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CN1241574A - Liver cancer-resisting monoclone antibody HAb25 and its single stranded antibody and bifunctional antibody - Google Patents

  • ️Wed Jan 19 2000
Liver cancer-resisting monoclone antibody HAb25 and its single stranded antibody and bifunctional antibody Download PDF

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Publication number
CN1241574A
CN1241574A CN 98112905 CN98112905A CN1241574A CN 1241574 A CN1241574 A CN 1241574A CN 98112905 CN98112905 CN 98112905 CN 98112905 A CN98112905 A CN 98112905A CN 1241574 A CN1241574 A CN 1241574A Authority
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China
Prior art keywords
scfv
antibody
liver cancer
hab25
puc19
Prior art date
1998-07-02
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CN 98112905
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Chinese (zh)
Inventor
刘彦仿
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Fourth Military Medical University FMMU
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Fourth Military Medical University FMMU
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1998-07-02
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1998-07-02
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2000-01-19
1998-07-02 Application filed by Fourth Military Medical University FMMU filed Critical Fourth Military Medical University FMMU
1998-07-02 Priority to CN 98112905 priority Critical patent/CN1241574A/en
2000-01-19 Publication of CN1241574A publication Critical patent/CN1241574A/en
Status Pending legal-status Critical Current

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Abstract

抗肝癌单克隆抗体HAb25及其基因工程单链抗体和双功能抗体,主要涉及基因工程技术。其主要技术特征是,首先利用杂交瘤技术制备出对人肝癌细胞具有特异结合活性的鼠源性单克隆抗体HAb25;进而通过基因工程技术将HAb25改造构建成抗肝癌单链抗体scFv25和抗肝癌双功能抗体pBV220－scFv25-PE40、pGEX4T－1－scFv25－TNFa。抗肝癌基因工程抗体具有分子量小、穿透能力强、分子稳定性好、毒副作用低、效力高和容易通过基因工程大量生产等特点,具有很大的临床应用潜力。The anti-liver cancer monoclonal antibody HAb25 and its genetically engineered single-chain antibody and bifunctional antibody mainly involve genetic engineering technology. Its main technical features are as follows: firstly, the mouse-derived monoclonal antibody HAb25, which has specific binding activity to human liver cancer cells, was prepared by hybridoma technology; and then HAb25 was transformed into anti-liver cancer single-chain antibody scFv 25 and anti-liver cancer antibody through genetic engineering technology. Bifunctional antibody pBV220-scFv 25 -PE40, pGEX4T-1-scFv 25 -TNFa. Anti-liver cancer genetically engineered antibodies have the characteristics of small molecular weight, strong penetrating ability, good molecular stability, low toxicity and side effects, high efficacy and easy mass production through genetic engineering, etc., and have great potential for clinical application.

Description

Liver cancer-resisting monoclone antibody HAb 25 and single-chain antibody thereof and bifunctional antibody

The present invention relates generally to genetically engineered.

According to the statistics of the World Health Organization, the dead and liver cancer of ten thousand patients surplus the whole world just has 100 every year, and the sickness rate of China's liver cancer accounts for global 40%.At present external Shouval and Dunk etc. and domestic Xie Hong etc. have reported liver cancer-resisting monoclone antibody, and the report of anti-ferritin monoclonal antibody, some monoclonal anti body and functions are wherein also arranged simultaneously 131Behind the I mark, carried out effective clinical liver cancer radio-immuno-image.Yet for genetic engineering antibody, though existing reports such as the genetically engineered of tumours such as resistive connection intestinal cancer, mammary cancer mouse/people's chimeric antibody, single-chain antibodies, the genetic engineering antibody of anti-liver cancer has not yet to see report both at home and abroad.

Monoclonal antibody is used for tumor treatment research already, but along with going deep into of studying, mouse monoclonal antibody exposes many weak points, mainly is: 1. the monoclonal antibody molecule amount is big, and penetrativity is low, is difficult for penetrating the tumour capillary vessel; 2. mouse monoclonal antibody immunogenicity height, human body are used and can be produced human antimouse antibody (HAMA), have limited its repeated use.

The objective of the invention is to utilize genetically engineered that monoclonal antibody is transformed into single-chain antibody, be used to overcome above-mentioned shortcoming, further make up bifunctional antibody and given antibody new function again, make it have the guiding killing activity, this makes has pushed ahead again a step the diagnosis of liver cancer and treatment research.

Technical scheme of the invention process

One. the preparation of liver cancer-resisting monoclone antibody HAb 25:

Fresh primary hepatocyte hepatocarcinoma sample preparations liver cancer cell suspension with surgical resection is the immunogen immune BALB/C mice, adopts the peritoneal immunity approach, has selected peritoneal macrophage or thymocyte as feeder cell.With mouse boosting cell after the immunity and SP2/0 cytogamy, obtain hybridoma, carry out the immunohistochemical method screening with paraffin section, all and normal liver cell paraffin section does not react and cuts into slices being kept of reaction with liver cancer, obtain the liver cancer-resisting monoclone antibody HAb 25 hybridoma cell strain of sustainable secretory antibody, the HAb25 hybridoma cell strain is inoculated in mouse peritoneal, collect ascites, albumen obtains the HAb25 activated protein through ammonium sulphate precipitation and cation normal-pressure liquid chromatography purifying in the ascites.HAb25 proves to have stronger specificity and higher avidity after testing, is in particular in:

1. hepatocellular carcinoma paraffin section positive reaction rate is 84% (79/94), with all livers of cancer, hepatitis, hepatic hemangioma, hepatic cyst and hepatic metastases knurl no cross reaction, has only 5.7% (3/53) cross reaction with liver cirrhosis.

2. behind colloid gold label, at 4 ℃ down and liver cancer cell effect after 1 hour, cultivate down after 0,5,10,20,30,60,120 minute for 37 ℃, electricity under Electronic Speculum, observe antibody by pinocytosis, lining cave in, fine hair fracture engulf, glycocalyx caves in and directly the diffusion path internalization to liver cancer cell

3.HAb25 warp 131Behind the I mark, inject lotus people liver cancer nude mice, 72 hours can be in the clear video picture of tumor locus (ECT), and the T/N value is 6.84.

4.HAb25 warp 131Injecting 5 with 3-5mci/ people behind the I mark, to doubt be the patient of liver cancer, but wherein 4 examples video picture under ECT.The immunoconjugate of HAb25 and medicine has certain therapeutic action to lotus people liver cancer nude mice.

Two, anti-liver cancer single-chain antibody (scFv 25) preparation:

Extract total RNA from the HAb25 hybridoma, reverse transcription synthesize cDNA, and the RT-PCR method amplifies light, the heavy chain variable region gene of monoclone antibody HAb 25, and (VL VH), is the IgV district gene of rearrangement by analysis.VL and VH are coupled together by the polypeptide Linker of synthetic, form the active antibody molecule that a molecular weight has only whole antibody 1/6.Specify as follows:

1. get the strain of HAb25 hybrid cell, extract total RNA, be the synthetic cDNA of primer reverse transcription through Oligo (dT), add light, heavy chain primer and go out VL and VH through pcr amplification, be cloned into the pUC19 plasmid vector and carry out sequential analysis, compare, higher homology is arranged with known IgV sequence, and the definitive variation of new antibodies arranged, successful acquisition light, heavy chain variable region gene VL, the VH of HAb25.

2. the structure of single-chain antibody: the small peptide Linker with synthetic is cloned into the pUC19 cloning vector earlier, be built into pUC19-Linker, further VL is cloned into pUC19-Linker, be built into pUC19-Linker-VL, again VH is cloned into pUC19-Linker-VL, be built into pUC19-VH-Linker-VL, anti-liver cancer single-chain antibody cloning vector pUC19-scFv promptly recombinates 25, then with scFv 25Subclone is gone into pET15b, is built into the recombinant prokaryotic expression vector pET15b-scFv of anti-liver cancer single-chain antibody 25

3.scFv 25Abduction delivering: the reorganization pET15b-scFv 25Prokaryotic expression carrier host bacterium is through IPTG (isopropylthio-) abduction delivering, and collection thalline → extraction inclusion body → sex change → renaturation → be further purified obtains anti-liver cancer single-chain antibody scFv 25Activated protein.

4.scFv 25Determination of activity: scFv 25With add parental antibody HAb25 behind the SMMC-7721 liver cancer cell effect blocking antigen, add the sheep anti-mouse igg of FITC mark after the effect, compare with parental antibody self and irrelevant antibody.The cells were tested by flow cytometry result shows, scFv 25Can seal the affine activity of parental antibody 53%.

Advantage: the single-chain antibody molecular weight is little, penetrativity is strong, immunogenicity is low, the transformation period is short, circulation of blood and whole body are cleaned up advantages such as fast and easy genetically engineered mass production, is a kind of genetic engineering antibody that has very much the clinical application potentiality.

Three, the preparation of anti-liver cancer single-chain bifunctional antibody:

Utilize genetic engineering means will have liver-cancer cell specific in conjunction with active HAb25 single-chain antibody and PE40 (false pseudomonas bacillus extracellular toxin) or huamn tumor necrosis factory alpha (TNF α, the tumor cytotoxicity factor of the immune species specificity that produces by monocyte and scavenger cell) gene coupling, make up anti-liver cancer bifunctional antibody, and be cloned into prokaryotic expression carrier and express.

1.pBV220-scFv 25The structure of the anti-liver cancer monochain immunotoxin of-PE40 is expressed and cytotoxicity

To keep parental antibody HAb25 to liver-cancer cell specific in conjunction with active scFv 25Gene excises the coupling of the amino acid whose muton PE40 of 4-252 gene with PE, makes up anti-liver cancer monochain immunotoxin scFv 25-PE40 is cloned into prokaryotic expression carrier pBV220 then, is built into reorganization pBV220-scFv 25The anti-liver cancer monochain immunotoxin of-PE40 prokaryotic expression carrier.Its concrete implementation step is as follows:

(1) structure of anti-liver cancer monochain immunotoxin

Be built into pBV220scFv 25, and then the PE40 gene clone gone into pBV220scFv 25In, make the PE40 gene be next to scFv 253 ' end, be built into pBV220scFv 25-PE40 prokaryotic expression carrier.

(2) pBV220scFv 25The abduction delivering of-PE40 and purifying

Reorganization pBV220scFv 25-PE40 expression vector is expressed through 42 ℃ of thermal inductions, collects thalline and extracts inclusion body, and again through sex change, renaturation is further purified, and obtains purity and reaches electrophoretically pure anti-liver cancer monochain immunotoxin scFv 25-PE40.

(3) anti-liver cancer monochain immunotoxin scFv 25The immunocompetence of-PE40 is measured

With scFv 25-PE40 is one anti-, is two anti-with mouse-anti PE40 antibody, is three anti-with the sheep anti-mouse igg of FITC mark, and HCC-9204 liver cancer cell smear is carried out indirect IF staining, and the result shows, scFv 25-PE40 can combine with liver cancer cell specificity.

(4) cytotoxicity experiment:

Carry out anti-liver cancer scFv by mtt assay 25-PE40 is to the toxicity test of HCC-9204 cell, and the result shows that anti-liver cancer scFv-PE40 has clear and definite selective killing effect to liver cancer cell.

2. pGEX 4T-1-scFv recombinates 25The structure of the anti-liver cancer single-chain bifunctional of-TNF α antibody is expressed and the targeted therapy effect

(1) pGEX 4T-1-scFv is gone in TNF α gene clone 25, make up reorganization pGEX 4T-1-scFv 25-TNF α prokaryotic expression carrier.

(2) anti-liver cancer scFv 25The purifying of-TNF α: reorganization pGEX 4T-1-scFv 25-TNF α host bacterium is through the IPTG abduction delivering, and collection thalline → extraction inclusion body → sex change → renaturation → GST affinitive layer purification → zymoplasm digests and is further purified, and obtains anti-liver cancer bifunctional antibody scFv 25-TNF alpha active albumen.

(3) the indirect IF staining result shows, scFv 25-TNF α has kept the specific binding activity of parental antibody preferably; MTT tests demonstration, scFv 25-TNF α is obvious to the cytotoxicity of liver cancer cell SMMC-7721; The targeted therapy experiment of carrying out in lotus people liver cancer nude mouse shows scFv 25-TNF α can make the liver cancer enclosed mass of 2mm diameter alleviate fully, so scFv 25-TNF α has bigger clinical application potentiality.

Anti cancer gene engineering recombinant immunotoxin has following characteristics: molecular weight is little, is easy to penetrate blood vessel and enters tumor center and play a role, and anti-knurl is renderd a service higher; Exist with the fusion rotein form, stability of molecule is good; Owing to got receptor binding domains except that toxin, only kept the bio-toxicity part, effectively reduce toxic side effect; Recombinant immunotoxin also has easily by the mass-produced characteristics of genetically engineered.Above-mentioned work is that further fundamental research and clinical application are laid a good foundation.

Claims (1)

1.抗肝癌单克隆抗体HAb25及其单链抗体和双功能抗体，其特征在于：1. Anti-hepatoma monoclonal antibody HAb25 and its single-chain antibody and bifunctional antibody, characterized in that: (1)抗肝癌单克隆抗体HAb25：以外科手术切除的新鲜的原发性肝细胞肝癌标本制备的肝癌细胞悬液为免疫原免疫BALB/C小鼠。将免疫后的小鼠脾细胞与SP2/0细胞融合，获得杂交瘤细胞，杂交瘤细胞用免疫组织化学方法筛选，得到可持续分泌抗体的抗肝癌单克隆抗体HAb25杂交瘤细胞株，将该细胞株接种于小鼠腹腔，收集腹水，腹水内蛋白经硫酸胺沉淀和阳离子常压液相色谱纯化，获得HAb25活性蛋白。(1) Anti-hepatoma monoclonal antibody HAb25: BALB/C mice were immunized with liver cancer cell suspension prepared from fresh surgically resected primary hepatocellular carcinoma specimens as the immunogen. The immunized mouse splenocytes were fused with SP2/0 cells to obtain hybridoma cells. The hybridoma cells were screened by immunohistochemistry to obtain the anti-hepatoma monoclonal antibody HAb25 hybridoma cell line that continuously secretes antibodies. The cells were The strain was inoculated into the peritoneal cavity of mice, and the ascites was collected, and the protein in the ascites was purified by ammonium sulfate precipitation and cationic atmospheric pressure liquid chromatography to obtain HAb25 active protein. (2).抗肝癌单链抗体(scFv25)：(2). Anti-liver cancer single-chain antibody (scFv 25 ): ①HAb25轻、重链可变区基因(VL，VH)的克隆：从上述杂交瘤细胞中提取总RNA，逆转录合成cDNA，RT-PCR法扩增出单克隆抗体HAb25的轻、重链可变区基因，经分析是重排的IgV区基因。① Cloning of HAb25 light and heavy chain variable region genes (VL, VH): total RNA was extracted from the above hybridoma cells, cDNA was synthesized by reverse transcription, and the light and heavy chain variable regions of monoclonal antibody HAb25 were amplified by RT-PCR. The region gene was analyzed to be a rearranged IgV region gene. ②抗肝癌单链抗体scFv25的构建：先将人工合成的短肽Linker克隆入pUC19克隆载体，构建成pUC19-Linker，进一步将VL克隆入pUC19-Linker，构建成pUC19-Linker-VL，再将VH克隆入pUC19-Linker-VL，构建成pUC19-VH-Linker-VL，即重组抗肝癌单链抗体克隆载体pUC19-scFv25，然后将scFv25亚克隆入pET15b，构建成分子量只有全抗体1/6的抗肝癌单链抗体的重组原核表达载体pET15b-scFv25。②Construction of anti-hepatoma single-chain antibody scFv 25 : first clone the synthetic short peptide Linker into pUC19 cloning vector to construct pUC19-Linker, further clone VL into pUC19-Linker to construct pUC19-Linker-VL, and then clone VH is cloned into pUC19-Linker-VL to construct pUC19-VH-Linker-VL, which is the recombinant anti-hepatoma single-chain antibody cloning vector pUC19-scFv 25 , and then scFv 25 is subcloned into pET15b to construct a molecular weight of only 1/ The recombinant prokaryotic expression vector pET15b-scFv 25 of the anti-liver cancer single-chain antibody of 6. ③pET15b-scFv25的诱导表达：重组pET15b-scFv25原核表达载体宿主菌经IPTG(异丙基硫代-β-D-半乳糖苷)诱导表达，收集菌体→提取包涵体→变性→复性→进一步纯化，获得抗肝癌单链抗体scFv25活性蛋白。③Induced expression of pET15b-scFv 25 : the recombinant pET15b-scFv 25 prokaryotic expression vector host bacteria were induced to express by IPTG (isopropylthio-β-D-galactoside), and the bacteria were collected → extraction of inclusion bodies → denaturation → renaturation → Further purification to obtain the active protein of anti-hepatoma single-chain antibody scFv 25 . (3)抗肝癌双功能抗体：(3) Anti-liver cancer bifunctional antibody: ①抗肝癌单链免疫毒素(scFv25-PE40)的构建、表达：将PE40基因克隆入pUC19-scFv25中，构建成pUC19-scFv25-PE40重组抗肝癌单链免疫毒素克隆载体，然后再将scFv25-PE40亚克隆入原核表达载体pBV220中，构建成pBV220-scFv25-PE40，即重组抗肝癌单链免疫毒素原核表达载体pBV220-scFv25-PE40。上述载体宿主菌经42℃诱导表达，收集菌体→提取包涵体→变性→复性→进一步纯化，获得抗肝癌单链免疫毒素scFv25-PE40活性蛋白。① Construction and expression of anti-hepatoma single-chain immunotoxin (scFv 25 -PE40): Cloning PE40 gene into pUC19-scFv 25 to construct pUC19-scFv 25 -PE40 recombinant anti-hepatoma single-chain immunotoxin cloning vector, and then The scFv 25 -PE40 was subcloned into the prokaryotic expression vector pBV220 to construct pBV220-scFv 25 -PE40, which is the prokaryotic expression vector pBV220-scFv 25 -PE40 of recombinant anti-hepatoma single-chain immunotoxin. The expression of the above vector host bacteria was induced at 42°C, the bacteria were collected → inclusion bodies were extracted → denatured → renatured → further purified to obtain the anti-hepatoma single-chain immunotoxin scFv 25 -PE40 active protein. ②抗肝癌双功能抗体(scFv25-TNFα)的构建、表达：将TNFα基因克隆入pUC19-scFv25中，构建成pUC19-scFv25-TNFα重组抗肝癌双功能抗体克隆载体，然后再将scFv25-TNFα亚克隆入原核表达载体pGEX 4T-1中，构建成pGEX4T-1-scFv25-TNFα，即重组抗肝癌单链双功能抗体原核表达载体pGEX 4T-1-scFv25-TNFα。上述载体宿主菌经IPTG诱导表达，收集菌体→提取包涵体→变性→复性→GST亲和层析纯化→凝血酶消化和进一步纯化，获得抗肝癌双功能抗体scFv25-TNFα活性蛋白。②Construction and expression of anti-liver cancer bifunctional antibody (scFv 25 -TNFα): clone TNFα gene into pUC19-scFv 25 to construct pUC19-scFv 25 -TNFα recombinant anti-liver cancer bifunctional antibody cloning vector, and then scFv 25 -TNFα was subcloned into the prokaryotic expression vector pGEX 4T-1 to construct pGEX4T-1-scFv 25 -TNFα, which is the prokaryotic expression vector pGEX 4T-1-scFv 25 -TNFα of the recombinant anti-liver cancer single-chain bifunctional antibody. The above vector host bacteria were induced and expressed by IPTG, the bacteria were collected → inclusion bodies were extracted → denaturation → renaturation → GST affinity chromatography purification → thrombin digestion and further purification, and the anti-hepatoma bifunctional antibody scFv 25 -TNFα active protein was obtained.

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CN100410274C (en) * 2006-04-21 2008-08-13 暨南大学 Specific anti-human liver cancer phage single-chain antibody HscFv4-16 gene and its uses
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Publication number Priority date Publication date Assignee Title
CN101537180B (en) * 2002-07-18 2016-02-10 莫鲁斯有限公司 The recombinant production of mixtures of antibodies
EP1694700A1 (en) * 2003-11-11 2006-08-30 National Cancer Center Neutralizable epitope of hgf and neutralizing antibody binding to the same
EP1694700A4 (en) * 2003-11-11 2007-10-31 Nat Cancer Ct NEUTRALIZED HGF EPITOPE AND NEUTRALIZING ANTIBODY RELATED THERETO
AU2004287743B2 (en) * 2003-11-11 2008-10-09 National Cancer Center Neutralizable epitope of HGF and neutralizing antibody binding to the same
CN100410274C (en) * 2006-04-21 2008-08-13 暨南大学 Specific anti-human liver cancer phage single-chain antibody HscFv4-16 gene and its uses
CN107001481A (en) * 2014-07-22 2017-08-01 奥隆制药 By making method that the antibody with completely immune globulin form is positioned in cytoplasm by antibody penetration cell film and application thereof
US10844136B2 (en) 2014-07-22 2020-11-24 Orum Therapeutics Inc. Method for positioning, in cytoplasm, antibody having complete immunoglobulin form by penetrating antibody through cell membrane, and use for same
US10851177B2 (en) 2014-07-22 2020-12-01 Orum Therapeutics Inc. Method for inhibiting intracellular activated RAS using intact immunoglobulin-type antibody having cytosol-penetrating ability and use thereof
US11155641B2 (en) 2016-05-27 2021-10-26 Orum Therapeutics Inc. Cytosol-penetrating antibody and use thereof
US10787487B2 (en) 2018-06-21 2020-09-29 Orum Therapeutics Inc. Cell/tissue-specific cell-penetrating antibodies

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