发明领域field of invention
本发明涉及新的抗PD-1抗体。The present invention relates to novel anti-PD-1 antibodies.
背景技术Background technique
越来越多的临床前和临床结果的证据表明，靶向免疫检查点正在成为最有希望的治疗癌症患者的方法。程序性细胞死亡分子1是免疫检查点蛋白之一，其在限制T细胞活性中起主要作用，所述T细胞提供了主要的免疫抵抗机制，通过这种限制作用肿瘤细胞能够躲过免疫监视。在活化的T细胞上表达的PD-1与在肿瘤细胞上表达的PD-L1的相互作用对免疫应答起负调节并减弱抗肿瘤免疫力。PD-L1在肿瘤上的表达与食管癌、胰腺癌和其它类型的癌症的生存率下降相关，突出了该通路可以作为新的有前途的肿瘤免疫治疗靶点。制药公司已经开发了多种针对PD-1通路的药物，如百时美施贵宝公司(BMS)、默克公司、罗氏公司和葛兰素史克(GSK)公司。临床试验的数据显示了在各种肿瘤类型的患者中持久的临床活性和良好的安全性的早期证据。Nivolumab是BMS开发的PD-1药物，其正被投入到下一代领域的中心阶段。目前在6个后期研究中，在研究的5个癌症组中的3个中，治疗促使了肿瘤的缩小，其中包括72例肺癌患者中的18％，98例黑色素瘤患者中的接近三分之一和33例肾癌患者中的27％。由默克公司研制的lambrolizumab是全人源单克隆IgG4抗体，其作用于PD-1，其在针对皮肤癌获得的令人印象深刻的IB数据后抓住了FDA的新突破指标。阶段性IB研究的结果显示在85例癌症患者中有51％的客观的抗肿瘤反应，并在9％的患者中出现完全的反应。罗氏公司的实验性MPDL3280A证明了其在140例携带各种大小的肿瘤的晚期癌症患者中缩小了29例(21％)患者的肿瘤。Mounting evidence of preclinical and clinical outcomes suggests that targeting immune checkpoints is emerging as the most promising approach for treating cancer patients. Programmed cell death molecule 1 is one of the immune checkpoint proteins that plays a major role in restricting the activity of T cells, which provide the main mechanism of immune resistance, by which tumor cells are able to evade immune surveillance. The interaction of PD-1 expressed on activated T cells with PD-L1 expressed on tumor cells negatively regulates the immune response and attenuates antitumor immunity. Tumor expression of PD-L1 is associated with decreased survival in esophageal, pancreatic, and other types of cancer, highlighting this pathway as a new promising target for tumor immunotherapy. Pharmaceutical companies such as Bristol-Myers Squibb (BMS), Merck & Co., Roche, and GlaxoSmithKline (GSK) have developed a variety of drugs targeting the PD-1 pathway. Data from clinical trials showed early evidence of durable clinical activity and a favorable safety profile in patients with various tumor types. Nivolumab is a PD-1 drug developed by BMS that is being put into the center stage of the next-generation field. In six late-stage studies now, treatment led to tumor shrinkage in three of five cancer groups studied, including 18 percent of 72 lung cancer patients and nearly one-third of 98 melanoma patients One and 27% of the 33 patients with kidney cancer. Lambrolizumab, developed by Merck, a fully human monoclonal IgG4 antibody targeting PD-1, is grabbing the FDA's new breakthrough indicator after impressive IB data against skin cancer. The results of the phase IB study showed an objective antitumor response in 51% of 85 cancer patients and a complete response in 9% of the patients. Roche's experimental MPDL3280A demonstrated that it shrank tumors in 29 (21%) of 140 patients with advanced cancer bearing tumors of various sizes.
然而，现有的治疗方法不都是尽如人意的，因此仍然需要新的抗PD-1的抗体。However, existing treatments are not all satisfactory, so new anti-PD-1 antibodies are still needed.
发明简述Brief description of the invention
本申请提供了新的抗PD-1单克隆抗体(特别是全人源抗体)、编码其的多核苷酸和使用其的方法。The present application provides novel anti-PD-1 monoclonal antibodies (especially fully human antibodies), polynucleotides encoding them and methods of using them.
在一个方面，本申请提供了一种分离的单克隆抗体或其抗原结合片段，其能够以不超过10-8M(例如，≤9x10-9M、≤8x10-9M、≤7x10-9M、≤6x10-9M、≤5x10-9M、≤4x10-9M、≤3x10-9M、≤2x10-9M、or≤10-9M)的Kd值特异性地与人PD-1结合，所述Kd值通过等离子共振结合法测定。In one aspect, the application provides an isolated monoclonal antibody or antigen- binding fragment thereof, which can beproduced at no more than10-8 M (e.g., , ≤6x10-9 M, ≤5x10-9 M, ≤4x10-9 M, ≤3x10-9 M, ≤2x10-9 M, or≤10-9 M) specific binding to human PD-1 , the Kd value is determined by plasmon resonance binding.
在某些实施方式中，所述的抗体或其抗原结合片段以不超过100nM或不超过10nM(如不超过50nM、40nM、30nM、20nM、10nM、9nM、8nM、7nM、6nM、5nM、4nM、3nM、2nM或1nM)的EC50与猴PD-1结合。在某些实施方式中，所述的抗体或其抗原结合片段不与小鼠PD-1结合，但以人PD-1相似的结合亲和性与猴PD-1结合。在某些实施方式中，所述的抗体或其抗原结合片段以不超过100nM(如不超过50nM、40nM、30nM、20nM、10nM、9nM、8nM、7nM、6nM、5nM、4nM、3nM、2nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM、0.09nM、0.08nM、0.07nM、0.06nM、0.05nM、0.04nM、0.03nM、0.02nM或0.01nM)的IC50高效地抑制人或猴PD-1与其配体(如PD-L1、PD-L2)的结合。在某些实施方式中，所述EC50或IC50通过荧光激活的细胞分选(FACS)分析进行测定。In certain embodiments, the antibody or antigen-binding fragment thereof is dosed at no more than 100 nM or no more than 10 nM (such as no more than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3nM, 2nM or 1nM)EC50 binding to monkey PD-1. In certain embodiments, the antibody or antigen-binding fragment thereof does not bind to mouse PD-1, but binds to monkey PD-1 with a similar binding affinity to human PD-1. In certain embodiments, the antibody or antigen-binding fragment thereof is dosed at no more than 100 nM (such as no more than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, IC50 of 0.02nM or 0.01nM) efficiently inhibits the binding of human or monkey PD-1 to its ligands (such as PD-L1, PD-L2). In certain embodiments, theEC50 orIC50 is determined by fluorescence activated cell sorting (FACS) analysis.
在某些实施方式中，所述的抗体或其抗原结合片段具有基本上减少的效应功能。在某些实施方式中，所述的抗体或其抗原结合片段不介导ADCC或CDC或两者均不介导。In certain embodiments, the antibody or antigen-binding fragment thereof has substantially reduced effector function. In certain embodiments, the antibody or antigen-binding fragment thereof does not mediate ADCC or CDC or both.
在某些实施方式中，本申请所述的抗体或其抗原结合片段包括重链CDR序列，所述序列选自：SEQ ID NO:1、3、5、13、15、21、23、25、33、35和37。。In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a heavy chain CDR sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37. .
在某些实施方式中，本申请所述的抗体或其抗原结合片段包括轻链CDR序列，所述序列选自：SEQ ID NO:7、9、11、17、19、27、29、31、39、41、43和65。In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a light chain CDR sequence selected from the group consisting of: SEQ ID NO: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, 43 and 65.
在某些实施方式中，本申请所述的抗体或其抗原结合片段包括选自SEQ ID NO:1、3、5、7、9和11；或选自SEQ ID NO:13、15、5、7、17和11；或选自SEQ ID NO:1、15、5、7、17和19；或选自SEQ ID NO:1、15、5、7、17和65；或选自SEQ ID NO:1、15、5、7、17和19；或选自SEQ ID NO:21、23、25、27、29和31；或选自SEQ ID NOs:33、35、37、39、41和43中的至少1个、2个、3个、4个、5个或6个CDR。In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises an antibody selected from SEQ ID NO: 1, 3, 5, 7, 9, and 11; or an antibody selected from SEQ ID NO: 13, 15, 5, 7, 17 and 11; Or selected from SEQ ID NO:1,15,5,7,17 and 19; Or selected from SEQ ID NO:1,15,5,7,17 and 65; Or selected from SEQ ID NO or selected from SEQ ID NOs: 21, 23, 25, 27, 29 and 31; or selected from SEQ ID NOs: 33, 35, 37, 39, 41 and 43 At least 1, 2, 3, 4, 5 or 6 CDRs in the CDR.
在某些实施方式中，本申请所述的抗体或其抗原结合片段包括重链可变区，所述重链可变区选自：In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region selected from the group consisting of:
a)重链可变区，其包括SEQ ID NO:1、SEQ ID NO:3和/或SEQ ID NO:5；a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3 and/or SEQ ID NO: 5;
b)重链可变区，其包括SEQ ID NO:13、SEQ ID NO:15和/或SEQ ID NO:5；b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15 and/or SEQ ID NO: 5;
c)重链可变区，其包括SEQ ID NO:1、SEQ ID NO:15和/或SEQ ID NO:5；c) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15 and/or SEQ ID NO: 5;
d)重链可变区，其包括SEQ ID NO:21、SEQ ID NO:23和/或SEQ ID NO:25；以及d) a heavy chain variable region comprising SEQ ID NO:21, SEQ ID NO:23 and/or SEQ ID NO:25; and
e)重链可变区，其包括SEQ ID NO:33、SEQ ID NO:35和/或SEQ ID NO:37。e) A heavy chain variable region comprising SEQ ID NO:33, SEQ ID NO:35 and/or SEQ ID NO:37.
在某些实施方式中，本申请所述的抗体或其抗原结合片段包括轻链可变区，所述轻链可变区选自：In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a light chain variable region selected from the group consisting of:
a)轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:9和/或SEQ ID NO:11；a) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:9 and/or SEQ ID NO:11;
b)轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:11；b) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO:11;
c)轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:19；c) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO:19;
d)轻链可变区，其包括SEQ ID NO:27、SEQ ID NO:29和/或SEQ ID NO:31；d) a light chain variable region comprising SEQ ID NO:27, SEQ ID NO:29 and/or SEQ ID NO:31;
e)轻链可变区，其包括SEQ ID NO:39、SEQ ID NO:41和/或SEQ ID NO:43；以及E) light chain variable region, it comprises SEQ ID NO:39, SEQ ID NO:41 and/or SEQ ID NO:43; And
f)轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:65。f) A light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO:65.
在某些实施方式中，本申请所述的抗体或其抗原结合片段包括：In certain embodiments, the antibodies or antigen-binding fragments thereof described herein include:
a)重链可变区，其包括SEQ ID NO:1、SEQ ID NO:3和/或SEQ ID NO:5；和轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:9和/或SEQ ID NO:11；A) heavy chain variable region, it comprises SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5; And light chain variable region, it comprises SEQ ID NO:7, SEQ ID NO:9 and/or SEQ ID NO: 11;
b)重链可变区，其包括SEQ ID NO:13、SEQ ID NO:15和/或SEQ ID NO:5；和轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:11；B) heavy chain variable region, it comprises SEQ ID NO:13, SEQ ID NO:15 and/or SEQ ID NO:5; And light chain variable region, it comprises SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO: 11;
c)重链可变区，其包括SEQ ID NO:1、SEQ ID NO:15和/或SEQ ID NO:5；和轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:19；c) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15 and/or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO: 19;
d)重链可变区，其包括SEQ ID NO:21、SEQ ID NO:23和/或SEQ ID NO:25；和轻链可变区，其包括SEQ ID NO:27、SEQ ID NO:29和/或SEQ ID NO:31；d) a heavy chain variable region comprising SEQ ID NO:21, SEQ ID NO:23 and/or SEQ ID NO:25; and a light chain variable region comprising SEQ ID NO:27, SEQ ID NO:29 and/or SEQ ID NO: 31;
e)重链可变区，其包括SEQ ID NO:33、SEQ ID NO:35和/或SEQ ID NO:37；和轻链可变区，其包括SEQ ID NO:39、SEQ ID NO:41和/或SEQ ID NO:43；以及E) heavy chain variable region, it comprises SEQ ID NO:33, SEQ ID NO:35 and/or SEQ ID NO:37; And light chain variable region, it comprises SEQ ID NO:39, SEQ ID NO:41 and/or SEQ ID NO:43; and
f)重链可变区，其包括SEQ ID NO:1、SEQ ID NO:15和/或SEQ ID NO:5；和轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:65。F) heavy chain variable region, it comprises SEQ ID NO:1, SEQ ID NO:15 and/or SEQ ID NO:5; And light chain variable region, it comprises SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO:65.
在某些实施方式中，本申请所述的抗体或其抗原结合片段包括重链可变区，所述重链可变区选自SEQ ID NO:45、SEQ ID NO:49、SEQ ID NO:53、SEQ ID NO:57和SEQ ID NO:61。In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region selected from the group consisting of SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO: 53. SEQ ID NO:57 and SEQ ID NO:61.
在某些实施方式中，本申请所述的抗体或其抗原结合片段包括轻链可变区，所述轻链可变区选自SEQ ID NO:47、SEQ ID NO:51、SEQ ID NO:55、SEQ ID NO:59、SEQ ID NO:63和SEQ ID NO:67。In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a light chain variable region selected from the group consisting of SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO: 55. SEQ ID NO:59, SEQ ID NO:63 and SEQ ID NO:67.
在某些实施方式中，本申请所述的抗体或其抗原结合片段包括：In certain embodiments, the antibodies or antigen-binding fragments thereof described herein include:
a)重链可变区，其包括SEQ ID NO:45；和轻链可变区，其包括SEQ ID NO:47；A) a heavy chain variable region comprising SEQ ID NO:45; and a light chain variable region comprising SEQ ID NO:47;
b)重链可变区，其包括SEQ ID NO:49；和轻链可变区，其包括SEQ ID NO:51；B) a heavy chain variable region comprising SEQ ID NO:49; and a light chain variable region comprising SEQ ID NO:51;
c)重链可变区，其包括SEQ ID NO:53；和轻链可变区，其包括SEQ ID NO:55；c) a heavy chain variable region comprising SEQ ID NO:53; and a light chain variable region comprising SEQ ID NO:55;
d)重链可变区，其包括SEQ ID NO:57；和轻链可变区，其包括SEQ ID NO:59；d) a heavy chain variable region comprising SEQ ID NO:57; and a light chain variable region comprising SEQ ID NO:59;
e)重链可变区，其包括SEQ ID NO:61；和轻链可变区，其包括SEQ ID NO:63；或E) a heavy chain variable region comprising SEQ ID NO:61; and a light chain variable region comprising SEQ ID NO:63; or
f)重链可变区，其包括SEQ ID NO:53；和轻链可变区，其包括SEQ ID NO:67。f) a heavy chain variable region comprising SEQ ID NO:53; and a light chain variable region comprising SEQ ID NO:67.
在某些实施方式中，本申请所述的抗体或其抗原结合片段包括例如1.7.3hAb、1.49.9hAb、1.103.11hAb、1.103.11-v2hAb、1.139.15hAb和1.153.7hAb。In certain embodiments, antibodies or antigen-binding fragments thereof described herein include, for example, 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, and 1.153.7hAb.
在某些实施方式中，本申请所述的抗体或其抗原结合片段与抗体1.7.3hAb、1.49.9hAb、1.103.11hAb、1.103.11-v2hAb、1.139.15hAb或1.153.7hAb竞争相同的表位。在某些实施方式中，本申请所述的抗体或其抗原结合片段结合的表位包括以下PD-L1氨基酸残基中的至少一个：V64、P83、D85、L128、A129、P130、K131、A132和Q133。In certain embodiments, an antibody or antigen-binding fragment thereof described herein competes for the same epitope as antibody 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, or 1.153.7hAb . In certain embodiments, the epitope bound by the antibody or antigen-binding fragment thereof described herein includes at least one of the following PD-L1 amino acid residues: V64, P83, D85, L128, A129, P130, K131, A132 and Q133.
在某些实施方式中，本申请所述的抗体或其抗原结合片段能够阻断人PD-1与其配体结合，并且因此提供以下活性中的至少一个：In certain embodiments, an antibody or antigen-binding fragment thereof described herein is capable of blocking the binding of human PD-1 to its ligand, and thus provides at least one of the following activities:
a)在CD4+T细胞中诱导IL-2的产生；a) Inducing IL-2 production in CD4+ T cells;
b)在CD4+T细胞中诱导IFNγ的产生；b) induction of IFNγ production in CD4+ T cells;
c)诱导CD4+T细胞的增殖；以及c) induce proliferation of CD4+ T cells; and
d)逆转T reg抑制功能。d) Reversal of T reg suppressive function.
在某些实施方式中，本申请所述抗体是单克隆抗体、全人源抗体、人源化抗体、嵌合抗体、重组抗体、双特异抗体、标记抗体、双价抗体或抗独特型抗体。在某些实施方式中，所述抗体或其抗原结合片段是全人源单克隆抗体，其任选地由转基因大鼠产生，例如，内源性大鼠免疫球蛋白基因表达被失活、且携带具有J基因座缺失和C-kappa突变的重组的人源免疫球蛋白基因座的转基因大鼠。In certain embodiments, the antibodies described herein are monoclonal antibodies, fully human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, labeled antibodies, diabodies or anti-idiotypic antibodies. In certain embodiments, the antibody or antigen-binding fragment thereof is a fully human monoclonal antibody, optionally produced by a transgenic rat, e.g., endogenous rat immunoglobulin gene expression is inactivated, and Transgenic rats carrying recombinant human immunoglobulin loci with J locus deletion and C-kappa mutation.
在某些实施方式中，本申请中提供的抗原结合片段是骆驼化单域抗体(camelizedsingle chain domain antibody)、双功能抗体(diabody)、scFv、scFv二聚体、BsFv、dsFv、(dsFv)2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds双功能抗体(ds diabody)、纳米抗体、域抗体或双价域抗体。In certain embodiments, the antigen-binding fragment provided in the present application is a camelized single domain antibody (camelized single chain domain antibody), diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv)2 , dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds diabody, nanobody, domain antibody or bivalent domain antibody.
在某些实施方式中，本申请所述的抗体或其抗原结合片段进一步包括免疫球蛋白恒定区。In certain embodiments, an antibody or antigen-binding fragment thereof described herein further comprises an immunoglobulin constant region.
在某些实施方式中，本申请所述的抗体或其抗原结合片段进一步包括缀合物。In certain embodiments, an antibody or antigen-binding fragment thereof described herein further comprises a conjugate.
在某些实施方式中，所述缀合物可以是可检测标记、药代动力学修饰部分或纯化部分。In certain embodiments, the conjugate may be a detectable label, a pharmacokinetically modified moiety, or a purified moiety.
在另一方面，本申请提供了分离的多核苷酸，其编码如本申请所述的抗体或其抗原结合片段。在某些实施方式中，本申请提供的多核苷酸编码如本申请所述的抗体或其抗原结合片段的氨基酸序列。在某些实施方式中，本申请提供了包括这些多核苷酸的载体。在某些实施方式中，本申请提供了表达本申请所述的一种或多种抗体或抗原结合片段的方法，其通过在载体中表达由多核苷酸编码的抗体或抗原结合片段的条件下培养宿主细胞实现。在某些实施方式中，本申请提供的多核苷酸在载体中与启动子如SV40启动子可操作地连接。在某些实施方式中，包括本申请提供的载体的宿主细胞是中国仓鼠卵巢细胞，或293F细胞。In another aspect, the application provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof as described herein. In certain embodiments, the polynucleotide provided herein encodes the amino acid sequence of an antibody or antigen-binding fragment thereof as described herein. In certain embodiments, the application provides vectors comprising these polynucleotides. In certain embodiments, the application provides a method for expressing one or more antibodies or antigen-binding fragments described herein by expressing an antibody or antigen-binding fragment encoded by a polynucleotide in a vector cultured host cells. In certain embodiments, the polynucleotide provided herein is operably linked to a promoter, such as the SV40 promoter, in a vector. In certain embodiments, the host cells comprising the vectors provided herein are Chinese hamster ovary cells, or 293F cells.
在另一方面，本申请提供了包括本申请所述的抗体或其抗原结合片段的试剂盒。In another aspect, the application provides kits comprising the antibodies or antigen-binding fragments thereof described herein.
在另一方面，本申请的PD-1抗体，例如1.7.3hAb、1.49.9hAb、1.103.11hAb、1.103.11-v2hAb、1.139.15hAb和1.153.7hAb在动物中具有良好的耐受性和较高的体内抗肿瘤活性。在某些实施方式中，与仅施用溶剂的具有相似基线肿瘤体积的对照组动物相比，施用本申请所述的PD-1抗体的患有肿瘤的动物的肿瘤体积减少至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、或至少95％。On the other hand, the PD-1 antibodies of the present application, such as 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb and 1.153.7hAb have good tolerance and comparative High antitumor activity in vivo. In certain embodiments, an animal bearing a tumor that is administered a PD-1 antibody described herein has a reduction in tumor volume of at least 20%, at least 30%, compared to a control group of animals having a similar baseline tumor volume that is administered vehicle alone. %, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
在另一方面，本申请提供了在个体中治疗与PD-1相关的状况的方法，其包括向所述个体施用治疗有效量的本申请所述的抗体或其抗原结合片段。在某些实施方式中，所述个体被鉴定为患有可能对PD-1拮抗剂响应的病症或状况。在某些实施方式中，所述个体被鉴定为在来自所述个体的待测生物样品中呈PD-L1阳性或PD-L1水平上调。In another aspect, the application provides a method of treating a condition associated with PD-1 in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein. In certain embodiments, the individual is identified as having a disorder or condition that is likely to be responsive to a PD-1 antagonist. In certain embodiments, the individual is identified as being positive for PD-L1 or having an upregulated level of PD-L1 in a test biological sample from the individual.
在另一方面，本申请提供了药物组合物，包括本申请所述的抗体或其抗原结合片段以及一种或多种药学上可接受的载体。在某些实施方式中，所述药学载体可以是例如稀释剂、抗氧化剂、辅剂、赋形剂或无毒的辅助物质。In another aspect, the present application provides a pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof described in the present application and one or more pharmaceutically acceptable carriers. In certain embodiments, the pharmaceutical carrier can be, for example, a diluent, an antioxidant, an adjuvant, an excipient, or a non-toxic auxiliary substance.
在另一方面，本申请提供了治疗会从上调的免疫响应获益的受试者的状况的方法，包括对所述受试者施用有效量的本申请所述的抗体或其抗原结合片段。在某些实施方式中，所述受试者具有上调的PD-L1表达，或被鉴定为PD-L1表达阳性。In another aspect, the application provides a method of treating a condition in a subject that would benefit from an upregulated immune response comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof described herein. In certain embodiments, the subject has upregulated PD-L1 expression, or is identified as positive for PD-L1 expression.
在另一方面，提供了本申请所述的抗体或其抗原结合片段在制备用于治疗会从上调的免疫响应中获益的状况的药物中的用途。在某些实施方式中，所述状况是癌症或慢性病毒感染。In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof described herein in the manufacture of a medicament for the treatment of a condition which would benefit from an up-regulated immune response. In certain embodiments, the condition is cancer or chronic viral infection.
附图简述Brief description of the drawings
图1显示了FACS分析测定的全人源抗-PD-1抗体与表达PD-1的CHO细胞的结合。Figure 1 shows the binding of fully human anti-PD-1 antibodies to PD-1 expressing CHO cells as determined by FACS analysis.
图2显示了FACS分析测定的全人源PD-1抗体与表达PD-1的CHO细胞以约2nM的EC50结合。Figure 2 shows the binding of fully human PD-1 antibody to PD-1 expressing CHO cells with an EC50 of approximately 2 nM as determined by FACS analysis.
图3显示了FACS分析测定的全人源抗PD-1抗体与表达PD-1的活化的CD4+T细胞)细胞的结合。Figure 3 shows the binding of fully human anti-PD-1 antibodies to activated CD4+ T cells expressing PD-1 as determined by FACS analysis.
图4显示了FACS分析测定的全人源抗PD-1抗体以约3-8nM的IC50阻断了PD-L1与转染了PD-1的CHO细胞的结合。Figure 4 shows that the fully human anti-PD-1 antibody determined by FACS analysis blocked the binding of PD-L1 to CHO cells transfected with PD-1 with an IC50 of about 3-8 nM.
图5显示了FACS分析测定的全人源抗PD-1抗体与PD-1特异性结合，而不与家族成员CD28和CTLA4结合。Figure 5 shows that fully human anti-PD-1 antibodies specifically bind to PD-1, but not to family members CD28 and CTLA4, as determined by FACS analysis.
图6显示了抗PD-1的全人源抗PD-1抗体与食蟹猴PD-1结合但不与鼠科PD-1结合。Figure 6 shows that fully human anti-PD-1 antibodies against PD-1 bind to cynomolgus monkey PD-1 but not to murine PD-1.
图7显示了通过等离子体共振法测定的PD-1抗体与人PD-1结合亲和性的完整动力学为从3.76E-9至1.76E-10mol/L。Figure 7 shows the complete kinetics of the binding affinity of PD-1 antibody to human PD-1 determined by plasmon resonance method from 3.76E-9 to 1.76E-10 mol/L.
图8显示了在混合的淋巴细胞反应(MLR)中全人源抗-PD-1抗体对IL-2的生产的影响。Figure 8 shows the effect of a fully human anti-PD-1 antibody on IL-2 production in a mixed lymphocyte reaction (MLR).
图9显示了在MLR中全人源抗-PD-1抗体对IFNγ的生产的影响。Figure 9 shows the effect of fully human anti-PD-1 antibody on IFNγ production in MLR.
图10显示了在MLR中全人源抗-PD-1抗体促进T细胞增殖。Figure 10 shows that a fully human anti-PD-1 antibody promotes T cell proliferation in the MLR.
图11显示了全人源抗-PD-1抗体在特异性T细胞响应中促进T细胞增殖。Figure 11 shows that a fully human anti-PD-1 antibody promotes T cell proliferation in a specific T cell response.
图12显示了抗PD-1抗体逆转了Treg抑制功能。Figure 12 shows that anti-PD-1 antibody reverses Treg suppressive function.
图13显示了抗PD-1抗体在活化的T细胞中缺少ADCC。Figure 13 shows that anti-PD-1 antibodies lack ADCC in activated T cells.
图14显示了抗PD-1抗体在活化的T细胞中缺少CDC。Figure 14 shows that anti-PD-1 antibodies lack CDC in activated T cells.
图15显示了ELISA测定的在不同缓冲液中1.103.11-v2hAb与人PD-1胞外结构域结合的亲和性相似。“在缓冲液中的1.103.11-v2hAb”是指所述抗体在制剂缓冲液中，且“在PBS中的1.103.11-v2hAb”是指所述抗体在1xPBS(pH 7.4)中。Figure 15 shows that the affinities of 1.103.11-v2hAb binding to human PD-1 extracellular domain in different buffers determined by ELISA are similar. "1.103.11-v2hAb in buffer" means that the antibody is in formulation buffer, and "1.103.11-v2hAb in PBS" means that the antibody is in 1xPBS (pH 7.4).
图16显示了FACS测定的在不同缓冲液中的1.103.11-v2hAb结合表达PD-1的CHO细胞的亲和性相似。“在缓冲液中的1.103.11-v2hAb”是指所述抗体在制剂缓冲液中，且“在PBS中的1.103.11-v2hAb”是指所述抗体在1xPBS(pH 7.4)中。Figure 16 shows that 1.103.11-v2hAb in different buffers binds PD-1 expressing CHO cells with similar affinities measured by FACS. "1.103.11-v2hAb in buffer" means that the antibody is in formulation buffer, and "1.103.11-v2hAb in PBS" means that the antibody is in 1xPBS (pH 7.4).
图17显示了在抗体结合的人PD-L1的晶体结构上的热点(hot-spot)残基(阴影区)。A显示了共同热点残基；B至D分别显示了1.103.11hAb、Keytruda和11.148.10hAb的热点残基。Figure 17 shows hot-spot residues (shaded areas) on the crystal structure of antibody-bound human PD-L1. A shows the common hotspot residues; B to D show the hotspot residues of 1.103.11hAb, Keytruda and 11.148.10hAb, respectively.
发明详述Detailed description of the invention
本申请的以下描述只为说明本申请的多种实施方式。因此，此处讨论的具体修改方式不应理解为对申请范围的限制。本领域的技术人员在不偏离本申请范围的情况下即可很容易地得出多种等同方式，变化和修改，应理解这样的等同实施方式包括在本发明范围内。在本申请中引用的所有文献，包括公开出版物、专利和专利申请都通过引用的方式全文并入。The following description of the application is merely to illustrate various embodiments of the application. Therefore, the specific ways of modification discussed here should not be construed as limiting the scope of the application. Those skilled in the art can easily find various equivalents, changes and modifications without departing from the scope of the present application, and it should be understood that such equivalent embodiments are included in the scope of the present invention. All documents, including publications, patents, and patent applications, cited in this application are hereby incorporated by reference in their entirety.
定义definition
本发明中的“抗体”一词包括任意可结合某特定抗原的免疫球蛋白、单克隆抗体、多克隆抗体、多特异性抗体或双特异性(双价)抗体。一个天然的完整抗体包含两条重链和两条轻链。每条重链由一可变区和第一、第二、第三恒定区组成；每条轻链由一可变区和一恒定区组成。哺乳动物的重链可分为α、δ、ε、γ和μ，哺乳动物的轻链可分为λ或κ。抗体呈“Y”型，“Y”型结构的颈部由两条重链的第二和第三恒定区组成，其通过二硫键结合。“Y”型结构的每条臂包括其中一条重链的可变区和第一恒定区，其与一条轻链的可变区和恒定区结合。轻链和重链的可变区决定抗原的结合。每条链的可变区均含有三个高变区，称互补决定区(CDR)(轻链(L)的CDR包含LCDR1、LCDR2、LCDR3，重链(H)的CDR包含HCDR1、HCDR2、HCDR3)。本发明中公开的抗体和抗原结合片段的CDR边界可通过Kabat，Chothia或Al-Lazikani命名法命名或识别。(Al-Lazikani,B.,Chothia,C.,Lesk,A.M.,J.Mol.Biol.,273(4),927(1997)；Chothia,C.等，J Mol Biol.Dec 5；186(3):651-63(1985)；Chothia,C.and Lesk,A.M.,J.Mol.Biol.,196,901(1987)；Chothia,C.等，Nature.Dec 21-28；342(6252):877-83(1989)；Kabat E.A.等，National Institutes of Health,Bethesda,Md.(1991))。其中，三个CDR由被称为框架区(FR)的侧面连续部分间隔开，框架区比CDR更加高度保守并形成一个支架支撑超变环。重链和轻链的恒定区与抗原结合无关，但具有多种效应功能。抗体依据重链恒定区的氨基酸序列可以分成几类。根据是否含有α、δ、ε、γ和μ重链，抗体可分别分为五个主要的分类或异构体：IgA、IgD、IgE、IgG和IgM。几个主要的抗体分类还可分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)等。The term "antibody" in the present invention includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds to a specific antigen. A natural intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions; each light chain consists of a variable region and a constant region. Mammalian heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian light chains can be classified as λ or κ. Antibodies are in the shape of a "Y", with the neck of the "Y" structure consisting of the second and third constant domains of the two heavy chains, joined by disulfide bonds. Each arm of the "Y" structure includes the variable region and the first constant region of one of the heavy chains in combination with the variable and constant regions of one of the light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3 ). The CDR boundaries of the antibodies and antigen-binding fragments disclosed in the present invention can be named or identified by Kabat, Chothia or Al-Lazikani nomenclature. (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5; 186 (3 ):651-63(1985); Chothia, C.and Lesk, A.M., J.Mol.Biol., 196,901(1987); Chothia, C. et al., Nature.Dec 21-28; 342(6252):877- 83 (1989); Kabat E.A. et al., National Institutes of Health, Bethesda, Md. (1991)). Of these, the three CDRs are separated by lateral contiguous portions called framework regions (FR), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but have various effector functions. Antibodies can be classified into several classes based on the amino acid sequence of the heavy chain constant region. Antibodies can be divided into five major classes or isoforms based on whether they contain alpha, delta, epsilon, gamma, and mu heavy chains: IgA, IgD, IgE, IgG, and IgM, respectively. Several major antibody classes can be further divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain) and so on.
本申请中的“抗原结合片段”一词，指由含有一个或多个CDR的抗体部分或者任何其他结合抗原但不具有完整抗体结构的抗体片段所形成的一种抗体片段。抗原结合片段的例子包括，但不限于，如双功能抗体(diabody)、Fab、Fab'、F(ab')2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双功能抗体(ds diabody)、单链抗体分子(scFv)、scFv二聚体(双价的双功能抗体)、双价单链抗体(BsFv)、多特异性抗体、骆驼化单域抗体(camelized single domain antibody)、纳米抗体、域抗体和双价域抗体。抗原结合片段可以与母体抗体结合相同的抗原。在某些实施方式中，抗原结合片段可以含有来自某特定人抗体的一个或多个CDR，移接至来自一个或多个不同人抗体的框架区。The term "antigen-binding fragment" in this application refers to an antibody fragment formed from an antibody portion containing one or more CDRs or any other antibody fragment that binds to an antigen but does not have a complete antibody structure. Examples of antigen-binding fragments include, but are not limited to, such as diabodies, Fab, Fab', F(ab')2 , Fv fragments, disulfide bond-stabilized Fv fragments (dsFv), (dsFv)2 , Bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), bivalent single chain antibody (BsFv), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies and bivalent domain antibodies. Antigen-binding fragments can bind the same antigen as the parent antibody. In certain embodiments, an antigen-binding fragment may contain one or more CDRs from a particular human antibody grafted into framework regions from one or more different human antibodies.
抗体的“Fab”片段是指由一条轻链(包括可变区和恒定区)和一条重链的可变区和恒定区经二硫键结合起来的那部分抗体分子。The "Fab" fragment of an antibody refers to that part of an antibody molecule that is disulfide-bonded by a light chain (including variable and constant regions) and a heavy chain's variable and constant regions.
“Fab'”片段是指包含了部分铰链区的Fab片段。A "Fab'" fragment refers to a Fab fragment that includes part of the hinge region.
“F(ab')2”指的是Fab的二聚体。"F(ab')2 " refers to a dimer of Fab.
抗体的“Fc”指的是由重链的第二、第三恒定区经二硫键结合组成的那部分抗体。抗体的Fc段负责多种不同的效应功能如ADCC和CDC，但不参与抗原的结合。The "Fc" of an antibody refers to that part of the antibody that is composed of the second and third constant domains of the heavy chain through disulfide bonds. The Fc portion of an antibody is responsible for various effector functions such as ADCC and CDC, but is not involved in antigen binding.
抗体的“Fv”段指的是含有完整抗原结合位点的最小抗体片段。Fv片段由一条轻链的可变区和一条重链的可变区组成。The "Fv" fragment of an antibody refers to the smallest fragment of an antibody that contains the complete antigen binding site. The Fv fragment consists of the variable region of one light chain and the variable region of one heavy chain.
“单链Fv抗体”或“scFv”是指由轻链可变区与重链可变区直接相连或通过一个肽链连接而成的工程抗体(Huston JS等，Proc Natl Acad Sci USA、85:5879(1988))。"Single-chain Fv antibody" or "scFv" refers to an engineered antibody in which the variable region of the light chain is linked directly to the variable region of the heavy chain or through a peptide chain (Huston JS et al., Proc Natl Acad Sci USA, 85: 5879 (1988)).
“单链抗体Fv-Fc”或“scFv-Fc”是指由连接到某抗体Fc段的scFv组成的工程抗体。"Single-chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody consisting of scFv linked to the Fc segment of an antibody.
“骆驼化单域抗体(Camelized single domain antibody)”、“重链抗体”或“HCAb(Heavy-chain-only antibodies，HCAb)”都是指含有两个VH域而不含有轻链的抗体(Riechmann L.和Muyldermans S.,J Immunol Methods.Dec 10；231(1-2):25-38(1999)；Muyldermans S.,J Biotechnol.Jun；74(4):277-302(2001)；WO94/04678；WO94/25591；U.S.Patent No.6,005,079)。重链抗体最初从驼科(骆驼、单峰驼和美洲驼)衍生得到。虽然缺失轻链，骆驼化抗体(camelized antibodies)有确证的抗原结合全部功能(Hamers-Casterman C.等，Nature.Jun 3；363(6428):446-8(1993)；Nguyen VK.等，“Heavy-chainantibodies in Camelidae；a case of evolutionary innovation,”Immunogenetics.Apr；54(1):39-47(2002)；Nguyen VK.等，Immunology.May；109(1):93-101(2003))。重链抗体的可变区(VHH域)是最小的已知的获得性免疫产生的抗原结合单位(Koch-Nolte F.等，FASEB J.Nov；21(13):3490-8.Epub 2007Jun 15(2007))。"Camelized single domain antibody (Camelized single domain antibody)", "heavy chain antibody" or "HCAb (Heavy-chain-only antibodies, HCAb)" all refer to antibodies containing twoVH domains without light chains ( Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38(1999); Muyldermans S., J Biotechnol. Jun; 74(4):277-302(2001); WO94/04678; WO94/25591; US Patent No. 6,005,079). Heavy chain antibodies were originally derived from camelids (camels, dromedaries and llamas). Although lacking light chains, camelized antibodies (camelized antibodies) have confirmed full function of antigen binding (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428):446-8 (1993); Nguyen VK. et al., " Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,"Immunogenetics.Apr;54(1):39-47(2002); Nguyen VK. et al., Immunology.May; 109(1):93-101(2003)) . The variable region (VHH domain) of a heavy chain antibody is the smallest known antigen-binding unit produced by adaptive immunity (Koch-Nolte F. et al., FASEB J. Nov; 21(13):3490-8. Epub 2007 Jun 15 (2007)).
“纳米抗体”是指一种抗体片段，其由一个来自重链抗体的VHH域和两个恒定区CH2和CH3组成。"Nanobody" refers to an antibody fragment consisting of a VHH domain from a heavy chain antibody and two constant regions CH2 and CH3.
“双功能抗体(diabody)”包括带有两个抗原结合位点的小抗体片段，其中该片段含有在同一条多肽链上相连的VH域和VL域(VH-VL或VH-VL)(请参见，Holliger P.等，Proc NatlAcad Sci U S A.Jul 15；90(14):6444-8(1993)；EP404097；WO93/11161)。两个域之间衔接物很短，使同一条链上的两个域不能互相配对，从而迫使两个域与另一条链的互补域配对，形成两个抗体结合位点。这两个抗体结合位点可靶向结合相同或不同的抗原(或抗原表位)。A "diabody" includes a small antibody fragment with two antigen-binding sites, wherein the fragment contains aVH domain and aVL domain (VH -VL orVH domain) connected on the same polypeptide chain. -VL ) (see, Holliger P. et al., Proc Natl Acad Sci US A. Jul 15; 90(14):6444-8 (1993); EP404097; WO93/11161). The linker between the two domains is so short that the two domains on the same chain cannot pair with each other, thus forcing the two domains to pair with the complementary domains of another chain, forming two antibody binding sites. The two antibody binding sites can be targeted to bind the same or different antigens (or antigenic epitopes).
“域抗体”是指仅含有一条重链可变区或一条轻链可变区的抗体片段。在某些情况下，两个或多个VH域由一个多肽衔接物共价结合并形成双价域抗体。双价域抗体的两个VH域可靶向作用于相同或不同的抗原。"Domain antibody" refers to an antibody fragment that contains only one heavy chain variable region or one light chain variable region. In certain instances, two or moreVH domains are covalently joined by a polypeptide linker and form a bivalent domain antibody. The twoVH domains of bivalent domain antibodies can target the same or different antigens.
在某些实施方式中，“(dsFv)2”含有三条肽链：两个VH基团间通过一条多肽衔接物相连，并通过二硫键与两个VL基团结合。In certain embodiments, "(dsFv)2 " contains three peptide chains: twoVH groups connected by a polypeptide linker, and twoVL groups connected by a disulfide bond.
在某些实施方式中，“双特异性ds双功能抗体”含有VL1-VH2(由一个多肽衔接物相连)和VH1-VL2(也是由一个多肽衔接物相连)，两者在VH1和VL1间通过二硫键结合。In certain embodiments, a "bispecific ds diabody" contains VL1 -VH2 (linked by a polypeptide linker) and VH1 -VL2 (also linked by a polypeptide linker), both of which are at VH1 and VL1 are combined by a disulfide bond.
“双特异性dsFv”或“dsFv-dsFv”含有三条多肽链：VH1-VH2基团，其中两者的重链通过多肽衔接物(如：长的弹性衔接物)相连，并通过二硫键分别与VL1和VL2基团结合，每对通过二硫键配对的重链轻链具有不同的抗原特异性。"Bispecific dsFv" or "dsFv-dsFv" contains three polypeptide chains: VH1 -VH2 groups, in which the heavy chains of the two are connected by a polypeptide linker (such as: a long elastic linker) and connected by a disulfide linker. The bonds are combined with the VL1 and VL2 groups, respectively, and each pair of heavy and light chains paired by disulfide bonds has a different antigen specificity.
在某些实施方式中，“scFv二聚体”是双价双功能抗体或双价单链抗体(BsFv)，含有二聚化的两个VH-VL(由多肽衔接物连接)基团，其中一个基团的VH与另一个基团的VL协作形成两个结合位点，这两个结合位点可靶向结合相同抗原(或抗原表位)或不同抗原(或抗原表位)。在另一些实施方式中，“scFv二聚体”是双特异性双功能抗体，含有相互连接的VL1-VH2(由多肽衔接物连接)和VH1-VL2(由多肽衔接物连接)，其中VH1和VL1协作，VH2和VL2协作，且每个协作的配对具有不同的抗原特异性。In certain embodiments, a "scFv dimer" is a bivalent diabody or a bivalent single chain antibody (BsFv) containing twoVH -VL (linked by a polypeptide linker) groups that are dimerized , where the VH of one group cooperates with the VL of the other to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes) ). In other embodiments, "scFv dimers" are bispecific, bifunctional antibodies comprising interconnected VL1 -VH2 (linked by a polypeptide linker) and VH1 -VL2 (linked by a polypeptide linker) , where VH1 and VL1 cooperate, VH2 and VL2 cooperate, and each cooperative pair has a different antigen specificity.
本申请中使用的术语“全人源”当用于抗体或抗原结合片段时，是指所述抗体或抗原结合片段具有某氨基酸序列或由所述氨基酸序列组成，所述氨基酸序列对应于由人或人免疫细胞生产的、或从例如利用人源抗体库的转基因非人动物等非人来源衍生的抗体的氨基酸序列，或者其他编码人源抗体的序列。在某些实施方式中，全人源抗体不包含来源于非人抗体的氨基酸残基(特别是抗原结合残基)。The term "fully human" used in this application, when used for an antibody or an antigen-binding fragment, means that the antibody or antigen-binding fragment has or consists of an amino acid sequence corresponding to the Or the amino acid sequence of antibodies produced by human immune cells, or derived from non-human sources such as transgenic non-human animals using human antibody libraries, or other sequences encoding human antibodies. In certain embodiments, fully human antibodies do not comprise amino acid residues (particularly antigen binding residues) derived from non-human antibodies.
本申请中使用的术语“人源化”当用于抗体或抗原结合片段时，是指包括来源于非人动物的CDR、来源于人的FR区，以及来源于人的恒定区(当适用时)的抗体或抗原结合片段。由于人源化的抗体或抗原结合片段具有降低的免疫原性，其在某些实施方式中可用作人的治疗剂。在一些实施方式中，所述非人动物是哺乳动物例如小鼠、大鼠、兔、山羊、绵羊、豚鼠或仓鼠。在一些实施方式中，所述人源化抗体或抗原结合片段除了CDR序列是非人源的以外，基本上全部由人源序列组成。在一些实施方式中，所述来源于人的FR区可以包括与其来自的人源抗体相同的氨基酸序列，或其可以包括一些氨基酸改变，例如，不超过10、9、8、7、6、5、4、3、2或1个氨基酸改变。在一些实施方式中，该氨基酸改变可以仅存在于重链FR区、仅存在于轻链FR区或同时存在于两个链中。在一些优选实施方式中，所述人源化抗体包括人源FR1-3和人源JH和Jκ。The term "humanized" as used in this application, when applied to antibodies or antigen-binding fragments, refers to the inclusion of CDRs derived from non-human animals, FR regions derived from humans, and constant regions (when applicable) derived from humans. ) antibody or antigen-binding fragment. Due to their reduced immunogenicity, humanized antibodies or antigen-binding fragments are useful as therapeutic agents in humans in certain embodiments. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen-binding fragment consists essentially entirely of human sequences, except that the CDR sequences are non-human. In some embodiments, the human-derived FR region may comprise the same amino acid sequence as the human antibody from which it was derived, or it may comprise some amino acid changes, e.g., no more than 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid changes. In some embodiments, the amino acid change may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains. In some preferred embodiments, the humanized antibody comprises human FR1-3 and human JH and Jκ.
本申请中使用的术语“嵌合”是指具有来源于一种物种的重链和/或轻链的一部分，和所述重链和/或轻链其余部分来源于不同物种的抗体或抗原结合片段。在一个示例性的例子中，嵌合抗体可以包括来源于人的恒定区和来源于非人动物例如小鼠的可变区。The term "chimeric" as used in this application refers to an antibody or antigen combination having a portion of a heavy chain and/or light chain derived from one species, and the rest of the heavy chain and/or light chain derived from a different species fragment. In one illustrative example, a chimeric antibody can include constant regions derived from a human and variable regions derived from a non-human animal such as a mouse.
本申请使用的“PD-1”是指程序性细胞死亡蛋白，其属于免疫球蛋白超家族并作为对免疫系统负性调节的共抑制抗体发挥作用。PD-1是CD28/CTLA-4家族的成员，具有两个抑制的配体，包括PD-L1和PD-L2。人源PD-1代表性的氨基酸序列由NCBI登记号NP_005009.2公开，且编码所述人源PD-1的代表性核酸序列由由NCBI登记号NP_005018.2公开。"PD-1" as used herein refers to a programmed cell death protein, which belongs to the immunoglobulin superfamily and functions as a co-inhibitory antibody to the negative regulation of the immune system. PD-1 is a member of the CD28/CTLA-4 family and has two inhibitory ligands, including PD-L1 and PD-L2. The representative amino acid sequence of human PD-1 is disclosed by NCBI Accession No. NP_005009.2, and the representative nucleic acid sequence encoding said human PD-1 is disclosed by NCBI Accession No. NP_005018.2.
本申请中使用的“PD-L1”是指程序性细胞死亡配体1(PD-L1,参见例如Freeman et al.(2000)J.Exp.Med.192:1027)。代表性的人源PD-L1的氨基酸序列为NCBI登记号:NP_054862.1，且代表性的人源PD-L1的核酸序列为NCBI登记号:NM_014143.3。PD-L1表达于胎盘，脾，淋巴结，胸腺，心脏，胎儿肝脏，并且还发现存在于许多肿瘤或癌细胞上。PD-L1与在活化的T细胞、B细胞和骨髓细胞上表达的受体PD-1或B7-1结合。PD-L1与其受体的结合可引发信号转导来抑制TCR介导的对细胞因子生产的激活和T细胞增殖。因此，PD-L1在特定事件中，例如在怀孕、自身免疫性疾病、组织移植中对抑制免疫系统起着主要作用，并且其被认为允许肿瘤或癌细胞规避免疫检查点并逃避免疫应答。"PD-L1" as used herein refers to programmed cell death ligand 1 (PD-L1, see eg Freeman et al. (2000) J. Exp. Med. 192:1027). The amino acid sequence of a representative human PD-L1 is NCBI accession number: NP_054862.1, and the nucleic acid sequence of a representative human PD-L1 is NCBI accession number: NM_014143.3. PD-L1 is expressed in the placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells. PD-L1 binds to the receptors PD-1 or B7-1 expressed on activated T cells, B cells and myeloid cells. Binding of PD-L1 to its receptor triggers signal transduction to inhibit TCR-mediated activation of cytokine production and T cell proliferation. Thus, PD-L1 plays a major role in suppressing the immune system in specific events, such as pregnancy, autoimmune diseases, tissue transplantation, and it is thought to allow tumor or cancer cells to circumvent immune checkpoints and escape immune responses.
本申请中使用的“抗-PD-1抗体”是指能够以足以提供诊断和/或治疗用途的亲和性与PD-1(例如人或猴PD-1)特异性结合的抗体。An "anti-PD-1 antibody" as used herein refers to an antibody capable of specifically binding to PD-1 (eg, human or monkey PD-1) with an affinity sufficient to provide diagnostic and/or therapeutic use.
本申请中的“特异性结合”或“特异性的结合”是指，指两分子间的非随机结合反应，如抗体和抗原间的反应。在某些实施方式中，本申请的抗体或其抗原结合片段与人和/或猴PD-1特异性结合，并且其结合亲和力(KD)≤10-6M(如：≤5x10-7M，≤2x10-7M，≤10-7M，≤5x10-8M，≤2x10-8M，≤10-8M，≤5x10-9M，≤2x10-9M，≤10-9M，≤10-10M)。本申请中的KD是指解离速度与结合速度的比值(koff/kon)，可通过表面等离子共振的方法测定，例如使用如Biacore的仪器。"Specific binding" or "specific binding" in this application refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen. In certain embodiments, the antibody or antigen-binding fragment thereof of the present application specifically binds to human and/or monkey PD-1, and its binding affinity (KD ) is ≤10-6 M (e.g.: ≤5x10-7 M , ≤2x10-7 M, ≤10-7 M, ≤5x10-8 M, ≤2x10-8 M, ≤10-8 M, ≤5x10-9 M, ≤2x10-9 M, ≤10-9 M, ≤10-10M ). KD in the present application refers to the ratio of dissociation velocity to association velocity (koff/kon), which can be determined by surface plasmon resonance, for example, using an instrument such as Biacore.
本申请中的“阻断结合”或“竞争性同样的表位”的能力是指抗体或其抗原结合片段将两个分子间结合(例如人PD-1和抗-PD-1抗体)的相互作用抑制到任何可检测的程度的能力。在某些实施方式中，阻断两个分子间结合的抗体或抗原结合片段可将两个分子间结合的相互作用抑制至少50％。在某些实施方式中，这样的抑制作用可以大于60％，大于70％，大于80％，或大于90％。The ability of "blocking binding" or "competing for the same epitope" in this application refers to the ability of an antibody or its antigen-binding fragment to bind two molecules (such as human PD-1 and anti-PD-1 antibody) to each other. The ability to inhibit the effect to any detectable extent. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In certain embodiments, such inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.
本申请中使用的“表位”是指抗原分子中与抗体结合的那部分氨基酸或原子基团。如果两种抗体表现出对抗原的竞争性结合，则可能结合抗原上的相同表位。例如，如果本申请提供的抗体或其抗原结合片段阻断示例抗体，例如1.7.3hAb、1.49.9hAb、1.103.11hAb、1.103.11-v2hAb、1.139.15hAb和1.153.7hAb与人PD-1的结合，那么所述抗体或其抗原结合片段可以被认为与那些示例的抗体结合相同的表位。The "epitope" used in this application refers to the amino acid or atomic group in the antigen molecule that binds to the antibody. If two antibodies exhibit competitive binding to the antigen, they are likely to bind the same epitope on the antigen. For example, if the antibodies or antigen-binding fragments thereof provided herein block the interaction of exemplary antibodies, such as 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb and 1.153.7hAb with human PD-1 binding, then the antibody or antigen-binding fragment thereof can be considered to bind the same epitope as those exemplified antibodies.
将在表位中特定的氨基酸残基通过例如丙氨酸扫描突变(alanine scanningmutagenesis)而进行突变，鉴定了降低或阻止蛋白结合的突变。“丙氨酸扫描突变”是可以用于鉴定影响表位与其他与其结合的化合物或蛋白质相互作用的蛋白质的某些残基或区域的方法。将蛋白质中的残基或一组目标残基通过中性或负性电荷氨基酸取代(最优选丙氨酸或多聚丙氨酸，或保守的氨基酸取代)。任何降低所述蛋白质的结合的氨基酸残基突变或编码其的密码子突变的程度超过阈值或与其他突变相比最大化降低所述蛋白质结合的突变都有可能在所述蛋白结合的表位中。在本申请的某些实施方式中，对PD-1抗体重要的表位包括以下氨基酸残基中的至少一个：V64、P83、D85、L128、A129、P130、K131、A132和Q133。Mutation of specific amino acid residues in the epitope by, for example, alanine scanning mutagenesis identifies mutations that reduce or prevent protein binding. "Alanine scanning mutagenesis" is a method that can be used to identify certain residues or regions of a protein that affect the interaction of an epitope with other compounds or proteins that bind it. A residue or a group of residues of interest in a protein is substituted with a neutral or negatively charged amino acid (most preferably alanine or polyalanine, or a conservative amino acid substitution). Any amino acid residue mutation that reduces binding of the protein or mutation of the codons encoding it to a degree that exceeds a threshold or that maximizes the reduction in binding of the protein compared to other mutations is likely to be in an epitope that the protein binds . In certain embodiments of the present application, the epitope important to the PD-1 antibody includes at least one of the following amino acid residues: V64, P83, D85, L128, A129, P130, K131, A132 and Q133.
本申请所述“1.7.3hAb”是指具有如SEQ ID NO：45所示的重链可变区、如SEQ ID NO：47所示的轻链可变区和人源IgG4同种型恒定区的全人源单克隆抗体。The "1.7.3hAb" described in this application refers to a heavy chain variable region as shown in SEQ ID NO: 45, a light chain variable region as shown in SEQ ID NO: 47, and a human IgG4 isotype constant region fully human monoclonal antibody.
本申请中使用的“1.49.9hAb”是指具有如SEQ ID NO:49所示的重链可变区、如SEQ IDNO:51所示的轻链可变区和人源IgG4同种型恒定区的全人源单克隆抗体。The "1.49.9hAb" used in this application refers to a heavy chain variable region as shown in SEQ ID NO:49, a light chain variable region as shown in SEQ ID NO:51 and a human IgG4 isotype constant region fully human monoclonal antibody.
本申请中使用的“1.103.11hAb”是指具有如SEQ ID NO:53所示的重链可变区、如SEQID NO:55所示的轻链可变区和人源IgG4同种型恒定区的全人源单克隆抗体。The "1.103.11hAb" used in this application refers to a heavy chain variable region as shown in SEQ ID NO:53, a light chain variable region as shown in SEQ ID NO:55 and a human IgG4 isotype constant region fully human monoclonal antibody.
本申请中使用的“1.103.11_v2hAb”是指具有如SEQ ID NO:53所示的重链可变区、如SEQ ID NO:67所示的轻链可变区和人源IgG4同种型恒定区的全人源单克隆抗体。本申请中使用的“1.139.15hAb”是指具有如SEQ ID NO:57所示的重链可变区、如SEQ ID NO:59所示的轻链可变区和人源IgG4同种型恒定区的全人源单克隆抗体。"1.103.11_v2hAb" as used in the present application refers to an antibody having a heavy chain variable region as shown in SEQ ID NO:53, a light chain variable region as shown in SEQ ID NO:67, and a human IgG4 isotype constant Fully human monoclonal antibodies to the region. "1.139.15hAb" as used in this application refers to an antibody having a heavy chain variable region as shown in SEQ ID NO:57, a light chain variable region as shown in SEQ ID NO:59, and a human IgG4 isotype constant Fully human monoclonal antibodies to the region.
本申请中使用的“1.153.7hAb”是指具有如SEQ ID NO:61所示的重链可变区、如SEQ IDNO:63所示的轻链可变区和人源IgG4同种型恒定区的全人源单克隆抗体。The "1.153.7hAb" used in this application refers to a heavy chain variable region as shown in SEQ ID NO:61, a light chain variable region as shown in SEQ ID NO:63 and a human IgG4 isotype constant region fully human monoclonal antibody.
在本申请中当“保守替代”用于氨基酸序列时，是指将一个氨基酸残基用另一个具有相似理化性质的侧链的氨基酸残基替代。例如，可以在疏水侧链氨基酸残基间(例如Met、Ala、Val、Leu和Ile)、中性亲水侧链残基间(例如Cys、Ser、Thr、Asn和Gln)、酸性侧链残基间(例如Asp、Glu)、碱性侧链氨基酸间(例如His、Lys和Arg)或方向侧链残基间(例如Trp、Tyr和Phe)进行保守替代。本领域已知保守替代通常不会引起蛋白构象结构的显著变化，因此能够保留蛋白质的生物活性。In this application, when "conservative substitution" is used for an amino acid sequence, it refers to replacing one amino acid residue with another amino acid residue with a side chain having similar physicochemical properties. For example, between hydrophobic side chain amino acid residues (such as Met, Ala, Val, Leu and Ile), between neutral hydrophilic side chain residues (such as Cys, Ser, Thr, Asn and Gln), acidic side chain residues Conservative substitutions are made between bases (such as Asp, Glu), between basic side chain amino acids (such as His, Lys and Arg) or between directional side chain residues (such as Trp, Tyr and Phe). It is known in the art that conservative substitutions usually do not cause significant changes in the conformational structure of the protein, and thus can retain the biological activity of the protein.
当“百分比序列同一性”用于氨基酸序列(或核酸序列)时，是指在进行序列比对，并且必要时引入间隔使相同氨基酸(或核酸)数目达到最多后，在候选序列中，与参比序列相同的氨基酸(或核酸)残基占所述候选序列的氨基酸(或核酸)残基的百分比。所述氨基酸残基的保守替代可以认为或可以不认为是相同残基。可以通过本领域公开的工具，例如BLASTN,BLASTp(美国国家生物技术信息中心网站(NCBI)，也可参见，Altschul S.F.等、J.Mol.Biol.，215:403–410(1990)；Stephen F.等，Nucleic Acids Res.，25:3389–3402(1997))、ClustalW2(欧洲生物信息研究所网站，可参见，Higgins D.G.等，Methods inEnzymology，266:383-402(1996)；Larkin M.A.等，Bioinformatics(Oxford、England)，23(21):2947-8(2007))和ALIGN或Megalign(DNASTAR)软件，对序列进行比对以确定氨基酸(或核酸)序列的百分比序列同一性。本领域技术人员可以使用所述工具的默认参数或根据比对的需要适当调整参数，例如通过挑选合适的算法。When "percentage sequence identity" is used for an amino acid sequence (or nucleic acid sequence), it means that, in the candidate sequence, the number of identical The percentage of amino acid (or nucleic acid) residues identical to the sequence to the amino acid (or nucleic acid) residues of the candidate sequence. Conservative substitutions of such amino acid residues may or may not be considered identical residues. can be obtained by tools published in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information website (NCBI), see also, Altschul S.F. et al., J. Mol. Biol., 215:403-410 (1990); Stephen F etc., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute website, see, Higgins D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software, the sequences are aligned to determine the percent sequence identity of the amino acid (or nucleic acid) sequences. Those skilled in the art can use the default parameters of the tool or adjust the parameters appropriately according to the needs of alignment, for example, by selecting a suitable algorithm.
本申请中使用的“T细胞”包括CD4+T细胞、CD8+T细胞、T辅助1型T细胞、T辅助2型T细胞、T辅助17型T细胞和抑制性T细胞。"T cells" as used in the present application include CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells and suppressor T cells.
本申请中使用的“效应功能”是指抗体的Fc区与其效应器例如C1复合物和Fc受体结合的生物活性。示例性的效应功能包括抗体与C1复合物上的C1q相互作用诱导的补体依赖性细胞毒性(CDC)、抗体的Fc区与效应细胞上的Fc受体结合诱导的抗体依赖性细胞介导的细胞毒性(ADCC)以及吞噬。"Effector function" as used herein refers to the biological activity of the Fc region of an antibody in binding to its effectors such as the Cl complex and Fc receptors. Exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by antibody interaction with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity induced by binding of the Fc region of the antibody to Fc receptors on effector cells Toxicity (ADCC) and phagocytosis.
本申请中的“癌症”或“癌状况”是指任何由肿瘤或恶性细胞生长、增殖或转移所介导，并引发实体瘤和非实体瘤如白血病的医学状况。本发明中的“肿瘤”是指肿瘤和/或恶性细胞的实体物质。"Cancer" or "cancerous condition" in this application refers to any medical condition mediated by tumor or malignant cell growth, proliferation or metastasis, and giving rise to solid tumors and non-solid tumors such as leukemia. "Tumor" in the present invention refers to the solid substance of tumor and/or malignant cells.
对某种状况的“治疗”或“疗法”包括预防或减轻某种状况，降低某种状况兴起或发展的速度，减少发展出某种状况的风险，预防或延迟与某种状况相关的症状发展，减少或终止与某种状况相关的症状，产生某种状况的完全或部分的逆转，治愈某种状况，或以上的组合。对于癌症来说，“治疗”或“疗法”可以指抑制或减缓肿瘤或恶性细胞生长，繁殖，或转移，或以上的某些组合。对于肿瘤来说，“治疗”或“疗法”包括清除全部或部分的肿瘤，抑制或减缓肿瘤生长和转移，预防或延缓肿瘤的发展，或以上的某些组合。"Treatment" or "therapy" for a condition includes preventing or alleviating a condition, reducing the rate at which a condition arises or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition , to reduce or cease symptoms associated with a condition, to produce complete or partial reversal of a condition, to cure a condition, or a combination of the above. With respect to cancer, "treating" or "therapy" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumor or malignant cells, or some combination thereof. With respect to tumors, "treatment" or "therapy" includes eradicating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor progression, or some combination of the above.
“被分离”的物质已经经人工由自然状态改变。如果自然界中出现某种“被分离”的物质或成分，那么其已经被改变或脱离其原始状态，或二者均有发生。例如，某一活体动物体内天然存在的多核苷酸或多肽是未被分离的，但如果这些多核苷酸或多肽与之在天然状态下共存的物质足够分离并以足够纯的状态存在，则可以认为是“被分离”。在某些实施方式中，抗体和抗原结合片段的纯度为至少90％、93％、95％、96％、97％、98％、99％，其由电泳方法(如SDS-PAGE、等电聚焦、毛细管电泳)，或色谱法(如离子交换色谱或反相HPLC)确定。"Isolated" is a substance that has been artificially altered from its natural state. If an "isolated" substance or component occurs in nature, it has been altered or removed from its original state, or both. For example, polynucleotides or polypeptides naturally occurring in a living animal are not isolated, but if these polynucleotides or polypeptides are sufficiently separated from the substances that coexist in the natural state and exist in a sufficiently pure state, they can be Considered to be "separated". In certain embodiments, antibodies and antigen-binding fragments are at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, , capillary electrophoresis), or chromatographic methods (such as ion-exchange chromatography or reversed-phase HPLC).
本发明中“载体”是指，可将编码某蛋白的多核苷酸操作性地插入其中并使该蛋白获得表达的一种运载工具。载体可用于转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。举例来说，载体包括：质粒、噬菌粒、柯斯质粒、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1衍生的人工染色体(PAC)、噬菌体如λ噬菌体或M13噬菌体，以及动物病毒等。用作载体的动物病毒种类有逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。载体可含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还可包括协助其进入细胞的成分，包括但不限于，病毒颗粒、脂质体或蛋白外壳。"Vector" in the present invention refers to a delivery tool into which a polynucleotide encoding a certain protein can be operatively inserted and the protein can be expressed. Vectors can be used to transform, transduce or transfect host cells, so that the genetic material elements carried by them can be expressed in the host cells. Vectors include, for example: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), phage such as lambda phage or M13 phage , and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. A vector may also include components to facilitate its entry into cells, including, but not limited to, viral particles, liposomes, or protein coats.
本发明中“宿主细胞”是指导入外源多核苷酸和/或载体的细胞。"Host cell" in the present invention refers to a cell into which exogenous polynucleotides and/or vectors have been introduced.
本发明中的“与PD-1相关或有关联的疾病”是指，任何由于PD-1(如：人PD-1)表达或活性升高或降低而导致、加剧或其他相关的状况。"PD-1-related or associated disease" in the present invention refers to any condition caused, exacerbated or otherwise related to the increase or decrease in the expression or activity of PD-1 (eg, human PD-1).
本发明中的“治疗有效量”或“有效剂量”是指，某种药物有效治疗与人PD-1相关疾病或状况的剂量或浓度。例如，对于本发明中公开的抗体或其抗原结合片段的用途来说，治疗有效量是在该剂量或浓度下，该抗体或抗原结合物可以清除全部或部分肿瘤、抑制或减缓肿瘤生长、抑制介导癌状况的细胞的生长或繁殖、抑制肿瘤细胞转移、减轻任何与肿瘤或癌状况相关的症状或标记，预防或延缓肿瘤或癌状况的发展，或以上的某些组合。"Therapeutically effective amount" or "effective dose" in the present invention refers to the dose or concentration of a drug that effectively treats a disease or condition related to human PD-1. For example, for the use of the antibody or antigen-binding fragment thereof disclosed in the present invention, the therapeutically effective amount is at the dosage or concentration, the antibody or antigen-binding compound can eliminate all or part of the tumor, inhibit or slow down tumor growth, inhibit Mediating the growth or proliferation of cells of a cancerous condition, inhibiting tumor cell metastasis, alleviating any symptoms or markers associated with a tumor or cancerous condition, preventing or delaying the development of a tumor or cancerous condition, or some combination of the above.
“药用可接受的”是指所指的载剂、溶媒、稀释剂、辅料和/或盐，总的来说在化学上和/或在物理上与制剂中的其他配料相兼容，并在生理上与接受者相兼容。"Pharmaceutically acceptable" means that the carrier, vehicle, diluent, excipient and/or salt in general is chemically and/or physically compatible with the other ingredients in the Physiologically compatible with the recipient.
抗-PD-1抗体anti-PD-1 antibody
在一个方面，本发明提供了抗-PD-1抗体和其抗原结合片段。PD-1，也称为CD279，是已知的由活化T细胞表达的关键免疫检查点受体，其调节免疫抑制作用。PD-1配体1(PD-L1)是表达在多种肿瘤细胞、基质细胞或两者上的40kDa的跨膜蛋白，其与PD-1结合。抑制PD-1和PD-L1间的相互作用能够提高T细胞应答由此介导抗癌活性。In one aspect, the invention provides anti-PD-1 antibodies and antigen-binding fragments thereof. PD-1, also known as CD279, is a key immune checkpoint receptor known to be expressed by activated T cells, which regulates immune suppression. PD-1 ligand 1 (PD-L1) is a 40 kDa transmembrane protein expressed on a variety of tumor cells, stromal cells, or both, which binds to PD-1. Inhibition of the interaction between PD-1 and PD-L1 can enhance T cell responses and thereby mediate anticancer activity.
在某些实施方式中，本申请提供了示例性的全人源单克隆抗体1.7.3hAb、1.49.9hAb、1.103.11hAb、1.103.11-v2hAb、1.139.15hAb和1.153.7hAb，其CDR序列如表1中所示，并且重链或轻链可变区序列也如下列出。In certain embodiments, the application provides exemplary fully human monoclonal antibodies 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb and 1.153.7hAb, the CDR sequences of which are as follows: are shown in Table 1, and the heavy or light chain variable region sequences are also listed below.
表1Table 1
1.7.3hAb-VH(23466-VH):(SEQ ID NO:45为氨基酸，SEQ ID NO:46为核酸)重链CDR1-3:SEQ ID NO:1、3、5为氨基酸序列和SEQ ID NO:2、4、6为核酸序列。1.7.3hAb-VH (23466-VH): (SEQ ID NO: 45 is amino acid, SEQ ID NO: 46 is nucleic acid) heavy chain CDR1-3: SEQ ID NO: 1, 3, 5 are amino acid sequence and SEQ ID NO : 2, 4, 6 are nucleic acid sequences.
V区段：IGHV4-39*01V section: IGHV4-39*01
D区段：IGHD1-26*01Section D: IGHD1-26*01
J区段：IGHJ4*02Section J: IGHJ4*02
1.7.3hAb-VL(23195-VL):(SEQ ID NO:47为氨基酸和SEQ ID NO:48为核酸)轻链CDR1-3：SEQ ID NOs:7、9、11为氨基酸序列和SEQ ID NO:8、10、12为核酸序列：1.7.3hAb-VL (23195-VL): (SEQ ID NO: 47 is amino acid and SEQ ID NO: 48 is nucleic acid) light chain CDR1-3: SEQ ID NOs: 7, 9, 11 are amino acid sequence and SEQ ID NO : 8, 10, 12 are nucleic acid sequences:
V区段：IGLV2-14*01V section: IGLV2-14*01
J区段：IGLJ3*02Section J: IGLJ3*02
1.49.9hAb-VH(20951-VH):(SEQ ID NO:49为氨基酸，SEQ ID NO:50为核酸)重链CDR1-3：SEQ ID NOs:13、15、5为氨基酸序列和SEQ ID NO:14、16、6为核酸序列：1.49.9hAb-VH (20951-VH): (SEQ ID NO: 49 is amino acid, SEQ ID NO: 50 is nucleic acid) heavy chain CDR1-3: SEQ ID NOs: 13, 15, 5 are amino acid sequence and SEQ ID NO : 14, 16, 6 are nucleic acid sequences:
V区段：IGHV4-39*01V section: IGHV4-39*01
D区段：IGHD1-26*01Section D: IGHD1-26*01
J区段：IGHJ4*02Section J: IGHJ4*02
1.49.9hAb-VL(21526-VL):(SEQ ID NO:51为氨基酸，SEQ ID NO:52为核酸)轻链CDR1-3：SEQ ID NOs:7、17、11为氨基酸序列和SEQ ID NO:8、18、12为核酸序列：1.49.9hAb-VL (21526-VL): (SEQ ID NO: 51 is amino acid, SEQ ID NO: 52 is nucleic acid) light chain CDR1-3: SEQ ID NOs: 7, 17, 11 are amino acid sequence and SEQ ID NO : 8, 18, 12 are nucleic acid sequences:
V区段：IGLV2-14*01V section: IGLV2-14*01
J区段：IGLJ3*02Section J: IGLJ3*02
1.103.11hAb-VH(20975-VH):(SEQ ID NO:53为氨基酸，SEQ ID NO:54为核酸)重链CDR1-3：SEQ ID NOs:1、15、5为氨基酸序列和SEQ ID NO:2、16、6为核酸序列：1.103.11hAb-VH (20975-VH): (SEQ ID NO: 53 is amino acid, SEQ ID NO: 54 is nucleic acid) heavy chain CDR1-3: SEQ ID NOs: 1, 15, 5 are amino acid sequence and SEQ ID NO : 2, 16, 6 are nucleic acid sequences:
V区段：IGHV4-39*01V section: IGHV4-39*01
D区段：IGHD1-26*01Section D: IGHD1-26*01
J区段：IGHJ4*02Section J: IGHJ4*02
1.103.11hAb-VL(21038-VL):(SEQ ID NO:55为氨基酸，SEQ ID NO:56为核酸)轻链CDR1-3：SEQ ID NOs:7、17、19为氨基酸序列和SEQ ID NO:8、18、20为核酸序列：1.103.11hAb-VL (21038-VL): (SEQ ID NO: 55 is amino acid, SEQ ID NO: 56 is nucleic acid) light chain CDR1-3: SEQ ID NOs: 7, 17, 19 are amino acid sequence and SEQ ID NO : 8, 18, 20 are nucleic acid sequences:
V区段：IGLV2-14*01V section: IGLV2-14*01
J区段：IGLJ3*02Section J: IGLJ3*02
1.139.15hAb-VH(23521-VH)：(SEQ ID NO:57为氨基酸，SEQ ID NO:58为核酸)重链CDR1-3：SEQ ID NOs:21、23、25为氨基酸序列和SEQ ID NO:22、24、26为核酸序列：1.139.15hAb-VH (23521-VH): (SEQ ID NO: 57 is amino acid, SEQ ID NO: 58 is nucleic acid) heavy chain CDR1-3: SEQ ID NOs: 21, 23, 25 are amino acid sequence and SEQ ID NO : 22, 24, 26 are nucleic acid sequences:
V区段：IGHV4-39*01V section: IGHV4-39*01
D区段：IGHD6-13*01Section D: IGHD6-13*01
J区段；IGHJ4*02Section J; IGHJ4*02
1.139.15hAb-VL(22895-VL):(SEQ ID NO:59为氨基酸，SEQ ID NO:60为核酸)轻链CDR1-3：SEQ ID NOs:27、29、31为氨基酸序列和SEQ ID NO:28、30、32为核酸序列：1.139.15hAb-VL (22895-VL): (SEQ ID NO: 59 is amino acid, SEQ ID NO: 60 is nucleic acid) light chain CDR1-3: SEQ ID NOs: 27, 29, 31 are amino acid sequence and SEQ ID NO : 28, 30, 32 are nucleic acid sequences:
V区段：IGLV2-18*02V section: IGLV2-18*02
J区段：IGLJ3*02Section J: IGLJ3*02
1.153.7hAb-VH(20942-VH):(SEQ ID NO:61为氨基酸，SEQ ID NO:62为核酸)重链CDR1-3：SEQ ID NOs:33、35、37为氨基酸序列和SEQ ID NO:34、36、38为核酸序列：1.153.7hAb-VH (20942-VH): (SEQ ID NO: 61 is amino acid, SEQ ID NO: 62 is nucleic acid) heavy chain CDR1-3: SEQ ID NOs: 33, 35, 37 are amino acid sequence and SEQ ID NO :34, 36, 38 are nucleic acid sequences:
V区段：IGHV3-23*01V section: IGHV3-23*01
D区段：IGHD7-27*01Section D: IGHD7-27*01
J区段：IGHJ4*02Section J: IGHJ4*02
1.153.7hAb-VL(21110-VL):(SEQ ID NO:63为氨基酸，SEQ ID NO:64为核酸)轻链CDR1-3：SEQ ID NOs:39、41、43为氨基酸序列和SEQ ID NO:40、42、44为核酸序列：1.153.7hAb-VL (21110-VL): (SEQ ID NO: 63 is amino acid, SEQ ID NO: 64 is nucleic acid) light chain CDR1-3: SEQ ID NOs: 39, 41, 43 are amino acid sequence and SEQ ID NO :40, 42, 44 are nucleic acid sequences:
V区段：IGLV3-9*01V section: IGLV3-9*01
J区段：IGLJ3*02Section J: IGLJ3*02
1.103.11-v2hAb-VH(20975-VH):(SEQ ID NO:53为氨基酸，SEQ ID NO:54为核酸)轻链CDR1-3：SEQ ID NOs:1、15、5为氨基酸序列和SEQ ID NO:2、16、6为核酸序列：1.103.11-v2hAb-VH (20975-VH): (SEQ ID NO: 53 is amino acid, SEQ ID NO: 54 is nucleic acid) light chain CDR1-3: SEQ ID NOs: 1, 15, 5 are amino acid sequence and SEQ ID NO: ID NO:2, 16, 6 are nucleic acid sequences:
V区段：IGHV4-39*01V section: IGHV4-39*01
D区段：IGHD1-26*01Section D: IGHD1-26*01
J区段：IGHJ4*02Section J: IGHJ4*02
1.103.11-v2hAb-VL(21038-2-VL):(SEQ ID NO:67为氨基酸，SEQ ID NO:68为核酸)轻链CDR1-3：SEQ ID NOs:7、17、65为氨基酸序列和SEQ ID NO:8、18、66为核酸序列：1.103.11-v2hAb-VL (21038-2-VL): (SEQ ID NO: 67 is amino acid, SEQ ID NO: 68 is nucleic acid) light chain CDR1-3: SEQ ID NOs: 7, 17, 65 are amino acid sequences And SEQ ID NO:8,18,66 is nucleic acid sequence:
V区段：IGLV2-14*01V section: IGLV2-14*01
J区段：IGLJ3*02Section J: IGLJ3*02
在一些实施方式中，所述抗-PD-1抗体和其抗原结合片段包括重链CDR序列，所述序列选自：SEQ ID NO:1、3、5、13、15、21、23、25、33、35和37。在一些实施方式中，所述抗-PD-1抗体和其抗原结合片段包括轻链CDR序列，所述序列选自：SEQ ID NO:7、9、11、17、19、27、29、31、39、41、43和65。在一些实施方式中，本申请所述的一个或多个CDR序列可以被修饰或改变以使获得的抗体在一个或多个性质上相对原来的抗体有所改进(例如改进的抗原结合、改进的糖基化模式、降低的CDR残基上的糖基化风险、增加的药代动力学半衰期、pH敏感性和对缀合的相容性)，或与原来的抗体相当(即除上述修饰和改变外具有相同CDR序列的抗体)，或至少实质上保留了原来的抗体的抗原结合特性。In some embodiments, the anti-PD-1 antibody and antigen-binding fragments thereof include a heavy chain CDR sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 13, 15, 21, 23, 25 , 33, 35 and 37. In some embodiments, the anti-PD-1 antibody and antigen-binding fragments thereof include a light chain CDR sequence selected from the group consisting of: SEQ ID NO: 7, 9, 11, 17, 19, 27, 29, 31 , 39, 41, 43 and 65. In some embodiments, one or more of the CDR sequences described herein may be modified or altered such that the resulting antibody is improved in one or more properties relative to the original antibody (e.g., improved antigen binding, improved glycosylation pattern, reduced risk of glycosylation on CDR residues, increased pharmacokinetic half-life, pH sensitivity, and compatibility with conjugation), or comparable to the original antibody (i.e., except for the above modifications and Altered antibodies with the same CDR sequences), or at least substantially retain the antigen-binding properties of the original antibody.
在一些实施方式中，所述抗-PD-1抗体和其抗原结合片段包括选自下组的重链可变区：重链可变区，其包括SEQ ID NO:1、SEQ ID NO:3、和/或SEQ ID NO:5；重链可变区，其包括SEQ ID NO:13、SEQ ID NO:15、和/或SEQ ID NO:5；重链可变区，其包括SEQ ID NO:1、SEQID NO:15、和/或SEQ ID NO:5；重链可变区，其包括SEQ ID NO:21、SEQ ID NO:23、和/或SEQID NO:25；以及重链可变区，其包括SEQ ID NO:33、SEQ ID NO:35、和/或SEQ ID NO:37。In some embodiments, the anti-PD-1 antibody and antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of: a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3 , and/or SEQ ID NO:5; heavy chain variable region comprising SEQ ID NO:13, SEQ ID NO:15, and/or SEQ ID NO:5; heavy chain variable region comprising SEQ ID NO : 1, SEQ ID NO: 15, and/or SEQ ID NO: 5; heavy chain variable region comprising SEQ ID NO: 21, SEQ ID NO: 23, and/or SEQ ID NO: 25; and heavy chain variable A region comprising SEQ ID NO:33, SEQ ID NO:35, and/or SEQ ID NO:37.
在一些实施方式中，所述抗-PD-1抗体和其抗原结合片段包括轻链可变区，其中所述轻链可变区选自：轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:9和/或SEQ ID NO:11；轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:11；轻链可变区，其包括SEQ IDNO:7、SEQ ID NO:17和/或SEQ ID NO:19；轻链可变区，其包括SEQ ID NO:27、SEQ ID NO:29和/或SEQ ID NO:31；轻链可变区，其包括SEQ ID NO:39、SEQ ID NO:41和/或SEQ ID NO:43；以及轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:65。In some embodiments, the anti-PD-1 antibody and antigen-binding fragments thereof comprise a light chain variable region, wherein the light chain variable region is selected from the group consisting of: a light chain variable region comprising SEQ ID NO:7 , SEQ ID NO:9 and/or SEQ ID NO:11; light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO:11; light chain variable region, which Comprising SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO:19; light chain variable region comprising SEQ ID NO:27, SEQ ID NO:29 and/or SEQ ID NO:31; light chain A variable region comprising SEQ ID NO:39, SEQ ID NO:41 and/or SEQ ID NO:43; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO:17 and/or SEQ ID NO:43; ID NO:65.
在一些实施方式中，所述抗-PD-1抗体和其抗原结合片段包括：a)重链可变区，其包括SEQ ID NO:1、SEQ ID NO:3、和/或SEQ ID NO:5；和轻链可变区，其包括SEQ ID NO:7、SEQID NO:9和/或SEQ ID NO:11；b)重链可变区，其包括SEQ ID NO:13、SEQ ID NO:15、和/或SEQ ID NO:5；和轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:11；c)重链可变区，其包括SEQ ID NO:1、SEQ ID NO:15、和/或SEQ ID NO:5；和轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:19；d)重链可变区，其包括SEQ ID NO:21、SEQID NO:23、和/或SEQ ID NO:25；和轻链可变区，其包括SEQ ID NO:27、SEQ ID NO:29和/或SEQ ID NO:31；e)重链可变区，其包括SEQ ID NO:33、SEQ ID NO:35、和/或SEQ ID NO:37；和轻链可变区，其包括SEQ ID NO:39、SEQ ID NO:41和/或SEQ ID NO:43；或f)重链可变区，其包括SEQ ID NO:1、SEQ ID NO:15、和/或SEQ ID NO:5；和轻链可变区，其包括SEQ ID NO:7、SEQ ID NO:17和/或SEQ ID NO:65。In some embodiments, the anti-PD-1 antibody and antigen-binding fragments thereof comprise: a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5; and light chain variable region, it comprises SEQ ID NO:7, SEQID NO:9 and/or SEQ ID NO:11; B) heavy chain variable region, it comprises SEQ ID NO:13, SEQ ID NO: 15. and/or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO:11; c) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15, and/or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and/or SEQ ID NO:19; D) heavy chain variable region, it comprises SEQ ID NO:21, SEQID NO:23 and/or SEQ ID NO:25; And light chain variable region, it comprises SEQ ID NO:27, SEQ ID NO:29 And/or SEQ ID NO:31; E) heavy chain variable region, it comprises SEQ ID NO:33, SEQ ID NO:35 and/or SEQ ID NO:37; And light chain variable region, it comprises SEQ ID NO:37; ID NO:39, SEQ ID NO:41 and/or SEQ ID NO:43; or f) heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15, and/or SEQ ID NO:5 and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17 and/or SEQ ID NO:65.
本领域技术人员应理解可以将表1中提供的CDR序列进行修饰以包含一个或更多氨基酸的取代，由此得到提高的生物学活性例如提高的与人PD-1的结合亲和性。例如，可以利用噬菌体展示技术生产并表达抗体变体库(例如Fab或FcFv变体)，随后筛选与人PD-1有亲和性的抗体。另一个例子中，可以用计算机软件模拟所述抗体与人PD-1的结合并鉴别抗体上形成结合界面的氨基酸残基。可以避免这些残基的替代以防止结合亲和性降低，或可以靶向这些残基进行替代以形成更强的结合。在某些实施方式中，CDR序列中的至少一个(或全部)取代是保守替代。Those skilled in the art will understand that the CDR sequences provided in Table 1 can be modified to include one or more amino acid substitutions, thereby resulting in improved biological activity such as increased binding affinity to human PD-1. For example, a library of antibody variants (such as Fab or FcFv variants) can be produced and expressed using phage display technology, followed by screening for antibodies with affinity to human PD-1. In another example, computer software can be used to simulate the binding of the antibody to human PD-1 and identify the amino acid residues on the antibody that form the binding interface. Substitution of these residues can be avoided to prevent loss of binding affinity, or can be targeted for substitution to result in stronger binding. In certain embodiments, at least one (or all) of the substitutions in the CDR sequences are conservative substitutions.
在某些实施方式中，所述抗体和抗原结合片段包括一个或多个CDR序列，这些序列具有与表1中所列的序列至少80％(例如至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)的序列同一性，并且同时保留了与其亲本抗体相似或甚至高于其的与人PD-1的结合亲和性，所述亲本抗体具有基本相同的序列，但其相应的CDR序列与表1所列的序列具有100％序列同一性。In certain embodiments, the antibodies and antigen-binding fragments include one or more CDR sequences that are at least 80% (e.g., at least 85%, 88%, 90%, 91% identical) to the sequences listed in Table 1 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity, and at the same time retains its parental antibody similar or even higher than its human PD-1 Binding affinity, the parental antibody has substantially the same sequence, but its corresponding CDR sequence has 100% sequence identity with the sequences listed in Table 1.
在某些实施方式中，所述抗-PD-1抗体和其抗原结合片段是全人源的。所述全人源抗体在人体中没有如经常在人源化的抗体中观察到的免疫原性或降低的结合亲和性等问题。In certain embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof are fully human. Such fully human antibodies do not have the problems of immunogenicity or reduced binding affinity in humans as often observed with humanized antibodies.
在一些实施方式中，所述全人源抗-PD-1抗体和其抗原结合片段包括重链可变区，其中所述重链可变区选自SEQ ID NO:45、SEQ ID NO:49、SEQ ID NO:53、SEQ ID NO:57、SEQ IDNO:61，和与之具有至少80％(例如至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列同一性的同源序列；和/或轻链可变区，其中所述轻链可变区选自SEQ ID NO:47、SEQ ID NO:51、SEQ ID NO:55、SEQ ID NO:59、SEQ ID NO:63、SEQ ID NO:67，和与之具有至少80％(例如至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列同一性的同源序列。这些全人源抗体保留了与人PD-1的结合亲和性，优选地与示例性抗体：1.7.3hAb、1.49.9hAb、1.103.11hAb、1.103.11-v2hAb、1.139.15hAb和1.153.7hAb的水平相似。In some embodiments, the fully human anti-PD-1 antibody and antigen-binding fragments thereof include a heavy chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NO:45, SEQ ID NO:49 , SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, and at least 80% (eg, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%) %, 96%, 97%, 98%, 99%) sequence identity homologous sequence; and/or light chain variable region, wherein said light chain variable region is selected from SEQ ID NO:47, SEQ ID NO : 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, and at least 80% thereof (for example at least 85%, 88%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity of homologous sequences. These fully human antibodies retain binding affinity to human PD-1, preferably with the exemplary antibodies: 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, and 1.153.7hAb levels are similar.
在一些实施方式中，所述全人源抗-PD-1抗体和其抗原结合片段包括a)重链可变区，其包括SEQ ID NO:45；和轻链可变区，其包括SEQ ID NO:47；b)重链可变区，其包括SEQ IDNO:49；和轻链可变区，其包括SEQ ID NO:51；c)重链可变区，其包括SEQ ID NO:53；和轻链可变区，其包括SEQ ID NO:55；d)重链可变区，其包括SEQ ID NO:57；和轻链可变区，其包括SEQ ID NO:59；e)重链可变区，其包括SEQ ID NO:61；和轻链可变区，其包括SEQ ID NO:63；以及f)重链可变区，其包括SEQ ID NO:53；和轻链可变区，其包括SEQ ID NO:67。In some embodiments, the fully human anti-PD-1 antibody and antigen-binding fragments thereof comprise a) a heavy chain variable region comprising SEQ ID NO: 45; and a light chain variable region comprising SEQ ID NO: 45; NO:47; b) heavy chain variable region, which includes SEQ ID NO:49; and light chain variable region, which includes SEQ ID NO:51; c) heavy chain variable region, which includes SEQ ID NO:53; and a light chain variable region comprising SEQ ID NO:55; d) a heavy chain variable region comprising SEQ ID NO:57; and a light chain variable region comprising SEQ ID NO:59; e) a heavy chain Variable region, it comprises SEQ ID NO:61; And light chain variable region, it comprises SEQ ID NO:63; And f) heavy chain variable region, it comprises SEQ ID NO:53; And light chain variable region , which comprises SEQ ID NO:67.
本申请还包括了与本申请抗-PD-1抗体和其抗原结合片段竞争相同表位的抗体和其抗原结合片段。在某些实施方式中，所述抗体以低于10-6M、低于10-7M、低于10-7.5M、低于10-8M、低于10-8.5M或低于10-9M或低于10-10M的IC50值(即半数抑制浓度)阻断1.7.3hAb、1.49.9hAb、1.103.11hAb、1.103.11-v2hAb、1.139.15hAb或1.153.7hAb与人或猴PD-1的结合。IC50值通过竞争性测试例如ELISA测定，放射性配体竞争结合测定法，和FACS分析确定。The present application also includes antibodies and antigen-binding fragments thereof that compete for the same epitope as the anti-PD-1 antibodies and antigen-binding fragments thereof of the present application. In certain embodiments, the antibody is present at less than 10−6 M, less than 10−7 M, less than 10−7.5 M, less than 10−8 M, less than 10−8.5 M or less than 10− 9 M or lower than 10-10 M IC50 value (ie half inhibitory concentration) blocking 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb or 1.153.7hAb and human or monkey Binding of PD-1.IC50 values are determined by competitive assays such as ELISA assays, radioligand competition binding assays, and FACS analysis.
在一些实施方式中，本申请所述抗-PD-1抗体和其抗原结合片段能够以≤10-6M(e.g.,≤5x10-7M,≤2x10-7M,≤10-7M,≤5x10-8M,≤2x10-8M,≤10-8M,≤5x10-9M,≤2x10-9M,≤10-9M,10-10M)的结合亲和性(Kd)与人PD-1特异性结合，其通过等离子共振结合法测量。结合亲和性可以用KD值表示，其通过当抗原和抗原结合分子的结合达到平衡时的解离速率与结合速率的比值(koff/kon)计算得到。所述抗原结合亲和性(例如KD)可以通过本领域已知的适宜方法适宜地确定，所述方法包括使用仪器如如Biacore的等离子共振结合法(参加例如Murphy,M.et al,Current protocols in protein science,Chapter 19,unit 19.14,2006)。In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein can be produced at ≤10-6 M (eg, ≤5x10-7 M, ≤2x10-7 M, ≤10-7 M, ≤ 5x10-8 M, ≤2x10-8 M, ≤10-8 M, ≤5x10-9 M, ≤2x10-9 M, ≤10-9 M, 10-10 M) binding affinity (Kd) with human PD-1 specific binding, measured by plasmon resonance binding. Binding affinity can be expressed as aKD value, which is calculated as the ratio of the off-rate to the on-rate (koff/kon) when the binding of the antigen to the antigen-binding molecule reaches equilibrium. The antigen binding affinity (e.g.KD ) may suitably be determined by suitable methods known in the art, including the use of instruments such as the plasmon resonance binding method such as Biacore (see e.g. Murphy, M. et al, Current protocols in protein science, Chapter 19, unit 19.14, 2006).
在某些实施方式中，本申请所述抗体和其抗原结合片段与人PD-1以0.1nM-100nM(例如0.1nM-50nM、0.1nM-30nM、0.1nM-20nM或0.1nM-10nM或0.1nM-1nM)的EC50(即半数结合浓度)结合。所述抗体与人PD-1的结合可以通过本领域已知的方法如夹心法如ELISA，Western印迹，FACS或其他结合试验测定。在示例性的例子中，将待测抗体(即一抗)与固定化的人PD-1或表达人PD-1的细胞结合，随后洗掉未结合抗体，引入标记的二抗，其能够与一抗结合因此能够检测出结合的一抗。当使用固定化的PD-1时可在酶标仪板上进行所述检测，或当使用表达人PD-1的细胞时可使用FACS分析进行所述检测。在某些实施方式中，本申请所述抗体和其抗原结合片段以1nM至10nM或1nM至5nM(使用FACS分析测定)的EC50(即50％的有效浓度)与人PD-1结合。In certain embodiments, the antibodies and antigen-binding fragments thereof described in the present application bind to human PD-1 at 0.1 nM-100 nM (such as 0.1 nM-50 nM, 0.1 nM-30 nM, 0.1 nM-20 nM or 0.1 nM-10 nM or 0.1EC50 (ie, half maximal binding concentration) of nM-1 nM) binding. The binding of the antibody to human PD-1 can be determined by methods known in the art such as sandwich methods such as ELISA, Western blotting, FACS or other binding assays. In an illustrative example, the antibody to be tested (i.e. primary antibody) is combined with immobilized human PD-1 or cells expressing human PD-1, then the unbound antibody is washed away, and a labeled secondary antibody is introduced, which can interact with Binding of the primary antibody thus enables detection of the bound primary antibody. The detection can be performed on a microplate reader plate when using immobilized PD-1, or using FACS analysis when using cells expressing human PD-1. In certain embodiments, the antibodies and antigen-binding fragments thereof described herein bind to human PD-1 with an EC50 (ie, 50% effective concentration) of 1 nM to 10 nM or 1 nM to 5 nM (determined using FACS analysis).
在某些实施方式中，本申请所述抗体和其抗原结合片段以0.2nM-100nM(例如0.2nM-50nM、0.2nM-30nM、0.2nM-20nM、0.2nM-10nM或1nM-10nM)的IC50抑制人PD-1与其配体的结合，其通过竞争性测试测得。In certain embodiments, the antibodies and antigen-binding fragments thereof described herein have an IC of 0.2 nM-100 nM (eg, 0.2 nM-50 nM, 0.2 nM-30 nM, 0.2 nM-20 nM, 0.2 nM-10 nM, or 1 nM-10 nM).50 inhibits the binding of human PD-1 to its ligand as measured by a competition assay.
在某些实施方式中，本申请所述抗体和其抗原结合片段抑制人PD-1与其配体的结合，并由此提供了包括例如诱导活化的T细胞产生细胞因子(如CD4+T细胞和CD8+T细胞)、诱导活化的T细胞的增殖(如CD4+T细胞和CD8+T细胞)和逆转调节性Treg的抑制性功能的生物学活性。示例性的细胞因子包括IL-2和IFNγ。术语“IL-2”是指白细胞介素2，其是细胞因子信号传导分子，在免疫系统中调节的白血细胞(例如白细胞)的活性的。术语“干扰素γ(IFNγ)”是由天然杀伤(NK)细胞，NK T细胞，CD4+和CD8+T细胞产生的细胞因子，其是巨噬细胞的重要的活化剂和主要组织相容性复合物诱导体(MHC)分子表达的诱发剂。细胞因子的产生可以通过本领域已知的方法确定，如ELISA。这些方法也可以用来检测T细胞增殖，包括[3H]胸苷掺入测定。In certain embodiments, the antibodies and antigen-binding fragments thereof described herein inhibit the binding of human PD-1 to its ligand, and thus provide a therapeutic effect including, for example, induction of activated T cells to produce cytokines (such as CD4+ T cells and CD8+ T cells), inducing the proliferation of activated T cells (such as CD4+ T cells and CD8+ T cells) and reversing the biological activity of the suppressive function of regulatory Treg. Exemplary cytokines include IL-2 and IFNγ. The term "IL-2" refers to interleukin 2, which is a cytokine signaling molecule that regulates the activity of white blood cells (eg, leukocytes) in the immune system. The term "Interferon gamma (IFNγ)" is a cytokine produced by natural killer (NK) cells, NK T cells, CD4+ and CD8+ T cells, which is an important activator and major histocompatibility of macrophages Inducer of complex inducer (MHC) molecule expression. Cytokine production can be determined by methods known in the art, such as ELISA. These methods can also be used to detect T cell proliferation, including [3 H]thymidine incorporation assays.
所述抗-PD-1抗体和其抗原结合片段是PD-1特异性的。在某些实施方式中，所述抗体和其抗原结合片段不与CD28和/或CTLA-4结合。例如，与CD28和/或CTLA-4的结合亲和性比PD-1的结合亲和性的15％、10％、9％、8％、7％、6％、5％、4％、3％、2％或1％还要低。The anti-PD-1 antibodies and antigen-binding fragments thereof are specific for PD-1. In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind CD28 and/or CTLA-4. For example, the binding affinity to CD28 and/or CTLA-4 is 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% that of PD-1 %, 2% or 1% lower.
在某些实施方式中，所述抗体和其抗原结合片段以不高于100nM，例如，不高于或约为10nM、9nM、8nM、7nM、6nM、5nM、4nM、3nM、2nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM、0.09nM、0.08nM、0.07nM、0.06nM、0.05nM、0.04nM、0.03nM、0.02nM或0.01nM的EC50(通过ELISA测定)与猴PD-1结合。在某些实施方式中，所述抗体和其抗原结合片段以约1nM-10nM的EC50与猴PD-1结合。In certain embodiments, the antibodies and antigen-binding fragments thereof are dosed at no more than 100 nM, for example, at no more than or about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 orEC50 (determined by ELISA) of 0.01 nM for binding to monkey PD-1. In certain embodiments, the antibodies and antigen-binding fragments thereof bind monkey PD-1 with an EC50 of about 1 nM-10 nM.
在某些实施方式中，所述抗体和其抗原结合片段不与鼠PD-1结合，但与猴PD-1以与人PD-1相似的结合亲和性结合。例如，示例性抗体1.7.3hAb、1.49.9hAb、1.103.11hAb、1.103.11-v2hAb、1.139.15hAb和1.153.7hAb与鼠PD-1的结合用常用的结合测定如ELISA或FACS分析无法检出，而ELISA或FACS检测出这些抗体与猴PD-1以与人PD-1相似的亲和性或EC50值结合。In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind murine PD-1, but bind monkey PD-1 with a similar binding affinity as human PD-1. For example, binding of exemplary antibodies 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, and 1.153.7hAb to murine PD-1 was not detectable using commonly used binding assays such as ELISA or FACS analysis , while ELISA or FACS detected that these antibodies bound to monkey PD-1 with similar affinities orEC50 values to human PD-1.
在一些实施方式中，所述的抗-PD-1抗体和其抗原结合片段具有降低的或消除的效应功能。在一些实施方式中，所述的抗-PD-1抗体和其抗原结合片段具有IgG4同种型的恒定区，其具有降低的或耗竭的效应功能。例如ADCC和CDC等效应功能能够导致对表达PD-1的细胞的细胞毒性。许多细胞如T细胞能够正常表达PD-1。为了避免对这些正常细胞的潜在的不希望的毒性，本发明所述的抗体和其抗原结合片段的某些实施方式具有降低的或甚至消除的效应功能。已知有许多测试用来评估ADCC或CDC活性，例如Fc受体结合试验、补体C1q结合实验和细胞裂解法，本领域技术人员能够容易选择。不希望受到理论的束缚，但据信具有降低的或消除的效应功能如ADCC和CDC的抗体不会引起对表达PD-1的细胞(例如那些T细胞)的细胞毒性或将之降低到最小程度，因此避免了不希望的副作用。而与此同时，阻断PD-1将会激发免疫系统治疗如癌症或慢性感染的状况。In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof have reduced or eliminated effector functions. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof have constant regions of the IgG4 isotype with reduced or depleted effector functions. Effector functions such as ADCC and CDC can lead to cytotoxicity of PD-1 expressing cells. Many cells such as T cells can normally express PD-1. To avoid potential undesired toxicity to these normal cells, certain embodiments of the antibodies and antigen-binding fragments thereof described herein have reduced or even eliminated effector functions. A number of assays are known for assessing ADCC or CDC activity, such as Fc receptor binding assays, complement Clq binding assays, and cell lysis methods, which can be readily selected by those skilled in the art. Without wishing to be bound by theory, it is believed that antibodies with reduced or abolished effector functions such as ADCC and CDC do not cause or minimize cytotoxicity to PD-1 expressing cells such as those T cells , thus avoiding undesired side effects. At the same time, blocking PD-1 would prime the immune system to treat conditions such as cancer or chronic infections.
在一些实施方式中，本申请所述的抗-PD-1抗体和其抗原结合片段具有降低的副作用。例如，本申请所述的抗-PD-1抗体和其抗原结合片段具有全人源IgG序列，因此其免疫原性低于人源化的抗体。再例如，本申请所述的抗-PD-1抗体和其抗原结合片段可以是IgG4形式以消除ADCC和CDC。In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein have reduced side effects. For example, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein have fully human IgG sequences and are therefore less immunogenic than humanized antibodies. As another example, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein can be in the form of IgG4 to eliminate ADCC and CDC.
在一些实施方式中，本申请所述的抗-PD-1抗体和其抗原结合片段的优势在于其能与具有免疫原性的物质联用，如肿瘤细胞、纯化的肿瘤抗原和用编码免疫刺激因子转染的细胞、肿瘤疫苗。此外，所述抗-PD-1抗体和其抗原结合片段可以包括在联用治疗中，包括标准化学疗法和放射疗法、基于靶点的小分子疗法、其他新兴免疫检查点调节剂疗法。在一些实施方式中，所述抗体和其抗原结合片段可以用作抗体-药物缀合物、双特异性或多价抗体的基础分子。In some embodiments, an advantage of the anti-PD-1 antibodies and antigen-binding fragments thereof described herein is that they can be used in combination with immunogenic substances, such as tumor cells, purified tumor antigens, and immunostimulatory proteins encoded with Factor-transfected cells, tumor vaccines. In addition, the anti-PD-1 antibodies and antigen-binding fragments thereof can be included in combination therapy, including standard chemotherapy and radiation therapy, target-based small molecule therapy, and other emerging immune checkpoint modulator therapies. In some embodiments, the antibodies and antigen-binding fragments thereof can be used as base molecules for antibody-drug conjugates, bispecific or multivalent antibodies.
本申请所述的抗-PD-1抗体和其抗原结合片段可以是单克隆抗体、多克隆抗体、全人源抗体、人源化抗体、嵌合抗体、重组抗体、双特异性抗体、标记抗体、二价抗体或抗独特型抗体。重组抗体是在体外使用重组方法而非动物制备的抗体。双特异性抗体或双价抗体是具有两种不同的单克隆抗体的片段的人工抗体，其能结合两种不同的抗原。“二价”的抗体和其抗原结合片段包括两个抗原结合位点。两个抗原结合位点可以结合相同抗原，或者可以各自结合到不同的抗原，在这种情况下，抗体或抗原结合片段为“双特异性”。The anti-PD-1 antibodies and antigen-binding fragments thereof described in this application can be monoclonal antibodies, polyclonal antibodies, fully human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, labeled antibodies , bivalent antibodies or anti-idiotypic antibodies. Recombinant antibodies are antibodies produced in vitro using recombinant methods rather than animals. Bispecific antibodies, or diabodies, are artificial antibodies that have fragments of two different monoclonal antibodies that bind two different antigens. "Bivalent" antibodies and antigen-binding fragments thereof comprise two antigen-binding sites. The two antigen binding sites may bind the same antigen, or may each bind to a different antigen, in which case the antibody or antigen-binding fragment is "bispecific".
在一些实施方式中，本申请所述的抗-PD-1抗体和其抗原结合片段是全人源抗体。在一些实施方式中，使用重组方法制备所述全人源抗体。例如，可以制备转基因动物如小鼠，使其携带人源免疫球蛋白基因的转基因或转染色体，并因此在用适宜的抗原如人源PD-1免疫后能够生产全人源抗体。全人源抗体可以从这样的转基因动物中分离，或另选地，可以通过杂交瘤技术制备，将所述转基因动物的脾细胞与永生细胞系融合以生成分泌所述全人源抗体的杂交瘤细胞。示例性的转基因动物包括但不限于，Omni大鼠，其内源性大鼠免疫球蛋白基因的表达被失活并同时被基因工程化以包含功能性的重组人源免疫球蛋白基因座；Omni小鼠，其内源性小鼠免疫球蛋白基因的表达被失活并同时被基因工程化以包含具有J-基因座缺失和C-kappa突变的重组人源免疫球蛋白基因座。OmniFilc，其为转基因大鼠，其内源性大鼠免疫球蛋白基因的表达被失活，并同时被基因工程化以包含具有单个的共有的、重组的VkJk轻链和功能性重链的重组人源免疫球蛋白基因座。具体信息请进一步参见：Osborn M.et al,Journal of Immunology,2013,190:1481-90；Ma B.et al,Journal ofImmunological Methods 400-401(2013)78-86；Geurts A.et al,Science,2009,325:433；美国专利8,907,157；欧洲专利2152880B1；欧洲专利2336329B1，其均通过引用整体并入本申请。也可使用其他适宜的转基因动物，例如，HuMab小鼠(具体参见Lonberg,N.etal.Nature 368(6474):856 859(1994)),Xeno-小鼠(Mendez et al.Nat Genet.,1997,15:146–156),TransChromo小鼠(Ishida et al.Cloning Stem Cells,2002,4:91–102)和VelocImmune小鼠(Murphy et al.Proc Natl Acad Sci USA,2014,111:5153–5158),Kymouse转基因小鼠(Lee et al.Nat Biotechnol,2014,32:356–363)，和转基因兔(Flisikowska et al.PLoS One,2011,6:e21045)。In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein are fully human antibodies. In some embodiments, the fully human antibody is produced using recombinant methods. For example, transgenic animals such as mice can be prepared to carry a transgene or transchromosome of human immunoglobulin genes and thus be able to produce fully human antibodies after immunization with an appropriate antigen such as human PD-1. Fully human antibodies can be isolated from such transgenic animals, or alternatively, can be produced by hybridoma technology by fusing the spleen cells of said transgenic animals with an immortal cell line to generate hybridomas secreting said fully human antibodies cell. Exemplary transgenic animals include, but are not limited to, Omni rats, whose expression of endogenous rat immunoglobulin genes has been inactivated and simultaneously engineered to contain functional recombinant human immunoglobulin loci; Mice whose expression of endogenous mouse immunoglobulin genes were inactivated and simultaneously genetically engineered to contain recombinant human immunoglobulin loci with J-locus deletions and C-kappa mutations. OmniFilc, which is a transgenic rat whose expression of endogenous rat immunoglobulin genes has been inactivated and simultaneously genetically engineered to contain a recombinant with a single shared, recombinant VkJk light chain and a functional heavy chain Human immunoglobulin loci. For specific information, please refer to: Osborn M. et al, Journal of Immunology, 2013, 190:1481-90; Ma B. et al, Journal of Immunological Methods 400-401 (2013) 78-86; Geurts A. et al, Science , 2009,325:433; US Patent 8,907,157; European Patent 2152880B1; European Patent 2336329B1, all of which are incorporated herein by reference in their entirety. Other suitable transgenic animals can also be used, for example, HuMab mice (see in particular Lonberg, N. et al. Nature 368 (6474): 856 859 (1994)), Xeno-mice (Mendez et al. Nat Genet., 1997 , 15:146–156), TransChromo mice (Ishida et al.Cloning Stem Cells,2002,4:91–102) and VelocImmune mice (Murphy et al.Proc Natl Acad Sci USA,2014,111:5153–5158 ), Kymouse transgenic mice (Lee et al. Nat Biotechnol, 2014, 32:356–363), and transgenic rabbits (Flisikowska et al. PLoS One, 2011, 6:e21045).
在一些实施方式中，本申请所述的抗-PD-1抗体和其抗原结合片段是骆驼化单域抗体(camelized single chain domain antibody)、双功能抗体(diabody)、scFv、scFv二聚体、BsFv、dsFv、(dsFv)2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds双功能抗体(dsdiabody)、纳米抗体、域抗体或双价域抗体。In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein are camelized single chain domain antibodies, diabodies, scFv, scFv dimers, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, dsdiabody, nanobody, domainbody or bivalent domainbody.
在一些实施方式中，本申请所述的抗-PD-1抗体和其抗原结合片段进一步包括免疫球蛋白恒定区。在一些实施方式中，免疫球蛋白恒定区包括重链和/或轻链恒定区。所述重链恒定区包括CH1、CH1-CH2或CH1-CH3区。在一些实施方式中，免疫球蛋白恒定区可以进一步包括一个或多个修饰以获得所需的性质。例如，可以将所述恒定区修饰以降低的或耗竭一种或多种效应功能以增强FcRn受体结合或引入一个或多个半胱氨酸残基。In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant regions include heavy and/or light chain constant regions. The heavy chain constant region includes a CH1, CH1-CH2 or CH1-CH3 region. In some embodiments, an immunoglobulin constant region may further comprise one or more modifications to obtain desired properties. For example, the constant region can be modified to reduce or deplete one or more effector functions to enhance FcRn receptor binding or to introduce one or more cysteine residues.
在某些实施方式中，所述抗-PD-1抗体及其抗原结合片段进一步包含缀合物。可以设想，本发明中的抗体或其抗原结合片段可与多种缀合物连接(见例如"ConjugateVaccines"、Contributions to Microbiology and Immunology、J.M.Cruse andR.E.Lewis、Jr.(eds.)、Carger Press、New York、(1989))。这些缀合物可以通过共价结合、亲和结合、嵌入、同等结合(coordinate binding)、络合、结合、混合或加入等其他方式与所述抗体或抗原结合物连接。在某些实施方式中，本发明公开的抗体和抗原结合片段可以通过工程的方法使其含有表位结合部分以外的特定位点，这些位点可用来结合一种或多种缀合物。例如，这样的位点可包含一种或多种反应性氨基酸残基，例如半胱氨酸残基和组氨酸残基，用于协助与结合物的共价连接。在某些实施方式中，抗体可间接连于缀合物，或通过另一个缀合物相连。例如，所述抗体或其抗原结合片段可结合生物素，然后间接结合第二个缀合物，其与亲和素相连。所述缀合物可以是可检测的标记、药代动力学修饰部分、纯化部分或细胞毒性部分。可检测的标记的例子可以包括荧光标记(例如荧光素、罗丹明、丹酰、藻红蛋白或德克萨斯红)、酶-底物标记物(例如辣根过氧化物酶、碱性磷酸酶、荧光素酶、葡糖淀粉酶、溶菌酶、糖氧化酶或β-D-半乳糖苷酶)、放射性同位素(例如、123I、124I、125I、131I、35S、3H、111In、112In、14C、64Cu、67Cu、86Y、88Y、90Y、177Lu、211At、186Re、188Re、153Sm、212Bi、and32P、其他镧系元素、发光标记)、发色团部分、地高辛、生物素/亲和素、DNA分子或金以进行检测。在某些实施方式中，所述缀合物可以是药代动力学修饰部分如PEG，其帮助延长抗体的半衰期。其他适宜的聚合物包括例如羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、乙二醇/丙二醇共聚物等。在某些实施方式中，所述缀合物可以是纯化部分例如磁珠。“细胞毒性部分”可以是对细胞有害的或可能损坏或杀死细胞的任何试剂。细胞毒性部分的示例包括，但不限于，紫杉醇、细胞松弛素B、短杆菌肽D、溴化乙锭、吐根碱、丝裂霉素、依托泊苷、替尼泊苷、长春新碱、长春碱、秋水仙碱、阿霉素、柔红霉素、二羟基炭疽菌素二酮、米托蒽醌、光神霉素、放线菌素D、1-去氢睾酮、糖皮质激素、普鲁卡因、丁卡因、利多卡因、普萘洛尔、嘌呤霉素及其类似物、抗代谢物(例如，甲氨蝶呤、6-巯基嘌呤、6-硫鸟嘌呤、阿糖胞苷、5-氟尿嘧啶达卡巴)、烷化剂(例如氮芥、塞替派苯丁酸氮芥、美法仑、卡莫司汀(BSNU)和洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲霉素、丝裂霉素C和顺-二氯二胺铂(II)(DDP)顺铂)、蒽环类抗生素(例如柔红霉素(以前的道诺霉素)和阿霉素)、抗生素(例如更生霉素(以前称为放线菌素)、博来霉素、光神霉素和氨茴霉素(AMC))以及抗有丝分裂剂(例如长春新碱和长春碱)。In certain embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof further comprise conjugates. It is contemplated that the antibodies of the invention, or antigen-binding fragments thereof, may be linked to a variety of conjugates (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and R.E. Lewis, Jr. (eds.), Carger Press , New York, (1989)). These conjugates can be linked to the antibody or antigen conjugate by other means such as covalent binding, affinity binding, intercalation, coordinate binding, complexation, binding, mixing or addition. In certain embodiments, the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites other than the epitope-binding portion that can be used to bind one or more conjugates. For example, such sites may contain one or more reactive amino acid residues, such as cysteine residues and histidine residues, to facilitate covalent attachment to the binder. In certain embodiments, the antibody may be attached to the conjugate indirectly, or through another conjugate. For example, the antibody or antigen-binding fragment thereof can bind biotin and then indirectly bind a second conjugate, which is linked to avidin. The conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purification moiety or a cytotoxic moiety. Examples of detectable labels may include fluorescent labels (such as fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (such as horseradish peroxidase, alkaline phosphate enzyme, luciferase, glucoamylase, lysozyme, sugar oxidase or β-D-galactosidase), radioactive isotopes (for example,123 I,124 I,125 I,131 I,35 S,3 H ,111 In,112 In,14 C,64 Cu,67 Cu,86 Y,88 Y,90 Y,177 Lu,211 At,186 Re,188 Re,153 Sm,212 Bi, and32 P, other lanthanides elements, luminescent labels), chromophore moieties, digoxigenin, biotin/avidin, DNA molecules or gold for detection. In certain embodiments, the conjugate may be a pharmacokinetic modifying moiety such as PEG, which helps to extend the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. In certain embodiments, the conjugates may be purification moieties such as magnetic beads. A "cytotoxic moiety" can be any agent that is harmful to cells or that may damage or kill cells. Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, Vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, Procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, arabinose Cytidine, 5-fluorouracil (dakaba), alkylating agents (eg, nitrogen mustard, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide , busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamminoplatinum(II) (DDP) cisplatin), anthracyclines (such as daunorubicin (formerly daunomycin) and doxorubicin), antibiotics such as dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and anthraninomycin (AMC)), and antimitotic agents ( eg vincristine and vinblastine).
多核苷酸和重组方法Polynucleotides and recombinant methods
本申请提供了编码抗-PD-1抗体和其抗原结合片段的分离的多核苷酸。在某些实施方式中，所述分离的多核苷酸包括一个或多个如表1中的核苷酸序列，其编码如表1中的CDR序列。The application provides isolated polynucleotides encoding anti-PD-1 antibodies and antigen-binding fragments thereof. In certain embodiments, the isolated polynucleotide comprises one or more nucleotide sequences as in Table 1, which encode the CDR sequences as in Table 1.
在一些实施方式中，所述分离的多核苷酸编码重链可变区并包括选自下组的序列：SEQID NO:46、SEQ ID NO:50、SEQ ID NO:54、SEQ ID NO:58、SEQ ID NO:62，以及与之具有至少80％(例如至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)的序列同一性的同源序列。在一些实施方式中，所述分离的多核苷酸编码轻链可变区并包括选自下组的序列：SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:56、SEQ ID NO:60、SEQ ID NO:64、SEQ ID NO:68，以及与之具有至少80％(例如至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)的序列同一性的同源序列。在某些实施方式中，所述一致性的百分比是源自遗传密码的简并性，而编码的蛋白序列保持不变。In some embodiments, the isolated polynucleotide encodes a heavy chain variable region and comprises a sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58 , SEQ ID NO: 62, and at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Homologous sequences with a sequence identity of 99%). In some embodiments, the isolated polynucleotide encodes a light chain variable region and comprises a sequence selected from the group consisting of SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO: 60. SEQ ID NO: 64, SEQ ID NO: 68, and at least 80% (eg, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%) , 97%, 98%, 99%) sequence identities of homologous sequences. In certain embodiments, the percent identity is derived from the degeneracy of the genetic code, while the encoded protein sequence remains unchanged.
使用本领域公知的重组技术，可以将包括编码所述抗-PD-1抗体和其抗原结合片段(例如包括表1所示的序列)的多核苷酸的载体引入宿主细胞用于克隆(扩增DNA)或基因表达。在另一实施方式中，所述抗体可通过本领域公知的同源重组的方法制得。编码所述单克隆抗体的DNA可以通过常规的方法分离和测序(如可以使用寡核苷酸探针，该探针可特异性与编码所述抗体的重链和轻链的基因结合)。多种载体可供选择。载体组分通常包括，但不限于，以下的一种或多种：信号序列、复制起始点、一种或多种标记基因、增强序列、启动子(例如：SV40、CMV、EF-1α)和转录终止序列。Using recombinant techniques well known in the art, vectors comprising polynucleotides encoding the anti-PD-1 antibodies and antigen-binding fragments thereof (for example comprising the sequences shown in Table 1) can be introduced into host cells for cloning (amplification) DNA) or gene expression. In another embodiment, the antibody can be produced by homologous recombination methods known in the art. The DNA encoding the monoclonal antibody can be isolated and sequenced by conventional methods (for example, oligonucleotide probes can be used, which can specifically bind to the genes encoding the heavy and light chains of the antibody). Various carriers are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (e.g., SV40, CMV, EF-1α) and Transcription termination sequence.
在一些实施方式中，所述载体系统包括哺乳动物、细菌、酵母系统等，并将包括质粒例如但不限于pALTER、pBAD、pcDNA、pCal、pL、pET、pGEMEX、pGEX、pCI、pCMV、pEGFP、pEGFT、pSV2、pFUSE、pVITRO,pVIVO、pMAL、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS420、pLexA、pACT2等其他可从实验室获得或市售的载体。适宜的载体可以包括质粒或病毒载体(例如，复制缺陷型逆转录病毒、腺病毒和腺相关病毒)。In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc., and will include plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2 and others can be obtained from the laboratory or the market carrier for sale. Suitable vectors may include plasmid or viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).
可以将包括编码所述抗体和其抗原结合片段的多核苷酸的载体引入宿主细胞用于克隆或基因表达。本发明中适用于克隆或表达所述载体中的DNA的宿主细胞为原核细胞、酵母或上述高级真核细胞。适用于本发明用途的原核细胞包括真细菌如，革兰氏阴性菌或革兰氏阳性菌，例如，肠杆菌科，如，大肠杆菌，肠杆菌属，欧文氏菌属，克雷白氏杆菌属，变形杆菌属，沙门氏菌属，如，鼠伤寒沙门(氏)杆菌，沙雷氏菌属，如，粘质沙雷氏菌，以及志贺氏菌属，及杆菌属如，枯草芽孢杆菌和地衣芽孢杆菌，假单胞菌如，绿脓杆菌和链霉菌。Vectors comprising polynucleotides encoding the antibodies and antigen-binding fragments thereof can be introduced into host cells for cloning or gene expression. The host cells suitable for cloning or expressing the DNA in the vector in the present invention are prokaryotic cells, yeast or the above-mentioned higher eukaryotic cells. Prokaryotic cells suitable for use according to the invention include eubacteria such as Gram-negative or Gram-positive bacteria, for example, Enterobacteriaceae, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella genera, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and Shigella, and Bacillus, e.g., Bacillus subtilis and Bacillus licheniformis, such as Pseudomonas, Pseudomonas aeruginosa and Streptomyces.
除了原核细胞以外，真核微生物如丝状真菌或酵母也可作宿主细胞克隆或表达编码抗PD-1抗体的载体。酿酒酵母，或面包酵母是最常用的低等真核宿主微生物。但是，许多其他属、种和株都比较常用且在本发明中适用，如粟酒裂殖酵母；克鲁维酵母属宿主如，乳酸克鲁维酵母、脆壁克鲁维酵母(ATCC 12,424)、保加利亚克鲁维酵母(ATCC 16,045)、魏氏克鲁维酵母(ATCC 24,178)、克鲁雄酵母(ATCC 56,500)、果蝇克鲁维酵母(ATCC 36,906)、耐热克鲁维酵母和马克斯克鲁维酵母；解脂耶氏酵母(EP 402,226)；巴斯德毕赤酵母(EP 183,070)；假丝酵母；里氏木霉(EP 244,234)；链孢霉；西方许旺酵母，如：西方许旺酵母；和丝状真菌，如：脉孢菌、青霉菌、弯颈霉和曲霉菌，如：钩巢曲霉和黑曲霉。In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast can also be used as host cell clones or express vectors encoding anti-PD-1 antibodies. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are commonly used and are suitable for use in the present invention, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424) , Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wilsonii (ATCC 24,178), Kluyveromyces (ATCC 56,500), Kluyveromyces drosophila (ATCC 36,906), Kluyveromyces thermotolerant and Max Kluyveromyces; Yarrowia lipolytica (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesei (EP 244,234); Neurospora; Schwannomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Curvularia, and Aspergillus such as Aspergillus haunches and Aspergillus niger.
本发明中提供的适用于表达糖基化抗体或其抗原结合片段的宿主细胞由多细胞生物衍生得到。无脊椎细胞的实例包括植物和昆虫细胞。已发现多种杆状病毒株(baculoviralstrains)及其变体以及对应的许可性昆虫宿主细胞(permissive insect host cells)，来自于诸如以下的宿主：草地夜蛾(毛虫)、埃及斑蚊(蚊子)、白纹伊蚊(蚊子)、黑腹果蝇(果蝇)及家蚕。多种用于转染的病毒株为公众可得，例如苜蓿银纹夜蛾核型多角体病毒和家蚕核型多角体病毒的Bm-5变种，这些病毒都可在本发明中使用，特别是用于转染草地夜蛾细胞。棉花、玉米、土豆、大豆、矮牵牛花、西红柿和烟草的植物细胞培养也可用作宿主。The host cells suitable for expressing glycosylated antibodies or antigen-binding fragments thereof provided in the present invention are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculoviral strains and their variants and corresponding permissive insect host cells have been found from hosts such as: Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito) , Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and silkworm. A variety of virus strains for transfection are publicly available, such as the Bm-5 variant of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which can be used in the present invention, especially Used to transfect Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.
但是，最感兴趣的是脊椎细胞，且脊椎细胞的培养(组织培养)已经成为常规操作。可用的哺乳动物宿主细胞实例有，SV40转化的猴肾细胞CV1系(COS-7,ATCC CRL1651)；人胚胎肾细胞系(293或悬浮培养的293细胞亚克隆，Graham et al.,J.Gen Virol.36:59(1977))；幼地鼠肾细胞(BHK，ATCC CCL 10)；中国仓鼠卵巢细胞/-DHFR(CHO，Urlaub et al.,Proc.Natl.Acad.Sci.USA 77:4216(1980))；小鼠睾丸支持细胞(TM4，Mather,Biol.Reprod.23:243-251(1980))；猴肾细胞(CV1ATCC CCL 70)；非洲绿猴肾细胞(VERO-76，ATCC CRL-1587)；人宫颈癌细胞(HELA，ATCC CCL 2)；犬肾细胞(MDCK，ATCC CCL 34)；布法罗大鼠肝细胞(BRL 3A，ATCC CRL 1442)；人肺细胞(W138，ATCC CCL 75)；人肝细胞(HepG2，HB 8065)；小鼠乳腺瘤(MMT060562，ATCC CCL51)；TRI细胞(Mather等，AnnalsN.Y.Acad.Sci.383:44-68(1982))；MRC 5细胞；FS4细胞；及人肝癌细胞系(Hep G2)。在某些优选的实施方式中，所述宿主细胞是293F细胞。However, vertebrate cells are of greatest interest and their culture (tissue culture) has become routine practice. Examples of usable mammalian host cells are, SV40-transformed monkey kidney cell line CV1 (COS-7, ATCC CRL1651); Virol.36:59(1977)); Young hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc.Natl.Acad.Sci.USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL -1587); Human cervical cancer cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human hepatocytes (HepG2, HB 8065); Mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). In certain preferred embodiments, the host cell is a 293F cell.
用上述的可产生抗PD-1抗体的表达或克隆载体转化宿主细胞，并将其在常规的营养培养基中培养，所述营养培养基经修饰后适宜于诱导启动子、选择转化细胞或扩增编码目的序列的基因。Transform host cells with the expression or cloning vectors that can produce anti-PD-1 antibodies, and culture them in a conventional nutrient medium that is modified to induce promoters, select transformed cells or expand Add the gene encoding the sequence of interest.
本发明中用于产生所述抗体或其抗原结合片段的宿主细胞可在多种培养基中培养。市售的培养基如Ham's F10(Sigma)、最低基本培液(MEM，(Sigma))、RPMI-1640(Sigma)及Dulbecco's Modified Eagle's Medium(DMEM)，Sigma)可用于培养所述宿主细胞。另外，任何在Ham et al.,Meth.Enz.58:44(1979),Barnes et al.,Anal.Biochem.102:255(1980),美国专利号4,767,704；4,657,866；4,927,762；4,560,655；或5,122,469；WO 90/03430；WO87/00195；或美国专利申请Re.30,985中说明的培养基都可以用作所述宿主细胞的培养基。这些培养基都可添加必要的激素和/或其他生长因子(如胰岛素、转铁蛋白或表皮生长因子)、盐类(如氯化钠、氯化钙、氯化镁和磷酸盐)、缓冲液(如HEPES)、核苷酸(如腺苷酸和胸腺嘧啶)、抗生素(如庆大霉素)、微量元素(定义为终浓度通常在微摩尔范围无机化合物)，和葡萄糖或与之等同的能量源。所述培养基还可含有本领域公知的适当浓度的任何其他必要的添加剂。所述培养基的条件，如温度、pH值等类似条件，为选择用于表达的宿主细胞此前所使用的条件，为普通技术人员所熟知。The host cells used in the present invention to produce the antibodies or antigen-binding fragments thereof can be cultured in various media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, (Sigma)), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) can be used for culturing the host cells. Additionally, any of Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; The medium described in WO 90/03430; WO 87/00195; or US Patent Application Re. 30,985 can be used as the medium for the host cells. These media can be supplemented with necessary hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride and phosphate), buffers (such as HEPES), nucleotides (such as adenylic acid and thymine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually at final concentrations in the micromolar range), and glucose or an equivalent energy source . The medium may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the culture medium, such as temperature, pH value and the like, are the conditions previously used to select host cells for expression, and are well known to those of ordinary skill.
在使用重组技术时，所述抗体可在胞内、壁膜空间生成，或直接分泌到培养基中。如果所述抗体在胞内生成，首先除去宿主细胞或裂解片断的颗粒残骸，例如，可通过离心或超声的方法。Carter et al.,Bio/Technology 10:163-167(1992)描述了将分泌到大肠杆菌壁膜空间的抗体分离的方法。简要地说，在醋酸钠(pH 3.5)、EDTA和苯甲磺酰氟(PMSF)存在的条件下化开细胞糊(cell paste)约30分钟以上。离心除去细胞碎片。如所述抗体分泌到培养基中，则通常首先使用市售的蛋白浓度过滤器，如Amicon或Millipore Pelliconultrafiltration unit，浓缩该表达系统的上清液。在任何前述的步骤中都可加入蛋白酶抑制剂如PMSF以抑制蛋白降解，以及抗生素以防止偶然污染物的生长。When recombinant techniques are used, the antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, particulate debris of host cells or lysed fragments are first removed, for example, by centrifugation or sonication. Carter et al., Bio/Technology 10:163-167 (1992) describe a method for the isolation of antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethanesulfonyl fluoride (PMSF) over about 30 minutes. Cell debris was removed by centrifugation. If the antibody is secreted into the medium, the supernatant of the expression system is usually first concentrated using a commercially available protein concentration filter, such as Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF to inhibit protein degradation, and antibiotics to prevent growth of incidental contaminants can be added during any of the preceding steps.
从所述细胞中制得的抗体可采用纯化方法进行纯化，例如羟磷灰石色谱、凝胶电泳、透析、DEAE-纤维素离子交换色谱柱、硫酸铵沉淀、盐析以及亲和色谱，其中亲合色谱为优选的纯化技术。所述抗体的种类以及所述抗体中存在任何免疫球蛋白的Fc结构域决定了蛋白A作为亲和配体是否适合。蛋白A可用于纯化基于人γ1，γ2或γ4重链的抗体(Lindmark etal.,J.Immunol.Meth.62:1-13(1983))。蛋白G适用于所有鼠源异构体和人γ3(Guss etal.,EMBO J.5:1567 1575(1986))。琼脂糖是最常用的亲和配体附着基质，但也可选用其他基质。机械力稳定的基质如可控孔度玻璃或聚(苯乙烯)苯与用琼脂糖相比可实现更快的流速和更短的处理时间。如该抗体含有CH3结构域，则可用Bakerbond ABX.TM树脂进行纯化(J.T.Baker,Phillipsburg,N.J.)。也可根据需要获得的抗体确定其他蛋白纯化的技术，如离子交换柱中的分馏、乙醇沉淀、反相HPLC、硅胶色谱、基于阴离子或阳离子交换树脂的肝素琼脂糖凝胶色谱(如聚天冬氨酸柱)、层析聚焦、SDS-PAGE、以及硫酸铵沉淀。Antibodies prepared from said cells can be purified using purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography column, ammonium sulfate precipitation, salting out, and affinity chromatography, wherein Affinity chromatography is the preferred purification technique. The class of the antibody and the presence of any immunoglobulin Fc domains in the antibody determine the suitability of protein A as an affinity ligand. Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is suitable for all murine isoforms and human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). Agarose is the most commonly used matrix for affinity ligand attachment, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrene)benzene allow faster flow rates and shorter processing times than with agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX.TM resin (J.T. Baker, Phillipsburg, N.J.). Other protein purification techniques such as fractionation in ion-exchange columns, ethanol precipitation, reverse-phase HPLC, silica gel chromatography, heparin-sepharose chromatography based on anion or cation exchange resins (such as polyaspartic acid) can also be determined according to the antibody obtained as needed. amino acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.
在任意初步纯化步骤之后，可用低pH疏水相互作用色谱的方法处理含有感兴趣的抗体和杂质的混合物，用pH约2.5-4.5的洗涤缓冲液，优选地在低盐浓度下进行(例如，从约0到0.25M盐浓度)。After any preliminary purification steps, the mixture containing the antibody of interest and impurities can be treated by means of low pH hydrophobic interaction chromatography with a wash buffer at a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., from about 0 to 0.25M salt concentration).
试剂盒Reagent test kit
本申请提供了包括所述抗-PD-1抗体和其抗原结合片段的试剂盒。在一些实施方式中，所述试剂盒用于检测在生物样品中的PD-1的存在情况或水平。所述生物样品可以包括细胞或组织。The present application provides kits comprising the anti-PD-1 antibody and antigen-binding fragments thereof. In some embodiments, the kit is used to detect the presence or level of PD-1 in a biological sample. The biological sample may comprise cells or tissues.
在一些实施方式中，所述试剂盒包括与可检测标记缀合的抗-PD-1抗体和其抗原结合片段。在一些实施方式中，所述试剂盒包括未标记的抗-PD-1抗体和其抗原结合片段，并进一步包括能够与未标记的抗-PD-1抗体和其抗原结合片段结合标记的二抗。所述试剂盒可以进一步包括使用说明和在试剂盒中将每个组件分隔开的包装。In some embodiments, the kit includes an anti-PD-1 antibody and antigen-binding fragments thereof conjugated to a detectable label. In some embodiments, the kit comprises an unlabeled anti-PD-1 antibody and an antigen-binding fragment thereof, and further comprises a labeled secondary antibody capable of binding to the unlabeled anti-PD-1 antibody and an antigen-binding fragment thereof . The kit may further include instructions for use and packaging separating each component within the kit.
在一些实施方式中，所述抗-PD-1抗体和其抗原结合片段与底物或仪器连接用于夹心测定如ELISA或免疫色谱测定。适用的底物或仪器可以是例如微孔板和试纸。In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof are linked to substrates or instruments for sandwich assays such as ELISA or immunochromatographic assays. Suitable substrates or instruments may be, for example, microwell plates and test strips.
药物组合物和治疗方法Pharmaceutical compositions and methods of treatment
本申请进一步提供了包括所述抗-PD-1抗体和其抗原结合片段的药物组合物和一个或多个药学上可接受的载体。The present application further provides a pharmaceutical composition comprising the anti-PD-1 antibody and its antigen-binding fragment and one or more pharmaceutically acceptable carriers.
用在本申请公开的药物组合物中的药用可接受载剂可包括，例如，药用可接受的液体、凝胶或固体载剂、水相介质、非水相介质、抗微生物物质、等渗物质、缓冲液、抗氧剂、麻醉剂、悬浮剂/分散剂、螯合剂、稀释剂、佐剂、辅料或无毒辅助物质，其他本领域公知的组分或以上的多种组合。Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed in the present application may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, etc. Osmotic substances, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, adjuvants or non-toxic auxiliary substances, other components known in the art or multiple combinations of the above.
适用的组分可包括，例如，抗氧剂、填充剂、粘合剂、崩解剂、缓冲液、防腐剂、润滑剂、搅味剂、增稠剂、着色剂、乳化剂或稳定剂例如糖和环糊精。适用的抗氧剂可包括，例如，甲硫氨酸、抗坏血酸、EDTA、硫代硫酸钠、铂、过氧化氢酶、柠檬酸、半胱氨酸、巯基甘油、巯基乙酸、巯基山梨醇、丁基甲基茴香醚、丁基化羟基甲苯和/或没食子酸丙酯。如本发明所公开，在一种含有本发明公开的抗体或其抗原结合片段的组合物中包括一种或多种抗氧剂如甲硫氨酸，可将降低所述抗体或其抗原结合片段的氧化。对氧化作用的减少可防止或减少结合亲和力的降低，从而提高抗体稳定性并延长保质期。因此，在某些实施方式中，本发明提供的组合物中含有一种或多种所述的抗体或其抗原结合片段以及一种或多种抗氧剂例如甲硫氨酸。本发明进一步提供了多种方法，通过将本发明中提供的抗体或其抗原结合片段与一种或多种抗氧剂混合，例如甲硫氨酸，可防止所述抗体或其抗原结合片段氧化、延长其保质期和/或提高其活性。Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorants, thickeners, colorants, emulsifiers or stabilizers such as Sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl Anisole, Butylated Hydroxytoluene and/or Propyl Gallate. As disclosed herein, including one or more antioxidants such as methionine in a composition containing an antibody or antigen-binding fragment thereof disclosed herein will reduce the Oxidation. The reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving antibody stability and extending shelf life. Accordingly, in certain embodiments, compositions provided herein comprise one or more of the antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The invention further provides methods for preventing oxidation of the antibodies or antigen-binding fragments thereof provided herein by admixing the antibodies or antigen-binding fragments thereof with one or more antioxidants, such as methionine , prolong their shelf life and/or increase their activity.
进一步的说，药用可接受的载剂可包括，例如，水相介质如氯化钠注射液、林格氏液注射液、等渗葡萄糖注射液、无菌水注射液、或葡萄糖和乳酸林格注射液、非水介质例如：植物来源的不挥发性油、棉花子油、玉米油、芝麻油、或者花生油、细菌抑制或真菌抑制浓度下的抗菌物质、等渗剂如：氯化钠或葡萄糖、缓冲液如：磷酸盐或枸橼酸酸盐缓冲液，抗氧化剂如：硫酸氢钠，局部麻醉剂如：盐酸普鲁卡因，助悬剂和分散剂如：羧甲基纤维素钠、羟丙基甲基纤维素或聚乙烯吡咯烷酮，乳化剂如：聚山梨醇酯80(吐温-80)、螯合试剂如EDTA(乙二胺四乙酸)或EGTA(乙二醇双(2-氨基乙基醚)四乙酸)、乙醇、聚乙二醇、丙二醇、氢氧化钠、盐酸、柠檬酸或乳酸。作为载剂的抗菌剂可加入多次剂量容器中的药物组合物中，其包括酚类或甲酚、汞制剂、苯甲醇、氯代丁醇、甲基和丙基对羟基苯甲酸酯、噻汞撒、氯苯甲烷铵和氯苯乙铵。适用的辅料可包括，例如，水、盐、葡萄糖、甘油或乙醇。适用的无毒辅助物质可包括，例如，乳化剂、pH值缓冲剂、稳定剂、增溶剂，或者醋酸钠、去水山梨糖醇月桂酸酯、三乙醇胺油酸酯或者环糊精之类的物质。Further, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution injection, isotonic glucose injection, sterile water injection, or glucose and lactate injection. injection, non-aqueous media such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose , buffers such as: phosphate or citrate buffer, antioxidants such as: sodium bisulfate, local anesthetics such as: procaine hydrochloride, suspending and dispersing agents such as: sodium carboxymethylcellulose, hydroxyl Propylmethylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-amino ethyl ether) tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents as carriers can be added to pharmaceutical compositions in multi-dose containers and include phenols or cresols, mercury agents, benzyl alcohol, chlorobutanol, methyl and propyl parabens, Thiamrosal, Methalkonium Chloride, and Ethonium Chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrins. substance.
所述药物组合物可以是液体溶液、悬浮液、乳剂、丸剂、胶囊、片剂、持续释放制剂或粉末。口服制剂可以包括标准载体如药物级的甘露醇、乳糖、淀粉、硬脂酸镁、聚乙烯吡咯烷酮、糖精钠、纤维素、碳酸镁等。The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.
在某些实施方式中，所述药物组合物被制剂成可注射的组合物。可注射的药物组合物可以任何常规的形式制备，例如，液体溶剂、悬浮剂、乳化剂或适用于产生液体溶剂、悬浮剂或乳化剂的固体形式。注射制剂可包括现用的无菌和/或无热原溶液、使用前现与溶剂结合的无菌干燥的可溶物，如冻干粉，包括皮下片、注射即用的无菌悬浮剂、使用前现与介质结合的无菌干燥不溶产品，和无菌和/或无热原的乳剂。溶剂可以为水相或非水相。In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, for example, liquid solvents, suspensions, emulsifications or solid forms suitable for liquid solvents, suspensions, or emulsifications. Preparations for injection may include ready-to-use sterile and/or pyrogen-free solutions, sterile dry solubles that are combined with solvents just before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, Sterile dry insoluble products, and sterile and/or pyrogen-free emulsions, to be combined with a vehicle just before use. The solvent can be aqueous or non-aqueous.
在某些实施方式中，单位剂量的注射制剂包装在一个安瓿、一支管或一支带有针的针筒中。本领域习知，所有注射给药的制剂应为无菌无热原。In certain embodiments, the unit dose injection formulation is packaged in an ampoule, a tube or a syringe with a needle. As is known in the art, all preparations for parenteral administration should be sterile and pyrogen-free.
在某些实施方式中，通过将本申请公开的抗体或其抗原结合片段溶解于某适当的溶剂中可制备无菌冻干的粉末。所述溶剂可含有一种可提高粉或由粉末制得的重组溶液的稳定性，或改善粉末或重组溶液的其他药理组分。适用的辅料包括，但不限于，水、葡萄糖、三梨糖醇、果糖、玉米糖浆、木糖醇、甘油、葡萄糖、蔗糖或其他适用的物质。溶剂可含有缓冲液，如枸橼酸缓冲液、磷酸钠或磷酸钾缓冲液或其他本技术熟练人员公知的缓冲液，在一种实施方式中，缓冲液的pH为中性。在本领域公知的标准条件下进行对所述溶解进行随后的过滤除菌，然后冻干制得理想的制剂。在一种实施方式中，将所得的溶剂分装至小管中冻干。每支小管可容纳单次剂量或多次剂量的所述抗-PD-1抗体或其抗原结合片段或其组合物。每支小管中的装入量可略微高于每次剂量所需或多次剂量所需(例如10％过量)，从而保证取样精确和给药精确。冻干粉可在适当的条件下储存，如在约4℃到室温范围。In certain embodiments, sterile lyophilized powders can be prepared by dissolving an antibody or antigen-binding fragment thereof disclosed herein in an appropriate solvent. The solvent may contain an additional pharmacological component that increases the stability of the powder or reconstituted solution prepared from the powder, or improves the powder or reconstituted solution. Suitable excipients include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, dextrose, sucrose, or other suitable substances. The solvent may contain a buffer, such as citrate buffer, sodium or potassium phosphate buffer or other buffers known to those skilled in the art, and in one embodiment, the pH of the buffer is neutral. Subsequent filter sterilization of the lyophilization followed by lyophilization yields the desired formulation under standard conditions well known in the art. In one embodiment, the resulting solvent is aliquoted into vials and lyophilized. Each vial can contain a single dose or multiple doses of the anti-PD-1 antibody or antigen-binding fragment or composition thereof. The amount loaded into each vial can be slightly higher than that required for each dose or multiple doses (eg, 10% excess) to ensure accurate sampling and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.
用注射用水将冻干粉重溶得到用于注射给药的制剂。在一种实施方式中，可将冻干粉加至无菌无热原水或其他适用的液体载剂中重溶。精确的量由选择的疗法决定，可根据经验值决定。The lyophilized powder is redissolved with water for injection to obtain a preparation for injection. In one embodiment, the lyophilized powder can be added to sterile pyrogen-free water or other suitable liquid carriers for reconstitution. The precise amount is determined by the chosen therapy and can be determined empirically.
还提供了治疗方法，包括将治疗有效量的本申请所述的抗体或其抗原结合片段施用给需要其的受试者，由此治疗或预防与PD-1相关的状况或病症。在另一方面，还提供了治疗会从上调的免疫响应获益的受试者的状况的方法，包括对所述需要其的受试者施用治疗有效量的本申请所述的抗体或其抗原结合片段。Also provided are methods of treatment comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein, thereby treating or preventing a condition or disorder associated with PD-1. In another aspect, there is also provided a method of treating a condition in a subject that would benefit from an upregulated immune response, comprising administering to said subject in need thereof a therapeutically effective amount of an antibody or antigen thereof described herein Combine fragments.
本申请中提供的抗体或其抗原结合片段的治疗有效剂量依赖于本领域公知的多种因素，例如体重、年龄、过往病史、现用治疗、对象的健康状况和交叉感染的潜力、过敏、超敏和副作用，以及给药途径和肿瘤发展的程度。本领域熟练人员(例如医生或兽医)可根据这些或其它条件或要求按比例降低或升高剂量。Therapeutically effective doses of the antibodies or antigen-binding fragments thereof provided herein depend on a variety of factors known in the art, such as body weight, age, past medical history, current therapy, the subject's health and potential for cross-infection, allergies, Sensitivity and side effects, as well as route of administration and extent of tumor development. Those skilled in the art (eg, physician or veterinarian) may proportionally reduce or increase dosages according to these or other conditions or requirements.
在某些实施方式中，本发明提供的抗体或其抗原结合片段可在治疗有效剂量约0.01mg/kg到约100mg/kg之间给药(例如，约0.01mg/kg、约0.5mg/kg、约1mg/kg、约2mg/kg、约5mg/kg、约10mg/kg、约15mg/kg、约20mg/kg、约25mg/kg、约30mg/kg、约35mg/kg、约40mg/kg、约45mg/kg、约50mg/kg、约55mg/kg、约60mg/kg、约65mg/kg、约70mg/kg、约75mg/kg、约80mg/kg、约85mg/kg、约90mg/kg、约95mg/kg或约100mg/kg)。在某些实施方式中，所述抗体或其抗原结合片段以约50mg/kg或更少的剂量给药，在某些实施方式中，给药剂量为10mg/kg或更少、5mg/kg或更少、1mg/kg或更少、0.5mg/kg或更少或0.1mg/kg或更少。某特定剂量可在多个间隔给药，例如每天一次、每天两次或更多、每月两次或更多、每周一次、每两周一次、每三周一次、每月一次或每两月或更多月一次。在某些实施方式中，给药剂量可随治疗进程变化。例如，在某些实施方式中，初始给药剂量可比后续给药剂量高。在某些实施方式中，给药剂量在治疗进程中根据给药对象的反应进行调整。In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein may be administered at a therapeutically effective dose of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg , about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg , about 45mg/kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, about 70mg/kg, about 75mg/kg, about 80mg/kg, about 85mg/kg, about 90mg/kg , about 95 mg/kg or about 100 mg/kg). In certain embodiments, the antibody or antigen-binding fragment thereof is administered at a dose of about 50 mg/kg or less, in certain embodiments, the dose administered is 10 mg/kg or less, 5 mg/kg or Less, 1 mg/kg or less, 0.5 mg/kg or less or 0.1 mg/kg or less. A given dose may be given at multiple intervals, such as once a day, twice a day or more, twice a month or more, once a week, every two weeks, every three weeks, once a month, or every two weeks Once a month or more. In certain embodiments, the dosage administered may vary over the course of the treatment. For example, in certain embodiments, the initial dose administered may be higher than the subsequent dose administered. In certain embodiments, the dose administered is adjusted during the course of treatment based on the response of the subject.
给药方案可通过调整达到最优反应(如治疗反应)。例如，可进行单剂量给药或在一段时间分多个分隔的剂量给药。Dosage regimens may be adjusted to achieve an optimal response (eg, a therapeutic response). For example, a single dose may be administered or multiple divided doses may be administered over time.
本发明中公开的抗体和抗原结合片段可通过本领域公知的给药方式给药，例如注射给药(如，皮下注射、腹腔注射、静脉注射，包括静脉滴注，肌肉注射或皮内注射)或非注射给药(如，口服给药、鼻腔给药、舌下给药、直肠给药或外用给药)。The antibodies and antigen-binding fragments disclosed in the present invention can be administered by administration methods known in the art, such as injection administration (such as subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection or intradermal injection) Or non-injection administration (eg, oral administration, nasal administration, sublingual administration, rectal administration or topical administration).
与PD-1相关的状况和病症可以是免疫相关的疾病或病症。在某些实施方式中，所述与PD-1相关的状况和病症包括肿瘤和癌症、例如非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycoses fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵隔大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。在某些实施方式中，所述肿瘤和癌症是转移性的，尤其是表达PD-L1的转移性肿瘤。在某些实施方式中，所述与PD-1相关的状况和病症包括自体免疫疾病、如系统性红斑狼疮(SLE)、银屑病、系统性硬皮病、自身免疫性糖尿病。在某些实施方式中，所述与PD-1相关的状况和病症包括慢性病毒感染，例如乙型肝炎，丙型肝炎，疱疹病毒，Epstein-Barr病毒，艾滋病毒，巨细胞病毒，单纯疱疹病毒I型，单纯疱疹病毒2型，人乳头状瘤病毒，腺病毒的病毒感染，卡波西西肉瘤相关的疱疹病毒流行病，薄环病毒(Torquetenovirus)，JC病毒或BK病毒等。Conditions and disorders associated with PD-1 can be immune-related diseases or disorders. In certain embodiments, the conditions and disorders associated with PD-1 include tumors and cancers, such as non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, Stomach cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mushroom mycoses fungoids, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, B cell-rich T cells/histiocytic Lymphoma, EBV-positive and negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion Lymphoma, Hodgkin's lymphoma, central nervous system (CNS) tumors such as primary CNS lymphoma, spine tumors, brainstem gliomas. In certain embodiments, the tumors and cancers are metastatic, especially PD-L1 expressing metastatic tumors. In certain embodiments, the conditions and disorders associated with PD-1 include autoimmune diseases such as systemic lupus erythematosus (SLE), psoriasis, systemic scleroderma, autoimmune diabetes. In certain embodiments, the conditions and disorders associated with PD-1 include chronic viral infections, such as hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus Type I, herpes simplex virus type 2, human papilloma virus, adenovirus infection, Kaposi's sarcoma-associated herpes virus epidemics, Torquetenovirus, JC virus or BK virus, etc.
使用方法Instructions
本申请进一步提供了使用所述抗-PD-1抗体或其抗原结合片段的方法。The present application further provides methods of using the anti-PD-1 antibodies or antigen-binding fragments thereof.
在一些实施方式中，本申请提供了在个体中治疗与PD-1相关的状况或病症的方法，包括施用治疗有效量的本申请所述的PD-1抗体或其抗原结合片段。在一些实施方式中，所述个体被鉴定为患有可能对PD-1拮抗剂响应的病症或状况。In some embodiments, the present application provides a method of treating a condition or disorder associated with PD-1 in an individual, comprising administering a therapeutically effective amount of a PD-1 antibody or an antigen-binding fragment thereof described herein. In some embodiments, the individual is identified as having a disorder or condition that is likely to be responsive to a PD-1 antagonist.
在目标生物组织中PD-L1的存在情况和水平可以指示所述生物样品来源的个体是否可能对PD-1拮抗剂响应。可以使用多种方法在来自所述个体的待测生物样品中确定PD-L1的存在情况或水平。例如，可以将所述待测生物样品暴露于抗-PD-L1抗体或其抗原结合片段，其与表达的PD-L1蛋白结合并检测表达的PD-L1蛋白。另选地，可以使用如qPCR、反转录PCR、微阵列、SAGE、FISH等在核酸表达水平检测PD-L1。在一些实施方式中，所述待测样品来源于癌细胞或组织，或进入肿瘤的免疫细胞。在一些实施方式中，在所述待测生物样品中PD-L1的存在或水平上调表示响应的可能性。本申请使用的术语“上调”是指与使用相同抗体检测的参照样品中PD-L1蛋白水平相比，使用本申请所述的抗体或其抗原结合片段在待测样品中检测的PD-L1蛋白水平的总的增加不少于10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％或更多。所述参照样品可以是从健康或无疾病的个体中获得的对照样品，或从待测样品来源的个体中获得的健康或无疾病的样品。例如，所述参照样品可以是在待测样品(如肿瘤)附近或相邻的无疾病样品。The presence and level of PD-L1 in the biological tissue of interest can indicate whether the individual from which the biological sample is derived is likely to respond to a PD-1 antagonist. The presence or level of PD-L1 in the test biological sample from the individual can be determined using a variety of methods. For example, the test biological sample can be exposed to an anti-PD-L1 antibody or antigen-binding fragment thereof, which binds to and detects expressed PD-L1 protein. Alternatively, PD-L1 can be detected at the nucleic acid expression level using, for example, qPCR, reverse transcription PCR, microarray, SAGE, FISH, etc. In some embodiments, the sample to be tested is derived from cancer cells or tissues, or immune cells that enter tumors. In some embodiments, the presence or increased level of PD-L1 in said test biological sample indicates the likelihood of a response. The term "up-regulation" as used herein refers to the PD-L1 protein detected in a test sample using an antibody or antigen-binding fragment thereof described herein, compared to the PD-L1 protein level in a reference sample detected using the same antibody The total increase in level is not less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more. The reference sample can be a control sample obtained from a healthy or disease-free individual, or a healthy or disease-free sample obtained from the individual from which the sample to be tested is derived. For example, the reference sample can be a disease-free sample near or adjacent to the sample to be tested (eg, a tumor).
本发明公开的抗体和抗原结合片段可单独给药或与一种或多种其他治疗手段或物质联合给药。例如，本发明公开的抗体和抗原结合片段可与化疗、放疗、癌症治疗手术(如肿瘤切除术)、一种或多种抗呕吐药或其他化疗导致的并发症的疗法、或任何其他用于癌症的治疗物质或任何由PD-1介导的病症的治疗物质进行联用。在某些这样的实施方式中，本发明公开的抗体和抗原结合片段与一种或多种治疗物质联用时，可与所述的一种或多种治疗物质同时给药，在某些这样的实施方式中，所述的抗体和抗原结合片段可作为同一个药物组合物的一部分同时给药。但是，与其他治疗物质“联用”的抗体和抗原结合物不需要同时给药或与该治疗物质在同一组合物中给药。本发明中“联用”的含义还包括在另一个治疗物质之前或之后给药的抗体和抗原结合物也被认为是与该治疗物质“联用”，即使所述抗体或其抗原结合片段与第二种物质通过不同给药方式给药。在可能的情况下，与本发明公开的抗体或其抗原结合片段联用的其他治疗物质可参照该其他治疗物质的产品说明书的方法用药，或参照外科医生的案头参考书2003(Physicians'Desk Reference，57th Ed；MedicalEconomics Company；ISBN:1563634457；第57版(2002年11月))，或参照其他本领域公知的方法。The antibodies and antigen-binding fragments disclosed herein may be administered alone or in combination with one or more other therapeutic means or substances. For example, the antibodies and antigen-binding fragments disclosed herein can be combined with chemotherapy, radiation therapy, cancer treatment surgery (such as tumor resection), one or more anti-emetic drugs or other therapy for complications caused by chemotherapy, or any other therapy for Therapeutic substance for cancer or any therapeutic substance mediated by PD-1. In certain such embodiments, when the antibodies and antigen-binding fragments disclosed herein are used in combination with one or more therapeutic substances, the one or more therapeutic substances may be administered concurrently, and in some such In an embodiment, the antibody and antigen-binding fragment may be administered simultaneously as part of the same pharmaceutical composition. Antibodies and antigen conjugates "in combination" with other therapeutic substances, however, need not be administered at the same time or in the same composition as the therapeutic substance. The meaning of "combined use" in the present invention also includes that antibodies and antigen conjugates administered before or after another therapeutic substance are also considered to be "combined with" the therapeutic substance, even if the antibody or antigen-binding fragment thereof is combined with the therapeutic substance. The second substance is administered by a different mode of administration. Where possible, other therapeutic substances used in combination with the antibodies or antigen-binding fragments thereof disclosed in the present invention can be administered by referring to the product instructions of the other therapeutic substances, or referring to the surgeon's desk reference book 2003 (Physicians' Desk Reference , 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)), or by reference to other methods known in the art.
在某些实施方式中，所述治疗物质能够诱导或增强针对癌症的免疫反应。例如，肿瘤疫苗可以用于诱导对某些肿瘤或癌症的免疫应答。细胞因子治疗可以用于提高将肿瘤抗原向免疫系统的递呈。细胞因子治疗的示例包括但不限于干扰素如干扰素α、β和γ，集落刺激因子如巨噬细胞CSF、粒细胞巨噬细胞CSF和粒细胞-CSF，白介素如IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11和IL-12，肿瘤坏死因子如TNF-α和TNF-β。还可以使用灭活免疫抑制目标的试剂，如TGF-β抑制剂、IL-10抑制剂和Fas配体抑制剂。另一组试剂包括激活针对肿瘤或癌细胞的免疫响应的那些试剂，例如，提高T细胞激活(如T细胞共刺激分子激动剂如CTLA-4、ICOS和OX-40)的那些，以及提高树突细胞功能和抗原递呈的那些。In certain embodiments, the therapeutic substance is capable of inducing or enhancing an immune response against cancer. For example, tumor vaccines can be used to induce an immune response to certain tumors or cancers. Cytokine therapy can be used to increase the presentation of tumor antigens to the immune system. Examples of cytokine therapy include, but are not limited to, interferons such as interferon alpha, beta, and gamma, colony-stimulating factors such as macrophage CSF, granulocyte-macrophage CSF, and granulocyte-CSF, interleukins such as IL-1, IL-1α , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, tumor necrosis factors such as TNF -α and TNF-β. Agents that inactivate immunosuppressive targets, such as TGF-beta inhibitors, IL-10 inhibitors, and Fas ligand inhibitors, can also be used. Another group of agents includes those that activate the immune response against tumor or cancer cells, e.g., those that increase T cell activation (such as T cell co-stimulatory molecular agonists such as CTLA-4, ICOS, and OX-40), and those that increase tree those of synaptic cell function and antigen presentation.
以下实施例旨在更好地说明本发明，且不应理解为限制本发明的范围。所有下述的特定组合物、材料和方法，其整体或部分，都在本发明的范围内。这些特定的组合物、材料和方法不是为了限制本发明，而只是为说明特定的实施方式在本发明的范围内。本领域熟练技术人员可不添加创造性及不偏离本发明范围而开发出等同的组合物、材料和方法。应理解，在对本发明的方法作出的多种改动可以仍然包括在本发明范围内。发明人意在将这样的变动包括在本发明的范围内。The following examples are intended to better illustrate the present invention and should not be construed as limiting the scope of the present invention. All specific compositions, materials and methods described below, in whole or in part, are within the scope of the invention. The particular compositions, materials and methods are not intended to limit the invention but merely to illustrate that particular embodiments are within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials and methods without adding inventive step and without departing from the scope of the present invention. It should be understood that various modifications to the methods of the present invention may still be included within the scope of the present invention. The inventors intend to include such variations within the scope of the present invention.
实施例1：抗体杂交瘤的生成Example 1: Generation of Antibody Hybridomas
1.1免疫原的生成：合成编码PD-1和PD-L1的ECD的或二者全长的DNA并插入表达载体pcDNA3.3。大量制备质粒DNA并经测序验证插入的DNA序列。PD-1ECD和PD-L1ECD的融合蛋白包含多种标记，包括人源Fc、小鼠Fc和His标记，所述融合蛋白通过将人PD-1ECD基因转染进入CHO-S或HEK293细胞制备。5天后，将从所述瞬时转染的细胞培养中收获的上清液用于蛋白纯化。将所述融合蛋白纯化并定量以用于免疫和筛选。1.1 Generation of immunogen: Synthesize the ECD encoding PD-1 and PD-L1 or both full-length DNAs and insert them into the expression vector pcDNA3.3. Plasmid DNA was prepared in bulk and the inserted DNA sequence was verified by sequencing. The fusion protein of PD-1ECD and PD-L1ECD contains multiple tags, including human Fc, mouse Fc and His tags, and the fusion protein is prepared by transfecting human PD-1ECD gene into CHO-S or HEK293 cells. After 5 days, supernatants harvested from the transiently transfected cell cultures were used for protein purification. The fusion protein was purified and quantified for immunization and screening.
1.2建立稳定细胞系。为了获得用于抗体筛选和验证的工具，我们建立了PD-1和PD-L1转染细胞系。简言之，根据厂商说明书使用Lipofectamine 2000转染试剂盒将含有全长PD-1或PD-L1的pCND3.3表达载体转染CHO-K1、293F或Ba/F3细胞。转染后48-72小时，在含有用于选择的杀稻瘟菌素(Blasticidin)或G418的培养基中培养所述转染细胞。一段时间后，会选择出在基因组DNA中稳定掺入了PD-1或PD-L1基因的细胞。同时，验证所述细胞是否具有目的基因PD-1和PD-L1的表达。一旦验证了所述表达，通过有限的稀释挑选目的单个克隆并放大到大容量。随后将建立的单克隆细胞系在含有低剂量抗生素杀稻瘟菌素(Blasticidin)或G418的培养基中维持。1.2 Establish a stable cell line. To obtain tools for antibody screening and validation, we established PD-1 and PD-L1 transfected cell lines. Briefly, CHO-K1, 293F or Ba/F3 cells were transfected with pCND3.3 expression vector containing full-length PD-1 or PD-L1 using Lipofectamine 2000 transfection kit according to the manufacturer's instructions. 48-72 hours after transfection, the transfected cells were cultured in media containing Blasticidin or G418 for selection. Over time, cells are selected for stably incorporating the PD-1 or PD-L1 gene into their genomic DNA. At the same time, verify whether the cells have the expression of target genes PD-1 and PD-L1. Once the expression was verified, single clones of interest were picked by limiting dilution and scaled up to large capacity. The established monoclonal cell lines were then maintained in media containing low doses of the antibiotic Blasticidin or G418.
1.3抗体杂交瘤的建立。1.3 Establishment of antibody hybridomas.
1.3.1免疫和细胞融合：使用8-10周龄OMT-大鼠(获自Open Monoclonal Technology,Inc.,Palo Alto,US)用10μg TiterMax中的人源PD-1ECD蛋白在足垫上进行初次激发免疫，每3天用铝剂配置的PD-1ECD重复进行免疫。每2周对大鼠取血收集血清并通过ELISA或FACS测试测定抗体滴度。当所述抗体滴度达到足够高时，对大鼠给予最后的不含佐剂的激发(加入100μl 1XPBS替代)，按如下步骤进行细胞融合：将从免疫的OMT-大鼠的淋巴结中分离的B淋巴细胞与骨髓瘤细胞进行细胞融合(以1:1比例)。用5-10ml ECF溶液洗涤并悬浮细胞混合物。加入ECF溶液将浓度调整至2x106细胞/ml。在细胞电融合后，将融合室中的细胞悬液立即转移进入含有更多体积培养基的无菌管中。在37℃培养超过24小时后，将所述细胞悬液混合并移液入96孔板(0.5x106细胞/板)。在37℃、5％CO2条件下培养细胞。当所述克隆足够大时，将100μl上清液从96孔板转移用于抗体筛选测试。1.3.1 Immunization and cell fusion: 8-10 week-old OMT-rats (obtained from Open Monoclonal Technology, Inc., Palo Alto, US) were first challenged on the footpad with 10 μg of human PD-1ECD protein in TiterMax Immunization was repeated every 3 days with PD-1ECD formulated with aluminum agent. Rats were bled every 2 weeks to collect serum and assay antibody titers by ELISA or FACS tests. When the antibody titer reached a high enough level, the rat was given a final challenge without adjuvant (100 μl 1XPBS was added instead), and cell fusion was carried out as follows: the lymph nodes isolated from the immunized OMT-rat B lymphocytes were fused with myeloma cells (at a 1:1 ratio). Wash and suspend the cell mixture with 5-10 ml ECF solution. Add ECF solution to adjust the concentration to 2x106 cells/ml. Immediately after cell electrofusion, transfer the cell suspension in the fusion chamber into a sterile tube containing a further volume of medium. After incubation at 37° C. for more than 24 hours, the cell suspension was mixed and pipetted into 96-well plates (0.5×106 cells/plate). Cells were cultured at 37°C, 5% CO2 . When the clones were large enough, 100 μl of the supernatant was transferred from the 96-well plate for antibody screening tests.
1.3.2杂交瘤上清液的第一轮和确认筛选：使用ELISA测试作为第一轮筛选方法以测试杂交瘤上清液与PD-1蛋白的结合。简言之，用1μg/ml的人源PD-1胞外结构域的可溶性蛋白在4℃包被平板过夜。在封闭和洗涤后，将所述杂交瘤上清液转移至所述包被的平板并在室温下孵育1小时。之后洗涤所述平板并随后用山羊抗大鼠IgG1HRP(Bethyl)和山羊抗大鼠IgG2b HRP(Bethyl)的二抗孵育45分钟。洗涤后，加入TMB底物并用2M HCl终止反应。使用酶标仪(Molecular Device)读取450nm处的吸收光值。为了确认PD-1抗体与在细胞膜上表达的构象PD-1分子的天然结合，在PD-1转染的CHO-S细胞系上进行FACS分析。以1x106细胞/ml的浓度将表达PD-1的CHO-S细胞转移至96孔U形底平板(BD)。随后将所述杂交瘤上清液转移至所述平板并在4℃下孵育1小时。用1XPBS/1％BSA洗涤后，加入山羊抗大鼠FITC(JacksonImmunoresearch Lab)二抗并在4℃下与细胞避光孵育1小时。之后洗涤所述细胞并在1XPBS/1％BSA中重悬或在4％福尔马林中固定所述细胞，并以流式细胞仪(BD)分析。使用相同方法进行抗体与母本CHO-S细胞系的结合。图1显示了抗人源PD-1抗体与表达PD-1的CHO细胞的结合。将转染了全长的人源PD-1的CHO细胞用来自大鼠杂交瘤的抗人源PD-1抗体结合，随后用FITC缀合的山羊抗大鼠-IgG Fc进行二抗染色并通过FACS分析。数据显示所述抗体特异性地与CHO细胞上表达的PD-1结合。1.3.2 First-round and confirmation screening of hybridoma supernatants: ELISA assay was used as the first-round screening method to test the binding of hybridoma supernatants to PD-1 protein. Briefly, plates were coated with 1 μg/ml soluble protein of human PD-1 extracellular domain overnight at 4°C. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubated for 1 hour at room temperature. The plates were then washed and subsequently incubated with secondary goat anti-rat IgG1 HRP (Bethyl) and goat anti-rat IgG2b HRP (Bethyl) antibodies for 45 minutes. After washing, TMB substrate was added and the reaction was stopped with 2M HCl. The absorbance value at 450 nm was read using a microplate reader (Molecular Device). To confirm the natural binding of the PD-1 antibody to conformational PD-1 molecules expressed on the cell membrane, FACS analysis was performed on the PD-1 transfected CHO-S cell line. Transfer PD-1-expressing CHO-S cells to 96-well U-bottom plates (BD) at a concentration of 1x106 cells/ml. The hybridoma supernatants were then transferred to the plate and incubated at 4°C for 1 hour. After washing with 1XPBS/1%BSA, goat anti-rat FITC (Jackson Immunoresearch Lab) secondary antibody was added and incubated with the cells at 4°C for 1 hour in the dark. The cells were then washed and resuspended in 1XPBS/1% BSA or fixed in 4% formalin and analyzed by flow cytometry (BD). Antibody binding to the parental CHO-S cell line was performed using the same method. Figure 1 shows the binding of anti-human PD-1 antibody to PD-1 expressing CHO cells. CHO cells transfected with full-length human PD-1 were bound with anti-human PD-1 antibody from rat hybridoma, followed by secondary antibody staining with FITC-conjugated goat anti-rat-IgG Fc and passed through FACS analysis. The data show that the antibody specifically binds to PD-1 expressed on CHO cells.
为了测试所述抗体与人源CD4+T细胞上表达的天然PD-1的结合亲和性，从在IL-2和OKT3中培养3天的PBMC培养物中获得人源CD4+T细胞，并用抗人源PD-1抗体与之结合。所述抗体与所述T细胞上的PD-1的结合通过FACS进行分析。如图3所示，FACS分析显示所述抗体特异性的与CD4+T细胞上表达的天然PD-1结合。In order to test the binding affinity of the antibody to native PD-1 expressed on human CD4+ T cells, human CD4+ T cells were obtained from PBMC cultures cultured in IL-2 and OKT3 for 3 days, and used Anti-human PD-1 antibody binds to it. Binding of the antibody to PD-1 on the T cells was analyzed by FACS. As shown in Figure 3, FACS analysis showed that the antibody specifically combined with natural PD-1 expressed on CD4+ T cells.
测试抗体的阻断活性，作为确认筛选以选择潜在的目标抗体。通过FACS分析，测试所选择的抗体对配体PD-L1与转染PD-1的CHO-S细胞的结合的阻断能力。以1x106细胞/ml的浓度将表达人源PD-1的CHO-S细胞转移至96孔U形底平板(BD)中。将抗体在洗涤缓冲液(1XPBS/1％BSA)中连续稀释并在4℃下孵育细胞1小时。洗涤后，加入人源Fc融合-人源PD-L1蛋白并在4℃下孵育1小时。将山羊抗人源IgG Fc FITC抗体(与大鼠IgG Fc没有交叉反应性，Jackson Immunoresearch Lab)的二抗与细胞在4℃下避光孵育1小时。之后洗涤所述细胞并在1XPBS/1％BSA中重悬或在4％福尔马林中固定，并通过流式细胞仪(BD)分析。Antibodies are tested for blocking activity as a confirmatory screen to select potential antibodies of interest. The selected antibodies were tested for their ability to block the binding of the ligand PD-L1 to PD-1 transfected CHO-S cells by FACS analysis. CHO-S cells expressing human PD-1 were transferred to a 96-well U-bottom plate (BD) at a concentration of 1x106 cells/ml. Antibodies were serially diluted in wash buffer (1XPBS/1%BSA) and cells were incubated at 4°C for 1 hour. After washing, human Fc fusion-human PD-L1 protein was added and incubated at 4°C for 1 hour. The secondary antibody of goat anti-human IgG Fc FITC antibody (no cross-reactivity with rat IgG Fc, Jackson Immunoresearch Lab) was incubated with the cells for 1 hour at 4°C in the dark. The cells were then washed and resuspended in 1XPBS/1%BSA or fixed in 4% formalin and analyzed by flow cytometry (BD).
1.3.3杂交瘤亚克隆：一旦通过第一轮和确认筛选验证了特异性结合和阻断，可以使用所述阳性杂交瘤细胞系进行亚克隆。简言之，对于每个杂交瘤细胞系，将细胞进行计数并在克隆培养基中稀释至5细胞/孔、1细胞/孔和0.5细胞/孔。将200μl/孔铺板入96孔板，一个平板为5细胞/孔，一个平板为1细胞/孔和四个平板为0.5细胞/孔。将所有平板置于37℃、5％CO2。孵育直至所有细胞系可以通过ELISA测试进行检查。1.3.3 Hybridoma subcloning: Once the specific binding and blocking are verified by the first round and confirmatory screening, the positive hybridoma cell line can be used for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted to 5 cells/well, 1 cell/well and 0.5 cells/well in cloning medium. 200 μl/well were plated into 96-well plates at 5 cells/well for one plate, 1 cell/well for one plate and 0.5 cells/well for four plates. All plates were placed at 37°C, 5%CO2 . Incubate until all cell lines can be checked by ELISA test.
实施例2：抗体杂交瘤细胞测序和全人源抗体表征Example 2: Sequencing of antibody hybridoma cells and characterization of fully human antibodies
2.1抗体杂交瘤细胞测序：用Trizol试剂从单克隆杂交瘤细胞中分离RNA。用以下方案扩增PD-1抗体的VH和VL：简言之，首先如本申请描述的使用反转录酶将RNA反转录成cDNA，反应系统(20μl)：2.1 Sequencing of antibody hybridoma cells: RNA was isolated from monoclonal hybridoma cells with Trizol reagent. Use the following protocol to amplify the VH and VL of the PD-1 antibody: Briefly, first use reverse transcriptase to reverse transcribe RNA into cDNA as described in this application, reaction system (20 μl):
反应条件Reaction conditions
使用所得的cDNA作为模板用于随后的使用针对目的基因的特异性引物的PCR扩增。所述PCR反应通过以下步骤实施：The resulting cDNA was used as template for subsequent PCR amplification using specific primers for the gene of interest. Described PCR reaction is implemented through the following steps:
反应条件：Reaction conditions:
取10μl的PCR反应产物进行与pMD18-T载体的连接反应。用10μl的连接产物转化Top10感受态细胞并将所述混合物转移至按照标准方案预热的2-YT+Cab平板上，孵育过夜。使用M13-48和M13-47引物通过PCR检查阳性克隆，随后进行测序。Take 10 μl of the PCR reaction product for ligation reaction with pMD18-T vector. Top10 competent cells were transformed with 10 μl of the ligation product and the mixture was transferred to 2-YT+Cab plates pre-warmed according to the standard protocol and incubated overnight. Positive clones were checked by PCR using M13-48 and M13-47 primers, followed by sequencing.
2.2全人源抗体分子构建：按上文的表述将PD-1抗体的VH和VL进行扩增。所述PCR反应产物通过PCR clean-up试剂盒进行纯化并用限制性酶Pme I和BssH II在37℃消化VL和pCI载体2小时。将所述反应产物在1％的琼脂糖凝胶中进行电泳并按照厂商说明书进行凝胶提取。用以下步骤连接经消化的VL和pCI载体：2.2 Fully human antibody molecule construction: Amplify the VH and VL of the PD-1 antibody as described above. The PCR reaction product was purified by a PCR clean-up kit, and the VL and pCI vectors were digested with restriction enzymes Pme I and BssH II at 37° C. for 2 hours. The reaction products were electrophoresed on a 1% agarose gel and gel extracted according to the manufacturer's instructions. Ligate the digested VL and pCI vectors using the following steps:
在16℃下孵育所述混合物30分钟。用10μl反应产物进行转化和克隆增长。使用确认的克隆进行质粒pCI-VL DNA的提取。随后将所述pCI-VL载体和VH片段用Xbal和Sal I进行消化并用T4DNA连接酶在16℃下连接纯化的经消化的VH和载体30分钟。一旦经测序验证插入的VL和VH的序列，使用包含全人源PD-1抗体的整个IgG的表达载体进行瞬时转染和建立稳定细胞系。The mixture was incubated at 16°C for 30 minutes. 10 μl of the reaction product was used for transformation and clonal growth. The confirmed clones were used for extraction of plasmid pCI-VL DNA. The pCI-VL vector and VH fragments were then digested with Xbal and Sal I and the purified digested VH and vector were ligated with T4 DNA ligase at 16°C for 30 minutes. Once the sequence of the inserted VL and VH was verified by sequencing, an expression vector containing the whole IgG of the fully human PD-1 antibody was used for transient transfection and establishment of stable cell lines.
实施例3：全人源抗体的表征Example 3: Characterization of Fully Human Antibodies
3.1表面等离子体共振(SPR)测定的全动力学结合亲和性：通过SPR法使用ProteOnXPR36(Bio-Rad)对抗体与PD-1的亲和性和结合动力学进行表征。将蛋白A蛋白(Sigma)通过胺偶联固定于GLM传感芯片上(Bio-Rad)。使纯化的抗体流过传感器芯片并被蛋白A捕获。将芯片旋转90°并用电泳缓冲液洗涤(1XPBS/0.01％Tween20,Bio-Rad)直至基线稳定。使5个浓度的人PD-1和电泳缓冲液以流速100μL/min流经所述抗体流动单元，先为结合相流动240s，随后解离相600s。在每次运行后用pH 1.7的H3PO4再生所述芯片。使用ProteOn软件将结合和解离曲线拟合至1:1的Langmiur结合模型。3.1 Full kinetic binding affinity determined by surface plasmon resonance (SPR): The affinity and binding kinetics of the antibody to PD-1 were characterized by SPR method using ProteOnXPR36 (Bio-Rad). Protein A protein (Sigma) was immobilized on a GLM sensor chip (Bio-Rad) by amine coupling. Purified antibodies are flowed over the sensor chip and captured by protein A. The chip was rotated 90° and washed with running buffer (1XPBS/0.01% Tween20, Bio-Rad) until the baseline stabilized. Five concentrations of human PD-1 and electrophoresis buffer flowed through the antibody flow cell at a flow rate of 100 μL/min, the binding phase flowed for 240 s first, and then the dissociation phase flowed for 600 s.The chip was regenerated with H3PO4 at pH 1.7 after each run. Association and dissociation curves were fitted to a 1:1 Langmiur binding model using ProteOn software.
如图7所示，通过使用表面等离子体共振检测的抗PD-1抗体对重组的人源PD-1的亲和性为从3.76E-9到1.76E-10mol/L。As shown in FIG. 7 , the affinity of anti-PD-1 antibody to recombinant human PD-1 detected by using surface plasmon resonance is from 3.76E-9 to 1.76E-10 mol/L.
3.2流式细胞仪(FACS)测定的PD-1抗体与细胞表面PD-1分子的结合亲和性：经FACS分析测试抗体与细胞表面PD-1的结合亲和性。以5x105细胞/ml的浓度将表达PD-1的CHO-S细胞转移至96孔U形底平板(BD)。待测抗体用洗涤缓冲液以1:2系列稀释(1XPBS/1％BSA)并在4℃下孵育1小时。加入二抗山羊抗-人IgG Fc FITC(3.0摩尔FITC每摩尔IgG，(JacksonImmunoresearch Lab))并在4℃下避光孵育1小时。随后洗涤一次细胞并在1XPBS/1％BSA中重悬，使用流式细胞术(BD)分析。基于被定量的小珠(QuantumTM MESF Kit(BangsLaboratories,Inc.)，将荧光强度转换为每个细胞上结合的分子。使用Graphpad Prism5计算KD。图2显示了全人源PD-1抗体(即1.7.3hAb、1.49.9hAb、1.103.11hAb、1.139.15hAb、和1.153.7hAb)与表达PD-1的CHO细胞的结合。使用抗人源PD-1的全人源抗体结合转染了PD-1的CHO细胞，且FACS分析显示全人源PD-1抗体以2nmol/L的EC50与PD-1特异性地结合。3.2 The binding affinity of the PD-1 antibody to the cell surface PD-1 molecule determined by flow cytometry (FACS): the binding affinity of the antibody to the cell surface PD-1 was tested by FACS analysis. CHO-S cells expressing PD-1 were transferred to a 96-well U-bottom plate (BD) at a concentration of 5x105 cells/ml. Antibodies to be tested were serially diluted 1:2 in wash buffer (1XPBS/1%BSA) and incubated at 4°C for 1 hour. Secondary antibody goat anti-human IgG Fc FITC (3.0 moles FITC per mole IgG, (Jackson Immunoresearch Lab)) was added and incubated for 1 hour at 4°C in the dark. Cells were then washed once and resuspended in 1XPBS/1%BSA and analyzed using flow cytometry (BD). Based on the quantified beads (QuantumTM MESF Kit (Bangs Laboratories, Inc.), the fluorescence intensity was converted to the molecules bound on each cell. The KD was calculated using GraphpadPrism5 . Figure 2 shows the fully human PD-1 antibody (i.e. 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb, and 1.153.7hAb) combined with CHO cells expressing PD-1. Using anti-human PD-1 fully human antibody combined with transfected PD- 1 CHO cells, and FACS analysis showed that the fully human PD-1 antibody specifically binds to PD-1 with an EC50 of 2 nmol/L.
将表达人PD-1的CHO细胞与不同浓度的抗PD-1抗体孵育，随后将小鼠Fc-标记的人PD-L1加入细胞中。使用FITC连接的山羊抗-小鼠IgG检测人PD-L1与表达PD-1的细胞的结合，随后通过FACS分析。如图4所示，抗PD-1抗体阻断了PD-L1与PD-1转染的CHO细胞的结合。CHO cells expressing human PD-1 were incubated with different concentrations of anti-PD-1 antibody, and mouse Fc-tagged human PD-L1 was subsequently added to the cells. Binding of human PD-L1 to PD-1 expressing cells was detected using FITC-conjugated goat anti-mouse IgG, followed by FACS analysis. As shown in Figure 4, anti-PD-1 antibody blocked the binding of PD-L1 to PD-1 transfected CHO cells.
3.3同源基因(跨物种)和同源物(跨家族)筛选：3.3 Homologous genes (cross-species) and homologs (cross-family) screening:
3.3.1与食蟹猴PD-1和鼠科PD-1的交叉反应性：经ELISA测定交叉反应性。平板(Nunc)用1μg/ml的食蟹猴PD-1(Sino Biological)和鼠科PD-1(Sino Biological)在4℃包被过夜。封闭和洗涤后，加入1μg/ml抗体至平板并在室温下孵育1小时。之后洗涤所述平板并随后用山羊抗大鼠IgG1HRP(Bethyl)和山羊抗大鼠IgG2bHRP(Bethyl)孵育45分钟。洗涤后，加入TMB底物并用2M HCl终止反应。使用酶标仪(Molecular Device)读取450nm吸收光值。3.3.1 Cross-reactivity with cynomolgus monkey PD-1 and murine PD-1: the cross-reactivity was determined by ELISA. Plates (Nunc) were coated overnight at 4°C with cynomolgus PD-1 (Sino Biological) and murine PD-1 (Sino Biological) at 1 μg/ml. After blocking and washing, 1 μg/ml antibody was added to the plate and incubated for 1 hour at room temperature. The plates were then washed and subsequently incubated with goat anti-rat IgGl HRP (Bethyl) and goat anti-rat IgG2bHRP (Bethyl) for 45 minutes. After washing, TMB substrate was added and the reaction was stopped with 2M HCl. The absorbance value at 450 nm was read using a microplate reader (Molecular Device).
跨物种实验的结果显示抗PD-1抗体与食蟹猴PD-1结合但不与鼠科PD-1结合(图6)。The results of cross-species experiments showed that the anti-PD-1 antibody binds to cynomolgus monkey PD-1 but not to murine PD-1 (Figure 6).
3.3.2与PD-1家族成员CD28、CTLA4和ICOS的交叉反应性：为了检测所述全人源抗体的跨家族结合活性，用抗体对表达PD-1、CD28、CTLA4或ICOS的细胞系进行染色，随后进行与山羊抗人-IgG Fc缀合的FITC的二抗结合。表达PD-1的细胞作为阳性对照。对应的亲本细胞系作为阴性对照。使用BD Biosciences FACSCanto II和FlowJo版本软件分析所述被结合的细胞。3.3.2 Cross-reactivity with PD-1 family members CD28, CTLA4 and ICOS: In order to detect the cross-family binding activity of the fully human antibody, the antibody was used to test the cell lines expressing PD-1, CD28, CTLA4 or ICOS Staining was followed by secondary antibody binding to goat anti-human-IgG Fc-conjugated FITC. Cells expressing PD-1 served as a positive control. The corresponding parental cell line served as a negative control. The bound cells were analyzed using BD Biosciences FACSCanto II and FlowJo version software.
图5显示了PD-1、或CD28转染的CHO细胞、和CTLA4转染的293F细胞用抗PD-1抗体染色并用FACS分析。所述结果显示，PD-1抗体特异性地与PD-1，但不与PD-1家族的CD28和CTLA4结合。Figure 5 shows that CHO cells transfected with PD-1, or CD28, and 293F cells transfected with CTLA4 were stained with anti-PD-1 antibody and analyzed by FACS. The results show that the PD-1 antibody specifically binds to PD-1, but not to CD28 and CTLA4 of the PD-1 family.
3.4表位分类(Binning)测试3.4 Epitope classification (Binning) test
3.4.1PD-1抗体的结合表位通过SPR测试使用ProteOn XPR36(Bio-Rad)进行针对基准抗体A和B的分类。将基准抗体A和B通过胺基偶联固定在GLC传感芯片(Bio-Rad)。将人PD-1溶液流过固定抗体的通路并用基准抗体捕获。随后将芯片旋转90°并用电泳缓冲液洗涤直至基线稳定。将选择的抗体流经所述传感器芯片。3.4.1 Binding epitopes of PD-1 antibodies were classified against reference antibodies A and B by SPR testing using ProteOn XPR36 (Bio-Rad). Reference antibodies A and B were immobilized on a GLC sensor chip (Bio-Rad) by amine coupling. Human PD-1 solution was flowed through the channel of immobilized antibody and captured with reference antibody. The chip was then rotated 90° and washed with running buffer until the baseline stabilized. Antibodies of choice are flowed through the sensor chip.
3.4.2PD-1抗体的结合表位通过FACS进行针对基准抗体A和B的分类。将在细胞表面表达人PD-1的CHO细胞以10μg/ml与基准抗体A和B孵育1小时。将所述细胞洗涤并加入本申请的PD-1抗体孵育1小时。加入抗大鼠IgG-FITC的二抗并在4℃孵育1小时。随后洗涤所述细胞1次并在1XPBS/1％BSA中重悬，经流式细胞仪(BD)分析。3.4.2 Binding epitopes of PD-1 antibodies were classified against reference antibodies A and B by FACS. CHO cells expressing human PD-1 on the cell surface were incubated with reference antibodies A and B at 10 μg/ml for 1 hour. The cells were washed and incubated with the PD-1 antibody of the present application for 1 hour. A secondary antibody against rat IgG-FITC was added and incubated at 4°C for 1 hour. The cells were then washed once and resuspended in 1XPBS/1%BSA, and analyzed by flow cytometry (BD).
分类测试的SPR测试和FACS结果表明全人源PD-1抗体(即1.7.3hAb、1.49.9hAb、1.103.11hAb、1.139.15hAb、和1.153.7hAb)在人源PD-1上的结合表位与已知PD-1抗体(即基准抗体A和B)不同。The SPR test and FACS results of the classification test indicate the binding epitopes of the fully human PD-1 antibodies (i.e. 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb, and 1.153.7hAb) on human PD-1 Different from known PD-1 antibodies (i.e. benchmark antibodies A and B).
3.5基于细胞的测试测定PD-1抗体的体外功能3.5 Cell-based assays to determine the in vitro function of PD-1 antibodies
3.5.1人源PD-1抗体对T细胞增殖的作用。使用同种异体反应测试PD-1抗体对T淋巴细胞增殖的作用。在96孔U底形组织培养板中在包含10％FCS和抗生素的200μl RPMI 1640中进行原代树突细胞(DC)-刺激的MLR。将DC与1X105的同种异体总CD4+T细胞以1:10和1:100的DC:T细胞的比例混合。在存在或不存在中和mAb的条件下进行培养：人PD-1抗体和基准抗体A和B的使用浓度为10μg/ml。孵育测试5天，在最后16小时过程中加入1uCi/孔的[3H]胸苷。通过闪烁计数测定[3H]胸苷的掺入，用三复孔的平均[3H]胸苷掺入(每分钟计数)表示增殖反应。仅有DC的计数常规为<1000cpm。显示的结果是进行了最少5次试验的代表性的示例。3.5.1 The effect of human PD-1 antibody on the proliferation of T cells. The effect of PD-1 antibody on T lymphocyte proliferation was tested using alloreactivity. Primary dendritic cell (DC)-stimulated MLR was performed in 96-well U-bottom tissue culture plates in 200 μl RPMI 1640 containing 10% FCS and antibiotics. Mix DCs with 1X10 total allogeneic CD4+ T cells at DC:T cell ratios of 1:10 and1 :100. Cultures were performed in the presence or absence of neutralizing mAbs: human PD-1 antibody and reference antibodies A and B were used at a concentration of 10 μg/ml. Assays were incubated for 5 days, with 1uCi/well of [3H ]thymidine added during the last 16 hours. [3 H]thymidine incorporation was determined by scintillation counting, and the proliferative response was expressed as the mean [3 H]thymidine incorporation (counts per minute) of triplicate wells. DC-only counts were routinely <1000 cpm. The results shown are representative examples from which a minimum of 5 experiments were performed.
人树突细胞(DC)和以上同种异体MLR中使用的CD4+T、CD8+T和总细胞以如下步骤从PBMC中生成：通过使用人单核细胞浓缩试剂盒(human monocyte enrichment cocktailkit)根据厂商(StemCell Meylan)的说明书经负性筛选从PBMC中纯化人单核细胞。简言之，使用Ficoll-Paque梯度从健康的供体血液中分离PBMC。用PBS洗涤细胞两次，随后在分离缓冲液中以1X108细胞/ml重悬，并用单核细胞浓缩Ab混合物在4℃下孵育30分钟。收集通过MACS柱的未标记的单核细胞。为生成iDC，将单核细胞在含有10％FCS和抗生素的RPMI 1640的培养基中与GM-CSF(PeproTech,Rocky Hill,NJ；800U/ml)和IL-4(PeproTech；500U/ml)培养，细胞浓度为2X106细胞/ml。每天用含有GM-CSF和IL-4的培养基置换一半的培养基。在第5天用LPS(026:B6；Sigma-Aldrich,St.Louis,MO；1μg/ml)额外刺激iDC 24小时以生成成熟DC。通过根据厂商说明书(Stemsep)将PBMC与人CD4+T、CD8+T和总T细胞浓缩混合物和磁性胶体孵育进行负性选择，纯化CD4+T、CD8+T和总T细胞。Human dendritic cells (DC) and CD4+ T, CD8+ T and total cells used in the above allogeneic MLR were generated from PBMCs by using the human monocyte enrichment cocktail kit according to Human monocytes were purified from PBMC by negative selection according to the manufacturer's (StemCell Meylan) instructions. Briefly, PBMCs were isolated from healthy donor blood using a Ficoll-Paque gradient. Cells were washed twice with PBS, subsequently resuspended at1X108 cells/ml in isolation buffer, and incubated with monocyte enriched Ab mix for 30 min at 4 °C. Unlabeled monocytes passing through the MACS column were collected. To generate iDCs, monocytes were cultured with GM-CSF (PeproTech, Rocky Hill, NJ; 800 U/ml) and IL-4 (PeproTech; 500 U/ml) in RPMI 1640 medium containing 10% FCS and antibiotics , the cell concentration is 2X106 cells/ml. Half of the medium was replaced daily with medium containing GM-CSF and IL-4. iDCs were stimulated with LPS (026:B6; Sigma-Aldrich, St. Louis, MO; 1 μg/ml) for an additional 24 hours on day 5 to generate mature DCs. CD4+ T, CD8+ T and total T cells were purified by negative selection by incubating PBMC with enriched mixtures of human CD4+ T, CD8+ T and total T cells and magnetic colloids according to the manufacturer's instructions (Stemsep).
在PD-1抗体1.7.3hAb、1.49.9hAb、1.103.11hAb、1.139.15hAb和1.153.7hAb存在或不存在的情况下，用同种异体DC刺激人CD4+T细胞。经[3H]胸苷的掺入评估CD4+T细胞的增殖。图10显示了1.7.3hAb、1.49.9hAb、1.103.11hAb、1.139.15hAb和1.153.7hAb提高了浓度依赖的T细胞增殖。Human CD4+ T cells were stimulated with allogeneic DCs in the presence or absence of PD-1 antibodies 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb, and 1.153.7hAb. Proliferation of CD4+ T cells was assessed by incorporation of [3 H]thymidine. Figure 10 shows that 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb and 1.153.7hAb increased concentration-dependent T cell proliferation.
3.5.2体外人源PD-1抗体对细胞因子IFNγ分泌的作用：为了评估人源PD-1抗体对细胞因子IFNγ的产生的阻断作用，我们在同种异体-MLR中进行了IFNγ的产生的实验。简言之，根据厂商的说明书用CD4+T细胞浓缩试剂盒(CD4+T cell enrichment cocktail kit)经负性筛选将人源CD4+T细胞从PBMC中纯化出来。在GM-CSF和IL-4中培养5天的单核细胞中生成未成熟DC，并用LPS以1μg/ml刺激过夜，分化成成熟的DC。将CD4+T细胞和iDC/mDC以10:1和100:1的T:DC比例混合。在存在或不存在人源PD-1抗体和基准抗体的情况下进行培养。5天后，收集每个培养物的上清液测定细胞因子IFNγ。上清液中的IFNγ水平通过ELISA测试测定。简言之，用在包被缓冲液中稀释的抗人IFNγmAb包被Maxisorp平板(0.75μg/ml；即稀释为1/1360)，50μl/孔(即对一个满的96孔板加入3.7μl的抗体至5ml的包被缓冲液中)并在4℃孵育过夜。加入200μl/孔的封闭缓冲液2小时，以封闭多余的蛋白结合能力。准备重组IFNγ稀释液作为标准液，用完全培养基从8000pg/ml进行两倍稀释直至125pg/ml，加上只有完全培养基的情况。洗涤平板，加入标准液和测试上清液(100μl/孔)，孵育2-4小时。加入在封闭缓冲液中的生物素化抗-IFNγmAb(1/1333)，之后加入额外亲和素过氧化物酶。加入TMB底物进行所述反应，用2M HCl终止反应。在450nm测定吸光值。3.5.2 Effect of human PD-1 antibody on cytokine IFNγ secretion in vitro: To evaluate the blocking effect of human PD-1 antibody on cytokine IFNγ production, we performed IFNγ production in allo-MLR experiment of. Briefly, human CD4+ T cells were purified from PBMCs by negative selection using a CD4+T cell enrichment cocktail kit according to the manufacturer's instructions. Immature DCs were generated from monocytes cultured for 5 days in GM-CSF and IL-4 and stimulated with LPS at 1 μg/ml overnight to differentiate into mature DCs. CD4+ T cells and iDC/mDC were mixed at T:DC ratios of 10:1 and 100:1. Incubation was performed in the presence or absence of human PD-1 antibody and reference antibody. After 5 days, the supernatants from each culture were collected and assayed for the cytokine IFNγ. IFNγ levels in supernatants were determined by ELISA assay. Briefly, Maxisorp plates were coated with anti-human IFNγmAb diluted in coating buffer (0.75 μg/ml; ie diluted 1/1360), 50 μl/well (ie, 3.7 μl for a full 96-well plate). antibody into 5ml of coating buffer) and incubated overnight at 4°C. Add 200 μl/well of blocking buffer for 2 hours to block excess protein binding capacity. Dilutions of recombinant IFNγ were prepared as standards, two-fold diluted from 8000 pg/ml up to 125 pg/ml with complete medium, plus only complete medium. Wash the plate, add standard solution and test supernatant (100 μl/well), and incubate for 2-4 hours. Biotinylated anti-IFNγ mAb (1/1333) in blocking buffer was added followed by additional avidin peroxidase. The reaction was carried out by adding TMB substrate and quenched with 2M HCl. Absorbance was measured at 450 nm.
图9显示了在存在或不存在1.7.3hAb、1.49.9hAb、1.103.11hAb、1.139.15hAb和1.153.7hAb抗体的情况下，用同种异体DC刺激人CD4+T细胞。用ELISA测定IFNγ水平。结果显示全人源PD-1抗体以剂量依赖的方式增加IFNγ的分泌。Figure 9 shows stimulation of human CD4+ T cells with allogeneic DCs in the presence or absence of 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb and 1.153.7hAb antibodies. IFNγ levels were determined by ELISA. The results showed that fully human PD-1 antibody increased IFNγ secretion in a dose-dependent manner.
3.5.3体外人源PD-1对白介素2(IL-2)的产生的作用：将CD4+T细胞和iDC/mDC以10:1和100:1的T:DC比例混合。在存在或不存在人源PD-1抗体和基准抗体的情况下进行培养。5天后，收集每个培养物的上清液测定细胞因子。上清液中的IL-2水平通过ELISA测试测定。3.5.3 Effect of human PD-1 on interleukin 2 (IL-2) production in vitro: CD4+ T cells and iDC/mDC were mixed at T:DC ratios of 10:1 and 100:1. Incubation was performed in the presence or absence of human PD-1 antibody and reference antibody. After 5 days, supernatants from each culture were collected for cytokine determination. IL-2 levels in supernatants were determined by ELISA assay.
图8显示了在存在或不存在本申请的抗体或对照抗体的情况下，用同种异体DC刺激人CD4+T细胞。用ELISA测定IL-2水平。结果显示全人源PD-1抗体以剂量依赖的方式增加IFNγ的分泌。所述结果显示抗PD-1抗体以剂量依赖的方式增加IL-2的分泌。Figure 8 shows the stimulation of human CD4+ T cells with allogeneic DCs in the presence or absence of antibodies of the present application or control antibodies. IL-2 levels were determined by ELISA. The results showed that fully human PD-1 antibody increased IFNγ secretion in a dose-dependent manner. The results show that anti-PD-1 antibodies increase IL-2 secretion in a dose-dependent manner.
3.5.4人源PD-1抗体通过自体抗原特异性免疫反应对细胞增殖和细胞因子的生产的作用：在本测定中，T细胞和DC细胞来自相同供体。简言之，从PBMC中纯化CD4+T细胞并在CMVpp65肽和低剂量的IL2(20U/ml)中培养，同时，从在GM-CSF和IL-4中的相同供体的PBMC中培养的单核细胞中生成DC。5天后，用将用CMV pp65肽处理的CD4+T细胞与DC共培养，所述DC在存在或不存在人源PD-1抗体和基准抗体(作为对照)的条件下脉冲式加入pp65肽。3.5.4 The effect of human PD-1 antibody on cell proliferation and cytokine production through autoantigen-specific immune response: In this assay, T cells and DC cells came from the same donor. Briefly, CD4+ T cells were purified from PBMCs and cultured in CMVpp65 peptide and low doses of IL2 (20 U/ml), while PBMCs from the same donor were cultured in GM-CSF and IL-4. DCs are generated from monocytes. After 5 days, CD4+ T cells treated with CMV pp65 peptide were co-cultured with DC pulsed with pp65 peptide in the presence or absence of human PD-1 antibody and reference antibody (as a control).
在第5天，从每个培养物中取100μl的上清液用于测定细胞因子IFNγ和IL-2。通过ELISA测试检测IFNγ和IL-2的产生的水平。针对脉冲式加入CMV pp65肽的DC的特异性T细胞增殖通过[3H]胸苷的掺入测定。On day 5, 100 [mu]l of supernatant from each culture was used for the assay of cytokines IFN[gamma] and IL-2. The levels of IFNγ and IL-2 production were detected by ELISA assay. Specific T cell proliferation against DC pulsed with CMV pp65 peptide was determined by incorporation of [3 H]thymidine.
如图11显示，PD-1抗体提高了由装载了CMV pp65肽的自体DC所刺激的浓度依赖的CMV+-CD4+T细胞的增殖。As shown in Figure 11, PD-1 antibody enhanced the concentration-dependent proliferation of CMV+ -CD4+ T cells stimulated by autologous DCs loaded with CMV pp65 peptide.
3.5.5人源PD-1抗体对调节性T细胞(Tregs)抑制功能的作用：Tregs是T细胞的一个亚群，其是关键的免疫调节子，在维持自体耐受中起到关键作用。3.5.5 The effect of human PD-1 antibody on the inhibitory function of regulatory T cells (Tregs): Tregs are a subset of T cells, which are key immune regulators and play a key role in maintaining self-tolerance.
CD4+CD25+调节性T细胞与肿瘤相关，因为在多种癌症病人中发现了增加的Tregs数量，且其与较差的预后相关。为了直接评估人源PD-1抗体对免疫抑制响应的作用，我们进行了Tregs实验。使用特异性抗-CD25微珠(Miltenyi Biotec,Auburn,CA)和阳性或负性选择，分别分离CD4+CD25+和CD4+CD25-T细胞。开始时，根据厂商说明书(Stemsep)，使用人CD4+T细胞浓缩混合物和磁性胶体孵育PBMC，经负性选择纯化CD4+T细胞。之后在MACS缓冲液中重悬CD4+T细胞，在冰上与CD25+微珠孵育30分钟，洗涤并装柱。从流出溶液中收集不与柱结合的CD4+CD25-T细胞，并在使用前洗涤。随后从所述柱中恢复CD4+CD25+T细胞并在使用前洗涤。在存在或不存在10μg/ml浓度的人源PD-1抗体的条件下，将Tregs与CD4+CD25-T细胞和DC(Treg:Teff比例为1:1)培养。不用抗体或使用同种型抗体作为阴性对照。在第5天取所述培养物的上清液用于ELISA检测细胞因子，通过以1uCi/孔的浓度加入[3H]胸苷并进一步培养18小时，检测细胞增殖。[3H]胸苷的掺入通过闪烁计数。如图12所示，所述PD-1抗体去除了Treg抑制性功能并恢复了响应的T细胞增殖和IFNγ分泌。CD4+ CD25+ regulatory T cells are associated with tumors, as increased numbers of Tregs are found in various cancer patients and are associated with poorer prognosis. To directly assess the effect of human PD-1 antibodies on immunosuppressive responses, we performed Tregs experiments. CD4+ CD25+ and CD4+CD25- T cells were isolated using specific anti-CD25 microbeads (Miltenyi Biotec, Auburn, CA) and positive or negative selection, respectively. Initially, CD4+ T cells were purified by negative selection by incubating PBMC with human CD4+ T cell enrichment mixture and magnetic colloid according to the manufacturer's instructions (Stemsep). CD4+ T cells were then resuspended in MACS buffer, incubated with CD25+ microbeads on ice for 30 minutes, washed and packed. CD4+ CD25- T cells not bound to the column were collected from the flow-through solution and washed before use. CD4+ CD25+ T cells were subsequently recovered from the column and washed before use. Tregs were cultured with CD4+ CD25- T cells and DCs (Treg:Teff ratio 1:1) in the presence or absence of human PD-1 antibody at a concentration of 10 μg/ml. No antibody or an isotype antibody was used as a negative control. On day 5, the supernatant of the culture was taken for ELISA to detect cytokines, and cell proliferation was detected by adding [3 H]thymidine at a concentration of 1uCi/well and further culturing for 18 hours. [3 H]thymidine incorporation was counted by scintillation. As shown in Figure 12, the PD-1 antibody abolished Treg suppressive function and restored responsive T cell proliferation and IFNγ secretion.
3.6ADCC/CDC测定：为了使健康的PD-1+细胞不需要的毒性降到最低，对选择的抗PD-1全人源抗体进行确认不含ADCC和CDC功能。3.6 ADCC/CDC assay: In order to minimize the unwanted toxicity of healthy PD-1+ cells, the selected anti-PD-1 fully human antibody was confirmed to be free of ADCC and CDC functions.
3.6.1ADCC：使用表达高水平细胞表面PD-1的活化T细胞作为靶细胞并用不同浓度的全人源抗体在96孔板中预孵育30分钟，随后以50:1的效应/靶细胞比例加入IL-2激活的PBMC(作为天然杀伤(NK)细胞源使用，即效应细胞)。[在37℃、5％CO2的温箱中孵育所述平板6小时。经细胞毒性检测试剂盒(Roche)测定靶细胞裂解。通过Molecular Devices SpectraMaxM5e酶标仪测定光密度。结果显示，测试的全人源抗PD-1抗体不介导ADCC(图13)。3.6.1ADCC: Activated T cells expressing high levels of cell surface PD-1 were used as target cells and pre-incubated with different concentrations of fully human antibodies in 96-well plates for 30 minutes, and then added at an effector/target cell ratio of 50:1 IL-2 activated PBMCs (used as a source of natural killer (NK) cells, ie effector cells). [Incubate the plate for 6 hours in an incubator at 37°C, 5%CO2 . Target cell lysis was measured by Cytotoxicity Assay Kit (Roche). Optical density was determined by a Molecular Devices SpectraMaxM5e microplate reader. The results showed that the tested fully human anti-PD-1 antibodies did not mediate ADCC (Figure 13).
3.6.2CDC：将靶细胞(活化T细胞)、稀释的人血清补体(Quidel-A112)和不同浓度的全人源PD-1抗体在96孔板中混合。在37℃、5％CO2的温箱中孵育所述平板4小时。经CellTiterglo(Promega-G7573)测定靶细胞裂解。Rituxan(Roche)和人B淋巴细胞细Raji(CD20阳性)作为阳性对照。数据显示PD-1抗体不介导CDC(图14)。3.6.2 CDC: Mix target cells (activated T cells), diluted human serum complement (Quidel-A112) and different concentrations of fully human PD-1 antibodies in a 96-well plate. The plates were incubated for 4 hours in an incubator at 37°C, 5%CO2 . Target cell lysis was measured by CellTiterglo (Promega-G7573). Rituxan (Roche) and human B lymphocyte Raji (CD20 positive) were used as positive controls. The data show that PD-1 antibody does not mediate CDC (Figure 14).
实施例4：全人抗体的表位鉴定Example 4: Epitope identification of fully human antibodies
为了确定本申请所述的抗体1.103.11hAb与Keytruda(现有的hPD-1抗体)之间的表位差异，使用1.103.11hAb、Keytruda和11.148.10(其为对照hPD-1抗体，其结合的表位不与1.103.11hAb或Keytruda的表位重叠)进行了针对hPD-1的丙氨酸扫描突变实验(alaninescanning experiment)和抗体结合的效果评估。To determine epitope differences between the antibody 1.103.11hAb described in this application and Keytruda (an existing hPD-1 antibody), 1.103.11hAb, Keytruda, and 11.148.10 (which is a control hPD-1 antibody that binds The epitope of hPD-1 does not overlap with the epitope of 1.103.11hAb or Keytruda). The alanine scanning mutation experiment (alaninescanning experiment) for hPD-1 and the effect of antibody binding were evaluated.
将hPD-1中的丙氨酸残基突变成甘氨酸密码子，且将所有其他残基突变为丙氨酸密码子。针对hPD-1胞外结构域(ECD)的每个残基，使用两步连续PCR(sequential PCR)进行氨基酸的点取代。将编码人PD-1的ECD和C末端His-标签的pcDNA3.3-hPD-1_ECD.His质粒作为模板，使用一套诱变引物作为第一步PCR，并使用QuikChangeLighting多点定点突变试剂盒(Agilent technologies,Palo Alto,CA)。使用Dpn I内切酶在突变联合成反应后消化母体模板。在第二步PCR中，线性DNA表达盒包含CMV启动子、PD-1胞外结构域(ECD)、His-标签和单纯疱疹病毒胸苷激酶(TK)的多聚腺苷酸化，将所述线性DNA表达盒进行扩增并在HEK293F细胞中瞬时表达(Life Technologies,Gaithersburg,MD)。Alanine residues in hPD-1 were mutated to glycine codons and all other residues were mutated to alanine codons. For each residue of the hPD-1 extracellular domain (ECD), two-step sequential PCR (sequential PCR) was used to perform amino acid point substitutions. The pcDNA3.3-hPD-1_ECD.His plasmid encoding the ECD of human PD-1 and the C-terminal His-tag was used as a template, a set of mutagenic primers was used as the first step of PCR, and the QuikChangeLighting multi-site-directed mutagenesis kit ( Agilent technologies, Palo Alto, CA). Dpn I endonuclease was used to digest the parental template after the mutagenesis reaction. In the second PCR step, a linear DNA expression cassette containing the CMV promoter, PD-1 extracellular domain (ECD), His-tag, and polyadenylation of herpes simplex virus thymidine kinase (TK), the A linear DNA expression cassette was amplified and expressed transiently in HEK293F cells (Life Technologies, Gaithersburg, MD).
将单克隆抗体1.103.11hAb、keytruda和11.148.10hAb包被在板上用于ELISA结合测定。在与包含定量的PD-1突变的上清液反应后，将HRP偶联的抗-His抗体(Rockland,Cat#200-303-382)作为检测抗体加入。根据对照突变的平均值对吸光度进行基准化。在对结合倍数变化(<0.55)进行额外临界值设定后，鉴定最终确定的表位残基。Monoclonal antibodies 1.103.11hAb, keytruda and 11.148.10hAb were coated on plates for ELISA binding assay. After reacting with the supernatant containing quantified PD-1 mutations, an HRP-conjugated anti-His antibody (Rockland, Cat# 200-303-382) was added as a detection antibody. Absorbance was normalized to the mean of control mutants. Finalized epitope residues were identified after additional cut-off setting for fold change in binding (<0.55).
在表2中列出了显著降低抗体结合的前30个点取代的hPD-1突变。在hPD-1晶体结构(PDB码3RRQ和4ZQK)上对所有这些残基的位置进行核对，显示出一些氨基酸(e.g.Val144,Leu142,Val110,Met108,Cys123等)被完全埋在蛋白质中，且不太可能直接与任何抗体接触。观察到的结合降低最可能是由于丙氨酸取代后的hPD-1结构的不稳定或甚至结构坍塌。为了避免将这些数据错译成表位热点，利用了对照抗体11.148.10hAb(其结合抗原上完全不同的位置，但期望对hPD-1结构的坍塌有所反应)。对两个抗体都有影响的突变被认为是错误的热点并从列表中移除。在对结合倍数变化(<0.55)进行额外临界值设定后，最终确定的表位残基列于表3。他们是针对1.103.11hAb的9个位置和针对Keytruda的5个位置，和针对对照抗体11.148.10hAb的10个残基。The top 30 point-substitution hPD-1 mutations that significantly decreased antibody binding are listed in Table 2. Checking the positions of all these residues on the hPD-1 crystal structure (PDB codes 3RRQ and 4ZQK) showed that some amino acids (e.g. Val144, Leu142, Val110, Met108, Cys123, etc.) Too likely to be in direct contact with any antibody. The observed decrease in binding is most likely due to destabilization or even structural collapse of the hPD-1 structure after alanine substitution. To avoid misinterpreting these data as epitope hotspots, a control antibody 11.148.10hAb (which binds to a completely different position on the antigen but is expected to respond to the collapse of the hPD-1 structure) was utilized. Mutations affecting both antibodies were considered false hotspots and removed from the list. The final identified epitope residues are listed in Table 3 after setting an additional cutoff for the fold change in binding (<0.55). They are 9 positions against 1.103.11hAb and 5 positions against Keytruda, and 10 residues against the control antibody 11.148.10hAb.
表2.PD-1点突变对抗体结合的影响Table 2. Effect of PD-1 point mutations on antibody binding
粗体：与对照11.148.10hAb重叠的用于结构维持的氨基酸被从所述热点列表中排除。Bold: amino acids for structure maintenance overlapping with the control 11.148.10 hAb were excluded from the hotspot list.
a结合中的倍数变化是相对于若干沉默的丙氨酸取代的结合而言。a Fold change in binding is relative to binding of several silent alanine substitutions.
表3.识别的潜在表位Table 3. Potential epitopes identified
临界值:倍数变化<0.55Cutoff value: fold change <0.55
*C,C’,C”,F,G,A’代表如图17中的hPD-1的晶体结构的肽链。在mPD-1中观察到的所述C”链在hPD-1结构中不存在。该β-片层在hPD-1上被替换为无结构的环。仅仅为了便于与mPD-1比较，仍然使用C”来标记这段区域。* C, C', C", F, G, A' represent the peptide chains of the crystal structure of hPD-1 in Figure 17. The C" chain observed in mPD-1 is in the hPD-1 structure does not exist. The β-sheet is replaced by an unstructured loop on hPD-1. Just for the convenience of comparison with mPD-1, C" is still used to mark this region.
对表3中1.103.11hAb和Keytruda的表位残基进行比较显示了两个重叠的热点残基。其他残基则看上去十分多样化，表明两个抗体可能在结合hPD-1和阻断hPD-L1方面具有非常不同的机制。阅读表3中的残基ID并不能直观地解读所述机制。因此，将表3中的所有数据以及所述的hPD-L1结合位点在hPD-1的晶体结构上作图以更好的进行视觉化和比较(图17)。A comparison of the epitope residues of 1.103.11hAb and Keytruda in Table 3 revealed two overlapping hotspot residues. Other residues appeared to be quite diverse, suggesting that the two antibodies may have very different mechanisms for binding hPD-1 and blocking hPD-L1. Reading the residue IDs in Table 3 does not intuitively decipher the mechanism. Therefore, all the data in Table 3 and the hPD-L1 binding site were mapped on the crystal structure of hPD-1 for better visualization and comparison ( FIG. 17 ).
如图17所示，负责hPD-L1结合的所述热点残基全部集中在C、F和G链的中间(图17A)。两个研究的抗体1.103.11hAb和Keytruda，尽管在结合hPD-1结合和阻断hPD-L1中都具有功能，但明显具有不同的表位(图17B为1.103.11hAb，图17C为Keytruda)。Keytruda的表位主要是由C’D环上的残基贡献(对应于mPD-1的C”链)，其完全不切断PD-L1结合位点。这提示了Keytruda阻断hPD-L1的功能更多依赖于由抗体的大小提供的空间位阻效应。相反，本申请的抗体1.103.11hAb的表位由分布在多个位置的热点构成，且与hPD-L1结合位点直接重叠(图17A和17B)。本申请的1.103.11hAb通过比hPD-L1与两者的共同结合位点具有更高的竞争性阻断了hPD-L1。因此，1.103.11hAb被期望在下游研发中更具功能性。As shown in Figure 17, the hotspot residues responsible for hPD-L1 binding were all concentrated in the middle of the C, F and G chains (Figure 17A). The two studied antibodies, 1.103.11hAb and Keytruda, although functional in both binding hPD-1 binding and blocking hPD-L1, clearly have different epitopes (1.103.11hAb in Figure 17B and Keytruda in Figure 17C). The epitope of Keytruda is mainly contributed by residues on the C'D loop (corresponding to the C" chain of mPD-1), which does not cut off the PD-L1 binding site at all. This suggests that Keytruda blocks the function of hPD-L1 More rely on the steric hindrance effect provided by the size of the antibody. On the contrary, the epitope of the antibody 1.103.11hAb of the present application consists of hot spots distributed in multiple positions, and directly overlaps with the hPD-L1 binding site (Fig. 17A and 17B). The 1.103.11hAb of the present application blocks hPD-L1 by having higher competition than hPD-L1 for the common binding site of the two. Therefore, the 1.103.11hAb is expected to be more functional in downstream research and development sex.
然而，抗体11.148.10hAb与两个功能抗体相比结合完全不同的位置(图17D)，其证明了其本身对于在进行丙氨酸取代中监测hPD-1的功能方面是很好的对照抗体。However, antibody 11.148.10hAb binds to a completely different position than the two functional antibodies (Fig. 17D), which proved itself to be a good control antibody for monitoring hPD-1 function in making alanine substitutions.
虽然本公开已经具体示出并参考具体实施方式(其中一些是优选的实施方式)进行了描述，但本领域的技术人员应理解如本申请所示可以在不脱离本发明的精神和范围内，可以进行各种形式上和细节上的改变。While the present disclosure has been shown and described with reference to specific embodiments, some of which are preferred, those skilled in the art will appreciate that, as shown herein, the invention may be, without departing from the spirit and scope of the invention, Various changes in form and details may be made.
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| US11643465B2 (en) | Anti-PD-1 antibodies | |
| US12180282B2 (en) | Anti-PD-L1 antibodies | |
| CN106432494B (en) | Novel anti-PD-1 antibody | |
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| HK1238649B (en) | Novel anti-pd-1 antibody | |
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