CN110003338A - Anti- OX40 antibody and its application - Google Patents

抗OX40抗体及其应用Anti-OX40 antibody and its application

技术领域technical field

本申请涉及抗体领域，更具体地，本申请涉及抗OX40的抗体及其应用。The present application relates to the field of antibodies, and more particularly, the present application relates to antibodies against OX40 and applications thereof.

背景技术Background technique

免疫系统识别并清除癌症细胞的概念最早是在100多年前提出的，在不同类型癌症病人血液中可以检测到T细胞对癌症相关抗原的反应性。在有效的抗肿瘤免疫过程中，T细胞作为核心的执行者，首先被T细胞受体(T cell receptor，TCR)介导的抗原识别信号激活，同时众多的共刺激信号和共抑制信号精细调节T细胞反应的强度和质量，这些抑制信号即为免疫检查点。在生理情况下，共刺激分子与免疫检查点分子保持平衡，从而最大程度减少对于周围正常组织的损伤，维持对自身组织的耐受、避免自身免疫反应。而肿瘤细胞可以通过此机制，异常上调共抑制分子及其相关配体，抑制T细胞激活，从而逃避免疫杀伤。针对免疫检查点的阻断是增强T细胞激活的有效策略之一，也是近些年抗肿瘤药物开发的热门靶点。到目前为止，已有3个免疫检查点抑制剂在美国获批，用于治疗多种癌症类型，靶点分别是CTLA-4和PD-1/PD-L1。The concept that the immune system recognizes and eliminates cancer cells was first proposed more than 100 years ago, and the reactivity of T cells to cancer-associated antigens can be detected in the blood of patients with different types of cancer. In the process of effective anti-tumor immunity, T cells, as the core executors, are first activated by antigen recognition signals mediated by T cell receptors (TCRs), and are finely regulated by numerous co-stimulatory and co-inhibitory signals. The strength and quality of T cell responses, these inhibitory signals are immune checkpoints. Under physiological conditions, costimulatory molecules are in balance with immune checkpoint molecules, thereby minimizing damage to surrounding normal tissues, maintaining tolerance to self-tissue, and avoiding autoimmune responses. Through this mechanism, tumor cells can abnormally up-regulate co-suppressor molecules and their related ligands to inhibit T cell activation, thereby evading immune killing. Blockade of immune checkpoints is one of the effective strategies to enhance T cell activation, and it is also a popular target for antitumor drug development in recent years. So far, three immune checkpoint inhibitors have been approved in the United States for the treatment of various cancer types, targeting CTLA-4 and PD-1/PD-L1.

研究表明CTLA-4和PD-1在T细胞激活过程中发挥免疫抑制作用，从而抑制T细胞对肿瘤细胞的免疫杀伤功能；而针对这两个靶点的阻断性单克隆抗体可以解除这种免疫抑制，恢复T细胞抗肿瘤免疫功能。除了抑制型免疫检查点，还有一类激活型免疫检查点，正逐渐成为药物研发的新靶标。Studies have shown that CTLA-4 and PD-1 play an immunosuppressive role in the process of T cell activation, thereby inhibiting the immune killing function of T cells to tumor cells; and blocking monoclonal antibodies against these two targets can relieve this effect. Immunosuppression, restore T cell anti-tumor immune function. In addition to inhibitory immune checkpoints, there is also a class of activated immune checkpoints, which are gradually becoming new targets for drug development.

激活型免疫检查点分子，主要指T细胞活化的共刺激信号分子-T细胞共刺激受体，其归属于肿瘤坏死因子受体(tumor necrosis factor receptor，TNFR)家族，作用是调节T细胞的增殖、激活和分化反应，包括OX40(TNFRSF4)、CD40(TNFRSF5)、4-1BB(T cellantigen4-1BB homologue，CD137)和GITR(Glucocorticoid-induced TNFR-relatedprotein，GITR)等。Activated immune checkpoint molecules mainly refer to the costimulatory signaling molecules of T cell activation-T cell costimulatory receptors, which belong to the tumor necrosis factor receptor (TNFR) family and play a role in regulating the proliferation of T cells. , activation and differentiation responses, including OX40 (TNFRSF4), CD40 (TNFRSF5), 4-1BB (T cellantigen4-1BB homologue, CD137) and GITR (Glucocorticoid-induced TNFR-related protein, GITR) and so on.

OX40，又称CD134、ACT45或TNFRSF4，属于TNFR超家族的一员，其基因位于人1号染色体上，编码一种50kD的I型跨膜糖蛋白。胞外区有191个氨基酸，包含了三个完整的以及一个稍短的富含半胱氨酸结构域(CRD)。OX40主要在活化的效应T细胞(Teff)和调节性T细胞(Treg)上表达，是活化的CD4⁺T、CD8⁺T细胞表面的激活性受体，目前处于被称为“T细胞共刺激”领域的前沿，同时也在NKT细胞、NK细胞和嗜中性粒细胞上表达。OX40信号可以激活下游的NF-κB、PI3K和PKB通路，这些通路的持续激活最终能够延长T细胞的存活时间，并扩展T细胞记忆，促进T细胞的细胞杀伤能力；另外，OX40还能通过抑制调节性T细胞(Treg)的分化和活性，改善肿瘤微环境中的免疫抑制作用，进一步增强效应T细胞的功能。OX40, also known as CD134, ACT45 or TNFRSF4, is a member of the TNFR superfamily, and its gene is located on human chromosome 1, encoding a 50kD type I transmembrane glycoprotein. The extracellular region is 191 amino acids long and contains three complete and one short cysteine-rich domain (CRD). OX40 is mainly expressed on activated effector T cells (Teff) and regulatory T cells (Treg), and is an activating receptor on the surface of activated CD4 ⁺ T and CD8 ⁺ T cells. ” is at the forefront of the field and is also expressed on NKT cells, NK cells and neutrophils. OX40 signaling can activate the downstream NF-κB, PI3K and PKB pathways, and the continuous activation of these pathways can ultimately prolong the survival time of T cells, expand T cell memory, and promote the cell killing ability of T cells; in addition, OX40 can also inhibit Differentiation and activity of regulatory T cells (Treg), improve immunosuppression in the tumor microenvironment, and further enhance the function of effector T cells.

因此，开发OX40靶向抗体将有望用于治疗肿瘤包括晚期恶性肿瘤等相关疾病。Therefore, the development of OX40-targeting antibodies is expected to be used to treat tumors, including advanced malignant tumors and other related diseases.

发明内容SUMMARY OF THE INVENTION

第一方面，本申请提供了一种特异性结合OX40的抗体或其抗原结合部分，其包含重链可变区，所述重链可变区包含HCDR1、HCDR2和/或HCDR3。In a first aspect, the application provides an antibody or antigen-binding portion thereof that specifically binds OX40, comprising a heavy chain variable region comprising HCDR1, HCDR2 and/or HCDR3.

在一些实施方案中，所述HCDR1序列包含SEQ ID NOs:1-4任一项所示的氨基酸序列。在一些实施方案中，所述HCDR2序列包含SEQ ID NOs:5-8任一项所示的氨基酸序列。在一些实施方案中，所述HCDR3序列包含SEQ ID NOs:9-12任一项所示的氨基酸序列。在可选的实施方案中，上述抗原结合部分选自Fab片段、Fab’片段、F(ab’)₂片段、Fv片段、scFv片段、Fd片段或单域抗体。In some embodiments, the HCDRl sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4. In some embodiments, the HCDR2 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5-8. In some embodiments, the HCDR3 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-12. In alternative embodiments, the aforementioned antigen binding moieties are selected from Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, scFv fragments, Fd fragments or single domain antibodies.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分还包含轻链可变区，其中所述轻链可变区包含LCDR1、LCDR2和/或LCDR3序列。In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 further comprises a light chain variable region, wherein the light chain variable region comprises an LCDR1, LCDR2 and/or LCDR3 sequence.

在某些实施方案中，所述LCDR1序列包含SEQ ID NOs:13-16任一项所示的氨基酸序列。在某些实施方案中，所述LCDR2序列包含SEQ ID NOs:17-19任一项所示的氨基酸序列。在某些实施方案中，所述LCDR3序列包含SEQ ID NOs:20-23任一项所示的氨基酸序列。In certain embodiments, the LCDR1 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 13-16. In certain embodiments, the LCDR2 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17-19. In certain embodiments, the LCDR3 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 20-23.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分的重链可变区包含选自SEQ ID NOs:24-30任一项所示的氨基酸序列或者与上述序列具有至少80％同源性的氨基酸序列；优选地，所述重链可变区包含SEQ ID NO:24或29所示的氨基酸序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding portion thereof that specifically binds OX40 comprises an amino acid sequence selected from the group consisting of amino acid sequences set forth in any one of SEQ ID NOs: 24-30 or has at least 80 amino acid sequence of % homology; preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24 or 29.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分的轻链可变区包含选自SEQ ID NOs:31-37任一项所示的氨基酸序列或者与上述序列具有至少80％同源性的氨基酸序列；优选地，所述轻链可变区包含选自SEQ ID NO:31或36所示的氨基酸序列。In some embodiments, the light chain variable region of the antibody that specifically binds OX40, or an antigen-binding portion thereof, comprises an amino acid sequence selected from the group consisting of amino acid sequences set forth in any one of SEQ ID NOs: 31-37 or has at least 80% of the above-mentioned sequence. amino acid sequence of % homology; preferably, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31 or 36.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分特异性结合于灵长类OX40。In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 specifically binds to primate OX40.

在一些实施方案中，所述特异性结合OX40的抗体为单克隆抗体。In some embodiments, the antibody that specifically binds OX40 is a monoclonal antibody.

在一些实施方案中，所述特异性结合OX40的抗体为鼠源化抗体。In some embodiments, the antibody that specifically binds OX40 is a murine antibody.

在一些具体的实施方案中，所述特异性结合OX40的抗体的重链可变区氨基酸序列如SEQ ID NO:24或29所示，以及所述特异性结合OX40的抗体的轻链可变区氨基酸序列如SEQ ID NO:31或36所示。In some specific embodiments, the amino acid sequence of the heavy chain variable region of the antibody that specifically binds OX40 is set forth in SEQ ID NO: 24 or 29, and the light chain variable region of the antibody that specifically binds OX40 The amino acid sequence is shown in SEQ ID NO:31 or 36.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分以1×10^-8至1×10^-7 M的KD特异性结合于OX40分子。In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 specifically binds to an OX40 molecule with a KD of 1×10 ⁻⁸ to 1×10 ⁻⁷ M.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分具有OX40激动剂功能。In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 has OX40 agonist function.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分能够刺激T细胞的活化及增殖。In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is capable of stimulating T cell activation and proliferation.

第二方面，本申请提供了核苷酸分子，其编码第一方面所述的特异性结合OX40的抗体或其抗原结合部分。In a second aspect, the application provides a nucleotide molecule encoding the antibody or antigen-binding portion thereof that specifically binds to OX40 according to the first aspect.

第三方面，本申请提供了表达载体，其包含第二方面所述的核苷酸分子。In a third aspect, the application provides an expression vector comprising the nucleotide molecule of the second aspect.

第四方面，本申请提供了宿主细胞，其包含第二方面所述的核苷酸分子或第三方面所述的表达载体。In a fourth aspect, the present application provides a host cell comprising the nucleotide molecule of the second aspect or the expression vector of the third aspect.

第五方面，本申请提供了药物组合物，其包含第一方面所述的特异性结合OX40的抗体或其抗原结合部分以及药学上可接受的载体。In a fifth aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding portion thereof that specifically binds OX40 as described in the first aspect and a pharmaceutically acceptable carrier.

在一些实施方案中，所述药物组合物还包含一种或多种其他活性成分。在一些实施方案中，所述活性成分为化疗剂、PD-1结合拮抗剂等。In some embodiments, the pharmaceutical composition further comprises one or more other active ingredients. In some embodiments, the active ingredient is a chemotherapeutic agent, a PD-1 binding antagonist, or the like.

第六方面，本申请提供了疫苗，其包含第一方面所述的特异性结合OX40的抗体或其抗原结合部分，以及任选的免疫佐剂。In a sixth aspect, the present application provides a vaccine comprising the antibody or antigen-binding portion thereof that specifically binds to OX40 according to the first aspect, and optionally an immunological adjuvant.

在一些实施方案中，第五方面所述的药物组合物或第六方面所述的疫苗用于治疗OX40相关的疾病，例如肿瘤。In some embodiments, the pharmaceutical composition of the fifth aspect or the vaccine of the sixth aspect is used to treat OX40-related diseases, such as tumors.

第七方面，本申请提供了第一方面所述的特异性结合OX40的抗体或其抗原结合部分，或者第五方面所述的药物组合物在制备用于抑制Treg功能、杀死表达OX40的细胞、引发T细胞介导的反应、提高效应T细胞的功能、提高记忆T细胞的功能、和/或有效抑制肿瘤生长的药物中的用途。In the seventh aspect, the present application provides the antibody or antigen-binding portion thereof that specifically binds to OX40 described in the first aspect, or the pharmaceutical composition described in the fifth aspect is prepared for inhibiting the function of Treg and killing cells expressing OX40. , eliciting a T cell-mediated response, enhancing effector T cell function, enhancing memory T cell function, and/or use in a drug that effectively inhibits tumor growth.

第八方面，本申请提供了第一方面所述的特异性结合OX40的抗体或其抗原结合部分，或者第五方面所述的药物组合物在制备用于预防和/或治疗OX40相关的疾病，例如肿瘤的药物中的用途。In the eighth aspect, the present application provides the antibody or antigen-binding portion thereof that specifically binds to OX40 described in the first aspect, or the pharmaceutical composition described in the fifth aspect prepared for the prevention and/or treatment of OX40-related diseases, For example, the use in the medicine of tumor.

在一些实施方案中，所述肿瘤选自结肠癌、黑色素瘤、间皮质瘤、肾细胞癌、淋巴瘤、晚期实体瘤以及上述的转移瘤。In some embodiments, the tumor is selected from colon cancer, melanoma, mesothelioma, renal cell carcinoma, lymphoma, advanced solid tumors, and metastases described above.

第九方面，本申请提供了检测试剂或者试剂盒，其包含第一方面所述的特异性结合OX40的抗体或其抗原结合部分。In a ninth aspect, the present application provides a detection reagent or kit comprising the antibody or antigen-binding portion thereof that specifically binds to OX40 as described in the first aspect.

在其他方面，本申请提供了预防和/或治疗OX40相关的疾病，例如肿瘤的方法，其包括给予有需要的个体第一方面所述的特异性结合OX40的抗体或其抗原结合部分、或第五方面所述的药物组合物、或第六方面所述的疫苗。任选地，所述方法还包括联合施用其他的治疗剂，例如化疗剂、PD-1结合拮抗剂等。In other aspects, the present application provides a method of preventing and/or treating an OX40-related disease, such as a tumor, comprising administering to an individual in need thereof the antibody or antigen-binding portion thereof that specifically binds OX40 according to the first aspect, or the first The pharmaceutical composition of the fifth aspect, or the vaccine of the sixth aspect. Optionally, the method also includes co-administration of other therapeutic agents, such as chemotherapeutic agents, PD-1 binding antagonists, and the like.

本申请的特异性结合OX40的抗体或其抗原结合部分能够特异性与OX40结合，具有以下的一种或多种效应：具有OX40激动剂功能，刺激T细胞的增殖活化，诱导OX40介导的抗肿瘤免疫应答，和/或抑制肿瘤生长等。The antibody that specifically binds to OX40 or its antigen-binding portion of the present application can specifically bind to OX40, and has one or more of the following effects: it has the function of an OX40 agonist, stimulates the proliferation and activation of T cells, and induces OX40-mediated anti-tumor effects. Tumor immune response, and/or inhibition of tumor growth, etc.

附图的简要说明Brief Description of Drawings

图1显示了通过NF-κB信号通路验证候选杂交瘤单克隆抗体的功能活性的结果：其中图1A为对30个单克隆抗体进行初筛的结果，图1B为筛选获得的7个候选单克隆抗体的结果。Figure 1 shows the results of verifying the functional activity of the candidate hybridoma monoclonal antibodies through the NF-κB signaling pathway: Figure 1A shows the results of preliminary screening of 30 monoclonal antibodies, and Figure 1B shows the 7 candidate monoclonal antibodies obtained by screening Antibody results.

图2显示了通过NFAT信号通路验证候选杂交瘤单克隆抗体的功能活性的结果。Figure 2 shows the results of validating the functional activity of candidate hybridoma monoclonal antibodies through the NFAT signaling pathway.

图3显示了Fortebio检测抗OX40单克隆抗体的KD值：其中图3A至图3G分别对应抗OX40单克隆抗体18B10、4F2、6H12、6A6、4B9、6C1和13F7的KD值。Figure 3 shows the KD values of anti-OX40 monoclonal antibodies detected by Fortebio: Figure 3A to Figure 3G correspond to the KD values of anti-OX40 monoclonal antibodies 18B10, 4F2, 6H12, 6A6, 4B9, 6C1 and 13F7, respectively.

图4显示了抗OX40单克隆抗体与人OX40及猴OX40结合的实验结果。Figure 4 shows the experimental results of anti-OX40 monoclonal antibody binding to human OX40 and monkey OX40.

图5显示了抗OX40单克隆抗体对人OX40与其配体OX40L结合的阻断作用。Figure 5 shows the blocking effect of anti-OX40 monoclonal antibodies on the binding of human OX40 to its ligand OX40L.

图6显示了抗OX40单克隆抗体的EC50值功能活性检测结果。Figure 6 shows the EC50 value functional activity assay results of anti-OX40 monoclonal antibodies.

图7显示了抗OX40单克隆抗体刺激人CD4⁺T细胞增殖及IL-2和IFN-γ分泌的结果：其中图7A为CFSE流式检测CD4⁺T细胞增殖的结果，图7B为ELISA检测IL-2分泌的结果，图7C为ELISA检测IFN-γ分泌的结果。Figure 7 shows the results of anti-OX40 monoclonal antibody stimulating the proliferation of human CD4 ⁺ T cells and the secretion of IL-2 and IFN-γ: Figure 7A is the results of CFSE flow cytometry detection of CD4 ⁺ T cell proliferation, and Figure 7B is the detection of IL by ELISA -2 secretion results, Figure 7C shows the results of ELISA detection of IFN-γ secretion.

图8显示了抗OX40单克隆抗体在人源化OX40的MC38荷瘤鼠中的体内药效及安全性研究：其中图8A为小鼠的肿瘤体积变化趋势图，图8B为小鼠的体重变化趋势图，图8C为ALT检测结果图，图8D为AST检测结果图，图8E为解剖后获得的肿瘤的照片，图8F为肿瘤重量占小鼠总体重的百分比。Figure 8 shows the in vivo efficacy and safety study of anti-OX40 monoclonal antibody in humanized OX40-bearing MC38 tumor-bearing mice: Figure 8A shows the trend of tumor volume changes in mice, and Figure 8B shows the body weight changes in mice Trend diagram, Figure 8C is the ALT detection result, Figure 8D is the AST detection result, Figure 8E is the photo of the tumor obtained after dissection, and Figure 8F is the percentage of the tumor weight to the total weight of the mouse.

具体实施方式Detailed ways

本申请提供了特异性结合于OX40的新型抗OX40抗体或其抗原结合部分。在优选实施方案中，本申请的抗体或其抗原结合部分结合于靶细胞表面的OX40分子并具有OX40激动剂功能。本申请还提供了编码该抗体或其抗原结合片段的核苷酸分子、包含所述核苷酸分子的表达载体、包含所述核苷酸分子或表达载体的宿主细胞、制备和纯化该抗体的方法以及所述抗体或其抗原结合片段的医学和生物学应用，例如预防或治疗OX40相关疾病或病症。本申请还涵盖使用所述抗体或其抗原结合片段来检测OX40及调节OX40活性的方法以及相关检测试剂或试剂盒。The application provides novel anti-OX40 antibodies or antigen-binding portions thereof that specifically bind to OX40. In a preferred embodiment, the antibody or antigen-binding portion thereof of the present application binds to an OX40 molecule on the surface of a target cell and functions as an OX40 agonist. The present application also provides a nucleotide molecule encoding the antibody or an antigen-binding fragment thereof, an expression vector comprising the nucleotide molecule, a host cell comprising the nucleotide molecule or the expression vector, and a method for preparing and purifying the antibody. Methods and medical and biological applications of the antibodies or antigen-binding fragments thereof, such as the prevention or treatment of OX40-related diseases or disorders. The present application also encompasses methods of using the antibodies or antigen-binding fragments thereof to detect OX40 and modulate the activity of OX40, and related detection reagents or kits.

为容易地理解本申请，首先定义本文中使用的某些术语。For ease of understanding of this application, certain terms used herein are first defined.

如本文所用的，术语“抗体”指包含四条多肽链，即通过双硫键互连的两条重链(H)及两条轻链(L)的免疫球蛋白分子，以及其多聚体(例如IgM)。各重链包含重链可变区(缩写为VH)及重链恒定区(缩写为CH)。重链恒定区包含三个域，即CH1、CH2及CH3。各轻链包含轻链可变区(缩写为VL)及轻链恒定区(缩写为CL)。轻链恒定区包含一个域(CL1)。VH及VL区可进一步细分成称为互补决定区(CDR)的高变区，其中穿插有称为构架区(FR)的保守区。在一些实施方案中，从N-末端至C-末端，轻链与重链可变结构域均包含FR1、CDR1、FR2、CDR2、FR3、CDR3与FR4。As used herein, the term "antibody" refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof ( such as IgM). Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region comprises three domains, namely CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL). The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with conserved regions called framework regions (FRs). In some embodiments, from the N-terminus to the C-terminus, both the light chain and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

如本文所用的，术语抗体的“抗原结合部分”是指负责结合抗原的完整抗体分子的一部分或区段。抗原结合域可以包含重链可变区(VH)、轻链可变区(VL)或上述两者。抗体的抗原结合片段可使用任何适合的标准技术从完整抗体分子制备，所述标准技术包括蛋白水解消化或重组遗传工程化技术等。抗原结合部分的非限制性实例包括：Fab片段、F(ab')₂片段、Fd片段、Fv片段、单链Fv(scFv)分子、单域抗体、dAb片段及由模拟抗体高变区的氨基酸残基组成的最小识别单元(例如分离的CDR)。术语“抗原结合部分”也包括其它工程化的分子，如双抗体、三抗体、四抗体及微型抗体等。例如，本文中所述Fd片段指由VH与CH1结构域组成的抗体片段；Fv片段由抗体的单臂中VL与VH结构域组成；dAb片段(Ward et al.，Nature 1989；341：544-546)由VH结构域组成。As used herein, the term "antigen-binding portion" of an antibody refers to the portion or segment of an intact antibody molecule responsible for binding an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen-binding fragments of antibodies can be prepared from intact antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, among others. Non-limiting examples of antigen-binding moieties include: Fab fragments, F(ab') ₂ fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, single-domain antibodies, dAb fragments, and amino acids that mimic the hypervariable regions of antibodies The smallest recognition unit composed of residues (eg, isolated CDRs). The term "antigen binding portion" also includes other engineered molecules such as diabodies, tribodies, tetrabodies, minibodies, and the like. For example, Fd fragments described herein refer to antibody fragments composed of VH and CH1 domains; Fv fragments are composed of VL and VH domains in a single arm of an antibody; dAb fragments (Ward et al., Nature 1989; 341:544- 546) consists of a VH domain.

本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即Kabat定义和Chothia定义，例如参见Kabat et al.,“Sequences of Proteins ofImmunological Interest”，National Institutes of Health,Bethesda，MD.(1991)；Al-Lazikani et al.，J Mol Biol 273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA 86:9268-9272(1989)。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。在本文中，重链可变区的CDR1、CDR2及CDR3分别简称为HCDR1、HCDR2及HCDR3；轻链可变区的CDR1、CDR2及CDR3分别简称为LCDR1、LCDR2及LCDR3。It is well known to those skilled in the art that the complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of antibodies. There are two common ways of defining the CDR sequences of VH or VL, the Kabat definition and the Chothia definition, see, for example, Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, MD. (1991); Al -Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). For the variable region sequence of a given antibody, the CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In embodiments of the present application, the CDR sequences are defined using Kabat. Herein, CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。For the variable region sequence of a given antibody, the CDR region sequences in the variable region sequence can be analyzed in various ways, for example, can be determined using the online software Abysis (http://www.abysis.org/).

如本文所用的，术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合，例如抗体以比其对非特异性抗原的亲和性大至少两倍的亲和性结合于特异性抗原的能力。然而应了解，抗体能够特异性结合于两种或更多种与其序列相关的抗原。例如，本发明的抗体可特异性结合于人类与非人类(例如小鼠或非人类灵长动物)的OX40。As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, eg, binding of an antibody to an epitope, eg, an antibody with an affinity that is at least two greater than its affinity for a non-specific antigen The ability to bind to specific antigens with fold greater affinity. It will be appreciated, however, that antibodies are capable of specifically binding to two or more antigens with which their sequences are related. For example, the antibodies of the invention can specifically bind to OX40 in humans and non-humans (eg, mice or non-human primates).

如本文所用的，术语“单克隆抗体”指由基本同质的抗体群体获得的抗体，即，除了可能在少量个体中存在自然发生的突变以外，组成群体的各个抗体是相同的。本文所述单克隆抗体特别包括“嵌合”抗体，其中重链和/或轻链的一部分与来源于具体物种或属于具体抗体类或亚类的抗体中的对应序列相同或同源，而重链和/或轻链的余下部分与来源于另一物种或属于另一抗体类或亚类的抗体中的对应序列相同或同源，并且还包括这样的抗体的片段，只要它们能表现出所期望的生物学活性(参见，美国专利号4,816,567；和Morrison et al.,Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in a small number of individuals. Monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while The remainder of the chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, provided they exhibit the desired (See, US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

如本文所用的，术语“鼠源化抗体”是指其中所有恒定结构域序列均为小鼠序列的任何抗体。此类抗体可通过杂交瘤产生。As used herein, the term "murinized antibody" refers to any antibody in which all constant domain sequences are mouse sequences. Such antibodies can be produced by hybridomas.

如本文所用的，术语“嵌合抗体”是指包含来自两种或多种不同抗体的区段的抗体。在一些实施方案中，一个或多个CDR衍生自小鼠抗OX40抗体。在另一些实施方案中，所有CDR均衍生自小鼠抗OX40抗体。在一些实施方案中，在嵌合抗体中组合来自一种以上小鼠抗OX40抗体的CDR。例如，嵌合抗体可包含来自第一种小鼠抗OX40抗体中轻链的CDR1、来自第二种小鼠抗OX40抗体中轻链的CDR2、与来自第三种小鼠抗OX40抗体中轻链的CDR3，以及来自重链的CDR可衍生自一种或多种其它抗OX40抗体。此外，构架区可来自相同抗OX40抗体或来自一个或多个不同的个体。As used herein, the term "chimeric antibody" refers to an antibody comprising segments from two or more different antibodies. In some embodiments, one or more CDRs are derived from a mouse anti-OX40 antibody. In other embodiments, all CDRs are derived from mouse anti-OX40 antibodies. In some embodiments, CDRs from more than one mouse anti-OX40 antibody are combined in a chimeric antibody. For example, a chimeric antibody can comprise CDR1 from the light chain of a first mouse anti-OX40 antibody, CDR2 from the light chain of a second mouse anti-OX40 antibody, and a light chain from a third mouse anti-OX40 antibody The CDR3, as well as the CDRs from the heavy chain can be derived from one or more other anti-OX40 antibodies. Furthermore, the framework regions can be from the same anti-OX40 antibody or from one or more different individuals.

可额外用于抗体方法中的合适的技术包括基于OX40的亲和纯化、非变性凝胶纯化、HPLC或RP-HPLC、分子排阻、在蛋白A柱上纯化、或这些技术的任何组合。可使用ELISA测定法测定OX40抗体同种型，例如可使用鼠Ig吸附的抗人Ig鉴定人Ig。Suitable techniques that may additionally be used in the antibody method include OX40-based affinity purification, native gel purification, HPLC or RP-HPLC, size exclusion, purification on a protein A column, or any combination of these techniques. OX40 antibody isotypes can be determined using an ELISA assay, eg, human Ig can be identified using murine Ig-adsorbed anti-human Ig.

可通过本领域已知的多种标准的蛋白纯化或重组表达技术中的任何一种来产生适于产生抗体的OX40。适于产生免疫应答的OX40的形式包括OX40子序列(例如免疫原性片段)。另外的OX40的形式包括OX40表达细胞、含有OX40的制品或细胞提取物或级分、部分纯化的OX40。OX40 suitable for antibody production can be produced by any of a variety of standard protein purification or recombinant expression techniques known in the art. Forms of OX40 suitable for generating an immune response include OX40 subsequences (eg, immunogenic fragments). Additional forms of OX40 include OX40 expressing cells, preparations or cell extracts or fractions containing OX40, partially purified OX40.

如本文所用的，术语“同源性”被定义为经过序列比对和引入空位后，氨基酸或核苷酸序列变体中相同的残基的百分比，如果需要，达到最大百分比的同源性。用于比对的方法和计算机程序在本领域内是公知的。本文所述的“至少80％同源性”是指同源性为80％至100％任一值，例如85％、90％、95％、99％等。As used herein, the term "homology" is defined as the percentage of identical residues in a variant of an amino acid or nucleotide sequence after sequence alignment and introduction of gaps, if desired, to a maximum percent homology. Methods and computer programs for alignment are well known in the art. As used herein, "at least 80% homology" refers to any value of 80% to 100% homology, eg, 85%, 90%, 95%, 99%, and the like.

如本文所用的，术语“OX40相关疾病”包括与OX40信号通路相关的疾病和/或症状。示例性OX40相关疾病或病症包括肿瘤，例如结肠癌、黑色素瘤、间皮质瘤、肾细胞癌、淋巴瘤、晚期实体瘤以及上述的转移瘤。As used herein, the term "OX40-related disease" includes diseases and/or symptoms associated with the OX40 signaling pathway. Exemplary OX40-related diseases or disorders include tumors such as colon cancer, melanoma, mesothelioma, renal cell carcinoma, lymphoma, advanced solid tumors, and metastases described above.

如本文所用的，术语“EC50”是指半数最大效应浓度(concentration for50％ofmaximal effect,EC50)，指能引起50％最大效应的浓度。As used herein, the term "EC50" refers to the concentration for 50% of maximal effect (EC50), which refers to the concentration that elicits 50% of the maximal effect.

第一方面，本申请提供了特异性结合OX40的抗体或其抗原结合部分，其包含重链可变区的HCDR1、HCDR2和/或HCDR3。下表1-4中示例性列出了适用于本申请公开的抗体的CDR、重链可变区和轻链可变区氨基酸序列。在某些实施方案中，抗OX40抗体或其抗原结合部分包含HCDR1、HCDR2和/或HCDR3序列，其独立地选自表1中所示的HCDR1、HCDR2和/或HCDR3序列中任一者。在某些实施方案中，本申请的抗OX40抗体可进一步包含LCDR1、LCDR2和/或LCDR3，其独立地选自表2中所示的LCDR1、LCDR2和/或LCDR3序列中任一者。举例而言，本申请的抗OX40抗体可包含表3中所示的重链可变区中的任一者，任选地与表4中所示的轻链可变区的任一者组合或配对。In a first aspect, the application provides an antibody or antigen-binding portion thereof that specifically binds OX40, comprising HCDR1, HCDR2 and/or HCDR3 of the heavy chain variable region. The CDR, heavy chain variable region, and light chain variable region amino acid sequences suitable for use with the antibodies disclosed herein are exemplified in Tables 1-4 below. In certain embodiments, the anti-OX40 antibody, or antigen-binding portion thereof, comprises HCDRl, HCDR2, and/or HCDR3 sequences independently selected from any of the HCDRl, HCDR2, and/or HCDR3 sequences shown in Table 1. In certain embodiments, the anti-OX40 antibodies of the present application may further comprise LCDR1, LCDR2 and/or LCDR3 independently selected from any of the LCDR1, LCDR2 and/or LCDR3 sequences shown in Table 2. For example, an anti-OX40 antibody of the present application may comprise any of the heavy chain variable regions shown in Table 3, optionally in combination with any of the light chain variable regions shown in Table 4 or pair.

表1：示例性抗OX40抗体的重链CDR氨基酸序列的序列号Table 1: Sequence numbers of heavy chain CDR amino acid sequences of exemplary anti-OX40 antibodies

表2：示例性抗OX40抗体的轻链CDR氨基酸序列的序列号Table 2: Sequence numbers of light chain CDR amino acid sequences of exemplary anti-OX40 antibodies

表3：示例性抗OX40抗体的重链可变区氨基酸序列的序列号Table 3: Sequence numbers of heavy chain variable region amino acid sequences of exemplary anti-OX40 antibodies

表4：示例性抗OX40抗体的轻链可变区氨基酸序列的序列号Table 4: Sequence numbers of light chain variable region amino acid sequences of exemplary anti-OX40 antibodies

在一些实施方案中，本文公开的抗体或其抗原结合部分的HCDR1的氨基酸序列如SEQ ID NOs:1-4任一项所示。在一些实施方案中，HCDR2的氨基酸序列如SEQ ID NOs:5-8任一项所示。在一些实施方案中，HCDR3的氨基酸序列如SEQ ID NOs:9-12任一项所示。In some embodiments, the amino acid sequence of HCDRl of an antibody or antigen-binding portion thereof disclosed herein is set forth in any one of SEQ ID NOs: 1-4. In some embodiments, the amino acid sequence of HCDR2 is set forth in any one of SEQ ID NOs: 5-8. In some embodiments, the amino acid sequence of HCDR3 is set forth in any one of SEQ ID NOs: 9-12.

在可选的实施方案中，所述抗原结合部分选自Fab片段、Fab’片段、F(ab’)₂片段、Fv片段、scFv片段、Fd片段或单域抗体。In alternative embodiments, the antigen binding moiety is selected from the group consisting of Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, scFv fragments, Fd fragments or single domain antibodies.

本文公开的抗体或其抗原结合部分在包含重链可变区的基础上还可以进一步包含轻链可变区。The antibody or antigen-binding portion thereof disclosed herein may further comprise a light chain variable region in addition to the heavy chain variable region.

在一些实施方案中，本文公开的抗体或其抗原结合部分的LCDR1的氨基酸序列如SEQ ID NOs:13-16任一项所示。在一些实施方案中，LCDR2的氨基酸序列如SEQ ID NOs:17-19任一项所示。在一些实施方案中，LCDR3的氨基酸序列如SEQ ID NOs:20-23任一项所示。In some embodiments, the amino acid sequence of LCDRl of an antibody or antigen-binding portion thereof disclosed herein is set forth in any one of SEQ ID NOs: 13-16. In some embodiments, the amino acid sequence of LCDR2 is set forth in any one of SEQ ID NOs: 17-19. In some embodiments, the amino acid sequence of LCDR3 is set forth in any one of SEQ ID NOs: 20-23.

在一些具体的实施方案中，本文公开的抗体或其抗原结合部分的重链可变区与选自SEQ ID NOs:24-30的氨基酸序列具有至少80％同源性，例如90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同源性。在一些具体的实施方案中，上述抗体重链可变区由选自SEQ ID NOs:24-30任一项的氨基酸序列组成。在一些更具体的实施放方案中，上述抗体重链可变区的氨基酸序列如SEQ ID NO:24或29所示。In some specific embodiments, the heavy chain variable region of an antibody or antigen-binding portion thereof disclosed herein has at least 80% homology, eg, 90%, 91%, to an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-30 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology. In some specific embodiments, the above-mentioned antibody heavy chain variable region consists of an amino acid sequence selected from any one of SEQ ID NOs: 24-30. In some more specific embodiments, the amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 24 or 29.

在一些具体的实施方案中，本文公开的抗体或其抗原结合部分的轻链可变区与选自SEQ ID NOs:31-37的序列具有至少80％同源性，例如具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同源性。在一些具体的实施方案中，所述抗体轻链可变区由选自SEQ ID NOs:31-37任一项的氨基酸序列组成。在一些更具体的实施方案中，上述抗体轻链可变区的氨基酸序列如SEQ ID NO:31或36所示。In some specific embodiments, the light chain variable region of an antibody disclosed herein, or antigen-binding portion thereof, has at least 80% homology, eg, 90%, 91%, to a sequence selected from the group consisting of SEQ ID NOs: 31-37 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology. In some specific embodiments, the antibody light chain variable region consists of an amino acid sequence selected from any one of SEQ ID NOs: 31-37. In some more specific embodiments, the amino acid sequence of the above-mentioned antibody light chain variable region is shown in SEQ ID NO: 31 or 36.

在一些实施方案中，本文公开的抗体的重链可变区或轻链可变区可以在上述所列举的各自对应的具体氨基酸序列的基础上取代、缺失或添加至少一个氨基酸，且得到的变体仍保持结合OX40的活性。In some embodiments, the heavy chain variable region or light chain variable region of the antibodies disclosed herein may have at least one amino acid substituted, deleted or added based on the respective specific amino acid sequences listed above, and the resulting changes The body still retains the activity of binding OX40.

在某些实施方案中，上述氨基酸取代、缺失或添加的数目为1-30个或1-30个之间的任一数值，优选为1-20个，更优选为1-10个。在优选的实施方案中，序列变体与原氨基酸序列相差约1、2、3、4、5、6、7、8、9或10个氨基酸的取代、缺失和/或添加。在更优选的实施方案中，序列变体与原氨基酸序列相差约1、2、3、4或5个氨基酸的取代、缺失或添加。在具体的实施方案中，所述氨基酸取代为保守性取代。In certain embodiments, the number of amino acid substitutions, deletions or additions mentioned above is 1-30 or any value between 1-30, preferably 1-20, more preferably 1-10. In preferred embodiments, the sequence variant differs from the original amino acid sequence by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and/or additions. In more preferred embodiments, the sequence variant differs from the original amino acid sequence by about 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions. In specific embodiments, the amino acid substitutions are conservative substitutions.

在优选的实施方案中，本文公开的抗体为抗体6A6或4F2，其中抗体6A6的重链可变区氨基酸序列如SEQ ID NO:29所示，轻链可变区氨基酸序列如SEQ ID NO:36所示；抗体4F2的重链可变区氨基酸序列如SEQ ID NO:24所示，轻链可变区氨基酸序列如SEQ ID NO:31所示。In a preferred embodiment, the antibody disclosed herein is antibody 6A6 or 4F2, wherein the heavy chain variable region amino acid sequence of antibody 6A6 is shown in SEQ ID NO:29, and the light chain variable region amino acid sequence is shown in SEQ ID NO:36 The amino acid sequence of the heavy chain variable region of antibody 4F2 is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 31.

本文公开的抗体或其抗原结合部分能够特异性结合OX40。在一些实施方案中，OX40抗体显示物种与分子选择性。在一些具体的实施方案中，所述抗体或其抗原结合部分特异性结合灵长类OX40，或者与灵长类OX40具有高度同源性的物种的OX40。在一些具体的实施方案中，所述抗体或其抗原结合部分特异性结合人OX40和猴OX40。在一些更具体的实施方案中，抗OX40抗体与人、猕猴或恒河猴OX40结合。在一些其它的实施方案中，抗OX40抗体不与小鼠、大鼠、狗或兔子OX40结合。The antibodies or antigen-binding portions thereof disclosed herein are capable of specifically binding OX40. In some embodiments, the OX40 antibody exhibits species and molecular selectivity. In some specific embodiments, the antibody or antigen-binding portion thereof specifically binds primate OX40, or OX40 of a species with high homology to primate OX40. In some specific embodiments, the antibody or antigen-binding portion thereof specifically binds human OX40 and monkey OX40. In some more specific embodiments, the anti-OX40 antibody binds to human, macaque or rhesus OX40. In some other embodiments, the anti-OX40 antibody does not bind to mouse, rat, dog or rabbit OX40.

如本文所用的，术语“KD”欲指平衡解离常数，其系自kd对ka的比率(亦即kd/ka)获得且以摩尔浓度(M)表示。可使用业内充分确立的方法测定抗体的KD值。测定抗体的KD的优选方法系通过使用表面等离子共振，优选使用生物传感器系统(例如SPR系统)或流式细胞术及Scatchard分析。As used herein, the term "KD" is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (ie, kd/ka) and expressed in molar concentration (M). The KD value of an antibody can be determined using well-established methods in the art. A preferred method of determining the KD of an antibody is by using surface plasmon resonance, preferably a biosensor system (eg SPR system) or flow cytometry and Scatchard analysis.

如本文所用的，术语对IgG抗体的“高亲和力”系指抗体对于靶抗原具有10^-8M或更小，优选10^-9M或更小，且更优选10^-10M或更小的KD。然而，对于其他抗体同型，“高亲和力”结合可能是变化的。举例而言，对于IgM同型的“高亲和力”结合系指抗体具有10^-7M或更小，优选10^-8M或更小的KD。As used herein, the term "high affinity" for an IgG antibody refers to an antibody having a KD for the target antigen of ^10-8 M or less, preferably ^10-9 M or less, and more preferably ^10-10 M or less . However, for other antibody isotypes, "high affinity" binding may vary. For example, "high affinity" binding for an IgM isotype means that the antibody has a KD of 10" ⁷ M or less, preferably 10" ⁸ M or less.

如本文所用的，术语“抑制OX40-L与OX40结合”的抗体是指抑制OX40-L与OX40的结合，例如在使用HEK::OX40 100B5细胞的结合测定中抑制OX40-L与OX40的结合的抗体，在本领域公认的方法(例如本文所述基于FACS的结合测定)中，一定抗体浓度下能够部分或全部阻断0.5μg的OX40-L与OX40的结合。As used herein, the term "antibody that inhibits the binding of OX40-L to OX40" refers to an antibody that inhibits the binding of OX40-L to OX40, eg, an antibody that inhibits the binding of OX40-L to OX40 in a binding assay using HEK::OX40 100B5 cells Antibodies that partially or fully block the binding of 0.5 μg of OX40-L to OX40 in art-recognized methods (eg, the FACS-based binding assays described herein) at certain antibody concentrations.

如本文所用的，术语“抑制”或“阻断”(例如，在提及细胞上OX40-L与OX40的结合的抑制/阻断时)可互换使用，且涵盖部分的和完全的抑制/阻断。在某些实施方案中，抗OX40抗体将OX40-L与OX40的结合抑制至少约50％，例如约60％、70％、80％、90％、95％、99％或100％。在某些实施方案中，抗OX40抗体将OX40-L与OX40的结合抑制不超过50％，例如约40％、30％、20％、10％、5％或1％。As used herein, the terms "inhibit" or "block" (eg, when referring to the inhibition/blocking of OX40-L binding to OX40 on a cell) are used interchangeably and encompass both partial and complete inhibition/blocking. block. In certain embodiments, the anti-OX40 antibody inhibits the binding of OX40-L to OX40 by at least about 50%, eg, about 60%, 70%, 80%, 90%, 95%, 99%, or 100%. In certain embodiments, the anti-OX40 antibody inhibits the binding of OX40-L to OX40 by no more than 50%, eg, about 40%, 30%, 20%, 10%, 5%, or 1%.

如本文所用的，术语“核苷酸分子”意在包括DNA分子及RNA分子。核苷酸分子可为单链或双链，且可为cDNA。As used herein, the term "nucleotide molecule" is intended to include DNA molecules as well as RNA molecules. Nucleotide molecules can be single-stranded or double-stranded, and can be cDNA.

在一些更具体的实施方案中，本文公开的抗体为抗人OX40单克隆抗体。OX40抗体类型与亚型可由本领域已知的任何方式确定。通常，抗体类型与亚型可使用特异于特定抗体类型与亚型的抗体确定。In some more specific embodiments, the antibodies disclosed herein are anti-human OX40 monoclonal antibodies. OX40 antibody types and subtypes can be determined by any means known in the art. In general, antibody types and subtypes can be determined using antibodies specific for a particular antibody type and subtype.

如本文所用的，术语“激动剂抗体”是指当将该抗体加至表达OX40的细胞、组织或生物体中时，可使一种或多种OX40活性提高至少约20％。在某些实施方案中，具有激动剂功能的抗体使OX40活性提高至少40％、50％或60％。活化型抗体可扩大或取代OX40L对OX40的效应。在一些实施方案中，活化型抗体基本上为OX40L的模拟物，且与OX40L竞争结合OX40。在本文中，术语“激动剂抗体”、“激动型抗体”和“活化型抗体”可以互换使用。As used herein, the term "agonist antibody" refers to an antibody that, when added to a cell, tissue or organism expressing OX40, increases the activity of one or more OX40s by at least about 20%. In certain embodiments, the antibody with agonist function increases OX40 activity by at least 40%, 50% or 60%. Activated antibodies can augment or replace the effects of OX40L on OX40. In some embodiments, the activating antibody is substantially a mimetic of OX40L and competes with OX40L for binding to OX40. Herein, the terms "agonist antibody", "agonist antibody" and "activating antibody" are used interchangeably.

在一些实施方案中，抗OX40活化型单克隆抗体可通过以下至少一种机制抑制或根除肿瘤：增强活化肿瘤特异性CD4⁺与CD8⁺淋巴细胞，抑制瘤内Treg细胞，并增强肿瘤杀伤性记忆细胞等。在一些实施方案中，其它抗肿瘤活性可受OX40信号传递的其它免疫加强效应介导(例如产生趋化因子与细胞因子，以及加强CTL与NK溶胞活性等)，以及通过诱发细胞凋亡或刺激针对ADCC的体液反应而直接消灭肿瘤等。In some embodiments, anti-OX40-activating monoclonal antibodies inhibit or eradicate tumors by at least one of the following mechanisms: enhanced activation of tumor-specific CD4 ⁺ and CD8 ⁺ lymphocytes, inhibition of intratumoral Treg cells, and enhanced tumor-killing memory cells etc. In some embodiments, other anti-tumor activities may be mediated by other immune-boosting effects of OX40 signaling (eg, production of chemokines and cytokines, and enhancement of CTL and NK lytic activity, etc.), as well as by induction of apoptosis or Stimulate a humoral response to ADCC to directly destroy tumors, etc.

在一些实施方案中，本文公开的抗体为抗OX40激动型抗体，其与现有的Genentech公司的IgG1亚型OX40抗体(Genentech公司编号：MOXR0916)相比较，在细胞功能活性及刺激人CD4⁺T细胞增殖方面具有一定的优势，且在小鼠体内可以快速刺激特异性T细胞的增殖活化，并抑制肿瘤细胞的生长。In some embodiments, the antibody disclosed herein is an anti-OX40 agonist antibody, which, compared to the existing Genentech IgG1 subtype OX40 antibody (Genentech No.: MOXR0916), exhibits increased cellular functional activity and stimulation of human CD4 ⁺ T It has certain advantages in cell proliferation, and can rapidly stimulate the proliferation and activation of specific T cells in mice and inhibit the growth of tumor cells.

在一些具体的实施方案中，本文公开的抗体能够阻断人OX40蛋白与其配体OX40L的结合，且具有一定的量效关系。In some specific embodiments, the antibodies disclosed herein are capable of blocking the binding of human OX40 protein to its ligand OX40L in a dose-response relationship.

在一些实施方案中，本文公开的抗体可以刺激CD4⁺T细胞增殖并增加IL-2和IFN-γ的分泌。例如，在一些具体的实施方案中，本文公开的抗体可以有效促进CD4⁺T细胞增殖及细胞因子的分泌，包括但不限于IL-2、IFN-γ等。In some embodiments, the antibodies disclosed herein can stimulate CD4 ⁺ T cell proliferation and increase the secretion of IL-2 and IFN-γ. For example, in some specific embodiments, the antibodies disclosed herein can effectively promote CD4 ⁺ T cell proliferation and cytokine secretion, including but not limited to IL-2, IFN-γ, and the like.

在一些实施方案中，本文公开的抗体可有效激活肿瘤杀伤性T细胞(例如激活hOX40 KI鼠体内特异性T细胞)，并抑制瘤内Treg细胞。In some embodiments, the antibodies disclosed herein are effective in activating tumor-killing T cells (eg, activating hOX40 KI murine specific T cells in vivo) and inhibiting intratumoral Treg cells.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分能够杀死表达OX40的细胞，例如表达高水平OX40的细胞。In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is capable of killing cells that express OX40, eg, cells that express high levels of OX40.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分能够引发T细胞介导的反应。In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is capable of eliciting a T cell-mediated response.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分能够提高效应T细胞的功能。In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is capable of enhancing effector T cell function.

在一些实施方案中，所述特异性结合OX40的抗体或其抗原结合部分能够提高记忆T细胞的功能。In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is capable of enhancing memory T cell function.

在一些实施方案中，本文公开的抗体可以抑制肿瘤的生长。例如，在一些具体的实施方案中，本文公开的抗体可以抑制hOX40 KI鼠的MC38皮下移植瘤的生长。在一些具体的实施方案中，上述抗体抑制肿瘤生长至少达80％、85％或90％。在一些实施方案中，在荷瘤个体接受本文公开的抗体处理后14天即可检测到抑制肿瘤生长的情形。在另一些实施方案中，在初次接受抗体处理后6天即可检测到抑制肿瘤生长的情形。In some embodiments, the antibodies disclosed herein can inhibit tumor growth. For example, in some specific embodiments, the antibodies disclosed herein can inhibit the growth of MC38 subcutaneous xenografts in hOX40 KI mice. In some specific embodiments, the aforementioned antibodies inhibit tumor growth by at least 80%, 85%, or 90%. In some embodiments, inhibition of tumor growth is detectable 14 days after treatment of the tumor-bearing individual with the antibodies disclosed herein. In other embodiments, inhibition of tumor growth is detectable 6 days after initial antibody treatment.

本申请还提供了编码本文公开的抗体或其抗原结合部分的核苷酸分子、包含所述核苷酸分子的表达载体、包含所述核苷酸分子或表达载体的宿主细胞、以及制备和纯化该抗体的方法。The application also provides nucleotide molecules encoding the antibodies or antigen-binding portions thereof disclosed herein, expression vectors comprising the nucleotide molecules, host cells comprising the nucleotide molecules or expression vectors, and preparation and purification method of the antibody.

在一些实施方案中，编码所述抗体或其抗原结合部分的核苷酸分子可操作地连接到调控序列，调控序列可以被用所述表达载体转化过的宿主细胞识别。In some embodiments, the nucleotide molecule encoding the antibody, or antigen-binding portion thereof, is operably linked to regulatory sequences that can be recognized by host cells transformed with the expression vector.

在一些实施方案中，任何合适的表达载体都可以用于本申请。例如，所述表达载体可以为pQK1、pTT5、pUC57、pDR1、pcDNA3.1(+)、pDHFF及pCHO1.0中的任一种。表达载体中可以包括连接有合适的转录和翻译调节序列的融合DNA序列。In some embodiments, any suitable expression vector can be used in the present application. For example, the expression vector may be any one of pQK1, pTT5, pUC57, pDR1, pcDNA3.1(+), pDHFF and pCHO1.0. Fusion DNA sequences ligated with appropriate transcriptional and translational regulatory sequences may be included in the expression vector.

在一些实施方案中，可用的宿主细胞为含有上述表达载体的细胞，可以是真核细胞，例如哺乳动物或昆虫宿主细胞培养系统均可用于本申请的抗体或其抗原结合部分的表达。例如，HEK 293细胞、COS细胞、CHO细胞、NS0细胞、sf9细胞及sf21细胞等均可适用于本发明。所述宿主细胞也可以为含有上述表达载体的原核细胞，例如可以为DH5α、BL21(DE3)或TG1等。In some embodiments, useful host cells are cells containing the above-described expression vectors, which can be eukaryotic cells, eg, mammalian or insect host cell culture systems can be used for the expression of the antibodies or antigen-binding portions thereof of the present application. For example, HEK 293 cells, COS cells, CHO cells, NSO cells, sf9 cells, and sf21 cells, etc. can be used in the present invention. The host cell may also be a prokaryotic cell containing the above-mentioned expression vector, such as DH5α, BL21(DE3) or TG1, etc.

在一些实施方案中，本文公开的抗OX40单克隆抗体的制备方法包括：在表达条件下，培养宿主细胞，从而表达抗OX40单克隆抗体；分离和纯化表达的抗OX40单克隆抗体。利用上述方法，可以将重组蛋白纯化为基本均一的物质，例如在SDS-PAGE电泳上为单一条带。In some embodiments, the method for preparing an anti-OX40 monoclonal antibody disclosed herein comprises: culturing a host cell under expression conditions, thereby expressing the anti-OX40 monoclonal antibody; and isolating and purifying the expressed anti-OX40 monoclonal antibody. Using the methods described above, the recombinant protein can be purified to a substantially homogeneous material, eg, as a single band on SDS-PAGE.

在一些实施方案中，可以利用亲和层析的方法对本文公开的抗OX40抗体进行分离纯化，根据所利用的亲和柱的特性，可以使用常规的方法例如高盐缓冲液、改变pH等方法洗脱结合在亲和柱上的抗OX40抗体。In some embodiments, the anti-OX40 antibodies disclosed herein can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffers, pH changes, etc. can be used. The anti-OX40 antibody bound to the affinity column is eluted.

在一些实施方案中，动物接种OX40抗原后，可自动物体内得到抗体和/或产生抗体的细胞。产生抗体的永生化细胞系可由经免疫的动物体中分离出的细胞制备。免疫接种后，杀死动物，取淋巴结和/或脾脏B细胞进行永生化处理，经过致癌性化合物及诱变化合物处理，与永生化细胞(例如骨髓瘤细胞)融合，去除肿瘤抑制基因的活性。若使用骨髓瘤细胞进行融合时，该骨髓瘤细胞最好不分泌免疫球蛋白多肽(非分泌性细胞系)。使用OX40、其部分或表达OX40的细胞筛选永生化细胞。在优选的实施方案中，初次筛选是利用酶联免疫分析法(ELISA)进行。选取产生抗OX40抗体的细胞例如杂交瘤进行克隆，再进一步筛选所需的特性，包括生长良好、抗体产量高以及具有所需抗体特性等。杂交瘤的筛选、克隆及扩增方法是本领域普通技术人员熟知的。在一些实施方案中，经免疫接种的动物为非人类动物，其中脾脏B细胞与来自与该非人类动物相同物种的骨髓瘤细胞系融合。在一些实施方案中，该经免疫接种的动物为Balb/c小鼠且骨髓瘤细胞系为非分泌性小鼠骨髓瘤细胞SP2/0。In some embodiments, antibodies and/or antibody-producing cells can be obtained in vivo following vaccination of an animal with the OX40 antigen. Immortalized cell lines producing antibodies can be prepared from cells isolated from immunized animals. After immunization, animals are killed, lymph node and/or spleen B cells are harvested for immortalization, treated with carcinogenic and mutagenic compounds, and fused with immortalized cells (eg, myeloma cells) to remove tumor suppressor gene activity. When myeloma cells are used for fusion, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Immortalized cells are screened using OX40, a portion thereof, or cells expressing OX40. In a preferred embodiment, the primary screening is performed using an enzyme-linked immunoassay (ELISA). Anti-OX40 antibody-producing cells, such as hybridomas, are selected for cloning and further screened for desired properties, including good growth, high antibody yield, and desired antibody properties. Methods for screening, cloning and expansion of hybridomas are well known to those of ordinary skill in the art. In some embodiments, the immunized animal is a non-human animal, wherein the splenic B cells are fused with a myeloma cell line from the same species as the non-human animal. In some embodiments, the immunized animal is a Balb/c mouse and the myeloma cell line is a non-secreting mouse myeloma cell SP2/0.

本申请提供了药物组合物，其包含本文公开的抗体或其抗原结合部分以及药学上可接受的载体。本文公开的上述抗OX40抗体(例如抗人OX40单克隆抗体)可以和药学上可接受的载体一起配制成药物制剂，从而更稳定地发挥疗效。在一些实施方案中，这些制剂可以保证本文公开的抗OX40抗体(例如抗人OX40单克隆抗体)的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱酰胺或氧化)。在一些实施方案中，对于液体制剂，通常可以在2℃-8℃条件下至少一年保存稳定。在一些实施方案中，对于冻干制剂，在30℃下至少六个月保持稳定。在一些实施方案中，所述药物组合物还包含一种或多种其他活性成分。在一些实施方案中，所述活性成分为抗肿瘤药物。The application provides pharmaceutical compositions comprising an antibody or antigen-binding portion thereof disclosed herein and a pharmaceutically acceptable carrier. The above-mentioned anti-OX40 antibodies (eg, anti-human OX40 monoclonal antibodies) disclosed herein can be formulated into pharmaceutical preparations together with a pharmaceutically acceptable carrier, so as to stably exert therapeutic effects. In some embodiments, these formulations can preserve the conformational integrity of the amino acid core sequences of the anti-OX40 antibodies disclosed herein (eg, anti-human OX40 monoclonal antibodies), while also protecting the multifunctional groups of the protein from degradation (including, but not limited to, aggregation) , deamidation or oxidation). In some embodiments, liquid formulations are generally shelf stable at 2°C-8°C for at least one year. In some embodiments, lyophilized formulations are stable at 30°C for at least six months. In some embodiments, the pharmaceutical composition further comprises one or more other active ingredients. In some embodiments, the active ingredient is an antineoplastic drug.

本申请提供了疫苗，其包含第一方面所述的特异性结合OX40的抗体或其抗原结合部分，以及任选的免疫佐剂。所述免疫佐剂包括但不限于：1)生物性佐剂，例如细菌或其产物(如分枝杆菌(结核杆菌、卡介苗)、短小棒状杆菌、百日咳杆菌、革兰阴性杆菌内毒素)、粒细胞-巨噬细胞集落刺激因子、白细胞介素-l、白细胞介素-2、干扰素-γ等；2)无机佐剂，例如氢氧化铝、明矾、磷酸铝等；3)人工合成佐剂，例如双链多聚肌苷酸、胞苷酸、双链多聚腺苷酸；4)油剂，例如花生油乳化佐剂、矿物油、植物油、羊毛脂等；5)弗氏佐剂，例如弗氏不完全佐剂和弗氏完全佐剂。The application provides a vaccine comprising the antibody or antigen-binding portion thereof that specifically binds OX40 as described in the first aspect, and optionally an immunological adjuvant. The immune adjuvants include but are not limited to: 1) biological adjuvants, such as bacteria or their products (such as mycobacteria (Mycobacterium tuberculosis, BCG), Corynebacterium pumilus, Bacillus pertussis, Gram-negative bacilli endotoxin), particles Cell-macrophage colony stimulating factor, interleukin-1, interleukin-2, interferon-γ, etc.; 2) Inorganic adjuvants, such as aluminum hydroxide, alum, aluminum phosphate, etc.; 3) Synthetic adjuvants , such as double-chain polyinosinic acid, cytidine acid, double-chain polyadenylic acid; 4) oil agents, such as peanut oil emulsifying adjuvant, mineral oil, vegetable oil, lanolin, etc.; 5) Freund's adjuvant, such as Incomplete Freund's adjuvant and complete Freund's adjuvant.

本申请还提供了在肿瘤免疫领域预防和/或治疗OX40相关的疾病，例如肿瘤的方法，其包括给予个体抗OX40抗体、或者包含抗OX40抗体(例如抗人OX40单克隆抗体)的药物组合物或疫苗。在一些实施方案中，在对包括人在内的动物给药后，抗肿瘤效果明显。具体地说，本文公开的抗OX40抗体能够有效地预防和/或治疗肿瘤，可以作为抗肿瘤药物使用。The present application also provides methods for preventing and/or treating OX40-related diseases, such as tumors, in the field of tumor immunity, comprising administering an anti-OX40 antibody to an individual, or a pharmaceutical composition comprising an anti-OX40 antibody (eg, an anti-human OX40 monoclonal antibody) or vaccines. In some embodiments, the anti-tumor effect is pronounced following administration to animals, including humans. Specifically, the anti-OX40 antibodies disclosed herein can effectively prevent and/or treat tumors, and can be used as anti-tumor drugs.

在一些实施方案中，可以将本申请的抗OX40抗体、或者包含抗OX40抗体(例如抗人OX40单克隆抗体)的药物组合物或疫苗与其他的治疗剂(例如化疗剂、PD-1结合拮抗剂等)联合施用。In some embodiments, an anti-OX40 antibody of the present application, or a pharmaceutical composition or vaccine comprising an anti-OX40 antibody (eg, an anti-human OX40 monoclonal antibody), can be combined with other therapeutic agents (eg, chemotherapeutic agents, PD-1 binding antagonists) agents, etc.) in combination.

本申请还提供了抗OX40抗体或其抗原结合部分，或者包含所述抗OX40抗体或其抗原结合部分的药物组合物在制备用于预防和/或治疗OX40相关的疾病，例如肿瘤的药物中的用途。The present application also provides an anti-OX40 antibody or an antigen-binding portion thereof, or a pharmaceutical composition comprising the anti-OX40 antibody or an antigen-binding portion thereof in the preparation of a medicament for preventing and/or treating OX40-related diseases, such as tumors use.

在一些实施方案中，所述肿瘤为结肠癌、黑色素瘤、间皮质瘤、肾细胞癌、淋巴瘤、晚期实体瘤或其转移瘤等。In some embodiments, the tumor is colon cancer, melanoma, mesothelioma, renal cell carcinoma, lymphoma, advanced solid tumor or metastases thereof, and the like.

本文公开的抗人OX40抗体及包含所述抗人OX40抗体的药物组合物在对包括人在内的动物给药时，给药剂量因个体的年龄和体重、疾病特性和严重性以及给药途径而异，可以参考动物实验的结果和综合情况，总给药量不能超过一定范围。When the anti-human OX40 antibody disclosed herein and the pharmaceutical composition comprising the anti-human OX40 antibody are administered to animals, including humans, the dosage to be administered depends on the age and weight of the individual, the nature and severity of the disease, and the route of administration However, you can refer to the results of animal experiments and the comprehensive situation, and the total dose cannot exceed a certain range.

抗体或包含所述抗体的药物组合物的施用剂量和频率可根据对疾病进行预防或治疗而变化。在预防性应用中，向尚未处于疾病状态的患者施用含有本申请的抗体或其混合物的组合物以增强患者抵抗力，此量定义为“预防性有效剂量”。在此用途中，具体的剂量又视患者健康状况及全身免疫性而定。通常以相对不频繁的间隔施用相对较低剂量较长时间。在治疗性应用中，有时需要以相对较短间隔施用相对较高剂量直至疾病进展减缓或终止为止，且优选直至患者显示疾病症状部分或完全改善为止。此后，可向患者施用预防性方案。本领域普通技术人员可以容易地根据实际需要掌握具体的剂量和频率。The dose and frequency of administration of the antibody or pharmaceutical composition comprising the antibody may vary depending on the prevention or treatment of the disease. In prophylactic applications, a composition containing an antibody of the present application or a mixture thereof is administered to a patient who is not already in a disease state to increase patient resistance, and this amount is defined as a "prophylactically effective dose." In this use, the specific dosage again depends on the patient's health and systemic immunity. Relatively lower doses are usually administered at relatively infrequent intervals for longer periods of time. In therapeutic applications, it is sometimes necessary to administer relatively high doses at relatively short intervals until disease progression is slowed or terminated, and preferably until the patient shows partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen can be administered to the patient. Those of ordinary skill in the art can easily grasp the specific dosage and frequency according to actual needs.

本申请还提供了包含本文公开的抗体或其抗原结合部分的检测试剂或试剂盒。The application also provides detection reagents or kits comprising the antibodies or antigen-binding portions thereof disclosed herein.

如本文所用的，术语“免疫反应”指脊椎动物内针对外来作用剂的生物反应，该反应保护生物体抵抗此类作用剂及由其引起的疾病。免疫反应系由免疫系统的细胞(例如，T淋巴细胞、B淋巴细胞、自然杀伤(NK)细胞、巨噬细胞、嗜酸性粒细胞、肥大细胞、树突细胞或嗜中性粒细胞)及由此类细胞中的任一者或由肝脏产生的可溶性大分子(包括抗体、细胞因子及补体)的作用介导，其导致选择性靶向、结合、损害、破坏和/或自脊椎动物身体消除侵入病原体、经病原体感染的细胞或组织、癌性或其他异常细胞或在自体免疫或病理炎症的情形下正常人类细胞或组织。免疫反应包括T细胞(例如效应T细胞)或Th细胞(例如CD4⁺或CD8⁺T细胞)的活化或抑制或Treg细胞的抑制。As used herein, the term "immune response" refers to a biological response within a vertebrate to a foreign agent that protects the organism against such agent and the disease caused by it. The immune response is composed of cells of the immune system (eg, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and by Mediated by the action of any of these cells or by liver-produced soluble macromolecules (including antibodies, cytokines, and complement) that result in selective targeting, binding, damage, destruction, and/or elimination from the vertebrate body Invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or normal human cells or tissues in the context of autoimmunity or pathological inflammation. The immune response includes activation or suppression of T cells (eg, effector T cells) or Th cells (eg, CD4 ⁺ or CD8 ⁺ T cells) or suppression of Treg cells.

如本文所用的，术语“T细胞介导的反应”系指由T细胞(包括效应T细胞(例如，CD8⁺细胞)及辅助T细胞(例如，CD4⁺细胞))介导的反应。T细胞介导的反应包括T细胞细胞毒性及增殖。As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (eg, CD8 ⁺ cells) and helper T cells (eg, CD4 ⁺ cells). T cell-mediated responses include T cell cytotoxicity and proliferation.

如本文所用的，术语“癌症”指以体内异常细胞不受控生长为特点的一大类疾病。失调的细胞分裂可形成侵袭相邻组织且可经由淋巴系统或血流转移至身体的远处部分的恶性肿瘤或细胞。As used herein, the term "cancer" refers to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Dysregulated cell division can form malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

如本文所用的，术语“抑制肿瘤的生长”包括肿瘤生长的任何可测量的减少，例如肿瘤生长抑制至少约10％，例如至少约20％、至少约30％、至少约40％、至少约50％、至少约60％、至少约70％、至少约80％、至少约90％、至少约99％或100％。As used herein, the term "inhibition of tumor growth" includes any measurable reduction in tumor growth, eg, tumor growth inhibition of at least about 10%, eg, at least about 20%, at least about 30%, at least about 40%, at least about 50% %, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%.

如本文所用的，术语“治疗”指对受试者实施的任何类型的介入或方法或向其施用活性剂，其中目的是逆转、缓和、改善、抑制或缓解或预防症状、并发症、病况或与疾病相关的生物化学标记的进展、发展、严重程度或复发。预防指对未患疾病的受试者施用，以防止疾病发生或使其影响(若存在)最小化。As used herein, the term "treatment" refers to any type of intervention or method performed on a subject or the administration of an active agent thereto, wherein the purpose is to reverse, alleviate, ameliorate, inhibit or alleviate or prevent a symptom, complication, condition or Progression, progression, severity or recurrence of disease-related biochemical markers. Prophylaxis refers to administration to subjects not suffering from the disease to prevent the occurrence of the disease or to minimize its effects, if any.

本说明书和权利要求书中，词语“包括”、“包含”和“含有”意指“包括但不限于”，且并非意图排除其他部分、添加物、组分或步骤。In this specification and claims, the words "including", "comprising" and "containing" mean "including but not limited to" and are not intended to exclude other parts, additions, components or steps.

应该理解，在本申请的特定方面、实施方案或实施例中描述的特征、特性、组分或步骤，可适用于本文所描述的任何其他的方面、实施方案或实施例，除非与之矛盾。It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application are applicable to any other aspect, embodiment or example described herein unless contradicted by it.

上述公开内容从总体上描述了本申请，以下实施例是对本申请作进一步的说明，不应理解为对本申请的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域具有普通技术的人员来说是众所周知的，并且在许多出版物中都有所描述，例如参见Sambrook,J.,Fritsch,EF.and Maniais，T.(1989)MolecularCloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。The above disclosure generally describes the present application, and the following examples are intended to further illustrate the present application and should not be construed as limiting the present application. The Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting protein-encoding genes into vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications, eg, see Sambrook, J., Fritsch, EF. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

本发明公开了特异性结合哺乳动物(人、灵长类动物等)OX40的抗体，且该抗体或抗体部分被用于调节受体活性，刺激T细胞的活化及增殖等。本发明提供此类蛋白质在治疗、筛选和检测等方面的应用，如在癌症治疗中的用途。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明的范围内。本领域技术人员能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明。The invention discloses an antibody that specifically binds to mammalian (human, primate, etc.) OX40, and the antibody or antibody part is used to regulate receptor activity, stimulate T cell activation and proliferation, and the like. The present invention provides applications of such proteins in therapy, screening and detection, such as in cancer therapy. Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications apparent to those skilled in the art are deemed to be included within the scope of the present invention. Those skilled in the art can make modifications or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the present invention.

为了使本领域的技术人员更好地理解本发明的技术方案，下面结合实施例对本发明作进一步的详细说明。In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the embodiments.

实施例Example

实施例1.产生抗OX40抗体的杂交瘤的制备Example 1. Preparation of hybridomas producing anti-OX40 antibodies

1.取8-10周大的Balb/c小鼠，经腹膜内接种OX40蛋白(10μg/剂/只)。人OX40(TNFRSF4)蛋白订购自义翘神州，货号为No.10481-H08H，DNA序列为NP_003318.1，蛋白序列为胞外区Met1-Ala 216，C末端含有His标签，通过SDS-PAGE验证其分子量在40-45kDa。在3-8周内，重复此剂量5-7次。进行融合前4天，为小鼠注射最后一剂OX40蛋白。1. Take 8-10-week-old Balb/c mice and inoculate OX40 protein (10 μg/dose/mice) intraperitoneally. Human OX40 (TNFRSF4) protein was ordered from Yiqiao Shenzhou, the product number is No.10481-H08H, the DNA sequence is NP_003318.1, the protein sequence is the extracellular region Met1-Ala 216, and the C-terminal contains a His tag, which was verified by SDS-PAGE. The molecular weight is 40-45kDa. Repeat this dose 5-7 times over 3-8 weeks. Four days before fusion, mice were injected with a final dose of OX40 protein.

2.进行融合前1天，取普通Balb/c小鼠腹腔内巨噬细胞作为滋养层，接种于96孔板中。2. One day before fusion, the intraperitoneal macrophages of ordinary Balb/c mice were taken as trophoblasts and inoculated into 96-well plates.

3.取来自经免疫接种的小鼠的脾与淋巴结的淋巴细胞与非分泌性骨髓瘤SP2/0细胞系融合，将细胞加入到提前铺有滋养层的96孔板中，对融合的细胞进行HAT选择(Galfreand Milstein,Methods Enzymol 1981；73：3-46)。3. Lymphocytes from the spleen and lymph nodes of the immunized mice were fused with the non-secretory myeloma SP2/0 cell line, and the cells were added to a 96-well plate plated with trophoblasts in advance. HAT selection (Galfre and Milstein, Methods Enzymol 1981;73:3-46).

4.回收一组均分泌抗OX40特异性抗体的杂交瘤细胞。初次筛选是利用酶联免疫分析法(ELISA)确定杂交瘤分泌的抗OX40抗体的滴度。4. A group of hybridoma cells secreting anti-OX40-specific antibodies were recovered. The primary screening was to determine the titer of anti-OX40 antibodies secreted by hybridomas using an enzyme-linked immunoassay (ELISA).

实施例2.通过NF-κB信号通路检测抗体活性Example 2. Detection of antibody activity by NF-κB signaling pathway

1.将收集的1瓶(T75)HEK::OX40 100B5细胞转移至15mL离心管中，用5mL含10％FBS(Gemini)的DMEM培养基(Gibco)洗1次，1000rpm离心5min；弃上清，加入5mL含2％FBS的PBS重悬细胞计数。1. Transfer 1 bottle of (T75) HEK::OX40 100B5 cells collected to a 15mL centrifuge tube, wash once with 5mL DMEM medium (Gibco) containing 10% FBS (Gemini), and centrifuge at 1000rpm for 5min; discard the supernatant , and add 5 mL of PBS containing 2% FBS to resuspend the cells for counting.

2.将细胞稀释至2×10⁵个细胞/mL，按照100μL/孔加入96孔板，放入5％CO₂的37℃细胞培养箱孵育30min。2. Dilute the cells to 2×10 ⁵ cells/mL, add 100 μL/well to a 96-well plate, and incubate in a 37°C cell incubator with 5% CO ₂ for 30 min.

3.将待检测的各单克隆抗体及对照抗体(罗氏公司在研抗体MOXR0916，IgG1亚型人源化抗体)，人的4-1BB配体(h-OX40L，百普赛斯)，同亚型抗体(小鼠IgG1抗体(mIgG1)，人IgG1抗体(hIgG1)，Biolegend)，分别按照以下浓度进行稀释：4μg/mL、0.4μg/mL和0.04μg/mL，按照50μL/孔加入96孔板中，设置复孔，使得反应体系单克隆抗体的终浓度分别为1μg/mL、0.1μg/mL和0.01μg/mL。同时加入完全不加抗体的DMEM培养基组，单加二抗的抗mIgG-Fc组作为背景值对照。3. Each monoclonal antibody and control antibody to be detected (Roche's research antibody MOXR0916, IgG1 subtype humanized antibody), human 4-1BB ligand (h-OX40L, Bepsis), the same subtype Type antibodies (mouse IgG1 antibody (mIgG1), human IgG1 antibody (hIgG1), Biolegend) were diluted according to the following concentrations: 4 μg/mL, 0.4 μg/mL and 0.04 μg/mL, and added to 96-well plate at 50 μL/well , duplicate wells were set up so that the final concentrations of monoclonal antibodies in the reaction system were 1 μg/mL, 0.1 μg/mL, and 0.01 μg/mL, respectively. At the same time, the DMEM medium group without antibody was added, and the anti-mIgG-Fc group with secondary antibody was added as the background value control.

4.抗鼠IgG Fc(Biolegend)及抗人IgG Fc(Biolegend)作为二抗加入，起到模拟体内Fc受体交联的作用，二抗终浓度是一抗的3倍，按照50μL/孔的体系进行配制，加入96孔板中。4. Anti-mouse IgG Fc (Biolegend) and anti-human IgG Fc (Biolegend) were added as secondary antibodies to simulate the cross-linking of Fc receptors in vivo. The final concentration of secondary antibodies was 3 times that of primary antibodies. The system was formulated and added to a 96-well plate.

5.将96孔板放入5％CO₂的37℃细胞培养箱中孵育20h；然后将待测96孔板室温1000rpm离心5min，取新的96孔板每孔加入160μL提前在37℃温箱孵育30min的Quanti Blue(Invivo Gen)，后取40μL细胞上清加入，于37℃中孵育1h。5. Put the 96-well plate into a 37°C cell incubator with 5% CO ₂ and incubate for 20 hours; then centrifuge the 96-well plate to be tested at room temperature at 1000 rpm for 5 minutes, and add 160 μL to each well of a new 96-well plate and incubate at 37°C in advance. Incubate with Quanti Blue (Invivo Gen) for 30 min, then add 40 μL of cell supernatant, and incubate at 37° C. for 1 h.

6.使用酶标仪在620nm下检测OD值，并选择GraphPad Prism作图分析数据。6. Use a microplate reader to detect the OD value at 620 nm, and select GraphPad Prism to plot and analyze the data.

部分结果如图1A所示，共对30个单克隆抗体进行初筛；进一步选择优势克隆验证，如图1B所示，获得7个候选单克隆抗体，分别标记为4F2、4B9、6C1、6H12、13F7、6A6和18B10。Part of the results are shown in Figure 1A, a total of 30 monoclonal antibodies were initially screened; the dominant clones were further selected for verification, as shown in Figure 1B, 7 candidate monoclonal antibodies were obtained, labeled 4F2, 4B9, 6C1, 6H12, 13F7, 6A6 and 18B10.

实施例3.通过NFAT信号通路检测抗体活性Example 3. Detection of antibody activity by NFAT signaling pathway

1.将收集的1瓶(T75)Jurkat OX40细胞转移至15mL离心管中，用5mL含10％FBS的1640培养基(Gibco)洗1次，850rpm离心5min，弃上清，加入5mL含1％FBS的RPMI 1640培养基重悬细胞并计数。1. Transfer 1 bottle (T75) of Jurkat OX40 cells collected to a 15mL centrifuge tube, wash once with 5mL 1640 medium (Gibco) containing 10% FBS, centrifuge at 850rpm for 5min, discard the supernatant, add 5mL containing 1% Cells were resuspended in RPMI 1640 medium in FBS and counted.

2.将细胞稀释至1.0×10⁶个细胞/mL，按照25μL/孔加入96孔板，放入5％CO₂的37℃细胞培养箱孵育30min。2. Dilute the cells to 1.0×10 ⁶ cells/mL, add 25 μL/well to a 96-well plate, and incubate in a 37°C cell incubator with 5% CO ₂ for 30 min.

3.同实施例2的对照组设计，加入对照抗体MOXR0916同亚型抗体(mIgG1和hIgG1，Biolegend)，将待检测的各抗体分别按照以下浓度进行稀释：3μg/ml、0.3μg/ml和0.03μg/ml，按照25μL/孔加入96孔板中，设置复孔。同时加入完全不加抗体的RPMI 1640培养基组(图中标注为：1640)，作为背景值对照。3. The same as the control group design in Example 2, the control antibody MOXR0916 isotype antibody (mIgG1 and hIgG1, Biolegend) was added, and each antibody to be detected was diluted according to the following concentrations: 3 μg/ml, 0.3 μg/ml and 0.03 μg/ml. μg/ml, add 25 μL/well to 96-well plate, and set up duplicate wells. At the same time, the RPMI 1640 medium group (marked as: 1640 in the figure) without antibody at all was added as a background value control.

4.抗鼠IgG Fc(Biolegend)及抗人IgG Fc(Biolegend)作为二抗终浓度是一抗的3倍，按照25μL/孔的体系进行配制加入96孔板中。4. The final concentration of anti-mouse IgG Fc (Biolegend) and anti-human IgG Fc (Biolegend) as secondary antibodies was 3 times that of primary antibodies, and were added to 96-well plates according to the system of 25 μL/well.

5.将96孔板放入5％CO₂的37℃细胞培养箱孵育6h。5. Incubate the 96-well plate in a 37°C cell incubator with 5% CO ₂ for 6h.

6.取平衡至室温的Bio-Glo(Promega)，按照75μL/孔加到该96孔板中，进行发光检测。6. Take Bio-Glo (Promega) equilibrated to room temperature and add 75 μL/well to the 96-well plate for luminescence detection.

结果如图2所示，获得7个候选单克隆抗体，分别标记为4F2、4B9、6C1、6H12、13F7、6A6和18B10。The results are shown in Figure 2. Seven candidate monoclonal antibodies were obtained, which were labeled as 4F2, 4B9, 6C1, 6H12, 13F7, 6A6 and 18B10, respectively.

实施例4.抗体的动力学参数测定Example 4. Determination of kinetic parameters of antibodies

1.使用Fortebio分子相互作用仪测定人OX40与抗OX40单克隆抗体(4F2、4B9、6C1、6H12、13F7、6A6和18B10)结合的动力学参数。1. The kinetic parameters of human OX40 binding to anti-OX40 monoclonal antibodies (4F2, 4B9, 6C1, 6H12, 13F7, 6A6 and 18B10) were determined using a Fortebio molecular interaction instrument.

2.用ProA传感器捕获固定各单克隆抗体，在PBS中平衡后，与抗原人OX40结合。2. Each monoclonal antibody was captured and immobilized with a ProA sensor, and after equilibration in PBS, it bound to the antigen human OX40.

3.将人OX40用PBS稀释为2个浓度，分别为400nM和200nM，于PBS中解离。3. Human OX40 was diluted with PBS to 2 concentrations, 400 nM and 200 nM, respectively, and dissociated in PBS.

参数检测结果如表1所示，动力学结合解离图如图3所示，所有候选抗体的亲和力均处于亚nM水平。The parameter detection results are shown in Table 1, and the kinetic binding and dissociation diagram is shown in Figure 3. The affinities of all candidate antibodies are at the sub-nM level.

表1.人OX40与抗OX40单克隆抗体结合的动力学参数Table 1. Kinetic parameters of human OX40 binding to anti-OX40 monoclonal antibodies

实施例5.候选抗OX40单克隆抗体与人OX40及猴OX40的结合实验Example 5. Binding experiments of candidate anti-OX40 monoclonal antibodies to human OX40 and monkey OX40

1.提前瞬时转染人OX40及猴OX40膜表达质粒于293T细胞中。1. Transient human OX40 and monkey OX40 membrane expression plasmids into 293T cells in advance.

2.选择未转染的293T细胞作为对照细胞，取表达人OX40的293T细胞与表达猴OX40的293T细胞作为实验组，用胰酶将三种细胞消化后，取含2％FBS的PBS清洗两遍，850rpm离心5min，弃上清，然后加入含2％FBS的PBS重悬细胞并计数。2. Select untransfected 293T cells as control cells, and take 293T cells expressing human OX40 and 293T cells expressing monkey OX40 as experimental groups. After digesting the three cells with trypsin, wash them with PBS containing 2% FBS. Repeat, centrifuge at 850 rpm for 5 min, discard the supernatant, then add PBS containing 2% FBS to resuspend the cells and count.

3.将细胞稀释至浓度为1.0×10⁷个细胞/mL，按照50μL/孔加入流式管中。3. Dilute the cells to a concentration of 1.0×10 ⁷ cells/mL, and add 50 μL/well to the flow tube.

4.将各单克隆抗体(4F2、4B9、6C1、6H12、13F7、6A6和18B10)用含2％FBS的PBS稀释至2μg/mL，按照50μL/孔加入已有50μL细胞的流式管中。4. Dilute each monoclonal antibody (4F2, 4B9, 6C1, 6H12, 13F7, 6A6 and 18B10) to 2 μg/mL with PBS containing 2% FBS, and add 50 μL/well to a flow tube containing 50 μL of cells.

5.4℃下孵育30min，用3mL含2％FBS的PBS洗2次，1000rpm离心5min。Incubate at 5.4°C for 30 min, wash twice with 3 mL of PBS containing 2% FBS, and centrifuge at 1000 rpm for 5 min.

6.弃上清，用100μL含2％FBS的1×PBS重悬细胞，加入APC抗鼠IgG Fc(Biolegend)作为二抗。6. Discard the supernatant, resuspend the cells in 100 μL of 1×PBS containing 2% FBS, and add APC anti-mouse IgG Fc (Biolegend) as the secondary antibody.

7.4℃下孵育30min，用3mL含2％FBS的PBS洗1次，1000rpm离心5min。Incubate at 7.4°C for 30 min, wash once with 3 mL of PBS containing 2% FBS, and centrifuge at 1000 rpm for 5 min.

8.弃上清，用100μL含2％FBS的PBS重悬细胞，流式机检测获得阳性率。8. Discard the supernatant, resuspend the cells with 100 μL of PBS containing 2% FBS, and obtain the positive rate by flow cytometry.

结果如图4所示，候选抗OX40单克隆抗体18B10、4F2、6C1、6H12和6A6与人OX40及猴OX40均具有相对较高的亲和力结合，4B9和13F7对猴OX40表现出较弱的亲和力，考虑到方便后续药物在猴体内的安全性评价，故舍弃4B9和13F7这两个候选抗体。The results are shown in Figure 4. The candidate anti-OX40 monoclonal antibodies 18B10, 4F2, 6C1, 6H12 and 6A6 have relatively high affinity binding to both human OX40 and monkey OX40, while 4B9 and 13F7 show weaker affinity to monkey OX40. Considering the convenience of subsequent drug safety evaluation in monkeys, two candidate antibodies, 4B9 and 13F7, were discarded.

实施例6.抗OX40单克隆抗体针对人OX40L(hFc)与人OX40的结合的阻断实验Example 6. Blocking experiment of anti-OX40 monoclonal antibody against the binding of human OX40L (hFc) to human OX40

1.将收集的1瓶(T75)HEK::OX40 100B5细胞转移至15mL离心管中，用8mL含2％FBS的PBS洗1次，1000rpm离心5min；弃上清，加入1mL含2％FBS的PBS重悬细胞并计数。1. Transfer 1 bottle of (T75) HEK::OX40 100B5 cells collected to a 15 mL centrifuge tube, wash once with 8 mL PBS containing 2% FBS, and centrifuge at 1000 rpm for 5 min; discard the supernatant and add 1 mL of PBS containing 2% FBS. Cells were resuspended in PBS and counted.

2.实验样本组设置：空白组、0μg人OX40 L(Speed biosystems)组、0.5μg人OX40 L组及各单克隆抗体(4F2、6C1、6H12、6A6和18B10)的四个浓度梯度组。2. Setting of experimental sample groups: blank group, 0 μg human OX40 L (Speed biosystems) group, 0.5 μg human OX40 L group and four concentration gradient groups of each monoclonal antibody (4F2, 6C1, 6H12, 6A6 and 18B10).

3.将细胞稀释至1×10⁷个细胞/mL，按照10μL/孔加入流式管中，将单克隆抗体用含2％FBS的PBS稀释，单克隆抗体的终浓度分别为0.01μg/mL、0.1μg/mL、1μg/mL及10μg/mL。3. Dilute the cells to 1×10 ⁷ cells/mL, add 10 μL/well to the flow tube, dilute the monoclonal antibody with PBS containing 2% FBS, and the final concentration of the monoclonal antibody is 0.01 μg/mL. , 0.1 μg/mL, 1 μg/mL and 10 μg/mL.

4.在单克隆抗体样本组中加入对应四个浓度梯度的稀释抗体各100μL，对应单克隆抗体量分别为1ng、10ng、100ng及1000ng。剩余组中加入100μL含2％FBS的PBS，保证所有反应体系均为110μL。4. To the monoclonal antibody sample group, add 100 μL of diluted antibodies corresponding to the four concentration gradients, and the corresponding monoclonal antibody amounts are 1 ng, 10 ng, 100 ng and 1000 ng respectively. Add 100 μL of PBS containing 2% FBS to the remaining groups to ensure that all reaction systems are 110 μL.

5.4℃结合30min，用3mL含2％FBS的PBS洗2次，1000rpm离心5min。Binding at 5.4°C for 30 min, washed twice with 3 mL of PBS containing 2% FBS, and centrifuged at 1000 rpm for 5 min.

6.弃上清，除空白组及0μg人OX40 L组外，其余样本管均加入浓度为5μg/mL的人OX40 L 100μL。6. Discard the supernatant, except the blank group and the 0μg human OX40 L group, add 100 μL of human OX40 L with a concentration of 5 μg/mL to the other sample tubes.

7.4℃结合30min，用3mL含2％FBS的PBS洗2次，1000rpm离心5min。Binding at 7.4°C for 30 min, washed twice with 3 mL of PBS containing 2% FBS, and centrifuged at 1000 rpm for 5 min.

8.用100μL含2％FBS的1×PBS重悬细胞，除空白组外，其余各组均加入APC抗人IgGFc(Biolegend)作为二抗。8. Resuspend the cells with 100 μL of 1×PBS containing 2% FBS, except the blank group, APC anti-human IgGFc (Biolegend) was added as the secondary antibody in the other groups.

9.4℃结合30min，用3mL的2％FBS的PBS洗1次，1000rpm离心5min。Binding at 9.4°C for 30 min, washed once with 3 mL of 2% FBS in PBS, and centrifuged at 1000 rpm for 5 min.

10.弃上清，用100μL含2％FBS的1×PBS重悬细胞，流式机检测获得阳性率。10. Discard the supernatant, resuspend the cells with 100 μL of 1×PBS containing 2% FBS, and obtain the positive rate by flow cytometry.

结果如图5所示，候选抗体用量在100ng及以上浓度时能够部分或完全阻断0.5μgOX40L与OX40蛋白的结合。其中候选抗体18B10及6C1阻断效果稍差。The results are shown in Fig. 5, the candidate antibody dosage of 100 ng and above can partially or completely block the binding of 0.5 μg of OX40L to OX40 protein. Among them, candidate antibodies 18B10 and 6C1 had slightly less blocking effect.

实施例7.抗OX40单克隆抗体的EC50值检测Example 7. Detection of EC50 value of anti-OX40 monoclonal antibody

EC50值检测实验采用NF-κB信号通路检测抗体活性EC50 value detection experiment uses NF-κB signaling pathway to detect antibody activity

1.将收集的1瓶(T75)HEK::OX40 100B5细胞转移至15mL离心管中，用5mL含10％FBS(Gemini)的DMEM培养基(Gibco)洗1次，1000rpm离心5min；弃上清，加入5mL含2％FBS的PBS重悬细胞并计数。1. Transfer 1 bottle of (T75) HEK::OX40 100B5 cells collected to a 15mL centrifuge tube, wash once with 5mL DMEM medium (Gibco) containing 10% FBS (Gemini), and centrifuge at 1000rpm for 5min; discard the supernatant , add 5 mL of PBS containing 2% FBS to resuspend the cells and count.

2.将细胞稀释至2×10⁵个细胞/mL，按照100μL/孔加入96孔板，放入5％CO₂的37℃细胞培养箱孵育30min。2. Dilute the cells to 2×10 ⁵ cells/mL, add 100 μL/well to a 96-well plate, and incubate in a 37°C cell incubator with 5% CO ₂ for 30 min.

3.候选5个单克隆抗体(4F2、6C1、6H12、6A6和18B10)以及对照抗体MOXR0916的配制浓度按照起始浓度200nM，3倍梯度降低稀释，共10个浓度点。按照50μL/孔加入96孔板中，即单克隆抗体的终浓度为50nM的起始浓度，3倍梯度降低，设置复孔。3. The preparation concentration of five candidate monoclonal antibodies (4F2, 6C1, 6H12, 6A6 and 18B10) and the control antibody MOXR0916 is based on the initial concentration of 200nM, and the dilution is 3-fold gradient reduction, with a total of 10 concentration points. Add 50 μL/well to the 96-well plate, that is, the final concentration of monoclonal antibody is the initial concentration of 50 nM, the gradient is reduced by 3 times, and the duplicate wells are set.

4.抗鼠IgG Fc(Biolegend)作为二抗的终浓度是一抗的3倍，按照50μL/孔的体系进行配制，加入96孔板中。4. The final concentration of anti-mouse IgG Fc (Biolegend) as the secondary antibody is 3 times that of the primary antibody. It is prepared according to the system of 50 μL/well and added to the 96-well plate.

5.将96孔板放入5％CO₂的37℃细胞培养箱孵育20h。5. Incubate the 96-well plate in a 37°C cell incubator with 5% CO ₂ for 20h.

6.待测96孔板室温1000rpm离心5min，取新的96孔板每孔加入160μL提前在37℃温箱孵育30min的Quanti Blue(Invivo Gen)，后取40μL细胞上清加入，于37℃中孵育1h。6. Centrifuge the 96-well plate to be tested at 1000 rpm for 5 minutes at room temperature. Add 160 μL of Quanti Blue (Invivo Gen) to each well of the new 96-well plate and incubate it in an incubator at 37 °C for 30 minutes. Incubate for 1h.

7.使用酶标仪在620nm下检测OD值，并选择GraphPad Prism作图分析数据。7. Use a microplate reader to detect the OD value at 620 nm, and select GraphPad Prism to plot and analyze the data.

抗OX40单克隆抗体的相应EC50值如表2所示，EC50值拟合图如图6所示。前期流式检测抗体与细胞水平的结合力，本实验考察抗体发挥作用的功能活性。结果显示，候选抗体6A6及4F2具有优于对照抗体的功能活性；6C1相对较差，故后续实验中舍弃该抗体。进一步选定的候选单克隆抗体为4F2、6H12、6A6和18B10。The corresponding EC50 values of anti-OX40 monoclonal antibodies are shown in Table 2, and the fitting graph of EC50 values is shown in Figure 6 . In the early stage, the binding force of the antibody to the cell level was detected by flow cytometry, and the functional activity of the antibody was investigated in this experiment. The results showed that the candidate antibodies 6A6 and 4F2 had better functional activities than the control antibody; 6C1 was relatively poor, so this antibody was discarded in subsequent experiments. Further selected candidate monoclonal antibodies were 4F2, 6H12, 6A6 and 18B10.

表2.候选抗OX40单克隆抗体的EC50值生物活性汇总表Table 2. Summary table of biological activities of candidate anti-OX40 monoclonal antibodies with EC50 values

实施例8.抗OX40单克隆抗体刺激人CD4⁺T细胞增殖的实验Example 8. Experiment of anti-OX40 monoclonal antibody to stimulate proliferation of human CD4 ⁺ T cells

1.CD3包被96孔板：100μL/孔，0.05μg/mL或0.01μg/mL的CD3(OKT3)，4℃孵育过夜。1. CD3-coated 96-well plate: 100 μL/well, 0.05 μg/mL or 0.01 μg/mL of CD3 (OKT3), incubate at 4°C overnight.

2.分选细胞：采用CD4⁺阴选的磁珠分选试剂盒(Miltenyi Biotec)从PBMC中获得CD4⁺细胞并进行计数。2. Sorting cells: CD4 ⁺ cells were obtained and counted from PBMCs using a CD4 ⁺ negative magnetic bead sorting kit (Miltenyi Biotec).

3.CFSE(MCE)标记：留取适量细胞，剩余细胞(10⁷个)重悬于1mL的含5％FBS的PBS中，加入CFSE到终浓度为2μM进行CFSE标记，室温孵育10min，用5mL含5％FBS的PBS洗涤3次终止染色，用含10％FBS的1640培养基重悬。3. CFSE (MCE) labeling: take an appropriate amount of cells, resuspend the remaining cells (10 ⁷ cells) in 1 mL of PBS containing 5% FBS, add CFSE to a final concentration of 2 μM for CFSE labeling, incubate at room temperature for 10 min, and use 5 mL of Staining was terminated by washing 3 times in PBS containing 5% FBS and resuspending in 1640 medium containing 10% FBS.

4.选择包被不同CD3浓度的96孔板铺板：100μL/孔，3×10⁵个细胞/孔的CD4⁺T细胞，2个复孔。4. Select 96-well plates coated with different concentrations of CD3: 100 μL/well, 3×10 ⁵ cells/well of CD4 ⁺ T cells, 2 duplicate wells.

5.加单克隆抗体：100μL/孔，5μg/mL(终浓度为2.5μg/mL)的抗体(4F2、6H12、6A6、18B10和对照抗体MOXR0916)，设置阴性抗体对照(mIgG1及hIgG1，Biolegend)及空白孔对照，37℃孵育。5. Add monoclonal antibody: 100 μL/well, 5 μg/mL (final concentration of 2.5 μg/mL) antibodies (4F2, 6H12, 6A6, 18B10 and control antibody MOXR0916), set negative antibody control (mIgG1 and hIgG1, Biolegend) and blank well control, incubated at 37°C.

6.孵育20h左右，离心取60μL上清进行IL-2的ELISA检测，结果如图7B所示。6. After incubation for about 20 hours, centrifuge to take 60 μL of the supernatant for ELISA detection of IL-2. The results are shown in Figure 7B.

7.将96孔板继续孵育5-7天，取100μL上清进行IFN-γ的ELISA检测，结果如图7C所示。7. Continue to incubate the 96-well plate for 5-7 days, take 100 μL of the supernatant for ELISA detection of IFN-γ, and the results are shown in Figure 7C.

8.收集细胞进行CD4的CFSE流式检测，结果如图7A所示。8. Collect cells for CFSE flow detection of CD4, and the results are shown in Figure 7A.

结果显示，4个候选单克隆抗体均有效刺激人CD4+T细胞的增殖，且有效增加IL-2及IFN-γ的分泌，在该实验条件下，候选抗体均优于对照抗体。再结合前几项实验结果，特别关注EC50值生物活性检测，综合考虑，选定6A6和4F2进行小鼠体内药效抑瘤实验研究。The results showed that the four candidate monoclonal antibodies all effectively stimulated the proliferation of human CD4+ T cells, and effectively increased the secretion of IL-2 and IFN-γ. Under the experimental conditions, the candidate antibodies were superior to the control antibodies. Combined with the results of the previous experiments, special attention was paid to the biological activity detection of EC50 value. Taking into account comprehensively, 6A6 and 4F2 were selected for the experimental study of drug efficacy and tumor inhibition in mice.

实施例9.抗人OX40单克隆抗体在OX40人源化小鼠体内的抑瘤实验Example 9. Anti-human OX40 monoclonal antibody in vivo tumor inhibition experiment in OX40 humanized mice

1.细胞培养：将小鼠结直肠癌MC38细胞在37℃、5％CO₂的培养箱中培养，培养基成分为含有10％FBS的DMEM培养基，细胞每隔2天分瓶传代1次。1. Cell culture: The mouse colorectal cancer MC38 cells were cultured in an incubator at 37°C and 5% CO ₂ , the medium was DMEM medium containing 10% FBS, and the cells were passaged in flasks every 2 days. .

2.小鼠接种细胞：将PBS重悬的MC38肿瘤细胞以5×10⁶个细胞/mL的浓度，0.1mL/只体积接种于OX40人源化小鼠(北京百奥赛图基因生物技术有限公司)的右侧胁肋部皮下，共转接26只小鼠。当平均肿瘤体积达到大约100mm³时，挑选个体肿瘤体积适中的小鼠入组，将动物按肿瘤体积随机分配到4个组别中，包括对照组(PBS组)及三个实验组(MOXR0916对照抗体组、6A6组及4F2组)，每组6只，分组当天开始给药，具体给药方案见表3：2. Mice inoculated with cells: MC38 tumor cells resuspended in PBS were inoculated into OX40 humanized mice (Beijing ^Biositu Gene Biotechnology Co., Ltd. ) subcutaneously in the right flank of 26 mice. When the average tumor volume reached about 100 ^mm3 , mice with moderate tumor volume were selected into the group, and the animals were randomly assigned to 4 groups according to the tumor volume, including the control group (PBS group) and three experimental groups (MOXR0916 control group) Antibody group, 6A6 group and 4F2 group), there are 6 animals in each group, and the administration starts on the day of grouping. The specific dosing schedule is shown in Table 3:

表3.抑瘤实验给药方案Table 3. Dosing regimen for tumor inhibition experiment

注：^a指每3天给药1次，共给药4次。Note: ^a refers to administration once every 3 days, for a total of 4 administrations.

3.给药后，对实验动物体重及肿瘤生长状态继续观察。每周测量两次肿瘤体积及动物体重，并记录测量值。肿瘤体积的测量结果如图8A所示，其中，MOXR0916对照抗体组与对照相比时，^*P<0.05，^**P<0.01；6A6组与对照相比时，^#P<0.05，^##P<0.01；4F2组与对照相比时，^&P<0.05。动物体重的测量结果如图8B所示。3. After administration, continue to observe the body weight and tumor growth state of the experimental animals. Tumor volume and animal body weight were measured twice weekly and the measurements were recorded. The measurement results of tumor volume are shown in Figure 8A, wherein, when the MOXR0916 control antibody group was compared with the control, ^* P<0.05, ^** P<0.01; when the 6A6 group was compared with the control, ^#P <0.05, ^## P <0.01;^&P<0.05 when the 4F2 group was compared with the control. Measurements of animal body weight are shown in Figure 8B.

4.给药后观察记录14天，对小鼠进行解剖，取血清进行ALT及AST生化检测，检测数据分别如8C和8D所示，结果显示，其数据均在正常范围内，未见明显肝毒性。解剖肿瘤、肝脏和脾脏组织，进行称重并进行数据统计，肿瘤解剖图如图8E所示。计算解剖瘤重占小鼠总体重的百分比，更加直观地考察了组间差异性，数据统计结果如图8F所示，实验组与对照组相比，统计学上差异非常显著，^***P<0.001。4. Observe and record for 14 days after administration, dissect the mice, and take serum for ALT and AST biochemical detection. The test data are shown in 8C and 8D respectively. The results show that the data are all within the normal range, and no obvious liver disease is found. toxicity. Tumor, liver, and spleen tissues were dissected, weighed, and counted. The tumor anatomy is shown in Figure 8E. The percentage of anatomical tumor weight in the total weight of mice was calculated, and the differences between groups were more intuitively investigated. The statistical results of the data are shown in Figure 8F. Compared with the control group, the statistical difference between the experimental group and the control group is very significant, ^*** P <0.001.

5.同步分析肿瘤抑制率(TGI)数据，结果如表4显示。给药后，候选抗体(6A6和4F2)显示出与对照抗体(MOXR0916)相当的抑瘤效果，且克隆6A6略优于4F2。5. Simultaneous analysis of tumor inhibition rate (TGI) data, the results are shown in Table 4. After administration, the candidate antibodies (6A6 and 4F2) showed comparable tumor inhibitory effect to the control antibody (MOXR0916), and clone 6A6 was slightly better than 4F2.

表4.实验组TGI统计Table 4. Experimental group TGI statistics

可以理解，尽管本申请以上述具体形式描述了所涉及的发明，但这些发明并不局限于这些具体形式描述的特定内容。对本领域的技术人员显而易见的是，在不偏离本申请所描述的发明精神的前提下，还可对其中所涉及的发明包含的技术特征进行各种等同变化，这些变化都应该属于所述发明的范围之内。It is to be understood that although this application describes the inventions involved in the specific forms described above, these inventions are not limited to the specific content described in these specific forms. It is obvious to those skilled in the art that, on the premise of not departing from the spirit of the invention described in this application, various equivalent changes can be made to the technical features contained in the invention involved, and these changes shall belong to the described invention. within the range.

序列表sequence listing

<110> 北京免疫方舟医药科技有限公司<110> Beijing Immune Ark Pharmaceutical Technology Co., Ltd.

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Gly Tyr Tyr Met AsnGly Tyr Tyr Met Asn

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<211> 16<211> 16

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Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys AsnTyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Asn

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Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro Ser Leu Lys AsnTyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro Ser Leu Lys Asn

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Glu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe LysGlu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys

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Arg Ile Tyr Pro Gly Asp Glu Asn Thr Asn Tyr Asn Gly Lys Phe LysArg Ile Tyr Pro Gly Asp Glu Asn Thr Asn Tyr Asn Gly Lys Phe Lys

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Gly Leu Tyr Ser Val GlnGly Leu Tyr Ser Val Gln

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<211> 11<211> 11

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Lys Ala Ser Gln Asp Val Ser Ser Ala Val AlaLys Ala Ser Gln Asp Val Ser Ser Ala Val Ala

1 5 101 5 10

<210> 14<210> 14

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<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

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Lys Ala Ser Gln Asp Val Asn Thr Ala Val AlaLys Ala Ser Gln Asp Val Asn Thr Ala Val Ala

1 5 101 5 10

<210> 15<210> 15

<211> 11<211> 11

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

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Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu SerArg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ser

1 5 101 5 10

<210> 16<210> 16

<211> 11<211> 11

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

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Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu AsnArg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 101 5 10

<210> 17<210> 17

<211> 7<211> 7

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

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Trp Ala Ser Ile Arg His ThrTrp Ala Ser Ile Arg His Thr

1 51 5

<210> 18<210> 18

<211> 7<211> 7

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 18<400> 18

Ala Ala Ser Thr Leu Asp SerAla Ala Ser Thr Leu Asp Ser

1 51 5

<210> 19<210> 19

<211> 7<211> 7

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 19<400> 19

Tyr Thr Ser Arg Leu His SerTyr Thr Ser Arg Leu His Ser

1 51 5

<210> 20<210> 20

<211> 9<211> 9

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 20<400> 20

Gln Gln His Tyr Ser Thr Pro Trp ThrGln Gln His Tyr Ser Thr Pro Trp Thr

1 51 5

<210> 21<210> 21

<211> 9<211> 9

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

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Gln Pro His Tyr Ser Thr Pro Trp ThrGln Pro His Tyr Ser Thr Pro Trp Thr

1 51 5

<210> 22<210> 22

<211> 9<211> 9

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

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Leu Gln Tyr Ala Ser Tyr Pro Leu ThrLeu Gln Tyr Ala Ser Tyr Pro Leu Thr

1 51 5

<210> 23<210> 23

<211> 9<211> 9

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 23<400> 23

Gln Gln Leu Asn Thr Leu Pro Trp ThrGln Gln Leu Asn Thr Leu Pro Trp Thr

1 51 5

<210> 24<210> 24

<211> 140<211> 140

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 24<400> 24

Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly IleMet Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile

1 5 10 151 5 10 15

Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys ProLeu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30 20 25 30

Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile ThrSer Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

35 40 45 35 40 45

Ser Gly Tyr Asn Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys LeuSer Gly Tyr Asn Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu

50 55 60 50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn ProGlu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro

65 70 75 8065 70 75 80

Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn GlnSer Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln

85 90 95 85 90 95

Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr TyrPhe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr

100 105 110 100 105 110

Tyr Cys Ala Arg Gln Gly Ser Ser Gly His Phe Tyr Tyr Ala Met AspTyr Cys Ala Arg Gln Gly Ser Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125 115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser SerTyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140 130 135 140

<210> 25<210> 25

<211> 140<211> 140

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 25<400> 25

Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly IleMet Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile

1 5 10 151 5 10 15

Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys ProLeu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30 20 25 30

Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile ThrSer Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

35 40 45 35 40 45

Ser Gly Tyr Asn Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys LeuSer Gly Tyr Asn Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu

50 55 60 50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn ProGlu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro

65 70 75 8065 70 75 80

Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn GlnSer Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln

85 90 95 85 90 95

Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr TyrPhe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr

100 105 110 100 105 110

Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met AspTyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125 115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser SerTyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140 130 135 140

<210> 26<210> 26

<211> 141<211> 141

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 26<400> 26

Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Thr Ala GlyMet Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Thr Ala Gly

1 5 10 151 5 10 15

Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val LysVal His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30 20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Thr Ala Ser Gly His Ala PhePro Gly Ala Ser Val Lys Ile Ser Cys Thr Ala Ser Gly His Ala Phe

35 40 45 35 40 45

Ser Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn ThrSer Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr

50 55 60 50 55 60

Leu Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr AsnLeu Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn

65 70 75 8065 70 75 80

Pro Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys AsnPro Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn

85 90 95 85 90 95

Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala ThrGln Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr

100 105 110 100 105 110

Tyr Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala MetTyr Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met

115 120 125 115 120 125

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser SerAsp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140 130 135 140

<210> 27<210> 27

<211> 140<211> 140

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 27<400> 27

Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly IleMet Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile

1 5 10 151 5 10 15

Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys ProLeu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30 20 25 30

Phe Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile ThrPhe Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

35 40 45 35 40 45

Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr LeuSer Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu

50 55 60 50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn ProGlu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro

65 70 75 8065 70 75 80

Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn GlnSer Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln

85 90 95 85 90 95

Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr TyrPhe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr

100 105 110 100 105 110

Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met AspTyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125 115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser SerTyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140 130 135 140

<210> 28<210> 28

<211> 140<211> 140

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 28<400> 28

Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly IleMet Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile

1 5 10 151 5 10 15

Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys ProLeu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30 20 25 30

Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile ThrSer Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

35 40 45 35 40 45

Ser Gly Tyr Asn Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys LeuSer Gly Tyr Asn Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu

50 55 60 50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn ProGlu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro

65 70 75 8065 70 75 80

Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn GlnSer Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln

85 90 95 85 90 95

Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr TyrPhe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr

100 105 110 100 105 110

Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met AspTyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125 115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser SerTyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140 130 135 140

<210> 29<210> 29

<211> 138<211> 138

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 29<400> 29

Met Gly Trp Asn Trp Ile Phe Ile Leu Ile Leu Ser Val Thr Thr GlyMet Gly Trp Asn Trp Ile Phe Ile Leu Ile Leu Ser Val Thr Thr Gly

1 5 10 151 5 10 15

Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val LysVal His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30 20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser PhePro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45 35 40 45

Thr Gly Tyr Tyr Met Asn Trp Val Lys Gln Ser Pro Glu Lys Ser LeuThr Gly Tyr Tyr Met Asn Trp Val Lys Gln Ser Pro Glu Lys Ser Leu

50 55 60 50 55 60

Glu Trp Ile Gly Glu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr AsnGlu Trp Ile Gly Glu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr Asn

65 70 75 8065 70 75 80

Gln Lys Phe Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser Ser SerGln Lys Phe Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95 85 90 95

Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala ValThr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110 100 105 110

Tyr Tyr Cys Ala Arg Ser Gly Leu Pro Tyr Tyr Asp Val Asp Tyr TrpTyr Tyr Cys Ala Arg Ser Gly Leu Pro Tyr Tyr Asp Val Asp Tyr Trp

115 120 125 115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser SerGly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135 130 135

<210> 30<210> 30

<211> 134<211> 134

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 30<400> 30

Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Thr Ala GlyMet Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Thr Ala Gly

1 5 10 151 5 10 15

Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val LysVal His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30 20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Thr Ala Ser Gly His Ala PhePro Gly Ala Ser Val Lys Ile Ser Cys Thr Ala Ser Gly His Ala Phe

35 40 45 35 40 45

Ser Asn Ser Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly LeuSer Asn Ser Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60 50 55 60

Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Glu Asn Thr Asn Tyr AsnGlu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Glu Asn Thr Asn Tyr Asn

65 70 75 8065 70 75 80

Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser SerGly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

85 90 95 85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala ValThr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val

100 105 110 100 105 110

Tyr Phe Cys Ala Arg Gly Leu Tyr Ser Val Gln Trp Gly Gln Gly ThrTyr Phe Cys Ala Arg Gly Leu Tyr Ser Val Gln Trp Gly Gln Gly Thr

115 120 125 115 120 125

Thr Leu Thr Val Ser SerThr Leu Thr Val Ser Ser

130 130

<210> 31<210> 31

<211> 131<211> 131

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 31<400> 31

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val PheMet Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 151 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala HisLeu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala His

20 25 30 20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys LysLys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45 35 40 45

Ala Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys ProAla Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60 50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His ThrGly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 8065 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr ThrGly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95 85 90 95

Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr CysLeu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys

100 105 110 100 105 110

Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys LeuGln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125 115 120 125

Glu Ile LysGlu Ile Lys

130 130

<210> 32<210> 32

<211> 131<211> 131

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 32<400> 32

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val PheMet Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 151 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala HisLeu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala His

20 25 30 20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys LysLys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45 35 40 45

Ala Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys ProAla Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60 50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His ThrGly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 8065 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr ThrGly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95 85 90 95

Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr CysLeu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys

100 105 110 100 105 110

Leu Gln Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Asn LeuLeu Gln Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Asn Leu

115 120 125 115 120 125

Glu Leu LysGlu Leu Lys

130 130

<210> 33<210> 33

<211> 131<211> 131

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 33<400> 33

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val PheMet Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 151 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala HisLeu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala His

20 25 30 20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys LysLys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45 35 40 45

Ala Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys ProAla Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60 50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His ThrGly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 8065 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr ThrGly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95 85 90 95

Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr CysLeu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys

100 105 110 100 105 110

Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys LeuGln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125 115 120 125

Glu Ile LysGlu Ile Lys

130 130

<210> 34<210> 34

<211> 127<211> 127

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 34<400> 34

Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe Leu Trp Leu SerMet Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe Leu Trp Leu Ser

1 5 10 151 5 10 15

Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met SerGly Val Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser

20 25 30 20 25 30

Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln AspThr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp

35 40 45 35 40 45

Val Asn Thr Ala Val Ala Trp Tyr Gln His Lys Pro Gly Gln Ser ProVal Asn Thr Ala Val Ala Trp Tyr Gln His Lys Pro Gly Gln Ser Pro

50 55 60 50 55 60

Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr Gly Val Pro AspLys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr Gly Val Pro Asp

65 70 75 8065 70 75 80

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile SerArg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser

85 90 95 85 90 95

Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Pro His TyrSer Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Pro His Tyr

100 105 110 100 105 110

Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile LysSer Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125 115 120 125

<210> 35<210> 35

<211> 131<211> 131

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 35<400> 35

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val PheMet Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 151 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser HisLeu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His

20 25 30 20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys LysLys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45 35 40 45

Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys ProAla Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60 50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His ThrGly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 8065 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr ThrGly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95 85 90 95

Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr CysLeu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys

100 105 110 100 105 110

Gln Pro His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys LeuGln Pro His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125 115 120 125

Glu Ile LysGlu Ile Lys

130 130

<210> 36<210> 36

<211> 129<211> 129

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 36<400> 36

Met Asp Met Arg Val Pro Ala His Val Phe Gly Phe Leu Leu Leu TrpMet Asp Met Arg Val Pro Ala His Val Phe Gly Phe Leu Leu Leu Trp

1 5 10 151 5 10 15

Phe Pro Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser SerPhe Pro Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30 20 25 30

Leu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala SerLeu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser

35 40 45 35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu His Gln Lys Pro Asp GlyGln Glu Ile Ser Gly Tyr Leu Ser Trp Leu His Gln Lys Pro Asp Gly

50 55 60 50 55 60

Thr Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly ValThr Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val

65 70 75 8065 70 75 80

Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu ThrPro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr

85 90 95 85 90 95

Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu GlnIle Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110 100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Asn Leu Glu LeuTyr Ala Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Asn Leu Glu Leu

115 120 125 115 120 125

LysLys

<210> 37<210> 37

<211> 127<211> 127

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 37<400> 37

Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe GlnMet Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln

1 5 10 151 5 10 15

Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Ser Ser Ser Leu SerGly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Ser Ser Ser Leu Ser

20 25 30 20 25 30

Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln AspAla Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp

35 40 45 35 40 45

Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Ile ValIle Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Ile Val

50 55 60 50 55 60

Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro SerLys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser

65 70 75 8065 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile SerArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser

85 90 95 85 90 95

Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Leu AsnAsn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Leu Asn

100 105 110 100 105 110

Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile LysThr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125 115 120 125