid vector. As used herein, the term "nucleic acid vector" is intended to mean any nucleic acid used to carry, contain or express a nucleic acid of interest. Nucleic acid vectors can have specific functions, such as expression, packaging, pseudo-patterning, transduction or sequencing. Nucleic acid vectors can also have, for example, manipulation functions, such as cloning or shuttle vectors. The structure of the vector can include any desired form that can be manufactured and required for a specific purpose. Such forms include, for example, circular forms (such as plasmids and phagemids) and linear or branched forms. Nucleic acid vectors can be composed of, for example, DNA or RNA, and partially or completely contain nucleotide derivatives, analogs and mimetics. Such nucleic acid vectors can be obtained from natural sources, recombinantly produced or chemically synthesized.

本發明之載體系統之非限制性實例包括逆轉錄病毒、慢病毒、泡沫病毒及睡美人轉位子。 A.逆轉錄病毒載體 Non-limiting examples of the vector systems of the present invention include retroviruses, lentiviruses, foamy viruses, and Sleeping Beauty transposons. A. Retroviral vectors

逆轉錄病毒包括慢病毒、γ-逆轉錄病毒及α-逆轉錄病毒,其各自可用於使用此項技術中已知之方法將多核苷酸遞送至細胞。慢病毒為複合逆轉錄病毒,除常見逆轉錄病毒基因gag、pol及env以外,該等慢病毒含有其他具有調節或結構性功能之基因。較高的複雜度使得病毒能夠調節其生命週期,如在潛伏感染過程中一樣。慢病毒之一些實例包括人類免疫缺乏病毒(HIV-1及HIV-2)及猿猴免疫缺乏病毒(Simian Immunodeficiency Virus;SIV)。逆轉錄病毒載體已藉由多次減弱HIV毒力基因產生,例如缺失基因env、vif、vpr、vpu及nef,從而使得載體生物學上安全。Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which can be used to deliver polynucleotides to cells using methods known in the art. Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. The greater complexity enables the virus to regulate its life cycle, as during latent infection. Some examples of lentiviruses include human immunodeficiency virus (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). Retroviral vectors have been generated by multiple attenuation of HIV virulence genes, such as deleting the genes env, vif, vpr, vpu, and nef, thereby rendering the vector biologically safe.

說明性慢病毒載體包括描述於以下中之彼等者:Naldini等人(1996) Science 272:263-7;Zufferey等人(1998) J. Virol. 72:9873-9880;Dull等人(1998) J. Virol. 72:8463-8471;美國專利第6,013,516號;及美國專利第5,994,136號,其各自以全文引用之方式併入本文中。一般而言,此等載體經組態以攜帶必需序列以用於選擇含有載體之細胞、用於將外來核酸併入慢病毒顆粒中及用於將核酸轉移至目標細胞中。Illustrative lentiviral vectors include those described in Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. In general, these vectors are configured to carry the necessary sequences for selection of cells containing the vector, for incorporation of foreign nucleic acids into lentiviral particles, and for transfer of nucleic acids into target cells.

常用慢病毒載體系統為所謂的第三代系統。第三代慢病毒載體系統包括四種質體。「轉移質體」編碼藉由慢病毒載體系統遞送至目標細胞的多核苷酸序列。轉移質體一般具有一或多個側接有長末端重複(LTR)序列之所關注轉殖基因序列,該等長末端重複序列促進將轉移質體序列整合至宿主基因體中。出於安全原因,轉移質體一般設計成使所得載體不能複製。舉例而言,轉移質體缺乏在宿主細胞中產生感染性顆粒所必需的基因元件。另外,轉移質體可設計成具有3' LTR缺失,使得病毒「自身失活」(SIN)。參見Dull等人(1998) J. Virol. 72:8463-71;Miyoshi等人(1998) J. Virol. 72:8150-57。病毒顆粒亦可包含3'非轉譯區(UTR)及5' UTR。UTR包含逆轉錄病毒調節元件,該等逆轉錄病毒調節元件支持在細胞被逆轉錄病毒顆粒接觸之後將前病毒基因體封裝、逆轉錄及整合至細胞中。Commonly used lentiviral vector systems are so-called third generation systems. Third generation lentiviral vector systems include four types of plasmids. The "transfer plasmid" encodes the polynucleotide sequence that is delivered to the target cell by the lentiviral vector system. The transfer plasmid generally has one or more sequences of the transgene of interest flanked by long terminal repeat (LTR) sequences that promote integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is generally designed so that the resulting vector cannot replicate. For example, the transfer plasmid lacks the genetic elements necessary to produce infectious particles in the host cell. In addition, the transfer plasmid can be designed to have a 3' LTR deletion, rendering the virus "self-inactive" (SIN). See Dull et al. (1998) J. Virol. 72:8463-71; Miyoshi et al. (1998) J. Virol. 72:8150-57. The viral particles may also contain a 3' untranslated region (UTR) and a 5' UTR. The UTR contains retroviral regulatory elements that support packaging, reverse transcription, and integration of the proviral genome into cells after exposure to the retroviral particles.

第三代系統一般亦包括兩種「封裝質體」及「套膜質體」。「套膜質體」一般編碼可操作地連接於啟動子之Env基因。在說明性第三代系統中,Env基因為VSV-G,且啟動子為CMV啟動子。第三代系統使用兩種封裝質體作為另一安全特徵,一種編碼gag及pol且另一種編碼rev,此係一項優於所謂的第二代系統之單一封裝質體的改善。儘管較安全,但由於添加額外質體,因此第三代系統使用起來可能更繁瑣且導致病毒力價較低。說明性封裝質體包括但不限於pMD2.G、pRSV-rev、pMDLG-pRRE及pRRL-GOI。Third generation systems also generally include two "encapsulation plasmids" and "encapsulation plasmids." The "encapsulation plasmid" generally encodes the Env gene operably linked to a promoter. In an illustrative third generation system, the Env gene is VSV-G and the promoter is the CMV promoter. As another safety feature, third generation systems use two encapsulation plasmids, one encoding gag and pol and the other encoding rev, an improvement over the single encapsulation plasmid of the so-called second generation system. Although safer, third generation systems may be more cumbersome to use and result in lower virulence due to the addition of an extra plasmid. Illustrative encapsulation plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

許多逆轉錄病毒載體系統依賴於使用「封裝細胞株」。一般而言,封裝細胞株為細胞能夠在轉移質體、封裝一或多種質體及套膜質體被引入細胞中時產生感染性逆轉錄病毒顆粒之細胞株。可使用將質體引入細胞中之各種方法,包括轉染或電穿孔。在一些情況下,封裝細胞株適合於將逆轉錄病毒載體系統高效封裝至逆轉錄病毒顆粒中。Many retroviral vector systems rely on the use of a "packaging cell line." Generally, a packaging cell line is a cell line whose cells are capable of producing infectious retroviral particles when a transfer plasmid, packaging plasmid or plasmids, and envelope plasmid are introduced into the cell. A variety of methods for introducing plasmids into cells can be used, including transfection or electroporation. In some cases, the packaging cell line is adapted to efficiently package the retroviral vector system into retroviral particles.

如本文所用,術語「逆轉錄病毒載體」或「慢病毒載體」意欲意謂編碼基因體封裝所需之逆轉錄病毒或慢病毒順式核酸序列以及一或多種待遞送至目標細胞中之多核苷酸序列的核酸。逆轉錄病毒顆粒及慢病毒顆粒一般包括RNA基因體(源於轉移質體)、包埋Env蛋白於其中之脂質雙層套膜及其他輔助蛋白(包括整合酶、蛋白酶及基質蛋白)。如本文所用,術語「逆轉錄病毒顆粒」及「慢病毒顆粒」係指包括套膜、具有一或多種慢病毒之特徵且能夠侵襲目標宿主細胞的病毒顆粒。此類特徵包括例如:感染非分裂宿主細胞;轉導非分裂宿主細胞;感染或轉導宿主免疫細胞;含有逆轉錄病毒或慢病毒病毒粒子,包括gag結構性多肽中之一或多者;含有逆轉錄病毒或慢病毒套膜,包括env編碼之醣蛋白中之一或多者;含有基因體,包括在複製、前病毒整合或轉錄方面起作用之一或多種逆轉錄病毒或慢病毒順式作用序列;含有編碼逆轉錄病毒或慢病毒蛋白酶、逆轉錄酶或整合酶之基因體;或含有編碼調節活性基因體,諸如Tat或Rev。轉移質體可包含美國專利第8,093,042號中所描述之cPPT序列。As used herein, the term "retrotransfer virus vector" or "lentivirus vector" is intended to mean a nucleic acid encoding a retrotransfer virus or lentivirus cis nucleic acid sequence required for genome packaging and one or more polynucleotide sequences to be delivered to a target cell. Retrotransfer virus particles and lentivirus particles generally include an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope in which the Env protein is embedded, and other accessory proteins (including integrase, protease, and matrix protein). As used herein, the term "retrotransfer virus particle" and "lentivirus particle" refer to a virus particle that includes an envelope, has one or more characteristics of a lentivirus, and is capable of invading a target host cell. Such characteristics include, for example: infection of non-dividing host cells; transduction of non-dividing host cells; infection or transduction of host immune cells; containing retroviral or lentiviral particles, including one or more of the gag structural polypeptides; containing a retroviral or lentiviral envelope, including one or more of the glycoproteins encoded by env; containing a genome, including one or more retroviral or lentiviral cis-acting sequences that function in replication, proviral integration or transcription; containing a genome encoding a retroviral or lentiviral protease, reverse transcriptase or integrase; or containing a genome encoding a regulatory activity, such as Tat or Rev. The transfer plasmid may comprise a cPPT sequence described in U.S. Patent No. 8,093,042.

系統之效率為載體工程改造中之重要問題。逆轉錄病毒或慢病毒載體系統之效率可以此項技術中已知之各種方式評定,包括量測載體複本數(vector copy number;VCN)或載體基因體(vector genome;vg) (諸如藉由定量聚合酶鏈反應(quantitative polymerase chain reaction;qPCR))、或以感染單位/毫升(IU/mL)為單位之病毒之力價。舉例而言,力價可使用對於所培養腫瘤細胞株HT1080進行之功能分析來評定,如Humbert等人Development of third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy 24:1237-1246 (2016)中所描述。當對於在不斷分裂之所培養細胞株評定力價時,不需要刺激且因此所量測力價不受逆轉錄病毒顆粒之表面工程改造影響。用於評定逆轉錄病毒載體系統之效率的其他方法在Gaererts等人Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006)中提供。The efficiency of the system is an important issue in vector engineering. The efficiency of a retroviral or lentiviral vector system can be assessed in a variety of ways known in the art, including measuring vector copy number (VCN) or vector genome (vg) (e.g., by quantitative polymerase chain reaction (qPCR)), or viral titer in infectious units/ml (IU/mL). For example, titer can be assessed using functional assays performed on the cultured tumor cell line HT1080, as described in Humbert et al. Development of third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy 24:1237-1246 (2016). When titers are assessed for dividing cultured cell lines, no stimulation is required and therefore the titers measured are not affected by surface engineering of the retroviral particles. Other methods for assessing the efficiency of retroviral vector systems are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

在一些實施例中,本發明之逆轉錄病毒顆粒及/或慢病毒顆粒包含含有編碼與閘控轉接子特異性結合之受體之序列的多核苷酸。在一些實施例中,編碼與閘控轉接子特異性結合之受體的序列可操作地連接於啟動子。說明性啟動子包括但不限於細胞巨大病毒(CMV)啟動子、CAG啟動子、SV40啟動子、SV40/CD43啟動子及MND啟動子。In some embodiments, the retroviral particles and/or lentiviral particles of the present invention comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a gating adapter. In some embodiments, the sequence encoding a receptor that specifically binds to a gating adapter is operably linked to a promoter. Illustrative promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, a SV40 promoter, a SV40/CD43 promoter, and a MND promoter.

在一些實施例中,逆轉錄病毒顆粒包含轉導強化子。在一些實施例中,逆轉錄病毒顆粒包含標記蛋白質。In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a marker protein.

在一些實施例中,逆轉錄病毒顆粒各包含多核苷酸,以5'至3'順序,該多核苷酸包含:(i) 5'長末端重複序列(LTR)或非轉譯區(UTR),(ii)啟動子,(iii)編碼與配體特異性結合之受體的序列,及(iv) 3' LTR或UTR。In some embodiments, each retroviral particle comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat sequence (LTR) or a non-translated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to a ligand, and (iv) a 3' LTR or UTR.

在一些實施例中,逆轉錄病毒顆粒包含與目標宿主細胞上之表面標記結合,從而允許宿主細胞轉導的細胞表面受體。病毒載體可包含異源病毒套膜醣蛋白,從而產生假模式化病毒載體。舉例而言,病毒套膜醣蛋白可來源於RD114或其變體中之一者、VSV-G、長臂猿白血病病毒(GALV),或為雙嗜性套膜、麻疹套膜或狒狒逆轉錄病毒套膜醣蛋白。在一些實施例中,細胞表面受體為來自科卡爾病毒(Cocal)株或其功能變體之VSV G蛋白。In some embodiments, the retroviral particles comprise a cell surface receptor that binds to a surface marker on a target host cell, thereby allowing transduction of the host cell. The viral vector may comprise a heterologous viral envelope glycoprotein, thereby generating a pseudotyped viral vector. For example, the viral envelope glycoprotein may be derived from RD114 or one of its variants, VSV-G, Gibbon Ape Leukemia Virus (GALV), or is an amphoteric envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is a VSV G protein from a Cocal virus strain or a functional variant thereof.

各種融合醣蛋白可用於使慢病毒載體假模式化。儘管最常用實例為來自水泡性口炎病毒(VSVG)之套膜醣蛋白,但亦使用許多其他病毒蛋白來使慢病毒載體假模式化。參見Joglekar等人Human Gene Therapy Methods 28:291-301 (2017)。本發明涵蓋各種融合醣蛋白之取代。值得注意的是,一些融合醣蛋白產生較高載體效率。Various fusion glycoproteins can be used to pseudotype lentiviral vectors. Although the most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), many other viral proteins are also used to pseudotype lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present invention encompasses the replacement of various fusion glycoproteins. Notably, some fusion glycoproteins produce higher vector efficiencies.

在一些實施例中,使融合醣蛋白或其功能變體假模式化促進特定細胞類型之靶向轉導,該等細胞類型包括但不限於先天性淋巴細胞、細胞毒性先天性淋巴細胞或NK細胞。在一些實施例中,融合醣蛋白或其功能變體為以下各者之一或多種全長多肽、一或多種功能片段、一或多種同源物或一或多種功能變體:人類免疫缺乏病毒(HIV) gp160;鼠白血病病毒(MLV) gp70;長臂猿白血病病毒(GALV) gp70;貓白血病病毒(RD114) gp70;雙嗜性逆轉錄病毒(兩性) gp70;10A1 MLV (10A1)gp70;親嗜性逆轉錄病毒(Eco) gp70;狒狒白血病病毒(BaEV) gp70;麻疹病毒(Measles virus;MV) H及F;尼帕病毒(NiV) H及F;狂犬病病毒(RabV) G;莫科拉病毒(Mokola virus) (MOKV) G;埃博拉扎伊爾病毒(Ebola Zaire virus) (EboZ) G;淋巴球性脈絡叢腦膜炎病毒(LCMV) GP1及GP2;桿狀病毒GP64;屈公病毒(Chikungunya virus) (CHIKV) E1及E2;羅斯河病毒(Ross River virus;RRV) E1及E2;勝利基森林病毒(Semliki Forest virus;SFV) E1及E2;辛德比斯病毒(Sindbis virus;SV) E1及E2;委內瑞拉馬腦炎病毒(Venezualan equine encephalitis virus;VEEV) E1及E2;西部馬腦炎病毒(Western equine encephalitis virus;WEEV) E1及E2;A、B、C或D型流感HA;雞瘟病毒(Fowl Plague Virus;FPV)HA;水泡性口炎病毒VSV-G;或章地埔拉病毒(Chandipura virus)及皮里病毒(Piry virus) CNV-G及PRV-G。In some embodiments, the fusion glycoprotein or a functional variant thereof is pseudopatterned to facilitate targeted transduction of specific cell types, including but not limited to innate lymphoid cells, cytotoxic innate lymphoid cells, or NK cells. In some embodiments, the fusion glycoprotein or a functional variant thereof is one or more full-length polypeptides, one or more functional fragments, one or more homologs, or one or more functional variants of the following: human immunodeficiency virus (HIV) gp160; murine leukemia virus (MLV) gp70; gibbon ape leukemia virus (GALV) gp70; feline leukemia virus (RD114) gp70; amphitropic retrovirus (amphitropic) gp70; 10A1 MLV (10A1) gp70; ecotropic retrovirus (Eco) gp70; baboon leukemia virus (BaEV) gp70; measles virus (MV) H and F; Nipah virus (NiV) H and F; rabies virus (RabV) G; Mokola virus (MOKV) G; Ebola Zaire virus (Ebola Zaire virus (EboZ) G; lymphocytic choroidal meningitis virus (LCMV) GP1 and GP2; baculovirus GP64; Chikungunya virus (CHIKV) E1 and E2; Ross River virus (RRV) E1 and E2; Semliki Forest virus (SFV) E1 and E2; Sindbis virus (SV) E1 and E2; Venezuelan equine encephalitis virus (VEEV) E1 and E2; Western equine encephalitis virus (WEEV) E1 and E2; influenza A, B, C, or D HA; Fowl Plague Virus (FPV) HA; vesicular stomatitis virus VSV-G; or Chandipura virus (Chandipura virus) and Piry virus CNV-G and PRV-G.

在一些實施例中,融合醣蛋白或其功能變體為以下各者之G蛋白之全長多肽、功能片段、同源物或功能變體:水泡性口炎阿拉戈斯病毒(Vesicular Stomatitis Alagoas Virus;VSAV)、卡拉加斯水泡病毒(Carajas Vesiculovirus) (CJSV)、金迪普拉水泡病毒(Chandipura Vesiculovirus) (CHPV)、科卡爾水泡病毒(Cocal Vesiculovirus) (COCV)、水泡性口炎印地安那病毒(Vesicular Stomatitis Indiana Virus;VSIV)、伊斯法罕水泡病毒(Isfahan Vesiculovirus) (ISFV)、馬拉巴水泡病毒(Maraba Vesiculovirus) (MARAV)、水泡性口炎紐澤西病毒(Vesicular Stomatitis New Jersey virus;VSNJV)、巴斯-剛果病毒(Bas-Congo Virus) (BASV)。在一些實施例中,融合醣蛋白或其功能變體為科卡爾病毒G蛋白。In some embodiments, the fusion glycoprotein or a functional variant thereof is a full-length polypeptide, a functional fragment, a homolog or a functional variant of the G protein of Vesicular Stomatitis Alagoas Virus (VSAV), Carajas Vesiculovirus (CJSV), Chandipura Vesiculovirus (CHPV), Cocal Vesiculovirus (COCV), Vesicular Stomatitis Indiana Virus (VSIV), Isfahan Vesiculovirus (ISFV), Maraba Vesiculovirus (MARAV), Vesicular Stomatitis New Jersey virus (VSNJV), Bas-Congo Virus (BASV). In some embodiments, the fusion glycoprotein or a functional variant thereof is Cocal virus G protein.

在一些實施例中,融合醣蛋白或其功能變體為以下各者之G蛋白之全長多肽、功能片段、同源物或功能變體:水泡性口炎阿拉戈斯病毒(VSAV)、卡拉加斯水泡病毒(CJSV)、金迪普拉水泡病毒(CHPV)、科卡爾水泡病毒(COCV)、水泡性口炎印地安那病毒(VSIV)、伊斯法罕水泡病毒(ISFV)、馬拉巴水泡病毒(MARAV)、水泡性口炎紐澤西病毒(VSNJV)、巴斯-剛果病毒(BASV)。在一些實施例中,融合醣蛋白或其功能變體為科卡爾病毒G蛋白。本發明進一步提供各種逆轉錄病毒載體,包括但不限於γ-逆轉錄病毒載體、α-逆轉錄病毒載體及慢病毒載體。在一些實施例中,載體可為病毒載體、逆轉錄病毒載體、慢病毒載體、γ-逆轉錄病毒載體。在一些實施例中,病毒載體包含VSV G蛋白或其功能變體。在一些實施例中,病毒載體包含科卡爾病毒G蛋白或其功能變體。In some embodiments, the fusion glycoprotein or its functional variant is a full-length polypeptide, functional fragment, homolog or functional variant of the G protein of the following: vesicular stomatitis Alagoas virus (VSAV), vesicular stomatitis virus of Caracas (CJSV), vesicular stomatitis virus of Chandipura (CHPV), vesicular stomatitis virus of Cocal (COCV), vesicular stomatitis Indiana virus (VSIV), vesicular stomatitis virus of Isfahan (ISFV), vesicular stomatitis virus of Malaba (MARAV), vesicular stomatitis New Jersey virus (VSNJV), Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or its functional variant is the G protein of Cocal virus. The present invention further provides various retroviral vectors, including but not limited to γ-retroviral vectors, α-retroviral vectors and lentiviral vectors. In some embodiments, the vector can be a viral vector, a retroviral vector, a lentiviral vector, a γ-retroviral vector. In some embodiments, the viral vector comprises VSV G protein or a functional variant thereof. In some embodiments, the viral vector comprises Cocal virus G protein or a functional variant thereof.

在一些實施例中,本文提供一種病毒載體,其包含本文提供之多順反子構築體中之任一者。在一些實施例中,病毒載體為慢病毒載體。在一些實施例中,病毒載體進一步包含一或多種表面T細胞活化劑。在一些實施例中,該一或多種表面T細胞活化劑包含CD58、抗CD3或CD80。 III.病毒顆粒 In some embodiments, provided herein is a viral vector comprising any of the polycistronic constructs provided herein. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector further comprises one or more surface T cell activators. In some embodiments, the one or more surface T cell activators comprise CD58, anti-CD3, or CD80. III. Viral particles

在一些實施例中,本文提供一種囊封本文所揭示之多順反子構築體的病毒顆粒。在一些實施例中,多順反子構築體中之任一者可作為在病毒顆粒產生中之有效負載提供。本文亦提供病毒顆粒,諸如慢病毒載體,其併入所提供多順反子構築體中之任一者用於將雷帕黴素活化細胞介素受體(RACR)系統之組分(包括FRB、合成細胞介素受體及CAR)遞送至目標細胞。在另外的實施例中,病毒顆粒可經工程改造以表現一或多種表面T細胞活化劑。In some embodiments, provided herein is a viral particle encapsulating a polycistronic construct disclosed herein. In some embodiments, any of the polycistronic constructs can be provided as an effective load in the production of viral particles. Also provided herein are viral particles, such as lentiviral vectors, which incorporate any of the polycistronic constructs provided for delivery of components of the rapamycin activated interleukin receptor (RACR) system (including FRB, synthetic interleukin receptor and CAR) to target cells. In other embodiments, the viral particles can be engineered to express one or more surface T cell activators.

如此項技術中所熟知,病毒顆粒為一種允許或促進將實體自一個環境轉移至另一環境之工具。根據本發明且藉助於實例,重組DNA技術中所使用之一些病毒顆粒允許將實體(諸如DNA區段)轉移至宿主細胞中。重組DNA技術中所使用之載體之實例包括但不限於質體、染色體、人工染色體或病毒。術語「表現載體」意謂能夠活體內或活體外/離體表現之構築體。 A.逆轉錄病毒顆粒 As is well known in the art, a viral particle is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention and by way of example, some viral particles used in recombinant DNA technology allow the transfer of entities (such as DNA segments) into host cells. Examples of vectors used in recombinant DNA technology include, but are not limited to, plasmids, chromosomes, artificial chromosomes, or viruses. The term "expression vector" means a construct capable of in vivo or in vitro/ex vivo expression. A. Retroviral Particles

在一些實施例中,病毒顆粒包含逆轉錄病毒顆粒。在一些實施例中,本發明提供一種用於製備病毒調配物之方法。在一些實施例中,病毒為逆轉錄病毒。已鑑別出大量不同逆轉錄病毒。逆轉錄病毒之實例包括但不限於:鼠白血病病毒(murine leukemia virus;MLV)、人類免疫缺乏病毒(human immunodeficiency virus;HIV)、人類T細胞白血病病毒(HTLV)、小鼠乳腺腫瘤病毒(mouse mammary tumor virus;MMTV)、勞斯肉瘤病毒(Rous sarcoma virus;RSV)、藤波肉瘤病毒(Fujinami sarcoma virus) (FuSV)、莫洛尼鼠類白血病病毒(Moloney murine leukemia virus) (Mo-MLV)、FBR鼠類骨肉瘤病毒(FBR MSV)、莫洛尼鼠類肉瘤病毒(Mo-MSV)、艾貝爾森鼠白血病病毒(Abelson murine leukemia virus) (A-MLV)、禽類髓細胞瘤病病毒-29 (MC29)及禽類紅血球母細胞增多症病毒(Avian erythroblastosis virus;AEV)。逆轉錄病毒之詳述清單可見於Coffin等人, 1997, 「Retroviruses」, Cold Spring Harbor Laboratory Press, 編者:JM Coffin, SM Hughes, HE Varmus, 第758-763頁中。In some embodiments, the viral particles comprise retroviral particles. In some embodiments, the invention provides a method for preparing a viral formulation. In some embodiments, the virus is a retrovirus. A large number of different retroviruses have been identified. Examples of retroviruses include, but are not limited to, murine leukemia virus (MLV), human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelomatosis virus-29 (MC29), and avian erythroblastosis virus (A-MLV). A detailed list of retroviruses can be found in Coffin et al., 1997, "Retroviruses", Cold Spring Harbor Laboratory Press, eds. JM Coffin, SM Hughes, HE Varmus, pp. 758-763.

逆轉錄病毒包括慢病毒、γ-逆轉錄病毒及α-逆轉錄病毒,其各自可用於使用此項技術中已知之方法將多核苷酸遞送至細胞。慢病毒為複合逆轉錄病毒,除常見逆轉錄病毒基因gag、pol及env以外,該等慢病毒含有其他具有調節或結構性功能之基因。較高的複雜度使得病毒能夠調節其生命週期,如在潛伏感染過程中一樣。慢病毒之一些實例包括人類免疫缺乏病毒(HIV-1及HIV-2)及猿猴免疫缺乏病毒(SIV)。逆轉錄病毒載體已藉由多次減弱HIV毒力基因產生,例如缺失基因env、vif、vpr、vpu及nef,從而使得載體生物學上安全。Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which can be used to deliver polynucleotides to cells using methods known in the art. Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. The greater complexity enables the virus to regulate its life cycle, as during latent infection. Some examples of lentiviruses include human immunodeficiency virus (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). Retroviral vectors have been generated by multiple attenuation of HIV virulence genes, such as deleting the genes env, vif, vpr, vpu, and nef, thereby rendering the vector biologically safe.

本發明之慢病毒載體可來源於(derived from/derivable from)任何合適的慢病毒。重組逆轉錄病毒載體顆粒能夠用所關注核苷酸(nucleotide of interest;NOI)轉導接受者細胞。一旦處於細胞內,則來自載體顆粒之RNA基因體逆轉錄成DNA且整合至接受者細胞之DNA中。在本發明之一些實施例中,可自病毒移除複製必需的一或多個蛋白質編碼區之至少一部分。此使得病毒載體複製缺陷。病毒基因體之部分亦可經NOI置換以產生包含NOI之載體,其能夠轉導目標非分裂宿主細胞及/或將其基因體整合至宿主基因體中。The lentiviral vectors of the present invention may be derived from/derivable from any suitable lentivirus. Recombinant retroviral vector particles are capable of transducing recipient cells with a nucleotide of interest (NOI). Once inside the cell, the RNA genome from the vector particles is reverse transcribed into DNA and integrated into the DNA of the recipient cell. In some embodiments of the present invention, at least a portion of one or more protein coding regions essential for replication may be removed from the virus. This renders the viral vector replication-defective. Portions of the viral genome may also be replaced with the NOI to produce a vector comprising the NOI, which is capable of transducing a target non-dividing host cell and/or integrating its genome into the host genome.

說明性慢病毒載體包括描述於以下中之彼等者:Naldini等人(1996) Science 272:263-7;Zufferey等人(1998) J. Virol. 72:9873-9880;Dull等人(1998) J. Virol. 72:8463-8471;美國專利第6,013,516號;及美國專利第5,994,136號,其各自以全文引用之方式併入本文中。一般而言,此等載體經組態以攜帶必需序列以用於選擇含有載體之細胞、用於將外來核酸併入慢病毒顆粒中及用於將核酸轉移至目標細胞中。Illustrative lentiviral vectors include those described in Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. In general, these vectors are configured to carry the necessary sequences for selection of cells containing the vector, for incorporation of foreign nucleic acids into lentiviral particles, and for transfer of nucleic acids into target cells.

常用慢病毒載體系統為所謂的第三代系統。第三代慢病毒載體系統包括四種質體。「轉移質體」編碼藉由慢病毒載體系統遞送至目標細胞的多核苷酸序列。轉移質體一般具有一或多個側接有長末端重複(LTR)序列之所關注轉殖基因序列,該等長末端重複序列促進將轉移質體序列整合至宿主基因體中。出於安全原因,轉移質體一般設計成使所得載體不能複製。舉例而言,轉移質體缺乏在宿主細胞中產生感染性顆粒所必需的基因元件。另外,轉移質體可設計成具有3' LTR缺失,使得病毒「自身失活」(SIN)。參見Dull等人(1998) J. Virol. 72:8463-71;Miyoshi等人(1998) J. Virol. 72:8150-57。病毒顆粒亦可包含3'非轉譯區(UTR)及5' UTR。UTR包含逆轉錄病毒調節元件,該等逆轉錄病毒調節元件支持在細胞被逆轉錄病毒顆粒接觸之後將前病毒基因體封裝、逆轉錄及整合至細胞中。Commonly used lentiviral vector systems are so-called third generation systems. Third generation lentiviral vector systems include four types of plasmids. The "transfer plasmid" encodes the polynucleotide sequence that is delivered to the target cell by the lentiviral vector system. The transfer plasmid generally has one or more sequences of the transgene of interest flanked by long terminal repeat (LTR) sequences that promote integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is generally designed so that the resulting vector cannot replicate. For example, the transfer plasmid lacks the genetic elements necessary to produce infectious particles in the host cell. In addition, the transfer plasmid can be designed to have a 3' LTR deletion, rendering the virus "self-inactive" (SIN). See Dull et al. (1998) J. Virol. 72:8463-71; Miyoshi et al. (1998) J. Virol. 72:8150-57. The viral particles may also contain a 3' untranslated region (UTR) and a 5' UTR. The UTR contains retroviral regulatory elements that support packaging, reverse transcription, and integration of the proviral genome into cells after exposure to the retroviral particles.

第三代系統一般亦包括兩種「封裝質體」及「套膜質體」。「套膜質體」一般編碼可操作地連接於啟動子之Env基因。在說明性第三代系統中,Env基因為VSV-G,且啟動子為CMV啟動子。第三代系統使用兩種封裝質體作為另一安全特徵,一種編碼gag及pol且另一種編碼rev,此係一項優於所謂的第二代系統之單一封裝質體的改善。儘管較安全,但由於添加額外質體,因此第三代系統使用起來可能更繁瑣且導致病毒力價較低。說明性封裝質體包括但不限於pMD2.G、pRSV-rev、pMDLG-pRRE及pRRL-GOI。Third generation systems also generally include two "encapsulation plasmids" and "encapsulation plasmids." The "encapsulation plasmid" generally encodes the Env gene operably linked to a promoter. In an illustrative third generation system, the Env gene is VSV-G and the promoter is the CMV promoter. As another safety feature, third generation systems use two encapsulation plasmids, one encoding gag and pol and the other encoding rev, an improvement over the single encapsulation plasmid of the so-called second generation system. Although safer, third generation systems may be more cumbersome to use and result in lower virulence due to the addition of an extra plasmid. Illustrative encapsulation plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

許多逆轉錄病毒載體系統依賴於使用「封裝細胞株」。一般而言,封裝細胞株為細胞能夠在轉移質體、封裝一或多種質體及套膜質體被引入細胞中時產生感染性逆轉錄病毒顆粒之細胞株。可使用將質體引入細胞中之各種方法,包括轉染或電穿孔。在一些情況下,封裝細胞株適合於將逆轉錄病毒載體系統高效封裝至逆轉錄病毒顆粒中。Many retroviral vector systems rely on the use of a "packaging cell line." Generally, a packaging cell line is a cell line whose cells are capable of producing infectious retroviral particles when a transfer plasmid, packaging plasmid or plasmids, and envelope plasmid are introduced into the cell. A variety of methods for introducing plasmids into cells can be used, including transfection or electroporation. In some cases, the packaging cell line is adapted to efficiently package the retroviral vector system into retroviral particles.

如本文所用,術語「逆轉錄病毒載體」或「慢病毒載體」意欲意謂編碼基因體封裝所需之逆轉錄病毒或慢病毒順式核酸序列以及一或多種待遞送至目標細胞中之多核苷酸序列的核酸。逆轉錄病毒顆粒及慢病毒顆粒一般包括RNA基因體(源於轉移質體)、包埋Env蛋白於其中之脂質雙層套膜及其他輔助蛋白(包括整合酶、蛋白酶及基質蛋白)。如本文所用,術語「逆轉錄病毒顆粒」及「慢病毒顆粒」係指包括套膜、具有一或多種慢病毒之特徵且能夠侵襲目標宿主細胞的病毒顆粒。此類特徵包括例如:感染非分裂宿主細胞;轉導非分裂宿主細胞;感染或轉導宿主免疫細胞;含有逆轉錄病毒或慢病毒病毒粒子,包括gag結構性多肽中之一或多者;含有逆轉錄病毒或慢病毒套膜,包括env編碼之醣蛋白中之一或多者;含有基因體,包括在複製、前病毒整合或轉錄方面起作用之一或多種逆轉錄病毒或慢病毒順式作用序列;含有編碼逆轉錄病毒或慢病毒蛋白酶、逆轉錄酶或整合酶之基因體;或含有編碼調節活性基因體,諸如Tat或Rev。轉移質體可包含美國專利第8,093,042號中所描述之cPPT序列。As used herein, the term "retrotransfer virus vector" or "lentivirus vector" is intended to mean a nucleic acid encoding a retrotransfer virus or lentivirus cis nucleic acid sequence required for genome packaging and one or more polynucleotide sequences to be delivered to a target cell. Retrotransfer virus particles and lentivirus particles generally include an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope in which the Env protein is embedded, and other accessory proteins (including integrase, protease, and matrix protein). As used herein, the term "retrotransfer virus particle" and "lentivirus particle" refer to a virus particle that includes an envelope, has one or more characteristics of a lentivirus, and is capable of invading a target host cell. Such characteristics include, for example: infection of non-dividing host cells; transduction of non-dividing host cells; infection or transduction of host immune cells; containing retroviral or lentiviral particles, including one or more of the gag structural polypeptides; containing a retroviral or lentiviral envelope, including one or more of the glycoproteins encoded by env; containing a genome, including one or more retroviral or lentiviral cis-acting sequences that function in replication, proviral integration or transcription; containing a genome encoding a retroviral or lentiviral protease, reverse transcriptase or integrase; or containing a genome encoding a regulatory activity, such as Tat or Rev. The transfer plasmid may comprise a cPPT sequence described in U.S. Patent No. 8,093,042.

系統之效率為載體工程改造中之重要問題。逆轉錄病毒或慢病毒載體系統之效率可以此項技術中已知之各種方式評定,包括量測載體複本數(VCN)或載體基因體(vg) (諸如藉由定量聚合酶鏈反應(qPCR))、或以感染單位/毫升(IU/mL)為單位之病毒之力價。舉例而言,力價可使用對於所培養腫瘤細胞株HT1080進行之功能分析來評定,如Humbert等人Development of third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy 24:1237-1246 (2016)中所描述。當對於在不斷分裂之所培養細胞株評定力價時,不需要刺激且因此所量測力價不受逆轉錄病毒顆粒之表面工程改造影響。用於評定逆轉錄病毒載體系統之效率的其他方法在Gaererts等人Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006)中提供。The efficiency of the system is an important issue in vector engineering. The efficiency of a retroviral or lentiviral vector system can be assessed in a variety of ways known in the art, including measuring vector copy number (VCN) or vector genome (vg) (e.g., by quantitative polymerase chain reaction (qPCR)), or viral titer in infectious units/ml (IU/mL). For example, titer can be assessed using functional assays performed on the cultured tumor cell line HT1080, as described in Humbert et al. Development of third-generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy 24:1237-1246 (2016). When titers are assessed for dividing cultured cell lines, no stimulation is required and therefore the titers measured are not affected by surface engineering of the retroviral particles. Other methods for assessing the efficiency of retroviral vector systems are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

在一些實施例中,本發明之逆轉錄病毒顆粒及/或慢病毒顆粒包含含有編碼與閘控轉接子特異性結合之受體之序列的多核苷酸。在一些實施例中,編碼與閘控轉接子特異性結合之受體的序列可操作地連接於啟動子。說明性啟動子包括但不限於細胞巨大病毒(CMV)啟動子、CAG啟動子、SV40啟動子、SV40/CD43啟動子及MND啟動子。In some embodiments, the retroviral particles and/or lentiviral particles of the present invention comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a gating adapter. In some embodiments, the sequence encoding a receptor that specifically binds to a gating adapter is operably linked to a promoter. Illustrative promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, a SV40 promoter, a SV40/CD43 promoter, and a MND promoter.

在一些實施例中,逆轉錄病毒顆粒包含轉導強化子。在一些實施例中,逆轉錄病毒顆粒包含標記蛋白質。In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a marker protein.

在一些實施例中,逆轉錄病毒顆粒各包含多核苷酸,以5'至3'順序,該多核苷酸包含:(i) 5'長末端重複序列(LTR)或非轉譯區(UTR),(ii)啟動子,(iii)編碼與配體特異性結合之受體的序列,及(iv) 3' LTR或UTR。In some embodiments, each retroviral particle comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat sequence (LTR) or a non-translated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to a ligand, and (iv) a 3' LTR or UTR.

在一些實施例中,逆轉錄病毒顆粒包含與目標宿主細胞上之表面標記結合,從而允許宿主細胞轉導的細胞表面受體。病毒載體可包含異源病毒套膜醣蛋白,從而產生假模式化病毒載體。舉例而言,病毒套膜醣蛋白可來源於RD114或其變體中之一者、VSV-G、長臂猿白血病病毒(GALV),或為雙嗜性套膜、麻疹套膜或狒狒逆轉錄病毒套膜醣蛋白。在一些實施例中,細胞表面受體為來自科卡爾病毒株或其功能變體之VSV G蛋白。In some embodiments, the retroviral particles comprise a cell surface receptor that binds to a surface marker on a target host cell, thereby allowing transduction of the host cell. The viral vector may comprise a heterologous viral envelope glycoprotein, thereby generating a pseudotyped viral vector. For example, the viral envelope glycoprotein may be derived from RD114 or one of its variants, VSV-G, Gibbon Ape Leukemia Virus (GALV), or is an amphoteric envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is a VSV G protein from a Kokar virus strain or a functional variant thereof.

在一些實施例中,病毒套膜包含病毒套膜蛋白。在一些實施例中,病毒套膜蛋白為VSV-G套膜蛋白、麻疹病毒套膜蛋白、尼帕病毒(nipha virus)套膜蛋白或科卡爾病毒G蛋白。在一些實施例中,病毒顆粒包含缺乏LDLR結合親和力之經修飾之VSV G蛋白。在一些實施例中,此等突變包含位置47 (例如K47Q)及/或354 (例如R354A)處之突變。In some embodiments, the viral envelope comprises a viral envelope protein. In some embodiments, the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a nipha virus envelope protein, or a Kokar virus G protein. In some embodiments, the viral particles comprise a modified VSV G protein lacking LDLR binding affinity. In some embodiments, these mutations comprise mutations at positions 47 (e.g., K47Q) and/or 354 (e.g., R354A).

在一些實施例中,病毒套膜蛋白質為來自科卡爾病毒株之蛋白質(科卡爾病毒醣蛋白)。在一些實施例中,蛋白質為含有位置354處之突變(R354)的科卡爾病毒套膜蛋白。在一些實施例中,蛋白質為含有位置47 (K47)處之突變的科卡爾病毒套膜蛋白。在一些實施例中,蛋白質為含有R354Q突變之科卡爾病毒套膜變體。在一些實施例中,蛋白質為含有K47Q突變之科卡爾病毒套膜變體。在一些實施例中,此變體可稱為「盲態」科卡爾病毒套膜。說明性科卡爾病毒套膜變體例如在US 2020/0216502 A1 (其以全文引用的方式併入本文中)中提供。In some embodiments, the viral envelope protein is a protein from a Kokar virus strain (Kokar virus glycoprotein). In some embodiments, the protein is a Kokar virus envelope protein containing a mutation at position 354 (R354). In some embodiments, the protein is a Kokar virus envelope protein containing a mutation at position 47 (K47). In some embodiments, the protein is a Kokar virus envelope variant containing an R354Q mutation. In some embodiments, the protein is a Kokar virus envelope variant containing a K47Q mutation. In some embodiments, this variant may be referred to as a "blind" Kokar virus envelope. Illustrative Kokar virus envelope variants are provided, for example, in US 2020/0216502 A1 (which is incorporated herein by reference in its entirety).

各種融合醣蛋白可用於使慢病毒載體假模式化。儘管最常用實例為來自水泡性口炎病毒(VSVG)之套膜醣蛋白,但亦使用許多其他病毒蛋白來使慢病毒載體假模式化。參見Joglekar等人Human Gene Therapy Methods 28:291-301 (2017)。本發明涵蓋各種融合醣蛋白之取代。值得注意的是,一些融合醣蛋白產生較高載體效率。Various fusion glycoproteins can be used to pseudotype lentiviral vectors. Although the most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), many other viral proteins are also used to pseudotype lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present invention encompasses the replacement of various fusion glycoproteins. Notably, some fusion glycoproteins produce higher vector efficiencies.

在一些實施例中,使融合醣蛋白或其功能變體假模式化促進對特定細胞類型(包括但不限於先天性淋巴細胞或NK細胞)之靶向轉導。在一些實施例中,融合醣蛋白或其功能變體為以下各者之一或多種全長多肽、一或多種功能片段、一或多種同源物或一或多種功能變體:人類免疫缺乏病毒(HIV) gp160;鼠白血病病毒(MLV) gp70;長臂猿白血病病毒(GALV) gp70;貓白血病病毒(RD114) gp70;雙嗜性逆轉錄病毒(兩性) gp70;10A1 MLV (10A1)gp70;親嗜性逆轉錄病毒(Eco) gp70;狒狒白血病病毒(BaEV) gp70;麻疹病毒(MV) H及F;尼帕病毒(NiV) H及F;狂犬病病毒(RabV) G;莫科拉病毒(MOKV) G;埃博拉扎伊爾病毒(EboZ) G;淋巴球性脈絡叢腦膜炎病毒(LCMV) GP1及GP2;桿狀病毒GP64;屈公病毒(CHIKV) E1及E2;羅斯河病毒(RRV) E1及E2;勝利基森林病毒(SFV) E1及E2;辛德比斯病毒(SV) E1及E2;委內瑞拉馬腦炎病毒(VEEV) E1及E2;西部馬腦炎病毒(WEEV) E1及E2;A、B、C或D型流感HA;雞瘟病毒(FPV)HA;水泡性口炎病毒VSV-G;或章地埔拉病毒及皮里病毒CNV-G及PRV-G。In some embodiments, the fusion glycoprotein or a functional variant thereof is pseudopatterned to facilitate targeted transduction of specific cell types, including but not limited to innate lymphoid cells or NK cells. In some embodiments, the fusion glycoprotein or a functional variant thereof is one or more full-length polypeptides, one or more functional fragments, one or more homologs, or one or more functional variants of the following: human immunodeficiency virus (HIV) gp160; murine leukemia virus (MLV) gp70; gibbon ape leukemia virus (GALV) gp70; feline leukemia virus (RD114) gp70; amphitropic retrovirus (amphitropic) gp70; 10A1 MLV (10A1) gp70; ecotropic retrovirus (Eco) gp70; baboon leukemia virus (BaEV) gp70; measles virus (MV) H and F; Nipah virus (NiV) H and F; rabies virus (RabV) G; Mokola virus (MOKV) G; Ebola Zaire virus (EboZ) G; lymphocytic choroidal meningitis virus (LCMV) GP1 and GP2; baculovirus GP64; Chikungunya virus (CHIKV) E1 and E2; Ross River virus (RRV) E1 and E2; Victory Forest virus (SFV) E1 and E2; Sindbis virus (SV) E1 and E2; Venezuelan equine encephalitis virus (VEEV) E1 and E2; Western equine encephalitis virus (WEEV) E1 and E2; influenza A, B, C or D HA; fowl plague virus (FPV) HA; vesicular stomatitis virus VSV-G; or Changdipura virus and Piri virus CNV-G and PRV-G.

在一些實施例中,融合醣蛋白或其功能變體為以下各者之G蛋白之全長多肽、功能片段、同源物或功能變體:水泡性口炎阿拉戈斯病毒(VSAV)、卡拉加斯水泡病毒(CJSV)、金迪普拉水泡病毒(CHPV)、科卡爾水泡病毒(COCV)、水泡性口炎印地安那病毒(VSIV)、伊斯法罕水泡病毒(ISFV)、馬拉巴水泡病毒(MARAV)、水泡性口炎紐澤西病毒(VSNJV)、巴斯-剛果病毒(BASV)。在一些實施例中,融合醣蛋白或其功能變體為科卡爾病毒G蛋白。In some embodiments, the fusion glycoprotein or its functional variant is a full-length polypeptide, functional fragment, homolog or functional variant of the G protein of the following: vesicular stomatitis Alagoas virus (VSAV), vesicular stomatitis virus of Caracas (CJSV), vesicular stomatitis virus of Chandipura (CHPV), vesicular stomatitis virus of Cokar (COCV), vesicular stomatitis Indiana virus (VSIV), vesicular stomatitis virus of Isfahan (ISFV), vesicular stomatitis virus of Malaba (MARAV), vesicular stomatitis New Jersey virus (VSNJV), Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or its functional variant is the G protein of Cokar virus.

在一些實施例中,融合醣蛋白或其功能變體為以下各者之G蛋白之全長多肽、功能片段、同源物或功能變體:水泡性口炎阿拉戈斯病毒(VSAV)、卡拉加斯水泡病毒(CJSV)、金迪普拉水泡病毒(CHPV)、科卡爾水泡病毒(COCV)、水泡性口炎印地安那病毒(VSIV)、伊斯法罕水泡病毒(ISFV)、馬拉巴水泡病毒(MARAV)、水泡性口炎紐澤西病毒(VSNJV)、巴斯-剛果病毒(BASV)。在一些實施例中,融合醣蛋白或其功能變體為科卡爾病毒G蛋白。In some embodiments, the fusion glycoprotein or its functional variant is a full-length polypeptide, functional fragment, homolog or functional variant of the G protein of the following: vesicular stomatitis Alagoas virus (VSAV), vesicular stomatitis virus of Caracas (CJSV), vesicular stomatitis virus of Chandipura (CHPV), vesicular stomatitis virus of Cokar (COCV), vesicular stomatitis Indiana virus (VSIV), vesicular stomatitis virus of Isfahan (ISFV), vesicular stomatitis virus of Malaba (MARAV), vesicular stomatitis New Jersey virus (VSNJV), Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or its functional variant is the G protein of Cokar virus.

本發明進一步提供各種逆轉錄病毒載體,包括但不限於γ-逆轉錄病毒載體、α-逆轉錄病毒載體及慢病毒載體。在一些實施例中,載體可為病毒載體、逆轉錄病毒載體、慢病毒載體、γ-逆轉錄病毒載體。在一些實施例中,病毒載體包含VSV G蛋白或其功能變體。在一些實施例中,病毒載體包含科卡爾病毒G蛋白或其功能變體。 B.經工程改造之病毒套膜 The present invention further provides various retroviral vectors, including but not limited to γ-retroviral vectors, α-retroviral vectors and lentiviral vectors. In some embodiments, the vector can be a viral vector, a retroviral vector, a lentiviral vector, or a γ-retroviral vector. In some embodiments, the viral vector comprises VSV G protein or a functional variant thereof. In some embodiments, the viral vector comprises Cokar virus G protein or a functional variant thereof. B. Engineered viral envelope

在一些實施例中,囊封本文提供之核苷酸載體之病毒顆粒可包含經工程改造之病毒套膜。在一些實施例中,病毒套膜包含轉導強化子。在一些實施例中,病毒套膜包含免疫細胞活化蛋白。在一些實施例中,病毒套膜包含共刺激分子。在一些實施例中,病毒套膜包含免疫細胞活化蛋白及共刺激分子。In some embodiments, the viral particles encapsulating the nucleotide vectors provided herein may include an engineered viral envelope. In some embodiments, the viral envelope includes a transduction enhancer. In some embodiments, the viral envelope includes an immune cell activation protein. In some embodiments, the viral envelope includes a co-stimulatory molecule. In some embodiments, the viral envelope includes an immune cell activation protein and a co-stimulatory molecule.

在一些實施例中,病毒套膜包含一或多種轉導強化子。在一些實施例中,轉導強化子包括T細胞活化受體、NK細胞活化受體及/或共刺激分子。在一些實施例中,一或多種轉導強化子包含抗CD3scFv、CD86、CD80及/或CD58中之一或多者。在一些實施例中,轉導強化子至少包含抗CD3 scFv及CD58。在一些實施例中,轉導強化子至少包含抗CD3 scFv及CD80。在一些實施例中,轉導強化子至少包含抗CD3 scFv及CD86。在一些實施例中,轉導強化子至少包含抗CD3 scFv、CD80及CD58。在一些實施例中,轉導強化子至少包含抗CD3 scFv、CD86及CD58。In some embodiments, the viral envelope comprises one or more transduction enhancers. In some embodiments, the transduction enhancers include T cell activation receptors, NK cell activation receptors and/or co-stimulatory molecules. In some embodiments, one or more transduction enhancers include one or more of anti-CD3 scFv, CD86, CD80 and/or CD58. In some embodiments, the transduction enhancers at least include anti-CD3 scFv and CD58. In some embodiments, the transduction enhancers at least include anti-CD3 scFv and CD80. In some embodiments, the transduction enhancers at least include anti-CD3 scFv and CD86. In some embodiments, the transduction enhancers at least include anti-CD3 scFv, CD80 and CD58. In some embodiments, the transduction enhancers at least include anti-CD3 scFv, CD86 and CD58.

在一些實施例中,病毒顆粒包含與所靶向宿主細胞上之配體結合,從而允許宿主細胞轉導之細胞表面受體。在一些實施例中,病毒顆粒包含異源病毒套膜醣蛋白,從而產生假模式化病毒顆粒。舉例而言,病毒套膜醣蛋白可來源於RD114或其變體中之一者、VSV-G、長臂猿白血病病毒(GALV),或為雙嗜性套膜、麻疹套膜或狒狒逆轉錄病毒套膜醣蛋白。在一些實施例中,病毒套膜醣蛋白為來自科卡爾病毒株之VSV G蛋白(科卡爾病毒醣蛋白)或其功能變體。In some embodiments, the viral particles comprise a cell surface receptor that binds to a ligand on a targeted host cell, thereby allowing transduction of the host cell. In some embodiments, the viral particles comprise a heterologous viral envelope glycoprotein, thereby generating a pseudotyped viral particle. For example, the viral envelope glycoprotein may be derived from RD114 or one of its variants, VSV-G, Gibbon Ape Leukemia Virus (GALV), or is an amphoteric envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is a VSV G protein from the Cocal virus strain (Cocal virus glycoprotein) or a functional variant thereof.

在一些實施例中,病毒套膜包含超過一種多肽在表面上。在一些實施例中,超過一種多肽與目標免疫細胞結合且複製免疫突觸。在一些實施例中,病毒套膜包含免疫細胞活化蛋白、共刺激分子及黏附分子,其中免疫細胞活化蛋白、共刺激分子及黏附分子各自結合所靶向免疫細胞。 1.免疫細胞活化劑 In some embodiments, the viral envelope comprises more than one polypeptide on the surface. In some embodiments, more than one polypeptide binds to the target immune cell and replicates the immune junction. In some embodiments, the viral envelope comprises immune cell activation proteins, co-stimulatory molecules, and adhesion molecules, wherein the immune cell activation proteins, co-stimulatory molecules, and adhesion molecules each bind to the targeted immune cell. 1. Immune cell activator

在一些實施例中,轉導強化子包含細胞分裂刺激物,其被併入逆轉錄病毒或慢病毒殼體中,以使得病毒活化且轉導T細胞。此移除添加載體及促分裂原之需求。在一些實施例中,轉導強化子包含細胞分裂跨膜蛋白及/或一或多種共刺激分子,當細胞分裂跨膜蛋白及/或一或多種共刺激分子自生產者/封裝細胞膜出芽時其被併入逆轉錄病毒中。在一些實施例中,轉導強化子作為個別細胞表面分子而非作為病毒套膜醣蛋白之一部分在生產細胞上表現。In some embodiments, the transduction enhancer comprises a cell division stimulator that is incorporated into the retroviral or lentiviral capsid so that the virus activates and transduces T cells. This removes the need to add vectors and mitogens. In some embodiments, the transduction enhancer comprises a cell division transmembrane protein and/or one or more co-stimulatory molecules that are incorporated into the retrovirus when the cell division transmembrane protein and/or one or more co-stimulatory molecules bud from the producer/encapsulating cell membrane. In some embodiments, the transduction enhancer is expressed on the producer cell as a separate cell surface molecule rather than as part of the viral envelope glycoprotein.

在一些實施例中,本文所描述之病毒載體包含細胞分裂轉導強化子在病毒套膜中。在一些實施例中,細胞分裂轉導強化子來源於在逆轉錄病毒載體產生期間之宿主細胞。在一些實施例中,細胞分裂轉導強化子係藉由封裝細胞製備且表現在細胞表面處。當新生逆轉錄病毒載體自宿主細胞膜出芽時,細胞分裂轉導強化子可作為封裝細胞源性脂質雙層之一部分而併入病毒套膜中。在一些實施例中,細胞分裂強化子為抗體或其片段。在一些實施例中,細胞分裂強化子為單域抗體,例如駱駝抗體。在一些實施例中,細胞分裂強化子為scFv。在一些實施例中,細胞分裂強化子為奈米抗體。In some embodiments, the viral vectors described herein include a cell division transduction enhancer in the viral envelope. In some embodiments, the cell division transduction enhancer is derived from host cells during the production of the retroviral vector. In some embodiments, the cell division transduction enhancer is prepared by encapsulating cells and is expressed at the cell surface. When the nascent retroviral vector buds from the host cell membrane, the cell division transduction enhancer can be incorporated into the viral envelope as part of the encapsulating cell-derived lipid bilayer. In some embodiments, the cell division enhancer is an antibody or a fragment thereof. In some embodiments, the cell division enhancer is a single domain antibody, such as a camel antibody. In some embodiments, the cell division enhancer is a scFv. In some embodiments, the cell division enhancer is a nanobody.

在一些實施例中,轉導強化子為宿主細胞源性的。術語「宿主細胞源性」指示,細胞分裂轉導強化子係來源於上文所描述之宿主細胞,且不係作為融合體或嵌合體而自病毒基因(諸如gag,其編碼主要結構蛋白;或env,其編碼套膜蛋白)中之一者製造。In some embodiments, the transduction enhancer is host cell-derived. The term "host cell-derived" indicates that the cell division transduction enhancer is derived from a host cell as described above and is not made as a fusion or chimera from one of the viral genes (such as gag, which encodes a major structural protein; or env, which encodes an envelope protein).

套膜蛋白係藉由兩種次單元形成:跨膜(TM)次單元,其將蛋白質錨定於脂膜中;及表面(SU)次單元,其與細胞受體結合。在一些實施例中,本發明之封裝細胞源性細胞分裂轉導強化子不包含表面套膜次單元(SU)。Envelope proteins are composed of two subunits: a transmembrane (TM) subunit, which anchors the protein in the lipid membrane; and a surface (SU) subunit, which binds to cell receptors. In some embodiments, the encapsulated cell-derived cell division transduction enhancer of the present invention does not include a surface envelope subunit (SU).

在一些實施例中,細胞分裂轉導強化子具有以下結構:M-S-TM,其中M為細胞分裂域;S為視情況存在之間隔子域;且TM為跨膜域。In some embodiments, the cell division transduction enhancer has the following structure: M-S-TM, wherein M is a cell division domain; S is an optional spacer domain; and TM is a transmembrane domain.

細胞分裂域為使T細胞活化之細胞分裂轉導強化子之一部分。其可直接地或間接地結合T細胞或以其他方式與其相互作用,從而導致T細胞活化。在一些實施例中,細胞分裂域結合T細胞表面抗原,諸如CD3、CD28、CD134及CD137。The cell division domain is part of a cell division transduction enhancer that activates T cells. It can directly or indirectly bind to T cells or interact with them in other ways, thereby causing T cell activation. In some embodiments, the cell division domain binds to T cell surface antigens such as CD3, CD28, CD134 and CD137.

CD3為T細胞輔受體。其為由四條不同鏈構成之蛋白質複合物。在哺乳動物中,複合物含有一條CD3y鏈、一條CD35鏈及兩條CD3e鏈。此等鏈與T細胞受體(TCR)及z鏈結合以在T淋巴球中產生活化訊號。TCR、z鏈及CD3分子一起構成(comprise) TCR複合物。在一些實施例中,細胞分裂域與CD3e鏈結合。CD3 is a T cell co-receptor. It is a protein complex composed of four different chains. In mammals, the complex contains one CD3y chain, one CD35 chain, and two CD3e chains. These chains bind to the T cell receptor (TCR) and the z chain to produce activation signals in T lymphocytes. The TCR, z chain, and CD3 molecules together comprise the TCR complex. In some embodiments, the cell division domain is bound to the CD3e chain.

在一些實施例中,細胞分裂域包含抗體或特異性結合T細胞表面抗原之其他分子之全部或一部分。在一些實施例中,抗體活化TCR或CD28。在一些實施例中,抗體結合TCR、CD3或CD28。此類抗體之實例包括:OKT3、15E8及TGN1412。其他合適抗體包括: 抗CD28:CD28.2、10F3 抗CD3/TCR:UCHT1、YTH12.5、TR66 In some embodiments, the cell division domain comprises all or part of an antibody or other molecule that specifically binds to a T cell surface antigen. In some embodiments, the antibody activates TCR or CD28. In some embodiments, the antibody binds TCR, CD3 or CD28. Examples of such antibodies include: OKT3, 15E8 and TGN1412. Other suitable antibodies include: Anti-CD28: CD28.2, 10F3 Anti-CD3/TCR: UCHT1, YTH12.5, TR66

在一些實施例中,細胞分裂域包含來自以下之結合域:OKT3、15E8、TGN1412、CD28.2、10F3、UCHT1、YTH12.5或TR66。In some embodiments, the cell division domain comprises a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

在一些實施例中,細胞分裂域包含共刺激分子(諸如OX40L及41BBL)之全部或一部分。舉例而言,細胞分裂域可包含來自OX40L或41BBL之結合域。In some embodiments, the cell division domain comprises all or part of a co-stimulatory molecule such as OX40L and 41BBL. For example, the cell division domain may comprise a binding domain from OX40L or 41BBL.

OKT3 (亦稱為莫羅單抗(Muromonab)-CD3)為靶向CD3e鏈之單株抗體。其在臨床上用於降低器官移植患者中之急性排斥。其為第一種經批准用於在人類中之臨床用途的單株抗體。OKT3 (also known as Muromonab-CD3) is a monoclonal antibody that targets the CD3 epsilon chain. It is used clinically to reduce acute rejection in organ transplant patients. It is the first monoclonal antibody approved for clinical use in humans.

在一些實施例中,病毒套膜包含免疫細胞活化蛋白。在一些實施例中,免疫細胞活化蛋白特異性結合免疫細胞上之受體。在一些實施例中,免疫細胞活化蛋白提供訊號1用於T細胞活化。In some embodiments, the viral envelope comprises an immune cell activation protein. In some embodiments, the immune cell activation protein specifically binds to a receptor on an immune cell. In some embodiments, the immune cell activation protein provides signal 1 for T cell activation.

在一些實施例中,免疫細胞活化蛋白特異性結合CD2、CD3、CD28H、LFA-1、DNAM-1、CD27、ICOS、LIGHT、GITR、CD30、SLAM、Ly-9、CD84、Ly108、NKG2D、NKp46、NKp44、NKp30、CD244或NKp80。在一些實施例中,免疫細胞活化蛋白特異性結合CD3γ、CD3δ或CD3ε。在一些實施例中,免疫細胞活化蛋白特異性結合CD3γ、CD3δ、CD3ε、CD9、CD5、CD22、CD33、CD37、CD64、CD45、CD28H、LFA-1、DNAM-1、CD27、ICOS、LIGHT、GITR、CD30、SLAM、Ly-9、CD84、Ly108、CD16、CD56、NKG2D、NKp46、NKp44、NKp30、CD244、NKp80、TCRα鏈、TCRβ鏈、TCRγ鏈或TCRδ鏈。在一些實施例中,免疫細胞活化蛋白特異性結合CD3γ、CD3δ或CD3ε。在一些實施例中,免疫細胞活化蛋白特異性結合CD3。In some embodiments, the immune cell activation protein specifically binds to CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244 or NKp80. In some embodiments, the immune cell activation protein specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activation protein specifically binds to CD3γ, CD3δ, CD3ε, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCRα chain, TCRβ chain, TCRγ chain or TCRδ chain. In some embodiments, the immune cell activation protein specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activation protein specifically binds to CD3.

在一些實施例中,免疫細胞活化蛋白為特異性結合免疫細胞上之受體的抗體或其抗原結合片段。在一些實施例中,免疫細胞活化蛋白為特異性結合以下各者之抗體或其抗原結合片段:CD28、CD2、CD3、CD28H、LFA-1、OX40、4-1BB、CD40L、DNAM-1、CD27、ICOS、LIGHT、GITR、CD30、SLAM、Ly-9、CD84、Ly108、NKG2D、NKp46、NKp44、NKp30、CD244或NKp80。在一些實施例中,免疫細胞活化蛋白為特異性結合以下各者之抗體或其抗原結合片段:CD28、CD2、CD3γ、CD3δ、CD3ε、CD4、CD8、CD9、CD5、CD22、CD33、CD37、CD64、CD45、CD28H、LFA-1、OX40、4-1BB、CD40L、DNAM-1、CD27、ICOS、LIGHT、GITR、CD30、SLAM、Ly-9、CD84、Ly108、CD16、CD56、NKG2D、NKp46、NKp44、NKp30、CD244、NKp80、TCRα鏈、TCRβ鏈、TCRγ鏈或TCRδ鏈。在一些實施例中,免疫細胞活化蛋白為特異性結合CD3γ、CD3δ或CD3ε之抗體或其抗原結合片段。在一些實施例中,免疫細胞活化蛋白為特異性結合CD3之抗體或其抗原結合片段。In some embodiments, the immune cell activating protein is an antibody or an antigen binding fragment thereof that specifically binds to a receptor on an immune cell. In some embodiments, the immune cell activating protein is an antibody or an antigen binding fragment thereof that specifically binds to the following: CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, or NKp80. In some embodiments, the immune cell activating protein is an antibody or an antigen-binding fragment thereof that specifically binds to CD28, CD2, CD3γ, CD3δ, CD3ε, CD4, CD8, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCRα chain, TCRβ chain, TCRγ chain or TCRδ chain. In some embodiments, the immune cell activating protein is an antibody or an antigen binding fragment thereof that specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activating protein is an antibody or an antigen binding fragment thereof that specifically binds to CD3.

靶向本文所描述之多肽的抗體為熟習此項技術者已知的。用於產生抗體之方法為熟習此項技術者已知的。Antibodies targeting the polypeptides described herein are known to those skilled in the art. Methods for generating antibodies are known to those skilled in the art.

在一些實施例中,病毒套膜包含抗CD3ε抗體或其抗原結合片段。在一些實施例中,抗CD3ε抗體或其抗原結合片段與跨膜域偶聯。說明性抗CD3ε抗體為OKT3。OKT3 (亦稱為莫羅單抗-CD3)為靶向CD3ε鏈之單株抗體。In some embodiments, the viral envelope comprises an anti-CD3ε antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD3ε antibody or an antigen-binding fragment thereof is coupled to a transmembrane domain. An illustrative anti-CD3ε antibody is OKT3. OKT3 (also known as muromonab-CD3) is a monoclonal antibody that targets the CD3ε chain.

在一些實施例中,病毒套膜包含抗CD3抗體之單鏈Fv片段(scFv)。 2.共刺激分子 In some embodiments, the viral envelope comprises a single-chain Fv fragment (scFv) of an anti-CD3 antibody. 2. Co-stimulatory molecules

在一些實施例中,病毒套膜包含至少一種共刺激分子。在一些實施例中,共刺激分子特異性結合免疫細胞上之受體。在一些實施例中,共刺激提供訊號2用於細胞活化。In some embodiments, the viral envelope comprises at least one co-stimulatory molecule. In some embodiments, the co-stimulatory molecule specifically binds to a receptor on an immune cell. In some embodiments, the co-stimulatory molecule provides signal 2 for cell activation.

如本文所用,術語「共刺激分子」係指能夠產生針對T細胞之共刺激訊號的分子。淋巴球(諸如T細胞及自然殺手(NK)細胞)通常需要若干訊號且與抗原呈現細胞(APC)相互作用以用於最佳啟動(priming)以獲得完全效應功能。對於T細胞而言,此等包括經由T細胞受體(TCR)、共刺激分子(諸如CD28及CD2)、細胞介素以及允許足夠時間用於適當突觸形成及訊號轉導所需之各種黏附分子進行傳訊。NK細胞需要類似的刺激類型,但可能依賴於不同活化性受體,諸如NKG2D、NKp46及DNAM-1。對於T細胞而言,除TCR刺激以外,適當共刺激對於有效啟動而言尤其重要,且許多研究已顯示單獨TCR刺激可能導致功能性無反應及不起反應。共刺激訊號藉由增強細胞代謝、細胞介素產生、分化及長期存留而加強T細胞及NK細胞功能。共刺激為對於細胞增殖、分化及存活而言之重要因子。在一些實施例中,共刺激分子包括但不限於CD45、CD2、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137及CD154。在一些實施例中,共刺激分子包括但不限於結合劑,諸如與本文所描述之共刺激分子中之任一者結合的scFv、抗體、單域抗體、抗體片段、奈米抗體。在一些實施例中,此等結合劑可包括抗CD28、抗CD2、抗CD45、抗CD4、抗CD5、抗CD8、抗CD9、抗CD16、抗CD22、抗CD33、抗CD37、抗CD64、抗CD80、抗CD86、抗CD137、抗CD154、抗CD28H、抗LFA-1、抗OX40、抗4-1BB、抗CD40L、抗DNAM-1、抗CD27、抗ICOS、抗LIGHT、抗GITR、抗CD30、抗SLAM、抗Ly-9、抗CD84、抗Ly108、抗NKG2D、抗NKp46、抗NKp44、抗NKp30、抗CD244、抗NKp80、抗TCRα鏈、抗TCRβ鏈、抗TCRγ鏈及抗TCRδ鏈藥劑。As used herein, the term "co-stimulatory molecule" refers to a molecule capable of producing co-stimulatory signals for T cells. Lymphocytes, such as T cells and natural killer (NK) cells, typically require several signals and interactions with antigen presenting cells (APCs) for optimal priming to achieve full effector function. For T cells, these include communication via the T cell receptor (TCR), co-stimulatory molecules such as CD28 and CD2, cytokines, and various adhesion molecules required to allow sufficient time for proper synapse formation and signal transduction. NK cells require similar types of stimulation, but may rely on different activating receptors, such as NKG2D, NKp46, and DNAM-1. For T cells, in addition to TCR stimulation, appropriate co-stimulation is particularly important for effective activation, and many studies have shown that TCR stimulation alone may lead to functional anergy and unresponsiveness. Co-stimulatory signals enhance T cell and NK cell function by enhancing cell metabolism, interleukin production, differentiation and long-term persistence. Co-stimulation is an important factor for cell proliferation, differentiation and survival. In some embodiments, co-stimulatory molecules include but are not limited to CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, the costimulatory molecule includes, but is not limited to, a binding agent such as a scFv, an antibody, a single domain antibody, an antibody fragment, a nanobody that binds to any of the costimulatory molecules described herein. In some embodiments, such binding agents may include anti-CD28, anti-CD2, anti-CD45, anti-CD4, anti-CD5, anti-CD8, anti-CD9, anti-CD16, anti-CD22, anti-CD33, anti-CD37, anti-CD64, anti-CD80, anti-CD86, anti-CD137, anti-CD154, anti-CD28H, anti-LFA-1, anti-OX40, anti-4-1BB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-Ly108, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, anti-NKp80, anti-TCRα chain, anti-TCRβ chain, anti-TCRγ chain and anti-TCRδ chain agents.

在一些實施例中,共刺激分子為CD28之配體。CD28為提供T細胞活化及存活所需之共刺激訊號的T細胞上表現之蛋白質中之一者。除T細胞受體(TCR)以外,經由CD28發生的T細胞刺激可以提供強效訊號以用於產生各種介白素(特定言之,IL-6)。在一些實施例中,共刺激分子為與CD28結合之抗體或其片段。此類抗體之實例包括:15E8及TGN1412。其他合適抗體包括:CD28.2及10F3。In some embodiments, the co-stimulatory molecule is a ligand for CD28. CD28 is one of the proteins expressed on T cells that provides co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation via CD28 can provide a potent signal for the production of various interleukins (specifically, IL-6). In some embodiments, the co-stimulatory molecule is an antibody or fragment thereof that binds to CD28. Examples of such antibodies include: 15E8 and TGN1412. Other suitable antibodies include: CD28.2 and 10F3.

在一些實施例中,共刺激分子為CD86。CD86 (亦稱為B7-2)為CD28之配體。在一些實施例中,CD28之配體為CD86。在一些實施例中,共刺激分子為CD80。CD80為CD28之另一配體。在一些實施例中,CD28之配體為CD80。在一些實施例中,CD28之配體為抗CD28抗體或抗CD28 scFv。在一些實施例中,抗CD28抗體或抗CD28 scFv與跨膜域偶聯以用於呈現於病毒套膜之表面上。In some embodiments, the co-stimulatory molecule is CD86. CD86 (also known as B7-2) is a ligand of CD28. In some embodiments, the ligand of CD28 is CD86. In some embodiments, the co-stimulatory molecule is CD80. CD80 is another ligand of CD28. In some embodiments, the ligand of CD28 is CD80. In some embodiments, the ligand of CD28 is anti-CD28 antibody or anti-CD28 scFv. In some embodiments, the anti-CD28 antibody or anti-CD28 scFv is coupled to a transmembrane domain for presentation on the surface of the viral envelope.

在一些實施例中,共刺激分子為CD86多肽,其包含SEQ ID NO: 76之胺基酸序列。在一些實施例中,共刺激分子為CD86多肽,其包含與SEQ ID NO: 76至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%一致之胺基酸序列。In some embodiments, the costimulatory molecule is a CD86 polypeptide comprising an amino acid sequence of SEQ ID NO: 76. In some embodiments, the costimulatory molecule is a CD86 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 76.

在一些實施例中,共刺激分子為CD80多肽,其包含SEQ ID NO: 77之胺基酸序列。在一些實施例中,共刺激分子為CD80多肽,其包含與SEQ ID NO: 77至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%一致之胺基酸序列。In some embodiments, the costimulatory molecule is a CD80 polypeptide comprising an amino acid sequence of SEQ ID NO: 77. In some embodiments, the costimulatory molecule is a CD80 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 77.

在一些實施例中,CD86多肽係由SEQ ID NO: 78之核苷酸序列編碼。在一些實施例中,CD86多肽係由與SEQ ID NO: 78至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%一致之核苷酸序列編碼。In some embodiments, the CD86 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 78. In some embodiments, the CD86 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 78.

在一些實施例中,CD80多肽係由SEQ ID NO: 79之核苷酸序列編碼。在一些實施例中,CD80多肽係由與SEQ ID NO: 79至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%一致之核苷酸序列編碼。In some embodiments, the CD80 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 79. In some embodiments, the CD80 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 79.

CD134 (亦稱為OX40)為在活化T細胞上表現之受體TNFR超家族之成員。OX40可促進細胞分裂及存活。OX40為輔助共刺激分子,在活化後24至72小時之後得以表現;其配體OX40L亦不在休眠抗原呈現細胞上表現,但在其活化之後表現。在一些實施例中,病毒顆粒包含OX40之配體或其功能片段,其與其天然跨膜域或異源跨膜域偶聯。CD134 (also known as OX40) is a member of the TNFR superfamily of receptors expressed on activated T cells. OX40 promotes cell division and survival. OX40 is an auxiliary co-stimulatory molecule that is expressed 24 to 72 hours after activation; its ligand OX40L is also not expressed on dormant antigen-presenting cells, but is expressed after their activation. In some embodiments, the viral particle comprises a ligand of OX40 or a functional fragment thereof, which is coupled to its native transmembrane domain or a heterologous transmembrane domain.

CD134 (亦稱為OX40)為在活化T細胞上表現之受體TNFR超家族之成員。OX40可促進細胞分裂及存活。OX40為輔助共刺激分子,在活化後24至72小時之後表現;其配體OX40L亦不在休眠抗原呈現細胞上表現,但在其活化之後表現。在一些實施例中,病毒顆粒包含OX40之配體或其功能片段,其與其天然跨膜域或異源跨膜域偶聯。CD134 (also known as OX40) is a member of the TNFR superfamily of receptors expressed on activated T cells. OX40 can promote cell division and survival. OX40 is an auxiliary co-stimulatory molecule that is expressed 24 to 72 hours after activation; its ligand OX40L is also not expressed on dormant antigen-presenting cells, but is expressed after their activation. In some embodiments, the viral particle comprises a ligand of OX40 or a functional fragment thereof, which is coupled to its native transmembrane domain or a heterologous transmembrane domain.

CD137 (亦稱為4-1BB)為腫瘤壞死因子(TNF)受體家族之成員。CD137在活化T細胞上表現。另外,在樹突狀細胞、濾泡樹突狀細胞、自然殺手細胞、顆粒球及發炎部位處之血管壁細胞上發現CD137表現。CD137之最佳表徵活性為其對於活化T細胞之共刺激活性。CD137之交聯增強T細胞增殖、IL-2分泌、存活及細胞溶解活性。在一些實施例中,病毒顆粒包含4-1BB之配體或其功能片段,其與其天然跨膜域或異源跨膜域偶聯。4-1BBL為屬於腫瘤壞死因子(TNF)配體家族的細胞介素。此跨膜細胞介素為用作4-1BB之配體雙向訊號轉導子,其為T淋巴球中之共刺激受體分子。除促進T淋巴球增殖以外,已顯示4-1BBL再活化無反應性T淋巴球。CD137 (also known as 4-1BB) is a member of the tumor necrosis factor (TNF) receptor family. CD137 is expressed on activated T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and vascular wall cells at sites of inflammation. The best characterizing activity of CD137 is its co-stimulatory activity for activated T cells. The cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. In some embodiments, the viral particles comprise a ligand of 4-1BB or a functional fragment thereof, which is coupled to its native transmembrane domain or a heterologous transmembrane domain. 4-1BBL is a cytokine belonging to the tumor necrosis factor (TNF) ligand family. This transmembrane interleukin is a bidirectional signal transducer that serves as a ligand for 4-1BB, a co-stimulatory receptor molecule in T lymphocytes. In addition to promoting T lymphocyte proliferation, 4-1BBL has been shown to reactivate anergic T lymphocytes.

包含一或多種活化或共刺激分子之病毒顆粒可藉由以下來製備:藉由由WO 2016/139463提供之方法工程改造封裝細胞株;或藉由自多順反子輔助載體表現一或多種T細胞活化或共刺激分子,如國際專利公開案第WO 2020/106992 A1號中所描述,該兩篇文獻均以全文引用之方式併入本文中。 3.黏附分子 Viral particles containing one or more activation or co-stimulatory molecules can be prepared by engineering a packaging cell line by the method provided by WO 2016/139463; or by expressing one or more T cell activation or co-stimulatory molecules from a polycistronic helper vector, as described in International Patent Publication No. WO 2020/106992 A1, both of which are incorporated herein by reference in their entirety. 3. Adhesion molecules

在一些實施例中,病毒顆粒包含黏附分子。如本文所用,術語「黏附分子」係指涉及細胞與其他細胞結合之細胞表面分子的子集。黏附分子可幫助在免疫細胞之間形成更穩定相互作用,諸如免疫突觸。免疫突觸為極化免疫效應細胞與抗原攜帶細胞之間的穩定黏附接合。在一些實施例中,黏附分子可向目標細胞提供共刺激訊號。在一些實施例中,黏附分子包括但不限於CD58、HHLA2、ICAM-1、OX40L、4-1BBL、CD40、CD155、CD70、HVEM、GITRL、ICOSL、CD30L、SLAM、Ly-9、CD84、Ly108、MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6及B7-H6。在一些實施例中,黏附分子包括但不限於結合劑,諸如與本文所描述之黏附或共刺激分子中之任一者結合的scFv、抗體、單域抗體、抗體片段及奈米抗體。在一些實施例中,此等結合劑可包括抗CD28、抗CD2、抗CD28H、抗LFA-1、抗OX40、抗4-1BB、抗CD40L、抗DNAM-1、抗CD27、抗ICOS、抗LIGHT、抗GITR、抗CD30、抗SLAM、抗Ly-9、抗CD84、抗Ly108、抗NKG2D、抗NKp46、抗NKp44、抗NKp30、抗CD244、抗NKp80、抗TCRα鏈、抗TCRβ鏈、抗TCRγ鏈及抗TCRδ鏈藥劑。In some embodiments, the viral particles comprise adhesion molecules. As used herein, the term "adhesion molecules" refers to a subset of cell surface molecules that are involved in the binding of cells to other cells. Adhesion molecules can help form more stable interactions between immune cells, such as immune junctions. Immune junctions are stable adhesive junctions between polarized immune effector cells and antigen-carrying cells. In some embodiments, adhesion molecules can provide co-stimulatory signals to target cells. In some embodiments, adhesion molecules include but are not limited to CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6 and B7-H6. In some embodiments, adhesion molecules include but are not limited to binding agents such as scFv, antibodies, single domain antibodies, antibody fragments and nanobodies that bind to any of the adhesion or co-stimulatory molecules described herein. In some embodiments, such binding agents may include anti-CD28, anti-CD2, anti-CD28H, anti-LFA-1, anti-OX40, anti-4-1BB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-Ly108, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, anti-NKp80, anti-TCRα chain, anti-TCRβ chain, anti-TCRγ chain and anti-TCRδ chain agents.

在一些實施例中,黏附分子與CD2結合。CD2亦稱為T11、LFA-2及紅血球玫瑰花式受體,且為在T淋巴球及NK細胞上表現之表面蛋白質。CD2為CD58之天然配體。除進行黏附功能以外,CD2之接合提供可增強活化及效應功能之共刺激訊號。在一些實施例中,慢病毒顆粒包含與CD2結合之分子。在一些實施例中,慢病毒顆粒包含對CD2具有特異性之抗體、單域抗體、抗體片段及/或奈米抗體。在一些實施例中,慢病毒顆粒包含與CD2結合之CD58或其功能部分。In some embodiments, the adhesion molecule binds to CD2. CD2 is also known as T11, LFA-2, and erythrocyte rosette receptor, and is a surface protein expressed on T lymphocytes and NK cells. CD2 is a natural ligand for CD58. In addition to performing adhesion functions, engagement of CD2 provides co-stimulatory signals that enhance activation and effector functions. In some embodiments, the lentiviral particles comprise a molecule that binds to CD2. In some embodiments, the lentiviral particles comprise an antibody, a single domain antibody, an antibody fragment, and/or a nanobody that is specific for CD2. In some embodiments, the lentiviral particles comprise CD58 or a functional portion thereof that binds to CD2.

在一些實施例中,黏附分子為CD58。在一些實施例中,共刺激分子為CD58多肽,其包含SEQ ID NO: 80之胺基酸序列。在一些實施例中,共刺激分子為CD58多肽,其包含與SEQ ID NO: 80至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%一致之胺基酸序列。In some embodiments, the adhesion molecule is CD58. In some embodiments, the costimulatory molecule is a CD58 polypeptide comprising an amino acid sequence of SEQ ID NO: 80. In some embodiments, the costimulatory molecule is a CD58 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 80.

在一些實施例中,CD58多肽係由SEQ ID NO: 81之核苷酸序列編碼。在一些實施例中,CD58多肽係由與SEQ ID NO: 81至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%一致之核苷酸序列編碼。 4.額外非病毒蛋白質 In some embodiments, the CD58 polypeptide is encoded by a nucleotide sequence of SEQ ID NO: 81. In some embodiments, the CD58 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 81. 4. Additional non-viral proteins

在一些實施例中,病毒顆粒包含至少一種非病毒蛋白質。在一些實施例中,除上文所描述之彼等者以外,病毒顆粒包含至少一種非病毒蛋白質。In some embodiments, the viral particles comprise at least one non-viral protein. In some embodiments, the viral particles comprise at least one non-viral protein in addition to those described above.

在一些實施例中,病毒顆粒包含靶向配體。在一些實施例中,病毒顆粒包含CD19或其功能片段,其與其天然跨膜域或異源跨膜域偶聯。在一些實施例中,CD19用作博納吐單抗(blinatumomab)之配體,從而提供用於經由博納吐單抗之抗CD3部分使顆粒與T細胞偶聯的轉接子。在一些實施例中,另一類型之顆粒表面配體可用於使用包含顆粒表面配體之結合部分之多特異性抗體而將適當表面工程改造之慢病毒顆粒與T細胞偶聯。在一些實施例中,多特異性抗體為雙特異性抗體,例如雙特異性T細胞接合分子(BiTE)。In some embodiments, the viral particles comprise a targeting ligand. In some embodiments, the viral particles comprise CD19 or a functional fragment thereof coupled to its native transmembrane domain or a heterologous transmembrane domain. In some embodiments, CD19 is used as a ligand for blinatumomab, thereby providing an adapter for coupling the particles to T cells via the anti-CD3 portion of blinatumomab. In some embodiments, another type of particle surface ligand can be used to couple appropriately surface engineered lentiviral particles to T cells using a multispecific antibody comprising a binding portion of a particle surface ligand. In some embodiments, the multispecific antibody is a bispecific antibody, such as a bispecific T cell engager (BiTE).

在一些實施例中,非病毒蛋白質為細胞介素。在一些實施例中,細胞介素可選自由以下組成之群:IL-2、IL-7、IL-12、IL-15、IL-18、IL-21及其任何組合。在所使用之非病毒蛋白質為可溶性蛋白質(諸如scFv或細胞介素)之情況下,其可藉由與跨膜域(諸如CD8之跨膜域)融合而繫栓至病毒顆粒之表面。或者,其可藉由使用經工程改造以結合可溶性蛋白質之跨膜蛋白而間接繫栓至慢病毒顆粒。進一步包括一或多種細胞質殘基可增加融合蛋白之穩定性。In some embodiments, the non-viral protein is a cytokine. In some embodiments, the cytokine can be selected from the group consisting of: IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 and any combination thereof. In the case where the non-viral protein used is a soluble protein (such as scFv or cytokine), it can be tethered to the surface of the viral particle by fusion with a transmembrane domain (such as the transmembrane domain of CD8). Alternatively, it can be indirectly tethered to the lentiviral particle by using a transmembrane protein engineered to bind to a soluble protein. Further inclusion of one or more cytoplasmic residues can increase the stability of the fusion protein.

細胞分裂轉導強化子及/或基於細胞介素之轉導強化子可包含「間隔子序列」以將抗原結合結構域與跨膜域連接。可撓性間隔子允許抗原結合域以不同方向取向以有助於結合。如本文所用,術語「與...偶聯」係指:化學鍵聯,一種兩個蛋白質之直接C端與N端融合;與非肽空間之化學鍵聯;與多肽空間之化學鍵聯;及經由與多肽間隔子(例如間隔子序列)之肽鍵,兩個蛋白質之C端與N端融合。Cell division transduction enhancers and/or cytokine-based transduction enhancers may include a "spacer sequence" to link the antigen binding domain to the transmembrane domain. Flexible spacers allow the antigen binding domain to be oriented in different directions to facilitate binding. As used herein, the term "coupled to" refers to: chemical bonding, a direct C-terminal to N-terminal fusion of two proteins; chemical bonding to non-peptide space; chemical bonding to polypeptide space; and C-terminal to N-terminal fusion of two proteins via a peptide bond to a polypeptide spacer (e.g., a spacer sequence).

間隔子序列可例如包含lgG1 Fc區、lgG1鉸鏈或人類CD8柄或小鼠CD8柄。間隔子或者可包含替代性連接子序列,其具有與lgG1 Fc區、lgG1鉸鏈或CD8柄類似的長度及/或域間隔特性。可改變人類lgG1間隔子以移除Fc結合模體。在一些實施例中,間隔子序列可來源於人類蛋白。The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge, or a human CD8 stalk or a mouse CD8 stalk. The spacer may alternatively comprise an alternative linker sequence having similar length and/or domain spacing properties as the IgG1 Fc region, the IgG1 hinge, or the CD8 stalk. The human IgG1 spacer may be altered to remove the Fc binding motif. In some embodiments, the spacer sequence may be derived from a human protein.

在一些實施例中,間隔子序列包含CD8源性鉸鏈。In some embodiments, the spacer sequence comprises a CD8-derived hinge.

在一些實施例中,間隔子序列包含『短』鉸鏈。短鉸鏈描述為相對於此項技術中已知之CAR鉸鏈區包含更少核苷酸之鉸鏈區。In some embodiments, the spacer sequence comprises a "short" hinge. A short hinge is described as a hinge region that comprises fewer nucleotides relative to the hinge region of CARs known in the art.

跨膜域為跨越膜之細胞分裂轉導強化子及/或基於細胞介素之轉導強化子的序列。跨膜域可包含疏水性α螺旋。跨膜域可來源於CD28。在一些實施例中,跨膜域來源於人類蛋白。The transmembrane domain is a sequence of a cell division transduction enhancer and/or a cytokine-based transduction enhancer that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28. In some embodiments, the transmembrane domain is derived from a human protein.

本發明之病毒顆粒可包含基於細胞介素之轉導強化子在病毒套膜中。在一些實施例中,基於細胞介素之轉導強化子來源於在病毒顆粒產生期間之宿主細胞。在一些實施例中,基於細胞介素之轉導強化子係由宿主細胞製備且表現在細胞表面處。當新生病毒顆粒自宿主細胞膜出芽時,基於細胞介素之轉導強化子可作為封裝細胞源性脂質雙層之一部分而併入病毒套膜中。The viral particles of the present invention may include a cytokine-based transduction enhancer in the viral envelope. In some embodiments, the cytokine-based transduction enhancer is derived from host cells during viral particle production. In some embodiments, the cytokine-based transduction enhancer is prepared by host cells and expressed at the cell surface. When the nascent viral particle buds from the host cell membrane, the cytokine-based transduction enhancer can be incorporated into the viral envelope as part of the encapsulating cell-derived lipid bilayer.

基於細胞介素之轉導強化子可包含細胞介素域及跨膜域。其可具有結構C-S-TM,其中C為細胞介素域,S為視情況存在之間隔子域(例如間隔子序列),且TM為跨膜域。間隔子域及跨膜域如上文所定義。The cytokine-based transduction enhancer may comprise a cytokine domain and a transmembrane domain. It may have the structure C-S-TM, wherein C is the cytokine domain, S is an optional spacer domain (e.g., a spacer sequence), and TM is a transmembrane domain. The spacer domain and the transmembrane domain are as defined above.

細胞介素域可包含諸如來自IL2、IL7及IL15之T細胞活化性細胞介素或其功能片段。如本文所用,細胞介素之「功能片段」為保留結合其特定受體且活化T細胞之能力的多肽片段。The interleukin domain may include T cell activating interleukins such as IL2, IL7 and IL15 or their functional fragments. As used herein, a "functional fragment" of an interleukin is a polypeptide fragment that retains the ability to bind to its specific receptor and activate T cells.

IL2為T細胞分泌的用於調節T細胞及某些B細胞之生長及分化的因子中之一者。IL2為誘導反應性T細胞增殖之淋巴激素。其係作為單一醣基化之多肽分泌出,且其活性需要訊號序列之裂解。溶液NMR表明,IL2之結構包含4螺旋(稱為A-D)束,其側接有2個較短螺旋及若干不充分定義的環。螺旋A中及螺旋A與B之間的環區中之殘基對於受體結合而言非常重要。 IV.方法及其用途 IL2 is one of the factors secreted by T cells that regulates the growth and differentiation of T cells and some B cells. IL2 is a lymphokine that induces proliferation of reactive T cells. It is secreted as a monoglycosylated polypeptide and its activity requires cleavage of a signal sequence. Solution NMR shows that the structure of IL2 consists of a 4-helical (called A-D) bundle flanked by 2 shorter helices and several poorly defined loops. Residues in helix A and in the loop region between helices A and B are important for receptor binding. IV. Methods and Uses

在一些實施例中,本文提供使用本文所揭示之多順反子構築體或核苷酸載體之方法。在一些實施例中,所提供之方法將多順反子構築體遞送至細胞以用於表現包括胞質FRB、合成細胞介素受體及CAR之所提供系統。在一些實施例中,多順反子構築體含於病毒載體內,且病毒載體用於轉導目標細胞。在一些實施例中,離體或活體外進行所提供之方法以用多順反子構築體工程改造目標細胞。在一些實施例中,向個體投與經工程改造之細胞。在一些實施例中,活體內進行所提供之方法,且將含有多順反子構築體之病毒載體引入個體中以用於在活體內將多順反子載體靶向遞送至目標細胞(諸如T細胞)。 A.轉導細胞之方法 In some embodiments, methods of using the polycistronic constructs or nucleotide vectors disclosed herein are provided herein. In some embodiments, the provided methods deliver polycistronic constructs to cells for expression of provided systems including cytoplasmic FRBs, synthetic interleukin receptors, and CARs. In some embodiments, the polycistronic constructs are contained in viral vectors, and the viral vectors are used to transduce target cells. In some embodiments, the provided methods are performed in vitro or in vitro to engineer target cells with polycistronic constructs. In some embodiments, engineered cells are administered to an individual. In some embodiments, the provided methods are performed in vivo, and a viral vector containing a polycistronic construct is introduced into an individual for targeted delivery of the polycistronic vector to target cells (such as T cells) in vivo. A. Methods of Transducing Cells

在一些實施例中,本文提供一種轉導細胞之方法,其包含使目標細胞與含有本文提供之多順反子構築體中之任一者的顆粒(諸如病毒載體)接觸。在一些實施例中,目標細胞包含幹細胞。在一些實施例中,幹細胞包含誘導性富潛能幹細胞(iPSC)。在一些實施例中,目標細胞包含前驅細胞。在一些實施例中,前驅細胞包含周邊血液單核細胞(PBMC)。在一些實施例中,目標細胞包含T細胞。在一些實施例中,T細胞包含CD4+ 或CD8+ T細胞。在一些實施例中,該方法進一步包含使目標細胞與以下接觸:(i)靶向內源基因中之目標位點的引導RNA (gRNA),及(ii) RNA引導核酸內切酶,從而將核苷酸序列插入內源基因中。In some embodiments, provided herein is a method of transducing cells, comprising contacting a target cell with a particle (such as a viral vector) containing any one of the polycistronic constructs provided herein. In some embodiments, the target cell comprises a stem cell. In some embodiments, the stem cell comprises an induced enriched potential stem cell (iPSC). In some embodiments, the target cell comprises a progenitor cell. In some embodiments, the progenitor cell comprises a peripheral blood mononuclear cell (PBMC). In some embodiments, the target cell comprises a T cell. In some embodiments, the T cell comprises a CD4+ or CD8+ T cell. In some embodiments, the method further comprises contacting the target cell with: (i) a guide RNA (gRNA) that targets a target site in an endogenous gene, and (ii) an RNA-guided endonuclease to insert the nucleotide sequence into the endogenous gene.

在一些態樣中,可將本文所描述之多核苷酸活體內遞送至細胞。在一些實施例中,經由投與包含多核苷酸之顆粒而直接向個體投與編碼本文所揭示之多順反子構築體之元件的多核苷酸。在一些實施例中,顆粒為病毒顆粒。在一些實施例中,病毒顆粒包含抗CD3 scFv及科卡爾病毒醣蛋白,且能夠將多核苷酸活體內遞送至細胞。在一些實施例中,多核苷酸編碼胞質FRB、合成細胞介素受體及嵌合抗原受體(CAR)。可向個體投與本文所揭示之多核苷酸,此允許在活體內產生各種構築體組分(例如FRB、合成細胞介素受體及CAR)。在一些實施例中,此類多核苷酸之投與在活體內產生與直接投與表現FRB、合成細胞介素受體及CAR之離體經工程改造之細胞類似的作用。在一些實施例中,此類多核苷酸之投與改良顆粒之活體內轉導效率。在一些實施例中,多核苷酸為mRNA。In some aspects, the polynucleotides described herein can be delivered to cells in vivo. In some embodiments, polynucleotides encoding elements of the polycistronic constructs disclosed herein are administered directly to an individual by administering particles comprising the polynucleotides. In some embodiments, the particles are viral particles. In some embodiments, the viral particles comprise anti-CD3 scFv and Cokar virus glycoprotein and are capable of delivering polynucleotides to cells in vivo. In some embodiments, the polynucleotides encode cytoplasmic FRBs, synthetic interleukin receptors, and chimeric antigen receptors (CARs). The polynucleotides disclosed herein can be administered to an individual, which allows the production of various construct components (e.g., FRBs, synthetic interleukin receptors, and CARs) in vivo. In some embodiments, administration of such polynucleotides produces in vivo effects similar to direct administration of ex vivo engineered cells expressing FRB, synthetic interleukin receptors, and CARs. In some embodiments, administration of such polynucleotides improves the transduction efficiency of particles in vivo. In some embodiments, the polynucleotide is mRNA.

本文所描述之多核苷酸亦可離體遞送至細胞。本文所描述之病毒顆粒可在習知細胞製造製程中或在例如國際專利公開案第WO 2022/072885 A1號中所描述之體外或床邊製程中離體使用。在一個實施例中,本發明提供一種轉導目標細胞之離體方法,其包含使目標細胞與根據本發明之顆粒接觸。在一些實施例中,本文所描述之顆粒可用於轉導先前尚未活化之細胞。舉例而言,本文所描述之顆粒可適用於轉導先前尚未與細胞活化珠粒或活化試劑(例如戴諾珠粒(Dynabead)或包含抗CD3及/或抗CD28抗體或其結合片段之其他試劑)接觸的細胞。The polynucleotides described herein can also be delivered to cells in vitro. The viral particles described herein can be used in vitro in a known cell manufacturing process or in an in vitro or bedside process as described, for example, in International Patent Publication No. WO 2022/072885 A1. In one embodiment, the present invention provides an in vitro method for transducing a target cell, comprising contacting the target cell with a particle according to the present invention. In some embodiments, the particles described herein can be used to transduce cells that have not been previously activated. For example, the particles described herein can be used to transduce cells that have not been previously contacted with cell activating beads or activating reagents (e.g., Dynabeads or other reagents comprising anti-CD3 and/or anti-CD28 antibodies or binding fragments thereof).

在一些實施例中,本發明提供一種在離體CAR T製造製程中將核酸遞送至細胞之方法。此類方法通常涉及經由白血球分離術(leukapheresis)自患者分離PBMC。洗滌此等細胞,且視情況經由一或多個選擇步驟進一步純化以分離特定的所關注T細胞群體。在一些態樣中,此等可包括CD4+及/或CD8+ T細胞。經洗滌及/或純化細胞可視情況活化且接著使用慢病毒載體轉導。活化步驟可包含使細胞與同受質結合之外源性活化劑(諸如抗CD3及抗CD28抗體)接觸或使用未結合抗體。說明性活化藥劑包括呈現抗CD3及抗CD28之珠粒及/或可溶性聚合物。在轉導之後,細胞可視情況進一步洗滌且培養直至收穫。製造經工程改造之細胞療法(包括CAR T細胞)之方法為此項技術中已知的(參見例如Abou-el-Enein, M.等人Blood Cancer Discov (2021), 第2卷(5): 408-422;Arcangeli, S.等人Front. Immunol (2020年6月19日), 第11卷(1217) 1-13;Ghassemi, S.等人Nat Biomed Eng (2022年2月), 第6卷(2): 118-128;Vormittag, P.等人Curr Opin Biotechnol (2018年10月), 第54卷: 164-181;其各自以引用之方式併入本文中)。自體CAR T製造之說明性方法揭示於美國專利公開案第2019/0269727號、第2016/0122782號、第2021/0163893號及第US 2017/0037369號中,其中之各者全文併入本文中。In some embodiments, the present invention provides a method for delivering nucleic acids to cells in an ex vivo CAR T manufacturing process. Such methods typically involve isolating PBMCs from a patient by leukapheresis. These cells are washed and further purified by one or more selection steps to isolate specific T cell populations of interest, as appropriate. In some embodiments, these may include CD4+ and/or CD8+ T cells. The washed and/or purified cells may be activated as appropriate and then transduced with a lentiviral vector. The activation step may include contacting the cells with exogenous activators bound to the same substrate (such as anti-CD3 and anti-CD28 antibodies) or using unbound antibodies. Illustrative activating agents include beads and/or soluble polymers displaying anti-CD3 and anti-CD28. Following transduction, cells can be further washed and cultured as appropriate until harvest. Methods for making engineered cell therapies, including CAR T cells, are known in the art (see, e.g., Abou-el-Enein, M. et al. Blood Cancer Discov (2021), Vol. 2(5): 408-422; Arcangeli, S. et al. Front. Immunol (June 19, 2020), Vol. 11(1217) 1-13; Ghassemi, S. et al. Nat Biomed Eng (February 2022), Vol. 6(2): 118-128; Vormittag, P. et al. Curr Opin Biotechnol (October 2018), Vol. 54: 164-181; each of which is incorporated herein by reference). Illustrative methods of autologous CAR T production are disclosed in U.S. Patent Publication Nos. 2019/0269727, 2016/0122782, 2021/0163893, and US 2017/0037369, each of which is incorporated herein in its entirety.

在一些實施例中,本發明提供一種在離體閉合迴路製造製程中將核酸遞送至細胞之方法。在一些實施例中,離體製造製程為體外製程。在例示性實施例中,本文所揭示之慢病毒載體允許在閉合迴路製程期間將核酸遞送至目標細胞。閉合迴路及/或體外製程之例示性方法揭示於美國專利公開案第2021/0244871號及第WO2022072885號中,該兩者以全文併入本文中。在一些實施例中,本文所揭示之慢病毒載體可用於離體轉導細胞。舉例而言,在例示性閉合迴路製造製程中,細胞自個體獲得,洗滌,與慢病毒顆粒一起培育及/或與其接觸,視情況再次洗滌,且在閉合迴路系統中輸注至個體中。在此類實施例中,本文所揭示之慢病毒顆粒適用,即使在無細胞先前活化之情況下,且能夠在短培育及/或接觸步驟中與細胞結合。在一些實施例中,培育及/或接觸步驟為大約或小於一小時。在一些實施例中,培育及/或接觸步驟為大約或小於兩小時、大約或小於三小時、大約或小於四小時、或大約或小於五小時。在一些實施例中,培育及/或接觸步驟為小於12小時或小於24小時。在一些實施例中,藉由用慢病毒載體轉導而將核酸遞送至細胞以使得核酸離體進入細胞。在一些實施例中,藉由使慢病毒載體與細胞表面接觸而將核酸遞送至細胞。在此類實施例中,核酸可在細胞(與慢病毒載體複合)輸注回個體中之後離體或活體內進入細胞。In some embodiments, the present invention provides a method for delivering nucleic acids to cells in an in vitro closed-loop manufacturing process. In some embodiments, the in vitro manufacturing process is an in vitro process. In exemplary embodiments, the lentiviral vectors disclosed herein allow nucleic acids to be delivered to target cells during the closed-loop process. Exemplary methods of closed-loop and/or in vitro processes are disclosed in U.S. Patent Publication Nos. 2021/0244871 and WO2022072885, both of which are incorporated herein in their entirety. In some embodiments, the lentiviral vectors disclosed herein can be used to transduce cells in vitro. For example, in an exemplary closed-loop manufacturing process, cells are obtained from an individual, washed, incubated and/or contacted with lentiviral particles, optionally washed again, and infused into an individual in a closed-loop system. In such embodiments, the lentiviral particles disclosed herein are applicable even without prior activation of the cells and are capable of binding to the cells in a short incubation and/or contacting step. In some embodiments, the incubation and/or contacting step is about or less than one hour. In some embodiments, the incubation and/or contacting step is about or less than two hours, about or less than three hours, about or less than four hours, or about or less than five hours. In some embodiments, the incubation and/or contacting step is less than 12 hours or less than 24 hours. In some embodiments, the nucleic acid is delivered to the cell by transduction with a lentiviral vector so that the nucleic acid enters the cell ex vivo. In some embodiments, the nucleic acid is delivered to the cell by contacting the lentiviral vector with the cell surface. In such embodiments, the nucleic acid can enter the cell ex vivo or in vivo after the cell (complexed with the lentiviral vector) is infused back into the individual.

在一些實施例中,本文所揭示之慢病毒載體消除對離體活化步驟之需求。在此類實施例中,經分離之細胞可在白血球分離術、洗滌或選擇之後直接轉導。經考慮,本文所描述之表面工程改造使得本文所揭示之慢病毒顆粒能夠在單步中活化且轉導細胞。在此類實施例中,本文所揭示之慢病毒顆粒可實現短或截短製造製程、藉由消除一或多個單元操作(例如在轉導之前活化)而降低離體製造所耗費的時間及/或降低轉導後細胞培養中所需要的時間量。 B.表現受體之方法 In some embodiments, the lentiviral vectors disclosed herein eliminate the need for an ex vivo activation step. In such embodiments, the isolated cells can be transduced directly after leukapheresis, washing or selection. It is contemplated that the surface engineering described herein enables the lentiviral particles disclosed herein to activate and transduce cells in a single step. In such embodiments, the lentiviral particles disclosed herein can achieve a short or truncated manufacturing process, reduce the time spent in ex vivo manufacturing by eliminating one or more unit operations (e.g., activation prior to transduction), and/or reduce the amount of time required in cell culture after transduction. B. Methods of expressing receptors

在一些實施例中,本文提供一種在目標細胞中表現嵌合抗原受體及/或合成細胞介素受體之方法。在一些實施例中,該方法包括使目標細胞與含有本文提供之多順反子構築體中之任一者的顆粒(諸如病毒載體)接觸。在一些實施例中,該接觸係在離體或活體外進行。在一些實施例中,該接觸係藉由向個體投與含有其之多核苷酸構築體或顆粒(諸如病毒載體)而在個體中在活體內進行。In some embodiments, provided herein is a method of expressing a chimeric antigen receptor and/or a synthetic interleukin receptor in a target cell. In some embodiments, the method comprises contacting the target cell with a particle (such as a viral vector) containing any of the polycistronic constructs provided herein. In some embodiments, the contacting is performed in vitro or in vitro. In some embodiments, the contacting is performed in vivo in an individual by administering a polynucleotide construct or particle (such as a viral vector) containing the same to the individual.

在一些實施例中,目標細胞包含幹細胞。In some embodiments, the target cells comprise stem cells.

在一些實施例中,幹細胞包含誘導性富潛能幹細胞(iPSC)。In some embodiments, the stem cells comprise induced enriched-potential stem cells (iPSCs).

在一些實施例中,目標細胞包含前驅細胞。In some embodiments, the target cells comprise progenitor cells.

在一些實施例中,前驅細胞包含周邊血液單核細胞(PBMC)。In some embodiments, the progenitor cells comprise peripheral blood mononuclear cells (PBMCs).

在一些實施例中,目標細胞包含T細胞。In some embodiments, the target cell comprises a T cell.

在一些實施例中,T細胞包含CD4+ 或CD8+ T細胞。In some embodiments, the T cells comprise CD4+ or CD8+ T cells.

在一些實施例中,該方法係離體或在活體外進行。In some embodiments, the method is performed ex vivo or in vitro.

在一些實施例中,該方法係在活體內進行。In some embodiments, the method is performed in vivo.

在一些實施例中,本文提供一種藉由本文所提供之方法中之任一者產生的細胞。 C.投與方法 In some embodiments, provided herein is a cell produced by any of the methods provided herein. C. Methods of Administration

本文亦提供向個體投與用所提供多順反子構築體工程改造之所提供細胞中之任一者的方法。本文亦提供向個體投與所提供顆粒(諸如病毒載體,例如慢病毒載體)中之任一者的方法。在一些實施例中,個體患有疾病或病狀,且該等投與方法係用於治療疾病或病狀。在主題方法中之任一者中,細胞或顆粒係以醫藥組合物形式投與。在一些實施例中,組合物係用於治療疾病或病狀。亦提供所提供之組合物用於治療個體之疾病或病狀的用途。此類方法及用途包括治療方法及用途,例如涉及向患有疾病或病狀之投與經工程改造之細胞或顆粒(例如病毒載體)或含有其之組合物。在一些情況下,疾病或病狀為腫瘤或癌症。在一些實施例中,細胞或其醫藥組合物係以實現疾病或病症之治療的有效量投與。用途包括細胞或其醫藥組合物在此類方法及治療中以及在製備用於進行此類治療方法之藥劑中的用途。在一些實施例中,該等方法藉此治療個體的疾病或病狀或病症。Also provided herein are methods of administering any of the provided cells engineered with the provided polycistronic constructs to an individual. Also provided herein are methods of administering any of the provided particles (such as viral vectors, e.g., lentiviral vectors) to an individual. In some embodiments, the individual suffers from a disease or condition, and the methods of administration are used to treat the disease or condition. In any of the subject methods, the cells or particles are administered in the form of a pharmaceutical composition. In some embodiments, the composition is used to treat a disease or condition. Also provided are uses of the provided compositions for treating a disease or condition in an individual. Such methods and uses include treatment methods and uses, for example, involving administration of engineered cells or particles (e.g., viral vectors) or compositions containing the same to a subject suffering from a disease or condition. In some cases, the disease or condition is a tumor or cancer. In some embodiments, the cells or pharmaceutical compositions thereof are administered in an effective amount to achieve treatment of the disease or condition. Uses include use of the cells or pharmaceutical compositions thereof in such methods and treatments and in the preparation of a medicament for performing such treatment methods. In some embodiments, the methods thereby treat a disease or condition or disorder in an individual.

在一些實施例中,可向個體投與含有編碼胞質FRB、合成細胞介素受體及嵌合抗原受體(CAR)之所提供多順反子構築體中之任一者的經工程改造之細胞以治療疾病或病狀。在一些實施例中,可直接向個體投與顆粒(諸如病毒載體(例如慢病毒載體))以用於將多順反子構築體活體內靶向遞送至目標細胞以用於活體內產生各種構築體組分(例如FRB、合成細胞介素受體及CAR)。視需要局部抑或全身性治療而定,所揭示之細胞或顆粒(例如病毒載體)可以多種方式投與。In some embodiments, engineered cells containing any of the provided polycistronic constructs encoding cytoplasmic FRBs, synthetic interleukin receptors, and chimeric antigen receptors (CARs) may be administered to a subject to treat a disease or condition. In some embodiments, particles such as viral vectors (e.g., lentiviral vectors) may be administered directly to a subject for targeted delivery of polycistronic constructs to target cells in vivo for in vivo production of various construct components (e.g., FRBs, synthetic interleukin receptors, and CARs). The disclosed cells or particles (e.g., viral vectors) may be administered in a variety of ways, depending on whether local or systemic treatment is desired.

在過繼性細胞療法之情況下,用於投與用於過繼性細胞療法之細胞的方法為已知的且可與所提供之方法及組合物結合使用。In the case of secondary cell therapy, methods for administering cells for use in secondary cell therapy are known and can be used in conjunction with provided methods and compositions.

一般而言,投與可為局部、非經腸或經腸。本發明之組合物通常適合於非經腸投與。如本文所用,醫藥組合物之「非經腸投與」包括特徵在於個體之組織之物理突破及經由突破組織投與醫藥組合物,因此通常導致直接投與至血流、肌肉或內部器官的任何投與途徑。因此,非經腸投與包括但不限於藉由注射組合物、藉由經由手術切口施加組合物、藉由經由組織穿透性非手術傷口施加組合物及類似方式來投與醫藥組合物。特定言之,非經腸投與預期包括但不限於皮下、腹膜內、肌肉內、腦幹內、靜脈內、動脈內、鞘內、腦室內、尿道內、顱內、腫瘤內、滑膜內注射或輸注;及腎透析輸注技術。在一實施例中,本發明之組合物之非經腸投與包含靜脈內投與。在一些實施例中,藉由腹膜內注射病毒顆粒來投與病毒顆粒。在一些實施例中,藉由結節內注射來投與病毒顆粒,即,可經由注射至淋巴結(諸如腹股溝淋巴結)中來投與病毒顆粒。在一些實施例中,藉由將病毒顆粒注射至腫瘤部位中(亦即腫瘤內)來投與病毒顆粒。在一些實施例中,皮下投與病毒顆粒。在一些實施例中,全身性投與病毒顆粒。在一些實施例中,靜脈內投與病毒顆粒。在一些實施例中,動脈內投與病毒顆粒。在一些實施例中,病毒顆粒為慢病毒顆粒。In general, administration can be topical, parenteral or enteral. The compositions of the present invention are generally suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breach of a subject's tissue and administration of the pharmaceutical composition through breached tissue, thus generally resulting in direct administration to the bloodstream, muscle or internal organs. Thus, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue penetrating non-surgical wound, and the like. Specifically, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intracerebral, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, intrasynovial injection or infusion; and dialysis infusion techniques. In one embodiment, parenteral administration of the compositions of the present invention comprises intravenous administration. In some embodiments, the viral particles are administered by intraperitoneal injection of the viral particles. In some embodiments, the viral particles are administered by intranodal injection, i.e., the viral particles may be administered by injection into lymph nodes (e.g., inguinal lymph nodes). In some embodiments, the viral particles are administered by injecting the viral particles into the tumor site (i.e., into the tumor). In some embodiments, the viral particles are administered subcutaneously. In some embodiments, the viral particles are administered systemically. In some embodiments, the viral particles are administered intravenously. In some embodiments, the viral particles are administered intraarterially. In some embodiments, the viral particles are lentiviral particles.

適合於非經腸投與之醫藥組合物的調配物典型地總體上包含活性成分與醫藥學上可接受之載劑(諸如無菌水或無菌等張鹽水)的組合。此類調配物可以適合於以推注投與或連續投與之形式製備、封裝或出售。可注射調配物可以單位劑型形式製備、封裝或出售,諸如以含有防腐劑的安瓿或多劑量容器形式。用於非經腸投與之調配物包括但不限於油性或水性媒劑中之懸浮液、溶液、乳液、糊劑及其類似者。此類調配物可進一步包含一或多種額外成分,包括但不限於懸浮劑、穩定劑或分散劑。在用於非經腸投與之調配物的一個實施例中,活性成分係以乾燥(亦即粉末或顆粒)形式提供,以便在非經腸投與經復原之組合物之前,用合適媒劑(例如無菌無熱原質水)復原。非經腸調配物亦包括水溶液,其可含有賦形劑,諸如鹽、碳水化合物及緩衝劑(較佳緩衝至pH 3至9),但在一些應用中,非經腸調配物更宜調配為無菌非水性溶液或待與合適媒劑(諸如無菌無熱原質水)結合使用的乾燥形式。說明性非經腸投與形式包括在無菌水溶液,例如丙二醇或右旋糖水溶液中之溶液或懸浮液。必要時,此類劑型可經適當緩衝。其他有用的可非經腸投與之調配物包括含在微晶形式或脂質體製劑中之活性成分的調配物。用於非經腸投與之調配物可調配成立即釋放型及/或修飾釋放型。調釋型調配物包括延遲釋放型、持續釋放型、脈衝釋放型、受控釋放型、靶向釋放型及程控釋放型。對於水溶液中之非經腸投與而言,例如必要時溶液應經適當緩衝且首先用充足鹽水或葡萄糖賦予液體稀釋劑等張性。此等特定水溶液尤其適合於靜脈內、肌肉內、皮下及腹膜內投與。在一些實施例中,意欲用於皮下投與之溶液包括玻尿酸酶。The formulation of a pharmaceutical composition suitable for parenteral administration typically generally comprises a combination of an active ingredient and a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged or sold in unit dosage form, such as in an ampoule or multi-dose container containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, pastes and the like in oily or aqueous vehicles. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions, which may contain excipients such as salts, carbohydrates, and buffers (preferably buffered to pH 3 to 9), but in some applications, parenteral formulations are more preferably formulated as sterile non-aqueous solutions or dry forms to be combined with a suitable vehicle (e.g., sterile pyrogen-free water). Illustrative parenteral administration forms include solutions or suspensions in sterile aqueous solutions, such as propylene glycol or dextrose solutions. If necessary, such dosage forms can be appropriately buffered. Other useful formulations that can be administered parenterally include formulations containing active ingredients in microcrystalline form or liposomal formulations. Formulations for parenteral administration can be formulated to be immediate release and/or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmable release. For parenteral administration in aqueous solutions, for example, the solution should be appropriately buffered if necessary and the liquid diluent should first be given isotonicity with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In some embodiments, the solution intended for subcutaneous administration includes hyaluronidase.

本發明之組合物可另外含有其他習知地見於醫藥組合物中之佐劑組分。因此,舉例而言,組合物可含有額外相容的醫藥學上活性材料,諸如止癢劑、收斂劑、局部麻醉劑或消炎劑,或可含有適用於物理調配本發明組合物之多種劑型的額外材料,諸如染料、調味劑、防腐劑、抗氧化劑、遮光劑、增稠劑及穩定劑。然而,此類材料在添加時不應不恰當地干擾本發明組合物之組分的生物活性。調配物可經滅菌,且需要時與例如潤滑劑、防腐劑、穩定劑、濕潤劑、乳化劑、用於影響滲透壓之鹽、緩衝劑、著色劑、調味劑及/或芳族物質及其類似者的助劑混合,該等助劑不會與調配物之一或多種核酸有害地相互作用。The compositions of the present invention may additionally contain other adjuvant components known in pharmaceutical compositions. Thus, for example, the compositions may contain additional compatible pharmaceutically active materials, such as antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials suitable for physically preparing various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, sunscreens, thickeners and stabilizers. However, such materials should not be added in a manner that would unduly interfere with the biological activity of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings and/or aromatic substances and the like which do not deleteriously interact with one or more nucleic acids of the formulation.

本發明之多順反子構築體可以有效治療或預防疾病或病狀之量(諸如治療有效或預防有效量)投與。在一些實施例中,治療或預防功效藉由定期評定所治療之個體來監測。對於歷經數日或更長時間之重複投與,治療將視病狀而定進行重複直至發生疾病症狀之所需抑制為止。然而,其他給藥方案可適用且可加以確定。所要劑量可藉由單次推注投與組合物、藉由多次推注投與組合物或藉由連續輸注投與組合物來遞送。The polycistronic constructs of the present invention can be administered in an amount effective to treat or prevent a disease or condition (e.g., a therapeutically effective or preventatively effective amount). In some embodiments, the therapeutic or preventive efficacy is monitored by periodic assessment of the treated individual. For repeated administrations over several days or longer, the treatment will be repeated, depending on the condition, until the desired suppression of disease symptoms occurs. However, other dosing regimens may be applicable and may be determined. The desired dose may be delivered by administering the composition as a single bolus, by administering the composition as multiple boluses, or by administering the composition as a continuous infusion.

在某些實施例中,在根據本發明輸注已分化細胞或轉殖基因分化細胞的情形下,向個體投與範圍為約一百萬至約1000億個細胞,諸如1百萬至約500億個細胞(例如約5百萬個細胞、約2500萬個細胞、約5億個細胞、約10億個細胞、約50億個細胞、約200億個細胞、約300億個細胞、約400億個細胞,或任何兩個前述值所限定之範圍),諸如約1千萬至約1000億個細胞(例如約2千萬個細胞、約3千萬個細胞、約4千萬個細胞、約6千萬個細胞、約7千萬個細胞、約8千萬個細胞、約9千萬個細胞、約100億個細胞、約250億個細胞、約500億個細胞、約750億個細胞、約900億個細胞,或任何兩個前述值所限定之範圍),且在一些情況下為約1億個細胞至約500億個細胞(例如約1.2億個細胞、約2.5億個細胞、約3.5億個細胞、約4.5億個細胞、約6.5億個細胞、約8億個細胞、約9億個細胞、約30億個細胞、約300億個細胞、約450億個細胞)或此等範圍之間的任何值,及/或以個體之每公斤體重計的此類細胞數目。舉例而言,在一些實施例中,細胞或細胞群體之投與可包含投與約10 3至約10 9個細胞/kg體重,包括彼等範圍內之細胞數目之所有整數值。 In certain embodiments, in the case of transfusion of differentiated cells or transgenic differentiated cells according to the present invention, the range of cells administered to an individual is about one million to about 100 billion, such as 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 100 million ... 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about In some cases, the number of cells is about 100 million to about 50 billion cells (e.g., about 120 million cells). , about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value therebetween, and/or such numbers of cells per kilogram of body weight of an individual. For example, in some embodiments, administration of cells or cell populations may comprise administration of about 10 3 to about 10 9 cells/kg body weight, including all integer values of cell numbers within those ranges.

在一些實施例中,本文提供一種向個體投與本文提供之細胞中之任一者的方法。In some embodiments, provided herein is a method of administering any of the cells provided herein to a subject.

在一些實施例中,本文提供一種向個體投與本文提供之病毒載體中之任一者的方法。In some embodiments, provided herein is a method of administering any of the viral vectors provided herein to a subject.

在一些實施例中,由所提供多順反子構築體編碼之CAR靶向與疾病或病狀相關之抗原,且該等方法包括向患有或疑似患有疾病或病狀之個體投與細胞(例如藉由過繼性細胞療法)或病毒載體(諸如慢病毒載體)。在一些實施例中,所提供之方法包括用於治療罹患癌症之個體的方法,其包括向個體投與用本發明之所提供多順反子構築體中之任一者工程改造之所提供細胞的步驟,其中治療個體之癌症。在一些實施例中,所提供之方法包括用於治療罹患癌症之個體之方法,其包括向個體投與併入本發明之所提供多順反子構築體中之任一者的所提供病毒載體(諸如慢病毒顆粒)的步驟,其中治療個體之癌症。In some embodiments, the CAR encoded by the provided polycistronic construct targets an antigen associated with a disease or condition, and the methods include administering cells (e.g., by replication cell therapy) or viral vectors (such as lentiviral vectors) to an individual suffering from or suspected of having a disease or condition. In some embodiments, the provided methods include methods for treating an individual suffering from cancer, comprising the step of administering to the individual a provided cell engineered with any one of the provided polycistronic constructs of the invention, wherein the cancer in the individual is treated. In some embodiments, provided methods include methods for treating an individual suffering from cancer, comprising the step of administering to the individual a provided viral vector (such as a lentiviral particle) incorporating any of the provided multicistronic constructs of the invention, wherein the cancer in the individual is treated.

在一些實施例中,癌症為實體腫瘤,諸如黑色素瘤、非小細胞肺癌或乳癌。本發明之方法可包括治療任何癌症,包括(但不限於)急性顆粒球性白血病、急性淋巴球性白血病、急性骨髓性白血病、腺癌、腺肉瘤、腎上腺癌、腎上腺皮質癌、肛門癌、退行性星形細胞瘤、血管肉瘤、闌尾癌、星形細胞瘤、基底細胞癌、B細胞淋巴瘤、膽管癌、膀胱癌、骨癌、骨髓癌、腸癌、腦癌、腦幹神經膠質瘤、腦瘤、乳癌、類癌瘤、子宮頸癌、膽管癌、軟骨肉瘤、慢性淋巴球性白血病、慢性骨髓性白血病、結腸癌、結腸直腸癌、顱咽管瘤、皮膚淋巴瘤、皮膚黑色素瘤、瀰漫性星形細胞瘤、乳管原位癌、子宮內膜癌、室管膜瘤、上皮樣肉瘤、食道癌、尤文氏肉瘤(Ewing sarcoma)、肝外膽管癌、眼癌、輸卵管癌、纖維肉瘤、膽囊癌、胃癌、胃腸癌、胃腸類癌癌症、胃腸道基質瘤、普遍性生殖細胞腫瘤、妊娠期滋養細胞疾病、多形性膠質母細胞瘤、神經膠質瘤、毛細胞白血病、頭頸癌、血管內皮瘤、霍奇金氏淋巴瘤(Hodgkin lymphoma)、霍奇金氏淋巴瘤、霍奇金氏疾病、下咽癌、浸潤性乳管癌、浸潤性小葉癌、發炎性乳癌、腸癌、肝內膽管癌、侵襲性/浸潤性乳癌、胰島細胞癌、頜部癌、卡波西氏肉瘤(kaposi sarcoma)、腎癌、喉癌、平滑肌肉瘤、軟腦膜轉移、白血病、唇癌、脂肪肉瘤、肝癌、小葉原位癌、低級星形細胞瘤、肺癌、淋巴結癌、淋巴瘤、男性乳癌、髓性癌、神經管胚細胞瘤、黑色素瘤、腦膜瘤、梅克爾細胞癌(Merkel cell carcinoma)、間葉軟骨肉瘤、間葉間皮瘤、轉移性乳癌、轉移性黑色素瘤、轉移性鱗狀頸癌、混合型神經膠質瘤、口腔癌、黏液性癌、黏膜黑色素瘤、多發性骨髓瘤、蕈狀肉芽腫、骨髓發育不良症候群、鼻腔癌、鼻咽癌、頸癌、神經母細胞瘤、神經內分泌腫瘤、非霍奇金氏淋巴瘤、非小細胞肺癌、燕麥狀細胞癌、眼癌、眼黑色素瘤、寡樹突神經膠質瘤、口部癌、口腔癌、口咽癌、骨原性肉瘤、骨肉瘤、卵巢癌、卵巢上皮癌、卵巢生殖細胞腫瘤、卵巢原發性腹膜癌、卵巢性索基質腫瘤、佩吉特氏病(Paget's disease)、胰臟癌、乳頭狀癌、副鼻竇癌、甲狀旁腺癌、骨盆癌、陰莖癌、周邊神經癌、腹膜癌、咽癌、嗜鉻細胞瘤、毛狀星細胞瘤、松果體區域腫瘤、松果體母細胞瘤、垂體腫瘤、原發性中樞神經系統、前列腺癌、直腸癌、腎細胞癌、腎盂癌、橫紋肌肉瘤、唾液腺癌、肉瘤、骨骼肉瘤、軟組織肉瘤、子宮竇癌、皮膚癌、小細胞肺癌、小腸癌、軟組織肉瘤、脊椎癌、脊柱癌、脊髓癌、脊椎腫瘤、鱗狀細胞癌、胃癌、滑膜肉瘤、T細胞淋巴瘤、睪丸癌、咽喉癌、胸腺瘤/胸腺癌(thymoma/thymic carcinoma)、甲狀腺癌、舌癌、扁桃體癌、移行細胞癌、三陰性乳癌、輸卵管癌(tubal cancer)、管狀癌、未確診的癌症、輸尿管癌、子宮腺癌、子宮癌、子宮肉瘤、陰道癌、外陰癌。In some embodiments, the cancer is a solid tumor, such as melanoma, non-small cell lung cancer, or breast cancer. The methods of the invention may include treating any cancer, including but not limited to acute myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal cancer, adrenal cortical cancer, anal cancer, anaplastic astrocytoma, angiosarcoma, coccygeal cancer, astrocytoma, basal cell carcinoma, B cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, intestinal cancer , brain cancer, brain stem neuroglioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, cranio-pharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing's sarcoma sarcoma), extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumor, universal germ cell tumor, gestational trophoblastic disease, multiforme glioblastoma, neuroglioma, hair cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma, Hodgkin's lymphoma, Hodgkin's disease, hypopharyngeal cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct carcinoma, invasive/invasive breast cancer, pancreatic islet cell carcinoma, jaw cancer, Kaposi's sarcoma sarcoma), kidney cancer, laryngeal cancer, leiomyosarcoma, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma carcinoma), mesenchymal chondrosarcoma, mesenchymal mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous cervical cancer, mixed neuroglioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, nasal cancer, nasopharyngeal cancer, cervical cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin's lymphoma, non-small cell lung cancer, oat cell carcinoma, eye cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian primary peritoneal cancer, ovarian sex cord stromal tumor, Paget's disease disease), pancreatic cancer, papillary cancer, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytoma, pineal region tumor, pinealoblastoma, pituitary tumor, primary central nervous system, prostate cancer, rectal cancer, renal cell cancer, renal pelvic cancer, striate muscle Sarcoma, salivary gland cancer, sarcoma, skeletal sarcoma, soft tissue sarcoma, uterine sinus cancer, skin cancer, small cell lung cancer, small intestinal cancer, soft tissue sarcoma, spinal cancer, spinal cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, stomach cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma/thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, triple negative breast cancer, fallopian tube cancer (tubal cancer), tubular carcinoma, undiagnosed cancer, ureteral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer.

在一些實施例中,由所提供多順反子構築體編碼之CAR為靶向能夠與同疾病或病狀相關之細胞表面上之抗原結合之配體的CAR。在一些實施例中,所提供實施例中之任一者的CAR為針對FITC之抗FITC CAR,且該配體為由FITC及能夠與目標細胞上之表面分子或受體結合之結合分子構成的雙官能配體。在一些實施例中,該方法進一步包括投與雙官能配體以標記個體中之癌細胞,其中雙官能配體特異性結合腫瘤上表現之分子。在一些實施例中,雙官能配體為FITC-葉酸。在一些實施例中,癌症為骨肉瘤。In some embodiments, the CAR encoded by the provided multi-cistronic construct is a CAR targeting a ligand capable of binding to an antigen on the surface of a cell associated with a disease or condition. In some embodiments, the CAR of any of the provided embodiments is an anti-FITC CAR directed against FITC, and the ligand is a bifunctional ligand composed of FITC and a binding molecule capable of binding to a surface molecule or receptor on a target cell. In some embodiments, the method further comprises administering a bifunctional ligand to mark cancer cells in an individual, wherein the bifunctional ligand specifically binds to a molecule expressed on a tumor. In some embodiments, the bifunctional ligand is FITC-folate. In some embodiments, the cancer is osteosarcoma.

在一些實施例中,該方法進一步包含投與雙官能配體以標記個體中之癌細胞,其中雙官能配體特異性結合腫瘤上表現之分子。在一些實施例中,雙官能配體包含FITC-葉酸。In some embodiments, the method further comprises administering a bifunctional ligand to label cancer cells in the individual, wherein the bifunctional ligand specifically binds to a molecule expressed on the tumor. In some embodiments, the bifunctional ligand comprises FITC-folate.

在任何實施例中之一些中,該方法進一步包括向個體投與非生理學配體。在一些實施例中,非生理學配體能夠與合成細胞介素受體結合且誘導細胞中之γ細胞介素傳訊。在一些實施例中,非生理學配體包括雷帕黴素或雷帕黴素類似物。 1.非生理學配體 In some of any of the embodiments, the method further comprises administering a non-physiological ligand to the individual. In some embodiments, the non-physiological ligand is capable of binding to a synthetic interleukin receptor and inducing interleukin-γ signaling in the cell. In some embodiments, the non-physiological ligand comprises rapamycin or a rapamycin analog. 1. Non-physiological ligands

在本發明之組合物及方法之各種實施例中,系統包含非生理學配體。適用作配體之說明性小分子包括但不限於:雷帕黴素、螢光素、螢光異硫氰酸鹽(FITC)、4-[(6-甲基吡𠯤-2-基)氧基]苯甲酸(aMPOB)、葉酸、玫瑰紅、乙醯偶氮胺及CA9配體。In various embodiments of the compositions and methods of the present invention, the system comprises a non-physiological ligand. Illustrative small molecules suitable for use as ligands include, but are not limited to, rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4-[(6-methylpyridin-2-yl)oxy]benzoic acid (aMPOB), folic acid, rose bengal, acetyl azoamine, and CA9 ligand.

在一些實施例中,合成細胞介素受體藉由配體活化。在一些實施例中,配體為非生理學配體。In some embodiments, the synthetic interleukin receptor is activated by a ligand. In some embodiments, the ligand is a non-physiological ligand.

在一些實施例中,非生理學配體為雷帕黴素類似物。In some embodiments, the non-physiological ligand is a rapamycin analog.

在一些實施例中,非生理學配體為雷帕黴素。In some embodiments, the non-physiological ligand is rapamycin.

在一些實施例中,非生理學配體為AP21967。In some embodiments, the non-physiological ligand is AP21967.

在一些實施例中,非生理學配體為FK506。In some embodiments, the non-physiological ligand is FK506.

在一些實施例中,非生理學配體為FK1012。在一些實施例中,非生理學配體為AP1510。在一些實施例中,非生理學配體為AP1903。在一些實施例中,非生理學配體為AP20187。在一些實施例中,非生理學配體為環孢素-A (CsA)。在一些實施例中,非生理學配體為庫馬黴素(coumermycin)。In some embodiments, the non-physiological ligand is FK1012. In some embodiments, the non-physiological ligand is AP1510. In some embodiments, the non-physiological ligand is AP1903. In some embodiments, the non-physiological ligand is AP20187. In some embodiments, the non-physiological ligand is cyclosporine-A (CsA). In some embodiments, the non-physiological ligand is coumermycin.

在一些實施例中,合成細胞介素受體複合物藉由葉酸、螢光素、aMPOB、乙醯偶氮胺、CA9配體、他克莫司(tacrolimus)、雷帕黴素、雷帕黴素類似物(rapalog/rapamycin analog)、CD28配體、poly(his)標籤、鏈球親生物素蛋白標籤、FLAG-標籤、VS-標籤、Myc-標籤、HA-標籤、NE-標籤、生物素、地高辛(digoxigenin)、二硝基苯酚或其衍生物活化。In some embodiments, the synthetic interleukin receptor complex is activated by folic acid, fluorescein, aMPOB, acetyl azodipine, CA9 ligand, tacrolimus, rapamycin, rapamycin analog, CD28 ligand, poly(his) tag, tethered globulin tag, FLAG-tag, VS-tag, Myc-tag, HA-tag, NE-tag, biotin, digoxigenin, dinitrophenol, or a derivative thereof.

在一些實施例中,非生理學配體可為小於1000道爾頓之無機或有機化合物。In some embodiments, the non-physiological ligand may be an inorganic or organic compound with a molecular weight of less than 1000 Daltons.

在一些實施例中,配體可為雷帕黴素或雷帕黴素類似物(rapamycin analog/rapalog)。在一些實施例中,雷帕黴素類似物包含相對於雷帕黴素具有以下修飾中之一或多者之雷帕黴素的變體:在C7、C42及/或C29處之甲氧基的去甲基化、消除或置換;在C13、C43及/或C28處之羥基的消除、衍生化或置換;C14、C24及/或C30處之酮的還原、消除或衍生化;6員哌可酸(pipecolate)環經5員脯胺醯基環置換;及環己基環上之替代性取代或環己基環經經取代之環戊基環置換。In some embodiments, the ligand may be rapamycin or a rapamycin analog (rapamycin analog / rapalog). In some embodiments, the rapamycin analog comprises a variant of rapamycin with one or more of the following modifications relative to rapamycin: demethylation, elimination or substitution of methoxy groups at C7, C42 and / or C29; elimination, derivatization or substitution of hydroxyl groups at C13, C43 and / or C28; reduction, elimination or derivatization of ketones at C14, C24 and / or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolinyl ring; and substitution substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring.

因此,在一些實施例中,雷帕黴素類似物為依維莫司(everolimus)、諾沃莫斯(novolimus)、吡美莫司(pimecrolimus)、地磷莫司(ridaforolimus)、他克莫司(tacrolimus)、替西羅莫司(temsirolimus)、烏米莫司(umirolimus)、佐他莫司(zotarolimus)、替西羅莫司(Temsirolimus) (CCI-779)、C20-烯丙基雷帕黴素(methallylrapamycin)、C16-(S)-3-甲基吲哚雷帕黴素、C16-(S)-3-甲基吲哚雷帕黴素(C16-iRap)、AP21967 (A/C異二聚體,Takara Bio®)、黴酚酸鈉、鹽酸貝尼地平(benidipine hydrochloride)、雷帕明(rapamine)、AP23573 (地磷莫司(Ridaforolimus))、AP1903 (雷米杜西(Rimiducid)),或其代謝物、衍生物及/或組合。Thus, in some embodiments, the rapamycin analog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, Temsirolimus (CCI-779), C20-methallylrapamycin, C16-(S)-3-methylindorapamycin, C16-(S)-3-methylindorapamycin (C16-iRap), AP21967 (A/C heterodimer, Takara Bio®), sodium mycophenolate, benidipine hydrochloride, or hydrochloride), rapamine, AP23573 (Ridaforolimus), AP1903 (Rimiducid), or their metabolites, derivatives and/or combinations.

在一些實施例中,配體包含FK1012 (FK506之半合成二聚體)、他克莫司(tacrolimus) (FK506)、FKCsA (FK506與環孢黴素之複合物)、雷帕黴素、庫馬黴素、赤黴素(gibberellin)、HaXS二聚體(dimerizer) (HaloTag及SNAP-標籤之化學二聚體)、TMP-HTag (曲美普林(trimethoprim)鹵及酶蛋白質二聚體)或ABT-737,或其功能衍生物。In some embodiments, the ligand comprises FK1012 (a semisynthetic dimer of FK506), tacrolimus (FK506), FKCsA (a complex of FK506 and cyclosporin), rapamycin, coumacin, gibberellin, HaXS dimerizer (a chemical dimer of HaloTag and SNAP-tag), TMP-HTag (a dimer of trimethoprim halogen and enzyme protein), or ABT-737, or a functional derivative thereof.

在一些實施例中,非生理學配體以0 nM至1000 nM之量存在或提供,諸如,0.05 nM、0.1 nM、0.5. nM、1.0 nM、5.0 nM、10.0 nM、15.0 nM、20.0 nM、25.0 nM、30.0 nM、35.0 nM、40.0 nM、45.0 nM、50.0 nM、55.0 nM、60.0 nM、65.0 nM、70.0 nM、75.0 nM、80.0 nM、90.0 nM、95.0 nM、100 nM、200 nM、300 nM、400 nM、500 nM、600 nM、700 nM、800 nM、900 nM或1000 nM,或任何兩個前述量所限定之範圍內的量。In some embodiments, the non-physiological ligand is present or provided in an amount of 0 nM to 1000 nM, e.g., 0.05 nM, 0.1 nM, 0.5. nM, 1.0 nM, 5.0 nM, 10.0 nM, 15.0 nM, 20.0 nM, 25.0 nM, 30.0 nM, 35.0 nM, 40.0 nM, 45.0 nM, 50.0 nM, 55.0 nM, 60.0 nM, 65.0 nM, 70.0 nM, 75.0 nM, 80.0 nM, 90.0 nM, 95.0 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM. nM, or an amount within the range defined by any two of the foregoing amounts.

在一些實施例中,非生理學配體為AP21967,且以10 nM存在或提供。在一些實施例中,非生理學配體為AP21967,且以20 nM存在或提供。在一些實施例中,非生理學配體為AP21967,且以50 nM存在或提供。在一些實施例中,非生理學配體為AP21967,且以100 nM存在或提供。In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 100 nM.

在一些實施例中,非生理學配體為雷帕黴素,且以1 nM存在或提供。在一些實施例中,非生理學配體為雷帕黴素,且以10 nM存在或提供。在一些實施例中,非生理學配體為雷帕黴素,且以20 nM存在或提供。在一些實施例中,非生理學配體為雷帕黴素,且以50 nM存在或提供。In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 50 nM.

在一些實施例中,非生理學配體為雷帕黴素類似物,且以1 nM存在或提供。在一些實施例中,非生理學配體為雷帕黴素類似物,且以10 nM存在或提供。在一些實施例中,非生理學配體為雷帕黴素類似物,且以20 nM存在或提供。在一些實施例中,非生理學配體為雷帕黴素類似物,且以50 nM存在或提供。在一些實施例中,非生理學配體為雷帕黴素類似物,且以100 nM存在或提供。In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 100 nM.

在一些實施例中,非生理學配體係以1 nM存在或提供。In some embodiments, the non-physiological ligand is present or provided at 1 nM.

在一些實施例中,非生理學配體係以10 nM存在或提供。In some embodiments, the non-physiological ligand is present or provided at 10 nM.

在一些實施例中,非生理學配體係以100 nM存在或提供。In some embodiments, the non-physiological ligand is present or provided at 100 nM.

在一些實施例中,非生理學配體係以1000 nM存在或提供。 V.定義 In some embodiments, the non-physiological ligand is present or provided at 1000 nM. V. Definitions

除非另有定義,否則本文所用之所有技術術語、標記法及其他技術及科學術語意欲具有與一般熟習所主張主題所屬技術者通常所理解相同的含義。在一些情況下,為了清楚及/或便於參考,本文定義具有通常所理解之含義的術語,且本文中包括此類定義不應必然解釋為表示與此項技術中一般理解之內容存在實質性差異。Unless otherwise defined, all technical terms, notations, and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from what is generally understood in the art.

如本文中所使用,除非上下文另外指示,否則單數形式「一(a)」、「一(an)」及「該(the)」亦包括複數形式。連詞「及/或」表示所列舉物件中之一或多者之所有可能組合。As used herein, the singular forms "a", "an" and "the" include the plural forms as well, unless the context indicates otherwise. The conjunction "and/or" refers to all possible combinations of one or more of the listed items.

如本文所用,術語「約」係指此技術領域中熟習此項技術者易於知曉之各別值的常見誤差範圍。本文中,提及「約」一個值或參數包括(及描述)針對該值或參數本身之實施例。As used herein, the term "about" refers to the common error range of the respective values that is readily known to those skilled in the art. Herein, reference to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter itself.

應理解,本文所描述之本發明之態樣及實施例包括「包含」態樣及實施例、「由」態樣及實施例「組成」及「基本上由」態樣及實施例「組成」。It should be understood that aspects and embodiments of the present invention described herein include "comprising" aspects and embodiments, "consisting of" aspects and embodiments, and "consisting essentially of" aspects and embodiments.

如本文所用,「視情況存在之(optional)」或「視情況(optionally)」意謂隨後描述之事件或情形可能發生或不發生,且該描述包括該事件或情形發生之情況及該事件或情形不發生之情況。舉例而言,視情況經取代之基團意謂該基團未經取代或經取代。As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally substituted group means that the group is either unsubstituted or substituted.

術語「組合物」係指兩種或更多種產物、物質或化合物(包括細胞或抗體)之任何混合物。其可為溶液、懸浮液、液體、粉末、糊劑、水溶液、非水溶液或其任何組合。製劑一般呈諸如允許活性成分(例如抗體)之生物活性有效的形式。The term "composition" refers to any mixture of two or more products, substances or compounds (including cells or antibodies). It can be a solution, suspension, liquid, powder, paste, aqueous solution, non-aqueous solution or any combination thereof. The formulation is generally in a form that allows the biological activity of the active ingredient (e.g., antibody) to be effective.

「醫藥學上可接受之載劑」係指醫藥調配物中之除活性成分外的對個體無毒的成分。醫藥學上可接受之載劑包括但不限於緩衝劑、賦形劑、穩定劑或防腐劑。"Pharmaceutically acceptable carriers" refer to ingredients in pharmaceutical formulations other than active ingredients that are non-toxic to the individual. Pharmaceutically acceptable carriers include but are not limited to buffers, excipients, stabilizers or preservatives.

如本文所用,組合係指兩個或更多個物件之間或當中之任何結合。組合可為兩個或更多個個別物件,諸如兩個組合物或兩個集合,可為其混合物,諸如兩個或更多個物件之單一混合物或其任何變化形式。組合之要素一般在功能上相關聯或有關。As used herein, a combination refers to any association between or among two or more items. A combination can be two or more individual items, such as two compositions or two sets, a mixture thereof, such as a single mixture of two or more items, or any variation thereof. The elements of a combination are generally functionally related or associated.

如本文所用,套組為用於包括但不限於治療用途之目的之經封裝組合,其視情況包括其他要素(諸如額外藥劑)及該組合或其要素的使用說明書。As used herein, a kit is a packaged combination for purposes including but not limited to therapeutic use, which may include other elements (such as additional agents) and instructions for use of the combination or its elements as appropriate.

如本文所用,「個體」係指多順反子構築體或其他藥劑之接受者。該術語包括哺乳動物,諸如靈長類動物、小鼠、大鼠、狗、貓、牛、馬、山羊、駱駝、綿羊或豬,較佳地人類。As used herein, "subject" refers to a recipient of a polycistronic construct or other agent. The term includes mammals, such as primates, mice, rats, dogs, cats, cows, horses, goats, camels, sheep or pigs, preferably humans.

如本文所用,「治療(Treat)」、「治療(treating)」或「治療(treatment)」係指賦予患有疾病或病症之個體益處的任何類型之操作或投與,該益處包括患者之病狀改善(亦即一或多個症狀之改善、減少或減輕,及對於治療之部分或完全反應)。As used herein, "treat," "treating," or "treatment" refers to any type of manipulation or administration that confers a benefit to an individual suffering from a disease or disorder, including improvement in the patient's condition (i.e., improvement, reduction, or alleviation of one or more symptoms, as well as partial or complete response to treatment).

術語「有效量」係指有效產生所需生物化學、細胞或生理反應之量。術語「治療有效量」係指有效引起所要治療作用之療法的量、劑量或劑量方案。如本文所用,「個體(individual)」或「個體(subject)」為哺乳動物。出於治療之目的,「哺乳動物」包括人類、家養動物及農畜,以及動物園、競技或寵物動物,諸如狗、馬、兔、牛、豬、倉鼠、沙鼠、小鼠、雪貂、大鼠、貓等。在一些實施例中,個體(individual/subject)為人類。The term "effective amount" refers to an amount effective to produce a desired biochemical, cellular or physiological response. The term "therapeutically effective amount" refers to an amount, dosage or dosage regimen of a treatment that is effective to cause the desired therapeutic effect. As used herein, an "individual" or "subject" is a mammal. For therapeutic purposes, "mammals" include humans, domestic animals and farm animals, as well as zoo, competitive or pet animals, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual/subject is a human.

如本文所用,「多核苷酸」係指由經由一個核苷酸之磷醯基與鏈中下一核苷酸之糖組分之羥基之間的酯鍵共價鍵結的兩個或更多個核苷酸單體構成之生物聚合物。DNA及RNA為多核苷酸之非限制性實例。As used herein, "polynucleotide" refers to a biopolymer composed of two or more nucleotide monomers covalently bonded via an ester bond between the phosphoyl group of one nucleotide and the hydroxyl group of the sugar component of the next nucleotide in the chain. DNA and RNA are non-limiting examples of polynucleotides.

如本文所用,「多肽」係指由藉由肽鍵鏈接在一起,從而形成蛋白質之一部分(或全部)的胺基酸殘基組成之聚合物。As used herein, "polypeptide" refers to a polymer composed of amino acid residues linked together by peptide bonds to form part (or all) of a protein.

熟習此項技術者應理解,由於遺傳密碼之簡併,大量不同多核苷酸及核酸可編碼相同的多肽。另外,應理解,熟習此項技術者可使用常規技術進行不影響由本文所描述之多核苷酸編碼之多肽序列的核苷酸取代以反映其中待表現多肽之任何特定宿主生物體之密碼子使用。Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, a large number of different polynucleotides and nucleic acids can encode the same polypeptide. In addition, it will be appreciated that those skilled in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any specific host organism in which the polypeptide is to be expressed.

核酸可包含DNA或RNA。其可為單股或雙股。其亦可為在其內包括合成或經修飾之核苷酸的多核苷酸。對寡核苷酸之多種不同類型之修飾為此項技術中已知的。此等包括甲基磷酸酯及硫代磷酸酯主鏈、在分子之3'及/或5'端添加吖啶或聚離胺酸鏈。出於本文所描述之用途之目的,應瞭解,多核苷酸可藉由此項技術中可獲得之任何方法修飾。可進行此類修飾以便增強所關注多核苷酸之活體內活性或壽命。Nucleic acids may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides that include synthetic or modified nucleotides therein. A variety of different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the uses described herein, it will be understood that polynucleotides may be modified by any method available in the art. Such modifications may be performed in order to enhance the in vivo activity or lifespan of the polynucleotide of interest.

術語「變體」意指,與參考多核苷酸或多肽相比,多核苷酸或多肽在其序列中具有至少一個取代、插入或缺失。「功能變體」為保留參考多核苷酸或多肽之一或多個功能的變體。The term "variant" means that a polynucleotide or polypeptide has at least one substitution, insertion or deletion in its sequence compared to a reference polynucleotide or polypeptide. A "functional variant" is a variant that retains one or more functions of a reference polynucleotide or polypeptide.

如本文所用,與多核苷酸或多肽序列有關之術語「序列一致性」或「一致性」係指兩個最佳比對之多核苷酸或多肽序列在參考序列之全長中比對中之各位置處的匹配程度。「一致性百分比」為最佳比對中之匹配位置數除以參考序列之長度加上比對中之參考序列中之任何間隙之長度的總和。最佳比對為產生最大一致性百分比之比對。用於測定一致性百分比之序列的比對可藉由多種熟知方法來完成,包括例如藉由使用數學演算法,諸如BLAST程式組或Clustal Omega序列分析程式中之彼等者。除非另外提及,否則申請專利範圍中之術語「序列一致性」係指藉由2.12.0版BLAST,使用預設參數計算之序列一致性。且除非另外提及,否則比對為在參考序列之整個全長中所關注多核苷酸或多肽序列之全部或一部分的比對。As used herein, the term "sequence identity" or "identity" in relation to polynucleotide or polypeptide sequences refers to the degree of match between two optimally aligned polynucleotide or polypeptide sequences at each position in the alignment over the full length of a reference sequence. "Percent identity" is the sum of the number of matching positions in the optimal alignment divided by the length of the reference sequence plus the length of any gaps in the reference sequence in the alignment. The optimal alignment is the alignment that produces the greatest percent identity. Alignment of sequences for determining percent identity can be accomplished by a variety of well-known methods, including, for example, by using mathematical algorithms, such as those in the BLAST suite of programs or the Clustal Omega sequence analysis program. Unless otherwise mentioned, the term "sequence identity" in the scope of the patent application refers to sequence identity calculated by BLAST version 2.12.0 using default parameters. And unless otherwise stated, an alignment is an alignment of all or a portion of a polynucleotide or polypeptide sequence of interest over the entire length of a reference sequence.

如本文所用,「小分子」係指低分子量(<1000道爾頓)有機化合物。小分子可結合特定生物學大分子,且可具有各種生物學功能或應用,包含但不限於充當細胞傳訊分子、藥物、次級代謝物或各種其他作用模式。As used herein, "small molecule" refers to low molecular weight (<1000 Dalton) organic compounds. Small molecules can bind to specific biological macromolecules and can have various biological functions or applications, including but not limited to acting as cell signaling molecules, drugs, secondary metabolites or various other modes of action.

與小分子有關之術語「類似物」係指具有與另一化合物類似的結構及/或功能但關於某一組分而言與其不同的化合物。類似物可在一或多個原子、官能基或子結構方面不同,其經其他原子、基團或子結構置換。儘管較高的結構及/或功能相似性,但類似物可具有不同的物理、化學、生理化學、生物化學或藥理學特性。The term "analog" in relation to small molecules refers to a compound that has a structure and/or function similar to another compound but differs from it with respect to a certain component. Analogs may differ in one or more atoms, functional groups or substructures, which are replaced by other atoms, groups or substructures. Despite the high structural and/or functional similarity, analogs may have different physical, chemical, physiochemical, biochemical or pharmacological properties.

術語「雷帕黴素類似物」為此項技術中公認的與雷帕黴素共用結構及功能相似性之一組雷帕黴素類似物。已知某些雷帕黴素類似物共用雷帕黴素之一些但並非所有的功能屬性。舉例而言,一些雷帕黴素類似物適合於用作非生理學配體,此係因為其促進二聚化但實質上不具有免疫抑制活性(例如AP21967、AP23102或iRAP)。The term "rapamycin analogs" is a group of rapamycin analogs recognized in the art that share structural and functional similarities with rapamycin. Some rapamycin analogs are known to share some, but not all, of the functional properties of rapamycin. For example, some rapamycin analogs are suitable for use as non-physiological ligands because they promote dimerization but have substantially no immunosuppressive activity (e.g., AP21967, AP23102, or iRAP).

本發明之說明性雷帕黴素類似物為AP21967 The illustrative rapamycin analog of the present invention is AP21967 .

本發明之說明性雷帕黴素類似物為AP23102 The illustrative rapamycin analog of the present invention is AP23102 .

本發明之說明性雷帕黴素類似物為iRAP The illustrative rapamycin analog of the present invention is iRAP .

術語「細胞群體」係指懸浮於溶液中、附著於基質或儲存於容器中之細胞的混合物。可利用對具有複數個細胞之樣品體積進行批量量測來研究作為整體之細胞群體的特徵。流式細胞分析技術方法可用以減少在批量細胞群體量測中遭遇之背景螢光的問題。The term "cell population" refers to a mixture of cells suspended in solution, attached to a matrix, or stored in a container. Batch measurements of sample volumes with multiple cells can be used to study the characteristics of the cell population as a whole. Flow cytometry techniques can be used to reduce the problem of background fluorescence encountered in batch cell population measurements.

如本文所用,術語「經工程改造」係指細胞已經異源多核苷酸穩定轉導或經歷基因編輯以引入、缺失或修飾細胞中之多核苷酸,或以在細胞中產生穩定表型變化之方式細胞暫時經多核苷酸轉導。 VI.例示性實施例 As used herein, the term "engineered" refers to cells that have been stably transduced with heterologous polynucleotides or have undergone gene editing to introduce, delete, or modify a polynucleotide in the cell, or cells that have been transiently transduced with a polynucleotide in a manner that produces a stable phenotypic change in the cell. VI. Exemplary Examples

所提供之實施例包括: 1. 一種多順反子構築體,其包含以5'至3'順序:(a)第一表現卡匣,其包含編碼FRB之核苷酸序列;(b)第二表現卡匣,其包含編碼合成細胞介素γ鏈多肽之核苷酸序列;(c)第三表現卡匣,其包含編碼合成細胞介素β鏈多肽之核苷酸序列;及(d)第四表現卡匣,其包含編碼嵌合抗原受體(CAR)之核苷酸序列,其中該等表現卡匣各藉由編碼裂解位點序列之核苷酸序列分隔開。 2. 如實施例1之構築體,其中編碼該FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。 3. 如實施例1或實施例2之構築體,其中編碼該FRB之核苷酸序列包含SEQ ID NO: 3、13或50的核苷酸序列。 4. 如實施例1至3中任一項之構築體,其中該FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 5. 如實施例1至4中任一項之構築體,其中該FRB包含SEQ ID NO: 4、14或51的胺基酸序列。 6. 如實施例1至5中任一項之構築體,其中編碼該合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。 7. 如實施例1至6中任一項之構築體,其中編碼該合成細胞介素γ鏈多肽之核苷酸包含SEQ ID NO: 15的核苷酸序列。 8. 如實施例1至7中任一項之構築體,其中該合成細胞介素γ鏈多肽包含介白素2受體次單元γ (IL2RG)。 9. 如實施例8之構築體,其中該IL2RG包含與SEQ ID NO: 16之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 10. 如實施例8或實施例9之構築體,其中該IL2RG包含SEQ ID NO: 16的胺基酸序列。 11. 如實施例1至10中任一項之構築體,其中該第二表現卡匣進一步包含編碼FRB的核苷酸序列。 12. 如實施例11之構築體,其中編碼該FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。 13. 如實施例11或實施例12之構築體,其中編碼該FRB之核苷酸序列包含SEQ ID NO: 13的核苷酸序列。 14. 如實施例11至13中任一項之構築體,其中該FRB包含與SEQ ID NO: 14之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 15. 如實施例1至14中任一項之構築體,其中該FRB包含SEQ ID NO: 14的胺基酸序列。 16. 如實施例1至15中任一項之構築體,其中該第二表現卡匣經密碼子最佳化。 17. 如實施例1至16中任一項之構築體,其中該第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 18. 如實施例1至17中任一項之構築體,其中該第二表現卡匣包含SEQ ID NO: 11的核苷酸序列。 19. 如實施例1至18中任一項之構築體,其中該第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 20. 如實施例1至19中任一項之構築體,其中該第二表現卡匣編碼包含SEQ ID NO: 12之序列的胺基酸序列。 21. 如實施例1至15中任一項之構築體,其中該第二表現卡匣進一步包含編碼FKBP12的核苷酸序列。 22. 如實施例21之構築體,其中編碼該FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。 23. 如實施例21或實施例22之構築體,其中編碼該FKBP12之核苷酸序列包含SEQ ID NO: 21或55的核苷酸序列。 24. 如實施例21至23中任一項之構築體,其中該FKBP12包含與SEQ ID NO: 22之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 25. 如實施例21至24中任一項之構築體,其中該FKBP12包含SEQ ID NO: 22的胺基酸序列。 26. 如實施例1至25中任一項之構築體,其中編碼該合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。 27. 如實施例1至26中任一項之構築體,其中編碼該合成細胞介素β鏈多肽之核苷酸包含SEQ ID NO: 23或61的核苷酸序列。 28. 如實施例1至27中任一項之構築體,其中該合成細胞介素β鏈多肽包含介白素2受體次單元β (IL2RB)。 29. 如實施例28之構築體,其中該IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 30. 如實施例28或實施例29之構築體,其中該IL2RB包含SEQ ID NO: 24或62的胺基酸序列。 31. 如實施例1至30中任一項之構築體,其中該第三表現卡匣進一步包含編碼FKBP12的核苷酸序列。 32. 如實施例31之構築體,其中編碼該FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。 33. 如實施例31或實施例32之構築體,其中編碼該FKBP12之核苷酸序列包含SEQ ID NO: 21的核苷酸序列。 34. 如實施例31至33中任一項之構築體,其中該FKBP12包含與SEQ ID NO: 20之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 35. 如實施例31至34中任一項之構築體,其中該FKBP12包含SEQ ID NO: 20的胺基酸序列。 36. 如實施例1至35中任一項之構築體,其中該第三表現卡匣經密碼子最佳化。 37. 如實施例1至36中任一項之構築體,其中該第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 38. 如實施例1至37中任一項之構築體,其中該第三表現卡匣包含SEQ ID NO: 19的核苷酸序列。 39. 如實施例1至38中任一項之構築體,其中該第三表現卡匣編碼與SEQ ID NO: 20之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 40. 如實施例1至39中任一項之構築體,其中該第三表現卡匣編碼包含SEQ ID NO: 20之序列的胺基酸序列。 41. 如實施例1至40中任一項之構築體,其中該第三表現卡匣進一步包含編碼FRB的核苷酸序列。 42. 如實施例41之構築體,其中編碼該FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。 43. 如實施例41或實施例42之構築體,其中編碼該FRB之核苷酸序列包含SEQ ID NO: 13的核苷酸序列。 44. 如實施例41至43中任一項之構築體,其中該FRB包含與SEQ ID NO: 14之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 45. 如實施例1至44中任一項之構築體,其中該FRB包含SEQ ID NO: 14的胺基酸序列。 46. 如實施例1至45中任一項之構築體,其中該CAR包含scFv域。 47. 如實施例46之構築體,其中該scFv域包含抗螢光異硫氰酸鹽(FITC) E2。 48. 如實施例46或實施例47之構築體,其中該scFv域包含輕鏈可變域(VL)、連接子及重鏈可變域(VH)。 49. 如實施例48之構築體,其中該scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 50. 如實施例48或實施例49之構築體,其中該scFv VL包含SEQ ID NO: 30或65的核苷酸序列。 51. 如實施例48至50中任一項之構築體,其中該scFv VL包含與SEQ ID NO: 31之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 52. 如實施例48至51中任一項之構築體,其中該scFv VL包含SEQ ID NO: 31的胺基酸序列。 53. 如實施例48至52中任一項之構築體,其中該scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 54. 如實施例48至53中任一項之構築體,其中該scFv VH包含SEQ ID NO: 34或67的核苷酸序列。 55. 如實施例48至54中任一項之構築體,其中該scFv VH包含與SEQ ID NO: 35之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 56. 如實施例48至55中任一項之構築體,其中該scFv VH包含SEQ ID NO: 35的胺基酸序列。 57. 如實施例48至56中任一項之構築體,其中該scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 58. 如實施例48至57中任一項之構築體,其中該scFv連接子包含SEQ ID NO: 32或66的核苷酸序列。 59. 如實施例48至58中任一項之構築體,其中該scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 60. 如實施例48至59中任一項之構築體,其中該scFv連接子包含SEQ ID NO: 33的胺基酸序列。 61. 如實施例46至60中任一項之構築體,其中該scFv包含與SEQ ID NO: 28或64之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 62. 如實施例46至49中任一項之構築體,其中該scFv包含SEQ ID NO: 28或64的核苷酸序列。 63. 如實施例46至62中任一項之構築體,其中該scFv包含與SEQ ID NO: 29之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 64. 如實施例46至63中任一項之構築體,其中該scFv包含SEQ ID NO: 29的胺基酸序列。 65. 如實施例1至64中任一項之構築體,其中該CAR包含鉸鏈域。 66. 如實施例65之構築體,其中該鉸鏈域包含短鉸鏈或中等鉸鏈域。 67. 如實施例65或66之構築體,其中該鉸鏈域包含CD8或IgG。 68. 如實施例67之構築體,其中該CD8鉸鏈包含CD8α鉸鏈。 69. 如實施例68之構築體,其中該CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 70. 如實施例68或實施例69之構築體,其中該CD8α鉸鏈包含SEQ ID NO: 38或114的核苷酸序列。 71. 如實施例68至70中任一項之構築體,其中該CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 72. 如實施例68至71中任一項之構築體,其中該CD8α鉸鏈包含SEQ ID NO: 39或115的胺基酸序列。 73. 如實施例1至72中任一項之構築體,其中該CAR包含跨膜域。 74. 如實施例73之構築體,其中該跨膜域包含CD8或CD28。 75. 如實施例74之構築體,其中該CD8跨膜域包含CD8α跨膜域。 76. 如實施例73至75中任一項之構築體,其中該跨膜域包含與SEQ ID NO: 40之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 77. 如實施例73至76中任一項之構築體,其中該跨膜域包含SEQ ID NO: 40的核苷酸序列。 78. 如實施例73至77中任一項之構築體,其中該跨膜域包含與SEQ ID NO: 41之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 79. 如實施例73至78中任一項之構築體,其中該跨膜域包含SEQ ID NO: 41的胺基酸序列。 80. 如實施例1至79中任一項之構築體,其中該CAR包含胞內域。 81. 如實施例80之構築體,其中該胞內域包含共刺激分子。 82. 如實施例80或實施例81之構築體,其中該胞內域包含4-1BB、CD3ζ及/或CD28。 83. 如實施例82之構築體,其中該4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 84. 如實施例82或實施例83之構築體,其中該4-1BB胞內域包含SEQ ID NO: 42或69的核苷酸序列。 85. 如實施例82至84中任一項之構築體,其中該4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 86. 如實施例82至85中任一項之構築體,其中該4-1BB胞內域包含SEQ ID NO: 43的胺基酸序列。 87. 如實施例82至86中任一項之構築體,其中該CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 88. 如實施例82至87中任一項之構築體,其中該CD3ζ胞內域包含SEQ ID NO: 46、70、100或118的核苷酸序列。 89. 如實施例82至88中任一項之構築體,其中該CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 90. 如實施例82至89中任一項之構築體,其中該CD3ζ胞內域包含SEQ ID NO: 47的胺基酸序列。 91. 如實施例1至90中任一項之構築體,其中該第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 92. 如實施例1至91中任一項之構築體,其中該第四表現卡匣包含SEQ ID NO: 26、63、71或82的核苷酸序列。 93. 如實施例1至92中任一項之構築體,其中該第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 94. 如實施例1至93中任一項之構築體,其中該第四表現卡匣編碼SEQ ID NO: 27、72或127的胺基酸序列。 95. 如實施例1之構築體,其中該等裂解位點序列各包含2A可裂解連接子序列。 96. 如實施例95之構築體,其中編碼2A可裂解連接子序列之各核苷酸不同。 97. 如實施例95或實施例96之構築體,其中該2A可裂解連接子獨立地為T2A、P2A、E2A或F2A裂解位點。 98. 如實施例95或97中任一項之構築體,其中該2A可裂解連接子獨立地為P2A或T2A。 99. 如實施例95至98中任一項之構築體,其中至少一個2A可裂解連接子為P2A,且編碼該P2A可裂解連接子之核苷酸序列包含與SEQ ID NO: 17、25、52或58至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。 100. 如實施例99之構築體,其中編碼該P2A可裂解連接子之核苷酸序列述於SEQ ID NO: 17、25、52或58中。 101. 如實施例97至100中任一項之構築體,其中該P2A可裂解連接子包含與SEQ ID NO: 18至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。 102. 如實施例101之構築體,其中該P2A可裂解連接子包含SEQ ID NO: 18中所述的序列。 103. 如實施例95至102中任一項之構築體,其中至少一個2A可裂解連接子為T2A,且編碼該T2A可裂解連接子之核苷酸序列包含與SEQ ID NO: 9至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。 104. 如實施例95至103之構築體,其中編碼該T2A可裂解連接子之核苷酸序列述於SEQ ID NO: 9中。 105. 如實施例97、98、103及104中任一項之構築體,其中該T2A可裂解連接子包含與SEQ ID NO: 10至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。 106. 如實施例95至105之構築體,其中該T2A可裂解連接子包含SEQ ID NO: 10中所述之序列。 107. 如實施例1至106中任一項之構築體,其中該等裂解位點序列中之至少一者包含弗林蛋白酶(furin)裂解位點序列。 108. 如實施例107之構築體,其中該弗林蛋白酶裂解位點序列位於該第一表現卡匣與該第二表現卡匣之間。 109. 如實施例107或實施例108之構築體,其中編碼該弗林蛋白酶裂解位點序列之核苷酸序列包含與SEQ ID NO: 7至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。 110. 如實施例107至109中任一項之構築體,其中編碼該弗林蛋白酶裂解位點序列之核苷酸序列包含SEQ ID NO: 7中所述的序列。 111. 如實施例107至110中任一項之構築體,其中該弗林蛋白酶裂解位點序列包含與SEQ ID NO: 8之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 112. 如實施例106至111中任一項之構築體,其中該弗林蛋白酶裂解位點序列包含SEQ ID NO: 8的胺基酸序列。 113. 如實施例1至112中任一項之構築體,其中該裂解位點序列包含弗林蛋白酶裂解位點序列及T2A裂解序列(furinT2A)。 114. 如實施例1至113中任一項之構築體,其中編碼該裂解位點序列之核苷酸序列與SEQ ID NO: 5之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。 115. 如實施例1至114中任一項之構築體,其中編碼該裂解位點序列之核苷酸序列包含SEQ ID NO: 5的核苷酸序列。 116. 如實施例1至115中任一項之構築體,其中該裂解位點序列包含與SEQ ID NO: 6之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。 117. 如實施例1至116中任一項之構築體,其中該裂解位點序列包含SEQ ID NO: 6的胺基酸序列。 118. 如實施例1至109中任一項之構築體,其中該第一表現卡匣及該第二表現卡匣藉由furinT2A分隔開,該第二表現卡匣及該第三表現卡匣藉由P2A分隔開,且該第三表現卡匣及該第四表現卡匣藉由P2A分隔開。 119. 如實施例1至118中任一項之構築體,其中該構築體包含與SEQ ID NO: 1之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 120. 如實施例1至119中任一項之構築體,其中該構築體包含SEQ ID NO: 1的核苷酸序列。 121. 如實施例1至120中任一項之構築體,其中該構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致之胺基酸序列的多肽。 122. 如實施例1至121中任一項之構築體,其中該構築體編碼包含SEQ ID NO: 2之胺基酸序列的多肽。 123. 如實施例1至118中任一項之構築體,其中該構築體包含與SEQ ID NO: 48之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。 124. 如實施例1至118或123中任一項之構築體,其中該構築體包含SEQ ID NO: 48的核苷酸序列。 125. 如實施例1至118、123或124中任一項之構築體,其中該構築體編碼包含與SEQ ID NO: 49之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致之胺基酸序列的多肽。 126. 如實施例1至118或123至125中任一項之構築體,其中該構築體編碼包含SEQ ID NO: 49之胺基酸序列的多肽。 127. 一種病毒載體,其包含如實施例1至126中任一項之多順反子構築體。 128. 如實施例127之病毒載體,其中該病毒載體為慢病毒載體。 129. 如實施例127或實施例128之病毒載體,其中該病毒載體進一步包含一或多種表面T細胞活化劑。 130. 如實施例129之病毒載體,其中該一或多種表面T細胞活化劑包含CD58、抗-CD3,或CD80。 131. 一種細胞,其包含如實施例127至130中任一項之病毒載體。 132. 如實施例131之細胞,其中該細胞包含幹細胞或前驅細胞。 133. 如實施例132之細胞,其中該幹細胞包含誘導性富潛能幹細胞(iPSC)。 134. 如實施例131之細胞,其中該前驅細胞包含周邊血液單核細胞(PBMC)。 135. 如實施例131之細胞,其中該細胞包含T細胞。 136. 如實施例131之細胞,其中該細胞包含細胞毒性先天性淋巴球(CIL)細胞。 137. 如實施例131之細胞,其中該細胞包含自然殺手(NK)細胞。 138. 一種轉導細胞之方法,其包含使目標細胞與如實施例1至126中任一項之多順反子構築體中之任一者接觸。 139. 如實施例138之方法,其中該目標細胞包含幹細胞。 140. 如實施例139之方法,其中該幹細胞包含誘導性富潛能幹細胞(iPSC)。 141. 如實施例138之方法,其中該目標細胞包含前驅細胞。 142. 如實施例141之方法,其中該前驅細胞包含周邊血液單核細胞(PBMC)。 143. 如實施例138之方法,其中該目標細胞包含T細胞。 144. 如實施例143之方法,其中該T細胞包含CD4+或CD8+ T細胞。 145. 如實施例138至144中任一項之方法,其進一步包含使該目標細胞與以下接觸:(i)靶向內源基因中之目標位點的引導RNA(gRNA),及(ii) RNA引導核酸內切酶,從而將該核苷酸序列插入該內源基因中。 146. 一種在目標細胞中表現嵌合抗原受體及/或合成細胞介素受體之方法。 147. 如實施例146之方法,其中該目標細胞包含幹細胞。 148. 如實施例147之方法,其中該幹細胞包含誘導性富潛能幹細胞(iPSC)。 149. 如實施例146之方法,其中該目標細胞包含前驅細胞。 150. 如實施例149之方法,其中該前驅細胞包含周邊血液單核細胞(PBMC)。 151. 如實施例146之方法,其中該目標細胞包含T細胞。 152. 如實施例151之方法,其中該T細胞包含CD4+或CD8+ T細胞。 153. 如實施例146至152中任一項之方法,其中該方法係離體或在活體外進行。 154. 如實施例146至152中任一項之方法,其中該方法係在活體內進行。 155. 一種細胞,其係藉由如實施例138至154中任一項之方法產生。 156. 一種向個體投與如實施例155之細胞之方法。 157. 一種向個體投與如實施例127至130中任一項之病毒載體之方法。 158. 如實施例157之方法,其進一步包含投與雙官能配體以標記該個體中之癌細胞,其中該雙官能配體特異性結合腫瘤上表現之分子。 159. 如實施例158之方法,其中該雙功能配體包含FITC-葉酸。 160. 如實施例157至159中任一項之方法,其進一步包含投與非生理學配體。 161. 如實施例160之方法,其中該非生理學配體包含雷帕黴素或雷帕黴素類似物。 VII.實例 The embodiments provided include: 1. A polycistronic construct comprising, in 5' to 3' order: (a) a first expression cassette comprising a nucleotide sequence encoding FRB; (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic interleukin γ chain polypeptide; (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic interleukin β chain polypeptide; and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence. 2. The construct of embodiment 1, wherein the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 3, 13 or 50. 3. The construct of embodiment 1 or embodiment 2, wherein the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 3, 13 or 50. 4. The construct of any one of embodiments 1 to 3, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4, 14 or 51. 5. The construct of any one of embodiments 1 to 4, wherein the FRB comprises an amino acid sequence of SEQ ID NO: 4, 14 or 51. 6. The construct of any one of embodiments 1 to 5, wherein the nucleotide sequence encoding the synthetic interleukin gamma chain polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 15. 7. The construct of any one of embodiments 1 to 6, wherein the nucleotide encoding the synthetic interleukin gamma chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 15. 8. The construct of any one of embodiments 1 to 7, wherein the synthetic interleukin gamma chain polypeptide comprises interleukin 2 receptor subunit gamma (IL2RG). 9. The construct of embodiment 8, wherein the IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16. 10. The construct of embodiment 8 or embodiment 9, wherein the IL2RG comprises the amino acid sequence of SEQ ID NO: 16. 11. The construct of any one of embodiments 1 to 10, wherein the second expression cassette further comprises a nucleotide sequence encoding FRB. 12. The construct of embodiment 11, wherein the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 13. 13. The construct of embodiment 11 or embodiment 12, wherein the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 13. 14. The construct of any one of embodiments 11 to 13, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. 15. The construct of any one of embodiments 1 to 14, wherein the FRB comprises the amino acid sequence of SEQ ID NO: 14. 16. The construct of any one of embodiments 1 to 15, wherein the second expression cassette is codon optimized. 17. The construct of any one of embodiments 1 to 16, wherein the second expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 11. 18. The construct of any one of embodiments 1 to 17, wherein the second expression cassette comprises the nucleotide sequence of SEQ ID NO: 11. 19. The construct of any one of embodiments 1 to 18, wherein the second expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 12. 20. The construct of any one of embodiments 1 to 19, wherein the second expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 12. 21. The construct of any one of embodiments 1 to 15, wherein the second expression cassette further comprises a nucleotide sequence encoding FKBP12. 22. The construct of embodiment 21, wherein the nucleotide sequence encoding the FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. 23. The construct of embodiment 21 or embodiment 22, wherein the nucleotide sequence encoding the FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21 or 55. 24. The construct of any one of embodiments 21 to 23, wherein the FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22. 25. The construct of any one of embodiments 21 to 24, wherein the FKBP12 comprises the amino acid sequence of SEQ ID NO: 22. 26. The construct of any one of embodiments 1 to 25, wherein the nucleotides encoding the synthetic interleukin β chain polypeptide are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. 27. The construct of any one of embodiments 1 to 26, wherein the nucleotide encoding the synthetic interleukin beta chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 23 or 61. 28. The construct of any one of embodiments 1 to 27, wherein the synthetic interleukin beta chain polypeptide comprises interleukin 2 receptor subunit beta (IL2RB). 29. The construct of embodiment 28, wherein the IL2RB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24 or 62. 30. The construct of embodiment 28 or embodiment 29, wherein the IL2RB comprises the amino acid sequence of SEQ ID NO: 24 or 62. 31. The construct of any one of embodiments 1 to 30, wherein the third expression cassette further comprises a nucleotide sequence encoding FKBP12. 32. The construct of embodiment 31, wherein the nucleotide sequence encoding the FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 21. 33. The construct of embodiment 31 or embodiment 32, wherein the nucleotide sequence encoding the FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21. 34. The construct of any one of embodiments 31 to 33, wherein the FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20. 35. The construct of any one of embodiments 31 to 34, wherein the FKBP12 comprises the amino acid sequence of SEQ ID NO: 20. 36. The construct of any one of embodiments 1 to 35, wherein the third expression cassette is codon optimized. 37. The construct of any one of embodiments 1 to 36, wherein the third expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 19. 38. The construct of any one of embodiments 1 to 37, wherein the third expression cassette comprises the nucleotide sequence of SEQ ID NO: 19. 39. The construct of any one of embodiments 1 to 38, wherein the third expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20. 40. The construct of any one of embodiments 1 to 39, wherein the third expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 20. 41. The construct of any one of embodiments 1 to 40, wherein the third expression cassette further comprises a nucleotide sequence encoding FRB. 42. The construct of embodiment 41, wherein the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 13. 43. The construct of embodiment 41 or embodiment 42, wherein the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 13. 44. The construct of any one of embodiments 41 to 43, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. 45. The construct of any one of embodiments 1 to 44, wherein the FRB comprises the amino acid sequence of SEQ ID NO: 14. 46. The construct of any one of embodiments 1 to 45, wherein the CAR comprises a scFv domain. 47. The construct of Example 46, wherein the scFv domain comprises anti-fluorescent isothiocyanate (FITC) E2. 48. The construct of Example 46 or Example 47, wherein the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH). 49. The construct of Example 48, wherein the scFv VL comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. 50. The construct of Example 48 or Example 49, wherein the scFv VL comprises the nucleotide sequence of SEQ ID NO: 30 or 65. 51. The construct of any one of embodiments 48 to 50, wherein the scFv VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31. 52. The construct of any one of embodiments 48 to 51, wherein the scFv VL comprises an amino acid sequence of SEQ ID NO: 31. 53. The construct of any one of embodiments 48 to 52, wherein the scFv VH comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. 54. The construct of any one of embodiments 48 to 53, wherein the scFv VH comprises a nucleotide sequence of SEQ ID NO: 34 or 67. 55. The construct of any one of embodiments 48 to 54, wherein the scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35. 56. The construct of any one of embodiments 48 to 55, wherein the scFv VH comprises an amino acid sequence of SEQ ID NO: 35. 57. The construct of any one of embodiments 48 to 56, wherein the scFv linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. 58. The construct of any one of embodiments 48 to 57, wherein the scFv linker comprises a nucleotide sequence of SEQ ID NO: 32 or 66. 59. The construct of any one of embodiments 48 to 58, wherein the scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 33. 60. The construct of any one of embodiments 48 to 59, wherein the scFv linker comprises an amino acid sequence of SEQ ID NO: 33. 61. The construct of any one of embodiments 46 to 60, wherein the scFv comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. 62. The construct of any one of embodiments 46 to 49, wherein the scFv comprises a nucleotide sequence of SEQ ID NO: 28 or 64. 63. The construct of any one of embodiments 46 to 62, wherein the scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29. 64. The construct of any one of embodiments 46 to 63, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 29. 65. The construct of any one of embodiments 1 to 64, wherein the CAR comprises a hinge domain. 66. The construct of embodiment 65, wherein the hinge domain comprises a short hinge or a medium hinge domain. 67. The construct of embodiment 65 or 66, wherein the hinge domain comprises CD8 or IgG. 68. The construct of embodiment 67, wherein the CD8 hinge comprises a CD8 alpha hinge. 69. The construct of embodiment 68, wherein the CD8 alpha hinge comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. 70. The construct of embodiment 68 or embodiment 69, wherein the CD8 alpha hinge comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. 71. The construct of any one of embodiments 68 to 70, wherein the CD8 alpha hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39 or 115. 72. The construct of any one of embodiments 68 to 71, wherein the CD8α hinge comprises the amino acid sequence of SEQ ID NO: 39 or 115. 73. The construct of any one of embodiments 1 to 72, wherein the CAR comprises a transmembrane domain. 74. The construct of embodiment 73, wherein the transmembrane domain comprises CD8 or CD28. 75. The construct of embodiment 74, wherein the CD8 transmembrane domain comprises a CD8α transmembrane domain. 76. The construct of any one of embodiments 73 to 75, wherein the transmembrane domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 40. 77. The construct of any one of embodiments 73 to 76, wherein the transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 40. 78. The construct of any one of embodiments 73 to 77, wherein the transmembrane domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41. 79. The construct of any one of embodiments 73 to 78, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 41. 80. The construct of any one of embodiments 1 to 79, wherein the CAR comprises an intracellular domain. 81. The construct of embodiment 80, wherein the intracellular domain comprises a costimulatory molecule. 82. The construct of embodiment 80 or embodiment 81, wherein the intracellular domain comprises 4-1BB, CD3ζ and/or CD28. 83. The construct of embodiment 82, wherein the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. 84. The construct of embodiment 82 or embodiment 83, wherein the 4-1BB intracellular domain comprises a nucleotide sequence of SEQ ID NO: 42 or 69. 85. The construct of any one of embodiments 82 to 84, wherein the 4-1BB intracellular domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43. 86. The construct of any one of embodiments 82 to 85, wherein the 4-1BB intracellular domain comprises the amino acid sequence of SEQ ID NO: 43. 87. The construct of any one of embodiments 82 to 86, wherein the CD3 zeta intracellular domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. 88. The construct of any one of embodiments 82 to 87, wherein the CD3 zeta intracellular domain comprises the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. 89. The construct of any one of embodiments 82 to 88, wherein the CD3 zeta intracellular domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47. 90. The construct of any one of embodiments 82 to 89, wherein the CD3 zeta intracellular domain comprises an amino acid sequence of SEQ ID NO: 47. 91. The construct of any one of embodiments 1 to 90, wherein the fourth expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82. 92. The construct of any one of embodiments 1 to 91, wherein the fourth expression cassette comprises the nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82. 93. The construct of any one of embodiments 1 to 92, wherein the fourth expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27, 72 or 127. 94. The construct of any one of embodiments 1 to 93, wherein the fourth expression cassette encodes the amino acid sequence of SEQ ID NO: 27, 72 or 127. 95. The construct of embodiment 1, wherein the cleavage site sequences each comprise a 2A cleavable linker sequence. 96. The construct of embodiment 95, wherein each nucleotide encoding the 2A cleavable linker sequence is different. 97. The construct of embodiment 95 or embodiment 96, wherein the 2A cleavable linker is independently a T2A, P2A, E2A or F2A cleavage site. 98. The construct of any one of embodiments 95 or 97, wherein the 2A cleavable linker is independently P2A or T2A. 99. The construct of any one of embodiments 95 to 98, wherein at least one 2A cleavable linker is P2A, and the nucleotide sequence encoding the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17, 25, 52 or 58. 100. The construct of embodiment 99, wherein the nucleotide sequence encoding the P2A cleavable linker is described in SEQ ID NO: 17, 25, 52 or 58. 101. The construct of any one of embodiments 97 to 100, wherein the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18. 102. The construct of embodiment 101, wherein the P2A cleavable linker comprises the sequence described in SEQ ID NO: 18. 103. The construct of any one of embodiments 95 to 102, wherein at least one 2A cleavable linker is T2A, and the nucleotide sequence encoding the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9. 104. The construct of any one of embodiments 95 to 103, wherein the nucleotide sequence encoding the T2A cleavable linker is described in SEQ ID NO: 9. 105. The construct of any one of embodiments 97, 98, 103 and 104, wherein the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10. 106. The construct of embodiments 95 to 105, wherein the T2A cleavable linker comprises the sequence set forth in SEQ ID NO: 10. 107. The construct of any one of embodiments 1 to 106, wherein at least one of the cleavage site sequences comprises a furin cleavage site sequence. 108. The construct of embodiment 107, wherein the furin cleavage site sequence is located between the first expression cassette and the second expression cassette. 109. The construct of embodiment 107 or embodiment 108, wherein the nucleotide sequence encoding the furin cleavage site sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7. 110. The construct of any one of embodiments 107 to 109, wherein the nucleotide sequence encoding the furin cleavage site sequence comprises the sequence set forth in SEQ ID NO: 7. 111. The construct of any one of embodiments 107 to 110, wherein the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8. 112. The construct of any one of embodiments 106 to 111, wherein the furin cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 8. 113. The construct of any one of embodiments 1 to 112, wherein the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furinT2A). 114. The construct of any one of embodiments 1 to 113, wherein the nucleotide sequence encoding the cleavage site sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 5. 115. The construct of any one of embodiments 1 to 114, wherein the nucleotide sequence encoding the cleavage site sequence comprises the nucleotide sequence of SEQ ID NO: 5. 116. The construct of any one of embodiments 1 to 115, wherein the cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 6. 117. The construct of any one of embodiments 1 to 116, wherein the cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 6. 118. The construct of any one of embodiments 1 to 109, wherein the first expression cassette and the second expression cassette are separated by furinT2A, the second expression cassette and the third expression cassette are separated by P2A, and the third expression cassette and the fourth expression cassette are separated by P2A. 119. The construct of any one of embodiments 1 to 118, wherein the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 1. 120. The construct of any one of embodiments 1 to 119, wherein the construct comprises the nucleotide sequence of SEQ ID NO: 1. 121. The construct of any one of embodiments 1 to 120, wherein the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2. 122. The construct of any one of embodiments 1 to 121, wherein the construct encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 2. 123. The construct of any one of embodiments 1 to 118, wherein the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 48. 124. The construct of any one of embodiments 1 to 118 or 123, wherein the construct comprises the nucleotide sequence of SEQ ID NO: 48. 125. The construct of any one of embodiments 1 to 118, 123 or 124, wherein the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 49. 126. The construct of any one of embodiments 1 to 118 or 123 to 125, wherein the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 49. 127. A viral vector comprising the polycistronic construct of any one of embodiments 1 to 126. 128. The viral vector of embodiment 127, wherein the viral vector is a lentiviral vector. 129. The viral vector of Example 127 or Example 128, wherein the viral vector further comprises one or more surface T cell activators. 130. The viral vector of Example 129, wherein the one or more surface T cell activators comprise CD58, anti-CD3, or CD80. 131. A cell comprising the viral vector of any one of Examples 127 to 130. 132. The cell of Example 131, wherein the cell comprises a stem cell or a progenitor cell. 133. The cell of Example 132, wherein the stem cell comprises an induced enriched-potential stem cell (iPSC). 134. The cell of embodiment 131, wherein the progenitor cell comprises a peripheral blood mononuclear cell (PBMC). 135. The cell of embodiment 131, wherein the cell comprises a T cell. 136. The cell of embodiment 131, wherein the cell comprises a cytotoxic innate lymphocyte (CIL) cell. 137. The cell of embodiment 131, wherein the cell comprises a natural killer (NK) cell. 138. A method of transducing a cell, comprising contacting a target cell with any of the polycistronic constructs of any of embodiments 1 to 126. 139. The method of embodiment 138, wherein the target cell comprises a stem cell. 140. The method of Example 139, wherein the stem cells comprise induced enriched-potential stem cells (iPSCs). 141. The method of Example 138, wherein the target cells comprise progenitor cells. 142. The method of Example 141, wherein the progenitor cells comprise peripheral blood mononuclear cells (PBMCs). 143. The method of Example 138, wherein the target cells comprise T cells. 144. The method of Example 143, wherein the T cells comprise CD4+ or CD8+ T cells. 145. The method of any one of embodiments 138 to 144, further comprising contacting the target cell with: (i) a guide RNA (gRNA) that targets a target site in an endogenous gene, and (ii) an RNA-guided endonuclease to insert the nucleotide sequence into the endogenous gene. 146. A method for expressing a chimeric antigen receptor and/or a synthetic interleukin receptor in a target cell. 147. The method of embodiment 146, wherein the target cell comprises a stem cell. 148. The method of embodiment 147, wherein the stem cell comprises an induced enriched-potential stem cell (iPSC). 149. The method of embodiment 146, wherein the target cell comprises a progenitor cell. 150. The method of embodiment 149, wherein the progenitor cells comprise peripheral blood mononuclear cells (PBMCs). 151. The method of embodiment 146, wherein the target cells comprise T cells. 152. The method of embodiment 151, wherein the T cells comprise CD4+ or CD8+ T cells. 153. The method of any one of embodiments 146 to 152, wherein the method is performed ex vivo or in vitro. 154. The method of any one of embodiments 146 to 152, wherein the method is performed in vivo. 155. A cell produced by the method of any one of embodiments 138 to 154. 156. A method of administering the cell of embodiment 155 to an individual. 157. A method of administering to a subject a viral vector as in any one of Examples 127 to 130. 158. The method as in Example 157, further comprising administering a bifunctional ligand to label cancer cells in the subject, wherein the bifunctional ligand specifically binds to a molecule expressed on a tumor. 159. The method as in Example 158, wherein the bifunctional ligand comprises FITC-folate. 160. The method as in any one of Examples 157 to 159, further comprising administering a non-physiological ligand. 161. The method as in Example 160, wherein the non-physiological ligand comprises rapamycin or a rapamycin analog. VII. Examples

以下實例僅出於說明性目的而包括在內且不意欲限制本發明之範疇。 實例1 RACR-TagCAR多核苷酸多順反子構築體之產生及評定 The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention. Example 1 Generation and evaluation of RACR-TagCAR polynucleotide polycistronic constructs

此實例描述多順反子構築體之產生及其RACR及CAR表現含量之評估以便說明載體取向對於TagCAR活性的影響。This example describes the generation of a polycistronic construct and the assessment of its RACR and CAR expression levels to illustrate the effect of vector orientation on TagCAR activity.

E1 2A 至圖 2B中所示,產生一系列8種多順反子構築體。在所有構築體中,TagCAR之多核苷酸編碼以N端至C端順序具有以下組分之CAR:scFv (例如抗FITC E2)、鉸鏈(間隔子)、跨膜域及具有共刺激傳訊域與CD3ζ傳訊域(Z)之胞內域。構築體在以下方面不同:鉸鏈(間隔子)域,為IgG4鉸鏈(IgG4H)或CD8α鉸鏈(CD8H);跨膜域,為CD28 TM或CD8 TM;及共刺激域,為41BB共刺激域或CD28共刺激域。構築體亦在TagCAR之位置方面不同,該TagCAR存在作為構築體轉殖基因序列之前端或末端。構築體之個別多核苷酸組分藉由2A裂解位點序列分隔開。 As shown in Table E1 and Figures 2A to 2B , a series of 8 polycistronic constructs were generated. In all constructs, the polynucleotide of TagCAR encodes a CAR having the following components in N-terminal to C-terminal order: scFv (e.g., anti-FITC E2), hinge (spacer), transmembrane domain, and intracellular domain with costimulatory signaling domain and CD3ζ signaling domain (Z). The constructs differ in the following aspects: hinge (spacer) domain, which is IgG4 hinge (IgG4H) or CD8α hinge (CD8H); transmembrane domain, which is CD28 TM or CD8 TM; and costimulatory domain, which is 41BB costimulatory domain or CD28 costimulatory domain. The constructs also differ in the location of the TagCAR, which is present as the front end or the end of the construct transgene sequence. The individual polynucleotide components of the construct are separated by a 2A cleavage site sequence.

表E1:多順反子構築體設計 構築體名稱 構築體設計 構築體A.1 (E2_IgG4H_CD28TM_41BBZ)-FRB-RACR 構築體A.2 FRB-RACR-(E2_IgG4H_CD28TM_41BBZ) 構築體B.1 (E2_IgG4H_CD28TM_28Z)-FRB-RACR 構築體B.2 FRB-RACR-(E2_IgG4H_CD28TM_28Z) 構築體C.1 (E2_CD8HTM_41BBz)-FRB-RACR 構築體C.2 FRB-RACR-(E2_CD8HTM_41BBz) 構築體D.1 (E2_CD8HTM_28Z)-FRB-RACR 構築體D.2 FRB-RACR-(E2_CD8HTM_28Z) Table E1: Polycistronic construct design Structure Name Structural design Structure A.1 (E2_IgG4H_CD28TM_41BBZ)-FRB-RACR Structure A.2 FRB-RACR-(E2_IgG4H_CD28TM_41BBZ) Structure B.1 (E2_IgG4H_CD28TM_28Z)-FRB-RACR Structure B.2 FRB-RACR-(E2_IgG4H_CD28TM_28Z) Structure C.1 (E2_CD8HTM_41BBz)-FRB-RACR Structure C.2 FRB-RACR-(E2_CD8HTM_41BBz) Structure D.1 (E2_CD8HTM_28Z)-FRB-RACR Structure D.2 FRB-RACR-(E2_CD8HTM_28Z)

對於慢病毒載體產生而言,293T生產細胞用表現病毒載體蛋白質(gag/pol、rev)的質體及編碼以上有效負載構築體中之各者的轉移質體轉染。慢病毒載體亦經工程改造以編碼CD58、抗CD3及CD80表面蛋白以介導T細胞活化。在病毒載體製備之後,將細胞培養物離心以使細胞集結(pellet)且收集含有粗病毒之上清液。For lentiviral vector production, 293T production cells were transfected with plasmids expressing viral vector proteins (gag/pol, rev) and transfer plasmids encoding each of the above payload constructs. Lentiviral vectors were also engineered to encode CD58, anti-CD3, and CD80 surface proteins to mediate T cell activation. Following viral vector preparation, cell cultures were centrifuged to pellet the cells and the supernatant containing crude virus was collected.

在第0天,以2×10 6個細胞/mL,將來自3名供體之PBMC接種於24孔盤中之含有50 U/mL IL-2之培養基中,且以2或10之MOI用慢病毒轉導。在第3天,洗滌出病毒,且細胞藉由流式細胞分析技術針對作為T細胞活化之標記之CD25進行染色,且亦拆分至個別培養盤中並且暴露於50 U/mL IL-2 + 10 nM雷帕黴素或無雷帕黴素。在第7天,將細胞染色以評定CAR表現,溶解用於西方墨點,或用於共培養殺傷及擴增分析。 On day 0, PBMCs from 3 donors were seeded at 2×10 6 cells/mL in medium containing 50 U/mL IL-2 in 24-well plates and transduced with lentivirus at an MOI of 2 or 10. On day 3, the virus was washed out and the cells were stained by flow cytometry for CD25 as a marker of T cell activation and also split into individual plates and exposed to 50 U/mL IL-2 + 10 nM rapamycin or no rapamycin. On day 7, cells were stained to assess CAR expression, lysed for Western blot, or used for co-culture killing and expansion analysis.

T細胞活化( 3A)及T細胞轉導( 3B)隨著顆粒劑量而增加,其中MOI 10 (淺條杠)比MOI 2 (暗條杠)存在更高的活化及轉導。 3A中之結果亦顯示,在所有慢病毒載體製劑中,T細胞活化(藉由CD8+或CD4+ T細胞之CD25+%所測定)均類似。同樣, 3B顯示,在所有慢病毒載體製劑中,T細胞轉導(藉由CD8+及CD4+ T細胞之CAR表現%所測定)亦均類似。總之,結果出人意料地指示,多核苷酸構築體中組分之取向不會影響活化CD8+及CD4+ T細胞之百分比或經轉導CD8+及CD4+ TagCAR T細胞之百分比。 T cell activation ( Figure 3A ) and T cell transduction ( Figure 3B ) increased with particle dose, with higher activation and transduction at MOI 10 (light bars) compared to MOI 2 (dark bars). The results in Figure 3A also show that T cell activation (measured by CD25+% of CD8+ or CD4+ T cells) was similar in all lentiviral vector preparations. Similarly, Figure 3B shows that T cell transduction (measured by CAR expression% of CD8+ and CD4+ T cells) was also similar in all lentiviral vector preparations. In summary, the results unexpectedly indicate that the orientation of the components in the polynucleotide construct does not affect the percentage of activated CD8+ and CD4+ T cells or the percentage of transduced CD8+ and CD4+ TagCAR T cells.

相比之下,溶解物之西方墨點分析顯示,將FRB置放於轉殖基因中之第一位置中引起較高FRB蛋白表現( 3C),無論細胞係在僅IL2而無雷帕黴素中還是在IL-2 +雷帕黴素中生長。此等結果確立,其中FRB位於相對於RACR CAR序列之前端位置的構築體對於確保FRB之高含量表現為優良的。 In contrast, Western blot analysis of lysates showed that placing FRB in the first position in the transgene resulted in higher FRB protein expression ( Figure 3C ), regardless of whether cells were grown in IL-2 alone without rapamycin or in IL-2 + rapamycin. These results establish that constructs in which FRB is located at a leading position relative to the RACR CAR sequence are superior for ensuring high-level expression of FRB.

基於FRB作為第一轉殖基因之優越性,在轉導T細胞之後,進一步分析具有構築體C.2及構築體D.2取向轉殖基因之慢病毒載體。在第0天,以2×10 6個細胞/mL,將來自3名供體之PBMC接種於24孔盤中之含有50 U/mL IL-2之培養基中,且以10之MOI用慢病毒轉導。在第3天,洗滌出病毒,且將細胞拆分至個別培養盤中並且暴露於50 U/mL IL-2、10 nM雷帕黴素或50 U/mL IL-2 +10 nM雷帕黴素。在第7、11及14天,將細胞染色以評定CAR表現。如 4中所示,用任一轉殖基因轉導之CAR T細胞經增濃且用雷帕黴素擴增。 Based on the superiority of FRB as the first transgene, lentiviral vectors with construct C.2 and construct D.2 tropism transgenes were further analyzed after transduction of T cells. On day 0, PBMCs from 3 donors were seeded in 24-well plates at 2×10 6 cells/mL in medium containing 50 U/mL IL-2 and transduced with lentivirus at an MOI of 10. On day 3, the virus was washed out, and the cells were split into individual culture plates and exposed to 50 U/mL IL-2, 10 nM rapamycin, or 50 U/mL IL-2 +10 nM rapamycin. On days 7, 11, and 14, cells were stained to assess CAR expression. As shown in FIG. 4 , CAR T cells transduced with either transgene were enriched and expanded with rapamycin.

在殺傷分析中分析來自在轉導之後第10天的經轉導T細胞。在前一天(第10天),將6×10 3個用mCherry標記之FRα+ MDA-MB-231目標細胞塗鋪在96孔盤中之無葉酸培養基中。在第11天,以1:1或1:4效應物:目標(E:T)比率將經轉導CAR-T細胞添加至目標細胞中。接著,以0、0.1或10 nM將葉酸-螢光素(FITC-葉酸)添加至各孔中。在第12天,收集上清液且用於細胞介素之分析。如 5A(IgG4鉸鏈)及 5B(CD8鉸鏈)中所示,用CD8α鉸鏈之CAR T細胞在所有供體中在低劑量之葉酸-螢光素下在消除FRα+目標細胞方面展現優良活性,與先前雷帕黴素中之擴增無關。更特定言之,在96小時時之腫瘤體面積(tumor object area)在與用以下構築體( 5A中之頂部至底部)轉導之細胞一起共培養時減少:構築體A.2 + FITC-葉酸,在僅IL-2中擴增;構築體A.1;構築體A.2;構築體A.1 + FITC-葉酸,在IL-2 + 10 nM雷帕黴素中擴增;構築體A.2 + FITC-葉酸,在IL-2 + 10 nM雷帕黴素中擴增;構築體A.1 + FITC-葉酸,在僅IL-2中擴增。在96小時時之腫瘤體面積在與用以下構築體( 5B中之頂部至底部)轉導之細胞一起共培養時減少:構築體B.2;構築體B.1;構築體B.2 + FITC-葉酸,在IL-2 + 10 nM雷帕黴素中擴增;構築體B.2 + FITC-葉酸,在僅IL-2中擴增;構築體B.1 + FITC-葉酸,在IL-2 + 10 nM雷帕黴素中擴增;構築體B.1 + FITC-葉酸,在僅IL-2中擴增。在96小時時之腫瘤體面積在與用以下構築體( 6A中之頂部至底部)轉導之細胞一起共培養時減少:構築體C.1;構築體C.2;構築體C.1 + FITC-葉酸,在僅IL-2中擴增;構築體C.2 + FITC-葉酸,在僅IL-2中擴增;構築體C.1 + FITC-葉酸,在IL-2與10 nM雷帕黴素中擴增;構築體C.2 + FITC-葉酸,在IL-2與10 nM雷帕黴素中擴增。在96小時時之腫瘤體面積在與用以下構築體( 6B中之頂部至底部)轉導之細胞一起共培養時減少:構築體D.2;構築體D.1;構築體D.1 + FITC-葉酸,在IL-2與10 nM雷帕黴素中擴增;構築體D.2 + FITC-葉酸,在IL-2與10 nM雷帕黴素中擴增;構築體D.1 + FITC-葉酸,在僅IL-2中擴增;構築體D.2 + FITC-葉酸,在僅IL-2中擴增。 Transduced T cells from day 10 after transduction were analyzed in a killing assay. On the previous day (day 10), 6×10 3 FRα+ MDA-MB-231 target cells labeled with mCherry were plated in 96-well plates in folic acid-free medium. On day 11, transduced CAR-T cells were added to the target cells at a 1:1 or 1:4 effector: target (E:T) ratio. Then, folate-fluorescein (FITC-folate) was added to each well at 0, 0.1, or 10 nM. On day 12, supernatants were collected and used for analysis of cytokines. As shown in Figure 5A (IgG4 hinge) and Figure 5B (CD8 hinge), CAR T cells with the CD8α hinge showed excellent activity in eliminating FRα+ target cells under low doses of folate-fluorescein in all donors, independent of previous expansion in rapamycin. More specifically, tumor object area at 96 hours was reduced when co-cultured with cells transduced with the following constructs (top to bottom in Figure 5A ): construct A.2 + FITC-folate, expanded in IL-2 alone; construct A.1; construct A.2; construct A.1 + FITC-folate, expanded in IL-2 + 10 nM rapamycin; construct A.2 + FITC-folate, expanded in IL-2 + 10 nM rapamycin; construct A.1 + FITC-folate, expanded in IL-2 alone. Tumor area at 96 h was reduced when co-cultured with cells transduced with the following constructs (top to bottom in Fig. 5B ): construct B.2; construct B.1; construct B.2 + FITC-folate, expanded in IL-2 + 10 nM rapamycin; construct B.2 + FITC-folate, expanded in IL-2 alone; construct B.1 + FITC-folate, expanded in IL-2 + 10 nM rapamycin; construct B.1 + FITC-folate, expanded in IL-2 alone. Tumor area at 96 h was reduced when co-cultured with cells transduced with the following constructs (top to bottom in Fig. 6A ): construct C.1; construct C.2; construct C.1 + FITC-folate, expanded in IL-2 alone; construct C.2 + FITC-folate, expanded in IL-2 alone; construct C.1 + FITC-folate, expanded in IL-2 and 10 nM rapamycin; construct C.2 + FITC-folate, expanded in IL-2 and 10 nM rapamycin. Tumor area at 96 h was reduced when co-cultured with cells transduced with the following constructs (top to bottom in Fig. 6B ): construct D.2; construct D.1; construct D.1 + FITC-folate, expanded in IL-2 and 10 nM rapamycin; construct D.2 + FITC-folate, expanded in IL-2 and 10 nM rapamycin; construct D.1 + FITC-folate, expanded in IL-2 alone; construct D.2 + FITC-folate, expanded in IL-2 alone.

在FITC-葉酸存在下在與CD8α鉸鏈CAR候選物一起共培養中之促炎性細胞介素產生及T細胞增殖亦增加(資料未顯示)。Pro-inflammatory interleukin production and T cell proliferation were also increased in co-culture with CD8α hinge CAR candidates in the presence of FITC-folate (data not shown).

結果亦顯示,用含有編碼具有4-1BB共刺激域之CAR之轉殖基因的慢病毒載體轉導之CAR T細胞亦證實在FITC-葉酸存在下在用FRα+腫瘤細胞反覆刺激之後持久性增強(資料現在顯示)。此外,藉由在FITC-葉酸存在下活體內投與編碼41BB胞內域CAR候選物之慢病毒載體處理的小鼠證實與用編碼CD28胞內域CAR候選物之慢病毒載體處理之小鼠相比,FRα+實體腫瘤之消退/控制改善、增加循環CAR-T細胞增濃/擴增及增加存活。The results also showed that CAR T cells transduced with a lentiviral vector containing a transgene encoding a CAR with a 4-1BB co-stimulatory domain also demonstrated enhanced persistence after repeated stimulation with FRα+ tumor cells in the presence of FITC-folate (data now shown). In addition, mice treated with a lentiviral vector encoding a 41BB intracellular domain CAR candidate in vivo in the presence of FITC-folate demonstrated improved regression/control of FRα+ solid tumors, increased circulating CAR-T cell enrichment/expansion, and increased survival compared to mice treated with a lentiviral vector encoding a CD28 intracellular domain CAR candidate.

此等結果另外指示,編碼具有CD8鉸鏈及4-1BB共刺激域之CAR的構築體亦為優良的。These results further indicate that constructs encoding CARs with CD8 hinges and 4-1BB co-stimulatory domains are also superior.

基於以上分析,選擇構築體C.2用於進一步分析。 實例2 具有不同FRB取向之RACR-TagCAR多核苷酸多順反子構築體之產生及評定 Based on the above analysis, construct C.2 was selected for further analysis. Example 2 Generation and evaluation of RACR-TagCAR polynucleotide polycistronic constructs with different FRB orientations

為了進一步評定FRB-RACR處於轉殖基因之前端位置之構築體的影響,將所選擇構築體C.2與其中FRB-RACR取向在轉殖基因之末端的構築體比較。本實例中探究之多核苷酸取向顯示於 E2中。 To further assess the impact of constructs with FRB-RACR at the front end of the transgene, the selected construct C.2 was compared with constructs in which the FRB-RACR orientation was at the end of the transgene. The polynucleotide orientations investigated in this example are shown in Table E2 .

表E2 多核苷酸構築體 N端 C端 構築體V (FRB-RACR在末端) CD19-CAR FRB RACR FKBP12-IL2Rγ FRB-IL2Rβ 構築體C.2 FRB RACR Tag-CAR FKBP12-IL2Rγ FRB-IL2Rβ Table E2 Polynucleotide construct N-terminal C-end Structure V (FRB-RACR at the end) CD19-CAR FRB RACR FKBP12-IL2Rγ FRB-IL2Rβ Structure C.2 FRB RACR Tag-CAR FKBP12-IL2Rγ FRB-IL2Rβ

在第0天,以1×10 6個細胞/mL,將來自3名供體之PBMC接種於24孔盤中之含有250 U/mL IL2的培養基中,且用編碼以上構築體之慢病毒轉導。在第3天,對細胞進行計數且進行CD25染色以確認藉由慢病毒載體進行之T細胞活化,或將細胞轉移至個別培養盤用於用IL2及雷帕黴素刺激。在第7天,對細胞再次進行計數且染色以評定CAR表現,溶解用於西方墨點;抑或用IL2 (250 U/mL)及雷帕黴素(10 nM)進行刺激。在第11、14及18天,對細胞進行計數,染色以評定CAR表現或用IL2 (250 U/mL)及雷帕黴素(10 nM)進行刺激。 On day 0, PBMCs from 3 donors were seeded at 1×10 6 cells/mL in medium containing 250 U/mL IL2 in 24-well plates and transduced with lentivirus encoding the above constructs. On day 3, cells were counted and stained for CD25 to confirm T cell activation by the lentiviral vector, or transferred to separate culture plates for stimulation with IL2 and rapamycin. On day 7, cells were counted again and stained to assess CAR expression, lysed for Western blot; or stimulated with IL2 (250 U/mL) and rapamycin (10 nM). On days 11, 14, and 18, cells were counted, stained to assess CAR expression, or stimulated with IL2 (250 U/mL) and rapamycin (10 nM).

用含有構築體V或構築體C.2之慢病毒載體轉導之PBMC均表現CAR ( 6A)。與實例1中之結果一致,與構築體V相比,藉由西方墨點,在用構築體C.2轉導之PBMC中觀測到FRB及RACR表現增強(資料未顯示)。來自各供體之經轉導細胞中FRB之表現增強與同構築體V相比,在雷帕黴素存在下用C.2構築體轉導的PBMC之增殖增強有關。( 6B)。此等資料證實,與含有構築體V之細胞相比,含有構築體C.2之細胞對於雷帕黴素刺激反應更佳。 實例3 其他多順反子構築體之產生及表徵 PBMCs transduced with lentiviral vectors containing construct V or construct C.2 all expressed CAR ( Figure 6A ). Consistent with the results in Example 1, enhanced expression of FRB and RACR was observed in PBMCs transduced with construct C.2 compared to construct V by Western blotting (data not shown). The enhanced expression of FRB in transduced cells from each donor was associated with enhanced proliferation of PBMCs transduced with construct C.2 in the presence of rapamycin compared to construct V. ( Figure 6B ). These data confirm that cells containing construct C.2 respond better to rapamycin stimulation than cells containing construct V. Example 3 Generation and Characterization of Other Polycistronic Constructs

儘管用含有構築體C.2之慢病毒載體轉導之細胞中之FRB表現似乎被拯救,但嘗試修飾構築體以進一步改良經轉導細胞中FRB以及由FRB-IL2Rγ及FKBP12-IL2Rβ構成之合成細胞介素受體(RACR)的表現。FRB及合成細胞介素受體之增加可增強雷帕黴素介導之CAR T細胞增濃及擴增。Although FRB expression appeared to be rescued in cells transduced with lentiviral vectors containing construct C.2, attempts were made to modify the construct to further improve the expression of FRB and synthetic interleukin receptors (RACRs) composed of FRB-IL2Rγ and FKBP12-IL2Rβ in transduced cells. Increased FRB and synthetic interleukin receptors enhanced rapamycin-mediated enrichment and expansion of CAR T cells.

藉由與構築體C.2相比重排多核苷酸之取向來產生多核苷酸構築體。特定言之,交換FRB及FKBP12以編碼由FRB與IL2Rγ融合構成之合成γ鏈多肽以及由FKBP12序列與IL2Rβ融合構成之合成β鏈多肽。此外,包括弗林蛋白酶T2A連接子替代構築體之第一與第二轉殖基因之間(即FRB與FRB:IL2Rγ之間)的P2A。最後,編碼合成細胞介素受體(RACR)組分之多核苷酸經密碼子最佳化。所得最佳化的多核苷酸構築體顯示為 7A中之構築體C.2U以及SEQ ID NO: 1。 Polynucleotide constructs were generated by rearranging the orientation of the polynucleotides compared to construct C.2. Specifically, FRB and FKBP12 were exchanged to encode a synthetic γ-chain polypeptide consisting of a fusion of FRB and IL2Rγ and a synthetic β-chain polypeptide consisting of a fusion of the FKBP12 sequence and IL2Rβ. In addition, a furin T2A linker was included to replace P2A between the first and second transgenes of the construct (i.e., between FRB and FRB:IL2Rγ). Finally, the polynucleotide encoding the synthetic interleukin receptor (RACR) component was codon optimized. The resulting optimized polynucleotide construct is shown as construct C.2U in Figure 7A and SEQ ID NO: 1.

評定用含有構築體C.2或構築體C.2U之慢病毒載體轉導之T細胞的表現及活性。一般如實例1中所描述製備慢病毒載體。Evaluation of the expression and activity of T cells transduced with lentiviral vectors containing construct C.2 or construct C.2U. Lentiviral vectors were prepared generally as described in Example 1.

在第0天,以1×10 6個細胞/mL,將來自五名不同供體之PBMC接種於24孔盤中且用含有構築體C.2多核苷酸或構築體C.2U多核苷酸之慢病毒轉導。在第3天,對細胞進行CD25染色。在第7天,對細胞進行染色以評定RACR-TagCAR (FITC-CAR)之表現在第8天,收集細胞且溶解用於西方墨點。 On day 0, PBMCs from five different donors were seeded in 24-well plates at 1×10 6 cells/mL and transduced with lentivirus containing construct C.2 polynucleotide or construct C.2U polynucleotide. On day 3, cells were stained for CD25. On day 7, cells were stained to assess the expression of RACR-TagCAR (FITC-CAR). On day 8, cells were collected and lysed for Western blot.

用具有構築體C.2或構築體C.2U轉殖基因之慢病毒載體轉導之T細胞之間的CD25+細胞百分比相當( 7B)。在第7天,與構築體C.2轉導的T細胞相比,構築體C.2U轉導的T細胞表現更高的CAR含量,兩者均呈表現CAR之T細胞的百分比( 7C)及平均螢光強度(mean fluorescence intensity;MFI) ( 7D)。 The percentage of CD25+ cells was comparable between T cells transduced with lentiviral vectors carrying either construct C.2 or construct C.2U transgenes ( Fig. 7B ). On day 7, T cells transduced with construct C.2U expressed higher levels of CAR compared to T cells transduced with construct C.2, both in terms of percentage of T cells expressing CAR ( Fig. 7C ) and mean fluorescence intensity (MFI) ( Fig. 7D ).

藉由西方墨點,用含有多核苷酸構築體C.2U之慢病毒轉導之T細胞展現較高的FRB ( 7E)及RACR (FRB-IL2Rγ及FKBP12-IL2Rβ) ( 7F)之可偵測表現。 By Western blotting, T cells transduced with lentivirus containing the polynucleotide construct C.2U showed higher detectable expression of FRB ( FIG. 7E ) and RACR (FRB-IL2Rγ and FKBP12-IL2Rβ) ( FIG. 7F ).

在個別實驗中,在轉導後第3天及第7天,藉由流動式細胞測量術評定閘控T細胞群體之活化及轉導( 8A)。在轉導之後,在活化之第3天呈CD25+細胞之百分比的CD8+及CD4+ T細胞之百分比增加( 8A,左圖)。此外,在以MOI 10轉導之後,百分比RACR-TagCAR+ CD4+或CD8+ T細胞增加( 8A,右圖)。在轉導後第7天之代表性流式圖(flow plot)顯示增加百分比之CD8+ T細胞表現RACR-TagCAR,尤其在MOI 10下( 8B)。 In individual experiments, activation and transduction of gated T cell populations were assessed by flow cytometry on days 3 and 7 after transduction ( FIG. 8A ). Following transduction, the percentage of CD8+ and CD4+ T cells as a percentage of CD25+ cells increased on day 3 of activation ( FIG. 8A , left panel). In addition, the percentage of RACR-TagCAR+ CD4+ or CD8+ T cells increased after transduction at MOI 10 ( FIG. 8A , right panel). Representative flow plots on day 7 after transduction show an increased percentage of CD8+ T cells expressing RACR-TagCAR, especially at MOI 10 ( FIG. 8B ).

為了評定對於經轉導細胞之功能活性的影響,在第0天,用含有多核苷酸構築體C.2或C.2U之慢病毒轉導24孔盤中處於1×10 6個細胞/mL之PBMC。在第3天,細胞用IL2 (250 U/mL)或雷帕黴素(10 nM)進行刺激且進行CD25染色。在第7天,細胞用僅IL2、僅雷帕黴素、僅AP21967 (50 nM)、或雷帕黴素與IL2進行刺激。 To assess the effect on the functional activity of transduced cells, PBMCs at 1×10 6 cells/mL in 24-well plates were transduced with lentivirus containing polynucleotide constructs C.2 or C.2U on day 0. On day 3, cells were stimulated with IL2 (250 U/mL) or rapamycin (10 nM) and stained for CD25. On day 7, cells were stimulated with IL2 alone, rapamycin alone, AP21967 alone (50 nM), or rapamycin and IL2.

用構築體C.2U轉殖基因慢病毒載體轉導之T細胞證實,與用構築體C.2轉殖基因慢病毒載體轉導之T細胞相比,在第3天開始,在IL2或雷帕黴素存在下CAR T細胞擴增增強。一名代表性供體之結果顯示於 9A中。構築體C.2U轉殖基因轉導的T細胞亦證實在雷帕黴素或雷帕黴素類似物AP21967存在下CAR T細胞擴增增強,如 9B中所示。 實例4 RACR-TagCAR T細胞之表徵 T cells transduced with the construct C.2U transgenic lentiviral vector demonstrated enhanced CAR T cell expansion in the presence of IL2 or rapamycin starting on day 3 compared to T cells transduced with the construct C.2 transgenic lentiviral vector. Results for one representative donor are shown in FIG9A . T cells transduced with the construct C.2U transgenic lentiviral vector also demonstrated enhanced CAR T cell expansion in the presence of rapamycin or the rapamycin analog AP21967, as shown in FIG9B . Example 4 Characterization of RACR-TagCAR T cells

此實例證實用實例3中描述之構築體C.2U轉殖基因慢病毒載體轉導的原代人類T細胞之表徵(例如活化、轉導、細胞毒性等)。This example demonstrates the characteristics (e.g., activation, transduction, cytotoxicity, etc.) of primary human T cells transduced with the construct C.2U transgenic lentiviral vector described in Example 3.

以感染倍率(MOI) 2或10,在不存在T細胞活化試劑下,用含有構築體C.2U轉殖基因之慢病毒載體活體外轉導來自3名健康供體的PBMC。PBMCs from three healthy donors were transduced in vitro with lentiviral vectors containing construct C.2U transgene at a multiplicity of infection (MOI) of 2 or 10 in the absence of T cell activating agents.

接下來,評定RACR-TagCART細胞之雷帕黴素介導的RACR活化。以MOI 10,將來自2名健康供體之PBMC與構築體C.2U轉殖基因慢病毒載體一起培養。將PBMC拆分成兩個共培養物。一個共培養物含有50 U/mL IL-2,且另一共培養物含有50 U/mL IL-2與10 nM雷帕黴素,其在共培養之第3天添加;且在第7、11及14天,藉由流式細胞分析技術量測T細胞之增濃及擴增( 10A)。在第11及14天中,T細胞之雷帕黴素介導的RACR活化引起表現RACR-TagCAR之T細胞的增濃增加( 10B,左圖)。在第11及14天中,T細胞之雷帕黴素介導的RACR活化亦引起表現RACR-TagCAR之T細胞的擴增增加( 10B,右圖)。 實例5 RACR-TagCAR T細胞展現活體外腫瘤殺傷 Next, RACR-TagCART cells were assessed for rapamycin-mediated RACR activation. PBMCs from 2 healthy donors were cultured with the construct C.2U transgenic lentiviral vector at an MOI of 10. PBMCs were split into two co-cultures. One co-culture contained 50 U/mL IL-2 and the other co-culture contained 50 U/mL IL-2 and 10 nM rapamycin, which were added on day 3 of co-culture; and on days 7, 11, and 14, the enrichment and expansion of T cells were measured by flow cytometry ( Figure 10A ). On days 11 and 14, rapamycin-mediated RACR activation of T cells caused an increase in the concentration of T cells expressing RACR-TagCAR ( Figure 10B , left). On days 11 and 14, rapamycin-mediated RACR activation of T cells also caused an increase in the expansion of T cells expressing RACR-TagCAR ( Figure 10B , right). Example 5 RACR-TagCAR T cells exhibit in vitro tumor killing

使用與表現葉酸受體(FR)之MDA-MB-231乳房癌細胞一起共培養的途徑評定T細胞抗腫瘤活性及持久性,且滴定FITC-葉酸之劑量。用構築體C.2U轉殖基因慢病毒載體轉導T細胞。在轉導後第11天,將雷帕黴素擴增的T細胞與mCherry+FRα+MDA-MB-231腫瘤細胞一起在0.1 nM FITC-葉酸或0.1 nM FITC-葉酸與10 nM雷帕黴素存在下共培養。使用Incucyte® 活細胞分析系統定量隨著時間推移之乳房癌細胞。在 11A 11B中用箭頭指示之時間點處,再引入乳房癌細胞(腫瘤再刺激)。RACR-TagCAR T細胞在FITC-葉酸存在下反覆殺傷FRα+ MDA-MB-231乳房癌細胞,其中在雷帕黴素處理後活性提高( 11A)。此外,RACR-TagCAR T細胞增殖在整個實驗時程中及各腫瘤再刺激時保持( 11B)。 實例6 TagCAR T細胞之雷帕黴素活化的細胞介素受體(RACR)活化(RACR-TagCAR T細胞)展現活體內腫瘤殺傷 T cell antitumor activity and persistence were assessed using a co-culture approach with MDA-MB-231 breast cancer cells expressing the folate receptor (FR), and the dose of FITC-folate was titrated. T cells were transduced with the construct C.2U transgenic lentiviral vector. On day 11 post-transduction, rapamycin-expanded T cells were co-cultured with mCherry+FRα+MDA-MB-231 tumor cells in the presence of 0.1 nM FITC-folate or 0.1 nM FITC-folate and 10 nM rapamycin. Breast cancer cells were quantified over time using the Incucyte® Live Cell Assay System. Breast cancer cells were reintroduced at the time points indicated by arrows in Figures 11A and 11B (tumor restimulation). RACR-TagCAR T cells repeatedly killed FRα + MDA-MB-231 breast cancer cells in the presence of FITC-folate, with increased activity after rapamycin treatment ( Figure 11A ). In addition, RACR-TagCAR T cell proliferation was maintained throughout the experimental period and upon restimulation of each tumor ( Figure 11B ). Example 6 Rapamycin-activated interleukin receptor (RACR) activation of TagCAR T cells (RACR-TagCAR T cells) exhibits in vivo tumor killing

在接受離體轉導的RACR-TagCAR T細胞之小鼠模型中,測試RACR-TagCAR T細胞之活體內控制腫瘤的能力( 12A)。為了評定活體內抗腫瘤活性,對NSG MHCI/II DKO小鼠注射FRα+ MDA-MB-231乳房癌細胞。一旦腫瘤達到100 mm 3之大小,則小鼠接受i.v.注射5e6或10e6 RACR-TagCAR T細胞,該等T細胞用含有編碼FRB-RACR-TagCAR之構築體C.2轉殖基因的CD58/CD3/CD80表面工程改造的慢病毒顆粒離體轉導。接著,每週對小鼠給藥FITC-葉酸兩次。藉由腫瘤消退,使用數位測徑器量測功效且作圖,如 12B中所示。投與5e6及10e6 RACR-TagCAR T細胞數目兩者均快速清除小鼠中之FRα+實體腫瘤( 12B)。 The ability of RACR-TagCAR T cells to control tumors in vivo was tested in a mouse model that received ex vivo transduction of RACR-TagCAR T cells ( Figure 12A ). To assess in vivo anti-tumor activity, NSG MHCI/II DKO mice were injected with FRα+ MDA-MB-231 breast cancer cells. Once tumors reached a size of 100 mm3, mice received an iv injection of 5e6 or 10e6 RACR-TagCAR T cells that were transduced ex vivo with CD58/CD3/CD80 surface engineered lentiviral particles containing the construct C.2 transgene encoding FRB-RACR-TagCAR. Mice were then dosed with FITC-folate twice a week. Efficacy was measured by tumor regression using a digital caliper and plotted as shown in Figure 12B . Administration of both 5e6 and 10e6 RACR-TagCAR T cell numbers rapidly eliminated FRα+ solid tumors in mice ( Figure 12B ).

在小鼠模型中,測試RACR-TagCAR慢病毒顆粒之活體內控制腫瘤的能力。對小鼠投與含有編碼FRB-RACR-TagCAR載體之構築體C.2U轉殖基因的CD58/CD3/CD80表面工程改造的慢病毒顆粒( 13A)。對雌性NSG MHCI/IIDKO小鼠在側腹植入FRα+MDA-MB-231腫瘤,且允許腫瘤生長直至100-200 mm 3大小。小鼠用健康人類供體PBMC (25e6或100e6)人源化,隨後投與含有編碼FRB-RACR-TagCAR載體之構築體C.2U轉殖基因的CD58/CD3/CD80表面工程改造的慢病毒顆粒。在此實驗中,以100e6轉染單位(TU) TagCAR載體或25e6 TU TagCAR載體之劑量投與慢病毒顆粒。小鼠每週用FITC-葉酸處理兩次,且藉由評定腫瘤消退以及利用流式細胞分析技術進行之來自每週血液抽取之血液中之循環RACR-TagCAR T細胞來測定功效。在早在第7天,偵測血液中之RACR-TagCAR T細胞( 13B)。投與慢病毒載體顆粒亦控制小鼠中之FRα+實體腫瘤,藉由腫瘤體積減小所證明( 13C)。 In a mouse model, the ability of RACR-TagCAR lentiviral particles to control tumors in vivo was tested. Mice were administered CD58/CD3/CD80 surface engineered lentiviral particles containing construct C.2U transgene encoding the FRB-RACR-TagCAR vector ( Figure 13A ). Female NSG MHCI/IIDKO mice were implanted with FRα+MDA-MB-231 tumors in the flank and allowed to grow until 100-200 mm3 in size. Mice were humanized with healthy human donor PBMC (25e6 or 100e6) and then administered CD58/CD3/CD80 surface engineered lentiviral particles containing construct C.2U transgene encoding the FRB-RACR-TagCAR vector. In this experiment, lentiviral particles were administered at a dose of 100e6 transfection units (TU) TagCAR vector or 25e6 TU TagCAR vector. Mice were treated twice weekly with FITC-folate, and efficacy was determined by assessing tumor regression and circulating RACR-TagCAR T cells in the blood from weekly blood draws using flow cytometry. RACR-TagCAR T cells were detected in the blood as early as day 7 ( Figure 13B ). Administration of lentiviral vector particles also controlled FRα+ solid tumors in mice, as evidenced by reduced tumor size ( Figure 13C ).

在個別實驗中,在 13A中所描述之小鼠模型中,測試RACR-TagCAR慢病毒顆粒之活體內控制腫瘤的能力。在此研究中,以0.2e6 TU TagCAR載體、1.0e6 TU TagCAR載體或5.0e6 TU TagCAR載體之劑量,對小鼠投與含有構築體C.2U的CD58/CD3/CD80表面工程改造的慢病毒顆粒。如上文所描述,藉由監測腫瘤消退及循環RACR-TagCAR T細胞測定功效。存在對實體腫瘤之劑量依賴性控制,且接受5.0e6 TU慢病毒顆粒之小鼠直至第25天具有最大的腫瘤減小( 14A)。另外,對於小鼠血液中偵測到之RACR-TagCAR T細胞之量存在劑量依賴性影響。在大約第6天偵測RACR-TagCAR T細胞,且直至第25天,接受5.0e6 TU慢病毒顆粒之小鼠具有最大的循環RACR-TagCAR T細胞數目( 14B)。結果與公認的實體腫瘤之劑量依賴性控制與活體內大量TagCART細胞之產生相關的觀測一致。 In separate experiments, the ability of RACR-TagCAR lentiviral particles to control tumors in vivo was tested in the mouse model described in FIG. 13A . In this study, mice were administered CD58/CD3/CD80 surface engineered lentiviral particles containing construct C.2U at a dose of 0.2e6 TU TagCAR vector, 1.0e6 TU TagCAR vector, or 5.0e6 TU TagCAR vector. Efficacy was determined by monitoring tumor regression and circulating RACR-TagCAR T cells as described above. There was dose-dependent control of solid tumors, and mice receiving 5.0e6 TU lentiviral particles had the greatest tumor reduction until day 25 ( FIG. 14A ). In addition, there was a dose-dependent effect on the amount of RACR-TagCAR T cells detected in the blood of mice. RACR-TagCAR T cells were detected at approximately day 6, and until day 25, mice that received 5.0e6 TU of lentiviral particles had the greatest number of circulating RACR-TagCAR T cells ( Figure 14B ). The results are consistent with the observation that dose-dependent control of solid tumors is associated with the generation of large numbers of TagCART cells in vivo.

此等結果確立,TagCAR T細胞可在投與慢病毒顆粒情況下在活體內產生,且在FITC-葉酸投與情況下介導腫瘤控制及消退。此處及上文中之研究確立TagCAR T細胞系統對於離體或活體內遞送至T細胞及投與腫瘤標籤之效用,其在額外靶向抗原之腫瘤標籤之情形下可提供效用。These results establish that TagCAR T cells can be generated in vivo with administration of lentiviral particles and mediate tumor control and regression with administration of FITC-folate. The studies herein and above establish the utility of the TagCAR T cell system for delivery of tumor tags to T cells and administration in vitro or in vivo, which may provide utility in the context of additional tumor tags targeting antigens.

本發明不意欲限制在特定揭示之實施例的範疇中,該等實施例被提供用於例如說明本發明之各種態樣。對組合物及方法之各種修改將由本文中之描述及教示而變得顯而易見。此類變化形式可在不脫離本發明之真正範疇及精神的情況下實踐且意欲落入本發明之範疇內。 VIII.序列 編號 序列 描述 1. ACATGTCTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATTGCCTGACGCGTAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTCAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGAGCCCACAACCCCTCACTCGGCGCGTGAACACAATTCTGCAGTCGAAGGCGTACCGTCACTTACGAGTCGGTAGCCTGCAGGGCCGCCACCATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGCTAGCAGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCCATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCCATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACCGGCAGCGGAGCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCCATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCCGGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGGCGAGGGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGGTGACGCCGGCGCTAGTGTCGACGTAGTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAATGTGTGTGTTGGTTTTTTGTGTGTCGTCGTCTCACTTGGATCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGGGAAATGTGTGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC 構築體C.2U完全載體核苷酸 2. MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKASRRKRGSGEGRGSLLTCGDVEENPGPMPLPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVGSGATNFSLLKQAGDVEENPGPLLLVTSLLLCELPHPAFLLIPSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 構築體C.2U多肽 3. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAG FRB核苷酸 4. MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK FRB多肽 5. AGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCC 弗林蛋白酶T2A核苷酸 6. RRKRGSGEGRGSLLTCGDVEENPGP 弗林蛋白酶T2A多肽 7. AGAAGAAAGAGA 弗林蛋白酶位點核苷酸 8. RRKR 弗林蛋白酶位點多肽 9. GAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCC T2A核苷酸 10. EGRGSLLTCGDVEENPGP T2A多肽 11. ATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACC FRB:IL2RG核苷酸 12. ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET FRB:IL2RG多肽 13. ATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAG FRB核苷酸 14. ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK FRB多肽 15. GGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACC IL2RG核苷酸 16. GSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET IL2RG多肽 17. GCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCC P2A 核苷酸 18. ATNFSLLKQAGDVEENPGP P2A多肽 19. GGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGGCGAGGGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTG FKBP12: IL2RB核苷酸 20. GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV FKBP12: IL2RB多肽 21. GGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTG FKBP12 核苷酸 22. GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKL FKBP12多肽 23. GGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTG IL2RB 核苷酸 24. GKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV IL2RB多肽 25. GCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCT P2A 核苷酸 26. CTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGGTGA TagCAR 核苷酸 27. LLLVTSLLLCELPHPAFLLIPSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR TagCAR多肽 28. TCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCC E2 scFv核苷酸 29. SVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSS E2 scFv多肽 30. TCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTG E2 scFv VL 核苷酸 31. SVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVL E2 scFv VL多肽 32. GGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGA E2 scFv連接子 核苷酸 33. GSTSGSGKPGSGEGSTKG E2 scFv連接子多肽 34. CAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCC E2 scFv VH 核苷酸 35. QVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSS E2 scFv VH多肽 36. ACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGC CD8 HTM核苷酸 37. TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC CD8 HTM多肽 38. ACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTAC CD8鉸鏈核苷酸 39. TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY CD8鉸鏈多肽 40. ATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGC CD8跨膜域核苷酸 41. IWAPLAGTCGVLLLSLVITLYC CD8跨膜域多肽 42. AAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTG 41BB 核苷酸 43. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 41BB多肽 44. ATGTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGCTATAGCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTG CD28核苷酸 45. MFWVLVVVGGVLACYSLLVTVAFIIFWV CD28多肽 46. AGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGG CD3ζ核苷酸 47. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3ζ多肽 48. ACATGTCTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATTGCCTGACGCGTAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTCAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGAGCCCACAACCCCTCACTCGGCGCGTGAACACAATTCTGCAGTCGAAGGCGTACCGTCACTTACGAGTCGGTAGCCTGCAGGGCCGCCACCATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGGCTCCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGACCTATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCAGGAGTGCAGGTGGAGACCATCTCCCCAGGCGACGGACGCACATTTCCAAAGAGGGGACAGACCTGCGTGGTGCACTACACAGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCCGCGATCGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGATGGGAGGAGGGAGTGGCACAGATGTCCGTGGGACAGAGGGCCAAGCTGACCATCTCTCCTGACTACGCCTATGGAGCAACAGGACACCCAGGAATCATCCCACCTCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGGCTCTAACACAAGCAAGGAGAATCCTTTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTCTATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTTTGGCTGGAGAGAACCATGCCAAGGATCCCCACACTGAAGAACCTGGAGGACCTGGTGACCGAGTATCACGGCAATTTCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTACTCTGAGCGGCTGTGCCTGGTGTCCGAGATCCCACCCAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCAGCCCTTGCAACCAGCACTCCCCATATTGGGCCCCTCCATGTTACACACTGAAGCCCGAGACCGGCTCTGGCGCCACAAACTTCAGCCTGCTGAAGCAAGCAGGCGACGTGGAAGAAAATCCAGGACCTATGGCACTGCCTGTGACCGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCTATCCTGTGGCACGAAATGTGGCATGAAGGCCTGGAGGAGGCAAGCAGGCTGTATTTTGGCGAGCGGAATGTGAAAGGAATGTTTGAAGTCCTGGAACCTCTGCATGCCATGATGGAAAGGGGACCACAGACACTGAAGGAGACCAGCTTTAACCAGGCCTATGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTACATGAAGTCCGGAAATGTGAAAGACCTGCTGCAGGCCTGGGATCTGTATTATCACGTGTTCAGGCGCATCTCTAAGGGCAAGGATACCATCCCCTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAATACCGGCCCTTGGCTGAAGAAGGTGCTGAAGTGTAACACACCAGACCCCTCCAAGTTCTTTTCTCAGCTGTCTAGCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCCTCTCCTTTTCCAAGCTCCTCTTTCAGCCCAGGAGGACTGGCACCAGAGATCTCCCCCCTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCCGAGCCTGCCAGCCTGAGCTCCAACCACAGCCTGACATCCTGCTTTACCAATCAGGGCTATTTCTTTTTCCACCTGCCAGACGCCCTGGAGATCGAGGCCTGTCAGGTGTACTTCACCTATGATCCATACTCCGAAGAGGACCCAGATGAGGGAGTGGCCGGCGCCCCCACAGGCTCTAGCCCACAGCCACTGCAGCCTCTGTCTGGAGAGGACGATGCCTACTGTACCTTTCCCAGCCGCGACGATCTGCTGCTGTTCAGCCCTTCCCTGCTGGGAGGACCATCTCCACCTAGCACAGCACCAGGAGGAAGCGGGGCAGGGGAGGAGCGGATGCCACCATCTCTGCAGGAGAGGGTGCCTAGGGACTGGGATCCTCAGCCACTGGGACCTCCAACCCCTGGAGTGCCAGACCTGGTGGATTTTCAGCCCCCTCCAGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCTGACGCAGGACCACGCGAGGGCGTGAGCTTTCCATGGTCCAGGCCACCTGGACAGGGAGAGTTCAGAGCCCTGAACGCCAGGCTGCCTCTGAATACAGACGCCTATCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCAACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGGTGACGCCGGCGCTAGTGTCGACGTAGTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAATGTGTGTGTTGGTTTTTTGTGTGTCGTCGTCTCACTTGGATCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGGGAAATGTGTGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC 構築體C.2完全載體核苷酸 49. MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVGSGATNFSLLKQAGDVEENPGPLLLVTSLLLCELPHPAFLLIPSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 構築體C.2多肽 50. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGGCTCCGGC FRB核苷酸 51. MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSG FRB多肽 52. GCCACCAACTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGACCT P2A核苷酸 53. ATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCAGGAGTGCAGGTGGAGACCATCTCCCCAGGCGACGGACGCACATTTCCAAAGAGGGGACAGACCTGCGTGGTGCACTACACAGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCCGCGATCGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGATGGGAGGAGGGAGTGGCACAGATGTCCGTGGGACAGAGGGCCAAGCTGACCATCTCTCCTGACTACGCCTATGGAGCAACAGGACACCCAGGAATCATCCCACCTCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGGCTCTAACACAAGCAAGGAGAATCCTTTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTCTATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTTTGGCTGGAGAGAACCATGCCAAGGATCCCCACACTGAAGAACCTGGAGGACCTGGTGACCGAGTATCACGGCAATTTCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTACTCTGAGCGGCTGTGCCTGGTGTCCGAGATCCCACCCAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCAGCCCTTGCAACCAGCACTCCCCATATTGGGCCCCTCCATGTTACACACTGAAGCCCGAGACCGGCTCTGGC FKBP12: IL2RG核苷酸 54. MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSG FKBP12: IL2RG多肽 55. GGAGTGCAGGTGGAGACCATCTCCCCAGGCGACGGACGCACATTTCCAAAGAGGGGACAGACCTGCGTGGTGCACTACACAGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCCGCGATCGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGATGGGAGGAGGGAGTGGCACAGATGTCCGTGGGACAGAGGGCCAAGCTGACCATCTCTCCTGACTACGCCTATGGAGCAACAGGACACCCAGGAATCATCCCACCTCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTG FKBP12 核苷酸 56. ATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCAGGAGTGCAGGTGGAGACCATCTCCCCAGGCGACGGACGCACATTTCCAAAGAGGGGACAGACCTGCGTGGTGCACTACACAGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCCGCGATCGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGATGGGAGGAGGGAGTGGCACAGATGTCCGTGGGACAGAGGGCCAAGCTGACCATCTCTCCTGACTACGCCTATGGAGCAACAGGACACCCAGGAATCATCCCACCTCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGGCTCTAACACAAGCAAGGAGAATCCTTTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTCTATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTTTGGCTGGAGAGAACCATGCCAAGGATCCCCACACTGAAGAACCTGGAGGACCTGGTGACCGAGTATCACGGCAATTTCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTACTCTGAGCGGCTGTGCCTGGTGTCCGAGATCCCACCCAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCAGCCCTTGCAACCAGCACTCCCCATATTGGGCCCCTCCATGTTACACACTGAAGCCCGAGACC FKBP12: IL2RG核苷酸 57. MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET FKBP12: IL2RG多肽 58. GCCACAAACTTCAGCCTGCTGAAGCAAGCAGGCGACGTGGAAGAAAATCCAGGACCT P2A核苷酸 59. ATGGCACTGCCTGTGACCGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCTATCCTGTGGCACGAAATGTGGCATGAAGGCCTGGAGGAGGCAAGCAGGCTGTATTTTGGCGAGCGGAATGTGAAAGGAATGTTTGAAGTCCTGGAACCTCTGCATGCCATGATGGAAAGGGGACCACAGACACTGAAGGAGACCAGCTTTAACCAGGCCTATGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTACATGAAGTCCGGAAATGTGAAAGACCTGCTGCAGGCCTGGGATCTGTATTATCACGTGTTCAGGCGCATCTCTAAGGGCAAGGATACCATCCCCTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAATACCGGCCCTTGGCTGAAGAAGGTGCTGAAGTGTAACACACCAGACCCCTCCAAGTTCTTTTCTCAGCTGTCTAGCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCCTCTCCTTTTCCAAGCTCCTCTTTCAGCCCAGGAGGACTGGCACCAGAGATCTCCCCCCTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCCGAGCCTGCCAGCCTGAGCTCCAACCACAGCCTGACATCCTGCTTTACCAATCAGGGCTATTTCTTTTTCCACCTGCCAGACGCCCTGGAGATCGAGGCCTGTCAGGTGTACTTCACCTATGATCCATACTCCGAAGAGGACCCAGATGAGGGAGTGGCCGGCGCCCCCACAGGCTCTAGCCCACAGCCACTGCAGCCTCTGTCTGGAGAGGACGATGCCTACTGTACCTTTCCCAGCCGCGACGATCTGCTGCTGTTCAGCCCTTCCCTGCTGGGAGGACCATCTCCACCTAGCACAGCACCAGGAGGAAGCGGGGCAGGGGAGGAGCGGATGCCACCATCTCTGCAGGAGAGGGTGCCTAGGGACTGGGATCCTCAGCCACTGGGACCTCCAACCCCTGGAGTGCCAGACCTGGTGGATTTTCAGCCCCCTCCAGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCTGACGCAGGACCACGCGAGGGCGTGAGCTTTCCATGGTCCAGGCCACCTGGACAGGGAGAGTTCAGAGCCCTGAACGCCAGGCTGCCTCTGAATACAGACGCCTATCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCAACCCACCTGGTG FRB:IL2RB核苷酸 60. MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV FRB:IL2RB多肽 61. GGGCAAGGATACCATCCCCTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAATACCGGCCCTTGGCTGAAGAAGGTGCTGAAGTGTAACACACCAGACCCCTCCAAGTTCTTTTCTCAGCTGTCTAGCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCCTCTCCTTTTCCAAGCTCCTCTTTCAGCCCAGGAGGACTGGCACCAGAGATCTCCCCCCTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCCGAGCCTGCCAGCCTGAGCTCCAACCACAGCCTGACATCCTGCTTTACCAATCAGGGCTATTTCTTTTTCCACCTGCCAGACGCCCTGGAGATCGAGGCCTGTCAGGTGTACTTCACCTATGATCCATACTCCGAAGAGGACCCAGATGAGGGAGTGGCCGGCGCCCCCACAGGCTCTAGCCCACAGCCACTGCAGCCTCTGTCTGGAGAGGACGATGCCTACTGTACCTTTCCCAGCCGCGACGATCTGCTGCTGTTCAGCCCTTCCCTGCTGGGAGGACCATCTCCACCTAGCACAGCACCAGGAGGAAGCGGGGCAGGGGAGGAGCGGATGCCACCATCTCTGCAGGAGAGGGTGCCTAGGGACTGGGATCCTCAGCCACTGGGACCTCCAACCCCTGGAGTGCCAGACCTGGTGGATTTTCAGCCCCCTCCAGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCTGACGCAGGACCACGCGAGGGCGTGAGCTTTCCATGGTCCAGGCCACCTGGACAGGGAGAGTTCAGAGCCCTGAACGCCAGGCTGCCTCTGAATACAGACGCCTATCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCAACCCACCTGGTG IL2RB核苷酸 62. GKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV IL2RB多肽 63. CTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGGTGA TagCAR 核苷酸 64. TCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCC E2 scFv核苷酸 65. TCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTG E2 scFv VL 核苷酸 66. GGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGA E2 scFv連接子 核苷酸 67. CAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCC E2 scFv VH 核苷酸 68. ACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGC CD8 HTM核苷酸 69. AAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTG 41BB 核苷酸 70. AGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGG CD3ζ核苷酸 71. ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTAGAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA TagCAR核苷酸 72. MALPVTALLLPLALLLHAARPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR TagCAR多肽 73. GCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTAGAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA 核苷酸插入 74. DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 不具有訊號肽之TagCAR胺基酸序列 75. MALPVTALLLPLALLLHAARP CAR胺基酸訊號肽 76. MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF CD86胺基酸 77. MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV CD80胺基酸 78. ATGGATCCCCAGTGCACTATGGGACTGAGTAACATTCTCTTTGTGATGGCCTTCCTGCTCTCTGGTGCTGCTCCTCTGAAGATTCAAGCTTATTTCAATGAGACTGCAGACCTGCCATGCCAATTTGCAAACTCTCAAAACCAAAGCCTGAGTGAGCTAGTAGTATTTTGGCAGGACCAGGAAAACTTGGTTCTGAATGAGGTATACTTAGGCAAAGAGAAATTTGACAGTGTTCATTCCAAGTATATGGGCCGCACAAGTTTTGATTCGGACAGTTGGACCCTGAGACTTCACAATCTTCAGATCAAGGACAAGGGCTTGTATCAATGTATCATCCATCACAAAAAGCCCACAGGAATGATTCGCATCCACCAGATGAACTCTGAACTGTCAGTGCTTGCTAACTTCAGTCAACCTGAAATAGTACCAATTTCTAATATAACAGAAAATGTGTACATAAATTTGACCTGCTCATCTATACACGGTTACCCAGAACCTAAGAAGATGAGTGTTTTGCTAAGAACCAAGAACTCAACTATCGAGTATGATGGTGTTATGCAGAAATCTCAAGATAATGTCACAGAACTGTACGACGTTTCCATCAGCTTGTCTGTTTCATTCCCTGATGTTACGAGCAATATGACCATCTTCTGTATTCTGGAAACTGACAAGACGCGGCTTTTATCTTCACCTTTCTCTATAGAGCTTGAGGACCCTCAGCCTCCCCCAGACCACATTCCTTGGATTACAGCTGTACTTCCAACAGTTATTATATGTGTGATGGTTTTCTGTCTAATTCTATGGAAATGGAAGAAGAAGAAGCGGCCTCGCAACTCTTATAAATGTGGAACCAACACAATGGAGAGGGAAGAGAGTGAACAGACCAAGAAAAGAGAAAAAATCCATATACCTGAAAGGTCTGATGAAGCCCAGCGTGTTTTTAAAAGTTCGAAGACATCTTCATGCGACAAAAGTGATACATGTTTT CD86核苷酸 79. ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTA CD80核苷酸 80. MVAGSDAGRALGVLSVVCLLHCFGFISCFSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSDEDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQCKRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPSSGHSRHRYALIPIPLAVITTCIVLYMNGILKCDRKPDRTNSN CD58胺基酸 81. ATGGTTGCTGGGAGCGACGCGGGGCGGGCCCTGGGGGTCCTCAGCGTGGTCTGCCTGCTGCACTGCTTTGGTTTCATCAGCTGTTTTTCCCAACAAATATATGGTGTTGTGTATGGGAATGTAACTTTCCATGTACCAAGCAATGTGCCTTTAAAAGAGGTCCTATGGAAAAAACAAAAGGATAAAGTTGCAGAACTGGAAAATTCTGAGTTCAGAGCTTTCTCATCTTTTAAAAATAGGGTTTATTTAGACACTGTGTCAGGTAGCCTCACTATCTACAACTTAACATCATCAGATGAAGATGAGTATGAAATGGAATCGCCAAATATTACTGATACCATGAAGTTCTTTCTTTATGTGCTTGAGTCTCTTCCATCTCCCACACTAACTTGTGCATTGACTAATGGAAGCATTGAAGTCCAATGCATGATACCAGAGCATTACAACAGCCATCGAGGACTTATAATGTACTCATGGGATTGTCCTATGGAGCAATGTAAACGTAACTCAACCAGTATATATTTTAAGATGGAAAATGATCTTCCACAAAAAATACAGTGTACTCTTAGCAATCCATTATTTAATACAACATCATCAATCATTTTGACAACCTGTATCCCAAGCAGCGGTCATTCAAGACACAGATATGCACTTATACCCATACCATTAGCAGTAATTACAACATGTATTGTGCTGTATATGAATGGTATTCTGAAATGTGACAGAAAACCAGACAGAACCAACTCCAAT CD58核苷酸 82. ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGG TagCAR核苷酸 83. ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGATATCGTGCTGACCCAGTCCCCCGCCATCCTGTCCGCCTCTCCTGGAGAGAAGGTGACCATGACATGTCGGGCCAGCTCCTCTGTGAACTACATGGACTGGTATCAGAAGAAGCCTGGCAGCTCCCCCAAGCCTTGGATCTACGCCACCTCCAATCTGGCCTCTGGAGTGCCAGCAAGATTCAGCGGATCCGGATCTGGCACAAGCTATTCCCTGACCATCTCCAGGGTGGAGGCAGAGGATGCAGCAACATACTATTGCCAGCAGTGGTCTTTCAACCCCCCTACATTTGGCGGCGGCACCAAGCTGGAGATCAAGGGCTCTACCAGCGGAGGAGGAAGCGGAGGAGGATCCGGAGGCGGCGGCTCTAGCGAGGTGCAGCTGCAGCAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCCTCTGTGAAGATGAGCTGTAAGGCCTCCGGCTACACCTTCACATCTTATAATATGCACTGGGTGAAGCAGACACCAGGACAGGGACTGGAGTGGATCGGAGCAATCTACCCTGGCAACGGCGACACCAGCTATAATCAGAAGTTTAAGGGCAAGGCCACCCTGACAGCCGATAAGTCCTCTAGCACAGCCTACATGCAGCTGTCCTCTCTGACCAGCGAGGACTCCGCCGATTACTATTGCGCCCGGTCCAACTACTATGGCAGCTCCTATTGGTTCTTTGACGTGTGGGGAGCAGGAACAACCGTGACCGTGTCTAGCGCTGCAGGAGGCGGAGGATCTGGAGGCGGCGGCGGAGACTACAAAGACGATGACGACAAGTTCGAAGCAAAGCCAACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACCTATGATGCCCTGCACATGCAGGCCCTGCCCCCTCGG CD20 CAR核苷酸(具有flag) 84. MLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSAAGGGGSGGGGGDYKDDDDKFEAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD20 CAR多肽 (具有flag序列) 85. ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGATATCGTGCTGACCCAGTCCCCCGCCATCCTGTCCGCCTCTCCTGGAGAGAAGGTGACCATGACATGTCGGGCCAGCTCCTCTGTGAACTACATGGACTGGTATCAGAAGAAGCCTGGCAGCTCCCCCAAGCCTTGGATCTACGCCACCTCCAATCTGGCCTCTGGAGTGCCAGCAAGATTCAGCGGATCCGGATCTGGCACAAGCTATTCCCTGACCATCTCCAGGGTGGAGGCAGAGGATGCAGCAACATACTATTGCCAGCAGTGGTCTTTCAACCCCCCTACATTTGGCGGCGGCACCAAGCTGGAGATCAAGGGCTCTACCAGCGGAGGAGGAAGCGGAGGAGGATCCGGAGGCGGCGGCTCTAGCGAGGTGCAGCTGCAGCAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCCTCTGTGAAGATGAGCTGTAAGGCCTCCGGCTACACCTTCACATCTTATAATATGCACTGGGTGAAGCAGACACCAGGACAGGGACTGGAGTGGATCGGAGCAATCTACCCTGGCAACGGCGACACCAGCTATAATCAGAAGTTTAAGGGCAAGGCCACCCTGACAGCCGATAAGTCCTCTAGCACAGCCTACATGCAGCTGTCCTCTCTGACCAGCGAGGACTCCGCCGATTACTATTGCGCCCGGTCCAACTACTATGGCAGCTCCTATTGGTTCTTTGACGTGTGGGGAGCAGGAACAACCGTGACCGTGTCTAGCGCAAAGCCAACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACCTATGATGCCCTGCACATGCAGGCCCTGCCCCCTCGG CD20 CAR核苷酸(不具有flag) 86. MLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD20 CAR多肽 (不具有flag序列) 87. GATATCGTGCTGACCCAGTCCCCCGCCATCCTGTCCGCCTCTCCTGGAGAGAAGGTGACCATGACATGTCGGGCCAGCTCCTCTGTGAACTACATGGACTGGTATCAGAAGAAGCCTGGCAGCTCCCCCAAGCCTTGGATCTACGCCACCTCCAATCTGGCCTCTGGAGTGCCAGCAAGATTCAGCGGATCCGGATCTGGCACAAGCTATTCCCTGACCATCTCCAGGGTGGAGGCAGAGGATGCAGCAACATACTATTGCCAGCAGTGGTCTTTCAACCCCCCTACATTTGGCGGCGGCACCAAGCTGGAGATCAAGGGCTCTACCAGCGGAGGAGGAAGCGGAGGAGGATCCGGAGGCGGCGGCTCTAGCGAGGTGCAGCTGCAGCAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCCTCTGTGAAGATGAGCTGTAAGGCCTCCGGCTACACCTTCACATCTTATAATATGCACTGGGTGAAGCAGACACCAGGACAGGGACTGGAGTGGATCGGAGCAATCTACCCTGGCAACGGCGACACCAGCTATAATCAGAAGTTTAAGGGCAAGGCCACCCTGACAGCCGATAAGTCCTCTAGCACAGCCTACATGCAGCTGTCCTCTCTGACCAGCGAGGACTCCGCCGATTACTATTGCGCCCGGTCCAACTACTATGGCAGCTCCTATTGGTTCTTTGACGTGTGGGGAGCAGGAACAACCGTGACCGTGTCTAGC CD20 scFv核苷酸 88. DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS CD20 scFv多肽 89. GATATCGTGCTGACCCAGTCCCCCGCCATCCTGTCCGCCTCTCCTGGAGAGAAGGTGACCATGACATGTCGGGCCAGCTCCTCTGTGAACTACATGGACTGGTATCAGAAGAAGCCTGGCAGCTCCCCCAAGCCTTGGATCTACGCCACCTCCAATCTGGCCTCTGGAGTGCCAGCAAGATTCAGCGGATCCGGATCTGGCACAAGCTATTCCCTGACCATCTCCAGGGTGGAGGCAGAGGATGCAGCAACATACTATTGCCAGCAGTGGTCTTTCAACCCCCCTACATTTGGCGGCGGCACCAAGCTGGAGATCAAG CD20 scFv VL核苷酸 90. DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK CD20 scFv VL多肽 91. GGCTCTACCAGCGGAGGAGGAAGCGGAGGAGGATCCGGAGGCGGCGGCTCTAGC 218連接子核苷酸 92. GSTSGGGSGGGSGGGGSS 218連接子多肽 93. GAGGTGCAGCTGCAGCAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCCTCTGTGAAGATGAGCTGTAAGGCCTCCGGCTACACCTTCACATCTTATAATATGCACTGGGTGAAGCAGACACCAGGACAGGGACTGGAGTGGATCGGAGCAATCTACCCTGGCAACGGCGACACCAGCTATAATCAGAAGTTTAAGGGCAAGGCCACCCTGACAGCCGATAAGTCCTCTAGCACAGCCTACATGCAGCTGTCCTCTCTGACCAGCGAGGACTCCGCCGATTACTATTGCGCCCGGTCCAACTACTATGGCAGCTCCTATTGGTTCTTTGACGTGTGGGGAGCAGGAACAACCGTGACCGTGTCTAGC CD20 scFv VH核苷酸 94. EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS CD20 scFv VH多肽 95. GACTACAAAGACGATGACGACAAG Flag核苷酸 96. DYKDDDDK Flag多肽 97. GCAAAGCCAACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGC CD8 HTM核苷酸 98. AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC CD8 HTM多肽 99. AAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTG 41BB核苷酸 100. CGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACCTATGATGCCCTGCACATGCAGGCCCTGCCCCCTCGG CD3ζ核苷酸 101. ACATGTCTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATTGCCTGACGCGTAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTCAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGAGCCCACAACCCCTCACTCGGCGCGTGAACACAATTCTGCAGTCGAAGGCGTACCGTCACTTACGAGTCGGTAGCCTGCAGGGCCGCCACCATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGCTAGCAGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCCATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCCATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACCGGCAGCGGAGCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCCATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCCGGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGGCGAGGGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACCTCCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGCGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTGAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGGTGACGCCGGCGCTAGTGTCGACGTAGTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAATGTGTGTGTTGGTTTTTTGTGTGTCGTCGTCTCACTTGGATCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGGGAAATGTGTGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC 構築體C.3U完全載體核苷酸 102. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGCTAGCAGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCCATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCCATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACCGGCAGCGGAGCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCCATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCCGGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGGCGAGGGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACCTCCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGCGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTGAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGG 構築體C.3U核苷酸 103. MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKASRRKRGSGEGRGSLLTCGDVEENPGPMPLPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVGSGATNFSLLKQAGDVEENPGPLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 構築體C.3U多肽 104. CTGCTGCTGGTGACCTCCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGCGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTGAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGG CD19 CAR核苷酸 105. LLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD19 CAR多肽 106. GACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGC CD19 scFv核苷酸 107. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS CD19 scFv多肽 108. GACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATC CD19 scFv VL核苷酸 109. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEI CD19 scFv VL多肽 110. ACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGA CD19 scFv連接子核苷酸 111. TGSTSGSGKPGSGEGSTKG CD19 scFv連接子多肽 112. GAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGC CD19 scFv VH核苷酸 113. EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS CD19 scFv VH多肽 114. GGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGAC CD8鉸鏈核苷酸 115. GAVHTRGLDFACD CD8鉸鏈多肽 116. TTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTG CD28跨膜域核苷酸 117. FWVLVVVGGVLACYSLLVTVAFIIFWV CD28跨膜域多肽 118. CGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGG CD3ζ域核苷酸 119. ESKYGPPSPPSPA 經修飾之IgG4鉸鏈 120. ESKYGPPSPPSP 經修飾之IgG4鉸鏈 121. PSPRPAGQFQTLV PD1鉸鏈 122. EPKSCDKTHTCP IgG1鉸鏈 123. GAVHTRGLDFACD CD8鉸鏈 124. LCPSPLFPGPSKP CD28鉸鏈 125. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGCTAGCAGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCCATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCCATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACCGGCAGCGGAGCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCCATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCCGGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGGCGAGGGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTcTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGG 構築體C.2U核苷酸 126. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGGCTCCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGACCTATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCAGGAGTGCAGGTGGAGACCATCTCCCCAGGCGACGGACGCACATTTCCAAAGAGGGGACAGACCTGCGTGGTGCACTACACAGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCCGCGATCGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGATGGGAGGAGGGAGTGGCACAGATGTCCGTGGGACAGAGGGCCAAGCTGACCATCTCTCCTGACTACGCCTATGGAGCAACAGGACACCCAGGAATCATCCCACCTCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGGCTCTAACACAAGCAAGGAGAATCCTTTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTCTATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTTTGGCTGGAGAGAACCATGCCAAGGATCCCCACACTGAAGAACCTGGAGGACCTGGTGACCGAGTATCACGGCAATTTCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTACTCTGAGCGGCTGTGCCTGGTGTCCGAGATCCCACCCAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCAGCCCTTGCAACCAGCACTCCCCATATTGGGCCCCTCCATGTTACACACTGAAGCCCGAGACCGGCTCTGGCGCCACAAACTTCAGCCTGCTGAAGCAAGCAGGCGACGTGGAAGAAAATCCAGGACCTATGGCACTGCCTGTGACCGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCTATCCTGTGGCACGAAATGTGGCATGAAGGCCTGGAGGAGGCAAGCAGGCTGTATTTTGGCGAGCGGAATGTGAAAGGAATGTTTGAAGTCCTGGAACCTCTGCATGCCATGATGGAAAGGGGACCACAGACACTGAAGGAGACCAGCTTTAACCAGGCCTATGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTACATGAAGTCCGGAAATGTGAAAGACCTGCTGCAGGCCTGGGATCTGTATTATCACGTGTTCAGGCGCATCTCTAAGGGCAAGGATACCATCCCCTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAATACCGGCCCTTGGCTGAAGAAGGTGCTGAAGTGTAACACACCAGACCCCTCCAAGTTCTTTTCTCAGCTGTCTAGCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCCTCTCCTTTTCCAAGCTCCTCTTTCAGCCCAGGAGGACTGGCACCAGAGATCTCCCCCCTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCCGAGCCTGCCAGCCTGAGCTCCAACCACAGCCTGACATCCTGCTTTACCAATCAGGGCTATTTCTTTTTCCACCTGCCAGACGCCCTGGAGATCGAGGCCTGTCAGGTGTACTTCACCTATGATCCATACTCCGAAGAGGACCCAGATGAGGGAGTGGCCGGCGCCCCCACAGGCTCTAGCCCACAGCCACTGCAGCCTCTGTCTGGAGAGGACGATGCCTACTGTACCTTTCCCAGCCGCGACGATCTGCTGCTGTTCAGCCCTTCCCTGCTGGGAGGACCATCTCCACCTAGCACAGCACCAGGAGGAAGCGGGGCAGGGGAGGAGCGGATGCCACCATCTCTGCAGGAGAGGGTGCCTAGGGACTGGGATCCTCAGCCACTGGGACCTCCAACCCCTGGAGTGCCAGACCTGGTGGATTTTCAGCCCCCTCCAGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCTGACGCAGGACCACGCGAGGGCGTGAGCTTTCCATGGTCCAGGCCACCTGGACAGGGAGAGTTCAGAGCCCTGAACGCCAGGCTGCCTCTGAATACAGACGCCTATCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCAACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTcTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGG 構築體C.2核苷酸 127. MLLLVTSLLLCELPHPAFLLIPSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR TagCAR多肽 128. GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET 不具有訊號肽之FKBP12: IL2RG多肽 129. ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV 不具有訊號肽之FRB:IL2RB多肽 The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the present invention. Various modifications to the compositions and methods will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the present invention and are intended to fall within the scope of the present invention. VIII. Sequence No. sequence describe 1. ACATGTCTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATTGCCTGACGCGTAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTCAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGAGCCCACAACCCCTCACTCGGCGCGTGAACACAATTCTGCAGTCGAAGGCGTACCGTCACTTACGAGTCGGTAGCCTGCAGGGCCGCCACCATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGCTAGCAGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCCATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCCATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACCGGCAGCGGAGCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCCATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCCGGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGGCGAGGGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGGTGACGCCGGCGCTAGTGTCGACGTAGTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAATGTGTGTGTTGGTTTTTTGTGTGTCGTCGTCTCACTTGGATCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGGGAAATGTGTGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC Construct C.2U complete vector nucleotides 2. MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKASRRKRGSGEGRGSLLTCGDVEENPGPMPLPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVGSGATNFSLLKQAGDVEENPGPLLLVTSLLLCELPHPAFLLIPSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Construct C.2U polypeptide 3. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTT TCGGAGAATCAGCAAG FRB nucleotides 4. MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK FRB peptide 5. AGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCC Furin T2A nucleotide 6. RRKRGSGEGRGSLLTCGDVEENPGP Furin T2A peptide 7. AGAAGAAAGAGA Furin site nucleotide 8. RRKR Furin site peptide 9. GAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCC T2A nucleotides 10. EGRGSLLTCGDVEENPGP T2A peptide 11. ATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACC FRB:IL2RG nucleotide 12. ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET FRB:IL2RG peptide 13. ATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACC ACGTGTTCAGAAGAATCAGCAAG FRB nucleotides 14. ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK FRB peptide 15. GGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTG AGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACC IL2RG nucleotide 16. GSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET IL2RG peptide 17. GCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCC P2A nucleotides 18. ATNFSLLKQAGDVEENPGP P2A peptide 19. GGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGGCGAGGGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTG FKBP12: IL2RB nucleotide 20. GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQ LLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV FKBP12: IL2RB peptide twenty one. GGCGTTCAAGTGGAGACTATCTCCCCGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCAT CCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTG FKBP12 Nucleotide twenty two. GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKL FKBP12 peptide twenty three. GGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTG IL2RB nucleotide twenty four. GKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSG AGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV IL2RB peptide 25. GCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCT P2A nucleotides 26. CTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGGTGA TagCAR Nucleotide 27. LLLVTSLLCELPHPAFLLIPSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKG RFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSY DSSGYWGHFYSYMDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR TagCAR peptide 28. TCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCC E2 scFv Nucleotide 29. SVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQM NSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSS E2 scFv peptide 30. TCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATT ATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTG E2 scFv VL Nucleotide 31. SVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVL E2 scFv VL polypeptide 32. GGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGA E2 scFv linker nucleotide 33. GSTSGSGKPGSGEGSTKG E2 scFv linker polypeptide 34. CAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAATA GCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCC E2 scFv VH Nucleotide 35. QVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSS E2 scFv VH polypeptide 36. ACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGC CD8 HTM nucleotides 37. TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC CD8 HTM peptide 38. ACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTAC CD8 hinge nucleotide 39. TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY CD8 hinge peptide 40. ATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGC CD8 transmembrane domain nucleotides 41. IWAPLAGTCGVLLLSLVITLYC CD8 transmembrane domain peptide 42. AAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTG 41BB nucleotide 43. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 41BB peptide 44. ATGTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGCTATAGCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTG CD28 Nucleotide 45. MFWVLVVVGGVLACYSLLVTVAFIIFWV CD28 peptide 46. AGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACA CGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGG CD3ζ nucleotides 47. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3ζ polypeptide 48. ACATGTCTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATTGCCTGACGCGTAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTCAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGAGCCCACAACCCCTCACTCGGCGCGTGAACACAATTCTGCAGTCGAAGGCGTACCGTCACTTACGAGTCGGTAGCCTGCAGGGCCGCCACCATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGGCTCCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGACCTATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCAGGAGTGCAGGTGGAGACCATCTCCCCAGGCGACGGACGCACATTTCCAAAGAGGGGACAGACCTGCGTGGTGCACTACACAGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCCGCGATCGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGATGGGAGGAGGGAGTGGCACAGATGTCCGTGGGACAGAGGGCCAAGCTGACCATCTCTCCTGACTACGCCTATGGAGCAACAGGACACCCAGGAATCATCCCACCTCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGGCTCTAACACAAGCAAGGAGAATCCTTTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTCTATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTTTGGCTGGAGAGAACCATGCCAAGGATCCCCACACTGAAGAACCTGGAGGACCTGGTGACCGAGTATCACGGCAATTTCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTACTCTGAGCGGCTGTGCCTGGTGTCCGAGATCCCACCCAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCAGCCCTTGCAACCAGCACTCCCCATATTGGGCCCCTCCATGTTACACACTGAAGCCCGAGACCGGCTCTGGCGCCACAAACTTCAGCCTGCTGAAGCAAGCAGGCGACGTGGAAGAAAATCCAGGACCTATGGCACTGCCTGTGACCGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCTATCCTGTGGCACGAAATGTGGCATGAAGGCCTGGAGGAGGCAAGCAGGCTGTATTTTGGCGAGCGGAATGTGAAAGGAATGTTTGAAGTCCTGGAACCTCTGCATGCCATGATGGAAAGGGGACCACAGACACTGAAGGAGACCAGCTTTAACCAGGCCTATGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTACATGAAGTCCGGAAATGTGAAAGACCTGCTGCAGGCCTGGGATCTGTATTATCACGTGTTCAGGCGCATCTCTAAGGGCAAGGATACCATCCCCTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAATACCGGCCCTTGGCTGAAGAAGGTGCTGAAGTGTAACACACCAGACCCCTCCAAGTTCTTTTCTCAGCTGTCTAGCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCCTCTCCTTTTCCAAGCTCCTCTTTCAGCCCAGGAGGACTGGCACCAGAGATCTCCCCCCTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCCGAGCCTGCCAGCCTGAGCTCCAACCACAGCCTGACATCCTGCTTTACCAATCAGGGCTATTTCTTTTTCCACCTGCCAGACGCCCTGGAGATCGAGGCCTGTCAGGTGTACTTCACCTATGATCCATACTCCGAAGAGGACCCAGATGAGGGAGTGGCCGGCGCCCCCACAGGCTCTAGCCCACAGCCACTGCAGCCTCTGTCTGGAGAGGACGATGCCTACTGTACCTTTCCCAGCCGCGACGATCTGCTGCTGTTCAGCCCTTCCCTGCTGGGAGGACCATCTCCACCTAGCACAGCACCAGGAGGAAGCGGGGCAGGGGAGGAGCGGATGCCACCATCTCTGCAGGAGAGGGTGCCTAGGGACTGGGATCCTCAGCCACTGGGACCTCCAACCCCTGGAGTGCCAGACCTGGTGGATTTTCAGCCCCCTCCAGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCTGACGCAGGACCACGCGAGGGCGTGAGCTTTCCATGGTCCAGGCCACCTGGACAGGGAGAGTTCAGAGCCCTGAACGCCAGGCTGCCTCTGAATACAGACGCCTATCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCAACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGGTGACGCCGGCGCTAGTGTCGACGTAGTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAATGTGTGTGTTGGTTTTTTGTGTGTCGTCGTCTCACTTGGATCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGGGAAATGTGTGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC Construct C.2 Complete vector nucleotides 49. MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVGSGATNFSLLKQAGDVEENPGPLLLVTSLLLCELPHPAFLLIPSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Construct C.2 polypeptide 50. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTT TCGGAGAATCAGCAAGGGCTCCGGC FRB nucleotides 51. MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSG FRB peptide 52. GCCACCAACTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGACCT P2A nucleotides 53. ATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCAGGAGTGCAGGTGGAGACCATCTCCCCAGGCGACGGACGCACATTTCCAAAGAGGGGACAGACCTGCGTGGTGCACTACACAGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCCGCGATCGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGATGGGAGGAGGGAGTGGCACAGATGTCCGTGGGACAGAGGGCCAAGCTGACCATCTCTCCTGACTACGCCTATGGAGCAACAGGACACCCAGGAATCATCCCACCTCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGGCTCTAACACAAGCAAGGAGAATCCTTTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTCTATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTTTGGCTGGAGAGAACCATGCCAAGGATCCCCACACTGAAGAACCTGGAGGACCTGGTGACCGAGTATCACGGCAATTTCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTACTCTGAGCGGCTGTGCCTGGTGTCCGAGATCCCACCCAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCAGCCCTTGCAACCAGCACTCCCCATATTGGGCCCCTCCATGTTACACACTGAAGCCCGAGACCGGCTCTGGC FKBP12: IL2RG nucleotide 54. MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLC LVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSG FKBP12: IL2RG peptide 55. GGAGTGCAGGTGGAGACCATCTCCCCAGGCGACGGACGCACATTTCCAAAGAGGGGACAGACCTGCGTGGTGCACTACACAGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCCGCGATCGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGATGGGAGGAGGGAGTGGCACAGATGTCCGTGGGACAGAGGGCCAAGCTGACCATCTCTCCTGACTACGCCTATGGAGCAACAGGACACCCCCC AGGAATCATCCCACCTCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTG FKBP12 Nucleotide 56. ATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCAGGAGTGCAGGTGGAGACCATCTCCCCAGGCGACGGACGCACATTTCCAAAGAGGGGACAGACCTGCGTGGTGCACTACACAGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCCGCGATCGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGATGGGAGGAGGGAGTGGCACAGATGTCCGTGGGACAGAGGGCCAAGCTGACCATCTCTCCTGACTACGCCTATGGAGCAACAGGACACCCAGGAATCATCCCACCTCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGGCTCTAACACAAGCAAGGAGAATCCTTTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTCTATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTTTGGCTGGAGAGAACCATGCCAAGGATCCCCACACTGAAGAACCTGGAGGACCTGGTGACCGAGTATCACGGCAATTTCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTACTCTGAGCGGCTGTGCCTGGTGTCCGAGATCCCACCCAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCAGCCCTTGCAACCAGCACTCCCCATATTGGGCCCCTCCATGTTACACACTGAAGCCCGAGACC FKBP12: IL2RG nucleotide 57. MPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLC LVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET FKBP12: IL2RG peptide 58. GCCACAAACTTCAGCCTGCTGAAGCAAGCAGGCGACGTGGAAGAAAATCCAGGACCT P2A nucleotides 59. ATGGCACTGCCTGTGACCGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCTATCCTGTGGCACGAAATGTGGCATGAAGGCCTGGAGGAGGCAAGCAGGCTGTATTTTGGCGAGCGGAATGTGAAAGGAATGTTTGAAGTCCTGGAACCTCTGCATGCCATGATGGAAAGGGGACCACAGACACTGAAGGAGACCAGCTTTAACCAGGCCTATGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTACATGAAGTCCGGAAATGTGAAAGACCTGCTGCAGGCCTGGGATCTGTATTATCACGTGTTCAGGCGCATCTCTAAGGGCAAGGATACCATCCCCTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAATACCGGCCCTTGGCTGAAGAAGGTGCTGAAGTGTAACACACCAGACCCCTCCAAGTTCTTTTCTCAGCTGTCTAGCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCCTCTCCTTTTCCAAGCTCCTCTTTCAGCCCAGGAGGACTGGCACCAGAGATCTCCCCCCTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCCGAGCCTGCCAGCCTGAGCTCCAACCACAGCCTGACATCCTGCTTTACCAATCAGGGCTATTTCTTTTTCCACCTGCCAGACGCCCTGGAGATCGAGGCCTGTCAGGTGTACTTCACCTATGATCCATACTCCGAAGAGGACCCAGATGAGGGAGTGGCCGGCGCCCCCACAGGCTCTAGCCCACAGCCACTGCAGCCTCTGTCTGGAGAGGACGATGCCTACTGTACCTTTCCCAGCCGCGACGATCTGCTGCTGTTCAGCCCTTCCCTGCTGGGAGGACCATCTCCACCTAGCACAGCACCAGGAGGAAGCGGGGCAGGGGAGGAGCGGATGCCACCATCTCTGCAGGAGAGGGTGCCTAGGGACTGGGATCCTCAGCCACTGGGACCTCCAACCCCTGGAGTGCCAGACCTGGTGGATTTTCAGCCCCCTCCAGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCTGACGCAGGACCACGCGAGGGCGTGAGCTTTCCATGGTCCAGGCCACCTGGACAGGGAGAGTTCAGAGCCCTGAACGCCAGGCTGCCTCTGAATACAGACGCCTATCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCAACCCACCTGGTG FRB:IL2RB nucleotide 60. MALPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQL LLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV FRB:IL2RB peptide 61. GGGCAAGGATACCATCCCCTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAATACCGGCCCTTGGCTGAAGAAGGTGCTGAAGTGTAACACACCAGACCCCTCCAAGTTCTTTTCTCAGCTGTCTAGCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCCTCTCCTTTTCCAAGCTCCTCTTTCAGCCCAGGAGGACTGGCACCAGAGATCTCCCCCCTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCCGAGCCTGCCAGCCTGAGCTCCAACCACAGCCTGACATCCTGCTTTACCAATCAGGGCTATTTCTTTTTCCACCTGCCAGACGCCCTGGAGATCGAGGCCTGTCAGGTGTACTTCACCTATGATCCATACTCCGAAGAGGACCCAGATGAGGGAGTGGCCGGCGCCCCCACAGGCTCTAGCCCACAGCCACTGCAGCCTCTGTCTGGAGAGGACGATGCCTACTGTACCTTTCCCAGCCGCGACGATCTGCTGCTGTTCAGCCCTTCCCTGCTGGGAGGACCATCTCCACCTAGCACAGCACCAGGAGGAAGCGGGGCAGGGGAGGAGCGGATGCCACCATCTCTGCAGGAGAGGGTGCCTAGGGACTGGGATCCTCAGCCACTGGGACCTCCAACCCCTGGAGTGCCAGACCTGGTGGATTTTCAGCCCCCTCCAGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCTGACGCAGGACCACGCGAGGGCGTGAGCTTTCCATGGTCCAGGCCACCTGGACAGGGAGAGTTCAGAGCCCTGAACGCCAGGCTGCCTCTGAATACAGACGCCTATCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCAACCCACCTGGTG IL2RB nucleotide 62. GKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSG AGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV IL2RB peptide 63. CTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGGTGA TagCAR Nucleotide 64. TCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCC E2 scFv Nucleotide 65. TCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATT ATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTG E2 scFv VL Nucleotide 66. GGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGA E2 scFv linker nucleotide 67. CAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAATA GCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCC E2 scFv VH Nucleotide 68. ACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGC CD8 HTM nucleotides 69. AAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTG 41BB nucleotide 70. AGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACA CGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGG CD3ζ nucleotides 71. ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTAGAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA TagCAR Nucleotide 72. MALPVTALLLPLALLLHAARPDVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEK GLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTG ASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR TagCAR peptide 73. GCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTAGAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA Nucleotide insertion 74. DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYET YYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQG TSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR TagCAR amino acid sequence without signal peptide 75. MALPVTALLLPLALLLHAARP CAR amino acid signal peptide 76. MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSF PDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF CD86 amino acid 77. MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKY GHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV CD80 amino acid 78. ATGGATCCCCAGTGCACTATGGGACTGAGTAACATTCTCTTTGTGATGGCCTTCCTGCTCTCTGGTGCTGCTCCTCTGAAGATTCAAGCTTATTTCAATGAGACTGCAGACCTGCCATGCCAATTTGCAAACTCTCAAAACCAAAGCCTGAGTGAGCTAGTAGTATTTTGGCAGGACCAGGAAAACTTGGTTCTGAATGAGGTATACTTAGGCAAAGAGAAATTTGACAGTGTTCATTCCAAGTATATGGGCCGCACAAGTTTTGATTCGGACAGTTGGACCCTGAGACTTCACAATCTTCAGATCAAGGACAAGGGCTTGTATCAATGTATCATCCATCACAAAAAGCCCACAGGAATGATTCGCATCCACCAGATGAACTCTGAACTGTCAGTGCTTGCTAACTTCAGTCAACCTGAAATAGTACCAATTTCTAATATAACAGAAAATGTGTACATAAATTTGACCTGCTCATCTATACACGGTTACCCAGAACCTAAGAAGATGAGTGTTTTGCTAAGAACCAAGAACTCAACTATCGAGTATGATGGTGTTATGCAGAAATCTCAAGATAATGTCACAGAACTGTACGACGTTTCCATCAGCTTGTCTGTTTCATTCCCTGATGTTACGAGCAATATGACCATCTTCTGTATTCTGGAAACTGACAAGACGCGGCTTTTATCTTCACCTTTCTCTATAGAGCTTGAGGACCCTCAGCCTCCCCCAGACCACATTCCTTGGATTACAGCTGTACTTCCAACAGTTATTATATGTGTGATGGTTTTCTGTCTAATTCTATGGAAATGGAAGAAGAAGAAGCGGCCTCGCAACTCTTATAAATGTGGAACCAACACAATGGAGAGGGAAGAGAGTGAACAGACCAAGAAAAGAGAAAAAATCCATATACCTGAAAGGTCTGATGAAGCCCAGCGTGTTTTTAAAAGTTCGAAGACATCTTCATGCGACAAAAGTGATACATGTTTT CD86 nucleotides 79. ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTA CD80 nucleotides 80. MVAGSDAGRALGVLSVVCLLHCFGFISCFSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSDEDEYEMESPNITDTMKFFLYVLESLPSPPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQCKRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPS SGHSRHRYALIPPIPLAVITTCIVLYMNGILKCDRKPDRTNSN CD58 amino acid 81. ATGGTTGCTGGGAGCGACGCGGGGCGGGCCCTGGGGGTCCTCAGCGTGGTCTGCCTGCTGCACTGCTTTGGTTTCATCAGCTGTTTTTCCCAACAAATATATGGTGTTGTGTATGGGAATGTAACTTTCCATGTACCAAGCAATGTGCCTTTAAAAGAGGTCCTATGGAAAAAACAAAAGGATAAAGTTGCAGAACTGGAAAATTCTGAGTTCAGAGCTTTCTCATCTTTTAAAAATAGGGTTTATTTAGACACTGTGTCAGGTAGCCTCACTATCTACAACTTAACATCATCAGATGAAGATGAGTATGAAATGGAATCGCCAAATATTACTGATACCATGAAGTTCTTTCTTTATGTGCTTGAGTCTCTTCCATCTCCCACACTAACTTGTGCATTGACTAATGGAAGCATTGAAGTCCAATGCATGATACCAGAGCATTACAACAGCCATCGAGGACTTATAATGTACTCATGGGATTGTCCTATGGAGCAATGTAAACGTAACTCAACCAGTATATATTTTAAGATGGAAAATGATCTTCCACAAAAAATACAGTGTACTCTTAGCAATCCATTATTTAATACAACATCATCAATCATTTTGACAACCTGTATCCCAAGCAGCGGTCATTCAAGACACAGATATGCACTTATACCCATACCATTAGCAGTAATTACAACATGTATTGTGCTGTATATGAATGGTATTCTGAAATGTGACAGAAAACCAGACAGAACCAACTCCAAT CD58 nucleotides 82. ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTCTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGG TagCAR Nucleotide 83. ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGATATCGTGCTGACCCAGTCCCCCGCCATCCTGTCCGCCTCTCCTGGAGAGAAGGTGACCATGACATGTCGGGCCAGCTCCTCTGTGAACTACATGGACTGGTATCAGAAGAAGCCTGGCAGCTCCCCCAAGCCTTGGATCTACGCCACCTCCAATCTGGCCTCTGGAGTGCCAGCAAGATTCAGCGGATCCGGATCTGGCACAAGCTATTCCCTGACCATCTCCAGGGTGGAGGCAGAGGATGCAGCAACATACTATTGCCAGCAGTGGTCTTTCAACCCCCCTACATTTGGCGGCGGCACCAAGCTGGAGATCAAGGGCTCTACCAGCGGAGGAGGAAGCGGAGGAGGATCCGGAGGCGGCGGCTCTAGCGAGGTGCAGCTGCAGCAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCCTCTGTGAAGATGAGCTGTAAGGCCTCCGGCTACACCTTCACATCTTATAATATGCACTGGGTGAAGCAGACACCAGGACAGGGACTGGAGTGGATCGGAGCAATCTACCCTGGCAACGGCGACACCAGCTATAATCAGAAGTTTAAGGGCAAGGCCACCCTGACAGCCGATAAGTCCTCTAGCACAGCCTACATGCAGCTGTCCTCTCTGACCAGCGAGGACTCCGCCGATTACTATTGCGCCCGGTCCAACTACTATGGCAGCTCCTATTGGTTCTTTGACGTGTGGGGAGCAGGAACAACCGTGACCGTGTCTAGCGCTGCAGGAGGCGGAGGATCTGGAGGCGGCGGCGGAGACTACAAAGACGATGACGACAAGTTCGAAGCAAAGCCAACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACCTATGATGCCCTGCACATGCAGGCCCTGCCCCCTCGG CD20 CAR Nucleotide (with flag) 84. MLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQK FKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVW GAGTTVTVSSAAGGGGSGGGGGDYKDDDDKFEAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD20 CAR peptide (with flag sequence) 85. ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGATATCGTGCTGACCCAGTCCCCCGCCATCCTGTCCGCCTCTCCTGGAGAGAAGGTGACCATGACATGTCGGGCCAGCTCCTCTGTGAACTACATGGACTGGTATCAGAAGAAGCCTGGCAGCTCCCCCAAGCCTTGGATCTACGCCACCTCCAATCTGGCCTCTGGAGTGCCAGCAAGATTCAGCGGATCCGGATCTGGCACAAGCTATTCCCTGACCATCTCCAGGGTGGAGGCAGAGGATGCAGCAACATACTATTGCCAGCAGTGGTCTTTCAACCCCCCTACATTTGGCGGCGGCACCAAGCTGGAGATCAAGGGCTCTACCAGCGGAGGAGGAAGCGGAGGAGGATCCGGAGGCGGCGGCTCTAGCGAGGTGCAGCTGCAGCAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCCTCTGTGAAGATGAGCTGTAAGGCCTCCGGCTACACCTTCACATCTTATAATATGCACTGGGTGAAGCAGACACCAGGACAGGGACTGGAGTGGATCGGAGCAATCTACCCTGGCAACGGCGACACCAGCTATAATCAGAAGTTTAAGGGCAAGGCCACCCTGACAGCCGATAAGTCCTCTAGCACAGCCTACATGCAGCTGTCCTCTCTGACCAGCGAGGACTCCGCCGATTACTATTGCGCCCGGTCCAACTACTATGGCAGCTCCTATTGGTTCTTTGACGTGTGGGGAGCAGGAACAACCGTGACCGTGTCTAGCGCAAAGCCAACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACCTATGATGCCCTGCACATGCAGGCCCTGCCCCCTCGG CD20 CAR Nucleotide (without flag) 86. MLLLVTSLLLCELPHPAFLLIPDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKF KGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNY YGSSYWFFDVWGAGTTVTVSSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD20 CAR peptide (without flag sequence) 87. GATATCGTGCTGACCCAGTCCCCCGCCATCCTGTCCGCCTCTCCTGGAGAGAAGGTGACCATGACATGTCGGGCCAGCTCCTCTGTGAACTACATGGACTGGTATCAGAAGAAGCCTGGCAGCTCCCCCAAGCCTTGGATCTACGCCACCTCCAATCTGGCCTCTGGAGTGCCAGCAAGATTCAGCGGATCCGGATCTGGCACAAGCTATTCCCTGACCATCTCCAGGGTGGAGGCAGAGGATGCAGCAACATACTATTGCCAGCAGTGGTCTTTCAACCCCCCTACATTTGGCGGCGGCACCAAGCTGGAGATCAAGGGCTCTACCAGCGGAGGAGGAAGCGGAGGAGGATCCGGAGGCGGCGGCTCTAGCGAGGTGCAGCTGCAGCAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCCTCTGTGAAGATGAGCTGTAAGGCCTCCGGCTACACCTTCACATCTTATAATATGCACTGGGTGAAGCAGACACCAGGACAGGGACTGGAGTGGATCGGAGCAATCTACCCTGGCAACGGCGACACCAGCTATAATCAGAAGTTTAAGGGCAAGGCCACCCTGACAGCCGATAAGTCCTCTAGCACAGCCTACATGCAGCTGTCCTCTCTGACCAGCGAGGACTCCGCCGATTACTATTGCGCCCGGTCCAACTACTATGGCAGCTCCTATTGGTTCTTTGACGTGTGGGGAGCAGGAACAACCGTGACCGTGTCTAGC CD20 scFv Nucleotide 88. DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAY MQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS CD20 scFv peptide 89. GATATCGTGCTGACCCAGTCCCCGCCATCCTGTCCGCCTCTCCTGGAGAGAAGGTGACCATGACATGTCGGGCCAGCTCCTCTGTGAACTACATGGACTGGTATCAGAAGAAGCCTGGCAGCTCCCCCAAGCCTTGGATCTACGCCACCTCCAATCTGGCCTCTGGAGTGCCAGCAAGATTCAGCGGATCCGGATCTGGCACAAGCTATTCCCTGACCATCTCCAGGGTGGAGGCAGAGGATGCAGCAACATACTATTGC CAGCAGTGGTCTTTCAACCCCCCTACATTTGCCGGCGGCACCAAGCTGGAGATCAAG CD20 scFv VL Nucleotide 90. DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK CD20 scFv VL peptide 91. GGCTCTACCAGCGGAGGAGGAAGCGGAGGAGGATCCGGAGGCGGCGGCTTCTAGC 218 linker nucleotides 92. GSTSGGGSGGGSGGGGSS 218 Linker Peptide 93. GAGGTGCAGCTGCAGCAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCCTCTGTGAAGATGAGCTGTAAGGCCTCCGGCTACACCTTCACATCTTATAATATGCACTGGGTGAAGCAGACACCAGGACAGGGACTGGAGTGGATCGGAGCAATCTACCCTGGCAACGGCGACACCAGCTATAATCAGAAGTTTAAGGGCAAGGCCACCCTGACAGCCGATAAGTCCTCTAGCACAGCCTACATGCAGCTGTCCTC TCTGACCAGCGAGGACTCCGCCGATTACTATTGCGCCCGGTCCAACTACTATGGCAGCTCCTATTGGTTCTTTGACGTGTGGGGAGCAGGAACAACCGTGACCGTGTCTAGC CD20 scFv VH Nucleotide 94. EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS CD20 scFv VH peptide 95. GACTACAAAGACGATGACGACAAG Flag nucleotide 96. DYKDDDDK Flag peptide 97. GCAAAGCCAACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGC CD8 HTM nucleotides 98. AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC CD8 HTM peptide 99. AAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTG 41BB nucleotide 100. CGCGTGAAGTTCAAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGA CACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACCTATGATGCCCTGCACATGCAGGCCCTGCCCCCTCGG CD3ζ nucleotides 101. ACATGTCTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATTGCCTGACGCGTAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTCAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGAGCCCACAACCCCTCACTCGGCGCGTGAACACAATTCTGCAGTCGAAGGCGTACCGTCACTTACGAGTCGGTAGCCTGCAGGGCCGCCACCATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGCTAGCAGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCCATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCCATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACCGGCAGCGGAGCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCCATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCCGGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGGCGAGGGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACCTCCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGCGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTGAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGGTGACGCCGGCGCTAGTGTCGACGTAGTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAATGTGTGTGTTGGTTTTTTGTGTGTCGTCGTCTCACTTGGATCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGGGAAATGTGTGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC Construct C.3U complete vector nucleotides 102. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGCTAGCAGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCCATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCCATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACCGGCAGCGGAGCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCCATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCCGGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGGCGAGGGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACCTCCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGCGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTGAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGG Construct C.3U nucleotide 103. MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKASRRKRGSGEGRGSLLTCGDVEENPGPMPLPVTALLLPLALLLHAARPILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETGSGATNFSLLKQAGDVEENPGPMPLGLLWLGLALLGALHAQAGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVGSGATNFSLLKQAGDVEENPGPLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Construct C.3U polypeptide 104. CTGCTGCTGGTGACCTCCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCGACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGCGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTGAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTTAAGCAGCCCTTCATGCGCCCTGTGCAGACCACACAGGAGGAGGACGGCTGCAGCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGCGAGCTGCGCGTGAAGTTCAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGG CD19 CAR Nucleotide 105. LLLVTSLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNS LQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR CD19 CAR peptide 106. GACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATCACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGC CD19 scFv Nucleotide 107. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLK MNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS CD19 scFv peptide 108. GACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGACAGAGTGACCATCTCCTGTAGGGCCTCTCAGGATATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGATGGCACAGTGAAGCTGCTGATCTACCACACCTCCAGGCTGCACTCTGGAGTGCCAAGCCGGTTCTCCGGATCTGGAAGCGGCACCGACTATTCCCTGACAATCTCTAACCTGGAGCAGGAGGATATCGCCACATACTTTTG CCAGCAGGGCAATACCCTGCCATATACATTCGGCGGAGGAACCAAGCTGGAGATC CD19 scFv VL Nucleotide 109. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEI CD19 scFv VL peptide 110. ACCGGATCCACATCTGGAAGCGGCAAGCCAGGAAGCGGAGAGGGATCCACAAAGGGA CD19 scFv linker nucleotide 111. TGSTSGSGKPGSGEGSTKG CD19 scFv linker peptide 112. GAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCATCCCAGTCTCTGAGCGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCTGACTACGGCGTGTCCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCTGAGACCACATACTATAATTCTGCCCTGAAGAGCCGCCTGACCATCATCAAGGACAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGC AGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACAGTGTCTAGC CD19 scFv VH Nucleotide 113. EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS CD19 scFv VH peptide 114. GGCGCTGTGCACAACCAGAGGACTGGATTTCGCCTGCGAC CD8 hinge nucleotide 115. GAVHTRGLDFACD CD8 hinge peptide 116. TTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTG CD28 transmembrane domain nucleotides 117. FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 transmembrane domain peptide 118. CGCGTGAAGTTCAAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGA CACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGG CD3 ζ domain nucleotides 119. ESKYGPPSPPSPA Modified IgG4 hinge 120. ESKYGPPSPPSP Modified IgG4 hinge 121. PSPRPAGQFQTLV PD1 Hinge 122. EPKSCDKTHTCP IgG1 hinge 123. GAVHTRGLDFACD CD8 Hinge 124. LCPSPLFPGPSKP CD28 Hinge 125. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGCTAGCAGAAGAAAGAGAGGCAGCGGCGAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCCATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCCATCCTGTGGCACGAGATGTGGCACGAGGGCCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAGACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAGAAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACTACCACGTGTTCAGAAGAATCAGCAAGGGCAGCAATACAAGCAAGGAAAACCCCTTCCTGTTCGCCCTGGAGGCCGTGGTGATCAGCGTGGGCAGCATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTCTGGCTGGAGAGAACCATGCCTAGAATCCCCACCCTGAAGAACCTGGAGGACCTGGTGACCGAGTACCACGGCAACTTCAGCGCCTGGAGCGGCGTGAGCAAGGGCCTGGCCGAGAGCCTGCAGCCCGACTACAGCGAGCGACTGTGCCTGGTGAGCGAGATTCCCCCTAAGGGCGGGGCCTTGGGTGAGGGACCCGGGGCAAGCCCGTGCAATCAGCACAGCCCCTACTGGGCCCCCCCCTGTTACACCCTGAAGCCCGAGACCGGCAGCGGAGCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCCATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCCGGCGTTCAAGTGGAGACTATCTCCCCCGGCGACGGCAGAACCTTCCCTAAGAGAGGGCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGAGGAGGGTGTGGCCCAAATGAGCGTGGGGCAGAGAGCCAAATTGACCATCTCACCCGACTACGCTTACGGGGCCACCGGCCATCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGGCGAGGGCAAGGACACCATCCCCTGGCTGGGCCACCTGCTGGTGGGCCTGAGCGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAACACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGCAACACCCCCGACCCTAGCAAGTTCTTCTCTCAGCTGAGCAGCGAGCACGGCGGCGACGTGCAGAAGTGGCTGAGCAGCCCATTTCCTAGCAGCAGCTTTAGCCCCGGCGGCCTAGCTCCCGAGATCAGCCCCCTGGAAGTACTGGAGAGAGATAAGGTGACACAGCTGCTGCTGCAGCAAGACAAGGTGCCCGAGCCCGCTAGCCTGAGCAGCAACCACAGCCTGACAAGCTGCTTCACCAACCAAGGCTACTTCTTCTTCCACCTGCCCGACGCCCTGGAGATCGAGGCCTGCCAAGTGTACTTCACCTACGATCCGTACAGCGAGGAAGACCCCGACGAGGGAGTGGCCGGCGCCCCTACCGGCAGCTCGCCACAACCGCTGCAACCTCTGAGCGGCGAGGACGACGCCTACTGCACCTTCCCTAGCAGAGATGATCTGCTGCTCTTTTCCCCTAGCTTGCTGGGGGGCCCTTCGCCTCCAAGTACTGCCCCCGGAGGTAGCGGGGCCGGCGAAGAAAGAATGCCCCCAAGCCTGCAAGAAAGAGTGCCTAGAGACTGGGACCCTCAACCACTGGGCCCACCCACTCCGGGCGTGCCCGACCTGGTAGATTTTCAGCCCCCTCCCGAGCTGGTGCTACGCGAGGCCGGCGAAGAGGTGCCCGACGCTGGGCCAAGAGAGGGTGTGTCATTCCCCTGGAGCCGACCCCCCGGTCAAGGCGAGTTCAGAGCCCTGAACGCTAGACTGCCCCTGAACACCGACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAAGACCCCACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTcTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGG Construct C.2U nucleotide 126. ATGGAGATGTGGCACGAGGGACTGGAGGAGGCAAGCAGACTGTACTTTGGCGAGAGGAACGTGAAGGGCATGTTCGAGGTGCTGGAGCCACTGCACGCAATGATGGAGAGGGGACCTCAGACACTGAAGGAGACCTCCTTCAACCAGGCCTATGGCAGAGACCTGATGGAGGCCCAGGAGTGGTGCAGGAAGTACATGAAGTCTGGCAATGTGAAGGACCTGCTGCAGGCCTGGGATCTGTACTATCACGTGTTTCGGAGAATCAGCAAGGGCTCCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGACCTATGCCACTGGGACTGCTGTGGCTGGGACTGGCCCTGCTGGGCGCCCTGCACGCCCAGGCAGGAGTGCAGGTGGAGACCATCTCCCCAGGCGACGGACGCACATTTCCAAAGAGGGGACAGACCTGCGTGGTGCACTACACAGGCATGCTGGAGGATGGCAAGAAGTTCGACAGCTCCCGCGATCGGAATAAGCCCTTTAAGTTCATGCTGGGCAAGCAGGAAGTGATCAGAGGATGGGAGGAGGGAGTGGCACAGATGTCCGTGGGACAGAGGGCCAAGCTGACCATCTCTCCTGACTACGCCTATGGAGCAACAGGACACCCAGGAATCATCCCACCTCACGCCACCCTGGTGTTTGATGTGGAGCTGCTGAAGCTGGGCGAGGGCTCTAACACAAGCAAGGAGAATCCTTTTCTGTTCGCACTGGAGGCAGTGGTCATCTCCGTGGGCTCTATGGGCCTGATCATCAGCCTGCTGTGCGTGTACTTTTGGCTGGAGAGAACCATGCCAAGGATCCCCACACTGAAGAACCTGGAGGACCTGGTGACCGAGTATCACGGCAATTTCAGCGCCTGGTCCGGCGTGTCTAAGGGACTGGCAGAGTCCCTGCAGCCAGATTACTCTGAGCGGCTGTGCCTGGTGTCCGAGATCCCACCCAAGGGAGGCGCCCTGGGAGAGGGACCAGGAGCCAGCCCTTGCAACCAGCACTCCCCATATTGGGCCCCTCCATGTTACACACTGAAGCCCGAGACCGGCTCTGGCGCCACAAACTTCAGCCTGCTGAAGCAAGCAGGCGACGTGGAAGAAAATCCAGGACCTATGGCACTGCCTGTGACCGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCTATCCTGTGGCACGAAATGTGGCATGAAGGCCTGGAGGAGGCAAGCAGGCTGTATTTTGGCGAGCGGAATGTGAAAGGAATGTTTGAAGTCCTGGAACCTCTGCATGCCATGATGGAAAGGGGACCACAGACACTGAAGGAGACCAGCTTTAACCAGGCCTATGGAAGGGACCTGATGGAGGCACAGGAGTGGTGCCGGAAGTACATGAAGTCCGGAAATGTGAAAGACCTGCTGCAGGCCTGGGATCTGTATTATCACGTGTTCAGGCGCATCTCTAAGGGCAAGGATACCATCCCCTGGCTGGGACACCTGCTGGTGGGACTGAGCGGAGCCTTTGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCAGAAATACCGGCCCTTGGCTGAAGAAGGTGCTGAAGTGTAACACACCAGACCCCTCCAAGTTCTTTTCTCAGCTGTCTAGCGAGCACGGCGGCGATGTGCAGAAGTGGCTGTCCTCTCCTTTTCCAAGCTCCTCTTTCAGCCCAGGAGGACTGGCACCAGAGATCTCCCCCCTGGAGGTGCTGGAGAGGGACAAGGTGACCCAGCTGCTGCTGCAGCAGGATAAGGTGCCCGAGCCTGCCAGCCTGAGCTCCAACCACAGCCTGACATCCTGCTTTACCAATCAGGGCTATTTCTTTTTCCACCTGCCAGACGCCCTGGAGATCGAGGCCTGTCAGGTGTACTTCACCTATGATCCATACTCCGAAGAGGACCCAGATGAGGGAGTGGCCGGCGCCCCCACAGGCTCTAGCCCACAGCCACTGCAGCCTCTGTCTGGAGAGGACGATGCCTACTGTACCTTTCCCAGCCGCGACGATCTGCTGCTGTTCAGCCCTTCCCTGCTGGGAGGACCATCTCCACCTAGCACAGCACCAGGAGGAAGCGGGGCAGGGGAGGAGCGGATGCCACCATCTCTGCAGGAGAGGGTGCCTAGGGACTGGGATCCTCAGCCACTGGGACCTCCAACCCCTGGAGTGCCAGACCTGGTGGATTTTCAGCCCCCTCCAGAGCTGGTGCTGCGGGAGGCAGGAGAGGAGGTGCCTGACGCAGGACCACGCGAGGGCGTGAGCTTTCCATGGTCCAGGCCACCTGGACAGGGAGAGTTCAGAGCCCTGAACGCCAGGCTGCCTCTGAATACAGACGCCTATCTGTCTCTGCAGGAGCTGCAGGGCCAGGATCCAACCCACCTGGTGGGATCCGGAGCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCTCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCCTCCGTGCTGACCCAGCCTAGCTCCGTGTCTGCCGCACCAGGACAGAAGGTGACAATCAGCTGTTCCGGCTCTACCAGCAACATCGGCAACAATTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGTCCAAGAGGCCATCTGGAGTGCCTGATCGGTTCTCCGGCTCTAAGAGCGGCAATTCCGCCTCTCTGGACATCAGCGGACTGCAGTCCGAGGACGAGGCAGATTACTATTGCGCCGCCTGGGACGATAGCCTGTCCGAGTTTCTGTTCGGCACCGGCACAAAGCTGACCGTGCTGGGCTCTACAAGCGGATCCGGCAAGCCAGGATCTGGAGAGGGCAGCACAAAGGGACAGGTGCAGCTGGTGGAGAGCGGAGGAAACCTGGTGCAGCCAGGAGGCTCCCTGCGCCTGTCTTGTGCCGCCAGCGGCTTTACCTTCGGCTCTTTTAGCATGTCCTGGGTGCGCCAGGCACCTGGAGGAGGACTGGAGTGGGTGGCCGGCCTGAGCGCCCGGTCTAGCCTGACACACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGATAACGCCAAGAATAGCGTGTACCTGCAGATGAATAGCCTGCGGGTGGAGGACACAGCCGTGTACTATTGCGCCAGGCGCTCCTATGATTCCTCTGGCTACTGGGGCCACTTTTACTCTTATATGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCACCACCACACCTGCTCCTAGACCACCTACACCCGCTCCTACCATCGCCAGCCAGCCTCTGTCTCTGAGACCTGAGGCCTGTAGACCTGCCGCTGGAGGCGCTGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCTCCTCTGGCTGGAACATGCGGCGTGCTGCTCCTGAGCCTGGTGATCACACTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTcTACATCTTTAAGCAGCCATTCATGCGCCCCGTGCAGACCACACAGGAGGAGGACGGCTGCTCCTGTCGGTTTCCAGAGGAGGAGGAGGGAGGATGTGAGCTGAGAGTGAAGTTCTCTAGGAGCGCCGATGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGG Construct C.2 Nucleotide 127. MLLLVTSLLLCELPHPAFLLIPSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGSTSGSGKPGSGEGSTKGQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKG RFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRS YDSSGYWGHFYSYMDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR TagCAR peptide 128. GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGEGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCN QHSPYWAPPCYTLKPET FKBP12:IL2RG peptide without signal peptide 129. ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSL TSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGPSPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV FRB:IL2RB peptide without signal peptide

1A顯示經工程改造以接合且活化T細胞以遞送有效負載之慢病毒顆粒表面,該有效負載含有自由FKBP12-雷帕黴素結合(FRB)、雷帕黴素活化的細胞介素受體(rapamycin activated cytokine receptor;RACR)及結合腫瘤標籤之嵌合抗原受體(CAR) (TagCAR)。雷帕黴素、FRB及RACR驅動經轉導TagCAR T細胞之擴增,同時亦抑制腫瘤增殖及針對慢病毒顆粒之免疫反應。 Figure 1A shows the surface of lentiviral particles engineered to bind and activate T cells to deliver a payload containing free FKBP12-rapamycin bound (FRB), rapamycin activated cytokine receptor (RACR), and a chimeric antigen receptor (CAR) conjugated to a tumor tag (TagCAR). Rapamycin, FRB, and RACR drive the expansion of transduced TagCAR T cells while also inhibiting tumor proliferation and immune responses to the lentiviral particles.

1B顯示TagCAR T系統如何靶向腫瘤細胞。雙特異性腫瘤標籤在一端上包含通用標籤抗原且在另一端上包含接合腫瘤或腫瘤微環境相關抗原之可調換的配體。一旦T細胞表現TagCAR,則TagCAR可結合通用標籤(例如FITC-葉酸)。 Figure 1B shows how the TagCAR T system targets tumor cells. The bispecific tumor tag contains a universal tag antigen on one end and a switchable ligand that binds to a tumor or tumor microenvironment-associated antigen on the other end. Once the T cell expresses the TagCAR, the TagCAR can bind to a universal tag (e.g., FITC-folate).

2A 至圖 2B顯示8種多順反子構築體。TagCAR之多核苷酸編碼以N端至C端順序具有以下組分之CAR:scFv (例如抗FITC E2)、鉸鏈(間隔子)、跨膜域及具有共刺激傳訊域與CD3ζ傳訊域(Z)的胞內域。構築體在以下方面不同:鉸鏈(間隔子)域,為IgG4鉸鏈(IgG4H)或CD8α鉸鏈(CD8H);跨膜域,為CD28 TM或CD8 TM;及共刺激域,為41BB共刺激域或CD28共刺激域。構築體亦在TagCAR之位置方面不同,該TagCAR存在作為構築體轉殖基因序列之前端或末端。構築體之個別多核苷酸組分藉由2A裂解位點序列分隔開。 Figures 2A to 2B show eight polycistronic constructs. The polynucleotide of TagCAR encodes a CAR having the following components in N-terminal to C-terminal order: scFv (e.g., anti-FITC E2), hinge (spacer), transmembrane domain, and intracellular domain with co-stimulatory signaling domain and CD3ζ signaling domain (Z). The constructs differ in the following aspects: hinge (spacer) domain, which is IgG4 hinge (IgG4H) or CD8α hinge (CD8H); transmembrane domain, which is CD28 TM or CD8 TM; and co-stimulatory domain, which is 41BB co-stimulatory domain or CD28 co-stimulatory domain. The constructs also differ in the location of the TagCAR, which is present as the front end or the end of the construct transgene sequence. The individual polynucleotide components of the construct are separated by the 2A cleavage site sequence.

3A 至圖 3B顯示以2或10之感染倍率(multiplicity of infection;MOI)與 2A 至圖 2B中之多核苷酸構築體一起培養之CD8+及CD4+ T細胞群體的第3天活化及第7天轉導。 Figures 3A - 3B show day 3 activation and day 7 transduction of CD8+ and CD4+ T cell populations cultured with the polynucleotide constructs in Figures 2A - 2B at a multiplicity of infection (MOI) of 2 or 10.

3C顯示當如 2A 2B中所描繪,FRB置放於轉殖基因中之第一或第二位時,T細胞中之FRB表現。 FIG. 3C shows FRB expression in T cells when FRB is placed in the first or second position in the transgene as depicted in FIGS . 2A - 2B .

4顯示當用構築體D.2或構築體C.2轉導且在單獨IL-2、單獨雷帕黴素或兩者中培養時所產生之TagCAR+ T細胞的百分比。 Figure 4 shows the percentage of TagCAR+ T cells generated when transduced with construct D.2 or construct C.2 and cultured in IL-2 alone, rapamycin alone, or both.

5A 至圖 5B顯示腫瘤細胞殺傷分析,其中PBMC用 2A 至圖 2B中揭示之構築體轉導且用僅IL-2或IL-2與雷帕黴素(rapa)處理。 Figures 5A - 5B show tumor cell killing assays in which PBMCs were transduced with the constructs disclosed in Figures 2A - 2B and treated with IL-2 alone or IL-2 and rapamycin (rapa).

6A顯示用編碼構築體V或構築體C.2多核苷酸之慢病毒轉導之PBMC中之CD19嵌合抗原受體(CAR)及TagCAR表現。 Figure 6A shows the expression of CD19 chimeric antigen receptor (CAR) and TagCAR in PBMCs transduced with lentivirus encoding construct V or construct C.2 polynucleotides.

6B顯示歷經11天,CD19-CAR+ (左圖)或FITC-葉酸+ (右圖) PBMC之數目。PBMC用編碼構築體V或構築體C.2多核苷酸之慢病毒轉導。 Figure 6B shows the number of CD19-CAR+ (left panel) or FITC-folate+ (right panel) PBMCs after day 11. PBMCs were transduced with lentivirus encoding construct V or construct C.2 polynucleotides.

7A顯示自N端至C端,多核苷酸構築體之取向。在構築體C.2中,FRB編碼在緊次於IL2Rβ。在構築體C.2U中,FRB編碼在緊次於IL2Rγ。 Figure 7A shows the orientation of the polynucleotide constructs from N-terminus to C-terminus. In construct C.2, FRB is encoded immediately after IL2Rβ. In construct C.2U, FRB is encoded immediately after IL2Rγ.

7B顯示在5個供體中,在用編碼構築體V或構築體C.2多核苷酸之慢病毒轉染之後3天,CD25 T細胞的百分比。 Figure 7B shows the percentage of CD25 T cells in 5 donors 3 days after transfection with lentivirus encoding construct V or construct C.2 polynucleotides.

7C顯示在第7天,TagCAR T細胞之百分比。PBMC用編碼構築體C.2或構築體C.2U多核苷酸之慢病毒轉導其在第7天進行染色。 Figure 7C shows the percentage of TagCAR T cells on day 7. PBMCs were transduced with lentivirus encoding construct C.2 or construct C.2U polynucleotides and stained on day 7.

7D顯示在第7天,T細胞中之TagCAR MFI。PBMC用編碼構築體C.2或構築體C.2U多核苷酸之慢病毒轉導。 Figure 7D shows TagCAR MFI in T cells on day 7. PBMCs were transduced with lentivirus encoding construct C.2 or construct C.2U polynucleotides.

7E顯示描繪T細胞中之FRB:IL2Rβ及FRB:IL2Rγ之表現的免疫墨點。PBMC用編碼構築體C.2或構築體C.2U多核苷酸之慢病毒轉導。使用針對FRB之兔pAb來偵測FRB。在第8天收集細胞用於西方墨點。 Figure 7E shows immunoblots depicting the expression of FRB:IL2Rβ and FRB:IL2Rγ in T cells. PBMCs were transduced with lentivirus encoding construct C.2 or construct C.2U polynucleotides. FRB was detected using a rabbit pAb against FRB. Cells were collected on day 8 for Western blots.

7F顯示描繪T細胞中之FKBP12:IL2Rβ及FKBP12:IL2Rγ之表現的免疫墨點。PBMC用編碼構築體C.2或構築體C.2U多核苷酸之慢病毒轉導。使用針對FKBP12之小鼠mAb來偵測FKBP12。在第8天收集細胞用於西方墨點。 Figure 7F shows immunoblots depicting the expression of FKBP12:IL2Rβ and FKBP12:IL2Rγ in T cells. PBMCs were transduced with lentivirus encoding construct C.2 or construct C.2U polynucleotides. FKBP12 was detected using a mouse mAb against FKBP12. Cells were collected on day 8 for Western blots.

8A顯示在1個供體中,歷經14天,用構築體C.2U或構築體C.2轉導之總Tag-CAR+ T細胞。Tag-CAR+ T細胞用IL-2 (250 U/mL)或雷帕黴素(10 nM)處理。 Figure 8A shows the total Tag-CAR+ T cells transduced with construct C.2U or construct C.2 over 14 days in one donor. Tag-CAR+ T cells were treated with IL-2 (250 U/mL) or rapamycin (10 nM).

8B顯示在1個供體中,歷經12天,用構築體C.2U或構築體C.2轉導之總Tag-CAR+ T細胞。Tag-CAR+ T細胞用IL-2 (250 U/mL)、雷帕黴素(10 nM)、雷帕黴素與IL-2、或AP21967 (50 nM)處理。 Figure 8B shows total Tag-CAR+ T cells transduced with construct C.2U or construct C.2 over 12 days in one donor. Tag-CAR+ T cells were treated with IL-2 (250 U/mL), rapamycin (10 nM), rapamycin and IL-2, or AP21967 (50 nM).

9A顯示以2或10之感染倍率(MOI),與構築體C.2一起培養之CD8+及CD4+ T細胞群體的第3天活化及第7天轉導。 Figure 9A shows day 3 activation and day 7 transduction of CD8+ and CD4+ T cell populations cultured with construct C.2 at a multiplicity of infection (MOI) of 2 or 10.

9B顯示在轉導後第7天,用構築體C.2轉導之表現TagCAR之CD8+ T細胞的代表性流式細胞分析技術圖。 Figure 9B shows representative flow cytometry analysis of CD8+ T cells expressing TagCAR transduced with construct C.2 at day 7 after transduction.

10A顯示用於量測T細胞活化及TagCAR T細胞豐度之活體外分析方法的時間軸。在第0天,PBMC用慢病毒顆粒轉導。在第3天,添加IL-2或IL-2與雷帕黴素以量測T細胞活化。在第7、11及14天,量測TagCAR T細胞豐度。 Figure 10A shows a timeline of the in vitro assay used to measure T cell activation and TagCAR T cell abundance. On day 0, PBMCs were transduced with lentiviral particles. On day 3, IL-2 or IL-2 and rapamycin were added to measure T cell activation. On days 7, 11, and 14, TagCAR T cell abundance was measured.

10B顯示歷經2週,TagCAR T細胞之增濃(左)及擴增(右)。藉由流式細胞分析技術量測增濃。藉由流式細胞分析技術與計數珠粒量測擴增。 Figure 10B shows the enrichment (left) and expansion (right) of TagCAR T cells over 2 weeks. Enrichment was measured by flow cytometry. Expansion was measured by flow cytometry and counting beads.

11A顯示描繪在將乳房癌細胞(MDA-MB-231或MDA)與雷帕黴素、TagCAR T細胞與FITC-葉酸、或TagCAR T細胞、FITC-葉酸與雷帕黴素一起培育之後腫瘤細胞生長的圖。未與TagCAR T細胞、FITC-葉酸或雷帕黴素一起培育之乳房癌細胞藉由箭頭顯示。在72小時、144小時及216小時再引入腫瘤細胞(亦即,腫瘤細胞再刺激)。 FIG. 11A shows a graph depicting tumor cell growth after incubation of breast cancer cells (MDA-MB-231 or MDA) with rapamycin, TagCAR T cells with FITC-folate, or TagCAR T cells, FITC-folate, and rapamycin. Breast cancer cells not incubated with TagCAR T cells, FITC-folate, or rapamycin are shown by arrows. Tumor cells were reintroduced at 72 hours, 144 hours, and 216 hours (i.e., tumor cell restimulation).

11B顯示描繪在將乳房癌細胞(MDA-MB-231或MDA)與雷帕黴素、TagCAR T細胞與FITC-葉酸、或TagCAR T細胞、FITC-葉酸與雷帕黴素(rapa)一起培育之後T細胞增殖的圖。未與TagCAR T細胞、FITC-葉酸或雷帕黴素一起培育之乳房癌細胞藉由箭頭顯示。在72小時、144小時及216小時再引入腫瘤細胞(亦即,腫瘤細胞再刺激)。 FIG. 11B shows a graph depicting T cell proliferation after incubation of breast cancer cells (MDA-MB-231 or MDA) with rapamycin, TagCAR T cells with FITC-folate, or TagCAR T cells, FITC-folate, and rapamycin (rapa). Breast cancer cells not incubated with TagCAR T cells, FITC-folate, or rapamycin are shown by arrows. Tumor cells were reintroduced at 72 hours, 144 hours, and 216 hours (i.e., tumor cell restimulation).

12A顯示乳癌之活體內小鼠模型之時間軸。在實驗開始之前十四天,對NSG MHCI/II DKO小鼠注射FRα+ MDA-MB-231細胞。在實驗之第一天(D0),對小鼠輸注離體產生之TagCAR T細胞。對小鼠皮下注射FITC-葉酸,每週兩次,持續四週。每週收集血液用於流式細胞分析技術。 Figure 12A shows the timeline of the in vivo mouse model of breast cancer. Fourteen days before the start of the experiment, NSG MHCI/II DKO mice were injected with FRα+ MDA-MB-231 cells. On the first day of the experiment (D0), mice were infused with ex vivo generated TagCAR T cells. Mice were injected subcutaneously with FITC-folate twice a week for four weeks. Blood was collected weekly for flow cytometry analysis.

12B顯示描繪歷經4週小鼠中之腫瘤體積的圖。對小鼠注射:(i) 10e6個未用慢病毒顆粒(Mock T細胞)與FITC-葉酸轉導的T細胞;(ii) 10e6個無FITC-葉酸之TagCAR T細胞;(iii) 5e6個TagCAR T細胞與FITC-葉酸;及(iv) 10e6個TagCAR T細胞與FITC-葉酸。使用測徑器來量測腫瘤體積。 Figure 12B shows a graph depicting tumor volume in mice over 4 weeks. Mice were injected with: (i) 10e6 T cells transduced with no lentiviral particles (Mock T cells) and FITC-folate; (ii) 10e6 TagCAR T cells without FITC-folate; (iii) 5e6 TagCAR T cells with FITC-folate; and (iv) 10e6 TagCAR T cells with FITC-folate. Tumor volume was measured using a caliper.

13A顯示乳癌之活體內小鼠模型之時間軸。在實驗開始之前十四天,對NSG MHCI/II DKO小鼠注射FRα+ MDA-MB-231細胞。在實驗之第一天(D0),小鼠用PBMC人源化且投與慢病毒顆粒。對小鼠皮下注射FITC-葉酸,每週兩次,持續7週。每週收集血液用於流式細胞分析技術。使用測徑器來量測腫瘤體積。 Figure 13A shows the timeline of the in vivo mouse model of breast cancer. Fourteen days before the start of the experiment, NSG MHCI/II DKO mice were injected with FRα+ MDA-MB-231 cells. On the first day of the experiment (D0), mice were humanized with PBMC and administered lentiviral particles. Mice were injected subcutaneously with FITC-folate twice a week for 7 weeks. Blood was collected weekly for flow cytometry. Tumor volume was measured using a caliper.

13B顯示藉由流式細胞分析技術進行之循環TagCAR T細胞偵測。左圖描繪在第7天,每CD3+ T細胞之TagCAR+的百分比。右圖描繪在第7天,每µL血液之CD3+/TagCAR+ T細胞之總數目。對小鼠注射:(i)無載體與FITC-葉酸;(ii) 100e6個TagCAR載體與FITC-葉酸之轉染單位(transfecting unit;TU);(iii) 25e6個TU TagCAR載體與FITC-葉酸;及(iv) 100e6個TU TagCAR載體與FITC-葉酸。 Figure 13B shows detection of circulating TagCAR T cells by flow cytometry. The left panel depicts the percentage of TagCAR+ per CD3+ T cell at day 7. The right panel depicts the total number of CD3+/TagCAR+ T cells per µL of blood at day 7. Mice were injected with: (i) no vector and FITC-folate; (ii) 100e6 transfecting units (TU) of TagCAR vector and FITC-folate; (iii) 25e6 TU TagCAR vector and FITC-folate; and (iv) 100e6 TU TagCAR vector and FITC-folate.

13C顯示描繪歷經7週小鼠中之腫瘤體積的圖。使用測徑器來量測腫瘤體積。 Figure 13C shows a graph depicting tumor volume in mice over 7 weeks. Tumor volume was measured using a caliper.

14A顯示描繪歷經25天小鼠中之腫瘤體積的圖。對小鼠注射:(i)單獨FITC-葉酸;(ii) 5.0e6個無FITC-葉酸之TagCAR載體的轉染單位(TU);(iii) 0.2e6個TagCAR載體與FITC-葉酸之TU;(iv) 1.0e6個TagCAR載體與FITC-葉酸之TU;及(v) 5.0e6個TagCAR載體與FITC-葉酸之TU。使用測徑器來量測腫瘤體積。 Figure 14A shows a graph depicting tumor volume in mice over 25 days. Mice were injected with: (i) FITC-folate alone; (ii) 5.0e6 transfection units (TU) of TagCAR vector without FITC-folate; (iii) 0.2e6 TU of TagCAR vector with FITC-folate; (iv) 1.0e6 TU of TagCAR vector with FITC-folate; and (v) 5.0e6 TU of TagCAR vector with FITC-folate. Tumor volume was measured using a caliper.

14B顯示藉由流式細胞分析技術偵測之每µL血液的循環CD3 TagCAR T細胞。對小鼠注射:(i)單獨FITC-葉酸(三角形);(ii) 5.0e6 TU單獨TagCAR載體;(iii) 0.2e6 TU TagCAR載體與FITC-葉酸;(iv) 1.0e6 TU TagCAR載體與FITC-葉酸;及(v) 5.0e6 TU TagCAR載體與FITC-葉酸。 Figure 14B shows circulating CD3 TagCAR T cells per µL of blood detected by flow cytometry. Mice were injected with: (i) FITC-Folic acid alone (triangles); (ii) 5.0e6 TU TagCAR vector alone; (iii) 0.2e6 TU TagCAR vector and FITC-Folic acid; (iv) 1.0e6 TU TagCAR vector and FITC-Folic acid; and (v) 5.0e6 TU TagCAR vector and FITC-Folic acid.

TW202434621A_112142462_SEQL.xmlTW202434621A_112142462_SEQL.xml

Claims (161)

一種多順反子構築體,其包含以5'至3'順序:(a)第一表現卡匣,其包含編碼FRB之核苷酸序列;(b)第二表現卡匣,其包含編碼合成細胞介素γ鏈多肽之核苷酸序列;(c)第三表現卡匣,其包含編碼合成細胞介素β鏈多肽之核苷酸序列;及(d)第四表現卡匣,其包含編碼嵌合抗原受體(CAR)之核苷酸序列,其中該等表現卡匣各藉由編碼裂解位點序列之核苷酸序列分隔開。A polycistronic construct comprises, in 5' to 3' order: (a) a first expression cassette comprising a nucleotide sequence encoding FRB; (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic interleukin γ chain polypeptide; (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic interleukin β chain polypeptide; and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence. 如請求項1之構築體,其中編碼該FRB之核苷酸序列與SEQ ID NO: 3、13或50之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。The construct of claim 1, wherein the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 3, 13 or 50. 如請求項1或請求項2之構築體,其中編碼該FRB之核苷酸序列包含SEQ ID NO: 3、13或50的核苷酸序列。The construct of claim 1 or claim 2, wherein the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 3, 13 or 50. 如請求項1至3中任一項之構築體,其中該FRB包含與SEQ ID NO: 4、14或51之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 1 to 3, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4, 14 or 51. 如請求項1至4中任一項之構築體,其中該FRB包含SEQ ID NO: 4、14或51的胺基酸序列。The construct of any one of claims 1 to 4, wherein the FRB comprises the amino acid sequence of SEQ ID NO: 4, 14 or 51. 如請求項1至5中任一項之構築體,其中編碼該合成細胞介素γ鏈多肽之核苷酸與SEQ ID NO: 15之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。The construct of any one of claims 1 to 5, wherein the nucleotide sequence encoding the synthetic interleukin gamma chain polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 15. 如請求項1至6中任一項之構築體,其中編碼該合成細胞介素γ鏈多肽之核苷酸包含SEQ ID NO: 15的核苷酸序列。The construct of any one of claims 1 to 6, wherein the nucleotide encoding the synthetic interleukin gamma chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 15. 如請求項1至7中任一項之構築體,其中該合成細胞介素γ鏈多肽包含介白素2受體次單元γ (IL2RG)。A construct as in any one of claims 1 to 7, wherein the synthetic interleukin gamma chain polypeptide comprises interleukin 2 receptor subunit gamma (IL2RG). 如請求項8之構築體,其中該IL2RG包含與SEQ ID NO: 16之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of claim 8, wherein the IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16. 如請求項8或請求項9之構築體,其中該IL2RG包含SEQ ID NO: 16的胺基酸序列。The construct of claim 8 or claim 9, wherein the IL2RG comprises the amino acid sequence of SEQ ID NO: 16. 如請求項1至10中任一項之構築體,其中該第二表現卡匣進一步包含編碼FRB的核苷酸序列。A construct as in any one of claims 1 to 10, wherein the second expression cassette further comprises a nucleotide sequence encoding FRB. 如請求項11之構築體,其中編碼該FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。The construct of claim 11, wherein the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 13. 如請求項11或請求項12之構築體,其中編碼該FRB之核苷酸序列包含SEQ ID NO: 13的核苷酸序列。The construct of claim 11 or claim 12, wherein the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 13. 如請求項11至13中任一項之構築體,其中該FRB包含與SEQ ID NO: 14之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 11 to 13, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. 如請求項1至14中任一項之構築體,其中該FRB包含SEQ ID NO: 14的胺基酸序列。The construct of any one of claims 1 to 14, wherein the FRB comprises the amino acid sequence of SEQ ID NO: 14. 如請求項1至15中任一項之構築體,其中該第二表現卡匣經密碼子最佳化。A construct as in any one of claims 1 to 15, wherein the second representation cassette is codon optimized. 如請求項1至16中任一項之構築體,其中該第二表現卡匣包含與SEQ ID NO: 11之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of any one of claims 1 to 16, wherein the second expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 11. 如請求項1至17中任一項之構築體,其中該第二表現卡匣包含SEQ ID NO: 11的核苷酸序列。The construct of any one of claims 1 to 17, wherein the second expression cassette comprises the nucleotide sequence of SEQ ID NO: 11. 如請求項1至18中任一項之構築體,其中該第二表現卡匣編碼與SEQ ID NO: 12之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 1 to 18, wherein the second expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 12. 如請求項1至19中任一項之構築體,其中該第二表現卡匣編碼包含SEQ ID NO: 12之序列的胺基酸序列。The construct of any one of claims 1 to 19, wherein the second expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 12. 如請求項1至15中任一項之構築體,其中該第二表現卡匣進一步包含編碼FKBP12的核苷酸序列。The construct of any one of claims 1 to 15, wherein the second expression cassette further comprises a nucleotide sequence encoding FKBP12. 如請求項21之構築體,其中編碼該FKBP12之核苷酸序列與SEQ ID NO: 21或55之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。The construct of claim 21, wherein the nucleotide sequence encoding the FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 21 or 55. 如請求項21或請求項22之構築體,其中編碼該FKBP12之核苷酸序列包含SEQ ID NO: 21或55的核苷酸序列。The construct of claim 21 or claim 22, wherein the nucleotide sequence encoding the FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21 or 55. 如請求項21至23中任一項之構築體,其中該FKBP12包含與SEQ ID NO: 22之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 21 to 23, wherein the FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22. 如請求項21至24中任一項之構築體,其中該FKBP12包含SEQ ID NO: 22的胺基酸序列。The construct of any one of claims 21 to 24, wherein the FKBP12 comprises the amino acid sequence of SEQ ID NO: 22. 如請求項1至25中任一項之構築體,其中編碼該合成細胞介素β鏈多肽之核苷酸與SEQ ID NO: 23或61之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。The construct of any one of claims 1 to 25, wherein the nucleotide sequence encoding the synthetic interleukin β chain polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. 如請求項1至26中任一項之構築體,其中編碼該合成細胞介素β鏈多肽之核苷酸包含SEQ ID NO: 23或61的核苷酸序列。The construct of any one of claims 1 to 26, wherein the nucleotide encoding the synthetic interleukin β chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 23 or 61. 如請求項1至27中任一項之構築體,其中該合成細胞介素β鏈多肽包含介白素2受體次單元β (IL2RB)。The construct of any one of claims 1 to 27, wherein the synthetic interleukin β chain polypeptide comprises interleukin 2 receptor subunit β (IL2RB). 如請求項28之構築體,其中該IL2RB包含與SEQ ID NO: 24或62之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of claim 28, wherein the IL2RB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24 or 62. 如請求項28或請求項29之構築體,其中該IL2RB包含SEQ ID NO: 24或62的胺基酸序列。The construct of claim 28 or claim 29, wherein the IL2RB comprises the amino acid sequence of SEQ ID NO: 24 or 62. 如請求項1至30中任一項之構築體,其中該第三表現卡匣進一步包含編碼FKBP12的核苷酸序列。The construct of any one of claims 1 to 30, wherein the third expression cassette further comprises a nucleotide sequence encoding FKBP12. 如請求項31之構築體,其中編碼該FKBP12之核苷酸序列與SEQ ID NO: 21之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。The construct of claim 31, wherein the nucleotide sequence encoding the FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 21. 如請求項31或請求項32之構築體,其中編碼該FKBP12之核苷酸序列包含SEQ ID NO: 21的核苷酸序列。The construct of claim 31 or claim 32, wherein the nucleotide sequence encoding the FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21. 如請求項31至33中任一項之構築體,其中該FKBP12包含與SEQ ID NO: 22之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 31 to 33, wherein the FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22. 如請求項31至34中任一項之構築體,其中該FKBP12包含SEQ ID NO: 22的胺基酸序列。The construct of any one of claims 31 to 34, wherein the FKBP12 comprises the amino acid sequence of SEQ ID NO: 22. 如請求項1至35中任一項之構築體,其中該第三表現卡匣經密碼子最佳化。A construct as in any one of claims 1 to 35, wherein the third representation cassette is codon optimized. 如請求項1至36中任一項之構築體,其中該第三表現卡匣包含與SEQ ID NO: 19之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of any one of claims 1 to 36, wherein the third expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 19. 如請求項1至37中任一項之構築體,其中該第三表現卡匣包含SEQ ID NO: 19的核苷酸序列。The construct of any one of claims 1 to 37, wherein the third expression cassette comprises the nucleotide sequence of SEQ ID NO: 19. 如請求項1至38中任一項之構築體,其中該第三表現卡匣編碼與SEQ ID NO: 20之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 1 to 38, wherein the third expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20. 如請求項1至39中任一項之構築體,其中該第三表現卡匣編碼包含SEQ ID NO: 20之序列的胺基酸序列。The construct of any one of claims 1 to 39, wherein the third expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 20. 如請求項1至40中任一項之構築體,其中該第三表現卡匣進一步包含編碼FRB的核苷酸序列。A construct as in any one of claims 1 to 40, wherein the third expression cassette further comprises a nucleotide sequence encoding FRB. 如請求項41之構築體,其中編碼該FRB之核苷酸序列與SEQ ID NO: 13之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。The construct of claim 41, wherein the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 13. 如請求項41或請求項42之構築體,其中編碼該FRB之核苷酸序列包含SEQ ID NO: 13的核苷酸序列。The construct of claim 41 or claim 42, wherein the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 13. 如請求項41至43中任一項之構築體,其中該FRB包含與SEQ ID NO: 14之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 41 to 43, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. 如請求項1至44中任一項之構築體,其中該FRB包含SEQ ID NO: 14的胺基酸序列。The construct of any one of claims 1 to 44, wherein the FRB comprises the amino acid sequence of SEQ ID NO: 14. 如請求項1至45中任一項之構築體,其中該CAR包含scFv域。The construct of any one of claims 1 to 45, wherein the CAR comprises a scFv domain. 如請求項46之構築體,其中該scFv域包含抗螢光異硫氰酸鹽(FITC) E2。The construct of claim 46, wherein the scFv domain comprises anti-fluorescent isothiocyanate (FITC) E2. 如請求項46或請求項47之構築體,其中該scFv域包含輕鏈可變域(VL)、連接子,及重鏈可變域(VH)。The construct of claim 46 or claim 47, wherein the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH). 如請求項48之構築體,其中該scFv VL包含與SEQ ID NO: 30或65之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of claim 48, wherein the scFv VL comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 30 or 65. 如請求項48或請求項49之構築體,其中該scFv VL包含SEQ ID NO: 30或65的核苷酸序列。The construct of claim 48 or claim 49, wherein the scFv VL comprises the nucleotide sequence of SEQ ID NO: 30 or 65. 如請求項48至50中任一項之構築體,其中該scFv VL包含與SEQ ID NO: 31之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 48 to 50, wherein the scFv VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31. 如請求項48至51中任一項之構築體,其中該scFv VL包含SEQ ID NO: 31的胺基酸序列。The construct of any one of claims 48 to 51, wherein the scFv VL comprises the amino acid sequence of SEQ ID NO: 31. 如請求項48至52中任一項之構築體,其中該scFv VH包含與SEQ ID NO: 34或67之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of any one of claims 48 to 52, wherein the scFv VH comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 34 or 67. 如請求項48至53中任一項之構築體,其中該scFv VH包含SEQ ID NO: 34或67的核苷酸序列。The construct of any one of claims 48 to 53, wherein the scFv VH comprises the nucleotide sequence of SEQ ID NO: 34 or 67. 如請求項48至54中任一項之構築體,其中該scFv VH包含與SEQ ID NO: 35之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 48 to 54, wherein the scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35. 如請求項48至55中任一項之構築體,其中該scFv VH包含SEQ ID NO: 35的胺基酸序列。The construct of any one of claims 48 to 55, wherein the scFv VH comprises the amino acid sequence of SEQ ID NO: 35. 如請求項48至56中任一項之構築體,其中該scFv連接子包含與SEQ ID NO: 32或66之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of any one of claims 48 to 56, wherein the scFv linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 32 or 66. 如請求項48至57中任一項之構築體,其中該scFv連接子包含SEQ ID NO: 32或66的核苷酸序列。The construct of any one of claims 48 to 57, wherein the scFv linker comprises the nucleotide sequence of SEQ ID NO: 32 or 66. 如請求項48至58中任一項之構築體,其中該scFv連接子包含與SEQ ID NO: 33之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 48 to 58, wherein the scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 33. 如請求項48至59中任一項之構築體,其中該scFv連接子包含SEQ ID NO: 33的胺基酸序列。The construct of any one of claims 48 to 59, wherein the scFv linker comprises the amino acid sequence of SEQ ID NO: 33. 如請求項46至60中任一項之構築體,其中該scFv包含與SEQ ID NO: 28或64之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of any one of claims 46 to 60, wherein the scFv comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 28 or 64. 如請求項46至61中任一項之構築體,其中該scFv包含SEQ ID NO: 28或64的核苷酸序列。The construct of any one of claims 46 to 61, wherein the scFv comprises the nucleotide sequence of SEQ ID NO: 28 or 64. 如請求項46至62中任一項之構築體,其中該scFv包含與SEQ ID NO: 29之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 46 to 62, wherein the scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29. 如請求項46至63中任一項之構築體,其中該scFv包含SEQ ID NO: 29的胺基酸序列。The construct of any one of claims 46 to 63, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 29. 如請求項1至64中任一項之構築體,其中該CAR包含鉸鏈域。A construct as in any one of claims 1 to 64, wherein the CAR comprises a hinge domain. 如請求項65之構築體,其中該鉸鏈域包含短鉸鏈或中等鉸鏈域。A construct as in claim 65, wherein the hinge domain comprises a short hinge domain or a medium hinge domain. 如請求項65或66之構築體,其中該鉸鏈域包含CD8或IgG。The construct of claim 65 or 66, wherein the hinge domain comprises CD8 or IgG. 如請求項67之構築體,其中該CD8鉸鏈包含CD8α鉸鏈。The construct of claim 67, wherein the CD8 hinge comprises a CD8α hinge. 如請求項68之構築體,其中該CD8α鉸鏈包含與SEQ ID NO: 38或114之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of claim 68, wherein the CD8α hinge comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 38 or 114. 如請求項68或請求項69之構築體,其中該CD8α鉸鏈包含SEQ ID NO: 38或114的核苷酸序列。The construct of claim 68 or claim 69, wherein the CD8α hinge comprises the nucleotide sequence of SEQ ID NO: 38 or 114. 如請求項68至70中任一項之構築體,其中該CD8α鉸鏈包含與SEQ ID NO: 39或115之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 68 to 70, wherein the CD8α hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39 or 115. 如請求項68至71中任一項之構築體,其中該CD8α鉸鏈包含SEQ ID NO: 39或115的胺基酸序列。The construct of any one of claims 68 to 71, wherein the CD8α hinge comprises the amino acid sequence of SEQ ID NO: 39 or 115. 如請求項1至72中任一項之構築體,其中該CAR包含跨膜域。The construct of any one of claims 1 to 72, wherein the CAR comprises a transmembrane domain. 如請求項73之構築體,其中該跨膜域包含CD8或CD28。The construct of claim 73, wherein the transmembrane domain comprises CD8 or CD28. 如請求項74之構築體,其中該CD8跨膜域包含CD8α跨膜域。The construct of claim 74, wherein the CD8 transmembrane domain comprises a CD8α transmembrane domain. 如請求項73至75中任一項之構築體,其中該跨膜域包含與SEQ ID NO: 40之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of any one of claims 73 to 75, wherein the transmembrane domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 40. 如請求項73至76中任一項之構築體,其中該跨膜域包含SEQ ID NO: 40的核苷酸序列。The construct of any one of claims 73 to 76, wherein the transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 40. 如請求項73至77中任一項之構築體,其中該跨膜域包含與SEQ ID NO: 41之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 73 to 77, wherein the transmembrane domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41. 如請求項73至78中任一項之構築體,其中該跨膜域包含SEQ ID NO: 41的胺基酸序列。A construct as in any one of claims 73 to 78, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 41. 如請求項1至79中任一項之構築體,其中該CAR包含胞內域。The construct of any one of claims 1 to 79, wherein the CAR comprises an intracellular domain. 如請求項80之構築體,其中該胞內域包含共刺激分子。The construct of claim 80, wherein the intracellular domain comprises a co-stimulatory molecule. 如請求項80或請求項81之構築體,其中該胞內域包含4-1BB、CD3ζ及/或CD28。The construct of claim 80 or claim 81, wherein the intracellular domain comprises 4-1BB, CD3ζ and/or CD28. 如請求項82之構築體,其中該4-1BB胞內域包含與SEQ ID NO: 42或69之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of claim 82, wherein the 4-1BB intracellular domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 42 or 69. 如請求項82或請求項83之構築體,其中該4-1BB胞內域包含SEQ ID NO: 42或69的核苷酸序列。The construct of claim 82 or claim 83, wherein the 4-1BB intracellular domain comprises the nucleotide sequence of SEQ ID NO: 42 or 69. 如請求項82至84中任一項之構築體,其中該4-1BB胞內域包含與SEQ ID NO: 43之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 82 to 84, wherein the 4-1BB intracellular domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43. 如請求項82至85中任一項之構築體,其中該4-1BB胞內域包含SEQ ID NO: 43的胺基酸序列。The construct of any one of claims 82 to 85, wherein the 4-1BB intracellular domain comprises the amino acid sequence of SEQ ID NO: 43. 如請求項82至86中任一項之構築體,其中該CD3ζ胞內域包含與SEQ ID NO: 46、70、100或118之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of any one of claims 82 to 86, wherein the CD3ζ intracellular domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. 如請求項82至87中任一項之構築體,其中該CD3ζ胞內域包含SEQ ID NO: 46、70、100或118的核苷酸序列。The construct of any one of claims 82 to 87, wherein the CD3ζ intracellular domain comprises the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. 如請求項82至88中任一項之構築體,其中該CD3ζ胞內域包含與SEQ ID NO: 47之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 82 to 88, wherein the CD3ζ intracellular domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47. 如請求項82至89中任一項之構築體,其中該CD3ζ胞內域包含SEQ ID NO: 47的胺基酸序列。A construct as in any one of claims 82 to 89, wherein the CD3ζ intracellular domain comprises the amino acid sequence of SEQ ID NO: 47. 如請求項1至90中任一項之構築體,其中該第四表現卡匣包含與SEQ ID NO: 26、63、71或82之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of any one of claims 1 to 90, wherein the fourth expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71 or 82. 如請求項1至91中任一項之構築體,其中該第四表現卡匣包含SEQ ID NO: 26、63或82的核苷酸序列。The construct of any one of claims 1 to 91, wherein the fourth expression cassette comprises the nucleotide sequence of SEQ ID NO: 26, 63 or 82. 如請求項1至92中任一項之構築體,其中該第四表現卡匣編碼與SEQ ID NO: 27、72或127之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 1 to 92, wherein the fourth expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27, 72 or 127. 如請求項1至93中任一項之構築體,其中該第四表現卡匣編碼SEQ ID NO: 27、72或127的胺基酸序列。The construct of any one of claims 1 to 93, wherein the fourth expression cassette encodes an amino acid sequence of SEQ ID NO: 27, 72 or 127. 如請求項1之構築體,其中該等裂解位點序列各包含2A可裂解連接子序列。The construct of claim 1, wherein each of the cleavage site sequences comprises a 2A cleavable linker sequence. 如請求項95之構築體,其中編碼2A可裂解連接子序列之各核苷酸不同。The construct of claim 95, wherein each nucleotide encoding the 2A cleavable linker sequence is different. 如請求項95或請求項96之構築體,其中該2A可裂解連接子獨立地為T2A、P2A、E2A或F2A裂解位點。The construct of claim 95 or claim 96, wherein the 2A cleavable linker is independently a T2A, P2A, E2A or F2A cleavage site. 如請求項95或97中任一項之構築體,其中該2A可裂解連接子獨立地為P2A或T2A。A construct as in any of claim 95 or 97, wherein the 2A cleavable linker is independently P2A or T2A. 如請求項95至98中任一項之構築體,其中至少一個2A可裂解連接子為P2A,且編碼該P2A可裂解連接子之核苷酸序列包含與SEQ ID NO: 17、25、52或58至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。The construct of any one of claims 95 to 98, wherein at least one 2A cleavable linker is P2A, and the nucleotide sequence encoding the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17, 25, 52 or 58. 如請求項99之構築體,其中編碼該P2A可裂解連接子之核苷酸序列述於SEQ ID NO: 17、25、52或58中。The construct of claim 99, wherein the nucleotide sequence encoding the P2A cleavable linker is described in SEQ ID NO: 17, 25, 52 or 58. 如請求項97至100中任一項之構築體,其中該P2A可裂解連接子包含與SEQ ID NO: 18至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。The construct of any one of claims 97 to 100, wherein the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18. 如請求項101之構築體,其中該P2A可裂解連接子包含SEQ ID NO: 18中所述的序列。The construct of claim 101, wherein the P2A cleavable linker comprises the sequence described in SEQ ID NO: 18. 如請求項95至102中任一項之構築體,其中至少一個2A可裂解連接子為T2A,且編碼該T2A可裂解連接子之核苷酸序列包含與SEQ ID NO: 9至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。The construct of any one of claims 95 to 102, wherein at least one 2A cleavable linker is T2A, and the nucleotide sequence encoding the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9. 如請求項95至103之構築體,其中編碼該T2A可裂解連接子之核苷酸序列述於SEQ ID NO: 9中。The construct of claim 95 to 103, wherein the nucleotide sequence encoding the T2A cleavable linker is described in SEQ ID NO: 9. 如請求項97、98、103及104中任一項之構築體,其中該T2A可裂解連接子包含與SEQ ID NO: 10至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。The construct of any one of claims 97, 98, 103 and 104, wherein the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10. 如請求項95至105之構築體,其中該T2A可裂解連接子包含SEQ ID NO: 10中所述之序列。The construct of claim 95 to 105, wherein the T2A cleavable linker comprises the sequence described in SEQ ID NO: 10. 如請求項1至106中任一項之構築體,其中該等裂解位點序列中之至少一者包含弗林蛋白酶(furin)裂解位點序列。The construct of any one of claims 1 to 106, wherein at least one of the cleavage site sequences comprises a furin cleavage site sequence. 如請求項107之構築體,其中該弗林蛋白酶裂解位點序列位於該第一表現卡匣與該第二表現卡匣之間。The construct of claim 107, wherein the furin cleavage site sequence is located between the first expression cassette and the second expression cassette. 如請求項107或請求項108之構築體,其中編碼該弗林蛋白酶裂解位點序列之核苷酸序列包含與SEQ ID NO: 7至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的序列。The construct of claim 107 or claim 108, wherein the nucleotide sequence encoding the furin cleavage site sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7. 如請求項107至109中任一項之構築體,其中編碼該弗林蛋白酶裂解位點序列之核苷酸序列包含SEQ ID NO: 7中所述的序列。The construct of any one of claims 107 to 109, wherein the nucleotide sequence encoding the furin cleavage site sequence comprises the sequence set forth in SEQ ID NO: 7. 如請求項107至110中任一項之構築體,其中該弗林蛋白酶裂解位點序列包含與SEQ ID NO: 8之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 107 to 110, wherein the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8. 如請求項106至111中任一項之構築體,其中該弗林蛋白酶裂解位點序列包含SEQ ID NO: 8的胺基酸序列。The construct of any one of claims 106 to 111, wherein the furin cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 8. 如請求項1至112中任一項之構築體,其中該裂解位點序列包含弗林蛋白酶裂解位點序列及T2A裂解序列(furinT2A)。The construct of any one of claims 1 to 112, wherein the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furinT2A). 如請求項1至113中任一項之構築體,其中編碼該裂解位點序列之核苷酸序列與SEQ ID NO: 5之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致。The construct of any one of claims 1 to 113, wherein the nucleotide sequence encoding the cleavage site sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 5. 如請求項1至114中任一項之構築體,其中編碼該裂解位點序列之核苷酸序列包含SEQ ID NO: 5的核苷酸序列。The construct of any one of claims 1 to 114, wherein the nucleotide sequence encoding the cleavage site sequence comprises the nucleotide sequence of SEQ ID NO: 5. 如請求項1至115中任一項之構築體,其中該裂解位點序列包含與SEQ ID NO: 6之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的胺基酸序列。The construct of any one of claims 1 to 115, wherein the cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 6. 如請求項1至116中任一項之構築體,其中該裂解位點序列包含SEQ ID NO: 6的胺基酸序列。The construct of any one of claims 1 to 116, wherein the cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 6. 如請求項1至109中任一項之構築體,其中該第一表現卡匣及該第二表現卡匣藉由furinT2A分隔開,該第二表現卡匣及該第三表現卡匣藉由P2A分隔開,且該第三表現卡匣及該第四表現卡匣藉由P2A分隔開。A structure as in any one of claims 1 to 109, wherein the first expression cassette and the second expression cassette are separated by furin T2A, the second expression cassette and the third expression cassette are separated by P2A, and the third expression cassette and the fourth expression cassette are separated by P2A. 如請求項1至118中任一項之構築體,其中該構築體包含與SEQ ID NO: 1之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of any one of claims 1 to 118, wherein the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 1. 如請求項1至119中任一項之構築體,其中該構築體包含SEQ ID NO: 1的核苷酸序列。The construct of any one of claims 1 to 119, wherein the construct comprises the nucleotide sequence of SEQ ID NO: 1. 如請求項1至120中任一項之構築體,其中該構築體編碼包含與SEQ ID NO: 2之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致之胺基酸序列的多肽。The construct of any one of claims 1 to 120, wherein the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2. 如請求項1至121中任一項之構築體,其中該構築體編碼包含SEQ ID NO: 2之胺基酸序列的多肽。The construct of any one of claims 1 to 121, wherein the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. 如請求項1至118中任一項之構築體,其中該構築體包含與SEQ ID NO: 48之核苷酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致的核苷酸序列。The construct of any one of claims 1 to 118, wherein the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 48. 如請求項1至118或123中任一項之構築體,其中該構築體包含SEQ ID NO: 48的核苷酸序列。The construct of any one of claims 1 to 118 or 123, wherein the construct comprises the nucleotide sequence of SEQ ID NO: 48. 如請求項1至118、123或124中任一項之構築體,其中該構築體編碼包含與SEQ ID NO: 49之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%、99%或100%一致之胺基酸序列的多肽。The construct of any one of claims 1 to 118, 123 or 124, wherein the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 49. 如請求項1至118或123至125中任一項之構築體,其中該構築體編碼包含SEQ ID NO: 49之胺基酸序列的多肽。The construct of any one of claims 1 to 118 or 123 to 125, wherein the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 49. 一種病毒載體,其包含如請求項1至126中任一項之多順反子構築體。A viral vector comprising the polycistronic construct of any one of claims 1 to 126. 如請求項127之病毒載體,其中該病毒載體為慢病毒載體。The viral vector of claim 127, wherein the viral vector is a lentiviral vector. 如請求項127或請求項128之病毒載體,其中該病毒載體進一步包含一或多種表面T細胞活化劑。A viral vector as claimed in claim 127 or claim 128, wherein the viral vector further comprises one or more surface T cell activators. 如請求項129之病毒載體,其中該一或多種表面T細胞活化劑包含CD58、抗-CD3,或CD80。The viral vector of claim 129, wherein the one or more surface T cell activators comprise CD58, anti-CD3, or CD80. 一種細胞,其包含如請求項127至130中任一項之病毒載體。A cell comprising the viral vector of any one of claims 127 to 130. 如請求項131之細胞,其中該細胞包含幹細胞或前驅細胞。The cell of claim 131, wherein the cell comprises a stem cell or a progenitor cell. 如請求項132之細胞,其中該幹細胞包含誘導性富潛能幹細胞(iPSC)。The cell of claim 132, wherein the stem cell comprises induced enriched-potential stem cells (iPSCs). 如請求項131之細胞,其中該前驅細胞包含周邊血液單核細胞(PBMC)。The cell of claim 131, wherein the progenitor cells comprise peripheral blood mononuclear cells (PBMC). 如請求項131之細胞,其中該細胞包含T細胞。The cell of claim 131, wherein the cell comprises a T cell. 如請求項131之細胞,其中該細胞包含細胞毒性先天性淋巴球(CIL)細胞。The cell of claim 131, wherein the cell comprises a cytotoxic innate lymphocyte (CIL) cell. 如請求項131之細胞,其中該細胞包含自然殺手(NK)細胞。The cell of claim 131, wherein the cell comprises a natural killer (NK) cell. 一種轉導細胞之方法,其包含使目標細胞與如請求項1至126中任一項之多順反子構築體中之任一者接觸。A method of transducing a cell, comprising contacting a target cell with any of the multicistronic constructs of any of claims 1 to 126. 如請求項138之方法,其中該目標細胞包含幹細胞。The method of claim 138, wherein the target cells comprise stem cells. 如請求項139之方法,其中該幹細胞包含誘導性富潛能幹細胞(iPSC)。The method of claim 139, wherein the stem cells comprise induced enriched-potential stem cells (iPSCs). 如請求項138之方法,其中該目標細胞包含前驅細胞。The method of claim 138, wherein the target cell comprises a progenitor cell. 如請求項141之方法,其中該前驅細胞包含周邊血液單核細胞(PBMC)。The method of claim 141, wherein the progenitor cells comprise peripheral blood mononuclear cells (PBMC). 如請求項138之方法,其中該目標細胞包含T細胞。The method of claim 138, wherein the target cell comprises a T cell. 如請求項143之方法,其中該T細胞包含CD4+或CD8+ T細胞。The method of claim 143, wherein the T cells comprise CD4+ or CD8+ T cells. 如請求項138至144中任一項之方法,其進一步包含使該目標細胞與以下接觸:(i)靶向內源基因中之目標位點的引導RNA(gRNA),及(ii) RNA引導核酸內切酶,從而將該核苷酸序列插入該內源基因中。A method as in any one of claims 138 to 144, further comprising contacting the target cell with: (i) a guide RNA (gRNA) that targets a target site in an endogenous gene, and (ii) an RNA-guided endonuclease, thereby inserting the nucleotide sequence into the endogenous gene. 一種在目標細胞中表現嵌合抗原受體及/或合成細胞介素受體之方法。A method for expressing chimeric antigen receptors and/or synthetic interleukin receptors in target cells. 如請求項146之方法,其中該目標細胞包含幹細胞。The method of claim 146, wherein the target cells comprise stem cells. 如請求項147之方法,其中該幹細胞包含誘導性富潛能幹細胞(iPSC)。The method of claim 147, wherein the stem cells comprise induced enriched-potential stem cells (iPSCs). 如請求項146之方法,其中該目標細胞包含前驅細胞。The method of claim 146, wherein the target cells comprise progenitor cells. 如請求項149之方法,其中該前驅細胞包含周邊血液單核細胞(PBMC)。The method of claim 149, wherein the progenitor cells comprise peripheral blood mononuclear cells (PBMC). 如請求項146之方法,其中該目標細胞包含T細胞。The method of claim 146, wherein the target cell comprises a T cell. 如請求項151之方法,其中該T細胞包含CD4+或CD8+ T細胞。The method of claim 151, wherein the T cells comprise CD4+ or CD8+ T cells. 如請求項146至152中任一項之方法,其中該方法係離體或在活體外進行。The method of any one of claims 146 to 152, wherein the method is performed ex vivo or in vitro. 如請求項146至152中任一項之方法,其中該方法係在活體內進行。The method of any one of claims 146 to 152, wherein the method is performed in vivo. 一種細胞,其係藉由如請求項138至154中任一項之方法產生。A cell produced by the method of any one of claims 138 to 154. 一種向個體投與如請求項155之細胞之方法。A method of administering the cell of claim 155 to an individual. 一種向個體投與如請求項127至130中任一項之病毒載體之方法。A method of administering the viral vector of any one of claims 127 to 130 to a subject. 如請求項157之方法,其進一步包含投與雙官能配體以標記該個體中之癌細胞,其中該雙官能配體特異性結合腫瘤上表現之分子。The method of claim 157, further comprising administering a bifunctional ligand to label cancer cells in the individual, wherein the bifunctional ligand specifically binds to a molecule expressed on the tumor. 如請求項158之方法,其中該雙功能配體包含FITC-葉酸。The method of claim 158, wherein the bifunctional ligand comprises FITC-folate. 如請求項157至159中任一項之方法,其進一步包含投與非生理學配體。The method of any one of claims 157 to 159, further comprising administering a non-physiological ligand. 如請求項160之方法,其中該非生理學配體包含雷帕黴素(rapamycin)或雷帕黴素類似物。The method of claim 160, wherein the non-physiological ligand comprises rapamycin or a rapamycin analog.

TW112142462A 2022-11-04 2023-11-03 Polynucleotide construct and related viral vectors and methods TW202434621A (en)

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