技术领域technical field
本发明涉及抗体以及抗体药物开发和生产领域，尤其是涉及获得抗人PD-1的全人源化单克隆抗体，本发明还涉及该单克隆抗体的制备方法及应用。The invention relates to the field of development and production of antibodies and antibody drugs, in particular to the acquisition of a fully humanized monoclonal antibody against human PD-1, and also relates to a preparation method and application of the monoclonal antibody.
背景技术Background technique
PD-1（CD279）是由 288 个氨基酸组成的免疫球蛋白超家族Ⅰ型跨膜糖蛋白，最初是从凋亡的小鼠T细胞杂交瘤2B4.11克隆，被认为与细胞凋亡相关而命名为程序性死亡-1（programmed death-1，PD-1）。最初发现时，因其结构类似于 CD28 分子，PD-1被认可是 CD28 超家族成员。然而PD-1 蛋白因缺乏介导 CD28/CTLA-4与B7.1/B7.2 相结合的 MYPPPY 序列，以及介导 ICOS 与 ICOS-L 相结合的 FDPPPF 序列，因而在结构上与 CD28、CTLA-4 及 ICOS 存在明显差异。因此，PD-1与配体的结合非常特异，不与其他的 B7 家族分子产生交叉结合。PD-1 (CD279) is an immunoglobulin superfamily type I transmembrane glycoprotein composed of 288 amino acids, which was originally cloned from the apoptotic mouse T cell hybridoma 2B4. Named programmed death-1 (programmed death-1, PD-1). When initially discovered, PD-1 was recognized as a member of the CD28 superfamily because of its structural resemblance to the CD28 molecule. However, PD-1 protein lacks the MYPPPY sequence that mediates the combination of CD28/CTLA-4 and B7.1/B7.2, and the FDPPPF sequence that mediates the combination of ICOS and ICOS-L, so it is structurally related to CD28, CTLA -4 and ICOS are significantly different. Therefore, the binding of PD-1 to the ligand is very specific and does not cross-link with other B7 family molecules.
PD-L1（B7-H1，CD274）和 PD-L2（B7-DC，CD273）是 PD-1 的两个配体，它们同属 B7 超家族，因而同其它成员一样，在蛋白结构上 PD-L1 和 PD-L2分子包含有 IgV 样区、IgC 样区、跨膜区和一个短而保守的胞浆区尾部。PD-L1 在活化的 T、B、单核细胞和多种类型的肿瘤细胞（如：肺癌、肝癌、乳腺癌、鳞状细胞癌及卵巢癌等）上诱导性表达。PD-L2 的蛋白表达谱相对于 PD-L1 要窄得多，主要表达于活化的巨噬细胞、树突状细胞、骨髓来源的基质细胞和个别肿瘤细胞株上。大量的研究表明，PD-L1与活化的 T 细胞上 PD-1 相互作用可显著抑制效应性 T 细胞的生物学功能，PD-1、PD-L1 或 PD-L2 的表达改变导致 PD-1/PD-L 抑制途径发生异常，进而机体发生免疫功能亢进/低下性疾病，PD-1 基因敲除小鼠可引发多种自身免疫性疾病。PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) are two ligands of PD-1, they belong to the B7 superfamily, so like other members, PD-L1 and PD-L2 molecules contain an IgV-like region, an IgC-like region, a transmembrane region, and a short and conserved cytoplasmic tail. PD-L1 is inducibly expressed on activated T, B, monocytes and various types of tumor cells (such as: lung cancer, liver cancer, breast cancer, squamous cell carcinoma and ovarian cancer, etc.). The protein expression profile of PD-L2 is much narrower than that of PD-L1, mainly expressed on activated macrophages, dendritic cells, bone marrow-derived stromal cells and individual tumor cell lines. A large number of studies have shown that the interaction between PD-L1 and PD-1 on activated T cells can significantly inhibit the biological function of effector T cells, and the expression changes of PD-1, PD-L1 or PD-L2 lead to PD-1/ Abnormalities in the PD-L inhibitory pathway lead to hyperimmune/hypoimmune diseases, and PD-1 knockout mice can induce a variety of autoimmune diseases.
研究表明，PD-1 信号通路也参与病毒性及微生物感染性疾病的进程。在 HIV、HCV 及 HBV慢性感染患者体内发现病毒特异性的细胞毒性 T细胞(CTL)为 PD-1 阳性，这群 PD-1 阳性的 CTL 位于记忆性的 T 细胞并具有活化标志，但不能分泌 IFN-γ 及穿孔素等抗病毒效应分子，表现出“功能衰竭”的特征。此外，PD-1 的表达与病毒载量呈正相关。而阻断 PD-1 信号则可逆转 CTL 的“功能衰竭”状态并清除病毒。在人源化的 BLT 小鼠身上发现，阻断 PD-1 信号可促进针对 HIV 病毒特异性的细胞及体液免疫。此外，在恒河猴的模型中，发现阻断 PD-1 信号可促进针对 SIV病毒特异性的细胞免疫及抗体的产生。这些结果都证实阻断 PD-1 信号有可能应用于 HIV 等病毒感染的临床治疗。Studies have shown that the PD-1 signaling pathway is also involved in the process of viral and microbial infectious diseases. In patients with HIV, HCV and HBV chronic infection, virus-specific cytotoxic T cells (CTLs) were found to be PD-1 positive. These PD-1 positive CTLs are located in memory T cells and have activation marks, but cannot secrete Antiviral effector molecules such as IFN-γ and perforin show the characteristics of "function failure". In addition, PD-1 expression was positively correlated with viral load. Blocking PD-1 signaling reversed the "dysfunctional" state of CTLs and cleared the virus. Blocking PD-1 signaling promotes HIV-specific cellular and humoral immunity in humanized BLT mice. In addition, in a rhesus monkey model, it was found that blocking PD-1 signaling can promote SIV virus-specific cellular immunity and antibody production. These results confirm that blocking PD-1 signaling may be applied to the clinical treatment of viral infections such as HIV.
肿瘤细胞表达的肿瘤抗原能够被宿主 T 细胞识别，却不能被及时地有效清除，肿瘤微环境中的抑制因素一方面削弱 T 细胞功能，另一方面促进肿瘤细胞迅速生长。研究发现，PD-1/PD-L1 抑制途径与肿瘤的发生、发展密切相关。研究发现 ， PD- L1广泛表达于各种肿瘤细胞 ，并通过 PD – 1/PD- L1途径诱导肿瘤的免疫逃避。PD - L1阳性的黑色瘤细胞诱导人 CD8 + CTL克隆凋亡的增加 ，几乎所有PD - L1阴性细胞在培养过程得到消除。将特异性针对 HLA - B7限制性 CEA表位的 CTL克隆与CEA阳性乳腺癌细胞 (组成表达 PD - L1)进行共培养 ，使用抗 PD - L1的单抗部分抑制特异性 CTL的凋亡。因此，PD-1/PD-L1 抑制途径对 CD8+T 细胞的活化增殖有明显的抑制作用，肿瘤细胞可通过此途径逃避 CTL 杀伤作用，减弱机体抗肿瘤免疫应答。PD - L1 过度表达可能抑制肝癌组织内 T细胞功能 ，促进肝癌细胞逃避免疫监控，并能显著增加人肝细胞性肝癌的恶化及术后的复发率。The tumor antigens expressed by tumor cells can be recognized by host T cells, but cannot be effectively removed in a timely manner. The inhibitory factors in the tumor microenvironment weaken the function of T cells on the one hand, and promote the rapid growth of tumor cells on the other hand. Studies have found that the PD-1/PD-L1 inhibitory pathway is closely related to the occurrence and development of tumors. Studies have found that PD-L1 is widely expressed in various tumor cells and induces tumor immune evasion through the PD-1/PD-L1 pathway. PD-L1-positive melanoma cells induced increased apoptosis in human CD8+ CTL clones, and almost all PD-L1-negative cells were eliminated during culture. CTL clones specifically targeting HLA-B7-restricted CEA epitopes were co-cultured with CEA-positive breast cancer cells (constitutively expressing PD-L1), and anti-PD-L1 monoclonal antibodies were used to partially inhibit the apoptosis of specific CTLs. Therefore, the PD-1/PD-L1 inhibitory pathway has a significant inhibitory effect on the activation and proliferation of CD8+ T cells, and tumor cells can escape the killing effect of CTL through this pathway and weaken the body's anti-tumor immune response. Overexpression of PD-L1 may inhibit the function of T cells in liver cancer tissue, promote liver cancer cells to escape immune surveillance, and significantly increase the deterioration and postoperative recurrence rate of human hepatocellular carcinoma.
同时，利用抗 PD-L1 单克隆抗体阻断 PD-1/PD-L1 信号可以通过上调 IFN-γ、IL-2、IL-10 的分泌，有效逆转 CD4+和 CD8+T 细胞的增殖受抑，同时 T 细胞的活化程度和杀伤能力显著增强。At the same time, blocking PD-1/PD-L1 signaling with anti-PD-L1 monoclonal antibody can effectively reverse the inhibition of CD4+ and CD8+ T cell proliferation by up-regulating the secretion of IFN-γ, IL-2, and IL-10, At the same time, the activation degree and killing ability of T cells were significantly enhanced.
综上所述，通过特异性阻断 PD-1/PD-L抑制信号可以使机体中失能的效应性细胞恢复生物学功能，促进肿瘤和病毒特异性 CD8+T 细胞的活化增殖与细胞因子的分泌，增强淋巴细胞对肿瘤抗原、外来入侵的病毒等的杀伤力，提高机体免疫力，及时清除肿瘤细胞和病毒。因此，PD-1/PD-L 有望成为肿瘤免疫治疗的有效靶分子，也为 HIV 和 HBV 等慢性病毒感染性疾病及自身免疫性疾病的治疗提供一个新的策略。To sum up, by specifically blocking the PD-1/PD-L inhibitory signal, the disabled effector cells in the body can restore their biological functions, and promote the activation and proliferation of tumor and virus-specific CD8+ T cells and the activation of cytokines. The secretion of lymphocytes can enhance the lethality of lymphocytes against tumor antigens and foreign invading viruses, improve the body's immunity, and remove tumor cells and viruses in time. Therefore, PD-1/PD-L is expected to become an effective target molecule for tumor immunotherapy, and it also provides a new strategy for the treatment of chronic viral infectious diseases such as HIV and HBV and autoimmune diseases.
发明内容Contents of the invention
本发明的目的在于提供一种对人PD-1分子具有高亲和力和低解离速率的全人源化抗PD-1单克隆抗体，该抗体从构建的大容量天然人源噬菌体抗体库中筛选获得；本发明还提供该抗体的制备方法，并公开了该抗体在制备治疗抗肿瘤、抗感染及自身免疫性疾病治疗药物中的用途。The purpose of the present invention is to provide a fully humanized anti-PD-1 monoclonal antibody with high affinity and low dissociation rate for human PD-1 molecules, which is screened from the constructed large-capacity natural human phage antibody library obtained; the present invention also provides a preparation method of the antibody, and discloses the use of the antibody in the preparation of therapeutic drugs for treating anti-tumor, anti-infection and autoimmune diseases.
为实现上述目的，本发明可采取下述技术方案：To achieve the above object, the present invention can take the following technical solutions:
本发明的全人源化抗PD-1单克隆抗体，它具有序列表中序列7所示的重链SEQ ID NO.7氨基酸序列和序列表中序列8所示的轻链SEQ ID NO.8氨基酸序列。本发明的抗体分子的重链和轻链为全人源的，尽管嵌合的或人源化的也可以使用，但是在用于人体时，全人源的可以避免很多副作用，如HAMA 和 HACA 反应引起的那些副作用。The fully humanized anti-PD-1 monoclonal antibody of the present invention has the heavy chain SEQ ID NO.7 amino acid sequence shown in sequence 7 in the sequence listing and the light chain SEQ ID NO.8 shown in sequence 8 in the sequence listing amino acid sequence. The heavy chain and light chain of the antibody molecule of the present invention are fully human, although chimeric or humanized can also be used, but when used in humans, fully human can avoid many side effects, such as HAMA and HACA Those side effects caused by the reaction.
本发明的全人源化抗PD-1单克隆抗体，其重链可变区含有的三个高变区为：SEQ ID NO．1 ：GFTFSSYDYYMH、SEQ ID NO．2 ：VIWYSGSNTYYNDSLKS和 SEQ ID NO.3 ：AYFGVDV ；轻链可变区含有的三 个 高 变 区 为 ：SEQ ID NO．4 ：RASQGIGNTLA、 SEQ ID NO．5 ：RASQGIGNTLA 和 SEQ ID NO．6 ：QQYDHVPLT。In the fully humanized anti-PD-1 monoclonal antibody of the present invention, the three hypervariable regions contained in the heavy chain variable region are: SEQ ID NO. 1: GFTFSSYDYYMH, SEQ ID NO. 2: VIWYSGSNTYYNDSLKS and SEQ ID NO.3: AYFGVDV; the three hypervariable regions contained in the light chain variable region are: SEQ ID NO. 4: RASQGIGNTLA, SEQ ID NO. 5: RASQGIGNTLA and SEQ ID NO. 6: QQYDHVPLT.
本发明的抗体包括由上述重链和轻链通过二硫键形成全长的抗体，以及形成的Fab、Fab′、F(ab′) 2 或 Fv 片段等抗体片段。The antibody of the present invention includes a full-length antibody formed by the above-mentioned heavy chain and light chain through a disulfide bond, and antibody fragments such as Fab, Fab', F(ab') 2 or Fv fragments formed.
本发明还公开了所述抗体的宿主细胞为中华仓鼠卵巢细胞（CHO），小仓鼠肾细胞（BHK）,COS细胞，小鼠NSO胸腺瘤细胞等哺乳动物细胞。The invention also discloses that the host cells of the antibody are Chinese hamster ovary cells (CHO), small hamster kidney cells (BHK), COS cells, mouse NSO thymoma cells and other mammalian cells.
本发明全人源化抗PD-1单克隆抗体的制备方法，包括以下步骤：The preparation method of the fully humanized anti-PD-1 monoclonal antibody of the present invention comprises the following steps:
a、从噬菌体人源抗体库筛选获得高亲和力全人源抗PD-1单链抗体；a. Obtain high-affinity fully human anti-PD-1 single chain antibody from the phage human antibody library;
b、构建全人源抗PD-1完整抗体分子真核表达载体；b. Construction of a eukaryotic expression vector for a fully human anti-PD-1 complete antibody molecule;
c、全人源抗PD-1完整抗体分子在 CHO 细胞中的表达；c. Expression of fully human anti-PD-1 complete antibody molecules in CHO cells;
d、全人源抗 PD-1完整抗体分子的纯化及活性测定。d. Purification and activity determination of fully human anti-PD-1 intact antibody molecules.
本发明所述的全人源化抗PD-1单克隆抗体可用来制备抗肿瘤（包括高表达 PD-1的肺癌、肝癌、乳腺癌、鳞状细胞癌、卵巢癌、结直肠癌、胃癌、胃肠道间质瘤、膀胱癌、甲状腺癌、黑色素瘤、颈癌、前列腺癌）药物、抗感染（感染性疾病包括HIV、HBV、HCV等病毒感染引起的疾病）药物及用来制备自身免疫性疾病（包括系统性红斑狼疮、类风湿性关节炎、系统性脉管炎、银屑病、多发性硬化症、溃疡性结肠炎）治疗的药物，其制备方法是以全人源化抗PD-1单克隆抗体为主要成分，加上制药学上可接受的辅料和/或添加剂，经过冻干处理，制备成药学上可接受的药剂。The fully humanized anti-PD-1 monoclonal antibody described in the present invention can be used to prepare anti-tumor (including lung cancer, liver cancer, breast cancer, squamous cell carcinoma, ovarian cancer, colorectal cancer, gastric cancer, Gastrointestinal stromal tumors, bladder cancer, thyroid cancer, melanoma, neck cancer, prostate cancer) drugs, anti-infection (infectious diseases including HIV, HBV, HCV and other viral infections) drugs and used to prepare autoimmune Drugs for the treatment of diseases (including systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, psoriasis, multiple sclerosis, ulcerative colitis), the preparation method of which is fully humanized anti-PD -1 Monoclonal antibody is the main component, plus pharmaceutically acceptable adjuvants and/or additives, after freeze-drying, it is prepared into a pharmaceutically acceptable medicament.
本发明的优点在于该抗体对PD-1有很高的亲和力和很低的免疫原性，在动物细胞中高效表达，可用于工业化生产。实验证明，本发明的全人源化抗PD-1单克隆抗体特异性阻断 PD-1/PD-L抑制信号可以使机体中失能的效应性细胞恢复生物学功能，促进肿瘤和病毒特异性 CD8+T 细胞的活化增殖与细胞因子的分泌，增强淋巴细胞对肿瘤抗原、外来入侵的病毒等的杀伤力，提高机体免疫力，及时清除肿瘤细胞和病毒。The advantage of the present invention is that the antibody has high affinity to PD-1 and low immunogenicity, is highly expressed in animal cells, and can be used for industrial production. Experiments have proved that the fully humanized anti-PD-1 monoclonal antibody of the present invention can specifically block the PD-1/PD-L inhibitory signal, which can restore the biological function of the disabled effector cells in the body, and promote tumor and virus specificity. The activation and proliferation of sexual CD8+ T cells and the secretion of cytokines can enhance the lethality of lymphocytes against tumor antigens and foreign invading viruses, improve the body's immunity, and eliminate tumor cells and viruses in time.
所以，本发明所述抗体对肿瘤、感染性疾病及自身免疫性疾病的治疗具有广泛的应用前景。Therefore, the antibody of the present invention has broad application prospects in the treatment of tumors, infectious diseases and autoimmune diseases.
附图说明Description of drawings
图1 是本发明PD-1抗体阻断 PD-1/PD-L1抑制途径促进 T 细胞增殖结果图。Figure 1 is a graph showing the results of the PD-1 antibody of the present invention blocking the PD-1/PD-L1 inhibitory pathway to promote T cell proliferation.
图2是本发明PD-1抗体对T细胞凋亡的影响结果图。Fig. 2 is a graph showing the effect of the PD-1 antibody of the present invention on T cell apoptosis.
图3是本发明PD-1抗体阻断 PD-1/PD-L1的相互结合结果图。Fig. 3 is a diagram showing the results of blocking the mutual binding of PD-1/PD-L1 by the PD-1 antibody of the present invention.
图4 是本发明PD-1抗体在体内抑制肿瘤生长结果图。Figure 4 is a graph showing the results of the PD-1 antibody of the present invention inhibiting tumor growth in vivo.
图5 是本发明PD-1抗体对CMV刺激的PBMC中IFNγ分泌的影响结果图。Figure 5 is a graph showing the effect of the PD-1 antibody of the present invention on the secretion of IFNγ in CMV-stimulated PBMCs.
具体实施方式Detailed ways
以下结合具体实施例和试验例对本发明做进一步的说明，但不应理解对本发明的限制。The present invention will be further described below in conjunction with specific examples and test examples, but it should not be understood as a limitation of the present invention.
1、cDNA的制备1. Preparation of cDNA
收集肝癌、肾癌和黑色素瘤以及健康人共50人的外周血各10毫升混合，肝素钠抗凝并采用密度梯度离心法分离单个核细胞（PBMC），用PD-1抗原进行体外致敏。PBMC体外致敏后，按4×106 PBMC加入1ml EBV培养上清，于37℃孵育3h，吸弃上清，加入含100ml/L FCS的RPMI 1640完全培基，培养2周后，换用含200ml/L NBS的RPMI 1640完全培基。转化细胞培养上清的筛选采用间接ELISA法，用PD-1作为肿瘤细胞抗原固相板，收集的B细胞培养上清作为一抗，HRP标记的羊抗人IgG/M(HRP-GAH-IgG/M)为二抗。将阳性孔的细胞扩大培养至106～107个细胞。然后用Invitrogen公司的Trizol试剂抽提转化后B细胞总RNA，并逆转录合成cDNA第1链，以此为模板扩增抗体可变区基因并连接成scFv，继而克隆入载体FUSE5构建噬菌体单链抗体库。Collect 10 ml of peripheral blood from 50 people with liver cancer, kidney cancer, melanoma, and healthy people, anticoagulate with heparin sodium, separate mononuclear cells (PBMC) by density gradient centrifugation, and use PD-1 antigen for in vitro sensitization. After PBMCs were sensitized in vitro, add 1ml of EBV culture supernatant to 4×106 PBMCs, incubate at 37°C for 3 hours, discard the supernatant, add RPMI 1640 complete medium containing 100ml/L FCS, culture for 2 weeks, replace with RPMI 1640 complete medium containing 200ml/L NBS. The culture supernatant of transformed cells was screened by indirect ELISA method, using PD-1 as the tumor cell antigen solid phase plate, the collected B cell culture supernatant as the primary antibody, HRP-labeled goat anti-human IgG/M (HRP-GAH-IgG /M) as the secondary antibody. Expand the cells in the positive wells to 106 -107 cells. Then use Invitrogen’s Trizol reagent to extract the total RNA of the transformed B cells, reverse transcribe to synthesize the first strand of cDNA, use this as a template to amplify the antibody variable region gene and connect it into scFv, and then clone it into the vector FUSE5 to construct a single-chain phage Antibody library.
2、VH和VL基因的PCR扩增与连接2. PCR amplification and connection of VH and VL genes
按文献（CaiXH， GarenA. PNAS， 1995.92(14): 6537-6541）设计合成扩增人抗体基因的引物。以cDNA第一链为模板，进行PCR扩增VH和VL基因。并通过编码连接肽(Gly4Ser)3使VH和VL基因连成ScFv基因。According to the literature (CaiXH, GarenA. PNAS, 1995.92(14): 6537-6541), the primers for synthesizing and amplifying human antibody genes were designed. Using the first strand of cDNA as a template, the VH and VL genes were amplified by PCR. And the VH and VL genes are connected into ScFv gene by encoding connecting peptide (Gly4Ser)3.
3、初级噬菌体抗体库的构建3. Construction of primary phage antibody library
以VH和VL基因片段为模板，将扩增产物以适当比例混合，通过重叠PCR拼接，扩增ScFv基因，克隆入pComb3XSS载体中，电穿孔转化XL1-Blue，计数克隆测定库容为107，细菌增殖后经辅助病毒超感染获噬菌体抗体库，沉淀浓缩后测定滴度为1013 CFU/ml。Using the VH and VL gene fragments as templates, the amplified products were mixed in an appropriate ratio, and the ScFv gene was amplified by overlapping PCR splicing, cloned into the pComb3XSS vector, transformed into XL1-Blue by electroporation, and the counting clone was determined to have a storage capacity of 107 After bacterial proliferation, the phage antibody library was obtained by superinfection with helper virus, and the titer was 1013 CFU/ml after precipitation and concentration.
4、构建大容量噬菌体抗体库及淘选4. Construction of large-capacity phage antibody library and panning
从初级噬菌体抗体库中取出5×1012CFU的噬菌体颗粒以高比例(MOI=100/l)感染BS1365菌，通过loxp-ere介导的定位重组使同一细胞内不同载体之间的轻/重链随机交换配对，所获噬菌体上清再低比例(MOI≤1)感染XL1-Blue菌，获得库容为1×1010的大容量人源噬菌体抗体库。以PD-1为抗原，对噬菌体单链抗体库进行了4轮亲和筛选过程，测定投入和产出的噬菌体的数目，产出/投入比增加了约500倍，说明特异性结合PD-1蛋白的ScFv得到了有效富集。Take 5×1012 CFU phage particles from the primary phage antibody library to infect BS1365 bacteria at a high ratio (MOI=100/l), and make the light/heavy ratio between different vectors in the same cell through loxp-ere-mediated recombination The chains were randomly exchanged and paired, and the obtained phage supernatant was infected with XL1-Blue bacteria at a low ratio (MOI ≤ 1) to obtain a large-capacity human phage antibody library with a storage capacity of 1×1010 . Using PD-1 as the antigen, four rounds of affinity screening were performed on the phage single-chain antibody library to determine the number of input and output phages. The output/input ratio increased by about 500 times, indicating that it specifically binds to PD-1 The ScFv of the protein was effectively enriched.
5、筛选结合PD-1的阳性噬菌体抗体单克隆5. Screen positive phage antibody monoclonal binding to PD-1
每轮随机挑选500个左右分隔良好的单克隆，挑取3-4轮淘选之后产出的共1500克隆进行噬菌体单克隆挽救，噬菌体抗体经ELISA检测，得到特异性结合PD-1的噬菌体单链抗体克隆。将经过ELISA鉴定阳性的噬菌体单克隆抗体，利用直标FITC-AntiM13，通过流式检测其与PD-1结合，得到特异性结合PD-1的噬菌体单链抗体克隆，对获得的阳性克隆进行序列测定，并对其进一步分析氨基酸序列和CDR区。About 500 well-segregated monoclonals were randomly selected in each round, and a total of 1,500 clones produced after 3-4 rounds of panning were selected for phage monoclonal rescue. Phage antibodies were detected by ELISA to obtain phage monoclonals that specifically bind to PD-1. Chain antibody cloning. The positive phage monoclonal antibody identified by ELISA was directly labeled with FITC-AntiM13, and its binding to PD-1 was detected by flow cytometry to obtain a phage single-chain antibody clone that specifically binds to PD-1, and the obtained positive clone was sequenced Determine and further analyze the amino acid sequence and CDR region.
6、噬菌体完整抗体的真核表达载体构建6. Construction of eukaryotic expression vectors for phage intact antibodies
以筛选到的3种阳性单克隆抗体的质粒为模板，利用设计好的引物扩增轻链可变区VL基因，约300bp。用扩增人抗体轻链恒定区CL基因，再以VL和CL为模板，利用overlopPCR扩增出L链基因，克隆到pMD19-T载体中，构建成pMD19-L，将pMD19-L以Hindlll和EcoRI酶切，与同酶切的质粒PcDNA3.1(+)进行连接，构建成真核表达载体。Using the plasmids of the three positive monoclonal antibodies screened as templates, the designed primers were used to amplify the VL gene of the light chain variable region, about 300 bp. Amplify theCL gene of the light chain constant region of the human antibody, and then use VL andCL as templates to amplify the L chain gene by overlopPCR, clone it into the pMD19-T vector, and construct pMD19-L. The pMD19-L Digest with Hindlll and EcoRI, and connect with the plasmid PcDNA3.1(+) digested with the same restriction enzymes to construct a eukaryotic expression vector.
从健康人淋巴细胞中提取总RNA，用RT-PCR合成cDNA，再扩增重链CH区基因，约1000bp，克隆到pMD19-T载体中，构建成pMD19-CH。以噬菌体单克隆抗体DNA为模板，扩增重链可变区VH基因，克隆到pMD19-T载体中，构建成pMD19-VH。将pMD19-CH和pMD19-VH分别用HindIII和NheI双酶切，将VH片断连接到pMD19-CH载体中。阳性克隆再通过HindIII和EcoRI酶切，连接到pcDNA3.1(+)中，构建真核表达载体。Total RNA was extracted from healthy human lymphocytes, cDNA was synthesized by RT-PCR, the heavy chain CH region gene was amplified, about 1000bp, cloned into pMD19-T vector, and pMD19-CH was constructed. Using the phage monoclonal antibody DNA as a template, the VH gene of the heavy chain variable region was amplified, cloned into the pMD19-T vector, and pMD19-VH was constructed. pMD19-CH and pMD19-VH were digested with HindIII and NheI respectively, and the VH fragment was ligated into the pMD19-CH vector. The positive clones were digested with HindIII and EcoRI, and connected to pcDNA3.1(+) to construct eukaryotic expression vectors.
7、抗体在CHO细胞中的表达7. Antibody expression in CHO cells
于3.5cm组织培养皿中接种3×105CHO细胞，细胞培养至90％-95％融合时进行转染，将上述载体进行线性化，转染CHO细胞。转染完毕，细胞在G418压力下进行选择，每浓度1次培养2周。然后转移至96孔板培养至70％覆盖度，换成选择培养基(含5％透析胎牛血清和适当水平的G418)进行筛选，并监控其生长情况，直至为转染的细胞死去，只留下转染的细胞。所述带有转染细胞的板生长大约4周，直至形成细胞团。镜检观察产生的细胞团，至适当大小(大于孔底面积的＞60％)，并确认每孔只有-个细胞团。根据对细胞上清的IgGκELISA检测结果，从960个转染子中筛选出80多个表达水平相对较高的，并对其进行了静态培养鉴定。对选出的细胞系在EX302无血清培养基中进行悬浮培养，用ELISA法进-步检定其表达水平并对细胞生长速率进行观察。根据收获上清的抗体浓度和可接受的生长特性，选出了两个最好的细胞系在EX302无血清培养基中进行批式摇瓶培养。Inoculate 3×105 CHO cells in a 3.5 cm tissue culture dish, and perform transfection when the cells are cultured to 90%-95% confluence, linearize the above vector, and transfect the CHO cells. After the transfection, the cells were selected under the pressure of G418, and each concentration was cultured for 2 weeks. Then transfer to a 96-well plate and cultivate to 70% coverage, replace with selection medium (containing 5% dialyzed fetal calf serum and G418 at an appropriate level) for screening, and monitor its growth until the transfected cells die, only Leave the transfected cells. The plates with transfected cells were grown for approximately 4 weeks until cell clumps formed. Microscopically observe the resulting cell clusters to an appropriate size (greater than >60% of the bottom area of the well), and confirm that there is only one cell cluster per well. According to the detection results of IgGκ ELISA on the cell supernatant, more than 80 transfectants with relatively high expression levels were screened out from 960 transfectants, and were identified by static culture. The selected cell lines were cultured in suspension in EX302 serum-free medium, and the expression level was further tested by ELISA method and the cell growth rate was observed. Based on the antibody concentration of the harvested supernatant and acceptable growth characteristics, the two best cell lines were selected for batch shake flask culture in EX302 serum-free medium.
8、抗体的纯化8. Antibody purification
用GE Healthcare的HiTrap Protein A层析柱对重组抗体进行纯化。Recombinant antibodies were purified using HiTrap Protein A columns from GE Healthcare.
9、本发明抗人PD-1单抗能促进T细胞增殖9. The anti-human PD-1 monoclonal antibody of the present invention can promote T cell proliferation
通过体外T细胞增殖实验观测该抗体阻断PD-L1负性信号后对T细胞的影响。CCK-8细胞染色结果显示，4天后与IgG对照组相比，本发明抗人PD-1单抗能进一步促进anti-CD3mAb+anti-CD28mAb单抗刺激的T细胞增殖（P<0.05），且随着剂量增加促增殖作用也逐渐增强，如图1所示。由此表明，抗人PD-1单抗能通过阻断PD-1/PD-L抑制途径显著促进T细胞增殖。The effect of the antibody on T cells after blocking the negative signal of PD-L1 was observed by T cell proliferation experiment in vitro. The results of CCK-8 cell staining showed that after 4 days, compared with the IgG control group, the anti-human PD-1 monoclonal antibody of the present invention could further promote the proliferation of T cells stimulated by anti-CD3mAb+anti-CD28mAb monoclonal antibody (P<0.05), and As the dose increases, the pro-proliferation effect is also gradually enhanced, as shown in Figure 1. This shows that anti-human PD-1 mAb can significantly promote T cell proliferation by blocking the PD-1/PD-L inhibitory pathway.
10、抗PD-1抗体对T细胞凋亡的影响10. Effect of anti-PD-1 antibody on T cell apoptosis
利用膜联蛋白V染色测试来测量抗PD-1抗体对诱导T细胞凋亡的影响。将T细胞在混合淋巴细胞反应中培养，向试管中加入浓度为25μg/ml的抗PD-1抗体。使用非特异性抗体作为对照。根据标准方案添加膜联蛋白V和碘化丙啶。将混合物在黑暗中于室温温育15分钟，然后使用流式细胞仪进行分析。结果见图2。The annexin V staining assay was used to measure the effect of anti-PD-1 antibody on the induction of T cell apoptosis. T cells were cultured in a mixed lymphocyte reaction, and anti-PD-1 antibody was added to the tube at a concentration of 25 μg/ml. A non-specific antibody was used as a control. Annexin V and propidium iodide were added according to standard protocols. The mixture was incubated for 15 minutes at room temperature in the dark and then analyzed using a flow cytometer. The results are shown in Figure 2.
结论：抗PD-1抗体对T细胞凋亡没有影响。Conclusion: Anti-PD-1 antibody has no effect on T cell apoptosis.
11、抗人PD-1抗体对PD-1/PD-L信号的阻断效应11. Blocking effect of anti-human PD-1 antibody on PD-1/PD-L signaling
将表达PD-1的CHO细胞悬浮于FACS缓冲液中。向细胞悬浮液中加入3种浓度的本发明抗PD-1抗体并于4℃温育30分钟。洗去未结合的抗体并将FITC标记的PD-L1融合蛋白加入到试管中并于4℃温育30分钟。使用FACScan流式细胞仪进行流式细胞计量分析。根据染色的平均荧光强度(MFI)的测量，抗PD-1单克隆抗体阻断PD-L1与表达PD-1的CHO细胞的结合。如图3所示，抗PD-1抗体能显著抑制PD-L1与细胞表面PD-1的相互作用。CHO cells expressing PD-1 were suspended in FACS buffer. Three concentrations of the anti-PD-1 antibody of the present invention were added to the cell suspension and incubated at 4°C for 30 minutes. Unbound antibody was washed away and FITC-labeled PD-L1 fusion protein was added to the tube and incubated at 4°C for 30 minutes. Flow cytometric analysis was performed using a FACScan flow cytometer. Anti-PD-1 mAbs blocked the binding of PD-L1 to PD-1-expressing CHO cells, as measured by the mean fluorescence intensity (MFI) of the staining. As shown in Figure 3, anti-PD-1 antibody can significantly inhibit the interaction of PD-L1 with cell surface PD-1.
12、使用抗PD-1抗体处理体内肿瘤模型12. Using anti-PD-1 antibody to treat tumor model in vivo
用抗PD-1抗体体内处理植入有癌性肿瘤的小鼠以检验抗体对肿瘤生长的体内影响。根据体重将6-8周龄之间的雌性AJ小鼠随机分成6组。在第0天将溶于200μlDMEM培养基的2×106个SA1/N纤维肉瘤细胞在右侧皮下植入小鼠。用PBS对照或10mg/kg的抗体处理小鼠。在第1、4、8和11天用大约200μl含有抗体的PBS或对照通过腹膜内注射给动物剂量给药。每组包含10只动物，各组组成如下：(1)PBS对照组，(2)对照小鼠IgG，(3)对照仓鼠IgG，(4)本发明抗PD-1抗体。对小鼠每周两次监测肿瘤生长，持续大约6周。使用电子测径器对肿瘤进行三维测量(高度×宽度×长度)并计算肿瘤体积。当肿瘤达到肿瘤终点(1500mm3)或显示大于15％的体重减少时将小鼠处死。结果显示于图4。抗PD-1抗体将达到肿瘤终点体积(1500mm3)的平均时间从对照组的-25天延长到-40天。因而，抗PD-1抗体处理对肿瘤生长具有直接的体内抑制效果。Mice implanted with cancerous tumors were treated in vivo with anti-PD-1 antibody to examine the in vivo effect of the antibody on tumor growth. Female AJ mice between 6-8 weeks of age were randomly divided into 6 groups according to body weight. On day 0, 2×106 SA1/N fibrosarcoma cells dissolved in 200 μl DMEM medium were subcutaneously implanted into mice on the right side. Mice were treated with PBS control or 10 mg/kg of antibody. Animals were dosed on days 1, 4, 8 and 11 by intraperitoneal injection with approximately 200 [mu]l of antibody-containing PBS or control. Each group contained 10 animals, and the composition of each group was as follows: (1) PBS control group, (2) control mouse IgG, (3) control hamster IgG, (4) anti-PD-1 antibody of the present invention. Mice were monitored for tumor growth twice weekly for approximately 6 weeks. Three-dimensional measurements (height x width x length) of tumors were performed using electronic calipers and tumor volumes were calculated. Mice were sacrificed when tumors reached tumor endpoint (1500 mm3 ) or showed greater than 15% body weight loss. The results are shown in Figure 4. Anti-PD-1 antibodies extended the mean time to tumor endpoint volume (1500 mm3 ) from -25 days in the control group to -40 days. Thus, anti-PD-1 antibody treatment had a direct in vivo inhibitory effect on tumor growth.
13、抗PD-1抗体对病毒刺激PBMC细胞的细胞因子分泌的影响13. Effect of anti-PD-1 antibody on cytokine secretion of virus-stimulated PBMC cells
从CMV阳性的供体分离外周血单个核细胞(PBMC)并在抗PD-1抗体存在或缺失的情况下暴露于CMV溶胞物以检验抗体对由抗原刺激的细胞因子分泌的影响。将105个来自CMV阳性供体的人PMBC在总体积200μl中培养，并与CMV感染细胞的溶胞物一起加到每个孔中。将各种浓度的抗PD-1抗体加到每个孔中达4天。第4天后，从每份培养物中取100μl培养基用于细胞因子测量。使用OptEIA ELISA试剂盒测量IFN-γ的水平。用3H-胸苷标记细胞，再培养18小时，并分析细胞增殖。使用CellTiter-Glo试剂分析细胞增殖。结果如图5所示。抗PD-1抗体以浓度依赖性方式提高IFNγ分泌。Peripheral blood mononuclear cells (PBMCs) were isolated from CMV-positive donors and exposed to CMV lysates in the presence or absence of anti-PD-1 antibodies to examine the effect of antibodies on antigen-stimulated cytokine secretion.105 human PMBCs from CMV-positive donors were cultured in a total volume of 200 μl and added to each well along with lysates of CMV-infected cells. Various concentrations of anti-PD-1 antibodies were added to each well for 4 days. After day 4, 100 μl of medium was taken from each culture for cytokine measurements. The level of IFN-γ was measured using the OptEIA ELISA kit. Cells were labeled with3 H- thymidine, incubated for an additional 18 hours, and analyzed for cell proliferation. Cell proliferation was analyzed using CellTiter-Glo reagent. The result is shown in Figure 5. Anti-PD-1 antibody increases IFNγ secretion in a concentration-dependent manner.
<110> 郑州大学<110> Zhengzhou University
<120> 一种全人源化抗PD-1单克隆抗体及其制备方法和应用<120> A fully humanized anti-PD-1 monoclonal antibody and its preparation method and application
<160> 8<160> 8
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<223> PD-1 抗体重链CDR1区<223> PD-1 antibody heavy chain CDR1 region
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PD-1 抗体轻链CDR1区PD-1 antibody light chain CDR1 region
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Gln Gln Tyr Asp His Val Pro Leu ThrGln Gln Tyr Asp His Val Pro Leu Thr
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<223> PD-1 抗体重链区<223> PD-1 antibody heavy chain region
<400> 7<400> 7
Gln Val Gln Leu Val Glu Ser Gly Pro Gly Val Lys Lys Pro Gly Ser Ser Leu Lys Leu Ser Cys Thr Val Ser Gly Phe Thr Phe Ser Ser Tyr Asp Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Met Ala Val Ile Trp Tyr Ser Gly Ser Asn Thr Tyr Tyr Asn Asp Ser Leu Lys Ser Arg Phe Ser Ile Thr Arg Asp Asn Ser Lys Asn Thr Ala Tyr Met Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Tyr Phe Gly Val Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerGln Val Gln Leu Val Glu Ser Gly Pro Gly Val Lys Lys Pro Gly Ser Ser Leu Lys Leu Ser Cys Thr Val Ser Gly Phe Thr Phe Ser Ser Tyr Asp Tyr Tyr Met His Trp M Trp Le Val Glu Glu Glnly Ala Pro Ala Val Ile Trp Ty Sergr Tyr Tyr Tyr Tyr Asr Leu lys Serg PHR ARG ARG ASN Ser Lysn Thr Met Gln Leu ARG Ala Glu Asp Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Ty Ala Tyr Phe Gly Val Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
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Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly Asp Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Gly Ile Gly Asn Thr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Arg Leu Leu Ile Tyr Arg Ala Ser Gln Gly Ile Gly Asn Thr Leu Ala Gly Val Pro Ala Arg Phe Ser Gly Asp Gly Asp Gly Thr Asp Phe Thr Leu Thr Ile Asp Asp Leu Glu Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysASP ILE Val Met THLN Ser Pro Ala Sering, Gly Gly Gly GLY ARG Ala Thr Ile Serg Ala Serge Gln GLY ASN THR Leu Ala Gln Gln LYS PRN ALA PRYSRG Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu ARG ALA Sern GLN GLE GLY Asn Thr Leu Ala Gly Val Pro Ala ARG PHE Ser Gly Asp Gly THR ASP PHR Leu Thr Ile Asp Pro GLU ALA TYR Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Cyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
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