HK1226951B - Methods of treating cancers using pd-1 axis binding antagonists and taxanes - Google Patents

Description

Translated fromChinese

用PD-1轴结合拮抗剂和紫杉烷治疗癌症的方法Methods for treating cancer using PD-1 axis binding antagonists and taxanes

相关申请的交叉参考CROSS-REFERENCE TO RELATED APPLICATIONS

本申请要求2013年12月17日提交的美国临时申请号61/917,264的优先权。This application claims priority to U.S. Provisional Application No. 61/917,264, filed December 17, 2013.

技术领域Technical Field

本发明涉及通过施用PD-1轴结合拮抗剂和紫杉烷(taxane)来在患有癌症的个体中治疗癌症和增强免疫功能的方法。The present invention relates to methods of treating cancer and enhancing immune function in individuals with cancer by administering a PD-1 axis binding antagonist and a taxane.

背景技术Background Art

向T细胞提供两种不同信号是抗原呈递细胞(APC)对静息T淋巴细胞进行淋巴细胞激活的广为接受的模型。此模型进一步提供了自身与非自身和免疫耐受的区分。初级信号或抗原特异性信号在识别在主要组织相容性复合体(MHC)背景中呈递的外来抗原肽之后通过T细胞受体(TCR)转导。次级信号或共刺激信号由表达在APC上的共刺激分子传递至T细胞，并诱导T细胞促进克隆扩充、细胞因子分泌和效应子功能。在缺乏共刺激的情况下，T细胞可变得耐受抗原刺激，其导致对外来抗原或内源抗原的致耐受性反应。Providing two different signals to T cells is a widely accepted model for the activation of resting T lymphocytes by antigen presenting cells (APCs). This model further provides the distinction between self and non-self and immune tolerance. Primary signals or antigen-specific signals are transduced by T cell receptors (TCRs) after identifying foreign antigen peptides presented in the context of the major histocompatibility complex (MHC). Secondary signals or co-stimulatory signals are delivered to T cells by co-stimulatory molecules expressed on APCs, and induce T cells to promote clonal expansion, cytokine secretion, and effector function. In the absence of co-stimulation, T cells can become tolerant to antigen stimulation, which results in a tolerogenic response to foreign or endogenous antigens.

在两信号模型中，T细胞收到正和负次级共刺激信号二者。这类正和负信号的调节对最大化宿主保护性免疫反应同时维持免疫耐受和防止自身免疫至关重要。负次级信号对诱导T细胞耐受似乎是必要的，而正信号促进T细胞激活。虽然简单的两信号模型仍为幼稚淋巴细胞提供了有效解释，但宿主免疫反应是动态过程，共刺激信号也可以提供给抗原暴露的T细胞。In the two-signal model, T cells receive both positive and negative secondary co-stimulatory signals. Regulation of these positive and negative signals is crucial for maximizing the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. Negative secondary signals appear to be necessary for inducing T cell tolerance, while positive signals promote T cell activation. Although the simple two-signal model still provides an effective explanation for naive lymphocytes, the host immune response is a dynamic process, and co-stimulatory signals can also be provided to antigen-exposed T cells.

共刺激的机制具有治疗意义，因为共刺激信号的操作提供了增强或终止细胞免疫反应的手段。T细胞功能异常或无反应性与抑制性受体(程序性死亡1多肽(PD-1)，其结合包括PD-L1和PD-L2的配体)的诱导和持续表达同时发生。PD-L1在许多癌症中过量表达，且通常与预后差相关。与正常组织中的T淋巴细胞和外周血T淋巴细胞不同，绝大多数肿瘤浸润T淋巴细胞显著表达PD-1，表明肿瘤反应性T细胞上PD-1的上调可以促成抗肿瘤免疫反应受损。这可以是由于利用了由表达PD-L1的肿瘤细胞与表达PD-1的T细胞相互作用介导的PD-L1信号发放，导致T细胞激活减弱和免疫监视逃避。因此，抑制PD-L1/PD-1相互作用可以增强CD8+T细胞介导的肿瘤杀伤。The mechanism of co-stimulation has therapeutic significance because manipulation of co-stimulatory signals provides a means to enhance or terminate cellular immune responses. T cell dysfunction or anergy occurs simultaneously with the induction and sustained expression of inhibitory receptors (programmed death 1 polypeptide (PD-1), which binds ligands including PD-L1 and PD-L2). PD-L1 is overexpressed in many cancers and is generally associated with poor prognosis. Unlike T lymphocytes in normal tissues and peripheral blood T lymphocytes, the vast majority of tumor-infiltrating T lymphocytes significantly express PD-1, indicating that upregulation of PD-1 on tumor-reactive T cells can contribute to impaired anti-tumor immune responses. This may be due to the use of PD-L1 signaling mediated by the interaction of PD-L1-expressing tumor cells with PD-1-expressing T cells, resulting in weakened T cell activation and evasion of immune surveillance. Therefore, inhibition of PD-L1/PD-1 interaction can enhance CD8+ T cell-mediated tumor killing.

最佳治疗处理可以将PD-1受体/配体相互作用的阻断与直接抑制肿瘤生长的物质(agent)组合。仍存在对这种用于治疗、稳定、预防和/或延迟多种癌症的发展的最佳治疗的需要。Optimal therapeutic treatment may combine blockade of the PD-1 receptor/ligand interaction with agents that directly inhibit tumor growth. There remains a need for such optimal treatments for treating, stabilizing, preventing and/or delaying the progression of various cancers.

发明概述SUMMARY OF THE INVENTION

本发明涉及通过施用PD-1轴结合拮抗剂和紫杉烷来在患有癌症的个体中治疗癌症和增强免疫功能的方法。The present invention relates to methods of treating cancer and enhancing immune function in individuals with cancer by administering a PD-1 axis binding antagonist and a taxane.

在一方面，本发明涉及用于在个体中治疗癌症或延迟癌症进展的方法，其包括对该个体施用有效量的人PD-1轴结合拮抗剂和紫杉烷。在一些实施方案中，该PD-1轴结合拮抗剂选自PD-1结合拮抗剂、PD-L1结合拮抗剂和PD-L2结合拮抗剂。In one aspect, the present invention relates to a method for treating cancer or delaying cancer progression in an individual, comprising administering to the individual an effective amount of a human PD-1 axis binding antagonist and a taxane. In some embodiments, the PD-1 axis binding antagonist is selected from a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.

在以上方面的一些实施方案中，该PD-1轴结合拮抗剂是PD-1结合拮抗剂。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与其配体结合配偶体的结合。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与PD-L1的结合。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与PD-L2的结合。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与PD-L1和PD-L2二者的结合。在一些实施方案中，该PD-1结合拮抗剂是抗体。在一些实施方案中，该PD-1结合拮抗剂选自MDX-1106(nivolumab)、MK-3475(lambrolizumab)、CT-011(pidilizumab)和AMP-224。In some embodiments of the above aspects, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 binding antagonist is selected from MDX-1106 (nivolumab), MK-3475 (lambrolizumab), CT-011 (pidilizumab) and AMP-224.

在以上方面的其他实施方案中，该PD-1轴结合拮抗剂是PD-L1结合拮抗剂。在一些实施方案中，该PD-L1结合拮抗剂抑制PD-L1与PD-1的结合。在一些实施方案中，该PD-L1结合拮抗剂抑制PD-L1与B7-1的结合。在一些实施方案中，该PD-L1结合拮抗剂抑制PD-L1与PD-1和B7-1二者的结合。在一些实施方案中，该PD-L1结合拮抗剂是抗体。在一些实施方案中，该抗体选自YW243.55.S70、MPDL3280A、MDX-1105和MEDI4736。在一些实施方案中，该抗体包含含有HVR-H1序列SEQ ID NO:19、HVR-H2序列SEQ ID NO:20和HVR-H3序列SEQ ID NO:21的重链，及含有HVR-L1序列SEQ ID NO:22、HVR-L2序列SEQ ID NO:23和HVR-L3序列SEQID NO:24的轻链。在一些实施方案中，该抗体包含含有氨基酸序列SEQ ID NO:26的重链可变区和含有氨基酸序列SEQ ID NO:4的轻链可变区。In other embodiments of the above aspects, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is selected from YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. In some embodiments, the antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO: 19, HVR-H2 sequence of SEQ ID NO: 20, and HVR-H3 sequence of SEQ ID NO: 21, and a light chain comprising HVR-L1 sequence of SEQ ID NO: 22, HVR-L2 sequence of SEQ ID NO: 23, and HVR-L3 sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

在以上方面的一些实施方案中，该PD-1轴结合拮抗剂是PD-L2结合拮抗剂。在一些实施方案中，该PD-L2结合拮抗剂是抗体。在一些实施方案中，该PD-L2结合拮抗剂是免疫黏附素。In some embodiments of the above aspects, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2 binding antagonist is an antibody. In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

在以上方面的任意前述实施方案中，该癌症可以是但不限于：乳腺癌(包括三阴乳腺癌(TNBC))、膀胱癌(包括膀胱上皮性膀胱癌(UBC)、肌层浸润性膀胱癌和BCG难治性非肌层浸润性膀胱癌)、结肠直肠癌、直肠癌、肺癌(包括非小细胞肺癌，其可为鳞状或非鳞状)、成胶质细胞瘤、非霍奇金淋巴瘤(NHL)、肾细胞癌(包括肾细胞瘤)、前列腺癌、肝癌、胰腺癌、软组织肉瘤、卡波西肉瘤、类癌(carcinoid carcinoma)、头颈癌、胃癌、食管癌、前列腺癌、子宫内膜癌、肾癌、卵巢癌、间皮瘤和血癌(heme malignancy)(包括骨髓增生异常综合征(MDS)和多发性骨髓瘤)。在具体实施方案中，该癌症可以是肺癌(包括非小细胞肺癌，其可为鳞状或非鳞状)、膀胱癌(包括UBC)、乳腺癌(包括TNBC)、肾细胞瘤、黑素瘤、结肠直肠癌和血癌(包括骨髓增生异常综合征(MDS)和多发性骨髓瘤)。在一些实施方案中，该肺癌是非小细胞肺癌，其可为鳞状或非鳞状。在一些实施方案中，该膀胱癌是UBC。在一些实施方案中，该乳腺癌是TNBC。在一些实施方案中，该血癌是MDS或多发性骨髓瘤。In any of the foregoing embodiments of the above aspects, the cancer can be, but is not limited to, breast cancer (including triple-negative breast cancer (TNBC)), bladder cancer (including urothelial bladder cancer (UBC), muscle-invasive bladder cancer, and BCG-refractory non-muscle-invasive bladder cancer), colorectal cancer, rectal cancer, lung cancer (including non-small cell lung cancer, which may be squamous or non-squamous), glioblastoma, non-Hodgkin lymphoma (NHL), renal cell carcinoma (including renal cell tumor), prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, gastric cancer, esophageal cancer, prostate cancer, endometrial cancer, kidney cancer, ovarian cancer, mesothelioma, and heme malignancy (including myelodysplastic syndrome (MDS) and multiple myeloma). In specific embodiments, the cancer can be lung cancer (including non-small cell lung cancer, which can be squamous or non-squamous), bladder cancer (including UBC), breast cancer (including TNBC), renal cell tumor, melanoma, colorectal cancer, and blood cancer (including myelodysplastic syndrome (MDS) and multiple myeloma). In some embodiments, the lung cancer is non-small cell lung cancer, which can be squamous or non-squamous. In some embodiments, the bladder cancer is UBC. In some embodiments, the breast cancer is TNBC. In some embodiments, the blood cancer is MDS or multiple myeloma.

在以上方面的任意前述实施方案中，该个体患有癌症或诊断患有癌症。在一些实施方案中，该个体中的癌细胞表达PD-L1。In any preceding embodiments of the above aspects, the individual has or is diagnosed with cancer. In some embodiments, the cancer cells in the individual express PD-L1.

在以上方面的任意前述实施方案中，该治疗可以在该个体中产生应答。在一些实施方案中，该反应是完全应答。在一些实施方案中，该反应是停止治疗后的持续应答。在一些实施方案中，该反应是停止治疗后仍持续的完全应答。In any of the foregoing embodiments of the above aspects, the treatment can produce a response in the individual. In some embodiments, the response is a complete response. In some embodiments, the response is a sustained response after cessation of treatment. In some embodiments, the response is a complete response that persists after cessation of treatment.

在以上方面的任意前述实施方案中，该紫杉烷在该PD-1轴结合拮抗剂之前、与该PD-1轴结合拮抗剂同时或在该PD-1轴结合拮抗剂之后施用。在一些实施方案中，该紫杉烷是纳米清蛋白结合紫杉醇(nab-paclitaxel)紫杉醇(paclitaxel)或多西紫杉醇(docetaxel)。在一些实施方案中，该紫杉烷是纳米清蛋白结合紫杉醇在一些实施方案中，该紫杉烷是紫杉醇。In any of the preceding embodiments of the above aspects, the taxane is administered before, simultaneously with, or after the PD-1 axis binding antagonist. In some embodiments, the taxane is nab-paclitaxel, paclitaxel, or docetaxel. In some embodiments, the taxane is nab-paclitaxel. In some embodiments, the taxane is paclitaxel.

在另一方面，本发明涉及在患有癌症的个体中增强免疫功能的方法，该方法包括施用有效量的PD-1轴结合拮抗剂和紫杉烷。在一些实施方案中，该个体中的CD8+T细胞相对于施用PD-1轴结合拮抗剂和紫杉烷之前具有增强的引发、激活、增殖和/或溶细胞活性。在一些实施方案中，CD8+T细胞的数目相对于施用该组合之前提高。在一些实施方案中，该CD8+T细胞是抗原特异性CD8+T细胞。在一些实施方案中，Treg功能相对于施用该组合之前受抑制。在一些实施方案中，T细胞衰竭相对于施用该组合之前减少。在一些实施方案中，该PD-1轴结合拮抗剂选自PD-1结合拮抗剂、PD-L1结合拮抗剂和PD-L2结合拮抗剂。In another aspect, the present invention relates to a method for enhancing immune function in an individual suffering from cancer, the method comprising administering an effective amount of a PD-1 axis binding antagonist and a taxane. In some embodiments, the CD8+ T cells in the individual have enhanced priming, activation, proliferation and/or cytolytic activity relative to before administration of the PD-1 axis binding antagonist and the taxane. In some embodiments, the number of CD8+ T cells is increased relative to before administration of the combination. In some embodiments, the CD8+ T cells are antigen-specific CD8+ T cells. In some embodiments, Treg function is suppressed relative to before administration of the combination. In some embodiments, T cell exhaustion is reduced relative to before administration of the combination. In some embodiments, the PD-1 axis binding antagonist is selected from a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.

在以上方面的一些实施方案中，该PD-1轴结合拮抗剂是PD-1结合拮抗剂。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与其配体结合配偶体的结合。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与PD-L1的结合。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与PD-L2的结合。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与PD-L1和PD-L2二者的结合。在一些实施方案中，该PD-1结合拮抗剂是抗体。在一些实施方案中，该PD-1结合拮抗剂选自MDX-1106(nivolumab)、MK-3475(lambrolizumab)、CT-011(pidilizumab)和AMP-224。In some embodiments of the above aspects, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 binding antagonist is selected from MDX-1106 (nivolumab), MK-3475 (lambrolizumab), CT-011 (pidilizumab) and AMP-224.

在以上方面的其他实施方案中，该PD-1轴结合拮抗剂是PD-L1结合拮抗剂。在一些实施方案中，该PD-L1结合拮抗剂抑制PD-L1与PD-1的结合。在一些实施方案中，该PD-L1结合拮抗剂抑制PD-L1与B7-1的结合。在一些实施方案中，该PD-L1结合拮抗剂抑制PD-L1与PD-1和B7-1二者的结合。在一些实施方案中，该PD-L1结合拮抗剂是抗体。在一些实施方案中，该抗体选自YW243.55.S70、MPDL3280A、MDX-1105和MEDI4736。在一些实施方案中，该抗体包含含有HVR-H1序列SEQ ID NO:19、HVR-H2序列SEQ ID NO:20和HVR-H3序列SEQ ID NO:21的重链，及含有HVR-L1序列SEQ ID NO:22、HVR-L2序列SEQ ID NO:23和HVR-L3序列SEQID NO:24的轻链。在一些实施方案中，该抗体包含含有氨基酸序列SEQ ID NO:26的重链可变区和含有氨基酸序列SEQ ID NO:4的轻链可变区。In other embodiments of the above aspects, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is selected from YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. In some embodiments, the antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO: 19, HVR-H2 sequence of SEQ ID NO: 20, and HVR-H3 sequence of SEQ ID NO: 21, and a light chain comprising HVR-L1 sequence of SEQ ID NO: 22, HVR-L2 sequence of SEQ ID NO: 23, and HVR-L3 sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

在以上方面的一些实施方案中，该PD-1轴结合拮抗剂是PD-L2结合拮抗剂。在一些实施方案中，该PD-L2结合拮抗剂是抗体。在一些实施方案中，该PD-L2结合拮抗剂是免疫黏附素。In some embodiments of the above aspects, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2 binding antagonist is an antibody. In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

在以上方面的任意前述实施方案中，该癌症可以是乳腺癌(包括三阴乳腺癌(TNBC))、膀胱癌(包括膀胱上皮性膀胱癌(UBC)、肌层浸润性膀胱癌和BCG难治性非肌层浸润性膀胱癌)、结肠直肠癌、直肠癌、肺癌(包括非小细胞肺癌，其可为鳞状或非鳞状)、成胶质细胞瘤、非霍奇金淋巴瘤(NHL)、肾细胞癌(包括肾细胞瘤)、前列腺癌、肝癌、胰腺癌、软组织肉瘤、卡波西肉瘤、类癌、头颈癌、胃癌、食管癌、前列腺癌、子宫内膜癌、肾癌、卵巢癌、间皮瘤和血癌(包括骨髓增生异常综合征和多发性骨髓瘤)。在具体实施方案中，该癌症可以是肺癌(包括非小细胞肺癌，其可为鳞状或非鳞状)、膀胱癌(包括UBC)、乳腺癌(包括TNBC)、肾细胞瘤、黑素瘤、结肠直肠癌和血癌(例如骨髓增生异常综合征(MDS)和多发性骨髓瘤)。在一些实施方案中，该肺癌是非小细胞肺癌，其可为鳞状或非鳞状。在一些实施方案中，该膀胱癌是UBC。在一些实施方案中，该乳腺癌是TNBC。在一些实施方案中，该血癌是MDS或多发性骨髓瘤。In any of the preceding embodiments of the above aspects, the cancer can be breast cancer (including triple-negative breast cancer (TNBC)), bladder cancer (including urothelial bladder cancer (UBC), muscle-invasive bladder cancer, and BCG-refractory non-muscle-invasive bladder cancer), colorectal cancer, rectal cancer, lung cancer (including non-small cell lung cancer, which can be squamous or non-squamous), glioblastoma, non-Hodgkin lymphoma (NHL), renal cell carcinoma (including renal cell tumor), prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid, head and neck cancer, gastric cancer, esophageal cancer, prostate cancer, endometrial cancer, kidney cancer, ovarian cancer, mesothelioma, and blood cancer (including myelodysplastic syndrome and multiple myeloma). In specific embodiments, the cancer can be lung cancer (including non-small cell lung cancer, which can be squamous or non-squamous), bladder cancer (including UBC), breast cancer (including TNBC), renal cell tumor, melanoma, colorectal cancer, and blood cancer (e.g., myelodysplastic syndrome (MDS) and multiple myeloma). In some embodiments, the lung cancer is non-small cell lung cancer, which can be squamous or non-squamous. In some embodiments, the bladder cancer is UBC. In some embodiments, the breast cancer is TNBC. In some embodiments, the blood cancer is MDS or multiple myeloma.

在一些实施方案中，该个体中的癌细胞表达PD-L1。在一些实施方案中，该紫杉烷是纳米清蛋白结合紫杉醇紫杉醇或多西紫杉醇。在一些实施方案中，该紫杉烷是纳米清蛋白结合紫杉醇在一些实施方案中，该紫杉烷是紫杉醇。In some embodiments, the cancer cells in the individual express PD-L1. In some embodiments, the taxane is nanoalbumin-bound paclitaxel or docetaxel. In some embodiments, the taxane is nanoalbumin-bound paclitaxel. In some embodiments, the taxane is paclitaxel.

在以上方面中任一个的一些实施方案中，该PD-1轴结合拮抗剂和/或该紫杉烷静脉内、肌内、皮下、局部、口服、经皮、腹腔内、眼眶内、通过植入、通过吸入、鞘内、心室内或鼻内施用。In some embodiments of any of the above aspects, the PD-1 axis binding antagonist and/or the taxane is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

在以上方面中任一个的一些实施方案中，该方法可以进一步包括施用有效量的化疗剂。在一些实施方案中，该化疗剂是基于铂的化疗剂。在一些实施方案中，该基于铂的化疗剂是卡铂(carboplatin)。In some embodiments of any of the above aspects, the method may further comprise administering an effective amount of a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a platinum-based chemotherapeutic agent. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin.

在另一方面，本发明涉及人PD-1轴结合拮抗剂在制备用于在个体中治疗癌症或延迟癌症进展的药物中的用途，其中该药物包含人PD-1轴结合拮抗剂和可选的可药用载体，其中该治疗包括施用该药物与包含紫杉烷和可选的可药用载体的组合物的组合。In another aspect, the present invention relates to the use of a human PD-1 axis binding antagonist in the preparation of a medicament for treating cancer or delaying cancer progression in an individual, wherein the medicament comprises a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, wherein the treatment comprises administering the medicament in combination with a composition comprising a taxane and an optional pharmaceutically acceptable carrier.

在另一方面，本发明涉及紫杉烷在制备用于在个体中治疗癌症或延迟癌症进展的药物中的用途，其中该药物包含紫杉烷和可选的可药用载体，其中该治疗包括施用该药物与包含人PD-1轴结合拮抗剂和可选的可药用载体的组合物的组合。In another aspect, the invention relates to the use of a taxane in the preparation of a medicament for treating cancer or delaying cancer progression in an individual, wherein the medicament comprises a taxane and optionally a pharmaceutically acceptable carrier, wherein the treatment comprises administering the medicament in combination with a composition comprising a human PD-1 axis binding antagonist and optionally a pharmaceutically acceptable carrier.

在另一方面，本发明涉及用于在个体中治疗癌症或延迟癌症进展的包含人PD-1轴结合拮抗剂和可选的可药用载体的组合物，其中该治疗包括施用该组合物与第二组合物的组合，其中该第二组合物包含紫杉烷和可选的可药用载体。In another aspect, the invention relates to a composition comprising a human PD-1 axis binding antagonist and, optionally, a pharmaceutically acceptable carrier for treating cancer or delaying cancer progression in an individual, wherein the treatment comprises administering the composition in combination with a second composition, wherein the second composition comprises a taxane and, optionally, a pharmaceutically acceptable carrier.

在另一方面，本发明涉及用于在个体中治疗癌症或延迟癌症进展的包含紫杉烷和可选的可药用载体的组合物，其中该治疗包括施用该组合物与第二组合物的组合，其中该第二组合物包含人PD-1轴结合拮抗剂和可选的可药用载体。In another aspect, the invention relates to a composition comprising a taxane and, optionally, a pharmaceutically acceptable carrier for treating cancer or delaying progression of cancer in an individual, wherein the treatment comprises administering the composition in combination with a second composition comprising a human PD-1 axis binding antagonist and, optionally, a pharmaceutically acceptable carrier.

在另一方面，本发明涉及药盒，该药盒包含含有PD-1轴结合拮抗剂和可选的可药用载体的药物及包装说明书，该包装说明书包含与含有紫杉烷和可选的可药用载体的组合物组合施用该药物来在个体中治疗癌症或延迟癌症进展的说明。In another aspect, the invention relates to a kit comprising a medicament comprising a PD-1 axis binding antagonist and, optionally, a pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament in combination with a composition comprising a taxane and, optionally, a pharmaceutically acceptable carrier, to treat cancer or delay cancer progression in an individual.

在另一方面，本发明涉及药盒，该药盒包含含有PD-1轴结合拮抗剂和可选的可药用载体的第一药物，及含有紫杉烷和可选的可药用载体的第二药物。在一些实施方案中，该药盒进一步包含包装说明书，该包装说明书含有施用该第一药物和该第二药物来在个体中治疗癌症或延迟癌症进展的说明。In another aspect, the invention relates to a kit comprising a first drug comprising a PD-1 axis binding antagonist and optionally a pharmaceutically acceptable carrier, and a second drug comprising a taxane and optionally a pharmaceutically acceptable carrier. In some embodiments, the kit further comprises a package insert comprising instructions for administering the first drug and the second drug to treat or delay cancer progression in an individual.

在另一方面，本发明涉及药盒，该药盒包含含有紫杉烷和可选的可药用载体的药物及包装说明书，该包装说明书包含与含有PD-1轴结合拮抗剂和可选的可药用载体的组合物组合施用该药物来在个体中治疗癌症或延迟癌症进展的说明。In another aspect, the invention relates to a kit comprising a medicament comprising a taxane and, optionally, a pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament in combination with a composition comprising a PD-1 axis binding antagonist and, optionally, a pharmaceutically acceptable carrier, to treat cancer or delay cancer progression in an individual.

在前述方面的任一个中，该PD-1轴结合拮抗剂选自PD-1结合拮抗剂、PD-L1结合拮抗剂和PD-L2结合拮抗剂。在一些实施方案中，该PD-1轴结合拮抗剂是PD-1结合拮抗剂。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与其配体结合配偶体的结合。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与PD-L1的结合。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与PD-L2的结合。在一些实施方案中，该PD-1结合拮抗剂抑制PD-1与PD-L1和PD-L2二者的结合。在一些实施方案中，该PD-1结合拮抗剂是抗体。在一些实施方案中，该PD-1结合拮抗剂选自MDX-1106(nivolumab)、MK-3475(lambrolizumab)、CT-011(pidilizumab)和AMP-224。在一些实施方案中，该PD-1轴结合拮抗剂是PD-L1结合拮抗剂。在一些实施方案中，该PD-L1结合拮抗剂抑制PD-L1与PD-1的结合。在一些实施方案中，该PD-L1结合拮抗剂抑制PD-L1与B7-1的结合。在一些实施方案中，该PD-L1结合拮抗剂抑制PD-L1与PD-1和B7-1二者的结合。在一些实施方案中，该PD-L1结合拮抗剂是抗体。在一些实施方案中，该抗体选自YW243.55.S70、MPDL3280A、MDX-1105和MEDI4736。在一些实施方案中，该抗体包含含有HVR-H1序列SEQ ID NO:19、HVR-H2序列SEQ ID NO:20和HVR-H3序列SEQ ID NO:21的重链，及含有HVR-L1序列SEQ ID NO:22、HVR-L2序列SEQ ID NO:23和HVR-L3序列SEQ ID NO:24的轻链。在一些实施方案中，该抗体包含含有氨基酸序列SEQ ID NO:26的重链可变区和含有氨基酸序列SEQ ID NO:4的轻链可变区。In any of the foregoing aspects, the PD-1 axis binding antagonist is selected from a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 binding antagonist is selected from MDX-1106 (nivolumab), MK-3475 (lambrolizumab), CT-011 (pidilizumab), and AMP-224. In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is selected from YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. In some embodiments, the antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO: 19, HVR-H2 sequence of SEQ ID NO: 20, and HVR-H3 sequence of SEQ ID NO: 21, and a light chain comprising HVR-L1 sequence of SEQ ID NO: 22, HVR-L2 sequence of SEQ ID NO: 23, and HVR-L3 sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

在前述方面的任一个中，该紫杉烷可以是纳米清蛋白结合紫杉醇紫杉醇或多西紫杉醇。在一些实施方案中，该紫杉烷是纳米清蛋白结合紫杉醇在一些实施方案中，该紫杉烷是紫杉醇。In any of the preceding aspects, the taxane can be nanoalbumin-bound paclitaxel, paclitaxel, or docetaxel. In some embodiments, the taxane is nanoalbumin-bound paclitaxel. In some embodiments, the taxane is paclitaxel.

应理解，本文所述多种实施方案的一种、一些或全部特性可以组合形成本发明的其他实施方案。本发明的这些及其他方面对本领域技术人员而言将变得显而易见。通过以下发明详述进一步描述本发明的这些及其他实施方案。It should be understood that one, some or all of the features of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description of the invention.

附图简述BRIEF DESCRIPTION OF THE DRAWINGS

图1是图表，显示与对照抗体或紫杉醇+卡铂单独相比，抗PD-L1抗体和紫杉醇+卡铂的联合治疗在C57BL/6小鼠同基因MC38结肠直肠肿瘤模型中显示协同抗肿瘤作用。图表显示各处理组肿瘤体积的三次样条拟合作为时间的函数。三次样条拟合是选择拟合每个处理组的所有数据的最佳平滑曲线的数学算法。用单剂腹腔内(IP)注射的80mg/kg卡铂加静脉内(IV)注射的10mg/kg紫杉醇和每周给药3次给药3周的10mg/kg抗gp120抗体或抗PD-L1(克隆25A1mIgG2a.DANA)处理已建立了约100-200mm³的皮下MC38肿瘤的小鼠。N＝10只小鼠/组。Figure 1 is a graph showing that the combination of anti-PD-L1 antibody and paclitaxel + carboplatin exhibits synergistic anti-tumor effects in a syngeneic MC38 colorectal tumor model in C57BL/6 mice compared to control antibody or paclitaxel + carboplatin alone. The graph shows a cubic spline fit of tumor volume for each treatment group as a function of time. A cubic spline fit is a mathematical algorithm that selects the best smooth curve that fits all the data for each treatment group. Mice with established subcutaneous MC38 tumors of approximately 100-200 mm 3 were treated with a single intraperitoneal (IP) injection of 80 mg/kg carboplatin plus intravenous (IV) injection of 10 mg/kg paclitaxel and 10 mg/kg anti-gp120 antibody or anti-PD-L1 (clone 25A1mIgG2a.DANA) administered three times a week for³ weeks. N = 10 mice/group.

图2A和2B是图表，显示地塞米松(dexamethasone)(Dex)废除抗PD-L1抗体(αPD-L1)单一治疗在C57BL/6小鼠同基因MC38结肠直肠肿瘤模型中的功效。图2A显示各处理组肿瘤体积的三次样条拟合，而图2B显示单只小鼠的曲线(Trellis曲线)(黑色曲线显示各处理组肿瘤体积的三次样条拟合)。图2B中的各图表包含代表对照组的三次样条拟合的虚线。对于图2B，约300mm³处的水平虚线是进展体积的参考(2x起始肿瘤体积)。低于32mm³(在图2B中表示为水平虚线)的肿瘤体积可见，但太小而不能准确测量。用单剂盐水或4mg/kg IV的地塞米松处理已建立了约100-200mm³的皮下MC38肿瘤的小鼠，12小时后，用每周给药3次给药3周的10mg/kg IP对照抗gp120抗体或抗PD-L1(克隆25A1.mIgG2a.DANA)处理。N＝10只小鼠/组。Figures 2A and 2B are graphs showing that dexamethasone (Dex) abolishes the efficacy of anti-PD-L1 antibody (αPD-L1) monotherapy in a syngeneic MC38 colorectal tumor model in C57BL/6 mice. Figure 2A shows a cubic spline fit of the tumor volume of each treatment group, while Figure 2B shows a curve (Trellis curve) for a single mouse (the black curve shows the cubic spline fit of the tumor volume of each treatment group). Each graph in Figure 2B contains a dashed line representing the cubic spline fit of the control group. For Figure 2B, the horizontal dashed line at approximately 300 mm³ is a reference for the progression volume (2x the starting tumor volume). Tumor volumes below 32 mm³ (represented as the horizontal dashed line in Figure 2B) are visible but too small to be accurately measured. Mice with established subcutaneous MC38 tumors of approximately 100-200^mm3 were treated with a single dose of saline or 4 mg/kg IV dexamethasone. Twelve hours later, they were treated with 10 mg/kg IP of a control anti-gp120 antibody or anti-PD-L1 (clone 25A1.mIgG2a.DANA) administered three times a week for three weeks. N = 10 mice/group.

图3是图表，显示地塞米松抑制OTI过继T细胞转移和接种模型中的抗原特异性T细胞反应。从OTI Thy1.1小鼠纯化CD8+T细胞，并按2.5x10⁶细胞/小鼠IV注射。次日用250ng与全长卵清蛋白融合的抗DEC205加单剂盐水或4mg/kg IV地塞米松接种受体小鼠。两天后，对小鼠实施安乐死，通过流式细胞术计数来自脾脏的总OTI CD8+细胞。N＝5只小鼠/组，每个符号代表单只小鼠。通过双尾非配对t检验计算P值。Figure 3 is a graph showing that dexamethasone inhibits antigen-specific T cell responses in an OTI adoptive T cell transfer and vaccination model. CD8+ T cells were purified from OTI Thy1.1 mice and injected IV at 2.5 x¹⁰⁶ cells/mouse. The following day, recipient mice were vaccinated with 250 ng of anti-DEC205 fused to full-length ovalbumin plus a single dose of saline or 4 mg/kg IV dexamethasone. Two days later, mice were euthanized, and total OTI CD8+ cells from the spleen were counted by flow cytometry. N = 5 mice/group, each symbol represents a single mouse. P values were calculated using a two-tailed unpaired t-test.

图4A和4B是图表，显示抗PD-L1抗体和纳米清蛋白结合紫杉醇(Abx)+卡铂(Carbo)的联合治疗在C57BL/6小鼠同基因MC38结肠直肠肿瘤模型中产生强协同抗肿瘤作用，并达到持续大于90天的持续完全应答(4/8小鼠)。图表显示肿瘤体积作为时间的函数。图4A显示各处理组肿瘤体积的三次样条拟合，而图4B显示单只小鼠的Trellis曲线(黑色曲线显示各处理组肿瘤体积的三次样条拟合)。图4B中的各图表包含代表对照组的三次样条拟合的虚线。对于图4B，约600mm³处的水平虚线是进展体积参考(2x起始肿瘤体积)。低于32mm³(在图4B中表示为水平虚线)的肿瘤体积可见，但太小而不能准确测量。按所示，用每周给药3次给药3周的IP注射施用的10mg/kg抗gp120对照抗体或抗PD-L1抗体(克隆YW243.55.S70mIgG2a.DANA)，加每周给药一次给药三周的盐水或75mg/kg IP卡铂，加每周给药一次给药三周的15mg/kg iv 处理已建立了约300mm³的皮下MC38肿瘤的小鼠。N＝8只小鼠/组。Figures 4A and 4B are graphs showing that the combination of anti-PD-L1 antibody and nanoalbumin combined with paclitaxel (Abx) + carboplatin (Carbo) produces a strong synergistic anti-tumor effect in a syngeneic MC38 colorectal tumor model in C57BL/6 mice, achieving a sustained complete response lasting greater than 90 days (4/8 mice). The graphs show tumor volume as a function of time. Figure 4A shows a cubic spline fit of the tumor volume for each treatment group, while Figure 4B shows a Trellis plot for a single mouse (the black curve shows the cubic spline fit of the tumor volume for each treatment group). Each graph in Figure 4B includes a dashed line representing the cubic spline fit of the control group. For Figure 4B, the horizontal dashed line at approximately 600 mm³ is the progression volume reference (2× the starting tumor volume). Tumor volumes below 32 mm³ (represented as the horizontal dashed line in Figure 4B) are visible but too small to be accurately measured. Mice with established subcutaneous MC38 tumors of approximately 300 mm3 were treated with 10 mg/kg anti-gp120 control antibody or anti-PD-L1 antibody (clone YW243.55.S70mIgG2a.DANA) administered by IP injection three times a week for three weeks, plus saline or 75 mg/kg IP carboplatin once a week for three weeks, plus 15 mg/kg iv once a week for^three weeks, as indicated. N = 8 mice/group.

图5A和5B是图表，显示之前用抗PD-L1抗体和纳米清蛋白结合紫杉醇+卡铂处理治愈MC38原发性肿瘤(图1A中所述的达到完全应答的小鼠)的小鼠产生抗肿瘤T细胞记忆反应。在用新的MC38肿瘤细胞进行二次再攻击时，肿瘤未能在100％(4/4)的已治愈小鼠中生长。图5A显示，二次攻击后7天收集的脾细胞具有与初次攻击的首次用于实验的小鼠相当的CD4+和CD8+T细胞数。图5B显示流式细胞术分析的结果，显示在用PMA加离子霉素体外刺激时，如通过胞内细胞因子染色所评估，与初次攻击的小鼠相比，来自已治愈小鼠的T细胞具有增强的干扰素-γ(IFN-γ)产生。误差棒表示n＝5(首次攻击的小鼠)或n＝4(已治愈的小鼠，二次攻击)的标准差，流式细胞术点阵图代表来自每个组的一只小鼠。通过双尾非配对t检验计算P值。Figures 5A and 5B are graphs showing that mice that had previously been cured of MC38 primary tumors (mice that achieved a complete response as described in Figure 1A) with anti-PD-L1 antibodies and nanoalbumin combined with paclitaxel + carboplatin produced anti-tumor T cell memory responses. When re-challenged for the second time with new MC38 tumor cells, tumors failed to grow in 100% (4/4) of the cured mice. Figure 5A shows that splenocytes collected 7 days after the second attack had CD4+ and CD8+ T cell counts comparable to those of mice that were first challenged. Figure 5B shows the results of flow cytometry analysis, showing that when stimulated in vitro with PMA plus ionomycin, as assessed by intracellular cytokine staining, T cells from cured mice had enhanced interferon-γ (IFN-γ) production compared to mice that were first challenged. Error bars represent the standard deviation of n=5 (mice attacked for the first time) or n=4 (cured mice, second attack), and the flow cytometry dot plots represent one mouse from each group. P values were calculated by two-tailed unpaired t-test.

图6A和6B是图表，显示来自测试抗PD-L1抗体(MPDL3280A)与紫杉烷和卡铂的联合治疗的有效性的1b期临床试验的结果。图6A是显示用MPDL3280A、纳米清蛋白结合紫杉醇和卡铂治疗后肿瘤大小随时间的变化的图表。客观反应率(ORR)为9/14名患者，有3名完全应答(CR)和6名部分反应(PR)。图6B是显示用MPDL3280A和紫杉醇+卡铂治疗后肿瘤大小随时间的变化的图表。ORR为2/6名患者(33％)，有2名部分反应。Figures 6A and 6B are graphs showing results from a Phase 1b clinical trial testing the effectiveness of a combination therapy of an anti-PD-L1 antibody (MPDL3280A) with a taxane and carboplatin. Figure 6A is a graph showing changes in tumor size over time after treatment with MPDL3280A, nanoalbumin-bound paclitaxel, and carboplatin. The objective response rate (ORR) was 9/14 patients, with 3 complete responses (CR) and 6 partial responses (PR). Figure 6B is a graph showing changes in tumor size over time after treatment with MPDL3280A and paclitaxel + carboplatin. The ORR was 2/6 patients (33%), with 2 partial responses.

发明详述Detailed Description of the Invention

I.定义I. Definition

在详细描述本发明之前，应理解，本发明不限于特定的组合物或生物系统，其当然可以变动。还应理解，本文所用的术语仅是为了描述具体实施方案的目的，并非旨在限制。Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

除非清楚地另有说明，本说明书及所附权利要求中所用的单数形式“一”、“一个”和“该”包括复数指代物。因此，例如，提到“一个分子”可选地包括两个或多个这类分子的组合，及其他类似情况。As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and so forth.

本文所用的术语“约”指本技术领域技术人员容易知道的该值通常的误差范围。本文提到“约”某个值或参数包括(和描述)了指向该值或参数本身的实施方案。The term "about" as used herein refers to the typical error range of the value readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself.

应理解，本文所述的本发明的方面和实施方案包括“含有该方面和实施方案”、“由该方面和实施方案组成”及“基本由该方面和实施方案组成”。It should be understood that aspects and embodiments of the present invention described herein include "comprising such aspects and embodiments," "consisting of such aspects and embodiments," and "consisting essentially of such aspects and embodiments."

术语“PD-1轴结合拮抗剂”指这样的分子，该分子抑制PD-1轴结合配偶体与其一种或多种结合配偶体的相互作用，以去除由PD-1信号发放轴上的信号发放引起的T细胞功能异常——结果恢复或增强T细胞功能(例如增殖、细胞因子产生和/或靶细胞杀伤)。本文所用的PD-1轴结合拮抗剂包括PD-1结合拮抗剂、PD-L1结合拮抗剂和PD-L2结合拮抗剂。The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners to remove T cell dysfunction caused by signaling on the PD-1 signaling axis - resulting in restoration or enhancement of T cell function (e.g., proliferation, cytokine production, and/or target cell killing). PD-1 axis binding antagonists, as used herein, include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

术语“PD-1结合拮抗剂”指这样的分子，该分子减少、阻断、抑制、废除或干扰由PD-1与其一种或多种结合配偶体如PD-L1和/或PD-L2的相互作用引起的信号转导。在一些实施方案中，该PD-1结合拮抗剂是抑制PD-1与其一种或多种结合配偶体的结合的分子。在具体方面，该PD-1结合拮抗剂抑制PD-1与PD-L1和/或PD-L2的结合。例如，PD-1结合拮抗剂包括抗PD-1抗体、其抗原结合片段、免疫黏附素、融合蛋白质、寡肽及其他减少、阻断、抑制、废除或干扰由PD-1与PD-L1和/或PD-L2的相互作用引起的信号转导的分子。在一个实施方案中，PD-1结合拮抗剂减少由或通过表达在通过PD-1介导信号发放的T淋巴细胞上的细胞表面蛋白质介导的负共刺激信号，从而使功能异常的T细胞的功能异常程度减弱(例如增强对抗原识别的效应子反应)。在一些实施方案中，该PD-1结合拮抗剂是抗PD-1抗体。在具体方面，PD-1结合拮抗剂是本文所述MDX-1106(nivolumab)。在另一具体方面，PD-1结合拮抗剂是本文所述MK-3475(lambrolizumab)。在另一具体方面，PD-1结合拮抗剂是本文所述CT-011(pidilizumab)。在另一具体方面，PD-1结合拮抗剂是本文所述AMP-224。The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abolishes or interferes with signal transduction caused by the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and/or PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, abolish or interfere with signal transduction caused by the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative co-stimulatory signals mediated by or on cell surface proteins expressed on T lymphocytes that mediate signaling through PD-1, thereby reducing the degree of functional abnormality of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab) described herein. In another specific aspect, the PD-1 binding antagonist is MK-3475 (lambrolizumab) described herein. In another specific aspect, the PD-1 binding antagonist is CT-011 (pidilizumab) described herein. In another specific aspect, the PD-1 binding antagonist is AMP-224 described herein.

术语“PD-L1结合拮抗剂”指这样的分子，该分子减少、阻断、抑制、废除或干扰由PD-L1与其一种或多种结合配偶体如PD-1和/或B7-1的相互作用引起的信号转导。在一些实施方案中，PD-L1结合拮抗剂是抑制PD-L1与其结合配偶体的结合的分子。在具体方面，该PD-L1结合拮抗剂抑制PD-L1与PD-1和/或B7-1的结合。在一些实施方案中，该PD-L1结合拮抗剂包括抗PD-L1抗体、其抗原结合片段、免疫黏附素、融合蛋白质、寡肽及其他减少、阻断、抑制、废除或干扰由PD-L1与其一种或多种结合配偶体如PD-1和/或B7-1的相互作用引起的信号转导的分子。在一个实施方案中，PD-L1结合拮抗剂减少由或通过表达在通过PD-L1介导信号发放的T淋巴细胞上的细胞表面蛋白质介导的负共刺激信号，从而使功能异常的T细胞的功能异常程度减弱(例如增强对抗原识别的效应子反应)。在一些实施方案中，PD-L1结合拮抗剂是抗PD-L1抗体。在具体方面，抗PD-L1抗体是本文所述YW243.55.S70。在另一具体方面，抗PD-L1抗体是本文所述MDX-1105。还在另一具体方面，抗PD-L1抗体是本文所述MPDL3280A。还在另一具体方面，抗PD-L1抗体是本文所述MEDI4736。The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abolishes or interferes with signal transduction caused by the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In specific aspects, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonist includes anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, abolish or interfere with signal transduction caused by the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and/or B7-1. In one embodiment, the PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or by a cell surface protein expressed on T lymphocytes that mediate signaling through PD-L1, thereby reducing the degree of functional abnormality of the dysfunctional T cells (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific aspect, the anti-PD-L1 antibody is YW243.55.S70 described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1105 described herein. In yet another specific aspect, the anti-PD-L1 antibody is MPDL3280A described herein. In yet another specific aspect, the anti-PD-L1 antibody is MEDI4736 described herein.

术语“PD-L2结合拮抗剂”指这样的分子，该分子减少、阻断、抑制、废除或干扰由PD-L2与其一种或多种结合配偶体如PD-1的相互作用引起的信号转导。在一些实施方案中，PD-L2结合拮抗剂是抑制PD-L2与其一种或多种结合配偶体的结合的分子。在具体方面，该PD-L2结合拮抗剂抑制PD-L2与PD-1的结合。在一些实施方案中，该PD-L2拮抗剂包括抗PD-L2抗体、其抗原结合片段、免疫黏附素、融合蛋白质、寡肽及其他减少、阻断、抑制、废除或干扰由PD-L2与其一种或多种结合配偶体如PD-1的相互作用引起的信号转导的分子。在一个实施方案中，PD-L2结合拮抗剂减少由或通过表达在通过PD-L2介导信号发放的T淋巴细胞上的细胞表面蛋白质介导的负共刺激信号，从而使功能异常的T细胞的功能异常程度减弱(例如增强对抗原识别的效应子反应)。在一些实施方案中，PD-L2结合拮抗剂是免疫黏附素。The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abolishes or interferes with signal transduction caused by the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, the PD-L2 antagonist includes anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, abolish or interfere with signal transduction caused by the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, a PD-L2 binding antagonist reduces a negative co-stimulatory signal mediated by or on a cell surface protein expressed on a T lymphocyte that mediates signaling through PD-L2, thereby reducing the degree of functional abnormality of a dysfunctional T cell (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

本文所用的“紫杉烷”是可以结合微管蛋白、促进微管组装和稳定化和/或防止微管解聚的双萜。本文中所包括的紫杉烷包括紫杉烷(taxoid)10-脱乙酰基巴卡丁III和/或其衍生物。示例性紫杉烷包括但不限于紫杉醇(即CAS#33069-62-4)、多西紫杉醇(即CAS#114977-28-5)、larotaxel、cabazitaxel、milataxel、tesetaxel和/或orataxel。在一些实施方案中，该紫杉烷是清蛋白包被的纳米颗粒(例如纳米清蛋白结合(nab)紫杉醇(即)和/或纳米清蛋白结合多西紫杉醇ABI-008)。在一些实施方案中，该紫杉烷是纳米清蛋白结合紫杉醇在一些实施方案中，该紫杉烷配制在中(例如)和/或吐温如聚山梨酸酯80中(例如)。在一些实施方案中，该紫杉烷是脂质体包封的紫杉烷。在一些实施方案中，该紫杉烷是紫杉烷的前体药物形式和/或缀合形式(例如DHA共价缀合至紫杉醇、聚谷紫杉醇和/或碳酸亚油醇酯紫杉醇)。在一些实施方案中，该紫杉醇配制为基本不含表面活性剂(例如，在缺乏CREMAPHOR和/或吐温的情况下，如紫杉醇)。As used herein, "taxane" is a diterpene that can bind to tubulin, promote microtubule assembly and stabilization, and/or prevent microtubule depolymerization. Taxoids encompassed herein include taxoid 10-deacetylbaccatin III and/or its derivatives. Exemplary taxoids include, but are not limited to, paclitaxel (i.e., CAS# 33069-62-4), docetaxel (i.e., CAS# 114977-28-5), larotaxel, cabazitaxel, milataxel, tesetaxel, and/or orataxel. In some embodiments, the taxane is an albumin-coated nanoparticle (e.g., nanoalbumin-bound (nab) paclitaxel (i.e., ABI-008) and/or nanoalbumin-bound docetaxel (ABI-008). In some embodiments, the taxane is nanoalbumin-bound paclitaxel. In some embodiments, the taxane is formulated in (e.g., Tween®) and/or Tween® such as polysorbate 80 (e.g., Tween®). In some embodiments, the taxane is a liposome-encapsulated taxane. In some embodiments, the taxane is a prodrug form and/or a conjugated form of a taxane (e.g., DHA covalently conjugated to paclitaxel, polysiglide, and/or linoleyl carbonate paclitaxel). In some embodiments, the paclitaxel is formulated to be substantially free of surfactant (e.g., in the absence of CREMAPHOR and/or Tween, as in paclitaxel).

免疫功能异常背景中的术语“功能异常”指对抗原刺激的免疫反应性降低。该术语包括“衰竭”和/或“无反应性”二者的共同要素，其中可以发生抗原识别，但随后的免疫反应对控制感染或肿瘤生长无效。The term "dysfunction" in the context of immune dysfunction refers to a decrease in immune responsiveness to antigenic stimulation. The term encompasses the common elements of both "exhaustion" and/or "anergy," in which antigen recognition can occur but the subsequent immune response is ineffective in controlling infection or tumor growth.

本文所用的术语“功能异常的”还包括对抗原识别耐受或无反应，具体而言，将抗原识别转化为下游T细胞效应子功能如增殖、细胞因子产生(例如IL-2)和/或靶细胞杀伤的能力受损。As used herein, the term "dysfunctional" also includes tolerance or anergy to antigen recognition, specifically, an impaired ability to translate antigen recognition into downstream T cell effector functions such as proliferation, cytokine production (e.g., IL-2), and/or target cell killing.

术语“无反应性”指由通过T细胞受体传递的信号不全或不足(例如胞内Ca⁺²在缺乏ras激活的情况下增加)引起的对抗原刺激无反应性的状态。T细胞无反应性也可以在缺乏共刺激的情况下用抗原刺激产生，导致细胞甚至在共刺激的背景中耐受随后的抗原激活。无反应性状态通常可以通过白介素-2的存在来推翻。无反应性T细胞不进行克隆扩充和/或获得效应子功能。The term "anergy" refers to a state of anergy to antigenic stimulation caused by incomplete or insufficient signals transmitted by the T cell receptor (e.g., intracellular Ca⁺² increases in the absence of ras activation). T cell anergy can also be produced by antigenic stimulation in the absence of costimulation, causing cells to tolerate subsequent antigenic activation even in the context of costimulation. The anergy state can usually be overturned by the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and/or acquire effector functions.

术语“衰竭”指产生自许多慢性感染和癌症期间发生的持续TCR信号发放的作为T细胞功能异常状态的T细胞衰竭。它与无反应性的区别在于，它不是通过不全或不足的信号发放产生，而是产生自持续信号发放。它定义为弱效应子功能、抑制性受体的持续表达和不同于功能性效应或记忆T细胞的转录状态。衰竭阻止了感染和肿瘤的最佳控制。衰竭可以由外在负调节途径(例如免疫调节细胞因子)以及细胞内在负调节(共刺激)途径(PD-1、B7-H3、B7-H4等)二者引起。The term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction caused by continuous TCR signaling that occurs during many chronic infections and cancers. It is different from anergy in that it is not produced by incomplete or insufficient signaling, but rather by continuous signaling. It is defined as weak effector function, persistent expression of inhibitory receptors, and a transcriptional state that is different from functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Exhaustion can be caused by both extrinsic negative regulatory pathways (such as immunomodulatory cytokines) and cellular intrinsic negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).

“增强T细胞功能”意指诱导、引起或刺激T细胞具有持续的或放大的生物学功能，或再生或再激活衰竭或失活的T细胞。增强T细胞功能的实例包括：相对于干预之前的这类水平增加来自CD8+T细胞的γ-干扰素分泌、增加增殖、提高抗原反应性(例如病毒、病原体或肿瘤清除)。在一个实施方案中，增强的水平为至少50％、备选地60％、70％、80％、90％、100％、120％、150％或200％增强。测量此增强的方式为本领域普通技术人员已知。"Enhancing T cell function" means inducing, causing or stimulating T cells to have a continuous or amplified biological function, or regenerating or reactivating exhausted or inactivated T cells. Examples of enhancing T cell function include: increasing secretion of gamma interferon from CD8+ T cells relative to such levels before intervention, increasing proliferation, improving antigen reactivity (e.g., virus, pathogen or tumor clearance). In one embodiment, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150% or 200% enhancement. The manner of measuring this enhancement is known to those of ordinary skill in the art.

“T细胞功能异常性障碍”是表征为对抗原刺激的反应性降低的T细胞障碍或病症。在具体实施方案中，T细胞功能异常性障碍是与通过PD-1的信号发放的不适当增加明确相关的障碍。在另一实施方案中，T细胞功能异常性障碍是这样的障碍，其中T细胞无反应性或分泌细胞因子、增殖或发挥溶细胞活性的能力降低。在具体方面，反应性降低导致表达免疫原的病原体或肿瘤的无效控制。表征为T细胞功能异常的T细胞功能异常性障碍的实例包括未解决的急性感染、慢性感染和肿瘤免疫。A "T cell dysfunction disorder" is a T cell disorder or condition characterized by reduced responsiveness to antigenic stimulation. In a specific embodiment, the T cell dysfunction disorder is a disorder clearly associated with an inappropriate increase in signaling through PD-1. In another embodiment, the T cell dysfunction disorder is a disorder in which T cells are anergic or have a reduced ability to secrete cytokines, proliferate, or exert cytolytic activity. In specific aspects, the reduced responsiveness results in ineffective control of pathogens or tumors expressing immunogens. Examples of T cell dysfunction disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity.

“肿瘤免疫”指肿瘤逃避免疫识别和清除的过程。因此，在减弱这种逃避且免疫系统识别和攻击肿瘤时，肿瘤免疫得到“治疗”。肿瘤识别的实例包括肿瘤结合、肿瘤收缩和肿瘤清除。"Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, when this evasion is reduced and the immune system recognizes and attacks the tumor, tumor immunity is "treated." Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

“免疫原性”指具体物质引起免疫反应的能力。肿瘤具有免疫原性，增强肿瘤免疫原性帮助通过免疫反应清除肿瘤细胞。增强肿瘤免疫原性的实例包括用PD-1轴结合拮抗剂和紫杉烷治疗。"Immunogenicity" refers to the ability of a substance to elicit an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity can help eliminate tumor cells through immune responses. Examples of enhancing tumor immunogenicity include treatment with PD-1 axis antagonists and taxanes.

“持续应答”指停止治疗后对减少肿瘤生长的持续效应。例如，与施用期开始时的大小相比，肿瘤大小可以保持相同或更小。在一些实施方案中，持续应答具有至少与治疗持续时间相同、治疗持续时间长度的至少1.5X、2.0X、2.5X或3.0X的持续时间。A "sustained response" refers to a persistent effect on reducing tumor growth after treatment has stopped. For example, the tumor size can remain the same or smaller than its size at the beginning of the administration period. In some embodiments, the sustained response has a duration that is at least the same as, at least 1.5X, 2.0X, 2.5X, or 3.0X the duration of treatment.

本文所用的“减少或抑制癌症复发”意指减少或抑制肿瘤或癌症复发或肿瘤或癌症进展。如本文所公开，癌症复发和/或癌症进展包括但不限于癌症转移。As used herein, "reducing or inhibiting cancer recurrence" means reducing or inhibiting tumor or cancer recurrence or tumor or cancer progression. As disclosed herein, cancer recurrence and/or cancer progression include, but are not limited to, cancer metastasis.

本文所用的“完全应答”或“CR”指所有靶病灶的消失。As used herein, "complete response" or "CR" refers to the disappearance of all target lesions.

本文所用的“部分反应”或“PR”指靶病灶的最长径总和(SLD)减少至少30％，取基线SLD作为参考。As used herein, a "partial response" or "PR" refers to a reduction of at least 30% in the sum of the longest diameters (SLD) of target lesions, taking the baseline SLD as a reference.

本文所用的“稳定疾病”或“SD”指靶病灶既未充分收缩至符合PR的条件，也未充分增加至符合PD的条件，取自治疗开始以来的最小SLD作为参考。As used herein, "stable disease" or "SD" means that the target lesion has neither shrunk sufficiently to qualify as a PR nor increased sufficiently to qualify as a PD, with the minimum SLD since the start of treatment being taken as a reference.

本文所用的“进行性疾病”或“PD”指靶病灶的SLD增加至少20％(取自治疗开始以来记录到的最小SLD作为参考)，或存在一个或多个新病灶。As used herein, "progressive disease" or "PD" refers to an increase in the SLD of a target lesion by at least 20% (taking as reference the minimum SLD recorded since the start of treatment), or the presence of one or more new lesions.

本文所用的“无进展存活期”(PFS)指治疗期间及之后所治疗的疾病(例如癌症)未恶化的时长。无进展存活期可以包括患者经历完全应答或部分反应的时间量，以及患者经历稳定疾病的时间量。As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment that the treated disease (e.g., cancer) does not worsen. Progression-free survival can include the amount of time a patient experiences a complete response or partial response, as well as the amount of time a patient experiences stable disease.

本文所用的“总反应率”或“客观反应率”(ORR)指完全应答(CR)率和部分反应(PR)率的总和。As used herein, "overall response rate" or "objective response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

本文所用的“总存活率”(OS)指一个组中可能在特定时期后存活的个体的百分比。As used herein, "overall survival" (OS) refers to the percentage of individuals in a group who are likely to survive after a specified period of time.

术语“药物制剂”指这样的制备物，其处于这样的形式，使得活性成分的生物活性有效，且不包含对将要施用该制剂的个体具有不可接受的毒性的附加成分。“可药用”赋形剂(载体、添加剂)是可以合理地对个体哺乳动物施用以提供所利用的活性成分的有效剂量的那些。The term "pharmaceutical formulation" refers to a preparation that is in such form that the biological activity of the active ingredient is effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered. "Pharmaceutically acceptable" excipients (carriers, additives) are those that can reasonably be administered to a mammalian subject to provide an effective dosage of the active ingredient employed.

本文所用的术语“处理/治疗(treatment)”指设计用于改变所处理的个体或细胞在临床病理过程中的自然过程的临床干预。希望得到的处理作用包括减慢疾病进展速率、改善或缓和疾病状态及缓解或改善的预后。例如，如果与癌症相关的一种或多种症状缓和或消除，则个体得到成功“处理”，该症状缓和或消除包括但不限于减少癌细胞的增殖或(破坏癌细胞)、减少疾病引起的症状、提高患有疾病的个体的生活质量、减少治疗疾病所需的其他药物的剂量、和/或延长个体存活。The term "treatment" as used herein refers to a clinical intervention designed to change the natural course of the individual or cell being treated during clinical pathology. The desired treatment effect includes slowing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis. For example, if one or more symptoms associated with cancer are alleviated or eliminated, the individual is successfully "treated," and this symptom alleviation or elimination includes, but is not limited to, reducing the proliferation of cancer cells or (destroying cancer cells), reducing the symptoms caused by the disease, improving the quality of life of the individual with the disease, reducing the dosage of other drugs required to treat the disease, and/or prolonging individual survival.

本文所用的“延迟疾病的进展”意指推迟、阻碍、减缓、减慢、稳定和/或延缓疾病(如癌症)的发展。取决于所治疗的疾病和/或个体的历史，此延迟可以具有不同的时长。如对本领域技术人员而言显而易见，充分或显著的延迟实际上可以涵盖预防，因为个体不发展该疾病。例如，可以延迟晚期癌症，如转移的发展。As used herein, "delaying the progression of a disease" means postponing, hindering, slowing down, slowing down, stabilizing, and/or delaying the development of a disease (e.g., cancer). This delay can be of varying lengths, depending on the disease being treated and/or the individual's history. As will be apparent to one skilled in the art, a sufficient or significant delay can actually encompass prevention, in that the individual does not develop the disease. For example, the development of advanced cancers, such as metastases, can be delayed.

“有效量”至少是达到具体障碍的可测量的改善或预防所需的最小量。本文的有效量可以根据诸如疾病状态、年龄、性别、患者体重和药物在个体中引出希望得到的反应的能力的因素而变。有效量还是其中治疗有益作用超过治疗的任何毒性作用或有害作用的量。对于预防用途，有益的或希望得到的结果包括诸如消除或降低风险、减轻严重度、或延迟疾病发病的结果，包括疾病的生物化学、组织学和/或行为症状、其并发症及疾病发展过程中存在的中间病理表型。对于治疗用途，有益的或希望得到的结果包括诸如减少疾病引起的一种或多种症状、提高患有疾病的个体的生活质量、减少治疗疾病所需的其他药物的剂量、增强另一药物的作用(如通过靶向)、延迟疾病的进展和/或延长存活的临床结果。在癌症或肿瘤的情况下，药物的有效量可以具有以下作用：减少癌细胞的数目、减少肿瘤大小、抑制(即在某种程度上减慢或希望阻止)癌细胞浸润入外周器官、抑制(即在某种程度上减慢或希望阻止)肿瘤转移、在某种程度上抑制肿瘤生长、和/或在某种程度上减轻与障碍相关的一种或多种症状。有效量可以在一次或多次施用中施用。为了本发明的目的，药物、化合物或药物组合物的有效量是足以直接或间接实现预防或治疗处理的量。如临床背景中所理解，药物、化合物或药物组合物的有效量可以与另一药物、化合物或药物组合物结合或不结合来达到。因此，在施用一种或多种治疗剂的背景中可以考虑“有效量”，如果与一种或多种其他治疗剂结合可以达到希望得到的结果，则可以考虑以有效量提供单一治疗剂。An "effective amount" is at least the minimum amount required to achieve a measurable improvement or prevention of a specific disorder. The effective amount herein can vary depending on factors such as disease state, age, sex, patient weight, and the ability of the drug to elicit the desired response in an individual. An effective amount is also an amount in which the beneficial effects of the treatment outweigh any toxic or deleterious effects of the treatment. For preventive uses, beneficial or desired results include results such as eliminating or reducing risk, reducing severity, or delaying the onset of the disease, including biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during the course of the disease. For therapeutic uses, beneficial or desired results include clinical results such as reducing one or more symptoms caused by the disease, improving the quality of life of individuals with the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug (such as through targeting), delaying the progression of the disease, and/or prolonging survival. In the case of cancer or tumors, an effective amount of a drug can have the following effects: reducing the number of cancer cells, reducing tumor size, inhibiting (i.e., slowing down or hopefully preventing to some extent) cancer cell infiltration into peripheral organs, inhibiting (i.e., slowing down or hoping to prevent to some extent) tumor metastasis, inhibiting tumor growth to some extent, and/or alleviating to some extent one or more symptoms associated with the disorder. The effective amount can be used in one or more administrations. For the purposes of the present invention, the effective amount of a drug, compound or pharmaceutical composition is the amount that is enough to directly or indirectly achieve prevention or treatment. As understood in the clinical context, the effective amount of a drug, compound or pharmaceutical composition can be combined with or not combined with another drug, compound or pharmaceutical composition to achieve. Therefore, in the context of administering one or more therapeutic agents, it is contemplated that an "effective amount" is provided, if combined with one or more other therapeutic agents to achieve the desired result, then it is contemplated that a single therapeutic agent is provided in an effective amount.

本文所用的“与…结合”指施用除另一种治疗方式外的一种治疗方式。因此，“与…结合”指在对个体施用另一治疗方式之前、期间或之后施用一种治疗方式。As used herein, "in combination with" refers to administering one therapeutic modality in addition to another therapeutic modality. Thus, "in combination with" refers to administering one therapeutic modality before, during, or after administering another therapeutic modality to a subject.

“障碍”是可从治疗受益的任意病症，包括但不限于慢性和急性障碍或疾病，包括使哺乳动物易感所讨论的障碍的那些病理状态。A "disorder" is any condition that would benefit from treatment, including but not limited to chronic and acute disorders or diseases, including those pathological conditions that predispose the mammal to the disorder in question.

术语“细胞增生性障碍”和“增生性障碍”指与某种程度的异常细胞增殖相关的障碍。在一个实施方案中，该细胞增生性障碍是癌症。在一个实施方案中，该细胞增生性障碍是肿瘤。The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

本文所用的术语“肿瘤”指所有恶性或良性的赘生性细胞生长和增殖，及所有癌变前细胞和组织及癌细胞和组织。本文提到的术语“癌症”、“癌性的”、“细胞增生性障碍”、“增生性障碍”和“肿瘤”不相互排斥。As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" as used herein are not mutually exclusive.

术语“癌症”和“癌性的”指或描述哺乳动物中通常表征为细胞生长失调的生理病症。癌症的实例包括但不限于癌、淋巴瘤、胚细胞瘤、肉瘤和白血病或恶性淋巴瘤。这类癌症更具体的实例包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、肺腺癌和肺鳞状细胞癌)、腹膜癌、肝细胞癌、胃癌(包括胃肠癌和胃肠间质癌)、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌(例如膀胱上皮性膀胱癌(UBC)、肌层浸润性膀胱癌(MIBC)和BCG难治性非肌层浸润性膀胱癌(NMIBC))、尿道癌、肝细胞瘤、乳腺癌(例如HER2+乳腺癌，及雌激素受体(ER-)、孕酮受体(PR-)和HER2(HER2-)阴性的三阴乳腺癌(TNBC))、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌或子宫癌、涎腺癌、肾癌(例如肾细胞癌(RCC))、前列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、黑素瘤、浅表扩散性黑素瘤、恶性雀斑样痣黑素瘤、肢端雀斑样痣黑素瘤、结节性黑素瘤、多发性骨髓瘤，以及B细胞淋巴瘤(包括低级/滤泡性非霍奇金淋巴瘤(NHL)、小淋巴细胞性(SL)NHL、中级/滤泡性NHL、中级弥漫性NHL、高级免疫母细胞性NHL、高级淋巴母细胞性NHL、高级小非裂细胞性NHL、巨块病性NHL、套细胞淋巴瘤、AIDS相关淋巴瘤和Waldenstrom巨球蛋白血症)、慢性淋巴细胞性白血病(CLL)、急性髓性白血病(ALL)、acute myologenous leukemia(AML)、多毛细胞白血病、慢性成髓细胞性白血病(CML)、移植后淋巴增生性障碍(PTLD)、骨髓增生异常综合征(MDS)、以及与瘢痣病相关的异常血管增生、水肿(如与脑肿瘤相关的水肿)、Meigs综合征、脑癌、以及头颈癌、及相关的转移。在某些实施方案中，可用本发明的方法和组合物治疗的癌症包括乳腺癌(例如三阴乳腺癌)、膀胱癌(例如UBC、MIBC和NMIBC)、结肠直肠癌、直肠癌、肺癌(例如非小细胞肺癌，其可为鳞状或非鳞状)、成胶质细胞瘤、非霍奇金淋巴瘤(NHL)、肾细胞癌(例如RCC)、前列腺癌、肝癌、胰腺癌、软组织肉瘤、卡波西肉瘤、类癌、头颈癌、卵巢癌、间皮瘤和血癌(例如MDS和多发性骨髓瘤)。在一些实施方案中，该癌症选自：小细胞肺癌、成胶质细胞瘤、成神经细胞瘤、黑素瘤、乳腺癌、胃癌、结肠直肠癌(CRC)和肝细胞癌。在其他实施方案中，该癌症选自：非小细胞肺癌、结肠直肠癌、成胶质细胞瘤和乳腺癌，包括那些癌症的转移形式。在具体实施方案中，该癌症选自肺癌(例如非小细胞肺癌，其可为鳞状或非鳞状)、膀胱癌(例如UBC)、乳腺癌(例如TNBC)、RCC、黑素瘤、结肠直肠癌和血癌(例如MDS和多发性骨髓瘤)。在一些实施方案中，该肺癌是非小细胞肺癌，其可为鳞状或非鳞状。在一些实施方案中，该膀胱癌是UBC。在一些实施方案中，该乳腺癌是TNBC。在一些实施方案中，该血癌是MDS或多发性骨髓瘤。The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoma. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer (e.g., urothelial bladder cancer (UBC), muscle invasive bladder cancer (MIBC), and BCG-refractory non-muscle invasive bladder cancer (NMIBC)), urethral cancer, hepatoma, breast cancer (e.g., HER2+ breast cancer, and triple-negative breast cancer (TNBC) that is negative for estrogen receptor (ER-), progesterone receptor (PR-), and HER2 (HER2-)), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, adenocarcinoma, renal cancer (e.g., renal cell carcinoma (RCC)), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma, nodular melanoma, multiple myeloma, and B-cell lymphomas (including low-grade/follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (ALL), acute Myologenous leukemia (AML), hairy cell leukemia, chronic myeloblastic leukemia (CML), post-transplant lymphoproliferative disorder (PTLD), myelodysplastic syndrome (MDS), and abnormal blood vessel proliferation associated with keloid disease, edema (such as edema associated with brain tumors), Meig's syndrome, brain cancer, and head and neck cancer, and related metastases. In certain embodiments, cancers that can be treated with the methods and compositions of the present invention include breast cancer (e.g., triple-negative breast cancer), bladder cancer (e.g., UBC, MIBC, and NMIBC), colorectal cancer, rectal cancer, lung cancer (e.g., non-small cell lung cancer, which can be squamous or non-squamous), glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma (e.g., RCC), prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid, head and neck cancer, ovarian cancer, mesothelioma, and blood cancers (e.g., MDS and multiple myeloma). In some embodiments, the cancer is selected from the group consisting of small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. In other embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including metastatic forms of those cancers. In specific embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, which may be squamous or non-squamous), bladder cancer (e.g., UBC), breast cancer (e.g., TNBC), RCC, melanoma, colorectal cancer, and blood cancer (e.g., MDS and multiple myeloma). In some embodiments, the lung cancer is non-small cell lung cancer, which may be squamous or non-squamous. In some embodiments, the bladder cancer is UBC. In some embodiments, the breast cancer is TNBC. In some embodiments, the blood cancer is MDS or multiple myeloma.

本文所用的术语“细胞毒剂”指对细胞有害(例如引起细胞死亡，抑制增殖，或以其他方式妨碍细胞功能)的任意物质。细胞毒剂包括但不限于放射性同位素(例如At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²和Lu的放射性同位素)；化疗剂；生长抑制剂；酶及其片段，如核溶解酶；毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，包括其片段和/或变体。示例性细胞毒剂可以选自抗微管剂、铂配位化合物、烷化剂、抗生剂、拓扑异构酶II抑制剂、抗代谢物、拓扑异构酶I抑制剂、激素和激素类似物、信号转导途径抑制剂、非受体酪氨酸激酶血管发生抑制剂、免疫治疗剂、促凋亡剂、LDH-A抑制剂、脂肪酸生物合成抑制剂、细胞周期信号发放抑制、HDAC抑制剂蛋白酶体抑制剂和癌症代谢抑制剂。在一个实施方案中，该细胞毒剂是基于铂的化疗剂。在一个实施方案中，该细胞毒剂是EGFR的拮抗剂。在一个实施方案中，该细胞毒剂是N-(3-乙炔基苯基)-6,7-双(2-甲氧基乙氧基)喹唑啉-4-胺(例如埃罗替尼(erlotinib)，TARCEVA^TM)。在一个实施方案中，该细胞毒剂是RAF抑制剂。在一个实施方案中，该RAF抑制剂是BRAF和/或CRAF抑制剂。在一个实施方案中，该RAF抑制剂是vemurafenib。在一个实施方案中，该细胞毒剂是PI3K抑制剂。As used herein, the term "cytotoxic agent" refers to any substance that is detrimental to cells (e.g., causes cell death, inhibits proliferation, or otherwise interferes with cell function). Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At²¹¹ , I¹³¹ , I¹²⁵ , Y⁹⁰ , Re¹⁸⁶ , Re¹⁸⁸ , Sm¹⁵³ , Bi²¹² , P³² , Pb²¹² , and radioisotopes of Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and/or variants thereof. Exemplary cytotoxic agents can be selected from anti-microtubule agents, platinum coordination compounds, alkylating agents, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutics, pro-apoptotic agents, LDH-A inhibitors, fatty acid biosynthesis inhibitors, cell cycle signaling inhibition, HDAC inhibitors proteasome inhibitors and cancer metabolism inhibitors. In one embodiment, the cytotoxic agent is a platinum-based chemotherapeutic agent. In one embodiment, the cytotoxic agent is an antagonist of EGFR. In one embodiment, the cytotoxic agent is N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g., erlotinib (erlotinib), TARCEVA^™ ). In one embodiment, the cytotoxic agent is a RAF inhibitor. In one embodiment, the RAF inhibitor is a BRAF and/or CRAF inhibitor. In one embodiment, the RAF inhibitor is vemurafenib. In one embodiment, the cytotoxic agent is a PI3K inhibitor.

本文所用的术语“化疗剂”包括用于治疗癌症的化合物。化疗剂的实例包括：埃罗替尼(Genentech/OSI Pharm.)、硼替佐米(bortezomib)(Millennium Pharm.)、双硫仑(disulfiram)、表没食子儿茶素没食子酸酯(epigallocatechin gallate)、salinosporamide A、carfilzomib、17-AAG(格尔德霉素)、根赤壳菌素(radicicol)、乳酸脱氢酶A(LDH-A)、氟维司群(fulvestrant)(AstraZeneca),sunitib(Pfizer/Sugen)、来曲唑(letrozole)(Novartis)、甲磺酸伊马替尼(imatinib mesylate)(Novartis)、finasunate(Novartis)、奥沙利铂(oxaliplatin)(Sanofi)、5-FU(5-氟尿嘧啶)、亚叶酸(leucovorin)、雷帕霉素(Sirolimus,Wyeth)、拉帕替尼(Lapatinib)(GSK572016,Glaxo Smith Kline)、lonafamib(SCH 66336)、索拉非尼(sorafenib)(Bayer Labs)、吉非替尼(gefitinib)(AstraZeneca)、AG1478；烷化剂，如噻替派(thiotepa)和环磷酰胺；烷基磺酸酯，如白消安(busulfan)、英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶(aziridine)，如benzodopa、卡波醌(carboquone)、meturedopa和uredopa；乙撑亚胺(ethylenimine)和methylamelamine，包括六甲蜜胺(altretamine)、三亚胺嗪(triethylenemelamine)、三亚乙基磷酰胺(triethylenephosphoramide)、三亚乙基硫代磷酰胺(triethylenethiophosphoramide)和trimethylomelamine；多聚乙酰(acetogenin)(尤其是番荔枝内酯(bullatacin)和布拉它辛酮(bullatacinone))；喜树碱(camptothecin)(包括拓扑替康(topotecan)和伊立替康(irinotecan))；苔藓虫素(bryostatin)；callystatin；CC-1065(包括其阿多来新(adozelesin)、卡折来新(carzelesin)和比折来新(bizelesin)合成类似物)；cryptophycin(尤其是cryptophycin 1和cryptophycin 8)；肾上腺类固醇(包括强的松和强的松龙)；环丙孕酮(cyproterone acetate)；5α-还原酶(包括非那雄胺(finasteride)和度他雄胺(dutasteride))；伏立诺他(vorinostat),罗米地辛(romidepsin)、帕比司他(panobinostat)、丙戊酸、mocetinostat、多拉司他汀(dolastatin)；阿地白介素(aldesleukin)、滑石粉、多卡霉素(包括合成类似物KW-2189和CB1-TM1)；eleutherobin；pancratistatin；sarcodictyin；spongistatin；氮芥(nitrogenmustard)，如苯丁酸氮芥(chlorambucil)、萘氮芥(chlomaphazine)、chlorophosphamide、磷雌氮芥(estramustine)、异环磷酰胺(ifosfamide)、氮芥(mechlorethamine)、盐酸氧氮芥(mechlorethamine oxide hydrochloride)、苯丙氨酸氮芥(melphalan)、新氮芥(novembichin)、苯芥胆甾醇(phenesterine)、松龙苯芥(prednimustine)、氯乙环磷酰胺(trofosfamide)、尿嘧啶氮芥(uracil mustard)；亚硝基脲(nitrosourea)，如卡莫司汀(carmustine)、氯脲菌素(chlorozotocin)、福莫司汀(fotemustine)、洛莫司汀(lomustine)、尼莫司汀(nimustine)和ranimnustine；抗生素，如烯二炔类抗生素(例如加利车霉素，尤其是加利车霉素γ1和加利车霉素ω1(Angew.Chem Intl.Ed.Engl.,33:183-186(1994))；dynemicin，包括dynemicin A；二膦酸盐，如氯膦酸二钠(clodronate)；esperamicin；以及新制癌菌素色基和相关色蛋白烯二炔类抗生素色基)、阿克拉霉素(aclacinomycin)、放线菌素(actinomycin)、安曲霉素(authramycin)、重氮丝氨酸(azaserine)、博来霉素(bleomycin)、放线菌素C(cactinomycin)、carabicin、洋红霉素(carminomycin)、嗜癌素(carzinophilin)、chromomycinis、更生霉素(dactinomycin)、柔红霉素(daunorubicin)、地托比星(detorubicin)、6-重氮-5-氧代-L-正亮氨酸、(多柔比星(doxorubicin))、吗啉基-多柔比星、氰基吗啉基-多柔比星、2-吡咯啉基-多柔比星和脱氧多柔比星、表柔比星(epirubicin)、依索比星(esorubicin)、伊达比星(idarubicin)、麻西罗霉素(marcellomycin)、丝裂霉素(mitomycin)(如丝裂霉素C)、霉酚酸、诺加霉素(nogalamycin)、橄榄霉素(olivomycins)、派来霉素(peplomycin)、泊非霉素(porfiromycin)、嘌呤霉素(puromycin)、三铁阿霉素(quelamycin)、罗多比星(rodorubicin)、链黑菌素(streptonigrin)、链脲霉素(streptozocin)、杀结核菌素(tubercidin)、乌苯美司(ubenimex)、新制癌菌素(zinostatin)、佐柔比星(zorubicin)；抗代谢剂，如氨甲喋呤和5-氟尿嘧啶(5-FU)；叶酸类似物，如二甲叶酸(denopterin)、氨甲喋呤、蝶酰三谷氨酸(pteropterin)、三甲曲沙(trimetrexate)；嘌呤类似物，如氟达拉滨(fludarabine)、6-巯基嘌呤、硫咪嘌呤(thiamiprine)、硫鸟嘌呤(thioguanine)；嘧啶类似物，如安西他滨As used herein, the term "chemotherapeutic agent" includes compounds used to treat cancer. Examples of chemotherapeutic agents include: erlotinib (Genentech/OSI Pharm.), bortezomib (Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (AstraZeneca), sunitib (Pfizer/Sugen), letrozole (Novartis), imatinib mesylate ( mesylate (Novartis), finasunate (Novartis), oxaliplatin (Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, Wyeth), lapatinib (GSK572016, Glaxo Smith Kline), lonafamib (SCH 66336), sorafenib (Bayer Labs), gefitinib (AstraZeneca), AG1478; alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimine and methylamelamines, including altretamine, triethylenemelamine, and triethylenephosphoramide , triethylenethiophosphoramide, and trimethylomelamine; polyacetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); adrenal steroids (including prednisone and prednisolone); cyproterone acetate; and acetaminophen. acetate); 5-alpha-reductase (including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat, dolastatin; aldesleukin, talc, duocarboxylic acid (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, and chlorambucil. hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1 and calicheamicin omega 1 (Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and the neocarcinogen and related chromoprotein enediyne antibiotics), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophicin, lin), chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin ycin), mitomycin (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenicin Ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine

<PN 161874>(ancitabine)、氮杂胞苷(azacitidine)、6-氮尿苷(6-azauridine)、卡莫氟(carmofur)、阿糖胞苷(cytarabine)、双脱氧尿苷(dideoxyuridine)、去氧氟脲苷(doxifluridine)、依诺他宾(enocitabine)、氟尿苷(floxuridine)；雄激素，如卡鲁睾酮(calusterone)、丙酸曲他雄酮(dromostanolone propionate)、环硫雄醇(epitiostanol)、美雄烷(mepitiostane)、睾内酯(testolactone)；抗肾上腺素(anti-adrenal)，如氨鲁米特(aminoglutethimide)、米托坦(mitotane)、曲洛司坦(trilostane)；叶酸补充剂，如亚叶酸；醋葡内酯(aceglatone)；醛磷酰胺糖苷(aldophosphamide glycoside)；氨基乙酰丙酸(aminolevulinic acid)；恩尿嘧啶(eniluracil)；安吖啶(amsacrine)；bestrabucil；比生群(bisantrene)；依达曲沙(edatraxate)；地磷酰胺(defofamine)；秋水仙胺(demecolcine)；地吖醌(diaziquone)；elfomithine；醋酸羟吡咔唑(elliptiniumacetate)；埃坡霉素(epothilone)；乙环氧啶(etoglucid)；硝酸镓；羟基脲；香菇多糖(lentinan)；氯尼达明(lonidainine)；美登木素生物碱(maytansinoid)，如美登素(maytansine)和美坦西醇(ansamitocin)；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；mopidamnol；nitraerine；喷司他丁(pentostatin)；蛋氨氮芥(phenamet)；吡柔比星(pirarubicin)；洛索蒽醌(losoxantrone)；鬼臼酸(podophyllinicacid)；2-乙肼；丙卡巴肼(procarbazine)；多糖复合物(JHS Natural Products,Eugene,Oreg.)；丙亚胺(razoxane)；根霉素(rhizoxin)；西佐喃(sizofuran)；螺旋锗(spirogermanium)；细格孢氮杂酸(tenuazonic acid)；三亚胺醌(triaziquone)；2,2',2"-三氯三乙胺；单端孢霉烯(trichothecene)(尤其是T-2毒素、verracurin A、杆孢菌素(roridin)A和anguidine)；乌拉坦(urethan)；脱乙酰长春花碱；达卡巴嗪(dacarbazine)；甘露莫司汀(mannomustine)；二溴甘露醇(mitobronitol)；二溴卫矛醇(mitolactol)；溴丙哌嗪(pipobroman)；gacytosine；阿糖胞苷(arabinoside)(“Ara-C”)；环磷酰胺(cyclophosphamide)；噻替派(thiotepa)；紫杉烷(taxane)；chloranmbucil；(吉西他滨(gemcitabine))；6-硫鸟嘌呤；巯基嘌呤；氨甲喋呤；长春花碱(vinblastine)；依托泊苷(etoposide)(VP-16)；异环磷酰胺(ifosfamide)；米托蒽醌(mitoxantrone)；长春花新碱(vincristine)；(长春烯碱(vinorelbine))；二羟蒽二酮(novantrone)；替尼泊苷(teniposide)；依达曲沙(edatrexate)；柔红霉素(daunomycin)；氨基喋呤(aminopterin)；卡培他滨(capecitabine)伊班磷酸(ibandronate)；CPT-11；拓扑异构酶抑制剂RFS 2000；二氟甲基鸟氨酸(DMFO)；类视黄醛，如视黄酸；及以上任一种的可药用盐、酸和衍生物。<PN 161874> ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenal drugs, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; aceglatone; and aldophosphamide glycosides. glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine e); maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazine; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vinblastine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxane; chloranmbucil (gemcitabine); 6-thioguanine ; mercaptopurine; methotrexate; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

化疗剂还包括“基于铂的”化疗剂，其包含含有铂的有机化合物作为分子的有机部分。通常，基于铂的化疗剂是铂的配位化合物。基于铂的化疗剂有时在本领域中称为“platin”。基于铂的化疗剂的实例包括但不限于卡铂、顺式铂氨和奥沙利铂。Chemotherapeutic agents also include "platinum-based" chemotherapeutic agents, which contain an organic compound containing platinum as the organic portion of the molecule. Typically, platinum-based chemotherapeutic agents are coordination compounds of platinum. Platinum-based chemotherapeutic agents are sometimes referred to in the art as "platins." Examples of platinum-based chemotherapeutic agents include, but are not limited to, carboplatin, cisplatin, and oxaliplatin.

化疗剂还包括：(i)发挥作用来调节或抑制激素对肿瘤的作用的抗激素剂，如抗雌激素和选择性雌激素受体调节剂(SERM)，包括例如他莫昔芬(tamoxifen)(包括枸缘酸他莫昔芬(tamoxifen citrate))、雷洛昔芬(raloxifene)、屈洛昔芬(droloxifene)、4-羟基他莫昔芬(4-hydroxytamoxifen)、曲沃昔芬(trioxifene)、雷洛昔芬盐酸盐(keoxifene)、LY117018、奥那司酮(onapristone)和(枸缘酸托瑞米芬(toremifine citrate))；(ii)抑制芳香酶(其调节肾上腺中的雌激素产生)的芳香酶抑制剂，例如，4(5)-咪唑、氨鲁米特(aminoglutethimide)、(醋酸甲地孕酮(megestrol acetate))、(依西美坦(exemestane)，辉瑞)、formestanie、法倔唑(fadrozole)、(伏氯唑(vorozole))、(来曲唑(letrozole)，诺华)和(阿那曲唑(anastrozole)，阿斯利康)；(iii)抗雄激素，如氟他胺(flutamide)、尼鲁米特(nilutamide)、比卡米特(bicalutamide)、醋酸亮丙瑞林(leuprolide)和戈舍瑞林(goserelin)、布舍瑞林(buserelin)、tripterelin、醋酸甲羟孕酮(medroxyprogesterone acetate)、己烯雌酚(diethylstilbestrol)、结合型雌激素(premarin)、氟羟甲基睾丸素(fluoxymesterone)、全反式维甲酸、维甲酰酚胺(fenretinide)、以及曲沙他滨(troxacitabine)(1,3-二氧戊环胞嘧啶核苷类似物)；(iv)蛋白激酶抑制剂；(v)脂质激酶抑制剂；(vi)反义寡核苷酸，尤其是抑制涉及异常细胞增殖的信号传导途径中的基因(例如PKC-α、Raf、H-Ras和)表达的那些；(vii)核酶，如VEGF表达抑制剂(例如)和HER2表达抑制剂；(viii)疫苗，如基因治疗疫苗，例如，和rIL-2，拓扑异构酶1抑制剂如rmRH；及(ix)以上任一种的可药用盐、酸和衍生物。Chemotherapeutic agents also include: (i) antihormonal agents that act to modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene citrate; (ii) aromatase inhibitors that inhibit the aromatase enzyme, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, megestrol acetate, and dapoxetine. acetate), (exemestane, Pfizer), formestanie, fadrozole, (vorozole), (letrozole, Novartis), and (anastrozole, AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide acetate, goserelin, buserelin, tripterelin, medroxyprogesterone acetate, and dapoxetine. acetate), diethylstilbestrol, conjugated estrogens (premarin), fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (a 1,3-dioxolane cytidine nucleoside analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, especially those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation (e.g., PKC-α, Raf, H-Ras and ); (vii) ribozymes, such as VEGF expression inhibitors (e.g., HER2 expression inhibitors); (viii) vaccines, such as gene therapy vaccines, for example, and rIL-2, topoisomerase 1 inhibitors such as rmRH; and (ix) pharmaceutically acceptable salts, acids, and derivatives of any of the above.

化疗剂还包括抗体，如阿伦单抗(alemtuzumab)(Campath)、贝伐单抗(bevacizumab)(Genentech)、西妥昔单抗(cetuximab)(Imclone)、帕尼单抗(panitumumab)(Amgen)、利妥昔单抗(rituximab)(Genentech/Biogen Idec)、帕妥珠单抗(pertuzumab)(2C4,Genentech)、曲妥珠单抗(trastuzumab)(Genentech)、托西莫单抗(tositumomab)(Bexxar,Corixia)及抗体药物缀合物吉妥珠单抗奥唑米星(gemtuzumabozogamicin)(Wyeth)。作为药物与本发明的化合物组合具有治疗潜能的其他人源化抗体包括：阿泊珠单抗(apolizumab)、aselizumab、atlizumab、bapineuzumab、bivatuzumab mertansine、cantuzumab mertansine、西利珠单抗(cedelizumab)、赛妥珠单抗(certolizumab pegol)、cidfusituzumab、cidtuzumab、达利珠单抗(daclizumab)、艾库组单抗(eculizumab)、依法利珠单抗(efalizumab)、依帕珠单抗(epratuzumab)、厄利珠单抗(erlizumab)、非维珠单抗(felvizumab)、芳妥珠单抗(fontolizumab)、妥珠单抗奥唑米星、inotuzumab ozogamicin、伊匹单抗(ipilimumab)、labetuzumab、林妥珠单抗(lintuzumab)、matuzumab、美泊利单抗(mepolizumab)、莫维珠单抗(motavizumab)、motovizumab、那他珠单抗(natalizumab)、尼妥珠单抗(nimotuzumab)、nolovizumab、numavizumab、ocrelizumab、奥马珠单抗(omalizumab)、帕利珠单抗(palivizumab)、pascolizumab、pecfusituzumab、pectuzumab、pexelizumab、ralivizumab、兰尼单抗(ranibizumab)、reslivizumab、reslizumab、resyvizumab、罗维珠单抗(rovelizumab)、ruplizumab、西罗珠单抗Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab (Genentech), cetuximab (Imclone), panitumumab (Amgen), rituximab (Genentech/Biogen Idec), pertuzumab (2C4, Genentech), trastuzumab (Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate gemtuzumab ozogamicin (Wyeth). Other humanized antibodies with therapeutic potential as drugs in combination with the compounds of the present invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, tocilizumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sirolizumab

<PN 161874>(sibrotuzumab)、siplizumab、sontuzumab、tacatuzumabtetraxetan、tadocizumab、talizumab、tefibazumab、托珠单抗(tocilizumab)、toralizumab、tucotuzumab celmoleukin、tucusituzumab、umavizumab、乌珠单抗(urtoxazumab)、优特克单抗(ustekinumab)、visilizumab和抗白介素12(ABT-874/J695,Wyeth Research和Abbott Laboratories)，抗白介素12是遗传修饰为识别白介素-12p40蛋白质的重组全人序列全长IgG₁λ抗体。<PN 161874> (sibrotuzumab), siplizumab, sontuzumab, tacatuzumabtetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories), a recombinant, fully human, full-length IgG₁ lambda antibody genetically modified to recognize the interleukin-12p40 protein.

化疗剂还包括“EGFR抑制剂”，EGFR抑制剂指结合EGFR或以其他方式与EGFR直接相互作用，并阻止或减少其信号发放活性的化合物，还称为“EGFR拮抗剂”。这类药物的实例包括结合EGFR的抗体和小分子。结合EGFR的抗体的实例包括：MAb 579(ATCC CRL HB 8506)、MAb 455(ATCC CRL HB8507)、MAb 225(ATCC CRL 8508)、MAb 528(ATCC CRL 8509)(参见美国专利号4,943,533)及其变体，如嵌合225(C225或西妥昔单抗；)和改造的人225(H225)(参见例如WO96/40210,Imclone Systems Inc.)；IMC-11F8全人EGFR靶向抗体(Imclone)；结合II型EGFR突变体的抗体(美国专利号5,212,290)；美国专利号中5,891,996所述的结合EGFR的人源化抗体和人抗体；结合EGFR的人抗体，如ABX-EGF帕尼单抗(参见WO98/50433,Abgenix/Amgen)；EMD 55900(Stragliotto等Eur.J.Cancer 32A:636-640(1996))；与EGF和TGF-α二者竞争结合EGFR的抗EGFR人源化EGFR抗体EMD7200(matuzumab)(EMD/Merck)；人EGFR抗体HuMax-EGFR(GenMab)；US 6,235,883中所述的称为E1.1、E2.4、E2.5、E6.2、E6.4、E2.11、E6.3和E7.6.3的全人抗体；MDX-447(Medarex Inc)；mAb 806或人源化mAb806(Johns等,J.Biol.Chem.279(29):30375-30384(2004))。抗EGFR抗体可以与细胞毒剂缀合，从而产生免疫缀合物(参见例如EP659439A2,Merck Patent GmbH)。EGFR拮抗剂包括小分子，如美国专利号5,616,582、5,457,105、5,475,001、5,654,307、5,679,683、6,084,095、6,265,410、6,455,534、6,521,620、6,596,726、6,713,484、5,770,599、6,140,332、5,866,572、6,399,602、Chemotherapeutic agents also include "EGFR inhibitors", which refer to compounds that bind to EGFR or otherwise interact directly with EGFR and prevent or reduce its signaling activity, also known as "EGFR antagonists". Examples of such drugs include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include: MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. Patent No. 4,943,533) and variants thereof, such as chimeric 225 (C225 or cetuximab;) and modified human 225 (H225) (see, for example, WO96/40210, Imclone Systems Inc.); IMC-11F8 fully human EGFR-targeting antibody (Imclone); antibodies that bind to type II EGFR mutants (U.S. Patent No. 5,212,290); humanized antibodies and human antibodies that bind to EGFR as described in U.S. Patent No. 5,891,996; human antibodies that bind to EGFR, such as ABX-EGF panitumumab (see WO98/50433, Abgenix/Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); anti-EGFR humanized EGFR antibody EMD7200 (matuzumab) (EMD/Merck) that competes with both EGF and TGF-α for binding to EGFR; human EGFR antibody HuMax-EGFR (GenMab); U.S. Patent No. 5,891,996; human antibodies that bind to EGFR, such as ABX-EGF panitumumab (see WO98/50433, Abgenix/Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); humanized anti-EGFR EGFR antibody EMD7200 (matuzumab) (EMD/Merck) that competes with both EGF and TGF-α for binding to EGFR; human EGFR antibody HuMax-EGFR (GenMab); Fully human antibodies designated E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3 as described in 6,235,883; MDX-447 (Medarex Inc); mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Anti-EGFR antibodies can be conjugated to cytotoxic agents to generate immunoconjugates (see, for example, EP 659439A2, Merck Patent GmbH). EGFR antagonists include small molecules, such as U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602,

<PN 161874>6,344,459、6,602,863、6,391,874、6,344,455、5,760,041、6,002,008和5,747,498以及PCT公开WO98/14451、WO98/50038、WO99/09016和WO99/24037中所述的化合物。具体的小分子EGFR拮抗剂包括：OSI-774(CP-358774,埃罗替尼,Genentech/OSI Pharmaceuticals)；PD 183805(CI1033,2-propenamide,N-[4-[(3-氯-4-氟苯基)氨基]-7-[3-(4-吗啉基)丙氧基]-6-喹唑啉基]-,二盐酸,辉瑞)；ZD1839,吉非替尼4-(3’-氯-4’-氟苯胺基)-7-甲氧基-6-(3-吗啉基丙氧基)喹唑啉基,阿斯利康)；ZM105180((6-氨基-4-(3-甲基苯基-氨基)-喹唑啉,Zeneca)；BIBX-1382(N8-(3-氯-4-氟-苯基)-N2-(1-甲基-派啶-4-基)-嘧啶并[5,4-d]嘧啶-2,8-二胺,勃林格殷格翰)；PKI-166((R)-4-[4-[(1-苯基乙基)氨基]-1H-吡咯并[2,3-d]嘧啶-6-基]-苯酚)；(R)-6-(4-羟基苯基)-4-[(1-苯基乙基)氨基]-7H-吡咯并[2,3-d]嘧啶)；CL-387785(N-[4-[(3-溴苯基)氨基]-6-喹唑啉基]-2-butynamide)；EKB-569(N-[4-[(3-氯-4-氟苯基)氨基]-3-氰基-7-乙氧基-6-喹啉基]-4-(二甲氨基)-2-butenamide)(惠氏)；AG1478(辉瑞)；AG1571(SU5271；辉瑞)；双重EGFR/HER2酪氨酸激酶抑制剂，如拉帕替尼(GSK572016或N-[3-氯-4-[(3-氟苯基)甲氧基]苯基]-6[5[[[2-甲磺酰基)乙基]氨基]甲基]-2-呋喃基]-4-quinazolinamine)。<PN 161874> Compounds described in 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and PCT publications WO98/14451, WO98/50038, WO99/09016, and WO99/24037. Specific small molecule EGFR antagonists include: OSI-774 (CP-358774, erlotinib, Genentech/OSI Pharmaceuticals); PD 183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholino)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer); ZD1839, gefitinib, 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazolinyl, AstraZeneca); ZM105180 ((6-amino -4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-b utenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU5271; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors such as lapatinib (GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furyl]-4-quinazolinamine).

化疗剂还包括“酪氨酸激酶抑制剂”，包括：前一段落中指出的EGFR靶向药物；可从Takeda获得的小分子HER2酪氨酸激酶抑制剂如TAK165；ErbB2受体酪氨酸激酶的口服选择性抑制剂CP-724,714(辉瑞和OSI)；优先结合EGFR但抑制过量表达HER2的细胞和过量表达EGFR的细胞二者的双重HER抑制剂如EKB-569(可从惠氏获得)；口服HER2和EGFR酪氨酸激酶抑制剂拉帕替尼(GSK572016，可从葛兰素史克获得)；PKI-166(可从诺华获得)；全HER抑制剂如卡奈替尼(CI-1033；Pharmacia)；抑制Raf-1信号发放的Raf-1抑制剂，如可从ISISPharmaceuticals获得的反义剂ISIS-5132；非HER靶向酪氨酸激酶抑制剂，如甲磺酸伊马替尼(可从葛兰素史克获得)；多重靶向酪氨酸激酶抑制剂，如舒尼替尼(sunitinib)(可从辉瑞获得)；VEGF受体酪氨酸激酶抑制剂，如vatalanib(PTK787/ZK222584,可从诺华/Schering AG获得)；MAPK胞外调节激酶I抑制剂CI-1040(可从Pharmacia获得)；喹唑啉，如PD 153035,4-(3-氯苯胺基)喹唑啉；吡啶并嘧啶；嘧啶并嘧啶；吡咯并嘧啶，如CGP 59326、CGP 60261和CGP 62706；吡唑并嘧啶，4-(苯基氨基)-7H-吡咯并[2,3-d]嘧啶；姜黄素(二阿魏酰甲烷,4,5-双(4-氟苯胺基)邻苯二甲酰亚胺)；含硝基噻吩部分的酪氨酸磷酸化抑制剂(tyrphostine)；PD-0183805(Warner-Lamber)；反义分子(例如与编码HER的核酸结合的反义分子)；喹噁啉(美国专利号5,804,396)；酪氨酸磷酸化抑制剂(美国专利号5,804,396)；ZD6474(阿斯利康)；PTK-787(诺华/Schering AG)；全HER抑制剂如CI-1033(辉瑞)；Affinitac(ISIS 3521；Isis/礼来)；甲磺酸伊马替尼PKI 166(诺华)；GW2016(葛兰素史克)；CI-1033(辉瑞)；EKB-569(惠氏)；Semaxinib(辉瑞)；ZD6474(阿斯利康)；PTK-787(诺华/Schering AG)；INC-1C11(Imclone)；雷帕霉素(sirolimus,)；或以下专利申请的任一个中所述的酪氨酸激酶抑制剂：美国专利号5,804,396、WO 1999/09016(美国氰氨公司)、WO 1998/43960(美国氰氨公司)、WO 1997/38983(Warner Lambert)、WO 1999/06378(WarnerLambert)、WO 1999/06396(Warner Lambert)、WO 1996/30347(辉瑞)、WO 1996/33978(Zeneca)、WO 1996/3397(Zeneca)和WO 1996/33980(Zeneca)。Chemotherapeutic agents also include "tyrosine kinase inhibitors," including: the EGFR-targeted drugs noted in the previous paragraph; small molecule HER2 tyrosine kinase inhibitors such as TAK165, available from Takeda; oral selective inhibitors of ErbB2 receptor tyrosine kinase, CP-724,714 (Pfizer and OSI); dual HER inhibitors such as EKB-569 (available from Wyeth) that preferentially bind to EGFR but inhibit both cells that overexpress HER2 and cells that overexpress EGFR; the oral HER2 and EGFR tyrosine kinase inhibitor lapatinib (GSK572016, available from GlaxoSmithKline); PKI-166 ( available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors that inhibit Raf-1 signaling, such as the antisense agent ISIS-5132 available from ISIS Pharmaceuticals; non-HER-targeted tyrosine kinase inhibitors such as imatinib mesylate (available from GlaxoSmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261, and CGP 62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrosine phosphorylation inhibitors containing a nitrothiophene moiety (tyrphostine); PD-0183805 (Warner-Lamber); antisense molecules (e.g., antisense molecules that bind to nucleic acids encoding HER); quinoxaline (U.S. Patent No. 5,804,396); tyrosine phosphorylation inhibitors (U.S. Patent No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Eli Lilly); imatinib mesylate PKI 166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); sirolimus; or a tyrosine kinase inhibitor described in any of the following patent applications: U.S. Patent No. 5,804,396, WO 1999/09016 (American Cyanamide Corporation), WO 1998/43960 (American Cyanamide Corporation), WO 1997/38983 (Warner Lambert), WO 1999/06378 (Warner Lambert), WO 1999/06396 (Warner Lambert), WO 1996/30347 (Pfizer), WO 1996/33978 (Zeneca), WO 1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

化疗剂还包括地塞米松、干扰素、秋水仙素、氯苯安啶(metoprine)、环孢菌素(cyclosporine)、两性霉素(amphotericin)、甲硝唑(metronidazole)、阿伦单抗、阿利维A酸(alitretinoin)、别嘌醇(allopurinol)、阿米斯丁(amifostine)、三氧化二砷、天冬酰胺酶、BCG活体、贝伐单抗、贝沙罗汀(bexarotene)、克拉屈滨(cladribine)、氯法拉滨(clofarabine)、阿法达贝泊汀(darbepoetin alfa)、地尼白介素(denileukin)、右丙亚胺(dexrazoxane)、阿法依泊汀(epoetin alfa)、厄洛替尼(elotinib)、非格司亭(filgrastim)、乙酸希司曲林(histrelin acetate)、ibritumomab、干扰素α-2a、干扰素α-2b、来那度胺(lenalidomide)、左旋咪唑(levamisole)、美司钠(mesna)、甲氧补骨脂素(methoxsalen)、诺龙(nandrolone)、奈拉滨(nelarabine)、nofetumomab、oprelvekin、palifermin、氨羟二磷酸二钠(pamidronate)、pegademase、聚乙二醇化天冬酰胺酶(pegaspargase)、聚乙二醇化非格司亭(pegfilgrastim)、培美曲塞二钠(pemetrexeddisodium)、普卡霉素(plicamycin)、卟吩姆钠(porfimer sodium)、米帕林(quinacrine)、拉布立酶(rasburicase)、沙格司亭(sargramostim)、替莫唑胺(temozolomide)、VM-26、6-TG、托瑞米芬(toremifene)、维A酸、ATRA、戊柔比星(valrubicin)、唑来膦酸二钠(zoledronate)、唑来膦酸(zoledronic acid)及其可药用盐。Chemotherapeutic agents also include dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG active, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erlotinib, filgrastim, histrelin acetate, acetate), ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium sodium), quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, zoledronic acid, and pharmaceutically acceptable salts thereof.

化疗剂还包括氢化可的松(hydrocortisone)、醋酸氢化可的松(hydrocortisoneacetate)、醋酸可的松(cortisone acetate)、tixocortol pivalate、醋酸曲安西龙(triamcinolone acetonide)、triamcinolone alcohol、莫米松(mometasone)、安西奈德(amcinonide)、布地奈德(budesonide)、地奈德(desonide)、氟西奈德(fluocinonide)、醋酸氟轻松(fluocinolone acetonide)、倍他米松(betamethasone)、倍他米松磷酸酯钠(betamethasone sodium phosphate)、地塞米松(dexamethasone)、地塞米松磷酸钠(dexamethasone sodium phosphate)、氟可龙(fluocortolone)、氢化可的松-17-丁酸酯(hydrocortisone-17-butyrate)、氢化可的松-17-戊酸酯(hydrocortisone-17-valerate)、二丙酸别氯地米松(aclometasone dipropionate)、戊酸倍他米松(betamethasone valerate)、二丙酸倍他米松(betamethasone dipropionate)、泼尼卡酯(prednicarbate)、氯倍他松-17-丁酸酯(clobetasone-17-butyrate)、氯倍他松-17-丙酸酯(clobetasol-17-propionate)、己酸氟可龙(fluocortolone caproate)、特戊酸氟可龙(fluocortolone pivalate)和醋酸氟甲叉龙(fluprednidene acetate)；免疫选择性抗炎肽(ImSAID)，如苯丙氨酸-谷氨酰胺-甘氨酸(FEG)及其D同分异构体形式(feG)(IMULANBioTherapeutics,LLC)；抗风湿药物，如硫唑嘌呤(azathioprine)、环孢霉素(ciclosporin)(环孢霉素A(cyclosporine A))、D-青霉胺(D-penicillamine)、金盐、羟氯喹林(hydroxychloroquine)、leflunomideminocycline、柳氮磺吡啶(sulfasalazine)；肿瘤坏死因子α(TNFα)阻断剂，如依那西普(etanercept)(Enbrel)、英夫利昔单抗(infliximab)(Remicade)、阿达木单抗(adalimumab)(Humira)、赛妥珠单抗(Cimzia)、戈利木单抗(golimumab)(Simponi)；白介素1(IL-1)阻断剂，如阿那白滞素(anakinra)(Kineret)；T细胞共刺激阻断剂，如阿巴西普(abatacept)(Orencia)；白介素6(IL-6)阻断剂，如托珠单抗白介素13(IL-13)阻断剂，如lebrikizumab；干扰素α(IFN)阻断剂，如rontalizumab；β7整联蛋白阻断剂，如rhuMAbβ7；IgE途径阻断剂，如抗-M1prime；分泌性同源三聚体LTa3和膜结合异源三聚体LTa1/β2阻断剂，如抗淋巴毒素α(LTa)；放射性同位素(例如At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²及Lu的放射性同位素)；多种研究性药物，如thioplatin、PS-341、丁酸苯酯、ET-18-OCH₃或法尼基转移酶抑制剂(L-739749、L-744832)；多酚，如栎精、白藜芦醇、白皮杉醇,epigallocatechinegallate、茶黄素(theaflavin)、黄烷醇(flavanol)、矢车菊苷配基(procyanidin)、白桦脂酸及其衍生物；自噬抑制剂，如氯喹啉(chloroquine)；δ-9-四氢大麻酚(屈大麻酚(dronabinol)，)；β-拉帕醌(β-lapachone)；拉帕醇(lapachol)；秋水仙素；白桦脂酸；乙酰喜树碱；莨菪亭和9-氨基喜树碱)；鬼臼毒素(podophyllotoxin)；替加氟(tegafur)贝沙罗汀(bexarotene)二膦酸盐，如氯膦酸二钠(clodronate)(例如或)、依替膦酸钠(etidronate)NE-58095、唑来膦酸(zoledronic acid)/唑来膦酸二钠(zoledronate)阿仑膦酸钠(alendronate)氨羟二磷酸二钠(pamidronate)替鲁膦酸钠(tiludronate)或利塞膦酸钠(risedronate)表皮生长因子受体(EGF-R)；疫苗，如疫苗；哌立福辛(perifosine)；COX-2抑制剂(例如塞来昔布(celecoxib)或依托考昔(etoricoxib))；蛋白酶体抑制剂(例如PS341)；CCI-779；tipifarnib(R11577)；orafenib、ABT510；Bcl-2抑制剂，如oblimersen sodium匹克生琼(pixantrone)；法尼基转移酶抑制剂，如lonafarnib(SCH6636、SARASAR^TM)；及上述任一种的可药用盐、酸或衍生物；以及以上两种或多种的组合，如CHOP(环磷酰胺、多柔比星、长春花新碱和强的松龙的联合治疗的缩写)和FOLFOX(奥沙利铂(ELOXATIN^TM)与5-FU和leucovovin组合的治疗方案的缩写)。Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, and dapoxetine. phosphate), fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate acetate); immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN Bio Therapeutics, LLC); antirheumatic drugs, such as azathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline, and sulfasalazine; tumor necrosis factor alpha (TNFα) blockers, such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), and golimumab (Simponi); interleukin-1 ( IL-1 blockers, such as anakinra (Kineret); T-cell costimulatory blockers, such as abatacept (Orencia); interleukin-6 (IL-6) blockers, such as tocilizumab; interleukin-13 (IL-13) blockers, such as lebrikizumab; interferon-alpha (IFN) blockers, such as rontalizumab; β7 integrin blockers, such as rhuMAb β7; IgE pathway blockers, such as anti-M1prime; secretory homotrimeric LTa3 and membrane-bound heterotrimeric LTa1/β2 blockers, such as antilymphotoxin α (LTa); radioisotopes (e.g., At²¹¹ , I¹³¹ , I¹²⁵ , Y⁹⁰ , Re¹⁸⁶ , Re¹⁸⁸ , Sm¹⁵³ , Bi²¹² , P³² , Pb²¹² , and radioisotopes of Lu); various investigational drugs, such as thioplatin, PS-341, phenyl butyrate, ET-18-OCH₃ , or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols, such as quercetin, resveratrol, piceatannol, epigallocatechinegallate, theaflavin, flavanol, procyanidin, betulinic acid and its derivatives; autophagy inhibitors, such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, ), β-lapachone, lapachol, colchicine, betulinic acid, acetylcamptothecin, scopoletin, and 9-aminocamptothecin, podophyllotoxin, tegafur, bexarotene, bisphosphonates such as clodronate (e.g., or), etidronate NE-58095, zoledronic acid, epidermal growth factor receptor (EGF-R); vaccines, such as vaccinia; perifosine; COX-2 inhibitors (e.g., celecoxib or etoricoxib); proteasome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium and pixantrone; farnesyltransferase inhibitors, such as lonafarnib (SCH6636, SARASAR^™ ); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more thereof, such as CHOP (abbreviation for combined therapy with cyclophosphamide, doxorubicin, vincristine and prednisolone) and FOLFOX (abbreviation for a treatment regimen combining oxaliplatin (ELOXATIN^™ ) with 5-FU and leucovovin).

化疗剂还包括具有镇痛、退热和抗炎作用的非类固醇抗炎药。NSAID包括环加氧酶的非选择性抑制剂。NSAID的具体实例包括阿司匹林；丙酸衍生物，如布洛芬(ibuprofen)、非诺洛芬(fenoprofen)、酮洛芬(ketoprofen)、氟比洛芬(flurbiprofen)、奥沙普秦(oxaprozin)和萘普生(naproxen)；醋酸衍生物，如吲哚美辛(indomethacin)、舒林酸(sulindac)、依托度酸(etodolac)、双氯芬酸(diclofenac)；烯醇酸衍生物，如吡罗昔康(piroxicam)、美洛昔康(meloxicam)、替诺昔康(tenoxicam)、哚昔康(droxicam)、氯诺昔康(lornoxicam)和伊索昔康(isoxicam)；灭酸衍生物，如甲灭酸(mefenamic acid)、甲氯灭酸(meclofenamic acid)、氟灭酸(flufenamic acid)、邻甲氯灭酸(tolfenamic acid)；COX-2抑制剂，如塞来昔布、依托考昔、罗美昔布(lumiracoxib)、帕瑞考昔(parecoxib)、罗非昔布(rofecoxib)、罗非昔布(rofecoxib)和伐地考昔(valdecoxib)。NSAID可以标示用于减轻以下病症的症状：类风湿性关节炎、骨关节炎、炎性关节病、关节强硬性脊椎病、银屑病关节炎、Reiter综合征、急性痛风、痛经、转移性骨痛、头痛和偏头痛、术后痛、炎症和组织损伤引起的轻度至中度疼痛、发热、肠梗阻和肾绞痛。Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs (NSAIDs) which have analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of cyclooxygenase. Specific examples of NSAIDs include aspirin; propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, and naproxen; acetic acid derivatives such as indomethacin, sulindac, etodolac, and diclofenac; enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam; fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and fenamic acid. NSAIDs are indicated for the relief of symptoms of rheumatoid arthritis, osteoarthritis, inflammatory joint disease, ankylosing spondylosis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headaches and migraines, postoperative pain, mild to moderate pain caused by inflammation and tissue damage, fever, intestinal obstruction, and renal colic.

在本文中使用时，“生长抑制剂”指在体外或体内抑制细胞生长的化合物或组合物。在一个实施方案中，生长抑制剂是阻止或减少细胞的增殖的生长抑制抗体，该细胞表达该抗体所结合的抗原。在另一实施方案中，该生长抑制剂可以是显著减少处于S期的细胞的百分比的物质。生长抑制剂的实例包括阻断细胞周期进程(处于S期之外的地方)的物质，如诱导G1阻滞和M期阻滞的物质。经典的M期阻断剂包括vincas(长春花新碱和长春花碱)、紫杉烷和拓扑异构酶II抑制剂，如多柔比星、表柔比星、柔红霉素、依托泊苷和博来霉素。阻滞G1期的那些物质也波及到S期阻滞，例如，DNA烷化剂，如他莫昔芬、强的松、达卡巴嗪、氮芥、顺式铂氨、氨甲蝶呤、5-氟尿嘧啶和ara-C。其他信息可见于Mendelsohn和Israel编辑,TheMolecular Basis of Cancer,第1章,标题“Cell cycle regulation,oncogenes,andantineoplastic drugs”by Murakami等(W.B.Saunders,Philadelphia,1995),例如第13页中。As used herein, "growth inhibitor" refers to a compound or composition that inhibits cell growth in vitro or in vivo. In one embodiment, a growth inhibitor is a growth inhibitory antibody that prevents or reduces the proliferation of cells expressing the antigen to which the antibody binds. In another embodiment, the growth inhibitor can be a substance that significantly reduces the percentage of cells in the S phase. Examples of growth inhibitors include substances that block cell cycle progression (outside the S phase), such as substances that induce G1 arrest and M phase arrest. Classical M phase blockers include vincas (vinblastine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Substances that arrest the G1 phase also affect S phase arrest, for example, DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled “Cell cycle regulation, oncogenes, and antimetoplastic drugs” by Murakami et al. (W. B. Saunders, Philadelphia, 1995), e.g., page 13.

“放射治疗”意指用定向γ射线或β射线来诱发对细胞的足够损伤，使得限制其正常发挥功能的能力或彻底破坏细胞。应理解，本领域存在确定处理的剂量和持续时间的许多已知方式。典型的处理作为一次性施用提供，典型的剂量在每天10至200单位(戈瑞)的范围内。"Radiotherapy" means the use of directed gamma or beta radiation to induce sufficient damage to cells to limit their ability to function normally or to completely destroy the cells. It will be appreciated that there are many known ways in the art to determine the dosage and duration of treatment. Typical treatments are provided as a one-time administration, with typical dosages ranging from 10 to 200 units (Gy) per day.

用于治疗目的的“对象”或“个体”指分类为哺乳动物的任意动物，包括人类，驯养动物和农场动物，及动物园动物、运动动物或宠物动物，如狗、马、猫、牛等。优选地，该哺乳动物是人。对象或个体可以是患者。A "subject" or "individual" for therapeutic purposes refers to any animal classified as a mammal, including humans, domesticated and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, and the like. Preferably, the mammal is a human. A subject or individual can be a patient.

术语“抗体”在本文中以最广泛的含义使用，且明确涵盖单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)和抗体片段，只要它们显示希望得到的生物学活性。The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

“分离的”抗体是已鉴定并从其天然环境的成分分开/取出的抗体。其天然环境的污染成分是干扰该抗体的研究、诊断或治疗用途的物质，且可以包括酶、激素和其他蛋白质或非蛋白质溶质。在一些实施方案中，将抗体纯化至：(1)通过例如Lowry法测定抗体高于95wt％，在一些实施方案中，高于99wt％；(2)足以通过使用例如旋杯式测序仪获得N端或内部氨基酸序列的至少15个残基的程度；或(3)例如考马斯蓝染色或银染的还原或非还原条件下的SDS-PAGE显示同质。分离的抗体包括重组细胞内的原位抗体，因为将不存在至少一种该抗体的天然环境的成分。但是，通常将通过至少一个纯化步骤纯化分离的抗体。An "isolated" antibody is one that has been identified and separated/removed from a component of its natural environment. Contaminating components of its natural environment are substances that interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to: (1) greater than 95% by weight, in some embodiments, greater than 99% by weight, as determined by, for example, the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using, for example, a spinning cup sequenator; or (3) to homogeneity as shown by SDS-PAGE under reducing or non-reducing conditions, such as Coomassie blue staining or silver staining. An isolated antibody includes the antibody in situ within recombinant cells because at least one component of the antibody's natural environment will not be present. However, the isolated antibody will generally be purified by at least one purification step.

“天然抗体”通常是由两条相同的轻(L)链和两条相同的重(H)链组成的约150,000道尔顿的异源四聚体糖蛋白。每条轻链通过一个共价二硫键与重链连接，而二硫键的数目在不同免疫球蛋白同种型的重链之间不同。每条重链和轻链还具有规则间隔开的链内二硫键。每条重链在一端具有可变结构域(V_L)，后面是许多恒定结构域。每条轻链在一端具有可变结构域(V_L)，在其另一端具有恒定结构域；轻链的恒定结构域与重链的第一恒定结构域对齐，轻链可变结构域与重链可变结构域对齐。认为特定氨基酸残基形成轻链和重链可变结构域之间的界面。"Native antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (_VL ) at one end, followed by a number of constant domains. Each light chain has a variable domain (_VL ) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the heavy chain variable domain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains.

术语“恒定结构域”指免疫球蛋白分子的相对于免疫球蛋白的另一部分(可变结构域，其包含抗原结合部位)而言具有更保守的氨基酸序列的部分。恒定结构域包含重链的C_H1、C_H2和C_H3结构域(统称CH)及轻链的CHL(或CL)结构域。The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin (the variable domain, which contains the antigen-binding site). The constant domain comprises the_CH1 ,_CH2 , and_CH3 domains (collectively referred to as CH) of the heavy chain and the CHL (or CL) domain of the light chain.

抗体的“可变区”或“可变结构域”指抗体的重链或轻链的氨基端结构域。重链可变区可称为“V_H”。轻链可变区可称为“V_L”。这些结构域通常是抗体的最可变部分，且包含抗原结合部位。An antibody "variable region" or "variable domain" refers to the amino-terminal domain of an antibody's heavy or light chain. The heavy chain variable region may be referred to as "_VH ." The light chain variable region may be referred to as "_VL ." These domains are generally the most variable parts of an antibody and contain the antigen-binding site.

术语“可变的”指这样的事实，可变结构域的某些部分在序列上在抗体间广泛不同，并用于各具体抗体对其具体抗原的结合和亲和力。但是，可变性并非在抗体的整个可变结构域内均匀分布。它在轻链和重链可变结构域中都集中在三个称为高变区(HVR)的区段中。可变结构域的更高度保守的部分称为构架区(FR)。天然重链和轻链的可变结构域各包含四个FR，其大致采用β-折叠构型，通过三个HVR连接，该HVR形成连接该β-折叠结构且在一些情况下形成该β-折叠结构的部分的环。每条链中的HVR通过FR区近距离保持在一起，并与来自另一条链的HVR一起促成抗体的抗原结合部位的形成(参见Kabat等,Sequences ofProteins of Immunological Interest,第5版,National Institute of Health,Bethesda,Md(1991))。恒定结构域不直接涉及抗体与抗原的结合，但显示多种效应子功能，如抗体在抗体依赖性细胞毒作用中的参与。The term "variable" refers to the fact that certain parts of the variable domain are widely different in sequence between antibodies and are used for the binding and affinity of each specific antibody to its specific antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FRs, which generally adopt a β-sheet configuration and are connected by three HVRs, which form loops that connect the β-sheet structure and, in some cases, form parts of the β-sheet structure. The HVRs in each chain are held together in close proximity by the FR region and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, National Institute of Health, Bethesda, Md (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

根据其恒定结构域的氨基酸序列，可以将来自任意哺乳动物物种的抗体(免疫球蛋白)的“轻链”分配至称为κ和λ的两个明显不同的类型之一。The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

本文所用的术语IgG“同种型”或“亚类”指通过其恒定区的化学和抗原特征定义的免疫球蛋白的任意亚类。As used herein, the term IgG "isotype" or "subclass" refers to any subclass of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.

取决于其重链恒定结构域的氨基酸序列，可以将抗体(免疫球蛋白)分配至不同种类。存在五个主要种类的免疫球蛋白：IgA、IgD、IgE、IgG和IgM，这些中的几个可以进一步划分为亚类(同种型)，例如IgG₁、IgG₂、IgG₃、IgG₄、IgA₁和IgA₂。对应于不同种类的免疫球蛋白的重链恒定结构域分别称为α、δ、ε、γ和μ。不同种类的免疫球蛋白的亚基结构和三维构型众所周知，且通常描述于例如Abbas等Cellular and Mol.Immunology,第4版(W.B.Saunders,Co.,2000)中。抗体可以是通过该抗体与一种或多种其他蛋白质或多肽的共价或非共价结合形成的更大的融合分子的一部分。Antibodies (immunoglobulins) can be assigned to different classes depending on the amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), such as IgG₁ , IgG₂ , IgG₃ , IgG₄ , IgA₁ and IgA_2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described in, for example, Cellular and Mol. Immunology, 4th edition (WB Saunders, Co., 2000) by Abbas et al. Antibodies can be part of a larger fusion molecule formed by the covalent or non-covalent binding of the antibody to one or more other proteins or polypeptides.

术语“全长抗体”、“完整抗体”和“全抗体”在本文中可互换使用，指处于其基本上完整的形式的抗体，不是下文定义的抗体片段。这些术语尤其指具有含Fc区的重链的抗体。The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, not an antibody fragment as defined below. These terms particularly refer to antibodies having heavy chains that contain an Fc region.

为了本文的目的，“裸抗体”是不与细胞毒性部分或放射性标记缀合的抗体。For purposes herein, a "naked antibody" is an antibody that is not conjugated to a cytotoxic moiety or a radiolabel.

“抗体片段”包含完整抗体的部分，优选包含其抗原结合区。在一些实施方案中，本文所述的抗体片段是抗原结合片段。抗体片段的实例包括Fab、Fab’、F(ab’)₂和Fv片段；双抗体；线性抗体；单链抗体分子；及从抗体片段形成的多特异性抗体。"Antibody fragments" comprise portions of intact antibodies, preferably comprising the antigen-binding region thereof. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')₂ , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

木瓜蛋白酶消化抗体产生两个相同的抗原结合片段(称为“Fab”片段，各具有单个抗原结合部位)和剩余的“Fc”片段(其名称反映其易结晶的能力)。胃蛋白酶处理产生F(ab’)₂片段，其具有两个抗原结合部位且仍能够交联抗原。Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment (whose name reflects its ability to crystallize readily). Pepsin treatment produces an F(ab')₂ fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

“Fv”是包含完整抗原结合部位的最小抗体片段。在一个实施方案中，双链Fv种类由紧密非共价结合的一个重链可变区结构域和一个轻链可变区结构域的二聚体组成。在单链Fv(scFv)种类中，一个重链可变结构域和一个轻链可变结构域可以通过柔性肽接头共价连接，使得轻链和重链可以以类似于双链Fv种类中的结构的“二聚体”结构结合。各可变结构域的三个HVR正是在此构型中相互作用来定义VH-VL二聚体表面上的抗原结合部位。六个HVR共同赋予抗体抗原结合特异性。但是，甚至单个可变结构域(或Fv的一半，其仅包含三个对抗原特异的HVR)也具有识别和结合抗原的能力，虽然亲和力低于整个结合部位。"Fv" is the smallest antibody fragment that contains a complete antigen-binding site. In one embodiment, the two-chain Fv species consists of a dimer of a heavy chain variable region domain and a light chain variable region domain that are tightly non-covalently bound. In the single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker, so that the light chain and the heavy chain can be combined in a "dimer" structure similar to the structure in the two-chain Fv species. The three HVRs of each variable domain interact in this configuration to define the antigen-binding site on the surface of the VH-VL dimer. The six HVRs together give the antibody antigen-binding specificity. However, even a single variable domain (or half of an Fv, which only contains three HVRs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than that of the entire binding site.

Fab片段包含重链和轻链可变结构域，还包含轻链的恒定结构域和重链的第一恒定结构域(CH1)。Fab’片段与Fab片段的不同在于，在重链CH1结构域的羧基端加入了几个残基，其包括一个或多个来自抗体铰链区的半胱氨酸。Fab’-SH是本文对其中恒定结构域的一个或多个半胱氨酸残基具有自由巯基的Fab’的命名。F(ab’)₂抗体片段最初作为其间具有铰合部半胱氨酸的Fab’片段对产生。还已知抗体片段的其他化学偶联。The Fab fragment comprises the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which one or more cysteine residues of the constant domains have free sulfhydryl groups. F(ab')₂ antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

“单链Fv”或“scFv”抗体片段包含抗体的VH和VL结构域，其中这些结构域存在于单条多肽链中。通常，scFv多肽进一步在VH和VL结构域之间包含多肽接头，该多肽接头使得scFv能够形成希望的结构用于抗原结合。scFv的综述参见Plückthun in ThePharmacology of Monoclonal Antibodies,113卷,Rosenburg和Moore编辑,(Springer-Verlag,纽约,1994)269-315页。"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, Volume 113, Rosenburg and Moore, eds., (Springer-Verlag, New York, 1994) pp. 269-315.

术语“双抗体”指具有两个抗原结合部位的抗体片段，该片段包含在同一条多肽链中与轻链可变结构域(VL)连接的重链可变结构域(VH)(VH-VL)。通过使用太短而不允许同一条链上的两个结构域之间配对的接头，迫使结构域与另一条链上的互补结构域配对，并产生两个抗原结合部位。双抗体可以是二价的或双特异性的。双抗体更充分地描述于例如EP404,097；WO 1993/01161；Hudson等,Nat.Med.9:129-134(2003)；及Hollinger等,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)中。三抗体和四抗体还描述于Hudson等,Nat.Med.9:129-134(2003)中。The term "diabody" refers to an antibody fragment with two antigen-binding sites, which fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and produce two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

本文所用的术语“单克隆抗体”指获自基本同质的抗体群体的抗体，例如，除了可以以较小的量存在的可能的突变(例如天然存在的突变)外，包含该群体的单种抗体是相同的。因此，修饰词“单克隆的”指抗体不是无关联抗体的混合物的性状。在某些实施方案中，这种单克隆抗体通常包含含有结合靶标的多肽序列的抗体，其中结合靶标的多肽序列通过包括从多个多肽序列选择单个结合靶标的多肽序列的方法获得。例如，选择方法可以是从多个克隆(例如，一系列杂交瘤克隆、噬菌体克隆或重组DNA克隆)选择独特克隆。应理解，可以进一步改变所选择的靶标结合序列，例如以改善对靶标的亲和力，人源化靶标结合序列，改善其在细胞培养中的产生，降低体内免疫原性，产生多特异性抗体等，包含改变的靶标结合序列的抗体也是本发明的单克隆抗体。与通常包含针对不同决定簇(表位)的不同抗体的多克隆抗体制备物不同，单克隆抗体制备物的每种单克隆抗体针对抗原上的单个决定簇。除它们的特异性外，单克隆抗体制备物的优势在于，它们通常未受其他免疫球蛋白污染。The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, for example, except for possible mutations (such as naturally occurring mutations) that may exist in smaller amounts, the single antibodies comprising the population are identical. Therefore, the modifier "monoclonal" refers to the fact that the antibody is not a mixture of unrelated antibodies. In certain embodiments, such monoclonal antibodies generally comprise antibodies containing a polypeptide sequence that binds to a target, wherein the polypeptide sequence that binds to a target is obtained by a method comprising selecting a single polypeptide sequence that binds to a target from a plurality of polypeptide sequences. For example, a selection method can be to select a unique clone from a plurality of clones (for example, a series of hybridoma clones, phage clones, or recombinant DNA clones). It should be understood that the selected target binding sequence can be further changed, for example, to improve affinity to the target, humanize the target binding sequence, improve its production in cell culture, reduce in vivo immunogenicity, produce multispecific antibodies, etc., and antibodies comprising the changed target binding sequence are also monoclonal antibodies of the present invention. Unlike polyclonal antibody preparations which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibody preparations is that they are generally not contaminated by other immunoglobulins.

修饰词“单克隆的”指抗体获自基本同质的抗体群体的特征，而不解释为需要通过任意具体方法来产生该抗体。例如，待按照本发明使用的单克隆抗体可以通过多种技术制备，包括例如，杂交瘤法(例如Kohler和Milstein.,Nature,256:495-97(1975)；Hongo等,Hybridoma,14(3):253-260(1995)；Harlow等,Antibodies:A Laboratory Manual,(ColdSpring Harbor Laboratory出版社,第2版1988)；Hammerling等,in:MonoclonalAntibodies and T-Cell Hybridomas 563-681(Elsevier,N.Y.,1981))、重组DNA法(参见例如美国专利号4,816,567)、噬菌体展示技术(参见例如Clackson等,Nature,352:624-628(1991)；Marks等,J.Mol.Biol.222:581-597(1992)；Sidhu等,J.Mol.Biol.338(2):299-310(2004)；Lee等,J.Mol.Biol.340(5):1073-1093(2004)；Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004)；及Lee等,J.Immunol.Methods284(1-2):119-132(2004))、及用于在具有部分或全部人免疫球蛋白基因座或编码人免疫球蛋白序列的基因的动物中产生人抗体或人样抗体的技术(参见例如WO1998/24893；WO1996/34096；WO 1996/33735；WO 1991/10741；Jakobovits等,Proc.Natl.Acad.Sci.USA90:2551(1993)；Jakobovits等,Nature 362:255-258(1993)；Bruggemann等,Year inImmunol.7:33(1993)；美国专利号5,545,807；5,545,806；5,569,825；5,625,126；5,633,425；5,661,016；Marks等,Bio/Technology 10:779-783(1992)；Lonberg等,Nature 368:856-859(1994)；Morrison,Nature 368:812-813(1994)；Fishwild等,NatureBiotechnol.14:845-851(1996)；Neuberger,Nature Biotechnol.14:826(1996)；及Lonberg和Huszar,Intern.Rev.Immunol.13:65-93(1995))。The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and techniques for producing human antibodies or human-like antibodies in animals having partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year 2004). in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

本文的单克隆抗体明确包括“嵌合”抗体，其中部分重链和/或轻链与源自特定物种或隶属于特定抗体种类或亚类的抗体中对应的序列相同或同源，而一条或多条链的其余部分与源自另一物种或隶属于另一抗体种类或亚类的抗体中对应的序列相同或同源，以及这类抗体的片段，只要它们显示希望得到的生物学活性(参见例如美国专利号4,816,567；及Morrison等,Proc.Natl.Acad.Sci.USA,81:6851-6855(1984))。嵌合抗体包括抗体，其中抗体的抗原结合区源自例如通过用目的抗原免疫猕猴而产生的抗体。The monoclonal antibodies herein specifically include "chimeric" antibodies, in which a portion of the heavy and/or light chains is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of one or more chains is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (see, e.g., U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies include antibodies in which the antigen-binding region of the antibody is derived from, for example, an antibody generated by immunizing a macaque with an antigen of interest.

非人(例如鼠)抗体的“人源化”形式是含有源自非人免疫球蛋白的最少序列的嵌合抗体。在一个实施方案中，人源化抗体是人免疫球蛋白(受体抗体)，其中用来自非人物种(如小鼠、大鼠、兔或非人灵长类)的具有希望得到的特异性、亲和力和/或能力的HVR(供体抗体)的残基取代来自受体的HVR的残基。在一些情况下，用对应的非人残基取代人免疫球蛋白的FR残基。此外，人源化抗体可以包含不见于受体抗体中或供体抗体中的残基。可以进行这些修饰来进一步改良抗体性能。通常，人源化抗体将包含至少一个(通常两个)可变结构域的基本上全部，其中全部或基本上全部高变环对应于非人免疫球蛋白的那些，且全部或基本上全部FR是具有人免疫球蛋白序列的那些。人源化抗体还将可选地包含至少部分免疫球蛋白恒定区(Fc)，通常是人免疫球蛋白的恒定区。进一步的细节参见Jones等,Nature321:522-525(1986)；Riechmann等,Nature 332:323-329(1988)；及Presta,Curr.Op.Struct.Biol.2:593-596(1992)。还参见例如Vaswani和Hamilton,Ann.Allergy,Asthma&Immunol.1:105-115(1998)；Harris,Biochem.Soc.Transactions 23:1035-1038(1995)；Hurle和Gross,Curr.Op.Biotech.5:428-433(1994)；及美国专利号6,982,321和7,087,409。The "humanized" form of non-human (e.g., mouse) antibodies is a chimeric antibody containing the minimum sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues of the HVR (donor antibody) with the desired specificity, affinity and/or ability from a non-human species (e.g., mouse, rat, rabbit, or non-human primate) are substituted for the residues from the HVR of the receptor. In some cases, the FR residues of the human immunoglobulin are substituted with corresponding non-human residues. In addition, the humanized antibody may include residues not found in the receptor antibody or in the donor antibody. These modifications may be performed to further improve antibody performance. Typically, the humanized antibody will comprise substantially all of at least one (typically two) variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FRs are those with human immunoglobulin sequences. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

“人抗体”是具有这样的氨基酸序列的抗体，该氨基酸序列对应于由人产生和/或用本文公开的用于制备人抗体的任意技术制备的抗体的氨基酸序列。此人抗体定义明确排除了包含非人抗原结合残基的人源化抗体。人抗体可以用本领域已知的多种技术产生，包括噬菌体展示文库。Hoogenboom和Winter,J.Mol.Biol.,227:381(1991)；Marks等,J.Mol.Biol.,222:581(1991)。还可以用Cole等,Monoclonal Antibodies and CancerTherapy,Alan R.Liss,77页(1985)；Boerner等,J.Immunol.,147(1):86-95(1991)中所述的方法制备人单克隆抗体。还参见van Dijk和van de Winkel,Curr.Opin.Pharmacol.,5:368-74(2001)。可以通过对转基因动物施用抗原来制备人抗体，该转基因动物已修饰为响应抗原攻击而产生这类抗体，但已使其内源基因座失能，例如免疫的xenomice(关于XENOMOUSE^TM技术，参见例如美国专利号6,075,181和6,150,584)。关于通过人B细胞杂交瘤技术产生人抗体，还参见Li等,Proc.Natl.Acad.Sci.USA,103:3557-3562(2006)。A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and/or prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Human monoclonal antibodies can also be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol.,5 : 368-74 (2001). Human antibodies can be prepared by administering antigens to transgenic animals that have been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, such as immune xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE^™ technology). See also Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006) for the production of human antibodies by human B cell hybridoma technology.

“物种依赖性抗体”是对来自第一哺乳动物物种的抗原具有比对来自第二哺乳动物物种的该抗原的同源物强的结合亲和力的抗体。通常，物种特异性抗体“特异性结合”人抗原(例如，具有不超过约1x10^-7M、优选不超过约1x10^-8M和优选不超过约1x10^-9M的结合亲和力(Kd))，但对来自第二非人哺乳动物的同源物的结合亲和力比其对人抗原的结合亲和力弱至少约50倍、或至少约500倍或至少约1000倍。物种依赖性抗体可以是上文定义的多种抗体类型中的任一种，但优选人源化抗体或人抗体。A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than for a homologue of that antigen from a second mammalian species. Typically, a species-specific antibody "specifically binds" to a human antigen (e.g., with a binding affinity (Kd) of no more than about^1x10-7 M, preferably no more than about^1x10-8 M, and preferably no more than about^1x10-9 M), but has a binding affinity for a homologue from a second non-human mammal that is at least about 50 times, or at least about 500 times, or at least about 1000 times weaker than its binding affinity for the human antigen. A species-dependent antibody can be any of the various antibody types defined above, but is preferably a humanized or human antibody.

在本文中使用时，术语“高变区”、“HVR”或“HV”指抗体可变结构域的序列上高变和/或形成结构确定的环的区域。通常，抗体包含六个HVR；三个在VH中(H1、H2、H3)，三个在VL中(L1、L2、L3)。在天然抗体中，H3和L3显示六个HVR中最大的多样性，尤其认为H3在赋予抗体良好的特异性中发挥独特作用。参见例如Xu等,Immunity 13:37-45(2000)；Johnson和Wu,in Methods in Molecular Biology 248:1-25(Lo编辑,Human出版社,Totowa,N.J.,2003)。实际上，天然存在的仅由重链组成的骆驼抗体在缺乏轻链的情况下具有功能且稳定。参见例如Hamers-Casterman等,Nature 363:446-448(1993)；Sheriff等,NatureStruct.Biol.3:733-736(1996)。As used herein, the term "hypervariable region", "HVR" or "HV" refers to the region of the antibody variable domain that is highly variable in sequence and/or forms a structurally defined loop. Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the greatest diversity of the six HVRs, and H3 is particularly believed to play a unique role in conferring good specificity on antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo ed., Human Press, Totowa, N.J., 2003). In fact, naturally occurring camel antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

许多HVR界定(delineation)在使用，并为本文所涵盖。Kabat互补决定区(CDR)基于序列可变性，且最常用((Kabat等,Sequences of Proteins of ImmunologicalInterest,第5版Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。而Chothia指结构环的位置(Chothia和Lesk J.Mol.Biol.196:901-917(1987))。AbM HVR代表了Kabat HVR和Chothia结构环之间的折中，且为Oxford Molecular's AbM抗体建模软件所使用。“接触”HVR基于可用的复合物晶体结构的分析。下文指出了来自这些HVR中的每一个的残基。Many HVR delineations are in use and are encompassed herein. The Kabat complementarity determining regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, on the other hand, refers to the positions of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on analysis of available complex crystal structures. The residues from each of these HVRs are indicated below.

HVR可以包含以下“扩展HVR”：VL中的24-36或24-34(L1)、46-56或50-56(L2)和89-97或89-96(L3)，VH中的26-35(H1)、50-65或49-65(H2)和93-102、94-102或95-102(H3)。对于这些定义中的每一个，按照Kabat等,上文编号可变结构域残基。An HVR may comprise the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al., supra.

“构架”或“FR”残基是除本文定义的HVR残基外的那些可变结构域残基。"Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

术语“如Kabat中的可变结构域残基编号”或“如Kabat中的氨基酸位置编号”及其变通形式指用于Kabat等,上文中的一系列抗体的重链可变结构域或轻链可变结构域的编号系统。使用此编号系统，实际的线性氨基酸序列可以包含较少的或附加的氨基酸，对应于可变结构域的FR或HVR的缩短或插入。例如，重链可变结构域可以包含H2的残基52后的单氨基酸插入(按照Kabat的残基52a)和重链FR残基82后的插入残基(例如按照Kabat的残基82a、82b和82c等)。可以通过在具有同源性的区域将抗体序列与“标准”Kabat编号序列比对来为给定的抗体确定残基的Kabat编号。The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system used for the heavy chain variable domain or light chain variable domain of a series of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of a FR or HVR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues for a given antibody can be determined by aligning the antibody sequence with the "standard" Kabat numbering sequence over regions of homology.

在提到可变结构域中的残基(约轻链的残基1-107和重链的残基1-113)时通常使用Kabat编号系统(例如Kabat等,Sequences of Immunological Interest.第5版PublicHealth Service,National Institutes of Health,Bethesda,Md.(1991))。在提到免疫球蛋白重链恒定区中的残基时通常使用“EU编号系统”或“EU指数”(例如Kabat等,上文中报道的EU指数)。“如Kabat中的EU指数”指人IgG1EU抗体的残基编号。The Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain). The "EU numbering system" or "EU index" (e.g., the EU index reported in Kabat et al., supra) is generally used when referring to residues in the constant region of the immunoglobulin heavy chain. The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

表述“线性抗体”指Zapata等(1995Protein Eng,8(10):1057-1062)中所述的抗体。简言之，这些抗体包含一对串联Fd区段(VH-CH1-VH-CH1)，该对串联Fd区段与互补的轻链多肽一起形成一对抗原结合区。线性抗体可以是双特异性的或单特异性的。The term "linear antibody" refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10): 1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

本文所用的术语“结合”、“特异性结合”或“对…特异”指可测量和可重现的相互作用，如靶标和抗体之间的结合，其决定靶标在包括生物分子的分子的异源群体存在下的存在。例如，结合或特异性结合靶标(其可以是表位)的抗体是以比它结合其他靶标更高的亲和力、抗体亲抗原性、更容易地和/或以更长的持续时间结合此靶标的抗体。在一个实施方案中，如通过例如放射免疫测定(RIA)测量，抗体与不相关靶标结合的程度小于该抗体与该靶标结合的约10％，。在某些实施方案中，特异性结合靶标的抗体具有≤1μM、≤100nM、≤10nM、≤1nM或≤0.1nM的解离常数(Kd)。在某些实施方案中，抗体特异性结合蛋白质上的表位，该表位在来自不同物种的该蛋白质间保守。在另一实施方案中，特异性结合可以包括但不需要专一性结合。As used herein, the terms "bind," "specifically bind," or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is one that binds to that target with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to that target, as measured by, for example, a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved between that protein from different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

II.PD-1轴结合拮抗剂II. PD-1 axis binding antagonists

本文提供用于在个体中治疗癌症或延迟癌症进展的方法，其包括对该个体施用有效量的PD-1轴结合拮抗剂和紫杉烷。本文还提供在患有癌症的个体中增强免疫功能的方法，其包括对该个体施用有效量的PD-1轴结合拮抗剂和紫杉烷。例如，PD-1轴结合拮抗剂包括PD-1结合拮抗剂、PD-L1结合拮抗剂和PD-L2结合拮抗剂。PD-1(程序性死亡1)在本领域中也称为“程序性细胞死亡1”、“PDCD1”、“CD279”和“SLEB2”。UniProtKB/Swiss-Prot检索号Q15116中显示示例性人PD-1。PD-L1(程序性死亡配体1)在本领域中也称为“程序性细胞死亡1配体1”、“PDCD1LG1”、“CD274”、“B7-H”和“PDL1”。UniProtKB/Swiss-Prot检索号Q9NZQ7.1中显示示例性人PD-L1。PD-L1(程序性死亡配体2)在本领域中也称为“程序性细胞死亡1配体2”、“PDCD1LG2”、“CD273”、“B7-DC”、“Btdc”和“PDL2”。UniProtKB/Swiss-Prot检索号Q9BQ51中显示示例性人PD-L2。在一些实施方案中，PD-1、PD-L1和PD-L2是人PD-1、PD-L1和PD-L2。Provided herein is a method for treating cancer or delaying cancer progression in an individual, comprising administering an effective amount of a PD-1 axis binding antagonist and a taxane to the individual. Also provided herein is a method for enhancing immune function in an individual suffering from cancer, comprising administering an effective amount of a PD-1 axis binding antagonist and a taxane to the individual. For example, the PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2." Exemplary human PD-1 is shown in UniProtKB/Swiss-Prot retrieval number Q15116. PD-L1 (programmed death ligand 1) is also referred to in the art as "programmed cell death 1 ligand 1," "PDCD1LG1," "CD274," "B7-H," and "PDL1." An exemplary human PD-L1 is shown in UniProtKB/Swiss-Prot accession number Q9NZQ7.1. PD-L1 (programmed death ligand 2) is also known in the art as "programmed cell death 1 ligand 2," "PDCD1LG2," "CD273," "B7-DC," "Btdc," and "PDL2." An exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot accession number Q9BQ51. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

在一些实施方案中，该PD-1结合拮抗剂是抑制PD-1与其配体结合配偶体的结合的分子。在具体方面，该PD-1配体结合配偶体是PD-L1和/或PD-L2。在另一实施方案中，PD-L1结合拮抗剂是抑制PD-L1与其结合配偶体的结合的分子。在具体方面，PD-L1结合配偶体是PD-1和/或B7-1。在另一实施方案中，该PD-L2结合拮抗剂是抑制PD-L2与其结合配偶体的结合的分子。在具体方面，PD-L2结合配偶体是PD-1。该拮抗剂可以是抗体、其抗原结合片段、免疫黏附素、融合蛋白质或寡肽。In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In specific aspects, the PD-1 ligand binding partner is PD-L1 and/or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In specific aspects, the PD-L1 binding partner is PD-1 and/or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In specific aspects, the PD-L2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

在一些实施方案中，该PD-1结合拮抗剂是抗PD-1抗体(例如人抗体、人源化抗体或嵌合抗体)。在一些实施方案中，该抗PD-1抗体选自MDX-1106(nivolumab)、MK-3475(lambrolizumab)和CT-011(pidilizumab)。在一些实施方案中，该PD-1结合拮抗剂是免疫黏附素(例如，包含与恒定区(例如免疫球蛋白序列的Fc区)融合的PD-L1或PD-L2的胞外或PD-1结合部分的免疫黏附素)。在一些实施方案中，该PD-1结合拮抗剂是AMP-224。在一些实施方案中，该PD-L1结合拮抗剂是抗PD-L1抗体。在一些实施方案中，该抗PD-L1抗体选自YW243.55.S70、MPDL3280A、MDX-1105和MEDI4736。抗体YW243.55.S70是WO2010/077634中描述的抗PD-L1。MDX-1105(也称为BMS-936559)是WO2007/005874中描述的抗PD-L1抗体。MEDI4736是WO2011/066389和US2013/034559中描述的抗PD-L1单克隆抗体。MDX-1106(也称为MDX-1106-04、ONO-4538、BMS-936558或nivolumab)是WO2006/121168中描述的抗PD-1抗体。MK-3475(也称为lambrolizumab)是WO2009/114335中描述的抗PD-1抗体。CT-011(也称为hBAT、hBAT-1或pidilizumab)是WO2009/101611中描述的抗PD-1抗体。AMP-224(也称为B7-DCIg)是WO2010/027827和WO2011/066342中描述的PD-L2-Fc融合可溶性受体。In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from MDX-1106 (nivolumab), MK-3475 (lambrolizumab), and CT-011 (pidilizumab). In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is selected from YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO2010/077634. MDX-1105 (also known as BMS-936559) is an anti-PD-L1 antibody described in WO2007/005874. MEDI4736 is an anti-PD-L1 monoclonal antibody described in WO2011/066389 and US2013/034559. MDX-1106 (also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab) is an anti-PD-1 antibody described in WO2006/121168. MK-3475 (also known as lambrolizumab) is an anti-PD-1 antibody described in WO2009/114335. CT-011 (also known as hBAT, hBAT-1, or pidilizumab) is an anti-PD-1 antibody described in WO2009/101611. AMP-224 (also known as B7-DCIg) is a soluble PD-L2-Fc fusion receptor described in WO2010/027827 and WO2011/066342.

在一些实施方案中，该PD-1轴结合拮抗剂是抗PD-L1抗体。在一些实施方案中，该抗PD-L1抗体能够抑制PD-L1和PD-1之间和/或PD-L1和B7-1之间的结合。在一些实施方案中，该抗PD-L1抗体是单克隆抗体。在一些实施方案中，该抗PD-L1抗体是选自Fab、Fab’-SH、Fv、scFv和(Fab’)₂片段的抗体片段。在一些实施方案中，该抗PD-L1抗体是人源化抗体。在一些实施方案中，该抗PD-L1抗体是人抗体。In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is capable of inhibiting the binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv and (Fab')₂ fragments. In some embodiments, the anti-PD-L1 antibody is a humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody.

用于本发明方法的抗PD-L1抗体及其制备方法的实例描述于PCT专利申请WO2010/077634、WO 2007/005874、WO 2011/066389和US2013/034559中，该专利申请在此引入作为参考。用于本发明的抗PD-L1抗体(包括含有这类抗体的组合物)可以与紫杉烷组合使用来治疗癌症。Examples of anti-PD-L1 antibodies useful in the methods of the present invention and methods for their preparation are described in PCT patent applications WO 2010/077634, WO 2007/005874, WO 2011/066389, and US 2013/034559, which are incorporated herein by reference. The anti-PD-L1 antibodies used in the present invention (including compositions containing such antibodies) can be used in combination with taxanes to treat cancer.

抗PD-1抗体Anti-PD-1 antibodies

在一些实施方案中，该抗PD-1抗体是MDX-1106。用于“MDX-1106”的备选名称包括MDX-1106-04、ONO-4538、BMS-936558或Nivolumab。在一些实施方案中，该抗PD-1抗体是nivolumab(CAS登记号：946414-94-4)。还在另一实施方案中，提供分离的抗PD-1抗体，其包含含有来自SEQ ID NO:1的重链可变区氨基酸序列的重链可变区和/或含有来自SEQ IDNO:2的轻链可变区氨基酸序列的轻链可变区。还在另一实施方案中，提供分离的抗PD-1抗体，其包含重链和/或轻链序列，其中：In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternative names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558, or Nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). In yet another embodiment, an isolated anti-PD-1 antibody is provided, comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO: 1 and/or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO: 2. In yet another embodiment, an isolated anti-PD-1 antibody is provided, comprising a heavy chain and/or light chain sequence, wherein:

(a)重链序列与以下重链序列具有至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性：(a) a heavy chain sequence that has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the heavy chain sequence of:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO：1)；和QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC PAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO: 1); and

(b)轻链序列与以下轻链序列具有至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性：(b) a light chain sequence that has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the light chain sequence of:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO：2)。EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 2).

抗PD-L1抗体Anti-PD-L1 antibodies

在一些实施方案中，该制剂中的抗体在重链和/或轻链序列中包含至少一个色氨酸(例如至少两个、至少三个、或至少四个)。在一些实施方案中，氨基酸色氨酸在该抗体的HVR区、构架区和/或恒定区中。在一些实施方案中，该抗体在HVR区中包含两个或三个色氨酸残基。在一些实施方案中，该制剂中的抗体是抗PD-L1抗体。PD-L1(程序性死亡配体1)也称为PDL1、B7-H1、B7-4、CD274和B7-H，是跨膜蛋白质，其与PD-1的相互作用抑制T细胞激活和细胞因子产生。在一些实施方案中，本文所述的抗PD-L1抗体结合人PD-L1。可以用于本文所述方法的抗PD-L1抗体的实例描述于PCT专利申请WO 2010/077634 A1和US 8,217,149中，该专利申请在此以其整体引入作为参考。In some embodiments, the antibody in the formulation comprises at least one tryptophan (e.g., at least two, at least three, or at least four) in the heavy chain and/or light chain sequence. In some embodiments, the amino acid tryptophan is in the HVR region, framework region, and/or constant region of the antibody. In some embodiments, the antibody comprises two or three tryptophan residues in the HVR region. In some embodiments, the antibody in the formulation is an anti-PD-L1 antibody. PD-L1 (programmed death ligand 1), also known as PDL1, B7-H1, B7-4, CD274, and B7-H, is a transmembrane protein whose interaction with PD-1 inhibits T cell activation and cytokine production. In some embodiments, the anti-PD-L1 antibodies described herein bind to human PD-L1. Examples of anti-PD-L1 antibodies that can be used in the methods described herein are described in PCT patent application WO 2010/077634 A1 and US 8,217,149, which are hereby incorporated by reference in their entirety.

在一些实施方案中，该抗PD-L1抗体能够抑制PD-L1和PD-1之间和/或PD-L1和B7-1之间的结合。在一些实施方案中，该抗PD-L1抗体是单克隆抗体。在一些实施方案中，该抗PD-L1抗体是选自Fab、Fab’-SH、Fv、scFv和(Fab’)2片段的抗体片段。在一些实施方案中，该抗PD-L1抗体是人源化抗体。在一些实施方案中，该抗PD-L1抗体是人抗体。In some embodiments, the anti-PD-L1 antibody is capable of inhibiting the binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv and (Fab')2 fragments. In some embodiments, the anti-PD-L1 antibody is a humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody.

WO 2010/077634 A1和US 8,217,149中所述的抗PD-L1抗体可以用于本文所述的方法。在一些实施方案中，该抗PD-L1抗体包含重链可变区序列SEQ ID NO:3和/或轻链可变区序列SEQ ID NO:4。还在另一实施方案中，提供分离的抗PD-L1抗体，其包含重链可变区和/或轻链可变区序列，其中：The anti-PD-L1 antibodies described in WO 2010/077634 A1 and US 8,217,149 can be used in the methods described herein. In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region sequence of SEQ ID NO: 3 and/or a light chain variable region sequence of SEQ ID NO: 4. In yet another embodiment, an isolated anti-PD-L1 antibody is provided, comprising a heavy chain variable region and/or a light chain variable region sequence, wherein:

(a)该重链序列与以下重链序列具有至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性：(a) the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence of:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA(SEQ ID NO:3)；和EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 3); and

(b)该轻链序列与以下轻链序列具有至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性：(b) the light chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the light chain sequence of:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:4)。DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

在一个实施方案中，该抗PD-L1抗体包含含有HVR-H1、HVR-H2和HVR-H3序列的重链可变区，其中：In one embodiment, the anti-PD-L1 antibody comprises a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

(a)该HVR-H1序列是GFTFSX₁SWIH(SEQ ID NO:5)；(a) the HVR-H1 sequence is GFTFSX₁ SWIH (SEQ ID NO: 5);

(b)该HVR-H2序列是AWIX₂PYGGSX₃YYADSVKG(SEQ ID NO:6)；(b) the HVR-H2 sequence is AWIX₂ PYGGSX₃ YYADSVKG (SEQ ID NO: 6);

(c)该HVR-H3序列是RHWPGGFDY(SEQ ID NO:7)；(c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 7);

此外，其中：X₁是D或G；X₂是S或L；X₃是T或S。在一个具体方面，X₁是D；X₂是S；X₃是T。Furthermore, wherein:_X1 is D or G;_X2 is S or L;_X3 is T or S. In a specific aspect,_X1 is D;_X2 is S;_X3 is T.

在另一方面，该多肽进一步包含按下式并置在HVR之间的可变区重链构架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)。还在另一方面，该构架区源自人共有构架序列。在另一方面，该构架序列是VH亚组III共有构架。还在另一方面，该构架序列中的至少一个是以下：In another aspect, the polypeptide further comprises a variable region heavy chain framework sequence juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another aspect, the framework regions are derived from a human consensus framework sequence. In another aspect, the framework sequence is a VH subgroup III consensus framework. In yet another aspect, at least one of the framework sequences is the following:

HC-FR1是EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:8)；HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8);

HC-FR2是WVRQAPGKGLEWV(SEQ ID NO:9)；HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 9);

HC-FR3是RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:10)；HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 10);

HC-FR4是WGQGTLVTVSA(SEQ ID NO:11)。HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 11).

还在另一方面，该重链多肽进一步与包含HVR-L1、HVR-L2和HVR-L3的可变区轻链组合，其中：In yet another aspect, the heavy chain polypeptide is further combined with a variable region light chain comprising HVR-L1, HVR-L2, and HVR-L3, wherein:

(a)该HVR-L1序列是RASQX₄X₅X₆TX₇X₈A(SEQ ID NO:12)；(a) the HVR-L1 sequence is RASQX₄ X₅ X₆ TX₇ X₈ A (SEQ ID NO: 12);

(b)该HVR-L2序列是SASX₉LX₁₀S(SEQ ID NO:13)；(b) the HVR-L2 sequence is SASX₉ LX₁₀ S (SEQ ID NO: 13);

(c)该HVR-L3序列是QQX₁₁X₁₂X₁₃X₁₄PX₁₅T(SEQ ID NO:14)；(c) the HVR-L3 sequence is QQX₁₁ X₁₂ X₁₃ X₁₄ PX₁₅ T (SEQ ID NO: 14);

其中：X₄是D或V；X₅是V或I；X₆是S或N；X₇是A或F；X₈是V或L；X₉是F或T；X₁₀是Y或A；X₁₁是Y、G、F或S；X₁₂是L、Y、F或W；X₁₃是Y、N、A、T、G、F或I；X₁₄是H、V、P、T或I；X₁₅是A、W、R、P或T。还在另一方面，X₄是D；X₅是V；X₆是S；X₇是A；X₈是V；X₉是F；X₁₀是Y；X₁₁是Y；X₁₂是L；X₁₃是Y；X₁₄是H；X₁₅是A。wherein:_X4 is D or V;_X5 is V or I;_X6 is S or N;_X7 is A or F;_X8 is V or L;_X9 is F or T;_X10 is Y or A;_X11 is Y, G, F, or S;_X12 is L, Y, F, or W;_X13 is Y, N, A, T, G, F, or I;_X14 is H, V, P, T, or I;_X15 is A, W, R, P, or T. In yet another aspect,_X4 is D;_X5 is V;_X6 is S;_X7 is A;_X8 is V;_X9 is F;_X10 is Y;_X11 is Y;_X12 is L;_X13 is Y;_X14 is H; and_X15 is A.

还在另一方面，该轻链进一步包含按下式并置在HVR之间的可变区轻链构架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。还在另一方面，该构架序列源自人共有构架序列。还在另一方面，该构架序列是VLκI共有构架。还在另一方面，该构架序列中的至少一个是以下：In yet another aspect, the light chain further comprises a variable region light chain framework sequence juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the framework sequence is a VLκI consensus framework. In yet another aspect, at least one of the framework sequences is the following:

LC-FR1是DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:15)；LC-FR1 is DIQMTQSPSSLSSASVGDRVTITC (SEQ ID NO:15);

LC-FR2是WYQQKPGKAPKLLIY(SEQ ID NO:16)；LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 16);

LC-FR3是GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:17)；LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17);

LC-FR4是FGQGTKVEIKR(SEQ ID NO:18)。LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 18).

在另一实施方案中，提供分离的抗PD-L1抗体或抗原结合片段，其包含重链和轻链可变区序列，其中：In another embodiment, an isolated anti-PD-L1 antibody or antigen-binding fragment is provided, comprising heavy and light chain variable region sequences, wherein:

(a)该重链包含HVR-H1、HVR-H2和HVR-H3，此外，其中：(a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, and further wherein:

(i)该HVR-H1序列是GFTFSX₁SWIH(SEQ ID NO:5)；(i) the HVR-H1 sequence is GFTFSX₁ SWIH (SEQ ID NO: 5);

(ii)该HVR-H2序列是AWIX₂PYGGSX₃YYADSVKG(SEQ ID NO:6)；(ii) the HVR-H2 sequence is AWIX₂ PYGGSX₃ YYADSVKG (SEQ ID NO: 6);

(iii)该HVR-H3序列是RHWPGGFDY(SEQ ID NO:7)；和(iii) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 7); and

(b)该轻链包含HVR-L1、HVR-L2和HVR-L3，此外，其中：(b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, and further, wherein:

(i)该HVR-L1序列是RASQX₄X₅X₆TX₇X₈A(SEQ ID NO:12)；(i) the HVR-L1 sequence is RASQX₄ X₅ X₆ TX₇ X₈ A (SEQ ID NO: 12);

(ii)该HVR-L2序列是SASX₉LX₁₀S(SEQ ID NO:13)；和(ii) the HVR-L2 sequence is SASX₉ LX₁₀ S (SEQ ID NO: 13); and

(iii)该HVR-L3序列是QQX₁₁X₁₂X₁₃X₁₄PX₁₅T(SEQ ID NO:14)；(iii) the HVR-L3 sequence is QQX₁₁ X₁₂ X₁₃ X₁₄ PX₁₅ T (SEQ ID NO: 14);

其中：X₁是D或G；X₂是S或L；X₃是T或S；X₄是D或V；X₅是V或I；X₆是S或N；X₇是A或F；X₈是V或L；X₉是F或T；X₁₀是Y或A；X₁₁是Y、G、F或S；X₁₂是L、Y、F或W；X₁₃是Y、N、A、T、G、F或I；X₁₄是H、V、P、T或I；X₁₅是A、W、R、P或T。在具体方面，X₁是D；X₂是S；X₃是T。在另一方面，X₄是D；X₅是V；X₆是S；X₇是A；X₈是V；X₉是F；X₁₀是Y；X₁₁是Y；X₁₂是L；X₁₃是Y；X₁₄是H；X₁₅是A。还在另一方面，X₁是D；X₂是S；X₃是T；X₄是D；X₅是V；X₆是S；X₇是A；X₈是V；X₉是F；X₁₀是Y；X₁₁是Y；X₁₂是L；X₁₃是Y；X₁₄是H；X₁₅是A。wherein:_X1 is D or G;_X2 is S or L;_X3 is T or S;_X4 is D or V;_X5 is V or I;_X6 is S or N;_X7 is A or F;_X8 is V or L;_X9 is F or T;_X10 is Y or A;_X11 is Y, G, F or S;_X12 is L, Y, F or W;_X13 is Y, N, A, T, G, F or I;_X14 is H, V, P, T or I;_X15 is A, W, R, P or T. In specific aspects,_X1 is D;_X2 is S; and_X3 is T. In another aspect,_X4 is D;_X5 is V;_X6 is S;_X7 is A; X8 is V;_X9 is F;_X10 is Y;_X11 is Y;_X12 is L;_X13 is Y;_X14 is H;_X15is A. In still another aspect,_X1 is D;_X2 is S;_X3 is T; X4 is D;_X5 is V;_X6 is S;_X7 is A;_X8 is V;_X9 is F;_X10 is Y;_X11 is Y;_X12 is L;_X13 is Y;_X14 is H;_X15is A.

在另一方面，该重链可变区包含按以下并置在HVR之间的一个或多个构架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)，且该轻链可变区包含按以下并置在HVR之间的一个或多个构架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。还在另一方面，该构架序列源自人共有构架序列。还在另一方面，该重链构架序列源自Kabat亚组I、II或III序列。还在另一方面，该重链构架序列是VH亚组III共有构架。还在另一方面，该重链构架序列中的一个或多个如SEQ ID NO:8、9、10和11所示。还在另一方面，该轻链构架序列源自KabatκI、II、II或IV亚组序列。还在另一方面，该轻链构架序列是VLκI共有构架。还在另一方面，该轻链构架序列中的一个或多个如SEQ ID NO:15、16、17和18所示。In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another aspect, the heavy chain framework sequences are VH subgroup III consensus frameworks. In yet another aspect, one or more of the heavy chain framework sequences are set forth in SEQ ID NOs: 8, 9, 10, and 11. In yet another aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences are set forth in SEQ ID NOs: 15, 16, 17, and 18.

还在另一具体方面，该抗体进一步包含人或鼠恒定区。还在另一方面，该人恒定区选自IgG1、IgG2、IgG2、IgG3、IgG4。还在另一具体方面，该人恒定区是IgG1。还在另一方面，该鼠恒定区选自IgG1、IgG2A、IgG2B、IgG3。还在另一方面，该鼠恒定区是IgG2A。还在另一具体方面，该抗体具有减少的或最小的效应子功能。还在另一具体方面，该最小的效应子功能源自“无效应子功能的Fc突变”或无岩藻糖基化。还在另一实施方案中，该无效应子功能的Fc突变是恒定区中的N297A或D265A/N297A取代。In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from IgG1, IgG2, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function is derived from an "Fc mutation with no effector function" or afucosylation. In yet another embodiment, the Fc mutation with no effector function is an N297A or D265A/N297A substitution in the constant region.

还在另一实施方案中，提供包含重链和轻链可变区序列的抗PD-L1抗体，其中：In yet another embodiment, an anti-PD-L1 antibody comprising heavy and light chain variable region sequences is provided, wherein:

(a)该重链进一步包含分别与GFTFSDSWIH(SEQ ID NO:19)、AWISPYGGSTYYADSVKG(SEQ ID NO:20)和RHWPGGFDY(SEQ ID NO:21)具有至少85％序列同一性的HVR-H1、HVR-H2和HVR-H3序列；或(a) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences that have at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20), and RHWPGGFDY (SEQ ID NO: 21), respectively; or

(b)该轻链进一步包含分别与RASQDVSTAVA(SEQ ID NO:22)、SASFLYS(SEQ ID NO:23)和QQYLYHPAT(SEQ ID NO:24)具有至少85％序列同一性的HVR-L1、HVR-L2和HVR-L3序列。(b) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences that have at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 22), SASFLYS (SEQ ID NO: 23), and QQYLYHPAT (SEQ ID NO: 24), respectively.

在具体方面，该序列同一性为86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％。In specific aspects, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

在另一方面，该重链可变区包含按以下并置在HVR之间的一个或多个构架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)，且该轻链可变区包含按以下并置在HVR之间的一个或多个构架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。还在另一方面，该构架序列源自人共有构架序列。还在另一方面，该重链构架序列源自Kabat亚组I、II或III序列。还在另一方面，该重链构架序列是VH亚组III共有构架。还在另一方面，该重链构架序列中的一个或多个如SEQ ID NO:8、9、10和11所示。还在另一方面，该轻链构架序列源自KabatκI、II、II或IV亚组序列。还在另一方面，该轻链构架序列是VLκI共有构架。还在另一方面，该轻链构架序列中的一个或多个如SEQ ID NO:15、16、17和18所示。In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another aspect, the heavy chain framework sequences are VH subgroup III consensus frameworks. In yet another aspect, one or more of the heavy chain framework sequences are set forth in SEQ ID NOs: 8, 9, 10, and 11. In yet another aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences are set forth in SEQ ID NOs: 15, 16, 17, and 18.

还在另一具体方面，该抗体进一步包含人或鼠恒定区。还在另一方面，该人恒定区选自IgG1、IgG2、IgG2、IgG3、IgG4。还在另一具体方面，该人恒定区是IgG1。还在另一方面，该鼠恒定区选自IgG1、IgG2A、IgG2B、IgG3。还在另一方面，该鼠恒定区是IgG2A。还在另一具体方面，该抗体具有减少的或最小的效应子功能。还在另一具体方面，该最小的效应子功能源自“无效应子功能的Fc突变”或无岩藻糖基化。还在另一实施方案中，该无效应子功能的Fc突变是恒定区中的N297A或D265A/N297A取代。In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from IgG1, IgG2, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function is derived from an "Fc mutation with no effector function" or afucosylation. In yet another embodiment, the Fc mutation with no effector function is an N297A or D265A/N297A substitution in the constant region.

在另一实施方案中，提供分离的抗PD-L1抗体，其包含重链和轻链可变区序列，其中：In another embodiment, an isolated anti-PD-L1 antibody is provided, comprising heavy and light chain variable region sequences, wherein:

(a)该重链序列与以下重链序列具有至少85％序列同一性：(a) the heavy chain sequence has at least 85% sequence identity with the following heavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS(SEQ ID NO:25)；和/或EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS(SEQ ID NO:25); and/or

(b)该轻链序列与以下轻链序列具有至少85％序列同一性：(b) the light chain sequence has at least 85% sequence identity with the following light chain sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:4)。DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

在具体方面，该序列同一性为86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％。在另一方面，该重链可变区包含按以下并置在HVR之间的一个或多个构架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)，且该轻链可变区包含按以下并置在HVR之间的一个或多个构架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。还在另一方面，该构架序列源自人共有构架序列。还在另一方面，该重链构架序列源自Kabat亚组I、II或III序列。还在另一方面，该重链构架序列是VH亚组III共有构架。还在另一方面，该重链构架序列中的一个或多个如SEQ ID NO:8、9、10和WGQGTLVTVSS(SEQ ID NO:27)所示。In specific aspects, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences are set forth in SEQ ID NOs: 8, 9, 10, and WGQGTLVTVSS (SEQ ID NO: 27).

还在另一方面，该轻链构架序列源自KabatκI、II、II或IV亚组序列。还在另一方面，该轻链构架序列是VLκI共有构架。还在另一方面，该轻链构架序列中的一个或多个如SEQ ID NO:15、16、17和18所示。In yet another aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences are set forth in SEQ ID NOs: 15, 16, 17, and 18.

还在另一具体方面，该抗体进一步包含人或鼠恒定区。还在另一方面，该人恒定区选自IgG1、IgG2、IgG2、IgG3、IgG4。还在另一具体方面，该人恒定区是IgG1。还在另一方面，该鼠恒定区选自IgG1、IgG2A、IgG2B、IgG3。还在另一方面，该鼠恒定区是IgG2A。还在另一具体方面，该抗体具有减少的或最小的效应子功能。还在另一具体方面，该最小的效应子功能源自在原核细胞中产生。还在另一具体方面，该最小的效应子功能源自“无效应子功能的Fc突变”或无岩藻糖基化。还在另一实施方案中，该无效应子功能的Fc突变是恒定区中的N297A或D265A/N297A取代。In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from IgG1, IgG2, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function is derived from production in prokaryotic cells. In yet another specific aspect, the minimal effector function is derived from an "Fc mutation with no effector function" or afucosylation. In yet another embodiment, the Fc mutation with no effector function is an N297A or D265A/N297A substitution in the constant region.

在另一方面，该重链可变区包含按以下并置在HVR之间的一个或多个构架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)，且该轻链可变区包含按以下并置在HVR之间的一个或多个构架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。还在另一方面，该构架序列源自人共有构架序列。还在另一方面，该重链构架序列源自Kabat亚组I、II或III序列。还在另一方面，该重链构架序列是VH亚组III共有构架。还在另一方面，该重链构架序列中的一个或多个是以下：In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another aspect, the heavy chain framework sequences are VH subgroup III consensus frameworks. In yet another aspect, one or more of the heavy chain framework sequences are:

还在另一方面，该轻链构架序列源自KabatκI、II、II或IV亚组序列。还在另一方面，该轻链构架序列是VLκI共有构架。还在另一方面，该轻链构架序列中的一个或多个是以下：In yet another aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences is the following:

还在另一具体方面，该抗体进一步包含人或鼠恒定区。还在另一方面，该人恒定区选自IgG1、IgG2、IgG2、IgG3、IgG4。还在另一具体方面，该人恒定区是IgG1。还在另一方面，该鼠恒定区选自IgG1、IgG2A、IgG2B、IgG3。还在另一方面，该鼠恒定区是IgG2A。还在另一具体方面，该抗体具有减少的或最小的效应子功能。还在另一具体方面，该最小的效应子功能源自“无效应子功能的Fc突变”或无岩藻糖基化。还在另一实施方案中，该无效应子功能的Fc突变是恒定区中的N297A或D265A/N297A取代。In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from IgG1, IgG2, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function is derived from an "Fc mutation with no effector function" or afucosylation. In yet another embodiment, the Fc mutation with no effector function is an N297A or D265A/N297A substitution in the constant region.

还在另一实施方案中，提供包含重链和轻链可变区序列的抗PD-L1抗体，其中：In yet another embodiment, an anti-PD-L1 antibody comprising heavy and light chain variable region sequences is provided, wherein:

(c)该重链进一步包含分别与GFTFSDSWIH(SEQ ID NO:19)、AWISPYGGSTYYADSVKG(SEQ ID NO:20)和RHWPGGFDY(SEQ IDNO:21)具有至少85％序列同一性的HVR-H1、HVR-H2和HVR-H3序列；和/或(c) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences that have at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20), and RHWPGGFDY (SEQ ID NO: 21), respectively; and/or

(d)该轻链进一步包含分别与RASQDVSTAVA(SEQ ID NO:22)、SASFLYS(SEQ ID NO:23)和QQYLYHPAT(SEQ ID NO:24)具有至少85％序列同一性的HVR-L1、HVR-L2和HVR-L3序列。(d) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences that have at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 22), SASFLYS (SEQ ID NO: 23), and QQYLYHPAT (SEQ ID NO: 24), respectively.

在具体方面，该序列同一性为86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％。In specific aspects, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

在另一方面，该重链可变区包含按以下并置在HVR之间的一个或多个构架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)，且该轻链可变区包含按以下并置在HVR之间的一个或多个构架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。还在另一方面，该构架序列源自人共有构架序列。还在另一方面，该重链构架序列源自Kabat亚组I、II或III序列。还在另一方面，该重链构架序列是VH亚组III共有构架。还在另一方面，该重链构架序列中的一个或多个如SEQ ID NO:8、9、10和WGQGTLVTVSSASTK(SEQ ID NO:31)所示。In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another aspect, the heavy chain framework sequences are VH subgroup III consensus frameworks. In yet another aspect, one or more of the heavy chain framework sequences are set forth in SEQ ID NOs: 8, 9, 10, and WGQGTLVTVSSASTK (SEQ ID NO: 31).

还在另一方面，该轻链构架序列源自KabatκI、II、II或IV亚组序列。还在另一方面，该轻链构架序列是VLκI共有构架。还在另一方面，该轻链构架序列中的一个或多个如SEQ ID NO:15、16、17和18所示。还在另一具体方面，该抗体进一步包含人或鼠恒定区。还在另一方面，该人恒定区选自IgG1、IgG2、IgG2、IgG3、IgG4。还在另一具体方面，该人恒定区是IgG1。还在另一方面，该鼠恒定区选自IgG1、IgG2A、IgG2B、IgG3。还在另一方面，该鼠恒定区是IgG2A。还在另一具体方面，该抗体具有减少的或最小的效应子功能。还在另一具体方面，该最小的效应子功能源自“无效应子功能的Fc突变”或无岩藻糖基化。还在另一实施方案中，该无效应子功能的Fc突变是恒定区中的N297A或D265A/N297A取代。In yet another aspect, the light chain framework sequence is derived from a Kabat κI, II, II, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VLκI consensus framework. In yet another aspect, one or more of the light chain framework sequences are set forth in SEQ ID NOs: 15, 16, 17, and 18. In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from IgG1, IgG2, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function is derived from an "effector-free Fc mutation" or afucosylation. In yet another embodiment, the Fc mutation without effector function is an N297A or D265A/N297A substitution in the constant region.

还在另一实施方案中，提供分离的抗PD-L1抗体，其包含重链和轻链可变区序列，其中：In yet another embodiment, an isolated anti-PD-L1 antibody is provided, comprising heavy and light chain variable region sequences, wherein:

(a)该重链序列与以下重链序列具有至少85％序列同一性：(a) the heavy chain sequence has at least 85% sequence identity with the following heavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK(SEQ ID NO:26)；或OR

(b)该轻链序列与以下轻链序列具有至少85％序列同一性：(b) the light chain sequence has at least 85% sequence identity with the following light chain sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:4)。DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

在一些实施方案中，提供分离的抗PD-L1抗体，其包含重链和轻链可变区序列，其中该轻链可变区序列与氨基酸序列SEQ ID NO:4具有至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性。在一些实施方案中，提供分离的抗PD-L1抗体，其包含重链和轻链可变区序列，其中该重链可变区序列与氨基酸序列SEQ ID NO：26具有至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性。在一些实施方案中，提供分离的抗PD-L1抗体，其包含重链和轻链可变区序列，其中该轻链可变区序列与氨基酸序列SEQ ID NO：4具有至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性，且该重链可变区序列与氨基酸序列SEQID NO：26具有至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性。在一些实施方案中，可以缺失、取代或修饰重链和/或轻链N端的一个、两个、三个、四个或五个氨基酸残基。In some embodiments, an isolated anti-PD-L1 antibody is provided, comprising heavy and light chain variable region sequences, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, an isolated anti-PD-L1 antibody is provided, comprising heavy and light chain variable region sequences, wherein the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, an isolated anti-PD-L1 antibody is provided, comprising heavy and light chain variable region sequences, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4, and the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, one, two, three, four, or five amino acid residues at the N-terminus of the heavy and/or light chain can be deleted, substituted, or modified.

还在另一实施方案中，提供分离的抗PD-L1抗体，其包含重链和轻链序列，其中：In yet another embodiment, an isolated anti-PD-L1 antibody is provided, comprising heavy and light chain sequences, wherein:

(a)该重链序列与以下重链序列具有至少85％序列同一性：(a) the heavy chain sequence has at least 85% sequence identity with the following heavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO：32)；和/或EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGK GLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 32); and/or

(b)该轻链序列与以下轻链序列具有至少85％序列同一性：(b) the light chain sequence has at least 85% sequence identity with the following light chain sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO：33)。DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 33).

在一些实施方案中，提供分离的抗PD-L1抗体，其包含重链和轻链序列，其中该轻链序列与氨基酸序列SEQ ID NO：33具有至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性。在一些实施方案中，提供分离的抗PD-L1抗体，其包含重链和轻链序列，其中该重链序列与氨基酸序列SEQ ID NO：32具有至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性。在一些实施方案中，提供分离的抗PD-L1抗体，其包含重链和轻链序列，其中该轻链序列与氨基酸序列SEQ ID NO：33具有至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性，且该重链序列与氨基酸序列SEQ ID NO：32具有至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性。In some embodiments, an isolated anti-PD-L1 antibody is provided that comprises a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, an isolated anti-PD-L1 antibody is provided that comprises a heavy chain and a light chain sequence, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, an isolated anti-PD-L1 antibody is provided, comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 33, and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 32.

在一些实施方案中，该分离的抗PD-L1抗体是无糖基化的。抗体的糖基化通常是N连接的或O连接的。N连接指糖类部分附着于天冬酰胺残基侧链。三肽序列天冬酰胺-X-丝氨酸和天冬酰胺-X-苏氨酸(其中X是除脯氨酸外的任意氨基酸)是酶促附着糖类部分至天冬酰胺侧链的识别序列。因此，这些三肽序列的任一个在多肽中的存在产生潜在的糖基化位点。O连接糖基化指糖N-乙酰半乳糖胺、半乳糖或木糖之一附着于羟基氨基酸，该羟基氨基酸最常是丝氨酸或苏氨酸，但也可以使用5-羟脯氨酸或5-羟赖氨酸。通过改变氨基酸序列，使得去除上述三肽序列之一(对于N连接糖基化位点)来方便地从抗体去除糖基化位点。该改变可以通过用另一氨基酸残基(例如甘氨酸、丙氨酸或保守取代)取代糖基化位点内的天冬酰胺、丝氨酸或苏氨酸残基来进行。In some embodiments, the isolated anti-PD-L1 antibody is aglycosylated. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid except proline) are recognition sequences for enzymatic attachment of carbohydrate moieties to asparagine side chains. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, which is most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used. Glycosylation sites are conveniently removed from antibodies by altering the amino acid sequence such that one of the above tripeptide sequences (for N-linked glycosylation sites) is removed. This alteration can be performed by replacing the asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or conservative substitution).

在本文的任意实施方案中，该分离的抗PD-L1抗体可以结合人PD-L1，例如UniProtKB/Swiss-Prot检索号Q9NZQ7.1中所示的人PD-L1，或其变体。In any of the embodiments herein, the isolated anti-PD-L1 antibody can bind to human PD-L1, such as the human PD-L1 shown in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof.

还在另一实施方案中，提供分离的编码本文所述任意抗体的核酸。在一些实施方案中，该核酸进一步包含适合用于表达编码之前所述的任意抗PD-L1抗体的核酸的载体。还在另一具体方面，该载体处于适合用于表达该核酸的宿主细胞中。还在另一具体方面，该宿主细胞是真核细胞或原核细胞。还在另一具体方面，该真核细胞是哺乳动物细胞，如中国仓鼠卵巢(CHO)细胞。In yet another embodiment, an isolated nucleic acid encoding any of the antibodies described herein is provided. In some embodiments, the nucleic acid further comprises a vector suitable for expressing the nucleic acid encoding any of the anti-PD-L1 antibodies described above. In yet another specific aspect, the vector is in a host cell suitable for expressing the nucleic acid. In yet another specific aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In yet another specific aspect, the eukaryotic cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

抗体或其抗原结合片段可以用本领域已知的方法制备，例如，通过包括以下的方法：在适于产生这种抗体或片段的条件下，培养包含处于适合用于表达的形式的编码之前所述任意抗PD-L1抗体或抗原结合片段的核酸的宿主细胞，并回收抗体或片段。The antibodies or antigen-binding fragments thereof can be prepared by methods known in the art, for example, by a method comprising culturing a host cell containing a nucleic acid encoding any of the previously described anti-PD-L1 antibodies or antigen-binding fragments in a form suitable for expression under conditions suitable for production of such antibodies or fragments, and recovering the antibody or fragment.

III.抗体制备III. Antibody Preparation

本文所述的抗体可以用本领域可用的用于产生抗体的技术制备，其示例性方法更详细地描述于以下章节中。The antibodies described herein can be prepared using techniques available in the art for generating antibodies, exemplary methods of which are described in more detail in the following sections.

该抗体针对目的抗原(例如PD-L1(如人PD-L1)、PD1(如人PD-L1)、PD-L2(如人PD-L2)等。优选地，该抗原是生物学上重要的多肽，对患有障碍的哺乳动物施用该抗体可以在该哺乳动物中产生治疗益处。The antibody is directed against an antigen of interest (e.g., PD-L1 (e.g., human PD-L1), PD1 (e.g., human PD-L1), PD-L2 (e.g., human PD-L2), etc. Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to a mammal suffering from a disorder can produce a therapeutic benefit in the mammal.

在某些实施方案中，本文提供的抗体具有≤1μM、≤150nM、≤100nM、≤50nM、≤10nM、≤1nM、≤0.1nM、≤0.01nM或≤0.001nM(例如10^-8M或更小，例如从10^-8M至10^-13M，例如从10^-9M至10^-13M)的解离常数(Kd)。In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≤1 μM, ≤150 nM, ≤100 nM, ≤50 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M).

在一个实施方案中，通过用以下测定所述的Fab形式的目的抗体及其抗原进行的放射性标记抗原结合测定(RIA)来测量Kd。通过在未标记抗原的滴定系列的存在下用最小浓度的(¹²⁵I)-标记抗原平衡Fab，然后用抗-Fab抗体包被的平板捕获结合的抗原来测量Fab对抗原的溶液结合亲和力(参见例如Chen等,J.Mol.Biol.293:865-881(1999))。为了确定用于测定的条件，用含5μg/ml捕获抗-Fab抗体(Cappel Labs)的50mM碳酸钠(pH 9.6)过夜包被多孔板(Thermo Scientific)，然后用含2％(w/v)牛血清白蛋白的PBS在室温(约23℃)封闭2至5小时。在非吸附平板(Nunc#269620)中，将100pM或26pM[¹²⁵I]-抗原与目的Fab的系列稀释液混合。然后过夜孵育目的Fab；但是，孵育可以持续更长时期(例如约65小时)，以确保达到平衡。然后，将混合物转移至捕获平板进行室温孵育(例如孵育1小时)。然后去除溶液，用含0.1％聚山梨酸酯20的PBS洗涤平板8次。平板干燥后，加入150μl/孔的闪烁体(MICROSCINT-20^TM；Packard)，在TOPCOUNT^TMγ计数器(Packard)上计数平板10分钟。选择给出小于或等于最大结合的20％的每种Fab的浓度用于竞争结合测定。In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) using a Fab form of the antibody of interest and its antigen as described in the following assay. Solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration of (¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen and then capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions for the assay, multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w/v) bovine serum albumin in PBS at room temperature (approximately 23° C.) for 2 to 5 hours. In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵ I]-antigen was mixed with a serial dilution of the Fab of interest. The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with PBS containing 0.1% polysorbate 20. After the plate is dried, 150 μl/well of scintillant (MICROSCINT-20^™ ; Packard) is added and the plate is counted on a TOPCOUNT^™ gamma counter (Packard) for 10 minutes. A concentration of each Fab that gives less than or equal to 20% of maximum binding is selected for competitive binding assays.

根据另一实施方案，用约10个响应单位(RU)的固定化抗原CM5芯片，在25℃下使用-2000或-3000(BIAcore,Inc.,Piscataway,NJ)，用表面等离振子共振测定来测量Kd。简言之，按照厂家说明书用N-乙基-N’-(3-二甲基氨基丙基)-碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)活化羧甲基化葡聚糖生物传感芯片(CM5,BIACORE,Inc.)。用10mM醋酸钠pH 4.8将抗原稀释至5μg/ml(约0.2μM)，然后按5μl/分钟的流速注入，以达到约10个响应单位(RU)的偶联蛋白质。注入抗原后，注入1M乙醇胺，以封闭未反应的基团。对于动力学测量，按约25μl/分钟的流速在25℃下注入两倍系列稀释于含0.05％聚山梨酸酯20(TWEEN20^TM)表面活性剂的PBS(PBST)中的Fab(0.78nM至500nM)。通过同时拟合结合和解离传感图，用简单的1:1Langmuir结合模型(EvaluationSoftware version 3.2)计算结合速率(k_on)和解离速率(k_off)。将平衡解离常数(Kd)计算为比值k_off/k_on。参见例如Chen等,J.Mol.Biol.293:865-881(1999)。如果通过以上表面等离振子共振测定测量的结合速率超过10⁶M^-1s^-1，则可以通过使用荧光淬灭技术来测定结合速率，如在分光计，如装配停流的分光光度计(Aviv Instruments)或具有搅拌杯的8000系列SLM-AMINCO^TM分光光度计(ThermoSpectronic)中测量，该技术在浓度递增的抗原存在下测量含20nM抗抗原的抗体(Fab形式)的PBS pH7.2在25℃下的荧光发射强度(激发＝295nm；发射＝340nm，16nm带通)的提高或降低。According to another embodiment, Kd is measured using a surface plasmon resonance assay using a CM5 chip with approximately 10 response units (RU) of immobilized antigen at 25°C using a -2000 or -3000 (BIAcore, Inc., Piscataway, NJ). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. Antigen is diluted to 5 μg/ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl/min to achieve approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS containing 0.05% polysorbate 20 (TWEEN20^™ ) surfactant (PBST) were injected at a flow rate of approximately 25 μl/min at 25° C. Association rates (k_on ) and dissociation rates (k_off ) were calculated using a simple 1:1 Langmuir binding model (Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) was calculated as the ratio k_off /k_on . See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate measured by the surface plasmon resonance assay above exceeds 10⁶ M^-1 s^-1 , the on-rate can be determined by using a fluorescence quenching technique, such as measured in a spectrometer, such as a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO^™ spectrophotometer with a stir cup (ThermoSpectronic), which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C in the presence of increasing concentrations of antigen.

(i)抗原制备(i) Antigen preparation

可以用可选地与其他分子缀合的可溶性抗原或其片段作为免疫原来产生抗体。对于跨膜分子，如受体，可以用这些的片段(例如受体的胞外域)作为免疫原。备选地，可以用表达该跨膜分子的细胞作为免疫原。这类细胞可以源自天然来源(例如癌细胞系)或可以是通过重组技术转化来表达该跨膜分子的细胞。用于制备抗体的其他抗原及其形式对本领域技术人员而言将显而易见。Antibodies can be produced using soluble antigens or fragments thereof that are optionally conjugated to other molecules as immunogens. For transmembrane molecules, such as receptors, fragments of these (e.g., the extracellular domain of a receptor) can be used as immunogens. Alternatively, cells expressing the transmembrane molecule can be used as immunogens. Such cells can be derived from natural sources (e.g., cancer cell lines) or can be cells transformed by recombinant technology to express the transmembrane molecule. Other antigens and forms thereof for the preparation of antibodies will be apparent to those skilled in the art.

(ii)某些基于抗体的方法(ii) Certain Antibody-Based Methods

多克隆抗体优选通过多次皮下(sc)或腹腔内(ip)注射相关抗原和佐剂来在动物中制备。用双功能剂或衍生剂(例如马来酰亚胺基苯甲酰基磺基琥珀酰亚胺酯(通过半胱氨酸残基缀合)、N-羟基琥珀酰亚胺(通过赖氨酸残基)、戊二醛、琥珀酐、SOCl₂或R¹N＝C＝NR，其中R和R¹是不同的烷基)将相关抗原与在待免疫的物种中具有免疫原性的多肽(例如匙孔槭血蓝蛋白、血清白蛋白、牛甲状腺球蛋白或大豆胰蛋白酶抑制剂)缀合可以是有用的。Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and^an adjuvant. It may be useful to conjugate the relevant antigen to a polypeptide that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin,_bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctional or derivatizing agent (e.g., maleimidobenzoylsulfosuccinimide ester (conjugated through cysteine residues), N-hydroxysuccinimide (conjugated through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2, or R 1 N═C═NR, wherein R and R¹ are different alkyl groups).

通过将例如100μg或5μg的蛋白质或缀合物(分别对于兔或小鼠)与3体积的弗氏完全佐剂组合，并在多个部位皮内注射该溶液来针对抗原、免疫原性缀合物或衍生物免疫动物。一个月后，通过在多个部位皮下注射来用弗氏完全佐剂中的初始量的1/5至1/10的肽或缀合物加强免疫动物。7至14天后，对动物进行采血，并测定血清的抗体效价。加强免疫动物，直至效价平台期。优选地，用同一抗原的缀合物加强免疫动物，但该抗原与不同的蛋白质缀合和/或通过不同的交联剂缀合。缀合物还可以作为蛋白质融合在重组细胞培养物中制备。另外，适宜地用诸如明矾的聚集剂来增强免疫反应。By combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites to immunize animals against antigens, immunogenic conjugates or derivatives. One month later, the animals are boosted with 1/5 to 1/10 of the initial amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animals are bled and the antibody titer of the serum is measured. The animals are boosted until the titer plateaus. Preferably, the animals are boosted with conjugates of the same antigen, but the antigen is conjugated to different proteins and/or conjugated by different cross-linking agents. The conjugates can also be prepared in recombinant cell cultures as protein fusions. In addition, the immune response is suitably enhanced with aggregating agents such as alum.

本发明的单克隆抗体可以用杂交瘤法制备，该杂交瘤法最先由Kohler等,Nature,256:495(1975)描述，并进一步描述于例如Hongo等,Hybridoma,14(3):253-260(1995)；Harlow等,Antibodies:A Laboratory Manual,(Cold Spring Harbor Laboratory出版社,第2版1988)；Hammerling等,in:Monoclonal Antibodies and T-Cell Hybridomas 563-681(Elsevier,N.Y.,1981)；及Ni,Xiandai Mianyixue,26(4):265-268(2006)(关于人-人杂交瘤)中。其他方法包括描述于例如美国专利号7,189,826(关于从杂交瘤细胞系产生单克隆人天然IgM抗体)中的那些。人杂交瘤技术(三元杂交瘤技术)描述于Vollmers和Brandlein,Histology and Histopathology,20(3):927-937(2005)及Vollmers和Brandlein,Methods and Findings in Experimental and Clinical Pharmacology,27(3):185-91(2005)中。The monoclonal antibodies of the present invention can be prepared using the hybridoma method, which was first described by Kohler et al., Nature, 256:495 (1975) and further described in, for example, Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981); and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (for human-human hybridomas). Other methods include those described in, for example, U.S. Pat. No. 7,189,826 (for the production of monoclonal human natural IgM antibodies from hybridoma cell lines). The human hybridoma technology (tri-oma technology) is described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

对于多种其他杂交瘤技术，参见例如美国专利公开号2006/258841、2006/183887(全人抗体)、2006/059575、2005/287149、2005/100546和2005/026229；及美国专利号7,078,492和7,153,507。用于用杂交瘤法产生单克隆抗体的示例性流程描述如下。在一个实施方案中，免疫小鼠或其他适宜的宿主动物(如仓鼠)，以引出产生或能够产生将特异性结合用于免疫的蛋白质的抗体的淋巴细胞。通过多次皮下(SC)或腹腔内(IP)注射本发明的多肽或其片段和佐剂(如单磷酰脂质A(MPL)/trehalose dicrynomycolate(TDM)(RibiImmunochem.Research,Inc.,Hamilton,MT))来在动物中制备抗体。本发明的多肽(例如抗原)或其片段可以用本领域公知的方法制备，如重组方法，其中一些在本文中进一步描述。针对抗抗原的抗体测定来自免疫动物的血清，可选地施用加强免疫。分离来自产生抗抗原的抗体的动物的淋巴细胞。备选地，可以体外免疫淋巴细胞。For various other hybridoma technologies, see, for example, U.S. Patent Publication Nos. 2006/258841, 2006/183887 (fully human antibodies), 2006/059575, 2005/287149, 2005/100546, and 2005/026229; and U.S. Patent Nos. 7,078,492 and 7,153,507. An exemplary process for producing monoclonal antibodies using the hybridoma method is described below. In one embodiment, a mouse or other suitable host animal (e.g., a hamster) is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies are prepared in animals by multiple subcutaneous (SC) or intraperitoneal (IP) injections of a polypeptide of the invention or a fragment thereof and an adjuvant (e.g., monophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT). The polypeptides (e.g., antigens) of the present invention or fragments thereof can be prepared using methods well known in the art, such as recombinant methods, some of which are further described herein. Serum from immunized animals can be assayed for antibodies against the antigen, and booster immunizations can be optionally administered. Lymphocytes from animals that produce antibodies against the antigen can be isolated. Alternatively, lymphocytes can be immunized in vitro.

然后用适宜的融合剂(如聚乙二醇)使淋巴细胞与骨髓瘤细胞融合形成杂交瘤细胞。参见例如Goding,Monoclonal Antibodies:Principles and Practice,59-103页(Academic出版社,1986)。可以使用高效融合、通过所选择的产抗体细胞支持抗体的稳定高水平产生、且对诸如HAT培养基的培养基敏感的骨髓瘤细胞。示例性骨髓瘤细胞包括但不限于鼠骨髓瘤细胞系，如可从Salk Institute Cell Distribution Center,San Diego,California USA获得的衍生自MOPC-21和MPC-11小鼠肿瘤的那些，及可从American TypeCulture Collection,Rockville,Md.USA获得的SP-2或X63-Ag8-653细胞。还针对人单克隆抗体的产生描述了人骨髓瘤和小鼠-人杂骨髓瘤(heteromyeloma)细胞系(Kozbor,J.Immunol.133:3001(1984)；Brodeur等,Monoclonal Antibody Production Techniquesand Applications,51-63页(Marcel Dekker,Inc.,纽约,1987))。Lymphocytes are then fused with myeloma cells using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells. See, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma cells that efficiently fuse, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to culture media such as HAT culture media can be used. Exemplary myeloma cells include, but are not limited to, mouse myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

将这样制备的杂交瘤细胞接种和培养在适宜的培养基中，例如包含一种或多种抑制未融合的亲本骨髓瘤细胞生长或存活的物质的培养基。例如，如果亲本骨髓瘤细胞缺乏次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT或HPRT)，则用于杂交瘤的培养基通常将包含次黄嘌呤、氨基蝶呤和胸苷(HAT培养基)，这些物质阻止缺乏HGPRT的细胞生长。优选地，按例如Even等,Trends in Biotechnology,24(3),105-108(2006)中所述，使用无血清杂交瘤细胞培养法，以减少动物来源的血清如胎牛血清的使用。The hybridoma cells thus prepared are inoculated and cultured in a suitable culture medium, such as a culture medium containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of cells lacking HGPRT. Preferably, a serum-free hybridoma cell culture method is used, such as described in Even et al., Trends in Biotechnology, 24(3), 105-108 (2006), to reduce the use of animal-derived serum, such as fetal bovine serum.

Franek,Trends in Monoclonal Antibody Research,111-122(2005)中描述了寡肽作为用于提高杂交瘤细胞培养物的生产力的工具。具体而言，标准培养基富含某些氨基酸(丙氨酸、丝氨酸、天冬酰胺、脯氨酸)，或富含蛋白质水解产物级分，由三至六个氨基酸残基组成的合成寡肽可以显著抑制凋亡。该肽以毫摩尔或更高浓度存在。Franek, Trends in Monoclonal Antibody Research, 111-122 (2005), describes oligopeptides as tools for increasing the productivity of hybridoma cell cultures. Specifically, standard culture media enriched with certain amino acids (alanine, serine, asparagine, proline) or enriched with protein hydrolysate fractions, and synthetic oligopeptides composed of three to six amino acid residues can significantly inhibit apoptosis. The peptides are present at millimolar or higher concentrations.

可以针对结合本发明的抗体的单克隆抗体的产生测定杂交瘤细胞在其中生长的培养基。由杂交瘤细胞产生的单克隆抗体的结合特异性可以通过免疫沉淀或通过体外结合测定如放射免疫测定(RIA)或酶联免疫吸附测定(ELISA)来测定。单克隆抗体的结合亲和力可以例如通过Scatchard分析来测定。参见例如Munson等,Anal.Biochem.,107:220(1980)。The culture medium in which the hybridoma cells are grown can be assayed for the production of monoclonal antibodies that bind to the antibodies of the present invention. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by Scatchard analysis. See, for example, Munson et al., Anal. Biochem., 107:220 (1980).

鉴定出产生具有希望得到的特异性、亲和力和/或活性的抗体的杂交瘤细胞后，可以通过有限稀释法亚克隆该克隆，并通过标准方法培养。参见例如Goding,上文。适合用于此目的的培养基包括例如D-MEM或RPMI-1640培养基。此外，可以在动物中作为腹水肿瘤体内培养杂交瘤细胞。通过常规免疫球蛋白纯化方法(例如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析或亲和层析)适宜地从培养基、腹水或血清分离由亚克隆分泌的单克隆抗体。US 2005/176122和美国专利号6,919,436中描述了一种用于从杂交瘤细胞分离蛋白质的方法。该方法包括在结合过程中使用最少的盐如易溶性盐，且优选还在洗脱过程中使用小量有机溶剂。After identifying hybridoma cells that produce antibodies with desired specificity, affinity and/or activity, the clone can be subcloned by limiting dilution and cultured by standard methods. See, for example, Goding, above. Culture medium suitable for this purpose includes, for example, D-MEM or RPMI-1640 culture medium. In addition, hybridoma cells can be cultured in animals as ascites tumors. Monoclonal antibodies secreted by subclones are suitably separated from culture medium, ascites or serum by conventional immunoglobulin purification methods (such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography). A method for separating proteins from hybridoma cells is described in US 2005/176122 and U.S. Patent number 6,919,436. The method includes using minimum salts such as soluble salts in the binding process, and preferably also using a small amount of organic solvents in the elution process.

(iii)文库衍生的抗体(iii) Library-derived antibodies

可以针对具有一种或多种希望得到的活性的抗体筛选组合文库来分离本发明的抗体。例如，本领域已知用于产生噬菌体展示文库并针对具有希望得到的结合特征的抗体筛选这类文库的多种方法。其他方法综述于例如Hoogenboom等in Methods in MolecularBiology 178:1-37(O’Brien等,编辑,Human出版社,Totowa,NJ,2001)中，并进一步描述于例如McCafferty等,Nature 348:552-554；Clackson等,Nature 352:624-628(1991)；Marks等,J.Mol.Biol.222:581-597(1992)；Marks和Bradbury,in Methods in MolecularBiology 248:161-175(Lo,编辑,Human出版社,Totowa,NJ,2003)；Sidhu等,J.Mol.Biol.338(2):299-310(2004)；Lee等,J.Mol.Biol.340(5):1073-1093(2004)；Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004)；及Lee等,J.Immunol.Methods 284(1-2):119-132(2004)中。Combinatorial libraries can be screened for antibodies with one or more desired activities to isolate the antibodies of the present invention. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. Other methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described in, for example, McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

在某些噬菌体展示法中，通过聚合酶链反应(PCR)分别克隆VH和VL基因库，并在噬菌体文库中随机组合，然后按Winter等,Ann.Rev.Immunol.,12:433-455(1994)中所述针对抗原结合噬菌体筛选该文库。噬菌体通常将抗体片段展示为单链Fv(scFv)片段或Fab片段。来自免疫来源的文库提供抗免疫原的高亲和力抗体而无需构建杂交瘤。备选地，可以按Griffiths等,EMBO J,12:725-734(1993)所述克隆(例如从人)首次用于实验的库来提供抗广范围的非自身以及自身抗原的抗体的单一来源而无需任何免疫。最后，还可以按Hoogenboom和Winter,J.Mol.Biol.,227:381-388(1992)所述，通过以下来合成制备首次用于实验的文库：从干细胞克隆未重排的V基因区段，用包含随机序列的PCR引物来编码高变的CDR3区，并实现体外重排。描述人抗体噬菌体文库的专利公开包括例如美国专利号5,750,373及美国专利公开号2005/0079574、2005/0119455、2005/0266000、2007/0117126、2007/0160598、2007/0237764、2007/0292936和2009/0002360。In some phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly combined in a phage library, which is then screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, a library that has been cloned (e.g., from humans) for the first time in an experiment as described in Griffiths et al., EMBO J, 12: 725-734 (1993) can be used to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization. Finally, naive libraries can also be prepared synthetically by cloning unrearranged V gene segments from stem cells, encoding hypervariable CDR3 regions with PCR primers containing random sequences, and achieving in vitro rearrangement as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent disclosures describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

本文将从人抗体文库分离的抗体或抗体片段视为人抗体或人抗体片段。Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

(iv)嵌合抗体、人源化抗体和人抗体(iv) Chimeric antibodies, humanized antibodies, and human antibodies

在某些实施方案中，本文提供的抗体是嵌合抗体。某些嵌合抗体描述于例如美国专利号4,816,567及Morrison等,Proc.Natl.Acad.Sci.USA,81:6851-6855(1984)中。在一个实例中，嵌合抗体包含非人可变区(例如源自小鼠、大鼠、仓鼠、兔、或非人灵长类(如猴)的可变区)和人恒定区。在另一实例中，嵌合抗体是“种类转换”抗体，其中种类或亚类已从亲本抗体的种类或亚类改变。嵌合抗体包括其抗原结合片段。In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described in, for example, U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

在某些实施方案中，嵌合抗体是人源化抗体。通常，人源化非人抗体来降低对人类的免疫原性，同时保留亲本非人抗体的特异性和亲和力。通常，人源化抗体包含一个或多个可变结构域，其中HVR例如CDR(或其部分)源自非人抗体，FR(或其部分)源自人抗体序列。人源化抗体还将可选地包含至少部分人恒定区。在一些实施方案中，用来自非人抗体(例如从其衍生HVR残基的抗体)的相应残基取代人源化抗体中的一些FR残基，例如以恢复或改善抗体特异性或亲和力。In certain embodiments, chimeric antibodies are humanized antibodies. Generally, humanized non-human antibodies reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR such as CDR (or part thereof) is derived from non-human antibodies, and FR (or part thereof) is derived from human antibody sequences. Humanized antibodies will also optionally include at least part of human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

人源化抗体和制备它们的方法综述于例如Almagro和Fransson,Front.Biosci.13:1619-1633(2008)中，并进一步描述于例如Riechmann等,Nature332:323-329(1988)；Queen等,Proc.Nat’l Acad.Sci.USA 86:10029-10033(1989)；美国专利号5,821,337、7,527,791、6,982,321和7,087,409；Kashmiri等,Methods 36:25-34(2005)(描述SDR(a-CDR)移植)；Padlan,Mol.Immunol.28:489-498(1991)(描述“表面重建”)；Dall’Acqua等,Methods36:43-60(2005)(描述“FR改组”)；Osbourn等,Methods 36:61-68(2005)；及Klimka等,Br.J.Cancer,83:252-260(2000)(描述FR改组的“指导选择”法)中。Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “surfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” method for FR shuffling).

可以用于人源化的人构架区包括但不限于：用“最适”法选择的构架区(参见例如Sims等J.Immunol.151:2296(1993))；源自具体亚组的轻链或重链可变区的人抗体的共有序列的构架区(参见例如Carter等Proc.Natl.Acad.Sci.USA,89:4285(1992)；及Presta等J.Immunol.,151:2623(1993))；人成熟(体细胞突变)构架区或人种系构架区(参见例如Almagro和Fransson,Front.Biosci.13:1619-1633(2008))；和源自筛选FR文库的构架区(参见例如Baca等,J.Biol.Chem.272:10678-10684(1997)和Rosok等,J.Biol.Chem.271:22611-22618(1996))。Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "optimal" method (see, e.g., Sims et al. J. Immunol. 151: 2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al. J. Immunol., 151: 2623 (1993)). ); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

在某些实施方案中，本文提供的抗体是人抗体。可以用本领域已知的多种技术产生人抗体。人抗体一般地描述于van Dijk和van de Winkel,Curr.Opin.Pharmacol.5:368-74(2001)及Lonberg,Curr.Opin.Immunol.20:450-459(2008)中。In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

可以通过对转基因动物施用免疫原来制备人抗体，该转基因动物已修饰为响应抗原攻击而产生完整人抗体或具有人可变区的完整抗体。这类动物通常包含全部或部分人免疫球蛋白基因座，其取代内源免疫球蛋白基因座，或其存在于染色体外，或随机整合入动物的染色体。在这类转基因小鼠中，内源免疫球蛋白基因座通常已失活。用于从转基因动物获得人抗体的方法的综述参见Lonberg,Nat.Biotech.23:1117-1125(2005)。还参见例如描述XENOMOUSE^TM技术的美国专利号6,075,181和US 6,150,584；描述技术的美国专利号5,770,429；描述K-M技术的美国专利号7,041,870；描述技术的美国专利申请公开号2007/0061900。可以例如通过与不同的人恒定区组合来进一步修饰来自这类动物所产生的完整抗体的人可变区。Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or it is present outside the chromosome, or randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and US 6,150,584 describing XENOMOUSE^™ technology; U.S. Patent No. 5,770,429 describing technology; U.S. Patent No. 7,041,870 describing KM technology; U.S. Patent Application Publication No. 2007/0061900 describing technology. The human variable regions of the complete antibodies produced from such animals can be further modified, for example, by combining with different human constant regions.

还可以通过基于杂交瘤的方法来制备人抗体。已描述了用于产生人单克隆抗体的人骨髓瘤和小鼠-人杂骨髓瘤细胞系。(参见例如Kozbor J.Immunol.,133:3001(1984)；Brodeur等,Monoclonal Antibody Production Techniques and Applications,51-63页(Marcel Dekker,Inc.,New York,1987)；和Boerner等,J.Immunol.,147:86(1991))。通过人B细胞杂交瘤技术产生的人抗体也描述于Li等,Proc.Natl.Acad.Sci.USA,103:3557-3562(2006)中。其他方法包括描述于例如美国专利号7,189,826(描述从杂交瘤细胞系产生单克隆人IgM抗体)和Ni,Xiandai Mianyixue,26(4):265-268(2006)(描述人-人杂交瘤)中的那些。人杂交瘤技术(三元杂交瘤技术)还描述于Vollmers和Brandlein,Histology andHistopathology,20(3):927-937(2005)及Vollmers和Brandlein,Methods and Findingsin Experimental and Clinical Pharmacology,27(3):185-91(2005)中。Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include those described in, for example, U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (tri-hybridoma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

还可以通过分离选自人衍生噬菌体展示文库的Fv克隆可变结构域序列来产生人抗体。然后可以将这类可变结构域序列与希望的人恒定结构域组合。下文描述用于从抗体文库选择人抗体的技术。Human antibodies can also be produced by isolating the Fv clone variable domain sequence selected from a human-derived phage display library. This type of variable domain sequence can then be combined with the desired human constant domain. The following describes the technology for selecting human antibodies from an antibody library.

(v)抗体片段(v) Antibody fragments

抗体片段可以通过传统手段(如酶消化)或通过重组技术来产生。在某些情况下，使用抗体片段而不是全抗体有优势。片段较小的尺寸允许快速清除，且可以导致对实体瘤的接近性改善。某些抗体片段的综述参见Hudson等(2003)Nat.Med.9:129-134。Antibody fragments can be produced by traditional means (such as enzymatic digestion) or by recombinant techniques. In some cases, there are advantages to using antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and can result in improved accessibility to solid tumors. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9: 129-134.

已发展了多种技术来产生抗体片段。通常，通过完整抗体的蛋白水解消化来衍生这些片段(参见例如Morimoto等,Journal of Biochemical and Biophysical Methods24:107-117(1992)；及Brennan等,Science 229:81(1985))。但是，现在可以通过重组宿主细胞直接产生这些片段。Fab、Fv和ScFv抗体片段全都可以在大肠杆菌(E.coli)中表达并从大肠杆菌分泌，从而允许轻易产生大量这些片段。可以从上文讨论的抗体噬菌体文库分离抗体片段。备选地，可以从大肠杆菌直接回收Fab’-SH片段，并化学偶联形成F(ab’)₂片段(Carter等,Bio/Technology 10:163-167(1992))。根据另一种方法，可以从重组宿主细胞培养物直接分离F(ab’)₂片段。美国专利号5,869,046中描述了包含挽救受体结合表位残基的具有增加的体内半衰期的Fab和F(ab')₂片段。用于产生抗体片段的其他技术对熟练的从业者而言显而易见。在某些实施方案中，抗体是单链Fv片段(scFv)。参见例如WO 93/16185、美国专利号5,571,894和美国专利号5,587,458。Fv和scFv是缺乏恒定区的具有完整结合部位的仅有的种类；因此，它们可适合用于减少体内使用期间的非特异性结合。可以构建scFv融合蛋白质来在scFv的氨基端或羧基端产生效应蛋白质的融合。参见AntibodyEngineering,Borrebaeck编辑,上文。抗体片段还可以是例如描述于例如美国专利号5,641,870中的“线性抗体”。这类线性抗体可以是单特异性的或双特异性的。A variety of techniques have been developed to produce antibody fragments. Typically, these fragments are derived by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science 229: 81 (1985)). However, these fragments can now be directly produced by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in Escherichia coli (E. coli) and secreted from E. coli, thereby allowing large quantities of these fragments to be easily produced. Antibody fragments can be isolated from the antibody phage library discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')₂ fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another method, F(ab')₂ fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab')₂ fragments with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments will be apparent to skilled practitioners. In certain embodiments, the antibody is a single-chain Fv fragment (scFv). See, for example, WO 93/16185, U.S. Patent No. 5,571,894, and U.S. Patent No. 5,587,458. Fv and scFv are the only species with complete binding sites that lack a constant region; therefore, they may be suitable for reducing nonspecific binding during in vivo use. scFv fusion proteins can be constructed to produce fusions of effector proteins at the amino or carboxyl termini of scFv. See Antibody Engineering, edited by Borrebaeck, supra. Antibody fragments may also be "linear antibodies," such as those described in, for example, U.S. Patent No. 5,641,870. Such linear antibodies may be monospecific or bispecific.

(vi)多特异性抗体(vi) Multispecific antibodies

多特异性抗体对至少两个不同表位具有结合特异性，其中该表位通常来自不同抗原。虽然这类分子通常将仅结合两个不同表位(即双特异性抗体BsAb)，但在本文中使用时，具有附加特异性的抗体如三特异性抗体也为此表述所涵盖。双特异性抗体可以作为全长抗体或抗体片段(例如F(ab')₂双特异性抗体)制备。Multispecific antibodies have binding specificity for at least two different epitopes, wherein the epitopes are generally from different antigens. Although such molecules will generally only bind to two different epitopes (i.e., bispecific antibodies, BsAbs), when used herein, antibodies with additional specificities, such as trispecific antibodies, are also encompassed by this expression. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')₂ bispecific antibodies).

用于制备双特异性抗体的方法为本领域已知。全长双特异性抗体的传统产生基于两个免疫球蛋白重链-轻链对的共表达，其中两条链具有不同的特异性(参见例如Millstein等,Nature 305:537-539(1983))。由于免疫球蛋白重链和轻链的随机分配，这些杂交瘤(四价体杂交瘤(quadromas))产生10种不同抗体分子的可能的混合物，其中只有一种具有正确的双特异性结构。正确分子的纯化(通常通过亲和层析步骤进行)非常麻烦，且产物产率低。WO 93/08829和Traunecker等,EMBO J.,10:3655-3659(1991)中公开了类似的方法。Methods for preparing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (see, for example, Millstein et al., Nature 305:537-539 (1983)). Due to the random distribution of immunoglobulin heavy chains and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule (usually carried out by affinity chromatography steps) is very troublesome, and the product yield is low. Similar methods are disclosed in WO 93/08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

本领域已知的一种用于制备双特异性抗体的方法是“杵入臼”或“凸起进入凹陷”法(参见美国专利号5,731,168)。在此方法中，两条免疫球蛋白多肽(例如重链多肽)各包含界面。一条免疫球蛋白多肽的界面与另一条免疫球蛋白多肽上对应的界面相互作用，从而允许两条免疫球蛋白多肽结合。可以这样改造这些界面，使得定位在一条免疫球蛋白多肽的界面中的“杵”或“凸起”(这些术语在本文中可互换使用)对应于定位在另一条免疫球蛋白多肽的界面中的“臼”或“凹陷”(这些术语在本文中可互换使用)。在一些实施方案中，该臼与该杵具有相同或相似的大小，适宜地放置，使得在两个界面相互作用时，一个界面的杵可定位在另一个界面对应的臼中。不希望受限于理论，认为这稳定了异源多聚体，偏向形成异源多聚体而不是其他种类例如同源多聚体。在一些实施方案中，此方法可用于促进两条不同免疫球蛋白多肽的异源多聚化，产生对不同表位具有结合特异性的包含两条免疫球蛋白多肽的双特异性抗体。One method known in the art for preparing bispecific antibodies is the "knob-in-hole" or "protrusion-into-cavity" approach (see U.S. Patent No. 5,731,168). In this approach, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each comprise an interface. The interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to bind. These interfaces can be engineered so that a "knob" or "protrusion" (these terms are used interchangeably herein) positioned in the interface of one immunoglobulin polypeptide corresponds to a "hole" or "cavity" (these terms are used interchangeably herein) positioned in the interface of the other immunoglobulin polypeptide. In some embodiments, the hole and the knob are of the same or similar size and are appropriately positioned so that, upon interaction of the two interfaces, the knob of one interface can be positioned in the corresponding hole of the other interface. Without wishing to be bound by theory, it is believed that this stabilizes the heteromultimers, favoring the formation of heteromultimers over other species, such as homomultimers. In some embodiments, this method can be used to promote heteromultimerization of two different immunoglobulin polypeptides to generate a bispecific antibody comprising two immunoglobulin polypeptides that has binding specificity for different epitopes.

在一些实施方案中，杵可以通过用较大的侧链取代小的氨基酸侧链来构建。在一些实施方案中，臼可以通过用较小的侧链取代大的氨基酸侧链来构建。杵或臼可以存在于原始界面中，或者它们可以合成引入。例如，通过改变编码界面的核酸序列以用至少一个“输入”氨基酸残基取代至少一个“原始”氨基酸残基，可以合成引入杵或臼。用于改变核酸序列的方法可以包括本领域公知的标准分子生物学技术。下表中显示多种氨基酸残基的侧链体积。在一些实施方案中，原始残基具有小的侧链体积(例如丙氨酸、天冬酰胺、天冬氨酸、甘氨酸、丝氨酸、苏氨酸或缬氨酸)，用于形成杵的输入残基是天然存在的氨基酸，且可以包括精氨酸、苯丙氨酸、酪氨酸和色氨酸。在一些实施方案中，原始残基具有大的侧链体积(例如精氨酸、苯丙氨酸、酪氨酸和色氨酸)，用于形成臼的输入残基是天然存在的氨基酸，且可以包括丙氨酸、丝氨酸、苏氨酸和缬氨酸。In some embodiments, a knob can be constructed by replacing a small amino acid side chain with a larger side chain. In some embodiments, a hole can be constructed by replacing a large amino acid side chain with a smaller side chain. The knob or hole can be present in the original interface, or they can be introduced synthetically. For example, a knob or hole can be introduced synthetically by altering the nucleic acid sequence encoding the interface to replace at least one "original" amino acid residue with at least one "import" amino acid residue. Methods for altering nucleic acid sequences can include standard molecular biology techniques known in the art. The side chain volumes of various amino acid residues are shown in the table below. In some embodiments, the original residue has a small side chain volume (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and the import residue used to form the knob is a naturally occurring amino acid and can include arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the original residue has a large side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan), and the import residue used to form the hole is a naturally occurring amino acid and can include alanine, serine, threonine, and valine.

表1：氨基酸残基的特性Table 1: Characteristics of amino acid residues

^a氨基酸分子量减去水的分子量。来自Handbook of Chemistry and Physics,第43版Cleveland,Chemical Rubber Publishing Co.,1961的值。^a The molecular weight of amino acids minus the molecular weight of water. Values from Handbook of Chemistry and Physics, 43rd edition, Cleveland, Chemical Rubber Publishing Co., 1961.

^b来自A.A.Zamyatnin,Prog.Biophys.Mol.Biol.24:107-123,1972的值。^b Values from AA Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972.

^c来自C.Chothia,J.Mol.Biol.105:1-14,1975的值。可接近的表面积在此参考文献的图6-20中定义。^c Values from C. Chothia, J. Mol. Biol. 105: 1-14, 1975. Accessible surface area is defined in Figures 6-20 of this reference.

在一些实施方案中，根据异源多聚体的三维结构鉴定用于形成杵或臼的原始残基。本领域已知的用于获得三维结构的技术可以包括X射线晶体分析法和NMR。在一些实施方案中，该界面是免疫球蛋白恒定结构域的CH3结构域。在这些实施方案中，人IgG1的CH3/CH3界面涉及定位在四个反向平行β-链上的每个结构域上的16个残基。不希望受限于理论，突变的残基优选定位在两个中央反向平行β-链上，以最小化杵可被周围溶剂而不是配偶CH3结构域中的补偿臼容纳的风险。在一些实施方案中，形成两条免疫球蛋白多肽中对应的杵和臼的突变对应于下表中提供的一对或多对。In some embodiments, the original residues for forming the knob or hole are identified based on the three-dimensional structure of the heteromultimer. Techniques known in the art for obtaining three-dimensional structures may include X-ray crystallography and NMR. In some embodiments, the interface is the CH3 domain of an immunoglobulin constant domain. In these embodiments, the CH3/CH3 interface of human IgG1 involves 16 residues on each domain located on four antiparallel β-strands. Without wishing to be bound by theory, the mutated residues are preferably located on the two central antiparallel β-strands to minimize the risk that the knob will be accommodated by the surrounding solvent rather than the compensating hole in the partner CH3 domain. In some embodiments, the mutations that form the corresponding knob and hole in the two immunoglobulin polypeptides correspond to one or more pairs provided in the table below.

表2：形成对应的杵和臼的突变的示例性组Table 2: Exemplary groups of mutations that form corresponding knobs and mortars

第一免疫球蛋白的CH3CH3 of the first immunoglobulin第二免疫球蛋白的CH3CH3 of the second immunoglobulinT366YT366YY407TY407TT366WT366WY407AY407AF405AF405AT394WT394WY407TY407TT366YT366YT366Y:F405AT366Y:F405AT394W:Y407TT394W:Y407TT366W:F405WT366W:F405WT394S:Y407AT394S:Y407AF405W:Y407AF405W:Y407AT366W:T394ST366W:T394SF405WF405WT394ST394S

突变表示为：原始残基，后随使用Kabat编号系统的位置，然后是输入残基(所有残基都以单字母氨基酸密码给出)。多个突变通过冒号分开。Mutations are indicated as: the original residue, followed by the position using the Kabat numbering system, and then the input residue (all residues are given in the single-letter amino acid code). Multiple mutations are separated by colons.

在一些实施方案中，免疫球蛋白多肽包含含有以上表2中所列的一个或多个氨基酸取代的CH3结构域。在一些实施方案中，双特异性抗体包含含有以上表2的左列中所列的一个或多个氨基酸取代的CH3结构域的第一免疫球蛋白多肽，及含有表2的右列中所列的一个或多个对应的氨基酸取代的CH3结构域的第二免疫球蛋白多肽。In some embodiments, the immunoglobulin polypeptide comprises a CH3 domain comprising one or more amino acid substitutions listed above in Table 2. In some embodiments, the bispecific antibody comprises a first immunoglobulin polypeptide comprising a CH3 domain comprising one or more amino acid substitutions listed above in the left column of Table 2, and a second immunoglobulin polypeptide comprising a CH3 domain comprising one or more corresponding amino acid substitutions listed above in the right column of Table 2.

按上文所讨论突变DNA后，可以用本领域已知的标准重组技术和细胞系统表达和纯化编码具有一个或多个形成对应的杵或臼的突变的修饰免疫球蛋白多肽的多核苷酸。参见例如美国专利号5,731,168；5,807,706；5,821,333；7,642,228；7,695,936；8,216,805；美国专利号2013/0089553；及Spiess等,Nature Biotechnology 31:753-758,2013。修饰的免疫球蛋白多肽可以用原核宿主细胞如大肠杆菌或真核宿主细胞如CHO细胞产生。具有对应的杵和臼的免疫球蛋白多肽可以在共培养的宿主细胞中表达，并一起纯化为异源多聚体，或者它们可以在单种培养物中表达，分别纯化，并体外组装。在一些实施方案中，用本领域已知的标准细菌培养技术共培养两株细菌宿主细胞(一株表达具有杵的免疫球蛋白多肽，另一株表达具有臼的免疫球蛋白多肽)。在一些实施方案中，两个菌株可以按特定比值混合，例如以在培养物中达到等同的表达水平。在一些实施方案中，两个菌株可以按50:50、60:40或70:30的比值混合。多肽表达后，可以一起裂解细胞，并可以提取蛋白质。本领域已知的允许测量同源多聚体对异源多聚体种类的丰度的标准技术可以包括大小排阻层析。在一些实施方案中，用标准重组技术分别表达各修饰免疫球蛋白多肽，并可以在体外将它们组装在一起。可以例如通过以下达到组装：纯化各修饰免疫球蛋白多肽，按相等质量一起混合和孵育它们，还原二硫键(例如通过用二硫苏糖醇处理)，浓缩，并再氧化多肽。所形成的双特异性抗体可以用包括阳离子交换层析的标准技术纯化，并用包括大小排阻层析的标准技术测量。对于这些方法的更详细描述，参见Speiss等,Nat Biotechnol 31:753-8,2013。在一些实施方案中，修饰的免疫球蛋白多肽可以分别在CHO细胞中表达，并用上述方法体外组装。After mutating the DNA as discussed above, polynucleotides encoding modified immunoglobulin polypeptides having one or more mutations that form the corresponding knob or hole can be expressed and purified using standard recombinant techniques and cell systems known in the art. See, for example, U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; U.S. Patent Nos. 2013/0089553; and Spiess et al., Nature Biotechnology 31:753-758, 2013. Modified immunoglobulin polypeptides can be produced using prokaryotic host cells such as E. coli or eukaryotic host cells such as CHO cells. Immunoglobulin polypeptides having the corresponding knob and hole can be expressed in co-cultured host cells and purified together as heteromultimers, or they can be expressed in single cultures, purified separately, and assembled in vitro. In some embodiments, two bacterial host cells (one expressing an immunoglobulin polypeptide with a knob and the other expressing an immunoglobulin polypeptide with a hole) are co-cultured using standard bacterial culture techniques known in the art. In some embodiments, the two strains can be mixed in a specific ratio, for example, to achieve equivalent expression levels in culture. In some embodiments, the two strains can be mixed in a ratio of 50:50, 60:40, or 70:30. After polypeptide expression, the cells can be lysed together and the protein can be extracted. Standard techniques known in the art that allow for measurement of the abundance of homo- versus hetero-multimeric species can include size exclusion chromatography. In some embodiments, each modified immunoglobulin polypeptide is expressed separately using standard recombinant techniques and can be assembled together in vitro. Assembly can be achieved, for example, by purifying each modified immunoglobulin polypeptide, mixing and incubating them together in equal masses, reducing disulfide bonds (e.g., by treatment with dithiothreitol), concentrating, and reoxidizing the polypeptides. The resulting bispecific antibodies can be purified using standard techniques including cation exchange chromatography and measured using standard techniques including size exclusion chromatography. For a more detailed description of these methods, see Speiss et al., Nat Biotechnol 31:753-8, 2013. In some embodiments, the modified immunoglobulin polypeptides can be expressed separately in CHO cells and assembled in vitro using the methods described above.

根据不同的方法，将具有希望得到的结合特异性(抗体-抗原结合部位)的抗体可变结构域与免疫球蛋白恒定结构域序列融合。该融合优选是与包含至少部分铰合部、CH2和CH3区的免疫球蛋白重链恒定结构域融合。通常使包含轻链结合所必需的部位的第一重链恒定区(CH1)存在于至少融合之一中。将编码免疫球蛋白重链融合和(如果希望)免疫球蛋白轻链的DNA插入分开的表达载体，并共转染入适宜的宿主生物。在将不等比值的三种多肽链用于构建可提供最佳产率的实施方案中，这提供了调整三种多肽片段的相互比例的极大灵活性。但是，在等比值表达至少两种多肽链产生高产率时或该比值没有具体意义时，可能将两种或全部三种多肽链的编码序列插入一个表达载体中。According to different methods, the antibody variable domain with the desired binding specificity (antibody-antigen binding site) is fused to the immunoglobulin constant domain sequence. The fusion is preferably fused to the immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Usually, the first heavy chain constant region (CH1) comprising the site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where three polypeptide chains of unequal ratios are used to construct an optimal yield, this provides great flexibility in adjusting the mutual ratio of the three polypeptide fragments. However, when expressing at least two polypeptide chains in equal ratios produces a high yield or when the ratio has no specific meaning, the coding sequences of two or all three polypeptide chains may be inserted into one expression vector.

在此方法的一个实施方案中，该双特异性抗体由一条臂中具有第一结合特异性的杂合免疫球蛋白重链和另一条臂中的杂合免疫球蛋白重链-轻链对(提供第二结合特异性)组成。发现此不对称结构便于从不想要的免疫球蛋白链组合分离希望得到的双特异性化合物，因为免疫球蛋白轻链仅存在于该双特异性分子的一半中提供了容易的分离方式。此方法公开于WO94/04690中。产生双特异性抗体的进一步详情参见例如Suresh等,Methods inEnzymology 121:210(1986)。In one embodiment of this method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations because the immunoglobulin light chain is only present in one half of the bispecific molecule, providing an easy separation method. This method is disclosed in WO94/04690. For further details of producing bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

根据WO96/27011中所述的另一种方法，可以改造一对抗体分子间的界面来最大化从重组细胞培养物回收的异二聚体的百分比。一个界面包含抗体恒定结构域的至少一部分C_H3结构域。在此方法中，用较大的侧链(例如酪氨酸或色氨酸)取代来自第一抗体分子的界面的一个或多个小的氨基酸侧链。通过用较小的氨基酸侧链(例如丙氨酸或苏氨酸)取代大的氨基酸侧链来在第二抗体分子的界面上产生大小与该一个或多个大的侧链相同或相似的补偿“凹陷”。这提供了增加异二聚体的产率超过其他不想要的终产物(如同二聚体)的机制。According to another method described in WO96/27011, the interface between a pair of antibody molecules can be transformed to maximize the percentage of heterodimers recovered from recombinant cell culture. An interface comprises at least a portion of the_CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "depressions" of a size identical or similar to the one or more large side chains are produced on the interface of the second antibody molecule by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers and exceeding other unwanted end products (e.g., homodimers).

双特异性抗体包括交联抗体或“异缀合物”抗体。例如，异缀合物中的抗体之一可以与抗生物素蛋白偶联，另一种与生物素偶联。例如，已提出这类抗体将免疫系统细胞靶向至不需要的细胞(美国专利号4,676,980)，并用于治疗HIV感染(WO 91/00360、WO 92/200373和EP 03089)。可以用任意方便的交联方法产生异缀合物抗体。适宜的交联剂为本领域公知，并连同许多交联技术一起公开于美国专利号4,676,980中。Bispecific antibodies include cross-linked antibodies or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. For example, it has been proposed that such antibodies target immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and are used to treat HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be produced using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, along with many cross-linking techniques.

文献中还描述了用于从抗体片段产生双特异性抗体的技术。例如，可以用化学连接制备双特异性抗体。Brennan等,Science 229:81(1985)描述了其中用蛋白酶解切割完整抗体来产生F(ab’)₂片段的方法。在二巯基络合剂亚砷酸钠的存在下还原这些片段，以稳定邻位二巯基并阻止分子间二硫键形成。然后将产生的Fab’片段转化为硫代硝基苯甲酸盐(TNB)衍生物。然后通过用巯基乙胺还原来将Fab’-TNB衍生物之一再转化为Fab’-巯基，并与等摩尔量另一Fab’-TNB衍生物混合形成双特异性抗体。可以将产生的双特异性抗体用作用于选择性固定酶的试剂。The technology for producing bispecific antibodies from antibody fragments is also described in the literature. For example, bispecific antibodies can be prepared by chemical linkage. Brennan et al., Science 229:81 (1985) described a method in which a complete antibody is cleaved by proteolysis to produce F(ab')₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the adjacent dithiol groups and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then converted into a Fab'-thiol group by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as a reagent for selective enzyme immobilization.

最近的进展便于从大肠杆菌直接回收Fab'-SH片段，该Fab'-SH片段可以化学偶联形成双特异性抗体。Shalaby等,J.Exp.Med.,175:217-225(1992)描述了全人源化双特异性抗体F(ab')₂分子的产生。各Fab'片段分别从大肠杆菌分泌，并在体外进行定向化学偶联来形成双特异性抗体。Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) described the generation of fully humanized bispecific antibody F(ab')_molecules . Separate Fab' fragments were secreted from E. coli and subjected to directed chemical coupling in vitro to form bispecific antibodies.

还已描述了用于直接从重组细胞培养物制备和分离双特异性抗体片段的多种技术。例如，已用亮氨酸拉链产生了双特异性抗体。Kostelny等,J.Immunol.148(5):1547-1553(1992)。通过基因融合将来自Fos和Jun蛋白质的亮氨酸拉链肽与两种不同抗体的Fab’部分连接。在铰链区还原抗体同二聚体形成单体，然后再氧化形成抗体异二聚体。还可以利用此方法来产生抗体同二聚体。Hollinger等,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)所述的“双抗体”技术提供了用于产生双特异性抗体片段的备选机制。片段包含通过接头与轻链可变结构域(V_L)连接的重链可变结构域(V_H)，该接头太短而不允许同一条链上的两个结构域间配对。因此，迫使一个片段的V_H和V_L结构域与另一片段的互补V_L和V_H结构域配对，从而形成两个抗原结合部位。还已报道了通过使用单链Fv(sFv)二聚体来制备双特异性抗体片段的另一策略。参见Gruber等,J.Immunol.152:5368(1994)。A variety of techniques have also been described for preparing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers, which are then oxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for producing bispecific antibody fragments. The fragments comprise a heavy chain variable domain (_VH ) connected to a light chain variable domain (_VL ) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the_VH and_VL domains of one fragment are forced to pair with the complementary_VL and_VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

另一种用于制备双特异性抗体片段的技术是“双特异性T细胞衔接物”或法(参见例如WO2004/106381、WO2005/061547、WO2007/042261和WO2008/119567)。此方法利用排列在单条多肽上的两个抗体可变结构域。例如，单条多肽链包含两个单链Fv(scFv)片段，每个片段具有通过多肽接头分开的可变重链(V_H)和可变轻链(V_L)，该多肽接头长度足以允许两个结构域间的分子内结合。此单条多肽进一步包含两个scFv片段间的多肽间隔序列。每个scFv识别不同表位，这些表位可以为不同细胞类型所特有，使得在各scFv与其关联表位衔接时使两种不同细胞类型的细胞紧密靠近或系在一起。此方法的一个具体实施方案包括识别由免疫细胞表达的细胞表面抗原(例如T细胞上的CD3多肽)的scFv，其与识别由靶细胞如恶性或肿瘤细胞表达的细胞表面抗原的另一scFv连接。Another technique for preparing bispecific antibody fragments is the "bispecific T cell engager" or approach (see, e.g., WO2004/106381, WO2005/061547, WO2007/042261, and WO2008/119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, the single polypeptide chain comprises two single-chain Fv (scFv) fragments, each having a variable heavy chain (_VH ) and a variable light chain (_VL ) separated by a polypeptide linker of sufficient length to allow intramolecular binding between the two domains. The single polypeptide further comprises a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, which may be specific for different cell types, such that upon engagement of each scFv with its cognate epitope, cells of two different cell types are brought into close proximity or tethered together. One embodiment of this method comprises a scFv that recognizes a cell surface antigen expressed by an immune cell (eg, CD3 polypeptide on a T cell) linked to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell.

由于它是单条多肽，双特异性T细胞衔接物可以用本领域已知的任意原核或真核细胞表达系统(例如CHO细胞系)表达。但是，可以需要具体纯化技术(参见例如EP1691833)来将单体双特异性T细胞衔接物从其他多聚体种类分开，该其他多聚体种类可以具有单体的预期活性之外的生物学活性。在一个示例性纯化方案中，包含分泌性多肽的溶液首先进行金属亲和层析，并用咪唑浓度梯度洗脱多肽。此洗脱物进一步用阴离子交换层析纯化，并用氯化钠浓度梯度洗脱多肽。最后，对此洗脱物进行大小排阻层析，以将单体从多聚体种类分开。Since it is a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic cell expression system known in the art (e.g., CHO cell line). However, specific purification techniques (see, e.g., EP1691833) may be required to separate the monomeric bispecific T cell engager from other multimeric species, which may have biological activities other than the expected activity of the monomer. In an exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted using an imidazole concentration gradient. This eluate is further purified using anion exchange chromatography, and the polypeptide is eluted using a sodium chloride concentration gradient. Finally, this eluate is subjected to size exclusion chromatography to separate the monomeric from the multimeric species.

考虑具有两价以上的抗体。例如，可以制备三特异性抗体。Tuft等.J.Immunol.147:60(1991)Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147:60 (1991)

(vii)单结构域抗体(vii) Single domain antibodies

在一些实施方案中，本发明的抗体是单结构域抗体。单结构域抗体是包含抗体的全部或部分重链可变结构域或全部或部分轻链可变结构域的单条多肽链。在某些实施方案中，单结构域抗体是人单结构域抗体(Domantis,Inc.,Waltham,MA；参见例如美国专利号6,248,516B1)。在一个实施方案中，单结构域抗体由抗体重链可变结构域的全部或部分组成。In some embodiments, the antibodies of the present invention are single-domain antibodies. A single-domain antibody is a single polypeptide chain comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516B1). In one embodiment, a single-domain antibody consists of all or part of the heavy chain variable domain of an antibody.

(viii)抗体变体(viii) Antibody variants

在一些实施方案中，考虑本文所述抗体的氨基酸序列修饰。例如，可以希望改善抗体的结合亲和力和/或其他生物学特性。可以通过在编码抗体的核苷酸序列中引入适当的改变或通过肽合成来制备抗体的氨基酸序列变体。这类修饰包括例如从抗体的氨基酸序列缺失残基和/或在抗体的氨基酸序列内插入残基和/或取代抗体的氨基酸序列内的残基。可以进行缺失、插入和取代的任意组合来达到最终构建体，只要最终构建体具有希望得到的特征。可以在制备该序列时在主题抗体氨基酸序列中引入氨基酸序列改变。In some embodiments, it is contemplated that the amino acid sequence of antibodies described herein is modified. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate changes in the nucleotide sequence encoding the antibody or by peptide synthesis. This type of modification includes, for example, deletions of residues from the amino acid sequence of the antibody and/or insertions of residues and/or substitutions of residues in the amino acid sequence of the antibody within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution may be performed to reach the final construct, as long as the final construct has the desired characteristics. Amino acid sequence changes may be introduced in the subject antibody amino acid sequence when preparing the sequence.

(ix)取代、插入和缺失变体(ix) Substitution, insertion and deletion variants

在某些实施方案中，提供具有一个或多个氨基酸取代的抗体变体。进行取代诱变的目的位点包括HVR和FR。表1中在“保守取代”的表头下显示保守取代。表1中在“示例性取代”的表头下显示更实质性的改变，如下文参考氨基酸侧链种类进一步描述。可以在目的抗体中引入氨基酸取代，并针对希望得到的活性(例如保留/改善抗原结合、降低免疫原性或改善ADCC或CDC)筛选产物。In certain embodiments, antibody variants having one or more amino acid substitutions are provided. The target sites for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown under the heading "Conservative Substitutions" in Table 1. More substantial changes are shown under the heading "Exemplary Substitutions" in Table 1, as further described below with reference to amino acid side chain types. Amino acid substitutions can be introduced into the target antibody and the product can be screened for the desired activity (e.g., retaining/improving antigen binding, reducing immunogenicity, or improving ADCC or CDC).

表3：示例性取代Table 3: Exemplary Substitutions

原残基original residue示例性取代Exemplary Substitutions优选的取代Preferred substitutionsAla(A)Ala(A)Val；Leu；IleVal; Leu; IleValValArg(R)Arg(R)Lys；Gln；AsnLys; Gln; AsnLysLysAsn(N)Asn(N)Gln；His；Asp,Lys；ArgGln; His; Asp, Lys; ArgGlnGlnAsp(D)Asp(D)Glu；AsnGlu; AsnGluGluCys(C)Cys(C)Ser；AlaSer; AlaSerSerGln(Q)Gln(Q)Asn；GluAsn；GluAsnAsnGlu(E)Glu(E)Asp；GlnAsp; GlnAspAspGly(G)Gly(G)AlaAlaAlaAlaHis(H)His(H)Asn；Gln；Lys；ArgAsn; Gln; Lys; ArgArgArgIle(I)Ile(I)Leu；Val；Met；Ala；Phe；正亮氨酸Leu; Val; Met; Ala; Phe; norleucineLeuLeuLeu(L)Leu(L)正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; PheIleIleLys(K)Lys(K)Arg；Gln；AsnArg; Gln; AsnArgArgMet(M)Met(M)Leu；Phe；IleLeu; Phe; IleLeuLeuPhe(F)Phe(F)Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; TyrTyrTyrPro(P)Pro(P)AlaAlaAlaAlaSer(S)Ser(S)ThrThrThrThrThr(T)Thr(T)Val；SerVal; SerSerSerTrp(W)Trp(W)Tyr；PheTyr; PheTyrTyrTyr(Y)Tyr(Y)Trp；Phe；Thr；SerTrp; Phe; Thr; SerPhePheVal(V)Val(V)Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; norleucineLeuLeu

氨基酸可以按照共同的侧链性质分组：Amino acids can be grouped according to common side chain properties:

a.疏水性：正亮氨酸、Met、Ala、Val、Leu、Ile；a. Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

b.中性亲水性：Cys、Ser、Thr、Asn、Gln；b. Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

c.酸性：Asp、Glu；c. Acidic: Asp, Glu;

d.碱性：His、Lys、Arg；d. Basic: His, Lys, Arg;

e.影响链取向的残基：Gly、Pro；e. Residues that affect chain orientation: Gly, Pro;

f.芳香族：Trp、Tyr、Phe。f. Aromatic: Trp, Tyr, Phe.

非保守取代将需要将这些种类之一的成员换为另一种类。Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

一类取代变体涉及取代亲本抗体(例如人源化抗体或人抗体)的一个或多个高变区残基。通常，所得到的选择用于进一步研究的一种或多种变体将相对于亲本抗体具有某些生物学特性(例如提高的亲和力、降低的免疫原性)的修饰(例如改善)，和/或将具有基本上保留的亲本抗体的某些生物学特性。示例性取代变体是亲和力成熟的抗体，其可以例如用基于噬菌体展示的亲和力成熟技术(如本文所述的那些)方便地产生。简言之，突变一个或多个HVR残基，将变体抗体展示在噬菌体上，并针对具体生物学活性(例如结合亲和力)进行筛选。A class of substitution variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variants selected for further study will have modifications (e.g., improvements) of certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will have certain biological properties of the parent antibody that are substantially retained. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently produced, for example, using affinity maturation techniques based on phage display (such as those described herein). In short, one or more HVR residues are mutated, the variant antibodies are displayed on phage, and screened for specific biological activity (e.g., binding affinity).

可以在HVR中进行改变(例如取代)，例如以改善抗体亲和力。可以在HVR“热点”(即由在体细胞成熟过程中以高频率发生突变的密码子编码的残基)(参见例如Chowdhury,Methods Mol.Biol.207:179-196(2008))和/或SDR(a-CDR)中进行这类改变，针对结合亲和力测试所得到的变体VH或VL。通过构建二级文库并从二级文库重新选择的亲和力成熟已描述于例如Hoogenboom等in Methods in Molecular Biology 178:1-37(O’Brien等,编辑,Human出版社,Totowa,NJ,(2001))中。在亲和力成熟的一些实施方案中，通过多种方法(例如易错PCR、链改组或寡核苷酸定点诱变)中的任一种来在选择用于成熟的可变基因中引入多样性。然后产生二级文库。然后筛选该文库来鉴定具有希望得到的亲和力的任意抗体变体。引入多样性的另一种方法涉及HVR定点途径，其中随机化几个HVR残基(例如每次4-6个残基)。可以例如用丙氨酸扫描诱变或模拟来明确地鉴定出涉及抗原结合的HVR残基。尤其是通常靶向CDR-H3和CDR-L3。Changes (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots" (i.e., residues encoded by codons that mutate at high frequency during somatic maturation) (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and/or SDRs (a-CDRs), and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by constructing a secondary library and reselecting from the secondary library has been described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves a site-directed approach to HVRs, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly commonly targeted.

在某些实施方案中，取代、插入或缺失可以发生在一个或多个HVR内，只要这类改变不实质性降低抗体结合抗原的能力。例如，可以在HVR中进行不实质性降低结合亲和力的保守改变(例如本文提供的保守取代)。这类改变可以在HVR“热点”或SDR之外。在上文提供的变体VH和VL序列的某些实施方案中，各HVR未改变，或包含不超过一个、两个或三个氨基酸取代。In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigens. For example, conservative changes (such as conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may be outside HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or comprises no more than one, two or three amino acid substitutions.

如Cunningham和Wells(1989)Science,244:1081-1085所述，用于鉴定可以靶向进行诱变的抗体残基或区域的方法称为“丙氨酸扫描诱变”。在此方法中，鉴定残基或靶残基组(例如带电荷残基，如Arg、Asp、His、Lys和Glu)，并用中性或带负电荷氨基酸(例如丙氨酸或多聚丙氨酸)取代，以确定抗体与抗原的相互作用是否受到影响。可以在证明对最初取代的功能敏感性的氨基酸位置引入其他取代。备选地或此外，用抗原-抗体复合物的晶体结构来鉴定抗体和抗原之间的接触点。这类接触残基和邻近残基可以作为取代的候选残基来靶向或消除。可以筛选变体来确定它们是否包含希望得到的特性。As described in Cunningham and Wells (1989) Science, 244:1081-1085, a method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis". In this method, residues or target residue groups (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively or in addition, the contact points between the antibody and the antigen can be identified using the crystal structure of the antigen-antibody complex. This type of contact residues and adjacent residues can be targeted or eliminated as candidate residues for substitution. Variants can be screened to determine whether they contain the desired characteristic.

氨基酸序列插入包括长度在一个残基至含有一百或更多个残基的多肽的范围内的氨基端和/或羧基端融合，以及单个或多个氨基酸残基的序列内插入。末端插入的实例包括具有N端蛋氨酰残基的抗体。抗体分子的其他插入变体包括抗体的N或C端与酶(例如，对于ADEPT)或增加抗体血清半衰期的多肽融合。Amino acid sequence insertions include amino-terminal and/or carboxyl-terminal fusions ranging in length from one residue to a polypeptide containing one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

(x)糖基化变体(x) Glycosylation variants

在某些实施方案中，改变本文提供的抗体以提高或降低该抗体的糖基化程度。通过改变氨基酸序列，使得产生或去除一个或多个糖基化位点，可以方便地实现抗体糖基化位点的加入或缺失。In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. By altering the amino acid sequence to create or remove one or more glycosylation sites, the addition or deletion of antibody glycosylation sites can be conveniently achieved.

在抗体包含Fc区时，可以改变附着于其上的糖类。哺乳动物细胞产生的天然抗体通常包含分枝的双触角寡糖，其一般通过N连接附着于Fc区CH2结构域的Asn297。参见例如Wright等TIBTECH 15:26-32(1997)。寡糖可以包括多种糖类，例如甘露糖、N-乙酰葡糖胺(GlcNAc)、半乳糖和唾液酸，以及在双触角寡糖结构的“茎”中附着于GlcNAc的岩藻糖。在一些实施方案中，可以进行本发明的抗体中的寡糖的修饰来产生具有某些改善的特性的抗体变体。When antibody comprises Fc district, can change the carbohydrate that is attached thereto.Natural antibody that mammalian cell produces usually comprises the biantennary oligosaccharide of branching, and it is generally connected to the Asn297 of Fc district CH2 domain by N.Referring to TIBTECH 15:26-32 (1997) such as Wright for example.Oligosaccharide can comprise multiple carbohydrate, for example mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose that is attached to GlcNAc in the " stem " of biantennary oligosaccharide structure.In some embodiments, can carry out the modification of the oligosaccharide in the antibody of the present invention to produce the antibody variant with some improved characteristic.

在一个实施方案中，提供包含Fc区的抗体变体，其中附着于Fc区的糖类结构具有减少的岩藻糖或缺乏岩藻糖，这可以改善ADCC功能。特别是，本文考虑相对于在野生型CHO细胞中产生的同一抗体上岩藻糖的量具有减少的岩藻糖的抗体。这就是说，它们的特征在于，具有比通过天然CHO细胞(例如，产生天然糖基化模式的CHO细胞，如包含天然FUT8基因的CHO细胞)产生时它们本该具有的岩藻糖量低的岩藻糖量。在某些实施方案中，该抗体是这样的抗体，其中其上N连接聚糖中的不到约50％、40％、30％、20％、10％或5％包含岩藻糖。例如，这种抗体中岩藻糖的量可以是1％至80％、1％至65％、5％至65％或20％至40％。在某些实施方案中，该抗体是这样的抗体，其中其上N连接聚糖中没有一种包含岩藻糖，即其中该抗体完全不含岩藻糖、或无岩藻糖或是无岩藻糖基化的。相对于通过例如WO 2008/077546中所述的MALDI-TOF质谱法测量的附着于Asn 297的所有糖结构(例如复合、杂合和高甘露糖结构)的总和，通过计算Asn297处的糖链内岩藻糖的平均量来测定岩藻糖的量。Asn297指定位在Fc区中的约297位(Fc区残基的EU编号)的天冬酰胺残基；但是，由于抗体中小的序列变异，Asn297也可以定位在297位上游或下游约±3个氨基酸，即在294位和300位之间。这类岩藻糖基化变体可以具有改善的ADCC功能。参见例如美国专利公开号US 2003/0157108(Presta,L.)；US2004/0093621(Kyowa Hakko Kogyo Co.,Ltd)。涉及“去岩藻糖基化”或“岩藻糖缺乏”的抗体变体的出版物的实例包括：US 2003/0157108；WO 2000/61739；WO 2001/29246；US 2003/0115614；US 2002/0164328；US 2004/0093621；US 2004/0132140；US 2004/0110704；US 2004/0110282；US 2004/0109865；WO 2003/085119；WO2003/084570；WO 2005/035586；WO 2005/035778；WO2005/053742；WO2002/031140；Okazaki等J.Mol.Biol.336:1239-1249(2004)；Yamane-Ohnuki等Biotech.Bioeng.87:614(2004)。能够产生去岩藻糖基化的抗体的细胞系的实例包括蛋白质岩藻糖基化缺陷的Lec13CHO细胞(Ripka等Arch.Biochem.Biophys.249:533-545(1986)；美国专利申请号US 2003/0157108 A1；及WO 2004/056312 A1，尤其是在实施例11处)和敲除细胞系，如α-1,6-岩藻糖基转移酶基因FUT8敲除的CHO细胞(参见例如Yamane-Ohnuki等Biotech.Bioeng.87:614(2004)；Kanda,Y.等,Biotechnol.Bioeng.,94(4):680-688(2006)；及WO2003/085107)。In one embodiment, an antibody variant comprising an Fc region is provided, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which can improve ADCC function. In particular, antibodies with reduced fucose relative to the amount of fucose on the same antibody produced in wild-type CHO cells are contemplated herein. That is, they are characterized in that they have a lower amount of fucose than the amount of fucose they would have if produced by natural CHO cells (e.g., CHO cells that produce a natural glycosylation pattern, such as CHO cells containing a natural FUT8 gene). In certain embodiments, the antibody is an antibody in which less than about 50%, 40%, 30%, 20%, 10% or 5% of the N-linked glycans thereon comprise fucose. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. In certain embodiments, the antibody is an antibody in which none of the N-linked glycans thereon comprises fucose, i.e., the antibody is completely fucose-free, afucose-free, or afucosylated. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry as described in, for example, WO 2008/077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region; however, due to small sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, eg, US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003/0157108 A1; and WO 2004/056312 A1, particularly at Example 11) and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003/085107).

还提供具有二等分寡糖的抗体变体，例如，其中附着于抗体Fc区的双触角寡糖被GlcNAc二等分。这类抗体变体可以具有减少的岩藻糖基化和/或改善的ADCC功能。这类抗体变体的实例描述于例如WO 2003/011878；美国专利号6,602,684；US 2005/0123546；及Ferrara等,Biotechnology and Bioengineering,93(5):851-861(2006)中。还提供在附着于Fc区的寡糖中具有至少一个半乳糖残基的抗体变体。这类抗体变体可以具有改善的CDC功能。这类抗体变体描述于例如WO 1997/30087；WO 1998/58964；及WO 1999/22764中。Also provided are antibody variants having bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; U.S. Patent No. 6,602,684; US 2005/0123546; and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

在某些实施方案中，包含本文所述Fc区的抗体变体能够结合FcγRIII。在某些实施方案中，与包含人野生型IgG1Fc区的同一抗体相比，包含本文所述Fc区的抗体变体在人效应细胞存在下具有ADCC活性，或在人效应细胞存在下具有提高的ADCC活性。In certain embodiments, the antibody variants comprising the Fc region described herein are capable of binding to FcγRIII. In certain embodiments, the antibody variants comprising the Fc region described herein have ADCC activity in the presence of human effector cells, or have increased ADCC activity in the presence of human effector cells, compared to the same antibody comprising a human wild-type IgG1 Fc region.

(xi)Fc区变体(xi) Fc region variants

在某些实施方案中，可以在本文提供的抗体的Fc区中引入一个或多个氨基酸修饰，从而产生Fc区变体。Fc区变体可以包含在一个或多个氨基酸位置上含有氨基酸修饰(例如取代)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

在某些实施方案中，本发明考虑具有一些但不是全部效应子功能的抗体变体，这使得它成为其中抗体的体内半衰期很重要但某些效应子功能(如补体和ADCC)不必要或有害的应用所希望的候选者。可以进行体外和/或体内细胞毒性测定来确认CDC和/或ADCC活性的降低/减损。例如，可以进行Fc受体(FcR)结合测定来确保抗体缺乏FcγR结合(因此可能缺乏ADCC活性)，但保留FcRn结合能力。介导ADCC的主要细胞NK细胞仅表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。造血细胞上的FcR表达总结在Ravetch和Kinet,Annu.Rev.Immunol.9:457-492(1991)的第464页上的表3中。评估目的分子的ADCC活性的体外测定的非限制性实例描述于美国专利号5,500,362(参见例如Hellstrom,I.等Proc.Nat’l Acad.Sci.USA 83:7059-7063(1986))和Hellstrom,I等,Proc.Nat’l Acad.Sci.USA 82:1499-1502(1985)；5,821,337(参见Bruggemann等,J.Exp.Med.166:1351-1361(1987))中。备选地，可以利用非放射性测定方法(参见例如用于流式细胞术的ACTI^TM非放射性细胞毒性测定(CellTechnology,Inc.Mountain View,CA)；和CytoTox非放射性细胞毒性测定(Promega,Madison,WI))。用于这类测定的效应细胞包括外周血单核细胞(PBMC)和天然杀伤(NK)细胞。备选地或此外，可以在体内，例如在诸如公开于Clynes等Proc.Nat’lAcad.Sci.USA 95:652-656(1998)中的动物模型的动物模型中评估目的分子的ADCC活性。还可以进行C1q结合测定来确认抗体不能结合C1q，并因此缺乏CDC活性。参见例如WO 2006/029879和WO 2005/100402中的C1q和C3c结合ELISA。为了评估补体活化，可以进行CDC测定(参见例如Gazzano-Santoro等,J.Immunol.Methods202:163(1996)；Cragg等,Blood 101:1045-1052(2003)；及Cragg等,Blood103:2738-2743(2004))。还可以用本领域已知的方法进行FcRn结合和体内清除/半衰期测定(参见例如Petkova等,Int’l.Immunol.18(12):1759-1769(2006))。In certain embodiments, the present invention contemplates antibody variants having some but not all effector functions, which makes it a desirable candidate for applications in which the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/impairment of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, e.g., Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be utilized (see, e.g., ACTI^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg et al., Blood 101: 1045-1052 (2003); and Cragg et al., Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

具有减少的效应子功能的抗体包括具有Fc区残基238、265、269、270、297、327和329中的一个或多个的取代的那些(美国专利号6,737,056)。这类Fc突变体包括在氨基酸位置265、269、270、297和327中的两个或多个处具有取代的Fc突变体，包括具有残基265和297至丙氨酸的取代的所谓“DANA”Fc突变体(美国专利号7,332,581)。Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine ( U.S. Patent No. 7,332,581 ).

描述了某些具有改善或减少的FcR结合的抗体变体(参见例如美国专利号6,737,056；WO 2004/056312；及Shields等,J.Biol.Chem.9(2):6591-6604(2001))。Certain antibody variants with improved or decreased FcR binding have been described (see, eg, US Pat. No. 6,737,056; WO 2004/056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

在某些实施方案中，抗体变体包含具有一个或多个改善ADCC的氨基酸取代(例如Fc区298、333和/或334位(残基的EU编号)的取代)的Fc区。在示例性实施方案中，该抗体在其Fc区中包含以下氨基酸取代：S298A、E333A和K334A。In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues). In an exemplary embodiment, the antibody comprises the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.

在一些实施方案中，例如，如美国专利号6,194,551、WO 99/51642及Idusogie等J.Immunol.164:4178-4184(2000)中所述，在Fc区中进行改变，该改变导致改变(即改善或减少)的C1q结合和/或依赖补体的细胞毒性(CDC)。In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or reduced) CIq binding and/or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

具有增加的半衰期和改善的新生儿Fc受体(FcRn)结合的抗体描述于US2005/0014934A1(Hinton等)中，新生儿Fc受体负责将母体IgG转移至胎儿(Guyer等,J.Immunol.117:587(1976)和Kim等,J.Immunol.24:249(1994))。那些抗体包含其中具有一个或多个取代的Fc区，该取代改善Fc区与FcRn的结合。这类Fc变体包括在Fc区残基238、256、265、272、286、303、305、307、311、312、317、340、356、360、362、376、378、380、382、413、424或434中的一个或多个处具有取代的那些，例如，Fc区残基434的取代(美国专利号7,371,826)。关于Fc区变体的其他实例，还参见Duncan&Winter,Nature 322:738-40(1988)；美国专利号5,648,260；美国专利号5,624,821；及WO 94/29351。Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US 2005/0014934A1 (Hinton et al.), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitution of Fc region residue 434 ( U.S. Patent No. 7,371,826 ). See also Duncan & Winter, Nature 322:738-40 (1988) for other examples of Fc region variants; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351.

(xii)抗体衍生物(xii) Antibody derivatives

可以进一步修饰本发明的抗体，以包含本领域已知且易于得到的附加非蛋白质部分。在某些实施方案中，适合用于衍生化抗体的部分是水溶性聚合物。水溶性聚合物的非限制性实例包括但不限于聚乙二醇(PEG)、乙二醇/丙二醇的共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯比咯烷酮、聚-1,3-二氧戊环、聚-1,3,6-三噁烷、乙烯/马来酸酐共聚物、聚氨基酸(同聚物或随机共聚物)、葡聚糖或聚(n-乙烯吡咯烷酮)聚乙二醇、propropyleneglycol同聚物、prolypropylene oxide/环氧乙烷共聚物、聚氧乙烯化多元醇(例如甘油)、聚乙烯醇及其混合物。由于其在水中的稳定性，聚乙二醇丙醛可以在制备中具有优势。聚合物可以具有任意分子量，且可以分枝或不分枝。附着于抗体的聚合物的数目可以不同，且如果附着超过一个聚合物，则它们可以是相同或不同的分子。一般而言，可以根据包括但不限于抗体要改善的具体特性或功能、抗体衍生物是否将在确定的条件下用于治疗等的考虑来确定用于衍生化的聚合物的数目和/或类型。The antibodies of the present invention can be further modified to include additional non-proteinaceous moieties known in the art and readily available. In certain embodiments, the part suitable for derivatizing the antibody is a water-soluble polymer. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde can have advantages in preparation. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can be different, and if more than one polymer is attached, they can be identical or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under a determined condition, and the like.

(xiii)载体、宿主细胞和重组方法(xiii) Vectors, host cells, and recombination methods

抗体也可以用重组方法产生。为了重组产生抗抗原的抗体，分离编码抗体的核酸，并插入复制载体进行进一步克隆(DNA的扩增)或进行表达。编码抗体的DNA可以用常规方法容易地分离(例如，通过使用能够特异性结合编码抗体重链和轻链的基因的寡核苷酸探针)。许多载体可用。载体成分通常包括但不限于以下一种或多种：信号序列、复制起点、一个或多个标记基因、增强子元件、启动子和转录终止序列。Antibodies can also be produced by recombinant methods. In order to recombinantly produce anti-antigen antibodies, the nucleic acid encoding the antibody is separated and inserted into a replication vector for further cloning (amplification of the DNA) or expression. The DNA encoding the antibody can be easily isolated using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally 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 element, a promoter, and a transcription termination sequence.

(a)信号序列成分(a) Signal sequence components

本发明的抗体不仅可以直接重组产生，还可以作为与异源多肽的融合多肽产生，该异源多肽优选在成熟蛋白质或多肽的N端具有特异性切割位点的信号序列或其他多肽。所选择的异源信号序列优选是宿主细胞识别和加工(例如通过信号肽酶切割)的信号序列。对于不识别和加工天然抗体信号序列的原核宿主细胞，用原核信号序列取代该信号序列，该原核信号序列选自例如碱性磷酸酶、青霉素酶、lpp或热稳定肠毒素II基前导序列。对于酵母分泌，可以用例如酵母转变酶前导序列、因子前导序列(包括酵母属(Saccharomyces)和克鲁维酵母属(Kluyveromyces)α-因子前导序列)或酸性磷酸酶前导序列、白假丝酵母(C.albicans)葡糖淀粉酶前导序列或WO90/13646中所述的信号序列取代天然信号序列。在哺乳动物细胞表达中，可用哺乳动物信号序列以及病毒分泌前导序列(例如单纯疱疹病毒gD信号)。The antibodies of the present invention can be produced not only by direct recombinant production but also as fusion polypeptides with heterologous polypeptides, preferably signal sequences or other polypeptides having specific cleavage sites at the N-terminus of the mature protein or polypeptide. The selected heterologous signal sequence is preferably a signal sequence that is recognized and processed by the host cell (e.g., by cleavage by a signal peptidase). For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is replaced with a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II-based leaders. For yeast secretion, the native signal sequence can be replaced with, for example, a yeast invertase leader, a factor leader (including Saccharomyces and Kluyveromyces α-factor leaders), an acid phosphatase leader, a Candida albicans glucoamylase leader, or a signal sequence described in WO 90/13646. For mammalian cell expression, mammalian signal sequences and viral secretion leaders (e.g., the herpes simplex virus gD signal) can be used.

(b)复制起点(b) Origin of replication

表达载体和克隆载体都包含使得该载体能够在所选择的一种或多种宿主细胞中复制的核酸序列。通常，在克隆载体中，此序列是使得该载体能够独立于宿主染色体DNA复制的序列，且包括复制起点或自主复制序列。对多种细菌、酵母和病毒而言，这类序列是公知的。来自质粒pBR322的复制起点适合用于大多数革兰氏阴性菌，2μ质粒起点适合用于酵母，多种病毒起点(SV40、多瘤病毒、腺病毒、VSV或BPV)用于哺乳动物细胞中的克隆载体。通常，哺乳动物表达载体不需要复制起点成分(通常可以使用SV40起点，但只是因为它包含早期启动子)。Expression vectors and cloning vectors all comprise a nucleotide sequence that enables the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is a sequence that enables the vector to replicate independently of the host chromosome DNA, and includes an origin of replication or an autonomously replicating sequence. For various bacteria, yeast and viruses, this type of sequence is well known. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the 2 μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma virus, adenovirus, VSV or BPV) are used for cloning vectors in mammalian cells. Typically, mammalian expression vectors do not require an origin of replication component (typically the SV40 origin can be used, but only because it comprises an early promoter).

(c)选择基因成分(c) Selecting genetic components

表达载体和克隆载体可以包含选择基因，也称为选择标记。典型的选择基因编码蛋白质，该蛋白质：(a)赋予对抗生素或其他毒素(例如氨苄青霉素、新霉素、氨甲喋呤或四环素)的抗性；(b)弥补营养缺陷；或(c)提供不能从复合培养基获得的关键营养物，例如编码芽孢杆菌D-丙氨酸消旋酶的基因。Expression vectors and cloning vectors can contain selection genes, also known as selectable markers. Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline); (b) compensate for auxotrophic deficiencies; or (c) provide critical nutrients that cannot be obtained from complex media, such as the gene encoding Bacillus D-alanine racemase.

选择方案的一个实例利用药物来阻断宿主细胞的生长。用异源基因成功转化的那些细胞产生赋予药物抗性的蛋白质，从而从该选择方案存活下来。这种显性选择的实例使用药物新霉素、霉酚酸和潮霉素。One example of a selection scheme utilizes a drug to block the growth of host cells. Those cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance, thereby surviving the selection scheme. Examples of this type of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

适合用于哺乳动物细胞的选择标记的另一实例是使得能够鉴定有能力摄取编码抗体的核酸的细胞的那些，如DHFR、谷氨酰胺合成酶(GS)、胸苷激酶、金属硫蛋白-I和-II(优选灵长类金属硫蛋白基因)、腺苷脱氨酶、鸟氨酸脱羧酶等。Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II (preferably primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, and the like.

例如，通过在包含氨甲喋呤(Mtx)(DHFR的竞争性拮抗剂)的培养基中培养转化体来鉴定用DHFR基因转化的细胞。在这些条件下，DHFR基因随同任意其他共转化的核酸扩增。可以使用缺乏内源DHFR活性的中国仓鼠卵巢(CHO)细胞系(例如ATCC CRL-9096)。For example, cells transformed with the DHFR gene are identified by culturing transformants in a culture medium comprising methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified along with any other co-transformed nucleic acids. A Chinese hamster ovary (CHO) cell line (e.g., ATCC CRL-9096) lacking endogenous DHFR activity can be used.

备选地，通过在包含L-蛋氨酸亚砜亚胺(Msx)(GS的抑制剂)的培养基中培养转化体来鉴定用GS基因转化的细胞。在这些条件下，GS基因随同任意其他共转化的核酸扩增。GS选择/扩增系统可以与上述DHFR选择/扩增系统组合使用。Alternatively, cells transformed with the GS gene are identified by culturing the transformants in a medium containing L-methionine sulfoximine (Msx), an inhibitor of GS. Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acids. The GS selection/amplification system can be used in combination with the DHFR selection/amplification system described above.

备选地，可以通过在包含用于选择标记的选择剂(如氨基糖苷抗生素，例如卡那霉素、新霉素或G418)的培养基中培养细胞来选择用编码目的抗体的DNA序列、野生型DHFR基因和另一选择标记(如氨基糖苷3’-磷酸转移酶(APH))转化或共转化的宿主细胞(尤其是包含内源DHFR的野生型宿主)。参见美国专利号4,965,199。Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) transformed or co-transformed with a DNA sequence encoding an antibody of interest, a wild-type DHFR gene, and another selectable marker (such as aminoglycoside 3'-phosphotransferase (APH)) can be selected by culturing the cells in a medium containing a selection agent for the selectable marker (such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418). See U.S. Patent No. 4,965,199.

适合用于酵母的选择基因是存在于酵母质粒YRp7中的trp1基因(Stinchcomb等,Nature 282:39(1979))。trp1基因为缺乏在色氨酸中生长的能力的突变体酵母菌株(例如ATCC No.44076或PEP4-1)提供了选择标记。Jones,Genetics 85:12(1977)。然后，酵母宿主细胞基因组中trp1损伤的存在为通过缺乏色氨酸情况下的生长来检测转化提供了有效的环境。类似地，通过已知的具有Leu2基因的质粒弥补了Leu2缺乏的酵母菌株(ATCC20,622或38,626)。A suitable selection gene for yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature 282:39 (1979)). The trp1 gene provides a selection marker for mutant yeast strains lacking the ability to grow in tryptophan (e.g., ATCC No. 44076 or PEP4-1). Jones, Genetics 85:12 (1977). The presence of a trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, yeast strains lacking Leu2 (ATCC 20,622 or 38,626) are complemented by known plasmids with the Leu2 gene.

此外，可以用衍生自1.6μm环状质粒pKD1的载体来转化克鲁维酵母属酵母。备选地，针对乳酸克鲁维酵母(K.lactis)报道了用于大规模产生重组小牛凝乳酶的表达系统。Van den Berg,Bio/Technology 8:135(1990)。还公开了用于通过克鲁维酵母属的工业菌株分泌成熟重组人血清白蛋白的稳定的多拷贝表达载体。Fleer等,Bio/Technology 9:968-975(1991)。In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used to transform Kluyveromyces yeast. Alternatively, an expression system for large-scale production of recombinant calf chymosin has been reported for Kluyveromyces lactis. Van den Berg, Bio/Technology 8:135 (1990). Stable multicopy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology 9:968-975 (1991).

(d)启动子成分(d) Promoter elements

表达载体和克隆载体通常包含宿主生物可识别且与编码抗体的核酸有效连接的启动子。适合用于原核宿主的启动子包括phoA启动子、β-内酰胺酶和乳糖启动子系统、碱性磷酸酶启动子、色氨酸(trp)启动子系统及杂合启动子(如tac启动子)。但是，其他已知的细菌启动子也是适宜的。用于细菌系统的启动子还将包含与编码抗体的DNA有效连接的Shine-Dalgarno(S.D.)序列。Expression vectors and cloning vectors typically contain a promoter that is recognizable by the host organism and operatively linked to the nucleic acid encoding the antibody. Promoters suitable for prokaryotic hosts include phoA promoters, beta-lactamase and lactose promoter systems, alkaline phosphatase promoters, tryptophan (trp) promoter systems, and hybrid promoters (such as the tac promoter). However, other known bacterial promoters are also suitable. Promoters for bacterial systems will also contain a Shine-Dalgarno (S.D.) sequence operatively linked to the DNA encoding the antibody.

对真核生物而言，启动子序列是已知的。基本上所有真核基因都具有定位在起始转录的位点上游约25至30个碱基的富含AT的区域。见于许多基因的转录起点上游70至80个碱基的另一序列是CNCAAT区，其中N可以是任意核苷酸。大多数真核基因的3’端是AATAAA序列，其可以是在编码序列的3’端加入poly A尾的信号。将所有这些序列适宜地插入真核表达载体中。For eukaryotic organisms, promoter sequences are known. Basically, all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream of the site of initial transcription. Another sequence found in the transcription start site upstream 70 to 80 bases of many genes is the CNCAAT district, in which N can be any nucleotide. The 3 ' end of most eukaryotic genes is an AATAAA sequence, which can be a signal that adds a poly A tail at the 3 ' end of the coding sequence. All of these sequences are suitably inserted into the eukaryotic expression vector.

适合用于酵母宿主的启动子序列的实例包括3-磷酸甘油酸激酶或其他糖酵解酶(如烯醇化酶、甘油醛-3-磷酸脱氢酶、己糖激酶、丙酮酸脱羧酶、磷酸果糖激酶、葡萄糖-6-磷酸异构酶、3-磷酸甘油酸变位酶、丙酮酸激酶、磷酸丙糖异构酶、磷酸葡萄糖异构酶和葡萄糖激酶)的启动子。Examples of suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

其他酵母启动子(其是具有通过生长条件控制转录的附加优势的诱导型启动子)是醇脱氢酶2、异细胞色素(isocytochrome)C、酸性磷酸酶、氮代谢相关降解酶、金属硫蛋白、甘油醛-3-磷酸脱氢酶及负责麦芽糖和半乳糖利用的酶的启动子区。适合用于酵母表达的载体和启动子进一步描述于EP 73,657中。还有利地与酵母启动子一起使用酵母增强子。Other yeast promoters (which are inducible promoters with the added advantage of being able to control transcription by growth conditions) are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, nitrogen metabolism-related degradation enzymes, metallothioneins, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Vectors and promoters suitable for yeast expression are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.

例如通过获自病毒(例如多瘤病毒、禽痘病毒、腺病毒(如腺病毒2)、牛乳头状病毒、禽肉瘤病毒、巨细胞病毒、反转录病毒、乙型肝炎病毒、猿猴病毒40(SV40))基因组、或获自异源哺乳动物启动子(例如肌动蛋白启动子或免疫球蛋白启动子)、或获自热休克启动子的启动子来控制哺乳动物宿主细胞中从载体转录抗体，只要这类启动子与宿主细胞系统相容。Transcription of the antibody from the vector in mammalian host cells is controlled, for example, by a promoter obtained from the genome of a virus (e.g., polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus, simian virus 40 (SV40)), or from a heterologous mammalian promoter (e.g., the actin promoter or the immunoglobulin promoter), or from a heat shock promoter, so long as such promoter is compatible with the host cell systems.

作为SV40限制片段方便地获得SV40病毒的早期和晚期启动子，该限制片段还包含SV40病毒复制起点。作为HindIII E限制片段方便地获得人巨细胞病毒的立即早期启动子。美国专利号4,419,446中公开了用牛乳头状病毒作为载体来在哺乳动物宿主中表达DNA的系统。美国专利号4,601,978中描述了此系统的改进。关于人β-干扰素cDNA在来自单纯疱疹病毒的胸苷激酶激动子控制下在小鼠细胞中的表达，还参见Reyes等,Nature 297:598-601(1982)。备选地，可以用劳斯肉瘤病毒长末端重复序列作为启动子。The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papillomavirus as a vector is disclosed in U.S. Patent No. 4,419,446. Improvements to this system are described in U.S. Patent No. 4,601,978. Regarding the expression of human beta-interferon cDNA in mouse cells under the control of a thymidine kinase activator from herpes simplex virus, see also Reyes et al., Nature 297:598-601 (1982). Alternatively, the Rous sarcoma virus long terminal repeat sequence can be used as a promoter.

(e)增强子元件成分(e) Enhancer element components

将增强子序列插入载体通常增加高等真核生物的编码本发明抗体的DNA的转录。现已知来自哺乳动物基因(珠蛋白、弹性蛋白酶、清蛋白、甲胎蛋白和胰岛素基因)的许多增强子序列。但是，通常将使用来自真核细胞病毒的增强子。实例包括复制起点晚期侧(bp100-270)的SV40增强子、巨细胞病毒早期启动子增强子、复制起点晚期侧的多瘤增强子和腺病毒增强子。关于用于激活真核启动子的增强元件，还参见Yaniv,Nature297:17-18(1982)。增强子可以在抗体编码序列5’或3’的位置剪接入载体，但优选定位在启动子5’的位点。Inserting an enhancer sequence into a vector generally increases transcription of the DNA encoding the antibodies of the present invention in higher eukaryotic organisms. Many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin genes) are now known. However, enhancers from eukaryotic cell viruses are generally used. Examples include the SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer on the late side of the replication origin (bp100-270). Regarding enhancing elements for activating eukaryotic promoters, see also Yaniv, Nature 297:17-18 (1982). The enhancer can be spliced into the vector at the position 5' or 3' of the antibody coding sequence, but is preferably located at the site 5' of the promoter.

(f)转录终止成分(f) Transcription termination element

用于真核宿主细胞(酵母、真菌、昆虫、植物、动物、人或来自其他多细胞生物的有核细胞)的表达载体还将包含终止转录和稳定mRNA所必需的序列。通常可从真核或病毒DNA或cDNA的5’和(偶尔)3’非翻译区获得这类序列。这些区域包含转录为编码抗体的mRNA的非翻译区中的多腺苷酸化片段的核苷酸区段。一种有用的转录终止成分是牛生长激素多腺苷酸化区域。参见WO 94/11026及其中公开的表达载体。Expression vectors for eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for terminating transcription and stabilizing the mRNA. Such sequences are typically obtained from the 5' and (occasionally) 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated region of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors disclosed therein.

(g)宿主细胞的选择和转化(g) Selection and transformation of host cells

适合用于克隆或表达本文载体中的DNA的宿主细胞是原核细胞、酵母细胞或高级真核细胞。适合用于此目的的原核生物包括真细菌，如革兰氏阴性或革兰氏阳性生物，例如肠杆菌科(Enterobacteriaceae)，如埃希氏菌属(Escherichia)，例如大肠杆菌；肠杆菌属(Enterobacter)；欧文氏菌属(Erwinia)；克雷伯氏菌属(Klebsiella)；变形杆菌属(Proteus)；沙门氏菌属(Salmonella)，例如鼠伤寒沙门氏菌(Salmonella typhimurium)；沙雷氏菌属(Serratia)，例如粘质沙雷氏菌(Serratia marcescans)；和志贺氏菌属(Shigella)；以及芽孢杆菌属(Bacilli)，如枯草芽孢杆菌(B.subtilis)和地衣芽孢杆菌(B.licheniformis)(例如1989年4月12日公开的DD 266,710中所公开的地衣芽孢杆菌41P)；假单胞菌属(Pseudomonas)，如铜绿假单胞菌(P.aeruginosa)；和链霉菌属(Streptomyces)。一种优选的大肠杆菌克隆宿主是大肠杆菌294(ATCC 31,446)，但其他菌株如大肠杆菌B、大肠杆菌X1776(ATCC 31,537)和大肠杆菌W3110(ATCC 27,325)也是适宜的。这些实例是说明性的而不是限制性的。Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast cells, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans; and Shigella; and Bacilli, such as B. subtilis and B. licheniformis (e.g., DD 12, published April 12, 1989). licheniformis 41P disclosed in 266,710); Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), but other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative and not limiting.

尤其是在不需要糖基化和Fc效应子功能时，如在治疗性抗体与本身在肿瘤细胞破坏中显示有效性的细胞毒性剂(例如毒素)缀合时，可以在细菌中产生全长抗体、抗体融合蛋白质和抗体片段。全长抗体具有更长的循环半衰期。大肠杆菌中的产生更快，且更节约成本。对于在细菌中表达抗体片段和多肽，参见例如美国专利号5,648,237(Carter等)、美国专利号5,789,199(Joly等)、美国专利号5,840,523(Simmons等)，其描述了用于优化表达和分泌的翻译起始区(TIR)和信号序列。还参见Charlton,Methods in Molecular Biology,248卷(B.K.C.Lo编辑,Humana出版社,Totowa,N.J.,2003),245-254页，其描述在大肠杆菌中表达抗体片段。表达后，从可溶性级分中的大肠杆菌细胞糊分离抗体，且可以通过例如A蛋白柱或G蛋白柱(取决于同种型)来纯化。可以与用于纯化在例如CHO细胞中表达的抗体的方法类似地进行最终的纯化。In particular, when glycosylation and Fc effector functions are not required, such as when therapeutic antibodies are conjugated to cytotoxic agents (e.g., toxins) that themselves show effectiveness in tumor cell destruction, full-length antibodies, antibody fusion proteins, and antibody fragments can be produced in bacteria. Full-length antibodies have longer circulation half-lives. Production in E. coli is faster and more cost-effective. For expressing antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent No. 5,648,237 (Carter et al.), U.S. Patent No. 5,789,199 (Joly et al.), U.S. Patent No. 5,840,523 (Simmons et al.), which describe translation initiation regions (TIR) and signal sequences for optimizing expression and secretion. Also see Charlton, Methods in Molecular Biology, 248 volumes (B.K.C.Lo, ed., Humana Press, Totowa, N.J., 2003), 245-254 pages, which describe expression of antibody fragments in E. coli. After expression, the antibody is isolated from the E. coli cell paste in the soluble fraction and can be purified, for example, by a protein A column or a protein G column (depending on the isotype). Final purification can be performed similarly to the method used to purify antibodies expressed in, for example, CHO cells.

除原核生物外，诸如丝状真菌或酵母的真核微生物也是适合用于编码抗体的载体的克隆或表达宿主。酿酒酵母(Saccharomyces cerevisiae)或常见的面包酵母是低等真核宿主微生物中最常用的。但是，许多其他属、种和菌株通常可得且在本文中使用，如粟酒裂殖酵母(Schizosaccharomyces pombe)；克鲁维酵母属宿主，如乳酸克鲁维酵母(K.lactis)、脆壁克鲁维酵母(K.fragilis)(ATCC 12,424)、保加利亚克鲁维酵母(K.bulgaricus)(ATCC16,045)、威克克鲁维酵母(K.wickeramii)(ATCC 24,178)、瓦尔提克鲁维酵母(K.waltii)(ATCC 56,500)、果蝇克鲁维酵母(K.drosophilarum)(ATCC36,906)、耐热克鲁维酵母(K.thermotolerans)和马克思克鲁维酵母(K.marxianus)；耶氏酵母属(yarrowia)(EP 402,226)；巴斯德毕赤酵母(Pichia pastoris)(EP 183,070)；假丝酵母属(Candida)；里氏木霉(Trichoderma reesia)(EP 244,234)；粗糙链孢霉(Neurosporacrassa)；许旺酵母属(Schwanniomyces)，如许旺酵母(Schwanniomyces occidentalis)；和丝状真菌，如脉孢霉属(Neurospora)、青霉属(Penicillium)、弯颈霉属(Tolypocladium)；和曲霉属(Aspergillus)宿主，如构巢曲霉(A.nidulans)和黑曲霉(A.niger)。讨论酵母和丝状真菌在产生治疗性蛋白质中的用途的综述参见例如Gerngross,Nat.Biotech.22:1409-1414(2004)。In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly available and used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium; and Aspergillus hosts, such as A. nidulans and A. niger. For a review discussing the use of yeast and filamentous fungi in producing therapeutic proteins, see, for example, Gerngross, Nat. Biotech. 22: 1409-1414 (2004).

可以选择某些真菌和酵母菌株，其中糖基化途径已“人源化”，导致产生具有部分或完全人糖基化模式的抗体。参见例如Li等,Nat.Biotech.24:210-215(2006)(描述巴斯德毕赤酵母中糖基化途径的人源化)；及Gerngross等,上文。Certain fungi and yeast strains can be selected in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describing humanization of the glycosylation pathway in Pichia pastoris); and Gerngross et al., supra.

适合用于表达糖基化抗体的宿主细胞源自多细胞生物(无脊椎动物和脊椎动物)。无脊椎动物细胞的实例包括植物细胞和昆虫细胞。已鉴定了来自诸如草地夜蛾(Spodoptera frugiperda)(毛虫)、埃及伊蚊(Aedes aegypti)(蚊子)、白纹伊蚊(Aedesalbopictus)(蚊子)、黑腹果蝇(Drosophila melanogaster)(果蝇)和家蚕(Bombyx mori)的宿主的许多杆状病毒株和变体及对应的受纳昆虫宿主细胞。多种用于转染的病毒株是公开可得的，例如苜蓿银纹夜蛾(Autographa californica)NPV的L-1变体和家蚕NPV的Bm-5病毒株，可以将这类病毒用作本发明的病毒，尤其是用于转染草地夜蛾细胞。Host cells suitable for expressing glycosylated antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains and variants and corresponding receptive insect host cells have been identified from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies), and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses of the present invention, particularly for transfecting Spodoptera frugiperda cells.

也可以用棉花、玉米、马铃薯、大豆、矮牵牛、番茄、浮萍(Leninaceae)、苜蓿(蒺藜苜蓿(M.truncatula))和烟草的植物细胞培养物作为宿主。参见例如美国专利号5,959,177、6,040,498、6,420,548、7,125,978和6,417,429(描述用于在转基因植物中产生抗体的PLANTIBODIES^TM技术)。Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES^™ technology for producing antibodies in transgenic plants).

可以用脊椎动物细胞作为宿主，繁殖培养(组织培养)的脊椎动物细胞已成为常规方法。有用的哺乳动物宿主细胞系的实例是SV40转化的猴肾CV1细胞系(COS-7，ATCC CRL1651)；人胚肾细胞系(293或为在悬浮培养物中生长亚克隆的293细胞，Graham等,J.Gen.Virol.36:59(1977))；幼仓鼠肾细胞(BHK，ATCC CCL 10)；小鼠支持细胞(TM4，Mather,Biol.Reprod.23:243-251(1980))；猴肾细胞(CVl ATCC 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 CCL34)；buffalo大鼠肝细胞(BRL 3A，ATCC CRL 1442)；人肺细胞(W138，ATCC CCL 75)；人肝细胞(Hep G2，HB8065)；小鼠乳腺肿瘤(MMT 060562，ATCC CCL51)；TRI细胞(Mather等,AnnalsN.Y.Acad.Sci.383:44-68(1982))；MRC 5细胞；FS4细胞；和人肝癌细胞系(Hep G2)。其他有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞(Urlaub等,Proc.Natl.Acad.Sci.USA 77:4216(1980))；骨髓瘤细胞系，如NS0和Sp2/0。某些适合用于抗体产生的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu,Methods in MolecularBiology,248卷(B.K.C.Lo编辑,Humana出版社,Totowa,NJ,2003),255-268页。Vertebrate cells can be used as hosts, and propagation and culture (tissue culture) of vertebrate cells has become a routine method. Examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL1651); human embryonic kidney cell line (293 or a subclone of 293 cells grown in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); myeloma cell lines, such as NS0 and Sp2/0. Certain mammalian host cell lines suitable for antibody production are reviewed in, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268.

用上述用于抗体产生的表达载体或克隆载体转化宿主细胞，并在根据诱导启动子、选择转化体或扩增编码希望的序列的基因的需要改进的常规营养培养基中培养。Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

(h)培养宿主细胞(h) Cultivating host cells

可在多种培养基中培养用来产生本发明的抗体的宿主细胞。诸如Ham's F10(Sigma)、Minimal Essential Medium(MEM)(Sigma)、RPMI-1640(Sigma)和Dulbecco'sModified Eagle's Medium(DMEM，Sigma)的市售培养基适合用于培养宿主细胞。此外，可以用Ham等,Meth.Enz.58:44(1979)；Barnes等,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Re.30,985中所述的任意培养基作为宿主细胞的培养基。可以根据需要向任意这些培养基中补充激素和/或其他生长因子(如胰岛素、转铁蛋白或表皮生长因子)、盐(如氯化钠、钙、镁和磷酸盐)、缓冲剂(如HEPES)、核苷酸(如腺苷和胸苷)、抗生素(如GENTAMYCIN^TM药物)、痕量元素(定义为通常以微摩尔范围内的终浓度存在的无机化合物)和葡萄糖或等同的能量来源。还可以按本领域技术人员已知的适当浓度包含任意其他必需的补充物。培养条件(如温度、pH等)是之前用于选择用于表达的宿主细胞的那些，且对普通技术人员而言将是显而易见的。Host cells used to produce the antibodies of the present invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 can be used as culture media for host cells. Hormones and/or other growth factors (such as insulin, transferrins or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN^™ drugs), trace elements (defined as inorganic compounds typically present at a final concentration in the micromolar range) and glucose or equivalent energy sources can be supplemented as needed in any of these culture media. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. Culture conditions (such as temperature, pH etc.) are those previously used to select the host cell for expression, and will be apparent to those of ordinary skill in the art.

(xiv)纯化抗体(xiv) Purification of antibodies

在使用重组技术时，抗体可以在胞内、在周质间隙中产生，或者直接分泌入培养基。如果抗体在胞内产生，作为第一步，例如通过离心或超滤去除颗粒碎片(宿主细胞或裂解的片段)。Carter等,Bio/Technology10:163-167(1992)描述了用于分离分泌至大肠杆菌的周质间隙的多肽的方法。简言之，在醋酸钠(pH 3.5)、EDTA和苯甲基磺酰氟(PMSF)的存在下融化细胞糊超过约30分钟。可以通过离心去除细胞碎片。在抗体分泌入培养基时，通常先用市售蛋白质浓缩滤器(例如Amicon或Millipore Pellicon超滤单元)浓缩来自这类表达系统的上清。可以在任意前述步骤中包含诸如PMSF的蛋白酶抑制剂来抑制蛋白水解，且可以包含抗生素来防止外来污染物的生长。When using recombinant technology, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particle debris (host cell or cracked fragments) is removed by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) have described a method for separating polypeptides secreted into the periplasmic space of Escherichia coli. In brief, the cell paste is melted in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for more than about 30 minutes. Cell debris can be removed by centrifugation. When the antibody is secreted into the culture medium, the supernatant from this type of expression system is usually first concentrated with a commercially available protein concentration filter (such as Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

可以用例如羟基磷灰石层析、疏水相互作用层析、凝胶电泳、透析和亲和层析来纯化从细胞制备的抗体组合物，亲和层析是通常优选的纯化步骤之一。A蛋白作为亲和配体的适宜性取决于存在于抗体中的任意免疫球蛋白Fc结构域的种类和同种型。A蛋白可以用于纯化基于人γ1、γ2或γ4重链的抗体(Lindmark等,J.Immunol.Meth.62:1-13(1983))。G蛋白推荐用于所有小鼠同种型及用于人γ3(Guss等,EMBO J.5:15671575(1986))。亲和配体所附着的基质最常见的是琼脂糖，但其他基质也可用。与用琼脂糖可以达到的流速和处理时间相比，机械稳定的基质(如可控孔度玻璃或聚(苯乙烯二乙烯)苯)允许更快的流速和更短的处理时间。在抗体包含CH3结构域时，用Bakerbond ABX^TM树脂(J.T.Baker,Phillipsburg,NJ)进行纯化。取决于要回收的抗体，用于蛋白质纯化的其他技术也可用，如离子交换柱上的分级分离、乙醇沉淀、反相HPLC、二氧化硅上的层析、肝素SEPHAROSE^TM上的层析、阴离子或阳离子交换树脂(如聚天冬氨酸柱)上的层析、层析聚焦、SDS-PAGE和硫酸铵沉淀。Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being one of the preferred purification steps. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. 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 recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The most common matrix to which the affinity ligand is attached is agarose, but other matrices are also available. Mechanically stable matrices (such as controlled pore glass or poly (styrene divinyl) benzene) allow faster flow rates and shorter processing times compared to the flow rates and processing times that can be achieved with agarose. When the antibody contains a CH3 domain, purification is performed using Bakerbond ABX^™ resin (JT Baker, Phillipsburg, NJ). Other techniques for protein purification may be used, depending on the antibody to be recovered, such as fractionation on an ion exchange column, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE^™ , chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

通常，用于制备用于研究、测试和临床应用的抗体的多种方法在本领域中是完善的，与上述方法一致，和/或本领域技术人员认为适合用于具体的目的抗体。In general, various methods for preparing antibodies for research, testing, and clinical applications are well established in the art and are consistent with the methods described above and/or as deemed appropriate by one skilled in the art for a particular antibody of interest.

(xv)选择具有生物学活性的抗体(xv) Selection of biologically active antibodies

可以对按上述产生的抗体进行一种或多种“生物学活性”测定，以选择从治疗角度看具有有益特性的抗体，或选择保持该抗体的生物学活性的制剂和条件。可以针对其结合抗原的能力测试抗体，该抗体针对该抗原制备。The antibodies generated as described above can be subjected to one or more "biological activity" assays to select antibodies with beneficial properties from a therapeutic perspective, or to select formulations and conditions that maintain the biological activity of the antibodies. Antibodies can be tested for their ability to bind to the antigen against which they were raised.

例如，对于抗PD-L1抗体，可以在检测结合PD-L1的能力的测定中评价抗体的抗原结合特性。在一些实施方案中，可以通过例如饱和结合、ELISA和/或竞争测定(例如RIA)来测定抗体的结合。另外，可以对抗体进行其他生物学活性测定，例如以评价其作为治疗剂的有效性。这类测定为本领域已知，且依赖于靶抗原及抗体的预期用途。例如，可以在CD8+T细胞、淋巴细胞性脉络丛脑膜炎病毒(LCMV)小鼠模型和/或同基因肿瘤模型中评估抗体阻断PD-L1的生物学效应，例如，如美国专利8,217,149中的所述。For example, for anti-PD-L1 antibodies, the antigen binding properties of the antibody can be evaluated in an assay for the ability to bind to PD-L1. In some embodiments, the binding of the antibody can be determined by, for example, saturation binding, ELISA, and/or competitive assays (e.g., RIA). In addition, other biological activity assays can be performed on the antibody, for example, to evaluate its effectiveness as a therapeutic agent. Such assays are known in the art and depend on the target antigen and the intended use of the antibody. For example, the biological effects of antibodies blocking PD-L1 can be evaluated in CD8+ T cells, lymphocytic choriomeningitis virus (LCMV) mouse models, and/or syngeneic tumor models, for example, as described in U.S. Patent No. 8,217,149.

为了筛选结合目的抗原上的特定表位的抗体(例如，阻断实施例的抗PD-L1抗体与PD-L1的结合的抗体)，可以进行常规交叉阻断测定，如Antibodies,A Laboratory Manual,Cold Spring Harbor Laboratory,Ed Harlow and David Lane(1988)中所述的交叉阻断测定。备选地，可以进行例如Champe等,J.Biol.Chem.270:1388-1394(1995)中所述的表位定位，以确定抗体是否结合目的表位。To screen for antibodies that bind to a specific epitope on an antigen of interest (e.g., antibodies that block the binding of the anti-PD-L1 antibodies of the Examples to PD-L1), a conventional cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping, such as that described in Champe et al., J. Biol. Chem. 270: 1388-1394 (1995), can be performed to determine whether the antibody binds to the epitope of interest.

IV.药物组合物和制剂IV. Pharmaceutical Compositions and Formulations

本文还提供包含本文所述的PD-1轴结合拮抗剂和/或抗体(如抗PD-L1抗体)和可药用载体的药物组合物和制剂。本发明还提供包含紫杉烷(例如纳米清蛋白结合紫杉醇紫杉醇或多西紫杉醇)的药物组合物和制剂。Also provided herein are pharmaceutical compositions and formulations comprising the PD-1 axis binding antagonists and/or antibodies (such as anti-PD-L1 antibodies) described herein and a pharmaceutically acceptable carrier. Also provided herein are pharmaceutical compositions and formulations comprising a taxane (such as nanoalbumin-bound paclitaxel, paclitaxel, or docetaxel).

可以通过将具有希望得到的纯度的活性成分(例如PD-1轴结合拮抗剂和/或紫杉烷)与一种或多种可选的可药用载体(Remington's Pharmaceutical Sciences第16版,Osol,A.编辑(1980))混合，以冻干制剂或水溶液的形式制备本文所述的药物组合物和制剂。可药用载体一般在所利用的剂量和浓度下对受体无毒性，且包括但不限于：缓冲剂，如磷酸、柠檬酸和其他有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(如十八烷基二甲基苄基氯化铵；氯己双铵；氯苄烷铵；苄索氯铵；苯酚；丁醇或苄醇；对羟基苯甲酸烷基酯，如对羟基苯甲酸甲酯或丙酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，如血清白蛋白、明胶或免疫球蛋白；亲水聚合物，如聚乙烯吡咯烷酮；氨基酸，如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其他糖类，包括葡萄糖、甘露糖或糊精；螯合剂，如EDTA；糖，如蔗糖、甘露醇、海藻糖或山梨醇；成盐抗衡离子，如钠；金属络合物(例如Zn-蛋白质络合物)；和/或非离子型表面活性剂，如聚乙二醇(PEG)。本文的示例性可药用载体进一步包括间质药物分散剂，如可溶性中性活性透明质酸酶糖蛋白(sHASEGP)，例如人可溶性PH-20透明质酸酶糖蛋白，如rHuPH20(Baxter International,Inc.)。包括rhuPH20的某些示例性sHASEGP和使用方法描述于美国专利公开号2005/0260186和2006/0104968中。在一方面，将sHASEGP与诸如软骨素酶的一种或多种附加的糖胺聚糖酶组合。The pharmaceutical compositions and formulations described herein can be prepared by mixing the active ingredient (e.g., PD-1 axis binding antagonist and/or taxane) having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polyols. Peptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other sugars including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (Baxter International, Inc.). Certain exemplary sHASEGPs, including rhuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

示例性冻干抗体制剂描述于美国专利号6,267,958中。水性抗体制剂包括描述于美国专利号6,171,586和WO 2006/044908中的那些，后一种制剂包含组氨酸-乙酸缓冲液。Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006/044908, the latter formulation comprising a histidine-acetate buffer.

本文的组合物和制剂还可以根据所治疗的具体适应症的需要包含一种以上活性成分，优选相互无不利影响的具有互补活性的那些。这类活性成分以对预期目的有效的量适宜地组合存在。The compositions and preparations herein may also contain more than one active ingredient as required for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably combined in amounts effective for the intended purpose.

活性成分可以包载(entrap)在例如通过凝聚技术或通过界面聚合制备的微囊(例如，分别为羟甲基纤维素微囊或明胶微囊和聚-(甲基丙烯酸甲酯)微囊)中、胶体药物递送系统(例如脂质体、白蛋白微球、微乳、纳米颗粒和纳米囊(nanocapsule))中或粗滴乳状液中。这类技术公开于Remington’s Pharmaceutical Sciences第16版,Osol,A.编辑(1980)中。The active ingredient can be entrapped in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively), prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

可以制备缓释制剂。缓释制剂的适宜实例包括含有抗体的固体疏水聚合物的半透性基质，该基质是成形物品的形式，例如膜或微囊。待用于体内施用的制剂通常是无菌的。无菌可以容易地例如通过滤过无菌滤膜来达到。Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of shaped articles, such as films or microcapsules. Formulations to be used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filter membrane.

IV.治疗方法IV. Treatment Methods

本文提供用于在个体中治疗癌症或延迟癌症进展的方法，其包括对该个体施用有效量的PD-1轴结合拮抗剂和紫杉烷(例如纳米清蛋白结合紫杉醇或紫杉醇)。在一些实施方案中，该治疗在该个体中产生治疗后的反应。在一些实施方案中，该反应是完全应答。在一些实施方案中，该治疗在该个体中产生停止治疗后的持续应答。本文所述的方法可用于治疗其中希望得到增强的免疫原性如提高肿瘤免疫原性用于治疗癌症的病症。本文还提供在患有癌症的个体中增强免疫功能的方法，其包括对该个体施用有效量的PD-1轴结合拮抗剂和紫杉烷(例如纳米清蛋白结合紫杉醇或紫杉醇)。本领域已知或本文描述的任意PD-1轴结合拮抗剂和紫杉烷都可用于该方法。在一些实施方案中，该方法进一步包括施用基于铂的治疗剂。在一些实施方案中，该基于铂的治疗剂是卡铂。Provided herein are methods for treating cancer or delaying cancer progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and a taxane (e.g., nanoalbumin combined with paclitaxel or paclitaxel). In some embodiments, the treatment produces a response in the individual after treatment. In some embodiments, the response is a complete response. In some embodiments, the treatment produces a sustained response in the individual after cessation of treatment. The methods described herein can be used to treat conditions in which enhanced immunogenicity is desired, such as increasing tumor immunogenicity for the treatment of cancer. Also provided herein are methods for enhancing immune function in an individual with cancer, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and a taxane (e.g., nanoalbumin combined with paclitaxel or paclitaxel). Any PD-1 axis binding antagonist and taxane known in the art or described herein can be used in this method. In some embodiments, the method further comprises administering a platinum-based therapeutic agent. In some embodiments, the platinum-based therapeutic agent is carboplatin.

在一些实施方案中，该个体是人。在一些实施方案中，该个体患有癌症。在一些实施方案中，该癌症是乳腺癌(例如三阴乳腺癌)、膀胱癌(例如UBC、MIBC和NMIBC)、结肠直肠癌、直肠癌、肺癌(例如非小细胞肺癌，其可为鳞状或非鳞状)、成胶质细胞瘤、非霍奇金淋巴瘤(NHL)、肾细胞癌(例如RCC)、前列腺癌、肝癌、胰腺癌、软组织肉瘤、卡波西肉瘤、类癌、头颈癌、胃癌、食管癌、前列腺癌、子宫内膜癌、肾癌、卵巢癌、间皮瘤和血癌(例如MDS和多发性骨髓瘤)。在一些实施方案中，该癌症选自：小细胞肺癌、成胶质细胞瘤、成神经细胞瘤、黑素瘤、乳腺癌、胃癌、结肠直肠癌(CRC)或肝细胞癌。在具体实施方案中，该癌症选自肺癌(例如非小细胞肺癌，其可为鳞状或非鳞状)、膀胱癌(例如UBC)、乳腺癌(例如TNBC)、RCC、黑素瘤、结肠直肠癌和血癌(例如MDS和多发性骨髓瘤)。在一些实施方案中，该肺癌是非小细胞肺癌，其可为鳞状或非鳞状。在一些实施方案中，该膀胱癌是UBC。在一些实施方案中，该乳腺癌是TNBC。在一些实施方案中，该血癌是MDS或多发性骨髓瘤。In some embodiments, the individual is a human. In some embodiments, the individual has cancer. In some embodiments, the cancer is breast cancer (e.g., triple-negative breast cancer), bladder cancer (e.g., UBC, MIBC, and NMIBC), colorectal cancer, rectal cancer, lung cancer (e.g., non-small cell lung cancer, which may be squamous or non-squamous), glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma (e.g., RCC), prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid, head and neck cancer, gastric cancer, esophageal cancer, prostate cancer, endometrial cancer, kidney cancer, ovarian cancer, mesothelioma, and blood cancer (e.g., MDS and multiple myeloma). In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma. In certain embodiments, the cancer is selected from lung cancer (e.g., non-small cell lung cancer, which may be squamous or non-squamous), bladder cancer (e.g., UBC), breast cancer (e.g., TNBC), RCC, melanoma, colorectal cancer, and blood cancers (e.g., MDS and multiple myeloma). In some embodiments, the lung cancer is non-small cell lung cancer, which may be squamous or non-squamous. In some embodiments, the bladder cancer is UBC. In some embodiments, the breast cancer is TNBC. In some embodiments, the blood cancer is MDS or multiple myeloma.

在一些实施方案中，该个体在PD-1轴结合拮抗剂和紫杉烷联合治疗之前已用癌症疗法治疗。在一些实施方案中，该个体患有对一种或多种癌症疗法有抗性的癌症。在一些实施方案中，对癌症疗法的抗性包括癌症复发或难治性癌症。复发可以指治疗后癌症在原部位或新部位重新出现。在一些实施方案中，对癌症疗法的抗性包括癌症在用抗癌疗法治疗期间进展。在一些实施方案中，对癌症疗法的抗性包括对治疗无反应的癌症。癌症可以在治疗开始时有抗性，或者它可以在治疗期间变得有抗性。在一些实施方案中，该癌症处于早期或晚期。In some embodiments, the individual has been treated with a cancer therapy prior to the combination therapy with a PD-1 axis binding antagonist and a taxane. In some embodiments, the individual has a cancer that is resistant to one or more cancer therapies. In some embodiments, resistance to cancer therapy includes cancer recurrence or refractory cancer. Recurrence can refer to the reappearance of cancer at the original site or a new site after treatment. In some embodiments, resistance to cancer therapy includes cancer progression during treatment with an anti-cancer therapy. In some embodiments, resistance to cancer therapy includes cancer that is unresponsive to treatment. Cancer can be resistant at the start of treatment, or it can become resistant during treatment. In some embodiments, the cancer is in the early or late stages.

在一些实施方案中，本发明的联合治疗包括施用PD-1轴结合拮抗剂和紫杉烷。PD-1轴结合拮抗剂和紫杉烷(例如纳米清蛋白结合紫杉醇或紫杉醇)可以以本领域已知的任意适宜的方式施用。例如，PD-1轴结合拮抗剂和紫杉烷可以顺次(在不同时间)或同时(在同一时间)施用。在一些实施方案，PD-1轴结合拮抗剂与紫杉烷处于分开的组合物中。在一些实施方案中，PD-1轴结合拮抗剂与紫杉烷处于同一组合物中。In some embodiments, the combination therapy of the present invention comprises administering a PD-1 axis binding antagonist and a taxane. The PD-1 axis binding antagonist and the taxane (e.g., nanoalbumin-bound paclitaxel or paclitaxel) can be administered in any suitable manner known in the art. For example, the PD-1 axis binding antagonist and the taxane can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the PD-1 axis binding antagonist and the taxane are in separate compositions. In some embodiments, the PD-1 axis binding antagonist and the taxane are in the same composition.

PD-1轴结合拮抗剂和紫杉烷可以通过相同的给药途径或通过不同的给药途径施用。在一些实施方案中，PD-1轴结合拮抗剂静脉内、肌内、皮下、局部、口服、经皮、腹腔内、眼眶内、通过植入、通过吸入、鞘内、心室内或鼻内施用。在一些实施方案中，紫杉烷静脉内、肌内、皮下、局部、口服、经皮、腹腔内、眼眶内、通过植入、通过吸入、鞘内、心室内或鼻内施用。可以施用有效量的PD-1轴结合拮抗剂和紫杉烷用于预防或治疗疾病。PD-1轴结合拮抗剂和/或紫杉烷的适当剂量可以根据待治疗的疾病类型、PD-1轴结合拮抗剂和紫杉烷的类型、疾病的严重度和病程、个体的临床病症、个体的临床病史和对治疗的反应及主治医生的判断来确定。The PD-1 axis binding antagonist and the taxane can be administered by the same route of administration or by different routes of administration. In some embodiments, the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implant, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the taxane is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implant, by inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of the PD-1 axis binding antagonist and the taxane can be administered for the prevention or treatment of a disease. The appropriate dosage of the PD-1 axis binding antagonist and/or taxane can be determined based on the type of disease to be treated, the type of PD-1 axis binding antagonist and taxane, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the judgment of the attending physician.

一般而言，无论一次还是多次施用，对人施用的抗体(例如抗PD-L1抗体)的治疗有效量将在约0.01至约50mg/kg患者体重的范围内。在一些实施方案中，所使用的抗体是例如每天施用约0.01至约45mg/kg、约0.01至约40mg/kg、约0.01至约35mg/kg、约0.01至约30mg/kg、约0.01至约25mg/kg、约0.01至约20mg/kg、约0.01至约15mg/kg、约0.01至约10mg/kg、约0.01至约5mg/kg、或约0.01至约1mg/kg。在一些实施方案中，按15mg/kg施用抗体。但是，可以使用其他剂量方案。在一个实施方案中，本文所述抗PD-L1抗体在21天循环的第1天按约100mg、约200mg、约300mg、约400mg、约500mg、约600mg、约700mg、约800mg、约900mg、约1000mg、约1100mg、约1200mg、约1300mg、约1400mg或约1500mg的剂量对人施用。在一些实施方案中，抗PD-L1抗体MPDL3280A每三周(q3w)按1200mg IV施用。剂量可以作为单个剂量或作为多个剂量(例如2或3个剂量)施用，如输注。与单一治疗相比，联合治疗中施用的抗体剂量可以降低。早期通过常规技术监测此疗法的进展。In general, whether administered once or multiple times, the therapeutically effective amount of an antibody (e.g., an anti-PD-L1 antibody) administered to a person will be in the range of about 0.01 to about 50 mg/kg of patient body weight. In some embodiments, the antibody used is, for example, administered daily at about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg. In some embodiments, the antibody is administered at 15 mg/kg. However, other dosage regimens may be used. In one embodiment, the anti-PD-L1 antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg on day 1 of a 21-day cycle. In some embodiments, the anti-PD-L1 antibody MPDL3280A is administered IV at 1200 mg every three weeks (q3w). The dose can be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as by infusion. The dose of the antibody administered in the combination therapy can be reduced compared to a monotherapy. The progress of this therapy is monitored early by conventional techniques.

一般而言，无论一次还是多次施用，对人施用的紫杉烷(例如纳米清蛋白结合紫杉醇或紫杉醇)的治疗有效量将在约25至约300mg/m²的范围内(例如约25mg/m²、约50mg/m²、约75mg/m²、约100mg/m²、约125mg/m²、约150mg/m²、约175mg/m²、约200mg/m²、约225mg/m²、约250mg/m²、约275mg/m²、或约300mg/m²)。例如，在一些实施方案中，施用约100mg/m²的纳米清蛋白结合紫杉醇在一些实施方案中，每周(q1w)按100mg/m²IV施用纳米清蛋白结合紫杉醇在一些实施方案中，施用约200mg/m²的紫杉醇。在一些实施方案中，每3周按200mg/m²IV施用紫杉醇。在一些实施方案中，紫杉烷(例如纳米清蛋白结合紫杉醇或紫杉醇)可以每周、每2周、每3周、每4周、在每个21天循环的第1、8和15天、或在每个28天循环的第1、8和15天施用。Generally, a therapeutically effective amount of a taxane (e.g., nanoalbumin-bound paclitaxel or paclitaxel) administered to a human, whether administered once or multiple times, will be in the range of about 25 to about 300 mg/m² (e.g., about 25 mg/m² , about 50 mg/m² , about 75 mg/m² , about 100 mg/m² , about 125 mg/m² , about 150 mg/m² , about 175 mg/m² , about 200 mg/m² , about 225 mg/m² , about 250 mg/m² , about 275 mg/m² , or about 300 mg/m² ). For example, in some embodiments, nanoalbumin-bound paclitaxel is administered at about 100 mg/m^2. In some embodiments, nanoalbumin-bound paclitaxel is administered weekly (q1w) at 100 mg/m² IV. In some embodiments, about 200 mg/m² paclitaxel is administered. In some embodiments, paclitaxel is administered at 200 mg/m² IV every 3 weeks. In some embodiments, the taxane (e.g., nanoalbumin-bound paclitaxel or paclitaxel) can be administered weekly, every 2 weeks, every 3 weeks, every 4 weeks, on days 1, 8, and 15 of each 21-day cycle, or on days 1, 8, and 15 of each 28-day cycle.

在一些实施方案中，该方法可以进一步包括附加治疗。该附加治疗可以是放射治疗、手术(例如肿块切除术和乳房切除术)、化疗、基因治疗、DNA治疗、病毒治疗、RNA治疗、免疫治疗、骨髓移植、纳米治疗(nanotherapy)、单克隆抗体治疗或前面这些的组合。该附加治疗可以是辅助治疗或新辅助治疗的形式。在一些实施方案中，该附加治疗是施用小分子酶抑制剂或抗转移剂。在一些实施方案中，该附加治疗是施用副作用限制剂(例如旨在减轻治疗副作用的发生和/或严重度的药物，如抗恶心剂等)。在一些实施方案中，该附加治疗是放射治疗。在一些实施方案中，该附加治疗是手术。在一些实施方案中，该附加治疗是放射治疗和手术的组合。在一些实施方案中，该附加治疗是γ照射。在一些实施方案中，该附加治疗是靶向PI3K/AKT/mTOR途径的治疗、HSP90抑制剂、微管蛋白抑制剂、凋亡抑制剂和/或化学预防剂。该附加治疗可以是上文所述的一种或多种化疗剂。In some embodiments, the method may further include additional treatment. The additional treatment can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy (nanotherapy), monoclonal antibody therapy, or a combination of the above. The additional treatment can be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional treatment is the administration of small molecule enzyme inhibitors or anti-metastatic agents. In some embodiments, the additional treatment is the administration of side effect limiters (e.g., drugs intended to reduce the occurrence and/or severity of treatment side effects, such as anti-nausea agents, etc.). In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiation therapy and surgery. In some embodiments, the additional treatment is gamma irradiation. In some embodiments, the additional treatment is the treatment of the targeted PI3K/AKT/mTOR pathway, HSP90 inhibitors, tubulin inhibitors, apoptosis inhibitors, and/or chemopreventive agents. The additional treatment can be one or more chemotherapeutics as described above.

在一些实施方案中，该方法进一步包括与PD-1轴结合拮抗剂和紫杉烷一起施用基于铂的化疗剂。在一些实施方案中，该基于铂的化疗剂是卡铂。卡铂的剂量和施用为本领域公知。按6mg/ml的目标曲线下面积(AUC)施用卡铂的示例性剂量。在一些实施方案中，每3周静脉内施用卡铂。In some embodiments, the method further comprises administering a platinum-based chemotherapeutic agent together with the PD-1 axis binding antagonist and the taxane. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin. Dosages and administration of carboplatin are well known in the art. An exemplary dose of carboplatin is administered at a target area under the curve (AUC) of 6 mg/ml. In some embodiments, carboplatin is administered intravenously every 3 weeks.

在一些实施方案中，该方法包括施用以下：每3周(q3w)IV施用的1200mg抗PD-L1抗体MPDL3280A，每周(q1w)100mg/m²IV的纳米清蛋白结合紫杉醇及每3周(q3w)6mg/ml目标AUC的卡铂。在一些实施方案中，该方法包括施用以下：每3周(q3w)IV施用的1200mg抗PD-L1抗体MPDL3280A，每3周200mg/m²IV的紫杉醇，及每3周(q3w)6mg/ml目标AUC的卡铂。In some embodiments, the method comprises administering the following: 1200 mg of the anti-PD-L1 antibody MPDL3280A administered IV every 3 weeks (q3w), 100 mg/m² of nanoalbumin-bound paclitaxel IV weekly (q1w), and a target AUC of 6 mg/ml carboplatin every 3 weeks (q3w). In some embodiments, the method comprises administering the following: 1200 mg of the anti-PD-L1 antibody MPDL3280A administered IV every 3 weeks (q3w), 200 mg/m² of paclitaxel IV every 3 weeks, and a target AUC of 6 mg/ml carboplatin every 3 weeks (q3w).

V.其他联合治疗V. Other combination therapies

本文还提供用于在个体中治疗癌症或延迟癌症进展的方法，其包括与另一抗癌剂或癌症疗法结合(conjunction)，对该个体施用人PD-1轴结合拮抗剂和紫杉烷。在一些实施方案中，该方法包括与另一抗癌剂或癌症疗法结合，对该个体施用人PD-1轴结合拮抗剂、紫杉烷和基于铂的化疗剂。Also provided herein are methods for treating cancer or delaying cancer progression in an individual comprising administering to the individual a human PD-1 axis binding antagonist and a taxane in conjunction with another anticancer agent or cancer therapy. In some embodiments, the method comprises administering to the individual a human PD-1 axis binding antagonist, a taxane, and a platinum-based chemotherapy agent in conjunction with another anticancer agent or cancer therapy.

在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与化疗或化疗剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与放疗或放疗剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与靶向治疗或靶向治疗剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与免疫治疗或免疫治疗剂(例如单克隆抗体)结合施用。In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with chemotherapy or a chemotherapeutic agent. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with radiotherapy or a radiotherapeutic agent. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with a targeted therapy or a targeted therapeutic agent. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an immunotherapy or an immunotherapeutic agent (e.g., a monoclonal antibody).

不希望受限于理论，认为通过促进激活性共刺激分子或通过抑制负共刺激分子增强T细胞刺激可以促进肿瘤细胞死亡，从而治疗癌症或延迟癌症进展。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与针对激活性共刺激分子的激动剂结合施用。在一些实施方案中，激活性共刺激分子可以包括CD40、CD226、CD28、OX40、GITR、CD137、CD27、HVEM或CD127。在一些实施方案中，针对激活性共刺激分子的激动剂是结合CD40、CD226、CD28、OX40、GITR、CD137、CD27、HVEM或CD127的激动抗体。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与针对抑制性共刺激分子的拮抗剂结合施用。在一些实施方案中，抑制性共刺激分子可以包括CTLA-4(也称为CD152)、PD-1、TIM-3、BTLA、VISTA、LAG-3、B7-H3、B7-H4、IDO、TIGIT、MICA/B或精氨酸酶。在一些实施方案中，针对抑制性共刺激分子的拮抗剂是结合CTLA-4(也称为CD152)、PD-1、TIM-3、BTLA、VISTA、LAG-3、B7-H3、B7-H4、IDO、TIGIT、MICA/B或精氨酸酶的拮抗抗体。Without wishing to be bound by theory, it is believed that by promoting activating co-stimulatory molecules or by inhibiting negative co-stimulatory molecules to enhance T cell stimulation, tumor cell death can be promoted, thereby treating cancer or delaying cancer progression. In some embodiments, the PD-1 axis binding antagonist and taxane can be combined with an agonist for activating co-stimulatory molecules for administration. In some embodiments, the activating co-stimulatory molecules may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM or CD127. In some embodiments, the agonist for activating co-stimulatory molecules is an agonist antibody that binds CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM or CD127. In some embodiments, the PD-1 axis binding antagonist and taxane can be combined with an antagonist for inhibitory co-stimulatory molecules for administration. In some embodiments, inhibitory costimulatory molecules can include CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B or arginase. In some embodiments, the antagonist to an inhibitory costimulatory molecule is an antagonistic antibody that binds to CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B or arginase.

在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与针对CTLA-4(也称为CD152)的拮抗剂(例如，阻断抗体)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与伊匹单抗(也称为MDX-010、MDX-101或)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与tremelimumab(也称为ticilimumab或CP-675,206)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与针对B7-H3(也称为CD276)的拮抗剂(例如，阻断抗体)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与MGA271结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与针对TGFβ的拮抗剂(例如，metelimumab(也称为CAT-192)、fresolimumab(也称为GC1008)或LY2157299)结合施用。In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an antagonist (e.g., a blocking antibody) against CTLA-4 (also known as CD152). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with ipilimumab (also known as MDX-010, MDX-101, or ). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with tremelimumab (also known as ticilimumab or CP-675,206). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an antagonist (e.g., a blocking antibody) against B7-H3 (also known as CD276). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with MGA271. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with an antagonist directed against TGFβ (eg, metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299).

在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与包括表达嵌合抗原受体(CAR)的T细胞(例如，细胞毒性T细胞或CTL)的过继转移的治疗结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与包括含有显性阴性TGFβ受体(例如，显性阴性TGFβII型受体)的T细胞的过继转移的治疗结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与包括HERCREEM流程(参见例如ClinicalTrials.gov Identifier NCT00889954)的治疗结合施用。In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in conjunction with a treatment comprising adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in conjunction with a treatment comprising adoptive transfer of T cells containing a dominant negative TGFβ receptor (e.g., a dominant negative TGFβ type II receptor). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in conjunction with a treatment comprising the HERCREEM process (see, e.g., ClinicalTrials.gov Identifier NCT00889954).

在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与针对CD137(也称为TNFRSF9、4-1BB或ILA)的激动剂(例如，激活抗体)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与urelumab(也称为BMS-663513)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与针对CD40的激动剂(例如，激活抗体)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与CP-870893结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与针对OX40(也称为CD134)的激动剂(例如，激活抗体)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与抗OX40抗体(例如AgonOX)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与针对CD27的激动剂(例如，激活抗体)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与CDX-1127结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与针对吲哚胺-2,3-双加氧酶(IDO)的拮抗剂结合施用。在一些实施方案中，该IDO拮抗剂是1-甲基-D-色氨酸(也称为1-D-MT)。In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an agonist (e.g., an activating antibody) for CD137 (also known as TNFRSF9, 4-1BB, or ILA). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with urelumab (also known as BMS-663513). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an agonist (e.g., an activating antibody) for CD40. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with CP-870893. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an agonist (e.g., an activating antibody) for OX40 (also known as CD134). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an anti-OX40 antibody (e.g., AgonOX). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an agonist for CD27 (e.g., an activating antibody). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with CDX-1127. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an antagonist for indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).

在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与抗体-药物缀合物结合施用。在一些实施方案中，该抗体-药物缀合物包含mertansine或monomethyl auristatin E(MMAE)。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与抗NaPi2b抗体-MMAE缀合物(也称为DNIB0600A或RG7599)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与trastuzumab emtansine(也称为T-DM1、ado-trastuzumab emtansine或Genentech)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与DMUC5754A结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与靶向内皮素B受体(EDNBR)的抗体-药物缀合物(例如，与MMAE缀合的抗EDNBR抗体)结合施用。In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in conjunction with an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in conjunction with an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in conjunction with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine or Genentech). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in conjunction with DMUC5754A. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in conjunction with an antibody-drug conjugate targeting the endothelin B receptor (EDNBR) (e.g., an anti-EDNBR antibody conjugated to MMAE).

在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与血管发生抑制剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与抗VEGF抗体(例如，VEGF-A)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与贝伐单抗(也称为Genentech)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与抗血管生成素2(也称为Ang2)抗体结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与MEDI3617结合施用。In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with an angiogenesis inhibitor. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with an anti-VEGF antibody (e.g., VEGF-A). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with bevacizumab (also known as Genentech). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with an anti-angiopoietin 2 (also known as Ang2) antibody. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with MEDI3617.

在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与抗肿瘤剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与靶向CSF-1R(也称为M-CSFR或CD115)的药物结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与抗CSF-1R(也称为IMC-CS4)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与干扰素(例如，干扰素α或干扰素γ)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与Roferon-A(也称为重组干扰素α-2a)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与GM-CSF(也称为重组人粒细胞巨噬细胞集落刺激因子、rhu GM-CSF、沙格司亭或)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与IL-2(也称为阿地白介素或)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与IL-12结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与靶向CD20的抗体结合施用。在一些实施方案中，该靶向CD20的抗体是obinutuzumab(也称为GA101或)或利妥昔单抗。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与靶向GITR的抗体结合施用。在一些实施方案中，该靶向GITR的抗体是TRX518。In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an anti-tumor agent. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with a drug targeting CSF-1R (also known as M-CSFR or CD115). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with anti-CSF-1R (also known as IMC-CS4). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an interferon (e.g., interferon alpha or interferon gamma). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with Roferon-A (also known as recombinant interferon alpha-2a). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim or). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with IL-2 (also known as aldesleukin or IL-2). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with IL-12. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an antibody targeting CD20. In some embodiments, the antibody targeting CD20 is obinutuzumab (also known as GA101 or IL-20) or rituximab. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an antibody targeting GITR. In some embodiments, the antibody targeting GITR is TRX518.

在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与癌症疫苗结合施用。在一些实施方案中，该癌症疫苗是肽癌症疫苗，其在一些实施方案中是个性化肽疫苗。在一些实施方案中，该肽癌症疫苗是多价长肽、多肽、肽混合物、杂合肽或肽脉冲处理的树突细胞疫苗(参见例如Yamada等,Cancer Sci,104:14-21,2013)。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与佐剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与包括TLR激动剂(例如，Poly-ICLC(也称为)、LPS、MPL或CpG ODN)的治疗结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与肿瘤坏死因子(TNF)α结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与IL-1结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与HMGB1结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与IL-10拮抗剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与IL-4拮抗剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与IL-13拮抗剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与HVEM拮抗剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与ICOS激动剂(例如，通过施用COS-L或抗ICOS的激动性抗体)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与靶向CX3CL1的治疗结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与靶向CXCL9的治疗结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与靶向CXCL10的治疗结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与靶向CCL5的治疗结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与LFA-1或ICAM1激动剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与选择蛋白激动剂结合施用。In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the peptide cancer vaccine is a multivalent long peptide, a polypeptide, a peptide mixture, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, for example, Yamada et al., Cancer Sci, 104: 14-21, 2013). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an adjuvant. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with a treatment including a TLR agonist (e.g., Poly-ICLC (also known as), LPS, MPL, or CpG ODN). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with tumor necrosis factor (TNF) α. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with IL-1. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with HMGB1. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an IL-10 antagonist. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an IL-4 antagonist. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an IL-13 antagonist. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an HVEM antagonist. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with an ICOS agonist (e.g., by administering COS-L or an agonist antibody against ICOS). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with a treatment targeting CX3CL1. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with a treatment targeting CXCL9. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with a therapy targeting CXCL10. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with a therapy targeting CCL5. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with an LFA-1 or ICAM1 agonist. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with a selectin agonist.

在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与靶向治疗结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与B-Raf抑制剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与vemurafenib(也称为)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与达拉非尼(dabrafenib)(也称为)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与埃罗替尼(也称为)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与MEK(如MEK1(也称为MAP2K1)或MEK2(也称为MAP2K2))抑制剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与cobimetinib(也称为GDC-0973或XL-518)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与曲美替尼(trametinib)(也称为)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与K-Ras抑制剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与c-Met抑制剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与onartuzumab(也称为MetMAb)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与Alk抑制剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与AF802(也称为CH5424802或alectinib)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与磷脂酰肌醇3-激酶(PI3K)抑制剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与BKM120结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与idelalisib(也称为GS-1101或CAL-101)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与哌立福辛(也称为KRX-0401)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与Akt抑制剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与MK2206结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与GSK690693结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与GDC-0941结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与mTOR抑制剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与sirolimus(也称为雷帕霉素)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与西罗莫司(temsirolimus)(也称为CCI-779或)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与依维莫司(everolimus)(也称为RAD001)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与ridaforolimus(也称为AP-23573、MK-8669或deforolimus)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与OSI-027结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与AZD8055结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与INK128结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与双重PI3K/mTOR抑制剂结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与XL765结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与GDC-0980结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与BEZ235(也称为NVP-BEZ235)结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与BGT226结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与GSK2126458结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与PF-04691502结合施用。在一些实施方案中，PD-1轴结合拮抗剂和紫杉烷可以与PF-05212384(也称为PKI-587)结合施用。In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with a targeted therapy. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with a B-Raf inhibitor. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with vemurafenib (also known as vemurafenib). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with dabrafenib (also known as dabrafenib). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with erlotinib (also known as erlotinib). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with a MEK (such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2)) inhibitor. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with cobimetinib (also known as GDC-0973 or XL-518). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with trametinib (also known as ). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with a K-Ras inhibitor. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with a c-Met inhibitor. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with onartuzumab (also known as MetMAb). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with an Alk inhibitor. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with AF802 (also known as CH5424802 or alectinib). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with a phosphatidylinositol 3-kinase (PI3K) inhibitor. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with BKM120. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with perifosine (also known as KRX-0401). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with an Akt inhibitor. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with MK2206. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with GSK690693. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with GDC-0941. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with an mTOR inhibitor. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with sirolimus (also known as rapamycin). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with temsirolimus (also known as CCI-779 or ). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with everolimus (also known as RAD001). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with ridaforolimus (also known as AP-23573, MK-8669, or deforolimus). In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with OSI-027. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with AZD8055. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with INK128. In some embodiments, the PD-1 axis binding antagonist and the taxane can be administered in combination with a dual PI3K/mTOR inhibitor. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with XL765. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with GDC-0980. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with BEZ235 (also known as NVP-BEZ235). In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with BGT226. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with GSK2126458. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with PF-04691502. In some embodiments, a PD-1 axis binding antagonist and a taxane can be administered in combination with PF-05212384 (also known as PKI-587).

VI.制成品或药盒VI. Finished products or medicine boxes

在本发明的另一实施方案中，提供包含PD-1轴结合拮抗剂和/或紫杉烷的制成品或药盒。在一些实施方案中，该制成品或药盒进一步包含包装说明书，该包装说明书包含用该PD-1轴结合拮抗剂与紫杉烷结合来在个体中治疗癌症或延迟癌症进展或在患有癌症的个体中增强免疫功能的说明。本文所述的任意PD-1轴结合拮抗剂和/或紫杉烷都可以包含在该制成品或药盒中。In another embodiment of the present invention, an article of manufacture or kit comprising a PD-1 axis binding antagonist and/or a taxane is provided. In some embodiments, the article of manufacture or kit further comprises a package insert comprising instructions for using the PD-1 axis binding antagonist in combination with a taxane to treat cancer or delay cancer progression in an individual, or to enhance immune function in an individual with cancer. Any PD-1 axis binding antagonist and/or taxane described herein may be included in the article of manufacture or kit.

在一些实施方案中，该PD-1轴结合拮抗剂和该紫杉烷处于相同的容器或分开的容器中。适宜的容器包括例如瓶、小管、袋和注射器。容器可以形成自多种材料，如玻璃、塑料(如聚氯乙烯或聚烯烃)或合金(如不锈钢或哈斯特镍合金)。在一些实施方案中，该容器容纳制剂，容器上或与容器结合的标签可以指示使用说明。该制成品或药盒可以进一步包含商业和用户角度希望的其他材料，包括其他缓冲液、稀释液、滤器、针头、注射器，及含有使用说明的包装说明书。在一些实施方案中，该制成品可以进一步包含一种或多种其他活性剂(例如，化疗剂和抗肿瘤剂)。适合用于该一种或多种活性剂的容器包括例如瓶、小管、袋和注射器。In some embodiments, the PD-1 axis binding antagonist and the taxane are in the same container or separate containers. Suitable containers include, for example, bottles, tubes, bags, and syringes. The container can be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin) or alloy (such as stainless steel or Hastelloy nickel alloy). In some embodiments, the container holds the formulation, and a label on or associated with the container can indicate instructions for use. The manufactured product or kit may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use. In some embodiments, the manufactured product may further include one or more other active agents (e.g., chemotherapeutic agents and anti-tumor agents). Suitable containers for the one or more active agents include, for example, bottles, tubes, bags, and syringes.

认为本说明书足以使得本领域技术人员能够实施本发明。对本领域技术人员而言，除本文所显示和描述的那些之外，本发明的多种修改将从前面的描述变得显而易见，且落在所附权利要求的范围之内。This description is considered to be sufficient to enable those skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

实施例Example

本发明将通过参考以下实施例得到更充分理解。但是，它们不应解释为限制本发明的范围。应理解，本文所述的实施例和实施方案仅是为了说明的目的，将向本领域技术人员提示其多种修改或改变，这些修改或改变也包括在本申请的精神和范围及所附权利要求的范围之内。The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only and are intended to suggest various modifications or variations thereof to those skilled in the art, which are intended to be within the spirit and scope of this application and the scope of the appended claims.

实施例1：抗PD-L1抗体和纳米清蛋白结合紫杉醇+卡铂联合治疗在MC38结肠直肠肿瘤模型中达到持续完全应答Example 1: Combination therapy of anti-PD-L1 antibody and nanoalbumin combined with paclitaxel + carboplatin achieves sustained complete response in the MC38 colorectal tumor model

材料和方法Materials and methods

体内肿瘤模型In vivo tumor models

MC38结肠直肠肿瘤细胞系维持在Genentech。用0.1x10⁶MC38细胞在右单侧胁腹皮下接种7-10周龄C57BL/6雌性小鼠(Charles River Laboratories；Hollister,CA)。肿瘤达到约100-300mm³的平均肿瘤体积时，招募小鼠，随机分入治疗组，并在随后第1天开始抗体和/或化学治疗。The MC38 colorectal tumor cell line is maintained at Genentech. Seven- to 10-week-old C57BL/6 female mice (Charles River Laboratories; Hollister, CA) were inoculated subcutaneously in the right flank with 0.1 x¹⁰ MC38 cells. Mice were enrolled and randomized into treatment groups when tumors reached an average tumor volume of approximately 100-300 mm³ . Antibody and/or chemotherapy treatment was initiated on day 1 of the subsequent study.

用混合建模法分析来自相同动物的肿瘤体积重复测量结果随时间的变化(Pinheiro等nmle:Linear and Nonlinear Mixed Effects Models.R.package version3.1-108(2013))。此方法处理研究结束前的重复测量结果和适度丢失二者。用三次回归样条拟合非线性特征为不同处理下的log2(肿瘤体积)的时程。使用nlme软件包3.1 108版(RFoundation for Statistical Computing；维也纳,奥地利)，通过2.15.2版R内的线性混合效应模型进行拟合。Repeated measurements of tumor volume from the same animals over time were analyzed using a mixed modeling approach (Pinheiro et al. nmle: Linear and Nonlinear Mixed Effects Models. R. package version 3.1-108 (2013)). This approach handles both repeated measurements and moderate dropout before the end of the study. A cubic regression spline was used to fit the time course of log2(tumor volume) under different treatments, with the nonlinear characteristic being the time course. Linear mixed effects models were fitted in R version 2.15.2 using the nlme package version 3.1.108 (R Foundation for Statistical Computing; Vienna, Austria).

对于图5A-5B中所示的MC38再攻击实验，用0.1x10⁶MC38细胞在初次肿瘤攻击的对侧胁腹皮下接种之前用抗PD-L1++卡铂组合处理治愈的小鼠。平行地，还用0.1x10⁶MC38细胞接种首次用于实验的雌性C57BL/6小鼠。7天后，对所有小鼠实施安乐死，收集脾脏进行流式细胞术分析。所有动物研究都按照动物福利法、实验动物护理和使用指南及实验动物护理和使用委员会(IACUC)指南中所述的指南和规定进行。For the MC38 rechallenge experiment shown in Figures 5A-5B, cured mice were treated with an anti-PD-L1++carboplatin combination prior to subcutaneous inoculation of 0.1x10⁶ MC38 cells in the contralateral flank of the initial tumor challenge. In parallel, naive female C57BL/6 mice were also inoculated with 0.1x10⁶ MC38 cells. After 7 days, all mice were euthanized and spleens were collected for flow cytometry analysis. All animal studies were performed in accordance with the guidelines and regulations described in the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee (IACUC).

再攻击小鼠的脾细胞培养物的体外刺激In vitro stimulation of splenocyte cultures from re-challenged mice

用10ng/ml 12-豆蔻酸13-乙酸佛波酯(PMA)和1μg/ml离子霉素(Sigma-Alrich；St.Louis,MO)加GOLGIPLUG^TM(布雷菲尔德菌素A)(BD Biosciences；San Jose,CA)将脾细胞按1x10⁶细胞/孔一式三份地在96孔U形底平板中37℃培养4小时。收集细胞，用表面标记CD4FITC(异硫氰酸荧光素)、CD3PE(藻红蛋白)和CD8PerCp-Cy5.5(BD Biosciences)染色，用4％多聚甲醛冰上固定30分钟。用1x透化缓冲液(BD Biosciences)透化细胞，用大鼠抗小鼠抗干扰素-γ(IFN-γ)-别藻蓝蛋白(APC)缀合抗体或大鼠IgG1-APC同种型对照抗体(BDBiosciences)染色，用FACSDIVA^TM软件在BD Biosciences LSRII上运行。用FlowJo软件(TreeStar)进行流式细胞术分析。Splenocytes were cultured in triplicate at 1 x 10 cells/well in 96-well U-bottom plates at 37°C for 4 hours with 10 ng/ml phorbol¹² -myristate 13-acetate (PMA) and 1 μg/ml ionomycin (Sigma-Alrich; St. Louis, MO) plus GOLGIPLUG^™ (Brefeldin A) (BD Biosciences; San Jose, CA). Cells were harvested, stained with surface markers CD4 FITC (fluorescein isothiocyanate), CD3 PE (phycoerythrin), and CD8 PerCp-Cy5.5 (BD Biosciences), and fixed with 4% paraformaldehyde on ice for 30 minutes. Cells were permeabilized with 1× permeabilization buffer (BD Biosciences) and stained with rat anti-mouse anti-interferon-γ (IFN-γ)-allophycocyanin (APC) conjugated antibody or rat IgG1-APC isotype control antibody (BD Biosciences) and run on a BD Biosciences LSRII using FACSDIVA^™ software. Flow cytometric analysis was performed using FlowJo software (TreeStar).

抗体和治疗Antibodies and treatments

所有治疗抗体都在Genentech产生。对照抗体是抗gp120鼠IgG1(mIgG1)克隆10E7.1D2。抗PD-L1为人/小鼠反向嵌合体克隆YW243.55.S70mIgG2a.DANA或全小鼠克隆25A1mIgG2a.DANA。获自Abraxis Bioscience,Inc.(Celgene所拥有；Summit,NJ)。卡铂获自Hospira,Inc.(Lake Forest,IL)。地塞米松获自West-WardPharmaceuticals(Eatontown,NJ)。给药时间表和给药途径如附图简述中所示。抗体稀释在PBS或20mM组氨酸醋酸盐、240mM蔗糖、0.02％聚山梨酸酯20、pH＝5.5中。化疗剂和地塞米松稀释在盐水中。All therapeutic antibodies were generated at Genentech. The control antibody was anti-gp120 murine IgG1 (mIgG1) clone 10E7.1D2. Anti-PD-L1 was either the human/mouse reverse chimera clone YW243.55.S70mIgG2a.DANA or the full mouse clone 25A1mIgG2a.DANA. They were obtained from Abraxis Bioscience, Inc. (owned by Celgene; Summit, NJ). Carboplatin was obtained from Hospira, Inc. (Lake Forest, IL). Dexamethasone was obtained from West-Ward Pharmaceuticals (Eatontown, NJ). Dosing schedules and routes of administration are as shown in the accompanying figures. Antibodies were diluted in PBS or 20 mM histidine acetate, 240 mM sucrose, 0.02% polysorbate 20, pH 5.5. Chemotherapeutic agents and dexamethasone were diluted in saline.

体内接种研究In vivo inoculation studies

用MACS CD8分离药盒(Miltenyi Biotec)通过阴性选择从供体OTI Thy1.1雌性脾脏和肠系膜淋巴结(Genentech colony)分离OTI Thy1.1CD8+T细胞。纯化的CD8+细胞用CFSE(Life Technologies；Grand Island,NY)标记，并将2.5x10⁶个细胞静脉内(IV)注入雌性C57BL/6雌性受体(Charles River Laboratories)。第二天，通过用250ng与全长卵清蛋白融合的抗DEC205(在Genentech产生)加盐水腹腔内注射(IP)，或用250ng与全长卵清蛋白融合的抗DEC205(在Genentech产生)加4mg/kg的地塞米松IV注射来接种小鼠。接种两天后，对小鼠实施安乐死，收集脾脏进行分析。使用活细胞事件与已知浓度的固定量的荧光小球(目录号9003-53-6,Polysciences,Inc.；Warrington,PA)的比值，通过流式细胞术测定脾细胞悬液的总细胞计数。通过用Thy1.1PE-Cy7和CD8Pacific Blue(BD Biosciences)染色并用FACSDIVA^TM软件在BD Biosciences LSRII上运行，通过流式细胞术鉴定OTI CD8+T细胞。用FlowJo软件(TreeStar)进行流式细胞术分析。OTI Thy1.1 CD8+ T cells were isolated from donor OTI Thy1.1 female spleens and mesenteric lymph nodes (Genentech colony) by negative selection using a MACS CD8 separation kit (Miltenyi Biotec). Purified CD8+ cells were labeled with CFSE (Life Technologies; Grand Island, NY) and^2.5x106 cells were injected intravenously (IV) into female C57BL/6 female recipients (Charles River Laboratories). The next day, mice were inoculated with 250ng of anti-DEC205 fused to full-length ovalbumin (produced at Genentech) plus saline intraperitoneal injection (IP), or with 250ng of anti-DEC205 fused to full-length ovalbumin (produced at Genentech) plus 4mg/kg of dexamethasone IV injection. Two days after inoculation, mice were euthanized and spleens were collected for analysis. Total cell counts of splenocyte suspensions were determined by flow cytometry using the ratio of live cell events to a fixed amount of fluorescent beads (Cat. No. 9003-53-6, Polysciences, Inc.; Warrington, PA) at known concentrations. OTI CD8+ T cells were identified by flow cytometry by staining with Thy1.1 PE-Cy7 and CD8 Pacific Blue (BD Biosciences) and running on a BD Biosciences LSRII using FACSDIVA^™ software. Flow cytometric analysis was performed using FlowJo software (TreeStar).

结果result

此研究在癌症治疗的背景中评价抗PD-L1抗体在临床前小鼠肿瘤模型中的功效。与对照抗体或紫杉醇+卡铂单独治疗相比，抗PD-L1抗体(克隆25A1mIgG2a.DANA)和紫杉醇+卡铂的联合治疗在同基因MC38结肠直肠肿瘤模型中产生协同抗肿瘤反应(图1)。与对照抗体或紫杉醇+卡铂单独治疗组中没有一只小鼠具有部分反应相比，10％的小鼠(1/10)对抗PD-L1和紫杉醇+卡铂联合治疗具有部分反应(图1)。导致肿瘤大小减小的强抗肿瘤反应记录为部分反应(FR)，在此实施例中定义为从初始肿瘤体积减小>50％且<100％，或记录为完全应答，在此实施例中定义为肿瘤体积减小100％。抗PD-L1抗体和紫杉醇+卡铂的联合治疗还延迟进展时间。对照抗体的进展时间(TTP)(在此实施例中定义为5x初始肿瘤体积)为11天，紫杉醇+卡铂为15.5天，抗PD-L1抗体和紫杉醇+卡铂的联合治疗为25天。This study evaluated the efficacy of anti-PD-L1 antibodies in preclinical mouse tumor models in the context of cancer treatment. Combination treatment with anti-PD-L1 antibody (clone 25A1mIgG2a.DANA) and paclitaxel + carboplatin produced a synergistic anti-tumor response in the syngeneic MC38 colorectal tumor model compared to control antibody or paclitaxel + carboplatin alone (Figure 1). 10% of mice (1/10) had a partial response to the combination treatment with anti-PD-L1 and paclitaxel + carboplatin, compared to none of the mice in the control antibody or paclitaxel + carboplatin alone groups (Figure 1). Strong anti-tumor responses that resulted in a decrease in tumor size were recorded as partial responses (FR), defined in this example as a decrease of >50% and <100% from the initial tumor volume, or as complete responses, defined in this example as a 100% decrease in tumor volume. Combination treatment with anti-PD-L1 antibody and paclitaxel + carboplatin also delayed time to progression. The time to progression (TTP) (defined in this example as 5x initial tumor volume) was 11 days for the control antibody, 15.5 days for paclitaxel + carboplatin, and 25 days for the combination of anti-PD-L1 antibody and paclitaxel + carboplatin.

在临床背景中，紫杉醇(其配制在具有潜在毒性的溶剂中)治疗通常涉及用糖皮质激素如地塞米松预先给药，以降低过敏反应的可能性。但是，糖皮质激素如地塞米松具有免疫抑制作用，可以抑制T细胞反应，其转而可以降低PD-1轴结合拮抗剂如抗PD-L1剂的活性。一致地，地塞米松的施用废除了单一活性剂抗PD-L1治疗在同基因MC38结肠直肠肿瘤模型中的功效(图2A和2B)。此外，地塞米松抑制OTI过继T细胞转移和接种模型中的抗原特异性T细胞反应(图3)。因此，不希望受限于理论，诸如地塞米松的糖皮质激素治疗可以抑制或抵消PD-1轴结合拮抗剂如抗PD-L1治疗的一些益处，从而减少T细胞功能的增强及其促进抗肿瘤反应(如CD8+T细胞介导的肿瘤杀伤)的能力。In the clinical setting, treatment with paclitaxel (which is formulated in a potentially toxic solvent) typically involves pre-administration with a glucocorticoid such as dexamethasone to reduce the likelihood of an allergic reaction. However, glucocorticoids such as dexamethasone have immunosuppressive effects and can suppress T cell responses, which in turn can reduce the activity of PD-1 axis binding antagonists such as anti-PD-L1 agents. Consistently, administration of dexamethasone abolished the efficacy of single-agent anti-PD-L1 therapy in a syngeneic MC38 colorectal tumor model (Figures 2A and 2B). In addition, dexamethasone inhibited antigen-specific T cell responses in the OTI adoptive T cell transfer and vaccination models (Figure 3). Therefore, without wishing to be bound by theory, glucocorticoid treatment such as dexamethasone can inhibit or counteract some of the benefits of PD-1 axis binding antagonists such as anti-PD-L1 therapy, thereby reducing the enhancement of T cell function and its ability to promote anti-tumor responses (such as CD8+ T cell-mediated tumor killing).

与对照抗体、单一活性剂抗PD-L1抗体或+卡铂单独治疗相比，抗PD-L1抗体(嵌合YW243.55.S70.mIgG2a.DANA)和纳米清蛋白结合紫杉醇+卡铂的联合治疗在同基因MC38结肠直肠肿瘤模型中产生意料之外的强协同抗肿瘤功效(图4A和4B)。抗PD-L1抗体和+卡铂联合治疗在4/8小鼠中达到持续超过90天的持续完全应答(图4A和4B)。此协同作用比作为抗PD-L1和紫杉醇+卡铂联合治疗的结果观察到的协同作用更强。对照抗体单独的TTP(5x初始肿瘤体积)为11.5天，抗PD-L1抗体单独为9天，+卡铂单独为13.5天，不适用于抗PD-L1抗体和+卡铂的联合治疗，其中4/8小鼠显示完全消退。这表明，抗PD-L1抗体和+卡铂的联合治疗以比抗PD-L1抗体和紫杉醇+卡铂联合治疗更大的程度强烈推迟进展时间。此外，来自抗PD-L1和+卡铂联合治疗的所有治愈鼠(即显示完全应答的小鼠)都能够完全排斥同一MC38肿瘤细胞系的二次攻击，表明治疗产生了T细胞记忆反应(图5A-5B)。与首次用于实验的首次攻击的小鼠相比，如通过来自CD4+T和CD8+T细胞二者的增强的干扰素-γ(IFN-γ)产生所观察，来自这些治愈小鼠的脾细胞的体外再刺激显示增强的T细胞效应子功能(图5A-5B)。Combination therapy of anti-PD-L1 antibody (chimeric YW243.55.S70.mIgG2a.DANA) and nanoalbumin-bound paclitaxel + carboplatin produced unexpectedly strong synergistic anti-tumor efficacy in the syngeneic MC38 colorectal tumor model compared to control antibody, single-agent anti-PD-L1 antibody, or + carboplatin alone (Figures 4A and 4B). Combination therapy of anti-PD-L1 antibody and + carboplatin achieved sustained complete responses lasting for more than 90 days in 4/8 mice (Figures 4A and 4B). This synergistic effect was stronger than that observed as a result of combination therapy with anti-PD-L1 and paclitaxel + carboplatin. The TTP (5x initial tumor volume) of the control antibody alone was 11.5 days, anti-PD-L1 antibody alone was 9 days, and + carboplatin alone was 13.5 days, which was not applicable to the combination therapy of anti-PD-L1 antibody and + carboplatin, where 4/8 mice showed complete regression. This shows that the combination of anti-PD-L1 antibody and + carboplatin strongly postpones the time to progression to a greater extent than the combination of anti-PD-L1 antibody and paclitaxel + carboplatin. In addition, all cured mice (i.e., mice that showed a complete response) from the combination of anti-PD-L1 and + carboplatin were able to completely reject the secondary attack of the same MC38 tumor cell line, indicating that the treatment produced a T cell memory response (Figures 5A-5B). Compared to the mice that were first attacked in the experiment, as observed by the enhanced interferon-γ (IFN-γ) production from both CD4+T and CD8+T cells, the in vitro restimulation of splenocytes from these cured mice showed enhanced T cell effector function (Figures 5A-5B).

惊人的强抗肿瘤协同活性及意料之外的获得完全应答和产生T细胞记忆反应的能力代表了PD-1轴结合拮抗剂和紫杉烷(如纳米清蛋白结合紫杉醇)联合治疗的重要治疗优势。此外，与紫杉醇治疗不同，纳米清蛋白结合紫杉醇治疗通常不涉及用糖皮质激素如地塞米松预先给药。本文提供的结果表明，PD-1轴结合拮抗剂(如抗PD-L1抗体)和纳米清蛋白结合紫杉醇联合治疗还使得能够实施更简单的治疗方案，该治疗方案可以避免使用糖皮质激素，从而降低潜在副作用的可能性。The surprisingly strong synergistic anti-tumor activity and the unexpected ability to obtain complete responses and generate T cell memory responses represent important therapeutic advantages of the combination therapy of PD-1 axis binding antagonists and taxanes (such as nanoalbumin combined with paclitaxel). In addition, unlike paclitaxel treatment, nanoalbumin combined with paclitaxel treatment does not usually involve pre-administration with glucocorticoids such as dexamethasone. The results provided herein indicate that the combination therapy of PD-1 axis binding antagonists (such as anti-PD-L1 antibodies) and nanoalbumin combined with paclitaxel also enables the implementation of a simpler treatment regimen that can avoid the use of glucocorticoids, thereby reducing the possibility of potential side effects.

实施例2：抗PD-L1抗体与纳米清蛋白结合紫杉醇和卡铂联合治疗在非小细胞肺癌患者的1b期临床试验中达到完全应答Example 2: Combination therapy of anti-PD-L1 antibody and nanoalbumin combined with paclitaxel and carboplatin achieves complete response in a phase 1b clinical trial in patients with non-small cell lung cancer

进行1b期临床研究，以评价抗PD-L1抗体(MPDL3280A)与紫杉烷(纳米清蛋白结合紫杉醇或紫杉醇)和卡铂组合的联合治疗对非小细胞肺癌(NSCLC)患者的功效。A Phase 1b clinical study was conducted to evaluate the efficacy of anti-PD-L1 antibody (MPDL3280A) in combination with a taxane (nab-paclitaxel or paclitaxel) and carboplatin in patients with non-small cell lung cancer (NSCLC).

此临床研究的给药方案如下：The dosing regimen for this clinical study is as follows:

1)MPDL3280A//卡铂联合治疗：(a)每3周(q3w)IV施用1200mgMPDL3280A；(b)每周(q1w)IV施用100mg/m²和(c)每3周(q3w)IV施用目标曲线下面积为6mg/ml的卡铂。1) MPDL3280A//carboplatin combination therapy: (a) 1200 mg MPDL3280A administered IV every 3 weeks (q3w); (b) 100 mg/^m2 administered IV weekly (q1w); and (c) carboplatin administered IV every 3 weeks (q3w) to a target area under the curve of 6 mg/ml.

2)MPDL3280A/紫杉醇/卡铂联合治疗：(a)每3周(q3w)IV施用1200mg MPDL3280A；(b)每3周(q3w)IV施用200mg/m²紫杉醇；和(c)每3周(q3w)IV施用目标AUC为6mg/ml的卡铂。2) MPDL3280A/paclitaxel/carboplatin combination therapy: (a) 1200 mg MPDL3280A administered IV every 3 weeks (q3w); (b) 200 mg/m² paclitaxel administered IV every 3 weeks (q3w); and (c) carboplatin administered IV every 3 weeks (q3w) with a target AUC of 6 mg/ml.

表4显示用MPDL3280A与和卡铂联合治疗的14名患者的研究结果。表5显示用MPDL3280A与紫杉醇和卡铂联合治疗的6名患者的研究结果。Table 4 shows the results of a study of 14 patients treated with MPDL3280A in combination with paclitaxel and carboplatin. Table 5 shows the results of a study of 6 patients treated with MPDL3280A in combination with paclitaxel and carboplatin.

表4：MPDL3280A//卡铂联合治疗的功效Table 4: Efficacy of MPDL3280A//Carboplatin Combination Therapy

结果result百分比(n/N)Percentage (n/N)客观反应率(ORR)Objective response rate (ORR)64.3％(9/14)64.3% (9/14)完全应答(CR)Complete response (CR)21.4％(3/14)21.4% (3/14)部分反应(PR)Partial response (PR)42.9％(6/14)42.9% (6/14)稳定疾病(SD)Stable disease (SD)28.6％(4/14)28.6% (4/14)进行性疾病(PD)Progressive disease (PD)7.1％(1/14)7.1% (1/14)

表5：MPDL3280A/紫杉醇/卡铂联合治疗的功效Table 5: Efficacy of MPDL3280A/paclitaxel/carboplatin combination therapy

结果result百分比(n/N)Percentage (n/N)客观反应率(ORR)Objective response rate (ORR)33.3％(2/6)33.3% (2/6)完全应答(CR)Complete response (CR)00部分反应(PR)Partial response (PR)33.3％(2/6)33.3% (2/6)稳定疾病(SD)Stable disease (SD)66.7％(4/6)66.7% (4/6)进行性疾病(PD)Progressive disease (PD)00

如表4和图6A中所示，MPDL3280A和纳米清蛋白结合紫杉醇+卡铂联合治疗产生意料之外的强抗肿瘤功效，客观反应率(ORR，CR+PR)为64.3％。令人惊奇地，21.4％(3/14)的用MPDL3280A和纳米清蛋白结合紫杉醇+卡铂联合治疗治疗的患者达到完全应答(即完全没有可检测到的瘤块)。42.9％(6/14)的患者经历部分反应。As shown in Table 4 and Figure 6A, the combination of MPDL3280A and nanoalbumin in combination with paclitaxel + carboplatin produced unexpectedly strong anti-tumor efficacy, with an objective response rate (ORR, CR + PR) of 64.3%. Surprisingly, 21.4% (3/14) of patients treated with the MPDL3280A and nanoalbumin combined with paclitaxel + carboplatin achieved a complete response (i.e., the complete absence of detectable tumor mass). 42.9% (6/14) of patients experienced a partial response.

MPDL3280A和紫杉醇+卡铂联合治疗也产生抗肿瘤功效，但在所测试的相对小的样本量中，在某种程度上没有MPDL3280A/纳米清蛋白结合紫杉醇+卡铂联合治疗那么强健(robust)。MPDL3280A和紫杉醇+卡铂联合治疗的ORR为33.3％，两名反应者都经历部分反应(表5和图6B)。The combination of MPDL3280A and paclitaxel + carboplatin also produced antitumor efficacy, but in the relatively small sample size tested, it was somewhat less robust than the MPDL3280A/nanoalbumin combined with paclitaxel + carboplatin combination. The ORR for the MPDL3280A and paclitaxel + carboplatin combination was 33.3%, with both responders experiencing partial responses (Table 5 and Figure 6B).

与实施例1中提供的临床前研究一致，惊人的强抗肿瘤活性和意料之外的获得持续完全应答的能力代表了PD-1轴结合拮抗剂(如抗PD-L1抗体)和紫杉烷(如纳米清蛋白结合紫杉醇)联合治疗的重要治疗优势。Consistent with the preclinical studies presented in Example 1, the surprisingly potent anti-tumor activity and the unexpected ability to achieve sustained complete responses represent important therapeutic advantages of combination therapy with a PD-1 axis binding antagonist (such as an anti-PD-L1 antibody) and a taxane (such as nanoalbumin combined with paclitaxel).

Claims (88)

Translated fromChinese

1.PD-L1结合拮抗剂在制备用于在个体中治疗癌症或延迟癌症进展的药物中的用途，其中PD-L1结合拮抗剂与紫杉烷和卡铂一起施用，其中紫杉烷是纳米清蛋白结合紫杉醇或紫杉醇，并且其中所述PD-L1结合拮抗剂是抗体。1. Use of a PD-L1 binding antagonist in the preparation of a medicament for treating cancer or delaying cancer progression in an individual, wherein the PD-L1 binding antagonist is administered with a taxane and carboplatin, wherein the taxane is nanoalbumin-bound paclitaxel or paclitaxel, and wherein the PD-L1 binding antagonist is an antibody.2.权利要求1的用途，其中PD-L1结合拮抗剂抑制PD-L1与PD-1的结合。2. The method of claim 1, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1.3.权利要求1的用途，其中PD-L1结合拮抗剂抑制PD-L1与B7-1的结合。3. The use of claim 1, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1.4.权利要求1的用途，其中PD-L1结合拮抗剂抑制PD-L1与PD-1和B7-1二者的结合。4. The use of claim 1, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.5.权利要求1-4中任一项的用途，其中抗体选自MPDL3280A、MDX-1105和MEDI4736。5. The use according to any one of claims 1 to 4, wherein the antibody is selected from the group consisting of MPDL3280A, MDX-1105 and MEDI4736.6.权利要求1-4中任一项的用途，其中抗体包含含有HVR-H1序列SEQ ID NO:19、HVR-H2序列SEQ ID NO:20和HVR-H3序列SEQ ID NO:21的重链，及含有HVR-L1序列SEQ ID NO:22、HVR-L2序列SEQ ID NO:23和HVR-L3序列SEQ ID NO:24的轻链。6. The use of any one of claims 1 to 4, wherein the antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO: 19, HVR-H2 sequence of SEQ ID NO: 20, and HVR-H3 sequence of SEQ ID NO: 21, and a light chain comprising HVR-L1 sequence of SEQ ID NO: 22, HVR-L2 sequence of SEQ ID NO: 23, and HVR-L3 sequence of SEQ ID NO: 24.7.权利要求1-4中任一项的用途，其中抗体包含含有氨基酸序列SEQ ID NO:25的重链可变区和含有氨基酸序列SEQ ID NO:4的轻链可变区。7. The use according to any one of claims 1 to 4, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.8.权利要求1-4中任一项的用途，其中癌症是肺癌、膀胱癌、乳腺癌、肾细胞癌、黑素瘤、结肠癌、直肠癌、卵巢癌或血癌。8. The use according to any one of claims 1 to 4, wherein the cancer is lung cancer, bladder cancer, breast cancer, renal cell carcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer or blood cancer.9.权利要求8的用途，其中癌症是肺癌。9. The use according to claim 8, wherein the cancer is lung cancer.10.权利要求9的用途，其中肺癌是非小细胞肺癌。10. The use according to claim 9, wherein the lung cancer is non-small cell lung cancer.11.权利要求10的用途，其中非小细胞肺癌是非鳞状非小细胞肺癌。11. The use according to claim 10, wherein the non-small cell lung cancer is non-squamous non-small cell lung cancer.12.权利要求10的用途，其中非小细胞肺癌是鳞状非小细胞肺癌。12. The use according to claim 10, wherein the non-small cell lung cancer is squamous non-small cell lung cancer.13.权利要求8的用途，其中癌症是卵巢癌。13. The use of claim 8, wherein the cancer is ovarian cancer.14.权利要求1-4中任一项的用途，其中个体患有癌症或诊断患有癌症。14. The use of any one of claims 1 to 4, wherein the individual has or is diagnosed with cancer.15.权利要求14的用途，其中个体中的癌细胞表达PD-L1。15. The use of claim 14, wherein the cancer cells in the individual express PD-L1.16.权利要求1-4中任一项的用途，其中治疗在所述个体中产生应答。16. The use of any one of claims 1 to 4, wherein the treatment produces a response in the individual.17.权利要求16的用途，其中反应是完全应答。17. The use of claim 16, wherein the response is a complete response.18.权利要求16的用途，其中反应是停止所述治疗后的持续应答。18. The use of claim 16, wherein the response is a sustained response after cessation of said treatment.19.权利要求1-4中任一项的用途，其中紫杉烷和/或卡铂在所述PD-L1结合拮抗剂之前、与所述PD-L1结合拮抗剂同时或在所述PD-L1结合拮抗剂之后施用。19. The use of any one of claims 1 to 4, wherein the taxane and/or carboplatin is administered before, simultaneously with, or after the PD-L1 binding antagonist.20.权利要求19的用途，其中紫杉烷和/或卡铂在所述PD-L1结合拮抗剂之前施用。20. The use of claim 19, wherein the taxane and/or carboplatin is administered before the PD-L1 binding antagonist.21.权利要求19的用途，其中紫杉烷和/或卡铂与所述PD-L1结合拮抗剂同时施用。21. The use of claim 19, wherein taxane and/or carboplatin are administered simultaneously with the PD-L1 binding antagonist.22.权利要求19的用途，其中紫杉烷和/或卡铂在所述PD-L1结合拮抗剂之后施用。22. The use of claim 19, wherein taxane and/or carboplatin is administered after the PD-L1 binding antagonist.23.权利要求1-4中任一项的用途，其中紫杉烷是纳米清蛋白结合紫杉醇。23. The use according to any one of claims 1 to 4, wherein the taxane is nanoalbumin-bound paclitaxel.24.权利要求1-4中任一项的用途，其中紫杉烷是紫杉醇。24. The use according to any one of claims 1 to 4, wherein the taxane is paclitaxel.25.PD-L1结合拮抗剂在制备用于在患有癌症的个体中增强免疫功能的药物中的用途，其中PD-L1结合拮抗剂与紫杉烷和卡铂组合施用，其中紫杉烷是纳米清蛋白结合紫杉醇或紫杉醇，并且其中所述PD-L1结合拮抗剂是抗体。25. Use of a PD-L1 binding antagonist in the preparation of a medicament for enhancing immune function in an individual suffering from cancer, wherein the PD-L1 binding antagonist is administered in combination with a taxane and carboplatin, wherein the taxane is nanoalbumin-bound paclitaxel or paclitaxel, and wherein the PD-L1 binding antagonist is an antibody.26.权利要求25的用途，其中相对于施用所述PD-L1结合拮抗剂、紫杉烷和/或卡铂之前，个体中的CD8+T细胞具有增强的引发、激活、增殖和/或溶细胞活性。26. The use of claim 25, wherein the CD8+ T cells in the individual have enhanced priming, activation, proliferation and/or cytolytic activity relative to before administration of the PD-L1 binding antagonist, taxane and/or carboplatin.27.权利要求25的用途，其中CD8+T细胞的数目相对于施用所述组合之前提高。27. The use of claim 25, wherein the number of CD8+ T cells is increased relative to that before administration of the combination.28.权利要求27的用途，其中CD8+T细胞是抗原特异性CD8+T细胞。28. The use according to claim 27, wherein the CD8+ T cells are antigen-specific CD8+ T cells.29.权利要求25的用途，其中Treg功能相对于施用所述组合之前受到抑制。29. The use of claim 25, wherein Treg function is suppressed relative to before administration of the combination.30.权利要求25的用途，其中T细胞衰竭相对于施用所述组合之前减少。30. The use of claim 25, wherein T cell exhaustion is reduced relative to before administration of the combination.31.权利要求25-30中任一项的用途，其中PD-L1结合拮抗剂抑制PD-L1与PD-1的结合。31. The use of any one of claims 25 to 30, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1.32.权利要求25-30中任一项的用途，其中PD-L1结合拮抗剂抑制PD-L1与B7-1的结合。32. The use of any one of claims 25 to 30, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1.33.权利要求25-30中任一项的用途，其中PD-L1结合拮抗剂抑制PD-L1与PD-1和B7-1二者的结合。33. The use of any one of claims 25 to 30, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.34.权利要求25-30中任一项的用途，其中抗体选自MPDL3280A、MDX-1105和MEDI4736。34. The use of any one of claims 25 to 30, wherein the antibody is selected from the group consisting of MPDL3280A, MDX-1105 and MEDI4736.35.权利要求25-30中任一项的用途，其中抗体包含含有HVR-H1序列SEQ ID NO:19、HVR-H2序列SEQ ID NO:20和HVR-H3序列SEQ ID NO:21的重链，及含有HVR-L1序列SEQ IDNO:22、HVR-L2序列SEQ ID NO:23和HVR-L3序列SEQ ID NO:24的轻链。35. The use of any one of claims 25 to 30, wherein the antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO: 19, HVR-H2 sequence of SEQ ID NO: 20, and HVR-H3 sequence of SEQ ID NO: 21, and a light chain comprising HVR-L1 sequence of SEQ ID NO: 22, HVR-L2 sequence of SEQ ID NO: 23, and HVR-L3 sequence of SEQ ID NO: 24.36.权利要求25-30中任一项的用途，其中抗体包含含有氨基酸序列SEQ ID NO:25的重链可变区和含有氨基酸序列SEQ ID NO:4的轻链可变区。36. The use of any one of claims 25 to 30, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.37.权利要求25-30中任一项的用途，其中癌症是肺癌、膀胱癌、乳腺癌、肾细胞癌、黑素瘤、结肠癌、直肠癌、卵巢癌或血癌。37. The use of any one of claims 25 to 30, wherein the cancer is lung cancer, bladder cancer, breast cancer, renal cell carcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, or blood cancer.38.权利要求37的用途，其中癌症是肺癌。38. The use of claim 37, wherein the cancer is lung cancer.39.权利要求38的用途，其中肺癌是非小细胞肺癌。39. The use of claim 38, wherein the lung cancer is non-small cell lung cancer.40.权利要求39的用途，其中非小细胞肺癌是非鳞状非小细胞肺癌。40. The use of claim 39, wherein the non-small cell lung cancer is non-squamous non-small cell lung cancer.41.权利要求39的用途，其中非小细胞肺癌是鳞状非小细胞肺癌。41. The use of claim 39, wherein the non-small cell lung cancer is squamous non-small cell lung cancer.42.权利要求37的用途，其中癌症是卵巢癌。42. The use of claim 37, wherein the cancer is ovarian cancer.43.权利要求25-30中任一项的用途，其中个体中的癌细胞表达PD-L1。43. The use of any one of claims 25 to 30, wherein the cancer cells in the individual express PD-L1.44.权利要求25-30中任一项的用途，其中紫杉烷是纳米清蛋白结合紫杉醇。44. The use according to any one of claims 25 to 30, wherein the taxane is nanoalbumin-bound paclitaxel.45.权利要求25-30中任一项的用途，其中紫杉烷是紫杉醇。45. The use according to any one of claims 25 to 30, wherein the taxane is paclitaxel.46.权利要求1-4和25-30中任一项的用途，其中PD-L1结合拮抗剂、紫杉烷和/或卡铂静脉内、肌内、皮下、局部、口服、经皮、腹腔内、眼眶内、通过植入、通过吸入、鞘内、心室内或鼻内施用。46. The use of any one of claims 1-4 and 25-30, wherein the PD-L1 binding antagonist, taxane and/or carboplatin are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly or intranasally.47.权利要求1-4和25-30中任一项的用途，其中药物进一步与血管发生抑制剂组合施用。47. The use of any one of claims 1 to 4 and 25 to 30, wherein the medicament is further administered in combination with an angiogenesis inhibitor.48.权利要求47的用途，其中血管发生抑制剂是抗VEGF抗体。48. The use of claim 47, wherein the angiogenesis inhibitor is an anti-VEGF antibody.49.权利要求48的用途，其中抗VEGF抗体是贝伐单抗。49. The use of claim 48, wherein the anti-VEGF antibody is bevacizumab.50.PD-L1结合拮抗剂的用途，用于制备用于在个体中治疗癌症或延迟癌症进展的药物，其中药物包含PD-L1结合拮抗剂和可药用载体，其中所述PD-L1结合拮抗剂是抗体，且其中治疗包括施用所述药物与(i)包含紫杉烷和可药用载体的组合物的组合，其中紫杉烷是纳米清蛋白结合紫杉醇或紫杉醇；和(ii)包含卡铂和可药用载体的组合物的组合。50. Use of a PD-L1 binding antagonist for the preparation of a medicament for treating cancer or delaying cancer progression in an individual, wherein the medicament comprises a PD-L1 binding antagonist and a pharmaceutically acceptable carrier, wherein the PD-L1 binding antagonist is an antibody, and wherein the treatment comprises administering the medicament in combination with (i) a composition comprising a taxane and a pharmaceutically acceptable carrier, wherein the taxane is nanoalbumin-bound paclitaxel or paclitaxel; and (ii) a composition comprising carboplatin and a pharmaceutically acceptable carrier.51.紫杉烷的用途，用于制备用于在个体中治疗癌症或延迟癌症进展的药物，其中药物包含紫杉烷和可药用载体，其中紫杉烷是纳米清蛋白结合紫杉醇或紫杉醇，且其中治疗包括施用所述药物与(i)包含PD-L1结合拮抗剂和可药用载体的组合物的组合，其中所述PD-L1结合拮抗剂是抗体；和(ii)包含卡铂和可药用载体的组合物的组合。51. Use of a taxane for the preparation of a medicament for treating cancer or delaying cancer progression in an individual, wherein the medicament comprises a taxane and a pharmaceutically acceptable carrier, wherein the taxane is nanoalbumin-bound paclitaxel or paclitaxel, and wherein the treatment comprises administering the medicament in combination with (i) a composition comprising a PD-L1 binding antagonist and a pharmaceutically acceptable carrier, wherein the PD-L1 binding antagonist is an antibody; and (ii) a composition comprising carboplatin and a pharmaceutically acceptable carrier.52.权利要求50或51的用途，其中紫杉烷是纳米清蛋白结合紫杉醇。52. The use of claim 50 or 51, wherein the taxane is nanoalbumin-bound paclitaxel.53.权利要求50或51的用途，其中紫杉烷是紫杉醇。53. The use of claim 50 or 51, wherein the taxane is paclitaxel.54.权利要求50或51的用途，其中癌症是肺癌、膀胱癌、乳腺癌、肾细胞癌、黑素瘤、结肠癌、直肠癌、卵巢癌或血癌。54. The use of claim 50 or 51, wherein the cancer is lung cancer, bladder cancer, breast cancer, renal cell carcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, or blood cancer.55.权利要求54的用途，其中癌症是肺癌。55. The use of claim 54, wherein the cancer is lung cancer.56.权利要求55的用途，其中肺癌是非小细胞肺癌。56. The use of claim 55, wherein the lung cancer is non-small cell lung cancer.57.权利要求56的用途，其中非小细胞肺癌是非鳞状非小细胞肺癌。57. The use of claim 56, wherein the non-small cell lung cancer is non-squamous non-small cell lung cancer.58.权利要求56的用途，其中非小细胞肺癌是鳞状非小细胞肺癌。58. The use of claim 56, wherein the non-small cell lung cancer is squamous non-small cell lung cancer.59.权利要求54的用途，其中癌症是卵巢癌。59. The use of claim 54, wherein the cancer is ovarian cancer.60.权利要求50或51的用途，其中药物与包含血管发生抑制剂和可药用载体的组合物组合施用。60. The use of claim 50 or 51, wherein the medicament is administered in combination with a composition comprising an angiogenesis inhibitor and a pharmaceutically acceptable carrier.61.权利要求60的用途，其中所述血管发生抑制剂是抗VEGF抗体。61. The use of claim 60, wherein the angiogenesis inhibitor is an anti-VEGF antibody.62.权利要求61的用途，其中抗VEGF抗体是贝伐单抗。62. The use of claim 61, wherein the anti-VEGF antibody is bevacizumab.63.包含PD-L1结合拮抗剂、紫杉烷和卡铂的组合物，用于在个体中治疗癌症或延迟癌症进展，其中紫杉烷是纳米清蛋白结合紫杉醇或紫杉醇，并且其中所述PD-L1结合拮抗剂是抗体。63. A composition comprising a PD-L1 binding antagonist, a taxane, and carboplatin for treating cancer or delaying cancer progression in an individual, wherein the taxane is nanoalbumin-bound paclitaxel or paclitaxel, and wherein the PD-L1 binding antagonist is an antibody.64.权利要求63的组合物，进一步包含可药用载体。64. The composition of claim 63, further comprising a pharmaceutically acceptable carrier.65.权利要求63或64的组合物在制备用于在个体中治疗癌症或延迟癌症进展的药物中的用途。65. Use of the composition of claim 63 or 64 in the preparation of a medicament for treating or delaying the progression of cancer in a subject.66.权利要求63或64的组合物，其中紫杉烷是纳米清蛋白结合紫杉醇。66. The composition of claim 63 or 64, wherein the taxane is nanoalbumin-bound paclitaxel.67.权利要求63或64的组合物，其中紫杉烷是紫杉醇。67. The composition of claim 63 or 64, wherein the taxane is paclitaxel.68.权利要求63或64的组合物，其中癌症是肺癌、膀胱癌、乳腺癌、肾细胞癌、黑素瘤、结肠癌、直肠癌、卵巢癌或血癌。68. The composition of claim 63 or 64, wherein the cancer is lung cancer, bladder cancer, breast cancer, renal cell carcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, or blood cancer.69.权利要求68的组合物，其中癌症是肺癌。69. The composition of claim 68, wherein the cancer is lung cancer.70.权利要求69的组合物，其中肺癌是非小细胞肺癌。70. The composition of claim 69, wherein the lung cancer is non-small cell lung cancer.71.权利要求70的组合物，其中非小细胞肺癌是非鳞状非小细胞肺癌。71. The composition of claim 70, wherein the non-small cell lung cancer is non-squamous non-small cell lung cancer.72.权利要求70的组合物，其中非小细胞肺癌是鳞状非小细胞肺癌。72. The composition of claim 70, wherein the non-small cell lung cancer is squamous non-small cell lung cancer.73.权利要求68的组合物，其中癌症是卵巢癌。73. The composition of claim 68, wherein the cancer is ovarian cancer.74.权利要求63或64的组合物，还包含血管发生抑制剂。74. The composition of claim 63 or 64, further comprising an angiogenesis inhibitor.75.权利要求74的组合物，其中所述血管发生抑制剂是抗VEGF抗体。75. The composition of claim 74, wherein the angiogenesis inhibitor is an anti-VEGF antibody.76.权利要求75的组合物，其中抗VEGF抗体是贝伐单抗。76. The composition of claim 75, wherein the anti-VEGF antibody is bevacizumab.77.药盒，其包含含有PD-L1结合拮抗剂的第一药物，含有紫杉烷的第二药物，及包含卡铂的第三药物，其中紫杉烷是纳米清蛋白结合紫杉醇或紫杉醇，并且其中所述PD-L1结合拮抗剂是抗体。77. A drug kit comprising a first medicament comprising a PD-L1 binding antagonist, a second medicament comprising a taxane, and a third medicament comprising carboplatin, wherein the taxane is nanoalbumin-bound paclitaxel or paclitaxel, and wherein the PD-L1 binding antagonist is an antibody.78.权利要求77的药盒，其中药盒进一步包含含有施用所述第一药物、所述第二药物和所述第三药物来在个体中治疗癌症或延迟癌症进展的说明的包装说明书。78. The kit of claim 77, wherein the kit further comprises a package insert containing instructions for administering the first drug, the second drug, and the third drug to treat cancer or delay progression of cancer in an individual.79.权利要求77或78的药盒，其中所述第一药物、所述第二药物或所述第三药物还包含可药用载体。79. The kit of claim 77 or 78, wherein the first drug, the second drug, or the third drug further comprises a pharmaceutically acceptable carrier.80.抗PD-L1抗体在制备用于治疗个体非小细胞肺癌或延迟个体非小细胞肺癌进展的药物中的用途，其中所述抗PD-L1抗体包含含有SEQ ID NO：25的氨基酸序列的重链可变区和含有SEQ ID NO：4的氨基酸序列的轻链可变区，并且其中所述抗PD-L1抗体与紫杉烷和卡铂组合施用，其中紫杉烷是纳米清蛋白结合紫杉醇或紫杉醇，并且其中非小细胞肺癌是非鳞状非小细胞肺癌或鳞状非小细胞肺癌。80. Use of an anti-PD-L1 antibody in the preparation of a medicament for treating or delaying progression of non-small cell lung cancer in an individual, wherein the anti-PD-L1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and wherein the anti-PD-L1 antibody is administered in combination with a taxane and carboplatin, wherein the taxane is nanoalbumin-bound paclitaxel or paclitaxel, and wherein the non-small cell lung cancer is non-squamous non-small cell lung cancer or squamous non-small cell lung cancer.81.权利要求80的用途，其中非小细胞肺癌是非鳞状非小细胞肺癌，紫杉烷是纳米清蛋白结合紫杉醇。81. The use of claim 80, wherein the non-small cell lung cancer is non-squamous non-small cell lung cancer and the taxane is nanoalbumin-bound paclitaxel.82.权利要求80的用途，其中非小细胞肺癌是鳞状非小细胞肺癌。82. The use of claim 80, wherein the non-small cell lung cancer is squamous non-small cell lung cancer.83.抗PD-L1抗体在制备用于治疗个体非小细胞肺癌或延迟个体非小细胞肺癌进展的药物中的用途，其中所述抗PD-L1抗体包含含有SEQ ID NO：25的氨基酸序列的重链可变区和含有SEQ ID NO：4的氨基酸序列的轻链可变区，并且其中抗PD-L1抗体与紫杉烷、卡铂和贝伐单抗组合施用，其中紫杉烷是纳米清蛋白结合紫杉醇或紫杉醇，并且其中非小细胞肺癌是非鳞状非小细胞肺癌。83. Use of an anti-PD-L1 antibody in the preparation of a medicament for treating or delaying progression of non-small cell lung cancer in an individual, wherein the anti-PD-L1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and wherein the anti-PD-L1 antibody is administered in combination with a taxane, carboplatin, and bevacizumab, wherein the taxane is nanoalbumin-bound paclitaxel or paclitaxel, and wherein the non-small cell lung cancer is non-squamous non-small cell lung cancer.84.权利要求83的用途，其中紫杉烷是紫杉醇。84. The use of claim 83, wherein the taxane is paclitaxel.85.抗PD-L1抗体在制备用于治疗个体卵巢癌或延迟个体卵巢癌进展的药物中的用途，其中所述抗PD-L1抗体包含含有SEQ ID NO：25的氨基酸序列的重链可变区和含有SEQ IDNO：4的氨基酸序列的轻链可变区，其中抗PD-L1抗体与紫杉烷、卡铂和贝伐单抗组合施用，并且其中紫杉烷是纳米清蛋白结合紫杉醇或紫杉醇。85. Use of an anti-PD-L1 antibody in the preparation of a medicament for treating ovarian cancer or delaying progression of ovarian cancer in an individual, wherein the anti-PD-L1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, wherein the anti-PD-L1 antibody is administered in combination with a taxane, carboplatin, and bevacizumab, and wherein the taxane is nanoalbumin-bound paclitaxel or paclitaxel.86.如权利要求85所述的用途，其中紫杉烷是紫杉醇。86. The use of claim 85, wherein the taxane is paclitaxel.87.权利要求80-86中任一项的用途，其中所述抗PD-L1抗体包含含有氨基酸SEQ IDNO：32的重链和含有氨基酸序列SEQ ID NO：33的轻链。87. The use of any one of claims 80-86, wherein the anti-PD-L1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain comprising the amino acid sequence of SEQ ID NO: 33.88.权利要求80-86中任一项的用途，其中所述抗PD-L1抗体以每三周1200mg的剂量静脉内施用。88. The use of any one of claims 80-86, wherein the anti-PD-L1 antibody is administered intravenously at a dose of 1200 mg every three weeks.