对相关申请的交叉引用Cross-reference to related applications
本申请要求临时美国申请2013年3月15日提交的No.61/802,296；2013年4月16日提交的No.61/812,678；2013年5月30日提交的No.61/829,236；和2013年9月26日提交的No.61/883,186的优先权权益，据此通过提及将其完整收录。This application claims the benefit of priority to Provisional U.S. Application Nos. 61/802,296, filed March 15, 2013; 61/812,678, filed April 16, 2013; 61/829,236, filed May 30, 2013; and 61/883,186, filed September 26, 2013, which are hereby incorporated by reference in their entirety.
发明领域Field of the Invention
本文中提供用于治疗病理疾患(诸如癌症)的生物标志物，及使用PD-L1/PD-1途径拮抗剂的方法。特别地，提供用于癌症中患者选择和预后的生物标志物，以及治疗性处理的方法、制品及其制备方法、诊断试剂盒、检测方法及其相关做广告的方法。Provided herein are biomarkers for treating pathological conditions such as cancer, and methods of using PD-L1/PD-1 pathway antagonists. In particular, provided herein are biomarkers for patient selection and prognosis in cancer, as well as methods of therapeutic treatment, articles and methods of preparing the same, diagnostic kits, detection methods, and methods of advertising the same.
发明背景Background of the Invention
癌症仍然是对人类健康的最致命威胁之一。在美国，癌症每年影响近130万新患者，而且是位于心脏病之后的第二位死因，占4例死亡中的大约1例。例如，肺癌是癌症的最常见形式，而且是美国女性中的首要癌症杀手。还预测癌症可能在5年内超越心血管疾病成为第一位死因。实体瘤对大多数上述死亡负有责任。虽然某些癌症的医学治疗中已经取得了重大进步，但是所有癌症的总体5年存活率在最近20年里只改进了约10％。癌症(或恶性肿瘤)以不受控制的方式快速生长和转移，使得及时检测和治疗极端困难。Cancer remains one of the most deadly threats to human health. In the United States, cancer affects nearly 1.3 million new patients each year and is the second leading cause of death after heart disease, accounting for approximately 1 in 4 deaths. For example, lung cancer is the most common form of cancer and the leading cancer killer among American women. It is also predicted that cancer may surpass cardiovascular disease to become the leading cause of death within 5 years. Solid tumors are responsible for most of the above-mentioned deaths. Although significant progress has been made in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has only improved by about 10% in the last 20 years. Cancer (or malignant tumors) grows and metastasizes rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult.
尽管癌症治疗获得了重大进展，但是仍然在寻找改良的疗法。Despite significant advances in cancer treatment, the search for improved therapies continues.
通过述及而完整收录本文中引用的所有参考文献(包括专利申请和出版物)。All references (including patent applications and publications) cited herein are incorporated by reference in their entirety.
发明概述SUMMARY OF THE INVENTION
本文中提供的是用于鉴定更有可能响应PD-L1轴结合拮抗剂治疗的具有疾病或病症的个体的方法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，其中该样品中存在PD-L1生物标志物指示该个体更有可能响应PD-L1轴结合拮抗剂治疗，并提供该个体会更有可能响应PD-L1轴结合拮抗剂治疗的推荐。Provided herein are methods for identifying an individual having a disease or condition who is more likely to respond to treatment with a PD-L1 axis binding antagonist, the method comprising: determining the presence of a PD-L1 biomarker in a sample from the individual, wherein the presence of the PD-L1 biomarker in the sample indicates that the individual is more likely to respond to treatment with a PD-L1 axis binding antagonist, and providing a recommendation that the individual will be more likely to respond to treatment with a PD-L1 axis binding antagonist.
本文中提供的是用于预测具有疾病或病症的个体对PD-L1轴结合拮抗剂治疗的响应性的方法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，其中该样品中存在PD-L1生物标志物指示该个体更有可能响应PD-L1轴结合拮抗剂治疗，并提供该个体会具有升高的可能性响应PD-L1轴结合拮抗剂治疗的推荐。Provided herein are methods for predicting responsiveness of an individual having a disease or condition to treatment with a PD-L1 axis binding antagonist, the method comprising: determining the presence of a PD-L1 biomarker in a sample from the individual, wherein the presence of the PD-L1 biomarker in the sample indicates that the individual is more likely to respond to treatment with a PD-L1 axis binding antagonist, and providing a recommendation that the individual will have an increased likelihood of responding to treatment with a PD-L1 axis binding antagonist.
本文中提供的是用于确定具有疾病或病症的个体会展现受益于PD-L1轴结合拮抗剂治疗的可能性的方法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，其中该样品中存在PD-L1生物标志物指示该个体具有升高的可能性受益于PD-L1轴结合拮抗剂治疗，并提供该个体会具有升高的可能性受益于PD-L1轴结合拮抗剂治疗的推荐。Provided herein are methods for determining a likelihood that an individual having a disease or condition will exhibit benefit from treatment with a PD-L1 axis binding antagonist, the method comprising: assaying a sample from the individual for the presence of a PD-L1 biomarker, wherein the presence of the PD-L1 biomarker in the sample indicates that the individual has an increased likelihood of benefiting from treatment with a PD-L1 axis binding antagonist, and providing a recommendation that the individual will have an increased likelihood of benefiting from treatment with a PD-L1 axis binding antagonist.
本文中提供的是用于为具有疾病或病症的个体选择疗法的方法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，并基于该样品中PD-L1生物标志物的存在提供为该个体选择的疗法包含PD-L1轴结合拮抗剂治疗的推荐。Provided herein are methods for selecting a therapy for an individual having a disease or condition, the method comprising: determining the presence of a PD-L1 biomarker in a sample from the individual, and providing a recommendation that the therapy selected for the individual comprises treatment with a PD-L1 axis binding antagonist based on the presence of the PD-L1 biomarker in the sample.
在一些实施方案中，该方法进一步包括将有效量的PD-L1轴结合拮抗剂施用于该个体。In some embodiments, the method further comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist.
本文中提供的是用于治疗个体中疾病或病症的方法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，并将有效量的PD-L1轴结合拮抗剂施用于该个体。Provided herein are methods for treating a disease or condition in an individual, the method comprising: determining the presence of a PD-L1 biomarker in a sample from the individual, and administering to the individual an effective amount of a PD-L1 axis binding antagonist.
本文中提供的是用于治疗个体中疾病或病症的方法，其包括对该个体施用有效量的PD-L1轴结合拮抗剂，其中治疗基于来自该个体的样品中PD-L1生物标志物的存在。Provided herein are methods for treating a disease or condition in an individual comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist, wherein the treatment is based on the presence of a PD-L1 biomarker in a sample from the individual.
本文中提供的是用于为PD-L1轴结合拮抗剂做广告的方法，其包括向目标受众推广基于PD-L1生物标志物的存在使用PD-L1轴结合拮抗剂来治疗具有疾病或病症的个体。Provided herein are methods for advertising a PD-L1 axis binding antagonist comprising promoting to a target audience the use of a PD-L1 axis binding antagonist to treat an individual having a disease or condition based on the presence of a PD-L1 biomarker.
本文中提供的是用于鉴定具有疾病或病症的个体来接受PD-L1轴结合拮抗剂的测定法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，并基于PD-L1生物标志物的存在推荐PD-L1轴结合拮抗剂。Provided herein are assays for identifying an individual with a disease or condition to receive a PD-L1 axis binding antagonist, the method comprising: determining the presence of a PD-L1 biomarker in a sample from the individual, and recommending a PD-L1 axis binding antagonist based on the presence of the PD-L1 biomarker.
本文中提供的是诊断试剂盒，其包括一种或多种用于测定来自具有疾病或病症的个体的样品中PD-L1生物标志物的存在的试剂，其中PD-L1生物标志物的存在意味着用PD-L1轴结合拮抗剂治疗个体时更高可能性的功效，且其中PD-L1生物标志物的缺失意味着用PD-L1轴结合拮抗剂治疗具有疾病的个体时更少可能性的功效。Provided herein are diagnostic kits comprising one or more reagents for determining the presence of a PD-L1 biomarker in a sample from an individual having a disease or condition, wherein the presence of the PD-L1 biomarker indicates a higher likelihood of efficacy when treating the individual with a PD-L1 axis binding antagonist, and wherein the absence of the PD-L1 biomarker indicates a lower likelihood of efficacy when treating the individual having the disease with a PD-L1 axis binding antagonist.
本文中还提供的是制品，其包括包装在一起的药学可接受载剂中的PD-L1轴结合拮抗剂和指示PD-L1轴结合拮抗剂基于PD-L1生物标志物的表达用于治疗具有疾病或病症的患者的包装插页。治疗方法包括本文中公开的任何治疗方法。又提供的用于制造制品的方法，其包括在一个包装中组合包含PD-L1轴结合拮抗剂的药物组合物和指示该药物组合物基于PD-L1生物标志物的表达用于治疗具有疾病或病症的患者的包装插页。Also provided herein are articles of manufacture comprising a PD-L1 axis binding antagonist packaged together in a pharmaceutically acceptable carrier and a package insert indicating that the PD-L1 axis binding antagonist is used to treat a patient having a disease or condition based on the expression of a PD-L1 biomarker. Methods of treatment include any of the methods disclosed herein. Also provided are methods for making an article of manufacture comprising combining, in a single package, a pharmaceutical composition comprising a PD-L1 axis binding antagonist and a package insert indicating that the pharmaceutical composition is used to treat a patient having a disease or condition based on the expression of a PD-L1 biomarker.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1生物标志物选自下组：PD-L1，PD-1，PD-L2及其任意组合。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is selected from the group consisting of PD-L1, PD-1, PD-L2, and any combination thereof.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1生物标志物是免疫相关标志物。在一些实施方案中，免疫相关标志物是T细胞相关标志物。在一些实施方案中，T细胞相关标志物选自下组：CD8A，IFN-g，EOMES，粒酶-A，CXCL9及其任意组合。在一些实施方案中，免疫相关标志物选自下组：CX3CL1，CD45RO，IDO1，半乳凝素9，MIC-A，MIC-B，CTLA-4及其任意组合。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is an immune-related marker. In some embodiments, the immune-related marker is a T-cell-related marker. In some embodiments, the T-cell-related marker is selected from the group consisting of CD8A, IFN-g, EOMES, granzyme-A, CXCL9, and any combination thereof. In some embodiments, the immune-related marker is selected from the group consisting of CX3CL1, CD45RO, IDO1, galectin-9, MIC-A, MIC-B, CTLA-4, and any combination thereof.
在任何方法、测定法和/或试剂盒的一些实施方案中，疾病或病症为增殖性疾病或病症。在任何方法、测定法和/或试剂盒的一些实施方案中，疾病或病症为免疫相关疾病或病症。在任何方法、测定法和/或试剂盒的一些实施方案中，疾病或病症为癌症。在一些实施方案中，癌症选自下组：非小细胞肺癌，小细胞肺癌，肾细胞癌，结肠直肠癌，卵巢癌，乳腺癌，胰腺癌，胃癌，膀胱癌，食道癌，间皮瘤，黑素瘤，头和颈癌，甲状腺癌，肉瘤，前列腺癌，成胶质细胞瘤，宫颈癌，胸腺癌，白血病，淋巴瘤，骨髓瘤，蕈样肉芽肿，梅克尔细胞癌，和其它血液学恶性肿瘤。In some embodiments of any method, assay and/or kit, the disease or condition is a proliferative disease or condition. In some embodiments of any method, assay and/or kit, the disease or condition is an immune-related disease or condition. In some embodiments of any method, assay and/or kit, the disease or condition is a cancer. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies.
在任何方法、测定法和/或试剂盒的一些实施方案中，其中自个体获得的样品选自下组：组织，全血，血浆，血清及其组合。在一些实施方案中，组织样品是肿瘤组织样品。在一些实施方案中，肿瘤组织样品包含肿瘤细胞，肿瘤浸润性免疫细胞，基质细胞及其任意组合。在一些实施方案中，组织样品是福尔马林固定和石蜡包埋的，存档的，新鲜的或冷冻的。在一些实施方案中，样品是全血。在一些实施方案中，全血包含免疫细胞，循环肿瘤细胞及其任意组合。In some embodiments of any of the methods, assays, and/or kits, the sample obtained from the individual is selected from the group consisting of tissue, whole blood, plasma, serum, and combinations thereof. In some embodiments, the tissue sample is a tumor tissue sample. In some embodiments, the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, and any combination thereof. In some embodiments, the tissue sample is formalin-fixed and paraffin-embedded, archived, fresh, or frozen. In some embodiments, the sample is whole blood. In some embodiments, the whole blood comprises immune cells, circulating tumor cells, and any combination thereof.
在任何方法、测定法和/或试剂盒的一些实施方案中，样品是在PD-L1轴结合拮抗剂治疗之前获得的。In some embodiments of any of the methods, assays, and/or kits, the sample is obtained prior to treatment with a PD-L1 axis binding antagonist.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1生物标志物的存在指示当用PD-L1轴结合拮抗剂治疗个体时该个体有可能具有增大的临床受益。在一些实施方案中，增大的临床受益包括下述一项或多项的相对增大：总体存活(OS)，无进展存活(PFS)，完全响应(CR)，部分响应(PR)及其组合。In some embodiments of any of the methods, assays, and/or kits, the presence of a PD-L1 biomarker indicates that the individual is likely to have an increased clinical benefit when treated with a PD-L1 axis binding antagonist. In some embodiments, the increased clinical benefit comprises a relative increase in one or more of: overall survival (OS), progression-free survival (PFS), complete response (CR), partial response (PR), and combinations thereof.
在任何方法、测定法和/或试剂盒的一些实施方案中，当0％的样品包含PD-L1生物标志物时，它是样品中缺失的。In some embodiments of any of the methods, assays, and/or kits, when 0% of the sample comprises the PD-L1 biomarker, it is absent from the sample.
在任何方法、测定法和/或试剂盒的一些实施方案中，当超过0％的样品包含PD-L1生物标志物时，它是样品中存在的。在一些实施方案中，至少1％的样品中存在PD-L1生物标志物。在一些实施方案中，至少5％的样品中存在PD-L1生物标志物。在一些实施方案中，至少10％的样品中存在PD-L1生物标志物。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is present in a sample when more than 0% of the sample contains it. In some embodiments, the PD-L1 biomarker is present in at least 1% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 5% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 10% of the sample.
在任何方法、测定法和/或试剂盒的一些实施方案中，使用选自下组的方法检测样品中的PD-L1生物标志物：FACS，Western印迹，ELISA，免疫沉淀，免疫组织化学，免疫荧光，放射免疫测定法，点印迹，免疫检测方法，HPLC，表面等离振子共振，光谱术，质谱术，HPLC，qPCR，RT-qPCR，多重qPCR或RT-qPCR，RNA-seq，微阵列分析，SAGE，MassARRAY技术，和FISH，及其组合。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof.
在任何方法、测定法和/或试剂盒的一些实施方案中，通过蛋白质表达检测样品中的PD-L1生物标志物。在一些实施方案中，通过免疫组织化学(IHC)测定蛋白质表达。在一些实施方案中，使用抗PD-L1抗体检测PD-L1生物标志物。在一些实施方案中，通过IHC以弱染色强度检测PD-L1生物标志物。在一些实施方案中，通过IHC以中等染色强度检测PD-L1生物标志物。在一些实施方案中，通过IHC以强染色强度检测PD-L1生物标志物。在一些实施方案中，在肿瘤细胞，肿瘤浸润性免疫细胞，基质细胞及其任意组合上检测PD-L1生物标志物。在一些实施方案中，染色是膜染色，胞质染色或其组合。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is detected in a sample by protein expression. In some embodiments, protein expression is determined by immunohistochemistry (IHC). In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 antibody. In some embodiments, the PD-L1 biomarker is detected by IHC with a weak staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC with a moderate staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC with a strong staining intensity. In some embodiments, the PD-L1 biomarker is detected on tumor cells, tumor-infiltrating immune cells, stromal cells, and any combination thereof. In some embodiments, the staining is membrane staining, cytoplasmic staining, or a combination thereof.
在任何方法、测定法和/或试剂盒的一些实施方案中，以样品中没有或无染色检测PD-L1生物标志物的缺失。在任何方法、测定法和/或试剂盒的一些实施方案中，以样品中的任何染色检测PD-L1生物标志物的存在。In some embodiments of any of the methods, assays, and/or kits, the absence of the PD-L1 biomarker is detected as an absence or lack of staining in the sample. In some embodiments of any of the methods, assays, and/or kits, the presence of the PD-L1 biomarker is detected as any staining in the sample.
在任何方法、测定法和/或试剂盒的一些实施方案中，通过核酸表达检测样品中的PD-L1生物标志物。在一些实施方案中，使用qPCR，RT-qPCR，多重qPCR或RT-qPCR，RNA-seq，微阵列分析，SAGE，MassARRAY技术，或FISH测定核酸表达。在一些实施方案中，在肿瘤细胞，肿瘤浸润性免疫细胞，基质细胞及其任意组合上检测PD-L1生物标志物。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is detected in a sample by nucleic acid expression. In some embodiments, nucleic acid expression is measured using qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, or FISH. In some embodiments, the PD-L1 biomarker is detected on tumor cells, tumor-infiltrating immune cells, stromal cells, and any combination thereof.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1轴结合拮抗剂选自下组：PD-L1结合拮抗剂和PD-1结合拮抗剂。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist and a PD-1 binding antagonist.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1轴结合拮抗剂是PD-L1结合拮抗剂。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合它的配体结合配偶。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合PD-1。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合B7-1。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合PD-1和B7-1二者。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to its ligand binding partner. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to PD-1. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to B7-1. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to both PD-1 and B7-1.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂是抗体。在任何方法、测定法和/或试剂盒的一些实施方案中，抗体是单克隆抗体。在任何方法、测定法和/或试剂盒的一些实施方案中，抗体是人抗体，人源化抗体或嵌合抗体。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist is an antibody. In some embodiments of any of the methods, assays, and/or kits, the antibody is a monoclonal antibody. In some embodiments of any of the methods, assays, and/or kits, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1轴结合拮抗剂是PD-1结合拮抗剂。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合它的配体结合配偶。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L1。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L2。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L1和PD-L2二者。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to its ligand binding partner. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to PD-L1. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to PD-L2. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to both PD-L1 and PD-L2.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂是抗体。在任何方法、测定法和/或试剂盒的一些实施方案中，抗体是单克隆抗体。在任何方法、测定法和/或试剂盒的一些实施方案中，抗体是人抗体，人源化抗体或嵌合抗体。In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist is an antibody. In some embodiments of any of the methods, assays, and/or kits, the antibody is a monoclonal antibody. In some embodiments of any of the methods, assays, and/or kits, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
在任何方法、测定法和/或试剂盒的一些实施方案中，进一步包含有效量的选自下组的第二治疗剂：细胞毒剂，化疗剂，生长抑制剂，放射疗法药剂，和抗血管发生剂，及其组合。In some embodiments of any of the methods, assays, and/or kits, further comprising an effective amount of a second therapeutic agent selected from the group consisting of a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, a radiotherapy agent, and an anti-angiogenic agent, and combinations thereof.
本文中提供的是用于评估个体对PD-L1轴结合拮抗剂的治疗响应的方法，该方法包括：(a)测定在施用PD-L1轴结合拮抗剂期间或之后的一个时间点自个体衍生的生物学样品中一种或多种生物标志物的水平；并(b)基于生物学样品中一种或多种生物标志物的水平与参照水平的比较维持，调节，或停止个体的治疗，其中生物学样品中一种或多种生物标志物的水平与参照水平相比的变化指示对PD-L1轴结合拮抗剂治疗的响应。Provided herein are methods for assessing a subject's response to treatment with a PD-L1 axis binding antagonist, the methods comprising: (a) determining the level of one or more biomarkers in a biological sample derived from the subject at a time point during or after administration of the PD-L1 axis binding antagonist; and (b) maintaining, adjusting, or discontinuing treatment of the subject based on a comparison of the level of the one or more biomarkers in the biological sample to a reference level, wherein a change in the level of the one or more biomarkers in the biological sample compared to the reference level indicates a response to treatment with the PD-L1 axis binding antagonist.
本文中提供的是用于监测用PD-L1轴结合拮抗剂治疗的个体的响应的方法，该方法包括：(a)测定在施用PD-L1轴结合拮抗剂期间或之后的一个时间点自个体衍生的生物学样品中一种或多种生物标志物的水平；并(b)将生物学样品中一种或多种生物标志物的水平与参照水平比较以监测经历PD-L1轴结合拮抗剂治疗的个体的响应。Provided herein are methods for monitoring the response of an individual treated with a PD-L1 axis binding antagonist, the methods comprising: (a) determining the level of one or more biomarkers in a biological sample derived from the individual at a time point during or after administration of the PD-L1 axis binding antagonist; and (b) comparing the level of the one or more biomarkers in the biological sample to a reference level to monitor the response of the individual undergoing treatment with the PD-L1 axis binding antagonist.
在一些实施方案中，一种或多种生物标志物的参照水平选自下组：(1)施用PD-L1轴结合拮抗剂之前来自个体的一种或多种生物标志物的水平；(2)来自参照群体的一种或多种生物标志物的水平；(3)一种或多种生物标志物的预指派水平；和(4)在第一时间点之前的第二时间点来自个体的一种或多种生物标志物的水平。In some embodiments, the reference level of one or more biomarkers is selected from the group consisting of: (1) the level of one or more biomarkers from the individual before administration of the PD-L1 axis binding antagonist; (2) the level of one or more biomarkers from a reference population; (3) a pre-assigned level of one or more biomarkers; and (4) the level of one or more biomarkers from the individual at a second time point prior to the first time point.
在一些实施方案中，生物学样品中一种或多种生物标志物的水平与参照水平相比的变化是水平的升高。In some embodiments, a change in the level of one or more biomarkers in a biological sample compared to a reference level is an increase in the level.
在一些实施方案中，生物学样品中一种或多种生物标志物的水平与参照水平相比的变化是水平的降低。In some embodiments, a change in the level of one or more biomarkers in a biological sample compared to a reference level is a decrease in the level.
在一些实施方案中，一种或多种生物标志物选自PD-L1，PD-1，PD-L2及其任意组合。In some embodiments, the one or more biomarkers are selected from PD-L1, PD-1, PD-L2, and any combination thereof.
在一些实施方案中，生物学样品中选自PD-L1，PD-1，PD-L2及其任意组合的一种或多种生物标志物与参照水平相比升高。在一些实施方案中，生物学样品中选自PD-L1，PD-1，PD-L2及其任意组合的一种或多种生物标志物与参照水平相比升高指示对治疗的积极响应。In some embodiments, one or more biomarkers selected from PD-L1, PD-1, PD-L2, and any combination thereof are elevated in a biological sample compared to a reference level. In some embodiments, an elevation in one or more biomarkers selected from PD-L1, PD-1, PD-L2, and any combination thereof in a biological sample compared to a reference level indicates a positive response to treatment.
在一些实施方案中，一种或多种生物标志物是免疫相关标志物。在一些实施方案中，一种或多种生物标志物是T细胞相关标志物。In some embodiments, the one or more biomarkers are immune-related markers. In some embodiments, the one or more biomarkers are T-cell-related markers.
在一些实施方案中，一种或多种生物标志物是T细胞活化标志物。In some embodiments, the one or more biomarkers are T cell activation markers.
在一些实施方案中，生物学样品中的T细胞活化标志物与参照水平相比升高。In some embodiments, the T cell activation marker is elevated in the biological sample compared to a reference level.
在一些实施方案中，T细胞活化标志物选自CD8，IFN-g，粒酶-A，TNF-a，穿孔蛋白及其任意组合。在一些实施方案中，生物学样品中选自CD8，IFN-g，粒酶-A，TNF-a，穿孔蛋白及其任意组合的T细胞活化标志物与参照水平相比升高指示对治疗的积极响应。In some embodiments, the T cell activation marker is selected from CD8, IFN-g, granzyme-A, TNF-a, perforin, and any combination thereof. In some embodiments, an increase in the T cell activation marker selected from CD8, IFN-g, granzyme-A, TNF-a, perforin, and any combination thereof in the biological sample compared to a reference level indicates a positive response to treatment.
在一些实施方案中，一种或多种生物标志物是活化的增殖T细胞。In some embodiments, the one or more biomarkers is activated, proliferating T cells.
在一些实施方案中，生物学样品中的活化的增殖T细胞与参照水平相比增多。In some embodiments, activated, proliferating T cells are increased in the biological sample compared to a reference level.
在一些实施方案中，活化的增殖T细胞是CD8+/Ki67+细胞，CD8+/HLA-DR+/Ki67+细胞及其任意组合。In some embodiments, the activated proliferating T cells are CD8+/Ki67+ cells, CD8+/HLA-DR+/Ki67+ cells, and any combination thereof.
在一些实施方案中，一种或多种生物标志物是IL-6。在一些实施方案中，生物学样品中的IL-6水平与参照水平相比降低。在一些实施方案中，生物学样品中的IL-6水平与参照水平相比降低指示对治疗的积极响应。在一些实施方案中，生物学样品中的IL-6水平与参照水平相比升高。在一些实施方案中，生物学样品中的IL-6水平与参照水平相比升高指示对治疗的消极响应。In some embodiments, the one or more biomarkers are IL-6. In some embodiments, the IL-6 level in the biological sample is reduced compared to the reference level. In some embodiments, a reduction in the IL-6 level in the biological sample compared to the reference level indicates a positive response to treatment. In some embodiments, the IL-6 level in the biological sample is increased compared to the reference level. In some embodiments, an increase in the IL-6 level in the biological sample compared to the reference level indicates a negative response to treatment.
在一些实施方案中，自个体衍生的生物学样品选自下组：细胞，组织，组织培养物，肿瘤，生物学流体及其组合。In some embodiments, the biological sample derived from the individual is selected from the group consisting of a cell, a tissue, a tissue culture, a tumor, a biological fluid, and combinations thereof.
在一些实施方案中，生物学流体选自下组：血浆，血清，全血，PBMC及其组合。In some embodiments, the biological fluid is selected from the group consisting of plasma, serum, whole blood, PBMCs, and combinations thereof.
在一些实施方案中，组织是肿瘤组织。在一些实施方案中，肿瘤组织选自下组：肿瘤细胞，肿瘤浸润性细胞，基质细胞及其任意组合。In some embodiments, the tissue is tumor tissue. In some embodiments, the tumor tissue is selected from the group consisting of tumor cells, tumor-infiltrating cells, stromal cells, and any combination thereof.
在一些实施方案中，细胞是循环肿瘤细胞(CTC)。In some embodiments, the cells are circulating tumor cells (CTCs).
在一些实施方案中，个体罹患增殖性疾病或病症。In some embodiments, the subject suffers from a proliferative disease or disorder.
在一些实施方案中，个体罹患癌或恶性肿瘤。在一些实施方案中，癌症或恶性肿瘤选自非小细胞肺癌，小细胞肺癌，肾细胞癌，结肠直肠癌，卵巢癌，乳腺癌，胰腺癌，胃癌，膀胱癌，食道癌，间皮瘤，黑素瘤，头和颈癌，甲状腺癌，肉瘤，前列腺癌，成胶质细胞瘤，宫颈癌，胸腺癌，白血病，淋巴瘤，骨髓瘤，蕈样肉芽肿，梅克尔细胞癌，和其它血液学恶性肿瘤。In some embodiments, the subject has a cancer or malignancy. In some embodiments, the cancer or malignancy is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies.
在一些实施方案中，个体罹患免疫相关疾病或病症。In some embodiments, the individual suffers from an immune-related disease or disorder.
在一些实施方案中，PD-L1轴结合拮抗剂是PD-L1结合拮抗剂。In some embodiments, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist.
在一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合它的配体结合配偶。In some embodiments, a PD-L1 binding antagonist inhibits the binding of PD-L1 to its ligand binding partner.
在一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合PD-1。在一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合B7-1。在一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合PD-1和B7-1二者。In some embodiments, the PD-L1 binding antagonist inhibits PD-L1 binding to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits PD-L1 binding to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits PD-L1 binding to both PD-1 and B7-1.
在一些实施方案中，PD-L1结合拮抗剂是抗体。在一些实施方案中，抗体是单克隆抗体。在一些实施方案中，抗体是人抗体，人源化抗体或嵌合抗体。In some embodiments, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
在一些实施方案中，PD-L1轴结合拮抗剂是PD-1结合拮抗剂。In some embodiments, the PD-L1 axis binding antagonist is a PD-1 binding antagonist.
在一些实施方案中，PD-1结合拮抗剂抑制PD-1结合它的配体结合配偶。In some embodiments, a PD-1 binding antagonist inhibits PD-1 from binding to its ligand binding partner.
在一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L1。在一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L2。在一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L1和PD-L2二者。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.
在一些实施方案中，PD-1结合拮抗剂是抗体。在一些实施方案中，抗体是单克隆抗体。在一些实施方案中，抗体是人抗体，人源化抗体或嵌合抗体。In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
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图1显示对照细胞样品的例示性IHC分析。(A)亲本HEK-293细胞的阴性对照IHC染色；(B)具有弱染色强度的用重组人PD-L1转染的HEK-293细胞的IHC染色；(C)具有中等染色强度的用重组人PD-L1转染的HEK-293细胞的IHC染色；(D)具有强染色强度的用重组人PD-L1转染的HEK-293细胞的IHC染色；(E)胎盘组织样品的阳性组织对照IHC染色；(F)扁桃体组织样品的阳性组织对照IHC染色。所有IHC染色是使用一种专利抗PD-L1抗体实施的。Figure 1 shows exemplary IHC analysis of control cell samples. (A) Negative control IHC staining of parental HEK-293 cells; (B) IHC staining of HEK-293 cells transfected with recombinant human PD-L1 with weak staining intensity; (C) IHC staining of HEK-293 cells transfected with recombinant human PD-L1 with moderate staining intensity; (D) IHC staining of HEK-293 cells transfected with recombinant human PD-L1 with strong staining intensity; (E) Positive tissue control IHC staining of placental tissue sample; (F) Positive tissue control IHC staining of tonsil tissue sample. All IHC staining was performed using a proprietary anti-PD-L1 antibody.
图2显示来自(A)三重阴性乳腺癌；(B)恶性黑素瘤；(C)NSCLC，腺癌的肿瘤样品的例示性PD-L1阳性IHC染色。FIG2 shows exemplary PD-L1 positive IHC staining of tumor samples from (A) triple negative breast cancer; (B) malignant melanoma; (C) NSCLC, adenocarcinoma.
图3显示肿瘤浸润性免疫细胞中的PD-L1表达与癌症患者中对抗PD-L1治疗的PD或PR/CR响应的关联。PD＝进行性疾病；PR＝部分响应；CR＝完全响应。(A)一个肿瘤样品区域内PD-L1+肿瘤浸润性免疫细胞的％，使用PD-L1IHC分析。(B)肿瘤样品内的总免疫浸润物内PD-L1+IC的％，使用PD-L1IHC分析。Figure 3 shows the correlation between PD-L1 expression in tumor-infiltrating immune cells and PD or PR/CR responses to anti-PD-L1 therapy in cancer patients. PD = progressive disease; PR = partial response; CR = complete response. (A) % of PD-L1+ tumor-infiltrating immune cells within a tumor sample region, analyzed using PD-L1 IHC. (B) % of PD-L1+ ICs within the total immune infiltrate within a tumor sample, analyzed using PD-L1 IHC.
图4显示肿瘤样品中的PD-L1基因表达与癌症患者中对抗PD-L1治疗的PD或PR/CR响应的关联，使用PD-L1qPCR分析。PD＝进行性疾病；PR＝部分响应；CR＝完全响应。Figure 4 shows the correlation between PD-L1 gene expression in tumor samples and PD or PR/CR response to anti-PD-L1 treatment in cancer patients using PD-L1 qPCR analysis. PD = progressive disease; PR = partial response; CR = complete response.
图5显示肿瘤样品中的PD-1基因表达与癌症患者中对抗PD-L1治疗的PD或PR/CR响应的关联。PD＝进行性疾病；PR＝部分响应；CR＝完全响应。Figure 5 shows the correlation between PD-1 gene expression in tumor samples and PD or PR/CR response to anti-PD-L1 treatment in cancer patients. PD = progressive disease; PR = partial response; CR = complete response.
图6显示肿瘤样品中的多种免疫基因表达与癌症患者中对抗PD-L1治疗的PD或PR响应的关联。PD＝进行性疾病；PR＝部分响应。Figure 6 shows the correlation of multiple immune gene expression in tumor samples with PD or PR response to anti-PD-L1 therapy in cancer patients. PD = progressive disease; PR = partial response.
图7显示来自用抗PD-L1抗体治疗的患者的系列治疗前/中肿瘤活检的示意图。评估来自罹患多种适应症(包括黑素瘤，肾细胞癌(RCC)，非小细胞肺癌(NSCLC)，头和颈癌(H&N)，结肠直肠癌(CRC)，胃，和乳腺癌)、用抗PD-L1抗体(n＝26)治疗的患者的成对基线(包括治疗前或存档肿瘤组织)和治疗中肿瘤活检。Figure 7 shows a schematic diagram of serial pre-treatment/interim tumor biopsies from patients treated with anti-PD-L1 antibodies. Paired baseline (including pre-treatment or archival tumor tissue) and on-treatment tumor biopsies from patients with multiple indications (including melanoma, renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck cancer (H&N), colorectal cancer (CRC), gastric, and breast cancer) treated with anti-PD-L1 antibodies (n=26) were evaluated.
图8显示(a)CD8+T细胞浸润的升高与来自响应抗PD-L1抗体治疗的患者的肿瘤样品中PD-L1表达的升高有关；和(b)用抗PD-L1抗体治疗之后，响应抗PD-L1抗体治疗的患者中T细胞活化标志物(包括粒酶A，穿孔蛋白，IFN-g，TNFa和CD8)的升高。Figure 8 shows (a) increased CD8+ T cell infiltration is associated with increased PD-L1 expression in tumor samples from patients who responded to anti-PD-L1 antibody treatment; and (b) increased T cell activation markers (including granzyme A, perforin, IFN-g, TNFa, and CD8) in patients who responded to anti-PD-L1 antibody treatment after treatment with anti-PD-L1 antibody.
图9汇总经历抗PD-L1抗体治疗的患者中PD-L1表达的变化。FIG9 summarizes changes in PD-L1 expression in patients undergoing anti-PD-L1 antibody treatment.
图10显示(a)血液中升高的增殖T细胞(鉴定为CD8+/Ki67+细胞)的频率；和(b)经历抗PD-L1抗体治疗的患者中升高的活化的增殖T细胞(鉴定为CD8+/HLA-DR+/Ki67+细胞)的频率。Figure 10 shows (a) increased frequency of proliferating T cells (identified as CD8+/Ki67+ cells) in blood; and (b) increased frequency of activated proliferating T cells (identified as CD8+/HLA-DR+/Ki67+ cells) in patients undergoing anti-PD-L1 antibody treatment.
图11显示血浆中IL-6水平的降低与响应抗PD-L1抗体治疗的患者有关，而且血浆中IL-6水平的升高与在抗PD-L1抗体治疗后进展的患者有关。FIG11 shows that decreased plasma IL-6 levels are associated with patients who respond to anti-PD-L1 antibody treatment, and increased plasma IL-6 levels are associated with patients who progress after anti-PD-L1 antibody treatment.
图12显示肿瘤样品中多种免疫基因表达与癌症患者中对抗PD-L1治疗的PD或PR/CR响应的关联。PD＝进行性疾病；PR＝部分响应；CR＝完全响应。Figure 12 shows the correlation between the expression of multiple immune genes in tumor samples and PD or PR/CR responses to anti-PD-L1 therapy in cancer patients. PD = progressive disease; PR = partial response; CR = complete response.
图13显示来自黑素瘤或NSCLC的肿瘤样品中的IDO1基因表达与癌症患者中对抗PD-L1治疗的PD或PR/CR响应的关联。PD＝进行性疾病；PR＝部分响应；CR＝完全响应。Figure 13 shows the correlation of IDO1 gene expression in tumor samples from melanoma or NSCLC with PD or PR/CR response to anti-PD-L1 treatment in cancer patients. PD = progressive disease; PR = partial response; CR = complete response.
图14显示自响应抗PD-L1抗体治疗的患者收集的血液中的循环T细胞上PD-L1表达的升高。PD＝进行性疾病；PR＝部分响应；CR＝完全响应。Figure 14 shows the increase in PD-L1 expression on circulating T cells in blood collected from patients responding to anti-PD-L1 antibody treatment. PD = progressive disease; PR = partial response; CR = complete response.
图15显示肿瘤样品中细胞毒性Th1细胞，IFN-g和T细胞运输标志物的基因表达与癌症患者中对抗PD-L1治疗的PD或PR/CR响应的关联。PD＝进行性疾病；PR＝部分响应；CR＝完全响应。Figure 15 shows the correlation between gene expression of cytotoxic Th1 cells, IFN-g, and T cell trafficking markers in tumor samples and PD or PR/CR response to anti-PD-L1 therapy in cancer patients. PD = progressive disease; PR = partial response; CR = complete response.
图16显示响应抗PD-L1抗体治疗的黑素瘤患者中T细胞活化标志物的升高。FIG16 shows elevation of T cell activation markers in melanoma patients in response to anti-PD-L1 antibody treatment.
图17显示不响应抗PD-L1抗体治疗的黑素瘤患者中肿瘤内T细胞的低频率和T细胞活化标志物的缺失。FIG17 shows the low frequency of intratumoral T cells and the absence of T cell activation markers in melanoma patients who do not respond to anti-PD-L1 antibody treatment.
图18显示响应抗PD-L1抗体治疗的患者的血液中CD8+/HLA-DR+/Ki-67+活化的T细胞频率的瞬时升高。Figure 18 shows a transient increase in the frequency of CD8+/HLA-DR+/Ki-67+ activated T cells in the blood of patients in response to anti-PD-L1 antibody treatment.
图19显示CD4+/ICOS+T细胞的波动，这个T细胞群体的延迟增多与响应有关和减少与疾病进展(在周期3后发生)有关。Figure 19 shows fluctuations in CD4+/ICOS+ T cells, with delayed increases in this T cell population associated with response and decreases associated with disease progression (occurring after cycle 3).
图20显示PD-L1表达的适应性升高在响应抗PD-L1抗体治疗的患者中是突出的。FIG20 shows that adaptive increases in PD-L1 expression are prominent in patients responding to anti-PD-L1 antibody treatment.
图21显示CTLA4表达与对抗PD-L1抗体治疗的响应的关联，而fractalkine/CX3CL1的表达与进展有关。Figure 21 shows that CTLA4 expression correlates with response to anti-PD-L1 antibody treatment, while fractalkine/CX3CL1 expression is associated with progression.
图22显示六种癌症适应症间与Teff(T效应)细胞，Treg(T调节)细胞，和Th17细胞有关的基因签名的关联。FIG22 shows the association of gene signatures related to Teff (T effector) cells, Treg (T regulatory) cells, and Th17 cells across six cancer indications.
图23显示不响应抗PD-L1治疗的患者中朝向更高的IL17F肿瘤基因表达的趋势。R＝响应者；nR＝非响应者。Figure 23 shows a trend toward higher IL17F tumor gene expression in patients who did not respond to anti-PD-L1 therapy. R = responder; nR = non-responder.
图24显示IL-17F的肿瘤基因表达在对抗PD-L1治疗具有晚期响应的患者中更高。FIG24 shows that tumor gene expression of IL-17F is higher in patients with late responses to anti-PD-L1 treatment.
图25显示经历抗PD-L1抗体治疗的患者中循环CD8+/HLA-DR+/Ki67+细胞的瞬时增多。(a)UBC患者中，(b)所有患者中。Figure 25 shows the transient increase in circulating CD8+/HLA-DR+/Ki67+ cells in patients treated with anti-PD-L1 antibodies: (a) UBC patients, (b) all patients.
图26显示经历抗PD-L1抗体治疗的患者中血浆IL-18的瞬时升高。而且，基线血浆MCP-1在对抗PD-L1治疗具有部分响应/完全响应(PR/CR)的患者中更低。IL-18和MCP-1均还在单核细胞中优势表达。Figure 26 shows a transient increase in plasma IL-18 in patients treated with anti-PD-L1 antibodies. Moreover, baseline plasma MCP-1 is lower in patients with partial response/complete response (PR/CR) to anti-PD-L1 treatment. Both IL-18 and MCP-1 are also predominantly expressed in monocytes.
图27显示来自在抗PD-L1抗体治疗之后进展的患者的治疗前肿瘤展示按比例更高的在髓样细胞(例如单核细胞，树突细胞)中优势表达的髓样基因签名(IL-8，CCL2，和IL1B)FIG27 shows that pre-treatment tumors from patients who progressed following anti-PD-L1 antibody treatment exhibited proportionally higher expression of a myeloid gene signature (IL-8, CCL2, and IL1B) that is predominantly expressed in myeloid cells (e.g., monocytes, dendritic cells)
图28显示响应抗PD-L1抗体治疗的患者的血液中可溶性PD-L1的关联。FIG28 shows the association of soluble PD-L1 in the blood of patients responding to anti-PD-L1 antibody treatment.
图29显示肿瘤浸润性免疫细胞(IC)中的PD-L1表达和对抗PD-L1治疗的响应之间的联系。(a)NSCLC中，(b)所有肿瘤中。Figure 29 shows the association between PD-L1 expression in tumor-infiltrating immune cells (ICs) and response to anti-PD-L1 therapy in (a) NSCLC and (b) all tumors.
图30显示肿瘤细胞中的PD-L1表达和对抗PD-L1治疗的响应之间的联系。(a)NSCLC中，(b)所有肿瘤中。Figure 30 shows the association between PD-L1 expression in tumor cells and response to anti-PD-L1 therapy: (a) NSCLC, (b) all tumors.
发明详述Detailed Description of the Invention
定义definition
术语“PD-L1轴结合拮抗剂”为如下的分子，其抑制PD-L1轴结合配偶与一种或多种它的结合配偶相互作用，从而去除源自PD-1信号传导轴上的信号传导的T细胞功能障碍–一项结果是恢复或增强T细胞功能。如本文中使用的，PD-L1轴结合拮抗剂包括PD-L1结合拮抗剂和PD-1结合拮抗剂以及干扰PD-L1和PD-1(例如PD-L2-Fc融合物)之间相互作用的分子。The term "PD-L1 axis binding antagonist" is a molecule that inhibits the interaction of a PD-L1 axis binding partner with one or more of its binding partners, thereby removing T cell dysfunction resulting from signaling on the PD-1 signaling axis - one result is restoration or enhancement of T cell function. As used herein, PD-L1 axis binding antagonists include PD-L1 binding antagonists and PD-1 binding antagonists, as well as molecules that interfere with the interaction between PD-L1 and PD-1 (e.g., PD-L2-Fc fusion).
术语“PD-L1结合拮抗剂”为如下的分子，其降低，阻断，抑制，消除或干扰源自PD-L1与一种或多种它的结合配偶(诸如PD-1，B7-1)相互作用的信号转导。在一些实施方案中，PD-L1结合拮抗剂是抑制PD-L1结合它的结合配偶的分子。在一个特定方面，PD-L1结合拮抗剂抑制PD-L1结合PD-1和/或B7-1。在一些实施方案中，PD-L1结合拮抗剂包括降低，阻断，抑制，消除或干扰源自PD-L1与一种或多种它的结合配偶(诸如PD-1，B7-1)相互作用的信号转导的抗PD-L1抗体，其抗原结合片段，免疫粘附素，融合蛋白，寡肽和其它分子。在一个实施方案中，PD-L1结合拮抗剂降低由或经由T淋巴细胞和其它细胞上表达的细胞表面蛋白质介导的负面信号(经由PD-L1或PD-1介导信号传导)，从而使得功能障碍性T细胞不太非功能障碍性。The term "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction derived from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding to its binding partner. In a specific aspect, the PD-L1 binding antagonist inhibits PD-L1 from binding 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, eliminate or interfere with signal transduction derived from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In one embodiment, the PD-L1 binding antagonist reduces negative signals (via PD-L1 or PD-1 mediated signaling) mediated by or via cell surface proteins expressed on T lymphocytes and other cells, thereby making dysfunctional T cells less non-dysfunctional.
术语“PD-1结合拮抗剂”为如下的分子，其降低，阻断，抑制，消除或干扰源自PD-1与一种或多种它的结合配偶(诸如PD-L1，PD-L2)相互作用的信号转导。在一些实施方案中，PD-1结合拮抗剂是抑制PD-1结合它的结合配偶的分子。在一个特定方面，PD-1结合拮抗剂抑制PD-1结合PD-L1和/或PD-L2。例如，PD-1结合拮抗剂包括降低，阻断，抑制，消除或干扰源自PD-1与PD-L1和/或PD-L2相互作用的信号转导的抗PD-1抗体，其抗原结合片段，免疫粘附素，融合蛋白，寡肽和其它分子。在一个实施方案中，PD-1结合拮抗剂降低由或经由T淋巴细胞和其它细胞上表达的细胞表面蛋白质介导的负面信号(经由PD-1或PD-L1介导信号传导)，从而使得功能障碍性T细胞不太非功能障碍性。The term "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction derived from the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2). In some embodiments, the PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its binding partner. In a specific aspect, the PD-1 binding antagonist inhibits PD-1 from binding 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, eliminate or interfere with signal transduction derived from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative signals (via PD-1 or PD-L1 mediated signaling) mediated by or via cell surface proteins expressed on T lymphocytes and other cells, thereby making dysfunctional T cells less non-dysfunctional.
术语“程序性死亡配体1”和“PD-L1”在本文中指天然序列PD-L1多肽、多肽变体及天然序列多肽和多肽变体的片段(其在本文中进一步定义)。本文中描述的PD-L1多肽可以是自多种来源，诸如自人组织类型或自另一种来源分离，或者通过重组或合成方法制备的PD-L1多肽。The terms "programmed death-ligand 1" and "PD-L1" herein refer to native sequence PD-L1 polypeptides, polypeptide variants, and fragments of native sequence polypeptides and polypeptide variants (which are further defined herein). The PD-L1 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
“天然序列PD-L1多肽”包括与从自然界衍生的相应PD-L1多肽具有相同氨基酸序列的多肽。A "native sequence PD-L1 polypeptide" includes a polypeptide having the same amino acid sequence as a corresponding PD-L1 polypeptide derived from nature.
“PD-L1多肽变体”或其变型意指与如本文中公开的任何天然序列PD-L1多肽序列具有至少约80％氨基酸序列同一性的PD-L1多肽，一般为活性PD-L1多肽，如本文中定义的。例如，此类PD-L1多肽变体包括如下的PD-L1多肽，其中在天然氨基酸序列的N或C端添加或删除一个或多个氨基酸残基。通常，PD-L1多肽变体与如本文中公开的天然序列PD-L1多肽序列会具有至少约80％氨基酸序列同一性，或者至少约81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％氨基酸序列同一性。通常，PD-L1变体多肽的长度是至少约10个氨基酸，或者长度为至少约20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、281、282、283、284、285、286、287、288、289个氨基酸，或更多个。任选地，与天然PD-L1多肽序列相比，PD-L1变体多肽会具有不超过1处保守氨基酸替代，或者与天然PD-L1多肽序列相比不超过2、3、4、5、6、7、8、9、或10处保守氨基酸替代。By "PD-L1 polypeptide variant" or variations thereof is meant a PD-L1 polypeptide, generally an active PD-L1 polypeptide, as defined herein, that has at least about 80% amino acid sequence identity to any native sequence PD-L1 polypeptide sequence as disclosed herein. For example, such PD-L1 polypeptide variants include PD-L1 polypeptides in which one or more amino acid residues are added to or deleted from the N- or C-terminus of the native amino acid sequence. Typically, a PD-L1 polypeptide variant will have at least about 80% amino acid sequence identity, or at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a native sequence PD-L1 polypeptide sequence as disclosed herein. Typically, the PD-L1 variant polypeptide is at least about 10 amino acids in length, or at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 amino acids in length, or more. Optionally, the PD-L1 variant polypeptide will have no more than 1 conservative amino acid substitution compared to the native PD-L1 polypeptide sequence, or no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to the native PD-L1 polypeptide sequence.
如本文中定义的，术语“PD-L1拮抗剂”指部分或完全阻断、抑制、或中和由天然序列PD-L1介导的生物学活性和/或功能的任何分子。在某些实施方案中，此类拮抗剂结合PD-L1。依照一个实施方案，拮抗剂是多肽。依照另一个实施方案，拮抗剂是抗PD-L1抗体。依照另一个实施方案，拮抗剂是小分子拮抗剂。依照另一个实施方案，拮抗剂是多核苷酸拮抗剂。As defined herein, the term "PD-L1 antagonist" refers to any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity and/or function mediated by native sequence PD-L1. In certain embodiments, such antagonists bind to PD-L1. According to one embodiment, the antagonist is a polypeptide. According to another embodiment, the antagonist is an anti-PD-L1 antibody. According to another embodiment, the antagonist is a small molecule antagonist. According to another embodiment, the antagonist is a polynucleotide antagonist.
“多核苷酸”或“核酸”在本文中可互换使用，指任何长度的核苷酸聚合物，包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、经过修饰的核苷酸或碱基、和/或其类似物，或者是可通过DNA或RNA聚合酶，或者通过合成反应掺入聚合物中的任何底物。多核苷酸可包含经过修饰的核苷酸，诸如甲基化核苷酸及其类似物。若存在的话，对核苷酸结构的修饰可以在装配聚合物之前或之后进行。核苷酸序列可以由非核苷酸组分中断。多核苷酸可以在合成后进一步修饰，诸如通过与标记物缀合。其它类型的修饰包括例如“帽”，将一个或多个天然存在的核苷酸用类似物替代，核苷酸间修饰诸如例如具有不带电荷连接(例如甲基膦酸酯、磷酸三酯、磷酰胺酯(phosphoamidate)、氨基甲酸酯等)和具有带电荷连接(例如硫代磷酸酯、二硫代磷酸酯等)的修饰，含有悬垂模块(pendant moiety)诸如例如蛋白质(例如核酸酶、毒素、抗体、信号肽、聚L-赖氨酸等)的修饰，具有嵌入剂(例如吖啶、补骨脂素等)的修饰，含有螯合剂(例如金属、放射性金属、硼、氧化性金属等)的修饰，含有烷化剂的修饰，具有经修饰连接(例如α端基异构核酸(anomeric nucleic acid)等)的修饰，以及未修饰形式的多核苷酸。另外，通常存在于糖类中的任何羟基基团可以用例如膦酸(phosphonate)基团、磷酸(phosphate)基团替换，用标准保护基团保护，或活化以制备与别的核苷酸的别的连接，或者可缀合至固体或半固体支持物。可磷酸化或者用胺或1-20个碳原子的有机加帽基团模块取代5’和3’末端OH。其它羟基也可衍生成标准保护基团。多核苷酸也可含有本领域普遍知道的核糖或脱氧核糖糖类的类似物形式，包括例如2’-氧-甲基-、2’-氧-烯丙基-、2’-氟-或2’-叠氮-核糖，碳环糖类似物，α-端基异构糖，差向异构糖诸如阿拉伯糖、木糖或来苏糖、吡喃糖、呋喃糖、景天庚酮糖，无环类似物及脱碱基核苷类似物诸如甲基核糖核苷。可用备选连接基团替换一个或多个磷酸二酯连接。这些备选连接基团包括但不限于以下实施方案，其中磷酸酯用P(O)S(“硫代酸酯”(thioate))、P(S)S(“二硫代酸酯”(dithioate))、(O)NR2(“酰胺酯”(amidate))、P(O)R、P(O)OR’、CO或CH2(“甲缩醛”(formacetal))替代，其中R或R’各自独立为H或者取代或未取代的烃基(1-20个C)，任选含有醚(-O-)连接、芳基、烯基、环烃基、环烯基或芳烃基(araldyl)。并非多核苷酸中的所有连接都必需是相同的。前述描述适用于本文中提及的所有多核苷酸，包括RNA和DNA。"Polynucleotide" or "nucleic acid" are used interchangeably herein to refer to nucleotide polymers of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, if any, can be made before or after assembly of the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps," replacement of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as, for example, modifications with uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoamidate, carbamate, etc.) and with charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), modifications containing pendant moieties such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications with intercalators (e.g., acridine, psoralen, etc.), modifications containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), modifications containing alkylating agents, modifications with modified linkages (e.g., α-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. In addition, any hydroxyl groups typically present in carbohydrates can be replaced with, for example, phosphonate groups, phosphate groups, protected with standard protecting groups, or activated to prepare additional linkages to other nucleotides, or can be conjugated to a solid or semi-solid support. The 5' and 3' terminal OH groups can be phosphorylated or replaced with amine or 1-20 carbon atom organic capping group modules. Other hydroxyl groups can also be derived into standard protecting groups. Polynucleotides can also contain analog forms of ribose or deoxyribose sugars generally known in the art, including, for example, 2'-oxy-methyl-, 2'-oxy-allyl-, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomers, epimers such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl ribonucleoside. One or more phosphodiester linkages can be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, orCH2 ("formacetal"), wherein R or R' is independently H or a substituted or unsubstituted hydrocarbon group (1-20 carbon atoms), optionally containing an ether (-O-) linkage, an aryl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, or an araldyl group. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.
如本文中所使用的，“寡核苷酸”一般指短的、单链的多核苷酸，其在长度上小于约250个核苷酸，但这不是必须的。寡核苷酸可以是合成的。术语“寡核苷酸”和“多核苷酸”并不互相排斥。上文关于多核苷酸的描述同样且完全可适用于寡核苷酸。As used herein, "oligonucleotide" generally refers to short, single-stranded polynucleotides that are less than about 250 nucleotides in length, but this is not necessary. Oligonucleotides can be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides.
术语“引物”指能够与核酸杂交并允许互补核酸的聚合作用(一般通过提供游离的3’-OH基团)的单链多核苷酸。The term "primer" refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid and allowing polymerization of complementary nucleic acid, typically by providing a free 3'-OH group.
术语“小分子”指具有约2000道尔顿或更小，优选约500道尔顿或更小的分子量的任何分子。The term "small molecule" refers to any molecule having a molecular weight of about 2000 Daltons or less, preferably about 500 Daltons or less.
术语“宿主细胞”、“宿主细胞系”、和“宿主细胞培养物”可互换使用，并且指已经导入有外源核酸的细胞，包括此类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞及自其衍生的后代而不考虑传代的次数。后代在核酸内容物上可以与亲本细胞不完全相同，而是可以含有突变。本文中包括具有与在初始转化细胞中筛选或选择的相同功能或生物学活性的突变体后代。The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the initial transformed cell are included herein.
如本文中使用的，术语“载体”指能够扩增与其连接的另一种核酸的核酸分子。该术语包括作为自身复制型核酸结构的载体及整合入接受其导入的宿主细胞的基因组中的载体。某些载体能够指导与其可操作连接的核酸的表达。此类载体在本文中称为“表达载体”。As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."
“分离的”抗体指已经与其天然环境的组分分开的抗体。在一些实施方案中，抗体纯化至大于95％或99％的纯度，如通过例如电泳(例如SDS-PAGE、等电聚焦(IEF)、毛细管电泳)或层析(例如离子交换或反相HPLC)测定的。关于用于评估抗体纯度的方法的综述，见例如Flatman et al.,J.Chromatogr.B 848:79-87(2007)。An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).
“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括通常含有核酸分子的细胞中含有的核酸分子，但是核酸分子在染色体外或在与其天然染色体位置不同的染色体位置处存在。An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
本文中的术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)、和抗体片段，只要它们展现出期望的抗原结合活性。The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.
术语“抗PD-L1抗体”和“结合PD-L1的抗体”指能够以足够亲和力结合PD-L1，使得该抗体可作为诊断剂和/或治疗剂用于靶向PD-L1的抗体。在一个实施方案中，根据例如通过放射免疫测定法(RIA)的测量，抗PD-L1抗体结合无关的、非PD-L1的蛋白质的程度小于该抗体对PD-L1的结合的约10％。在某些实施方案中，抗PD-L1抗体结合在来自不同物种的PD-L1中保守的PD-L1表位。The terms "anti-PD-L1 antibody" and "antibody that binds to PD-L1" refer to an antibody that binds to PD-L1 with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent to target PD-L1. In one embodiment, the extent to which the anti-PD-L1 antibody binds to unrelated, non-PD-L1 proteins is less than about 10% of the binding of the antibody to PD-L1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the anti-PD-L1 antibody binds to a PD-L1 epitope that is conserved among PD-L1 from different species.
“阻断性”抗体或“拮抗性”抗体是抑制或降低其结合的抗原的生物学活性的抗体。优选的阻断性抗体或拮抗性抗体实质性或完全抑制抗原的生物学活性。A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
“亲和力”指分子(例如抗体)的单一结合位点与其结合配偶(例如抗原)之间全部非共价相互作用总和的强度。除非另有指示，如本文中使用的，“结合亲和力”指反映结合对的成员(例如抗体和抗原)之间1:1相互作用的内在结合亲和力。分子X对其配偶Y的亲和力通常可以用解离常数(Kd)来表述。亲和力可以通过本领域知道的常用方法来测量，包括本文中所描述的方法。下文描述了用于测量结合亲和力的具体的说明性和例示性的实施方案。"Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
“亲和力成熟的”抗体指在一个或多个高变区(HVR)中具有一处或多处改变的抗体，与不拥有此类改变的亲本抗体相比，此类改变导致该抗体对抗原的亲和力改善。An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) which result in improved affinity of the antibody for antigen, compared to a parent antibody which does not possess such alteration(s).
“抗体片段”指与完整抗体不同的分子，其包含完整抗体的如下部分，该部分结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fv、Fab、Fab’、Fab’-SH、F(ab’)2；双抗体；线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。"Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
与参照抗体“结合相同表位的抗体”指在竞争测定法中将参照抗体对其抗原的结合阻断50％或更多的抗体，且相反，参照抗体在竞争测定法中将该抗体对其抗原的结合阻断50％或更多。本文中提供了例示性的竞争测定法。An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.
术语“嵌合”抗体指其中的重和/或轻链的一部分自特定的来源或物种衍生，而重和/或轻链的剩余部分自不同来源或物种衍生的抗体。The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
抗体的“类”指其重链拥有的恒定域或恒定区的类型。抗体有5大类：IgA、IgD、IgE、IgG、和IgM，并且这些中的几种可以进一步分成亚类(同种型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1、和IgA2。与不同类免疫球蛋白对应的重链恒定域分别称作α、δ、ε、γ、和μ。The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), for example,IgG1 ,IgG2 ,IgG3 ,IgG4 ,IgA1 , andIgA2 . The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.
术语“全长抗体”、“完整抗体”、和“全抗体”在本文中可互换使用，指与天然抗体结构具有基本上类似的结构或者具有含有如本文中所限定的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 that has a structure substantially similar to a native antibody structure or has heavy chains that contain an Fc region as defined herein.
如本文中使用的，术语“单克隆抗体”指从一群基本上同质的抗体获得的抗体，即构成群体的各个抗体是相同的和/或结合相同表位，除了例如含有天然存在的突变或在单克隆抗体制备物的生成期间发生的可能的变体抗体外，此类变体一般以极小量存在。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个单克隆抗体针对抗原上的单一决定簇。如此，修饰语“单克隆”指示抗体自一群基本上同质的抗体获得的特性，而不应解释为要求通过任何特定方法来生成抗体。例如，可以通过多种技术来生成要依照本发明使用的单克隆抗体，包括但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法、和利用含有所有或部分人免疫球蛋白基因座的转基因动物的方法，本文中描述了用于生成单克隆抗体的此类方法和其它例示性方法。As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical and/or bind to the same epitope, except for example containing naturally occurring mutations or possible variant antibodies occurring during the generation of monoclonal antibody preparations, such variants are generally present in very small amounts. Unlike polyclonal antibody preparations that typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a group of substantially homogeneous antibodies, and should not be interpreted as requiring the generation of antibodies by any particular method. For example, the monoclonal antibody to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and such methods and other exemplary methods for generating monoclonal antibodies are described herein.
“人抗体”指拥有与由人或人细胞生成的或利用人抗体全集或其它人抗体编码序列自非人来源衍生的抗体的氨基酸序列对应的氨基酸序列的抗体。人抗体的此定义明确排除包含非人抗原结合残基的人源化抗体。A "human antibody" is an antibody that possesses an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody repertoire or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
“人源化”抗体指包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在某些实施方案中，人源化抗体会包含至少一个，通常两个基本上整个可变域，其中所有或基本上所有HVR(例如CDR)对应于非人抗体的那些，且所有或基本上所有FR对应于人抗体的那些。任选地，人源化抗体可以至少包含自人抗体衍生的抗体恒定区的一部分。抗体，例如非人抗体的“人源化形式”指已经经历人源化的抗体。"Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, typically two, substantially entire variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. Optionally, a humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.
“免疫缀合物”指与一种或多种异源分子，包括但不限于细胞毒剂缀合的抗体。An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.
关于参考多肽序列的“百分比(％)氨基酸序列同一性”定义为比对序列并在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分时，候选序列中与参考多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的比对可以本领域技术范围内的多种方式进行，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定用于比对序列的适宜参数，包括对所比较序列全长实现最大比对所需的任何算法。然而，为了本文中的目的，％氨基酸序列同一性值是使用序列比较计算机程序ALIGN-2生成的。ALIGN-2序列比较计算机程序由Genentech公司编写，源代码已经连同用户文档一起提交给美国版权局(US Copyright Office，Washington D.C.，20559)，并以美国版权注册号TXU510087注册。公众可自Genentech公司(South San Francisco，California)得到ALIGN-2程序，或者可以自源代码编译。ALIGN-2程序应当编译成在UNIX操作系统，包括数码UNIX V4.0D上使用。所有序列比较参数由ALIGN-2程序设定且不变。"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and, if necessary, introducing gaps to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for purposes herein, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, and its source code has been submitted to the U.S. Copyright Office (US Copyright Office, Washington D.C., 20559) along with user documentation, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is available to the public from Genentech (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于(to)、与(with)、或针对(against)给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：In the case of using ALIGN-2 to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (or which can be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
分数X/Y乘100Multiply the fraction X/Y by 100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基数，且其中Y是B中的氨基酸残基总数。可以领会，若氨基酸序列A的长度与氨基酸序列B的长度不相等，则A相对于B的％氨基酸序列同一性将不等于B相对于A的％氨基酸序列同一性。除非另有具体说明，本文中所使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.
术语“检测”包括任何检测手段，包括直接和间接检测。The term "detecting" includes any means of detection, including direct and indirect detection.
如本文中使用的，术语“生物标志物”指可在样品中检测的指示物，例如预测性、诊断性和/或预后性的指示物。生物标志物可以充当由特定的分子、病理学、组织学和/或临床特征表征的特定疾病或病症(例如癌症)亚型的指示物。在一些实施方案中，生物标志物是一种基因。生物标志物包括但不限于，多核苷酸(例如DNA和/或RNA)、多核苷酸拷贝数目改变(例如DNA拷贝数)、多肽、多肽和多核苷酸修饰(例如翻译后修饰)、碳水化合物和/或基于糖脂的分子标志物。As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, such as a predictive, diagnostic, and/or prognostic indicator. A biomarker can serve as an indicator of a specific disease or condition (e.g., cancer) subtype characterized by specific molecular, pathological, histological, and/or clinical features. In some embodiments, a biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), changes in polynucleotide copy number (e.g., DNA copy number), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based molecular markers.
术语“生物标志物签名”、“签名”、“生物标志物表达签名”或“表达签名”在本文中可互换使用，指其表达为指示物，例如预测性、诊断性和/或预后性指示物的一种或一组生物标志物。生物标志物签名可以充当由特定的分子、病理学、组织学和/或临床特征表征的特定疾病或病症(例如癌症)亚型的指示物。在一些实施方案中，生物标志物签名是“基因签名”。术语“基因签名”与“基因表达签名”可互换使用，指其表达为指示物，例如预测性、诊断性和/或预后性指示物的一种或一组多核苷酸。在一些实施方案中，生物标志物签名是“蛋白质签名”。术语“蛋白质签名”与“蛋白质表达签名”可互换使用，指其表达为指示物，例如预测性、诊断性和/或预后性指示物的一种或一组多肽。The terms "biomarker signature," "signature," "biomarker expression signature," or "expression signature" are used interchangeably herein to refer to one or a group of biomarkers whose expression is an indicator, e.g., a predictive, diagnostic, and/or prognostic indicator. A biomarker signature can serve as an indicator of a particular disease or disorder (e.g., cancer) subtype characterized by specific molecular, pathological, histological, and/or clinical features. In some embodiments, the biomarker signature is a "gene signature." The terms "gene signature" and "gene expression signature" are used interchangeably to refer to one or a group of polynucleotides whose expression is an indicator, e.g., a predictive, diagnostic, and/or prognostic indicator. In some embodiments, the biomarker signature is a "protein signature." The terms "protein signature" and "protein expression signature" are used interchangeably to refer to one or a group of polypeptides whose expression is an indicator, e.g., a predictive, diagnostic, and/or prognostic indicator.
生物标志物与个体增加的临床益处有关的“量”或“水平”是生物学样品中的可检测水平。这些可通过本领域技术人员已知的及本文中公开的方法来测量。所评估的生物标志物的表达水平或量可用于确定对治疗的响应。The "amount" or "level" of a biomarker that correlates with an increased clinical benefit in an individual is the detectable level in a biological sample. These can be measured by methods known to those skilled in the art and disclosed herein. The expression level or amount of the assessed biomarker can be used to determine the response to treatment.
术语“表达的水平”或“表达水平”一般可互换使用，一般指生物学样品中生物标志物的量。“表达”一般指信息(例如基因编码和/或表观遗传)转化成细胞中存在并运行的结构的过程。因此，如本文中使用的，“表达”可以指转录成多核苷酸、翻译成多肽、或甚至多核苷酸和/或多肽修饰(例如多肽的翻译后修饰)。转录的多核苷酸的、翻译的多肽的、或多核苷酸和/或多肽修饰(例如多肽的翻译后修饰)的片段也应视为表达的，无论它们是源自通过可变剪接生成的转录物或经过降解的转录物，或者是源自多肽的翻译后加工(例如通过蛋白水解)。“表达的基因”包括转录成多核苷酸(如mRNA)、然后翻译成多肽的基因，还有转录成RNA但不翻译成多肽的基因(例如转运和核糖体RNA)。The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., genetically encoded and/or epigenetic) is converted into a structure present and operating in a cell. Therefore, as used herein, "expression" can refer to transcription into polynucleotides, translation into polypeptides, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of polypeptides). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered expressed, whether or not they are derived from transcripts generated by alternative splicing or from degraded transcripts, or from post-translational processing (e.g., by proteolysis) of polypeptides."Expressed genes" include genes that are transcribed into polynucleotides (e.g., mRNA) and then translated into polypeptides, as well as genes that are transcribed into RNA but not translated into polypeptides (e.g., transport and ribosomal RNA).
“升高的表达”、“升高的表达水平”或“升高的水平”指相对于对照如不具有疾病或病症(例如癌症)的个体或内部对照(例如持家型生物标志物)，个体中生物标志物的增加的表达或增加的水平。"Elevated expression," "elevated expression level," or "elevated level" refers to increased expression or increased levels of a biomarker in an individual relative to a control, such as an individual without a disease or disorder (e.g., cancer) or an internal control (e.g., a housekeeping biomarker).
“降低的表达”、“降低的表达水平”或“降低的水平”指相对于对照诸如没有罹患疾病或病症(例如癌症)的个体或内部对照(例如持家型生物标志物)，个体中生物标志物的降低的表达或降低的水平。"Decreased expression," "decreased expression level," or "decreased level" refers to decreased expression or decreased levels of a biomarker in an individual relative to a control, such as an individual not suffering from a disease or disorder (e.g., cancer) or an internal control (e.g., a housekeeping biomarker).
术语“持家型生物标志物”指通常相似地存在于所有细胞类型中的一种生物标志物或一组生物标志物(例如多核苷酸和/或多肽)。在一些实施方案中，持家型生物标志物是“持家基因”。“持家基因”在本文中指一种基因或一组基因，其编码活性对于细胞功能维持而言必要的蛋白质，且通常相似地存在于所有细胞类型中。The term "housekeeping biomarker" refers to a biomarker or a group of biomarkers (e.g., polynucleotides and/or polypeptides) that are generally similarly present in all cell types. In some embodiments, the housekeeping biomarker is a "housekeeping gene." "Housekeeping gene" herein refers to a gene or a group of genes that encodes a protein whose activity is necessary for the maintenance of cellular function and is generally similarly present in all cell types.
如本文中使用的，“扩增”一般指生成多拷贝的期望序列的过程。“多拷贝”指至少2个拷贝。“拷贝”不必然意味着与模板序列有完全的序列互补性或同一性。例如，拷贝可以包含核苷酸类似物诸如脱氧肌苷、有意的序列变化(诸如经由包含与模板可杂交但不互补的序列的引物引入的序列变化)和/或扩增期间发生的序列错误。As used herein, "amplification" generally refers to the process of generating multiple copies of a desired sequence. "Multiple copies" refers to at least two copies. "Copies" does not necessarily mean that there is complete sequence complementarity or identity with the template sequence. For example, copies can contain nucleotide analogs such as deoxyinosine, intentional sequence changes (such as sequence changes introduced via primers containing sequences that are hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.
术语“多重PCR”指出于在单个反应中扩增两种或更多种DNA序列的目的，使用超过一套引物在自单一来源(例如个体)获得的核酸上进行的单个PCR反应。The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acid obtained from a single source (eg, an individual) using more than one set of primers for the purpose of amplifying two or more DNA sequences in a single reaction.
杂交反应的“严格性”可以由本领域中的普通技术人员容易地确定，而且通常根据探针长度、洗涤温度和盐浓度凭经验计算。一般而言，较长的探针要求较高的温度以正确退火，而较短的探针需要较低的温度。杂交通常依赖于当互补链存在于低于其解链温度的环境中时变性DNA重新退火的能力。探针与可杂交序列之间的期望同源性程度越高，可使用的相对温度也越高。结果，可推出较高相对温度将趋向于使反应条件更为严格，而较低温度也就较不严格。关于杂交反应严格性的其它细节和解释，参见Ausubel et al.,CurrentProtocols in Molecular Biology,Wiley Interscience Publishers,(1995)。The " stringency " of hybridization reaction can be easily determined by those of ordinary skill in the art, and is usually calculated empirically according to probe length, washing temperature and salt concentration. Generally speaking, longer probes require higher temperatures to correctly anneal, while shorter probes require lower temperatures. Hybridization usually depends on the ability of denatured DNA to anneal again when the complementary chain is present in an environment lower than its melting temperature. The higher the degree of homology expected between the probe and the hybridizable sequence, the higher the relative humidity that can be used. As a result, it can be deduced that higher relative humidity will tend to make reaction conditions more stringent, while lower temperatures are also less stringent. For other details and explanations about the stringency of hybridization reaction, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
如本文中所定义的，“严格条件”或“高严格条件”可由以下鉴定：(1)对于洗涤采用低离子强度和高温，例如0.015M氯化钠/0.0015M柠檬酸钠/0.1％十二烷基硫酸钠，50℃；(2)在杂交期间使用变性剂，诸如甲酰胺，例如50％(v/v)甲酰胺及0.1％牛血清清蛋白/0.1％Ficoll/0.1％聚乙烯吡咯烷酮/50mM磷酸钠缓冲液pH 6.5及750mM氯化钠、75mM柠檬酸钠，42℃；或(3)在使用50％甲酰胺、5x SSC(0.75M NaCl、0.075M柠檬酸钠)、50mM磷酸钠(pH6.8)、0.1％焦磷酸钠、5x登哈特(Denhardt)氏溶液、超声处理的鲑精DNA(50μg/ml)、0.1％SDS和10％硫酸右旋糖苷的溶液中在42℃过夜杂交，在42℃于0.2x SSC(氯化钠/柠檬酸钠)洗涤10分钟，然后在55℃进行由含有EDTA的0.1x SSC组成的10分钟高严格洗涤。As defined herein, "stringent conditions" or "high stringency conditions" can be identified by: (1) low ionic strength and high temperature for washes, e.g., 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) use of a denaturing agent such as formamide during hybridization, e.g., 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH 6.5 and 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) use of a denaturing agent such as 50% formamide, 5x SSC (0.75 M Hybridization was performed overnight at 42°C in a solution of 50 mM NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate, followed by a 10-minute high-stringency wash at 42°C in 0.2x SSC (sodium chloride/sodium citrate), followed by a 10-minute high-stringency wash at 55°C in 0.1x SSC containing EDTA.
“中等严格条件”可以如Sambrook et al.,Molecular Cloning:A LaboratoryManual,New York:Cold Spring Harbor Press,1989所描述的来定义，而且包括使用与上文所述那些相比较不太严格的洗涤溶液和杂交条件(例如温度、离子强度和％SDS)。中等严格条件的一个例子是在含：20％甲酰胺、5x SSC(150mM NaCl、15mM柠檬酸三钠)、50mM磷酸钠(pH7.6)、5x登哈特氏溶液、10％硫酸右旋糖苷和20mg/ml变性的剪切的鲑精DNA的溶液中于37℃温育过夜，然后于约37-50℃在1x SSC中洗涤滤膜。熟练技术人员会认识到如何根据适应诸如探针长度等因素的需要来调节温度、离子强度等。"Moderately stringent conditions" can be defined as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of wash solutions and hybridization conditions (e.g., temperature, ionic strength, and % SDS) that are less stringent than those described above. An example of moderately stringent conditions is incubation overnight at 37°C in a solution containing: 20% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA, followed by washing the filter in 1x SSC at approximately 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as needed to accommodate factors such as probe length.
“聚合酶链式反应”或“PCR”技术在用于本文时一般指其中如1987年7月28日公告的美国专利第4,683,195号中所记载的，扩增微量的核酸、RNA和/或DNA特定片段的流程。通常，需要获知目的区域末端或以外的序列信息，从而可设计寡核苷酸引物；这些引物在序列上将与待扩增模板的相对链相同或相似。两条引物的5’末端核苷酸可与所扩增物质的末端一致。PCR可用于从总基因组DNA和由总细胞RNA转录的cDNA、噬菌体或质粒序列等扩增特定RNA序列、特定DNA序列。一般参见Mullis等,Cold Spring Harbor Symp.Quant.Biol.51:263(1987)；Erlich ed.,PCR Technology,Stockton Press,NY,1989。在用于本文时，PCR视为用于扩增核酸测试样品的核酸聚合酶反应方法的一个但非唯一的例子，包括使用已知核酸(DNA或RNA)作为引物并利用核酸聚合酶来扩增或生成特定核酸片段，或者扩增或生成与特定核酸互补的特定核酸片段。As used herein, the term "polymerase chain reaction" or "PCR" refers generally to a process for amplifying trace amounts of specific fragments of nucleic acids, RNA, and/or DNA, as described in U.S. Patent No. 4,683,195, issued July 28, 1987. Typically, sequence information at or beyond the termini of the region of interest is required so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may correspond to the termini of the material being amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences, and the like from total genomic DNA, cDNA transcribed from total cellular RNA, phage or plasmid sequences, and the like. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987); Erlich ed., PCR Technology, Stockton Press, NY, 1989. As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, including using a known nucleic acid (DNA or RNA) as a primer and utilizing a nucleic acid polymerase to amplify or generate a specific nucleic acid fragment, or amplify or generate a specific nucleic acid fragment that is complementary to a specific nucleic acid.
“定量实时聚合酶链式反应”或“qRT-PCR”指PCR的一种形式，其中在PCR反应的每个步骤测量PCR产物的量。这种技术已经记载于多份出版物，包括Cronin等,Am.J.Pathol.164(1):35-42(2004)；Ma等,Cancer Cell.5:607-616(2004)。"Quantitative real-time polymerase chain reaction" or "qRT-PCR" refers to a form of PCR in which the amount of PCR product is measured at each step of the PCR reaction. This technique has been described in several publications, including Cronin et al., Am. J. Pathol. 164(1):35-42 (2004); Ma et al., Cancer Cell. 5:607-616 (2004).
术语“微阵列”指可杂交阵列元素，优选多核苷酸探针在基片上的有序排列。The term "microarray" refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.
术语“多核苷酸”在以单数或复数使用时一般指任何多聚核糖核苷酸或多聚脱氧核糖核苷酸，可以是未经修饰的RNA或DNA或者是经过修饰的RNA或DNA。如此，例如，本文中所定义的多核苷酸包括但不限于单链和双链DNA、包含单链区和双链区的DNA、单链和双链RNA、及包含单链区和双链区的RNA，包含DNA和RNA的杂合分子，它可以是单链的，或者更典型的是双链的，或者包含单链区和双链区。另外，术语“多核苷酸”在用于本文时指包含RNA或DNA或RNA和DNA二者的三链区。此类区中的链可来自相同分子或来自不同分子。所述区可包含一种或多种分子的整个，但是更典型的是只包含有些分子的一个区。三股螺旋区的分子之一常常是寡核苷酸。术语“多核苷酸”明确包括cDNA。该术语包括包含一种或多种经修饰碱基的DNA(包括cDNA)和RNA。如此，主链为稳定性或其它原因而修饰的DNA或RNA也是“多核苷酸”该术语在本文中的意图所在。此外，包含罕见碱基诸如肌苷或经修饰碱基诸如氚化碱基的DNA或RNA也包括在本文中所定义的术语“多核苷酸”内。一般而言，术语“多核苷酸”涵盖未修饰多核苷酸的所有化学、酶学和/或代谢修饰形式，以及病毒和细胞，包括简单和复杂细胞的特征性的DNA和RNA的化学形式。The term "polynucleotide," when used in the singular or plural, generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, polynucleotides as defined herein include, but are not limited to, single-stranded and double-stranded DNA, DNA comprising single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA comprising single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA, which may be single-stranded or, more typically, double-stranded, or comprise single-stranded and double-stranded regions. Additionally, the term "polynucleotide," as used herein, refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The strands in such a region may be from the same molecule or from different molecules. The region may comprise the entirety of one or more molecules, but more typically comprises only a region of some molecules. One of the molecules in a triple-helical region is often an oligonucleotide. The term "polynucleotide" explicitly includes cDNA. The term encompasses DNA (including cDNA) and RNA comprising one or more modified bases. Thus, DNAs or RNAs whose backbones are modified for stability or other reasons are also "polynucleotides" for the purpose of this term. In addition, DNAs or RNAs containing unusual bases such as inosine or modified bases such as tritiated bases are also included within the term "polynucleotide" as defined herein. In general, the term "polynucleotide" encompasses all chemically, enzymatically, and/or metabolically modified forms of unmodified polynucleotides, as well as chemical forms of DNA and RNA that are characteristic of viruses and cells, including simple and complex cells.
术语“寡核苷酸”指相对较短的多核苷酸，包括但不限于单链脱氧核糖核苷酸、单链或双链核糖核苷酸、RNA:DNA杂合物、及双链DNA。寡核苷酸，诸如单链DNA探针寡核苷酸，常常通过化学方法来合成，例如使用商品化的自动化寡核苷酸合成仪。然而，寡核苷酸可通过多种其它方法来制备，包括体外重组DNA介导的技术和通过DNA在细胞和生物体中的表达。The term "oligonucleotide" refers to relatively short polynucleotides, including but not limited to single-stranded deoxyribonucleotides, single-stranded or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNA. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are usually synthesized by chemical methods, for example, using the automated oligonucleotide synthesizer of commercialization. However, oligonucleotides can be prepared by a variety of other methods, including the technology of in vitro recombinant DNA mediation and the expression of DNA in cells and organisms.
术语“诊断”在本文中用于指对分子或病理学状态、疾病或疾患(例如癌症)的鉴定或分类。例如，“诊断”可以指特定癌症类型的鉴定。“诊断”还可以指特定癌症亚型的分类，例如通过组织病理学标准或分子特征(例如由一种或一组生物标志物(例如特定基因或由所述基因编码的蛋白质)的表达表征的亚型)。The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease, or condition, such as cancer. For example, "diagnosis" can refer to the identification of a particular type of cancer. "Diagnosis" can also refer to the classification of a particular cancer subtype, such as by histopathological criteria or molecular signatures, such as a subtype characterized by the expression of one or a panel of biomarkers, such as a particular gene or protein encoded by said gene.
术语“帮助做出诊断”在本文中用于指帮助进行关于特定类型的疾病或病症(例如癌症)的症状或状况的存在或性质的临床确定的方法。例如，帮助做出疾病或状况(例如癌症)诊断的方法可以包括在来自个体的生物学样品中测量特定的生物标志物。The term "aiding diagnosis" is used herein to refer to methods that aid in making a clinical determination of the presence or nature of symptoms or conditions of a particular type of disease or disorder (e.g., cancer). For example, a method of aiding in making a diagnosis of a disease or condition (e.g., cancer) may comprise measuring a particular biomarker in a biological sample from an individual.
术语“样品”在用于本文时指获得自或衍生自感兴趣的受试者和/或个体的组合物，其包含有待根据例如物理、生化、化学和/或生理特点来表征和/或鉴定的细胞和/或其它分子实体。例如，短语“疾病样品”或其变体指得自感兴趣的受试者的任何样品，预计或已知其包含待表征的细胞和/或分子实体。样品包括但不限于，原代或培养的细胞或细胞系、细胞上清、细胞裂解物、血小板、血清、血浆、玻璃体液、淋巴液、滑液、滤泡液(follicularfluid)、精液、羊水、乳、全血、血液衍生的细胞、尿液、脑脊髓液、唾液、痰、泪、汗液、粘液、肿瘤裂解物、和组织培养液(tissue culture medium)、组织提取物如匀浆化的组织、肿瘤组织、细胞提取物、及其组合。The term "sample" as used herein refers to a composition obtained from or derived from a subject and/or individual of interest that contains cells and/or other molecular entities to be characterized and/or identified based on, for example, physical, biochemical, chemical, and/or physiological characteristics. For example, the phrase "disease sample" or variations thereof refers to any sample obtained from a subject of interest that is expected or known to contain cells and/or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.
“组织样品”或“细胞样品”意指从受试者或个体的组织获得的相似细胞的集合。组织或细胞样品的来源可以是实体组织如来自新鲜、冷冻和/或保存的器官，组织样品，活组织检查和/或抽吸物；血液或任何血液成分如血浆；体液如脑脊髓液、羊水、腹膜液或间隙液(interstitial fluid)；来自受试者的妊娠或发育中任意时间的细胞。组织样品还可以是原代或培养的细胞或细胞系。任选地，组织或细胞样品自患病的组织/器官获得。组织样品可以含有在自然界中不天然与组织混杂的化合物，如防腐剂、抗凝血剂、缓冲剂、固定剂、营养物、抗生素等。"Tissue sample" or "cell sample" means a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample can be solid tissue such as from fresh, frozen and/or preserved organs, tissue samples, biopsies and/or aspirates; blood or any blood component such as plasma; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from any time during the subject's pregnancy or development. The tissue sample can also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ. The tissue sample may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.
如本文中使用的，“参照样品”、“参照细胞”、“参照组织”、“对照样品”、“对照细胞”或“对照组织”指用于比较目的的样品、细胞、组织、标准、或水平。在一个实施方案中，参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织自同一受试者或个体的健康和/或无疾病身体部位(例如组织或细胞)获得。例如，临近于患病细胞或组织的健康和/或无疾病细胞或组织(例如临近肿瘤的细胞或组织)。在另一个实施方案中，参照样品自同一受试者或个体的身体的未治疗组织和/或细胞获得。在又一个实施方案中，参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织自不是该受试者或个体的个体的健康和/或无疾病身体部位(例如组织或细胞)获得。在再一个实施方案中，参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织自不是该受试者或个体的个体身体的未治疗组织和/或细胞获得。As used herein, "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, tissue, standard, or level for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or disease-free body part (e.g., tissue or cell) of the same subject or individual. For example, a healthy and/or disease-free cell or tissue (e.g., a cell or tissue adjacent to a diseased cell or tissue) is obtained. In another embodiment, a reference sample is obtained from untreated tissues and/or cells of the body of the same subject or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or disease-free body part (e.g., tissue or cell) of an individual that is not the subject or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from untreated tissues and/or cells of the body of an individual that is not the subject or individual.
为本发明目的，组织样品的“切片”意指一块或一片组织样品，例如从组织样品上切下来的一薄片组织或细胞。应当了解，可以制作多片组织样品切片并进行分析，只要理解可以将组织样品的同一切片用于形态学和分子两个水平的分析或者针对多肽和多核苷酸二者进行分析。For purposes of the present invention, a "section" of a tissue sample refers to a piece or slice of a tissue sample, such as a thin slice of tissue or cells cut from a tissue sample. It should be understood that multiple sections of a tissue sample can be prepared and analyzed, as long as it is understood that the same section of a tissue sample can be used for analysis at both the morphological and molecular levels or for analysis of both polypeptides and polynucleotides.
“关联”或“联系”意指以任何方式将第一分析或方案的性能和/或结果与第二分析或方案的性能和/或结果进行比较。例如，可以将第一分析或方案的结果用于实施第二方案，和/或，可以使用第一分析或方案的结果来决定是否应当实施第二分析或方案。就多肽分析或方案的实施方案而言，可以使用多肽表达分析或方案的结果来决定是否应当实施特定治疗方案。就多核苷酸分析或方案的实施方案而言，可以使用多核苷酸表达分析或方案的结果来决定是否应当实施特定治疗方案。"Correlation" or "association" means comparing the performance and/or results of a first analysis or protocol to the performance and/or results of a second analysis or protocol in any manner. For example, the results of a first analysis or protocol can be used to implement a second protocol, and/or the results of a first analysis or protocol can be used to decide whether the second analysis or protocol should be implemented. With respect to embodiments of polypeptide analysis or protocols, the results of a polypeptide expression analysis or protocol can be used to decide whether a particular treatment protocol should be implemented. With respect to embodiments of polynucleotide analysis or protocols, the results of a polynucleotide expression analysis or protocol can be used to decide whether a particular treatment protocol should be implemented.
“个体响应”或“响应”可使用指示对个体益处的任何终点评估，包括但不限于：(1)一定程度地抑制疾病进展(例如癌症进展)，包括减缓和完全阻滞；(2)缩小肿瘤尺寸；(3)抑制(即减轻、减缓或完全终止)癌细胞浸润入临近周围器官和/或组织；(4)抑制(即减轻、减缓或完全终止)转移；(5)一定程度地减轻与疾病或病症(例如癌症)有关的一种或多种症状；(6)存活(包括总体存活和无进展存活)的长度延长或扩展；和/或(9)治疗后给定时间点的死亡率降低。"Individual response" or "response" can be assessed using any endpoint indicative of benefit to the individual, including but not limited to: (1) some inhibition of disease progression (e.g., cancer progression), including slowing and complete arrest; (2) reduction in tumor size; (3) inhibition (i.e., reduction, slowing, or complete cessation) of cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition (i.e., reduction, slowing, or complete cessation) of metastasis; (5) some alleviation of one or more symptoms associated with a disease or condition (e.g., cancer); (6) extension or extension of the length of survival (including overall survival and progression-free survival); and/or (9) reduction in mortality at a given time point after treatment.
患者对药物治疗的“有效响应”或“响应性”及类似用语指对处于疾病或病症(诸如癌症)风险或患有疾病或病症(诸如癌症)的患者给予的临床或治疗好处。在一个实施方案中，此类好处包括如下任何一项或多项：延长存活(包括总体存活和无进展存活)；导致客观响应(包括完全响应或部分响应)；或改善癌症体征或症状。在一个实施方案中，使用生物标志物(例如PD-L1表达，例如使用IHC测定的)来鉴定预测具有相对于不表达该生物标志物的患者升高的可能性响应药物(例如抗PD-L1抗体)治疗的患者。在一个实施方案中，使用生物标志物(例如PD-L1表达，例如使用IHC测定的)来鉴定预测具有相对于不以相同水平表达该生物标志物的患者升高的可能性响应药物(例如抗PD-L1抗体)治疗的患者。在一个实施方案中，使用生物标志物的存在来鉴定相对于不存在该生物标志物的患者更有可能响应药物治疗的患者。在另一个实施方案中，使用生物标志物的存在来确定患者会具有相对于不存在该生物标志物的患者升高的可能性受益于药物治疗。"Effective response" or "responsiveness" and similar terms of a patient to a drug treatment refer to a clinical or therapeutic benefit given to a patient at risk for or suffering from a disease or condition (such as cancer). In one embodiment, such benefits include any one or more of the following: prolonging survival (including overall survival and progression-free survival); causing an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer. In one embodiment, a biomarker (e.g., PD-L1 expression, e.g., determined using IHC) is used to identify patients predicted to have an increased likelihood of responding to a drug (e.g., an anti-PD-L1 antibody) treatment relative to patients who do not express the biomarker. In one embodiment, a biomarker (e.g., PD-L1 expression, e.g., determined using IHC) is used to identify patients predicted to have an increased likelihood of responding to a drug (e.g., an anti-PD-L1 antibody) treatment relative to patients who do not express the biomarker at the same level. In one embodiment, the presence of a biomarker is used to identify patients who are more likely to respond to a drug treatment relative to patients in the absence of the biomarker. In another embodiment, the presence of a biomarker is used to determine that a patient will benefit from a drug treatment with an increased likelihood relative to a patient in the absence of the biomarker.
“存活”(survival)指患者保持存活，而且包括总体存活(overall survival)和无进展存活(progress free survival)。"Survival" means that the patient remains alive and includes overall survival and progression-free survival.
“总体存活”指保持一定时期诸如1年、5年等存活的患者，自诊断或治疗时起计算。"Overall survival" refers to patients who remain alive for a certain period of time, such as 1 year, 5 years, etc., measured from the time of diagnosis or treatment.
“无进展存活”指保持存活且癌症没有进展或恶化的患者。"Progression-free survival" refers to patients who remain alive and whose cancer has not progressed or gotten worse.
“延长存活”意味着使接受治疗的患者的总体存活或无进展存活相对于未接受治疗的患者(即相对于未用药物治疗的患者)，或相对于不以指定水平表达生物标志物的患者，和/或相对于用已获批准的抗肿瘤剂治疗的患者有延长。"Prolonging survival" means that the overall survival or progression-free survival of treated patients is prolonged relative to untreated patients (i.e., relative to patients not treated with the drug), or relative to patients who do not express the biomarker at a specified level, and/or relative to patients treated with an approved anti-tumor agent.
“客观响应”(objective response)指可测量的响应，包括完全响应(CR)或部分响应(PR)。"Objective response" refers to a measurable response, including complete response (CR) or partial response (PR).
“完全响应”(complete response)或“CR”指癌症的所有体征响应治疗而消失。这并不总是意味着癌症已经治愈。A "complete response" or "CR" means all signs of cancer disappear in response to treatment. This does not always mean the cancer is cured.
“部分响应”(partial response)或“PR”指一处或多处肿瘤或损害的大小或体内癌症的程度响应治疗而减小。A "partial response" or "PR" refers to a decrease in the size of one or more tumors or lesions, or the extent of cancer in the body, in response to treatment.
术语“基本上相同”在用于本文时表示两个数值之间足够高的相似程度，以致本领域技术人员将认为在用所述数值(例如Kd值或表达)所测量的生物学特性背景内两个数值之间的差异具有很小的或没有生物学和/或统计学显著性。作为参照/比较值的函数，所述两个数值之间的差异例如小于约50％，小于约40％，小于约30％，小于约20％，和/或小于约10％。The term "substantially identical" as used herein refers to a sufficiently high degree of similarity between two values that one skilled in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological property being measured using the value (e.g., Kd value or expression). As a function of a reference/comparison value, the difference between the two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10%.
短语“实质性不同”在用于本文时表示两个数值之间足够高的差异程度，以致本领域技术人员将认为在用所述数值(例如Kd值)所测量的生物学特性背景内两个数值之间的差异具有统计学显著性。作为参照/比较分子值的函数，所述两个数值之间的差异例如大于约10％，大于约20％，大于约30％，大于约40％，和/或大于约50％。The phrase "substantially different" as used herein refers to a sufficiently high degree of difference between two values that one skilled in the art would consider the difference between the two values to be statistically significant within the context of the biological property being measured by the value (e.g., Kd value). For example, the difference between the two values is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the reference/comparator molecule value.
词语“标记物”在用于本文时指可检测的化合物或组合物。标记物通常与试剂如多核苷酸探针或抗体直接或间接缀合或融合，而且协助对其所缀合或融合的试剂的检测。标记物可以是通过自身就可检测的(例如放射性同位素标记物或荧光标记物)，或者在酶标记物的情况中，可催化产生可检测产物的底物化合物或组合物的化学改变。The term "label" as used herein refers to a detectable compound or composition. A label is typically conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or an antibody, and facilitates detection of the reagent to which it is conjugated or fused. A label can be detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzyme label, can catalyze the chemical alteration of a substrate compound or composition that produces a detectable product.
“有效量”指在必需的剂量和时间段上有效实现期望的治疗或预防结果的量。An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
“治疗有效量”指治疗剂在哺乳动物中治疗或预防疾病或病症的量。在癌症的情况中，治疗有效量的治疗剂可减少癌细胞的数目；缩小原发性肿瘤的尺寸；抑制(即一定程度的减缓，优选阻止)癌细胞浸润入周围器官；抑制(即一定程度的减缓，优选阻止)肿瘤转移；一定程度的抑制肿瘤生长；和/或一定程度的减轻一种或多种与病症有关的症状。根据药物可阻止现有癌细胞生长和/或杀死现有癌细胞的程度，它可以是细胞抑制性的和/或细胞毒性的。对于癌症疗法，体内功效可以通过例如评估存活持续时间、距疾病进展的时间(TTP)、响应率(RR)、响应持续时间、和/或生活质量来测量。"Therapeutically effective amount" refers to the amount of a therapeutic agent to treat or prevent a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of a therapeutic agent can reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow down to some extent, preferably prevent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to some extent, preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with the condition to some extent. Depending on the extent to which a drug can prevent the growth of existing cancer cells and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy can be measured by, for example, assessing duration of survival, time to disease progression (TTP), response rate (RR), duration of response, and/or quality of life.
术语“癌(症)”和“癌(性)的”指向或描述哺乳动物中典型的以不受调节的细胞生长为特征的生理疾患。此定义中包括良性和恶性癌症。“早期癌症”或“早期肿瘤”指非侵入性的或转移性的，或者归为0期、I期、或II期癌症的癌症。癌症的例子包括但不限于癌、淋巴瘤、母细胞瘤(包括髓母细胞瘤和视网膜母细胞瘤)、肉瘤(包括脂肪肉瘤和滑膜细胞肉瘤)、神经内分泌肿瘤(包括类癌瘤、胃泌素瘤和胰岛细胞癌)、间皮瘤、施旺氏细胞瘤(包括听神经瘤)、脑膜瘤、腺癌、黑素瘤、和白血病或淋巴样恶性肿瘤。此类癌症的更具体例子包括鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌包括小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、肺的腺癌和肺的鳞癌、腹膜癌、肝细胞癌、胃癌(gastric或stomach cancer)包括胃肠癌、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌(liver cancer或hepatic carcinoma)、膀胱癌、肝瘤(hepatoma)、乳腺癌(包括转移性乳腺癌)、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌(kidney或renal cancer)、前列腺癌、外阴癌、甲状腺癌、肛门癌、阴茎癌、梅克尔细胞癌、蕈样肉芽肿、睾丸癌、食管癌、胆管肿瘤、及头和颈癌和造血恶性肿瘤。在一些实施方案中，癌症是三重阴性转移性乳腺癌，包括任何经组织学确认的具有局部复发性或转移性疾病的三重阴性(ER-、PR-、HER2-)乳腺癌(其中局部复发性疾病不适合具有治愈目的的切除术)。The terms "cancer" and "cancerous" refer to or describe a physiological condition typically characterized by unregulated cell growth in mammals. Both benign and malignant cancers are included in this definition. "Early stage cancer" or "early stage tumor" refers to a cancer that is non-invasive or metastatic, or classified as a stage 0, stage I, or stage II cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesothelioma, Schwann cell tumors (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (hepatic carcinoma), bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer (renal cancer), prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, Merkel cell carcinoma, mycosis fungoides, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancers and hematopoietic malignancies. In some embodiments, the cancer is triple-negative metastatic breast cancer, including any histologically confirmed triple-negative (ER-, PR-, HER2-) breast cancer with locally recurrent or metastatic disease (wherein the locally recurrent disease is not amenable to resection with curative intent).
术语“药物配制剂”指其形式容许其中含有的活性成分的生物学活性是有效的，且不含对会施用该配制剂的受试者产生不可接受的毒性的别的成分的制剂。The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that would be unacceptably toxic to a subject to which the formulation would be administered.
“药学可接受载体”指药物配制剂中除了活性成分外对受试者无毒的成分。药学可接受载体包括但不限于缓冲剂、赋形剂、稳定剂、或防腐剂。"Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical formulation that are non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
如本文中使用的，“治疗/处理”(及其语法变型)指试图改变所治疗个体的自然进程的临床干预，可以是为了预防或在临床病理学的进程中进行。治疗的期望效果包括但不限于预防疾病的发生或复发、缓解症状、削弱疾病的任何直接或间接病理学后果、预防转移、减缓疾病进展的速率、改善或减轻疾病状态、及免除或改善预后。在一些实施方案中，使用抗体来延迟疾病的形成或减缓疾病的进展。As used herein, "treatment" (and grammatical variations thereof) refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, antibodies are used to delay the development of disease or slow the progression of disease.
术语“抗癌疗法”指在治疗癌症中有用的疗法。抗癌治疗剂的例子包括但不限于例如化疗剂、生长抑制剂、细胞毒剂、放射疗法中所使用的药剂、抗血管发生剂、凋亡剂、抗微管蛋白剂、和其它治疗癌症的药剂，抗CD20抗体、血小板衍生生长因子抑制剂(例如GleevecTM(Imatinib Mesylate))、COX-2抑制剂(例如celecoxib)、干扰素、细胞因子、结合一种或多种以下靶物的拮抗剂(例如中和性抗体)(PDGFR-β、BlyS、APRIL、BCMA受体、TRAIL/Apo2)、和其它生物活性和有机化学剂，等。本发明还包括它们的组合。The term "anti-cancer therapy" refers to a therapy useful in treating cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, anti-CD20 antibodies, platelet-derived growth factor inhibitors (e.g., Gleevec™ (Imatinib Mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets (PDGFR-β, BlyS, APRIL, BCMA receptor, TRAIL/Apo2), and other biologically active and organic chemical agents, etc. The present invention also includes combinations thereof.
术语“细胞毒剂”在用于本文时指抑制或防止细胞的功能和/或引起细胞破坏的物质。该术语意图包括放射性同位素(例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32和Lu的放射性同位素)、化疗剂，例如甲氨蝶呤(methotrexate)、阿霉素(adriamicin)、长春花生物碱类(vinca alkaloids)(长春新碱(vincristine)、长春碱(vinblastine)、依托泊苷(etoposide))、多柔比星(doxorubicin)、美法仑(melphalan)、丝裂霉素(mitomycin)C、苯丁酸氮芥(chlorambucil)、柔红霉素(daunorubicin)或其它嵌入剂、酶及其片段、诸如溶核酶、抗生素、和毒素，诸如小分子毒素或者细菌、真菌、植物或动物起源的酶活毒素，包括其片段和/或变体；及下文披露的各种抗肿瘤药或抗癌药。下文记载了其它细胞毒剂。杀肿瘤药引起肿瘤细胞的破坏。The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes cell destruction. The term is intended to include radioisotopes (e.g., At211 , I131 , I125 , Y90 , Re186 , Re188 , Sm153 , Bi212 , P32 and radioisotopes of Lu), chemotherapeutic agents such as methotrexate, adriamicin, vinca alkaloids, [0013] In some embodiments, the present invention includes but is not limited to: alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof, such as nucleolytic enzymes, antibiotics, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer drugs disclosed below. Other cytotoxic agents are described below. Tumoricidal drugs cause destruction of tumor cells.
“化疗剂”指可用于治疗癌症的化学化合物。化疗剂的实例包括烷化剂类(alkylating agents)，诸如塞替派(thiotepa)和环磷酰胺(cyclophosphamide)磺酸烷基酯类(alkyl sulfonates)，诸如白消安(busulfan)、英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶类(aziridines)，诸如苯佐替派(benzodopa)、卡波醌(carboquone)、美妥替派(meturedopa)和乌瑞替派(uredopa)；乙撑亚胺类(ethylenimines)和甲基蜜胺类(methylamelamines)，包括六甲蜜胺(altretamine)、三乙撑蜜胺(triethylenemelamine)、三乙撑磷酰胺(triethylenephosphoramide)、三乙撑硫代磷酰胺(triethylenethiophosphoramide)和三羟甲蜜胺(trimethylomelamine)；番荔枝内酯类(acetogenin)(尤其是布拉他辛(bullatacin)和布拉他辛酮(bullatacinone))；δ-9-四氢大麻酚(tetrahydrocannabinol)(屈大麻酚(dronabinol)，)；β-拉帕醌(lapachone)；拉帕醇(lapachol)；秋水仙素类(colchicines)；白桦脂酸(betulinicacid)；喜树碱(camptothecin)(包括合成类似物托泊替康(topotecan)CPT-11(伊立替康(irinotecan)，)、乙酰喜树碱、东莨菪亭(scopolectin)和9-氨基喜树碱)；苔藓抑素(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)；鬼臼毒素(podophyllotoxin)；鬼臼酸(podophyllinic acid)；替尼泊苷(teniposide)；隐藻素类(cryptophycins)(特别是隐藻素1和隐藻素8)；多拉司他汀(dolastatin)；duocarmycin(包括合成类似物，KW-2189和CB1-TM1)；艾榴塞洛素(eleutherobin)；pancratistatin；sarcodictyin；海绵抑素(spongistatin)；氮芥类(nitrogen mustards)，诸如苯丁酸氮芥(chlorambucil)、萘氮芥(chlornaphazine)、胆磷酰胺(chlorophosphamide)、雌莫司汀(estramustine)、异环磷酰胺(ifosfamide)、双氯乙基甲胺(mechlorethamine)、盐酸氧氮芥(mechlorethamine oxide hydrochloride)、美法仑(melphalan)、新氮芥(novembichin)、苯芥胆甾醇(phenesterine)、泼尼莫司汀(prednimustine)、曲磷胺(trofosfamide)、尿嘧啶氮芥(uracil mustard)；亚硝脲类(nitrosoureas)，诸如卡莫司汀(carmustine)、氯脲菌素(chlorozotocin)、福莫司汀(fotemustine)、洛莫司汀(lomustine)、尼莫司汀(nimustine)和雷莫司汀(ranimnustine)；抗生素类，诸如烯二炔类抗生素(enediyne)(如加利车霉素(calicheamicin)，尤其是加利车霉素γ1I和加利车霉素ωI1(参见例如Nicolaou et al.,Angew.Chem Intl.Ed.Engl.,33:183-186(1994))；CDP323，一种口服α-4整联蛋白抑制剂；蒽环类抗生素(dynemicin)，包括dynemicin A；埃斯波霉素(esperamicin)；以及新制癌素(neocarzinostatin)发色团和相关色蛋白烯二炔类抗生素发色团)、阿克拉霉素(aclacinomysin)、放线菌素(actinomycin)、氨茴霉素(authramycin)、偶氮丝氨酸(azaserine)、博来霉素(bleomycin)、放线菌素C(cactinomycin)、carabicin、洋红霉素(carminomycin)、嗜癌霉素(carzinophilin)、色霉素(chromomycin)、放线菌素D(dactinomycin)、柔红霉素(daunorubicin)、地托比星(detorubicin)、6-二氮-5-氧-L-正亮氨酸、多柔比星(doxorubicin)(包括吗啉代多柔比星、氰基吗啉代多柔比星、2-吡咯代多柔比星、盐酸多柔比星脂质体注射剂脂质体多柔比星TLC D-99PEG化脂质体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)、丝裂霉素类(mitomycins)诸如丝裂霉素C、霉酚酸(mycophenolicacid)、诺拉霉素(nogalamycin)、橄榄霉素(olivomycin)、培洛霉素(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)、链佐星(streptozocin)、杀结核菌素(tubercidin)、乌苯美司(ubenimex)、净司他丁(zinostatin)、佐柔比星(zorubicin)；抗代谢物类，诸如甲氨蝶呤、吉西他滨(gemcitabine)替加氟(tegafur)卡培他滨(capecitabine)埃坡霉素(epothilone)和5-氟尿嘧啶(5-FU)；叶酸类似物，诸如二甲叶酸(denopterin)、甲氨蝶呤、蝶酰三谷氨酸(pteropterin)、三甲曲沙(trimetrexate)；嘌呤类似物，诸如氟达拉滨(fludarabine)、6-巯基嘌呤(mercaptopurine)、硫咪嘌呤(thiamiprine)、硫鸟嘌呤(thioguanine)；嘧啶类似物，诸如安西他滨(ancitabine)、阿扎胞苷(azacitidine)、6-氮尿苷、卡莫氟(carmofur)、阿糖胞苷(cytarabine)、双脱氧尿苷(dideoxyuridine)、去氧氟尿苷(doxifluridine)、依诺他滨(enocitabine)、氟尿苷(floxuridine)；雄激素类，诸如卡鲁睾酮(calusterone)、丙酸屈他雄酮(dromostanolone propionate)、表硫雄醇(epitiostanol)、美雄烷(mepitiostane)、睾内酯(testolactone)；抗肾上腺类，诸如氨鲁米特(aminoglutethimide)、米托坦(mitotane)、曲洛司坦(trilostane)；叶酸补充剂，诸如亚叶酸(frolinic acid)；醋葡醛内酯(aceglatone)；醛磷酰胺糖苷(aldophosphamideglycoside)；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)；地美可辛(demecolcine)；地吖醌(diaziquone)；elfornithine；依利醋铵(elliptinium acetate)；epothilone；依托格鲁(etoglucid)；硝酸镓；羟脲(hydroxyurea)；香菇多糖(lentinan)；氯尼达明(lonidainine)；美登木素生物碱类(maytansinoids)，诸如美登素(maytansine)和安丝菌素(ansamitocin)；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；莫哌达醇(mopidanmol)；二胺硝吖啶(nitraerine)；喷司他丁(pentostatin)；蛋氨氮芥(phenamet)；吡柔比星(pirarubicin)；洛索蒽醌(losoxantrone)；2-乙基酰肼(ethylhydrazide)；丙卡巴肼(procarbazine)；多糖复合物(JHS Natural Products，Eugene，OR)；雷佐生(razoxane)；根霉素(rhizoxin)；西索菲兰(sizofiran)；螺旋锗(spirogermanium)；细交链孢菌酮酸(tenuazonic acid)；三亚胺醌(triaziquone)；2,2’,2”-三氯三乙胺；单端孢菌素类(trichothecenes)(尤其是T-2毒素、疣孢菌素(verracurin)A、杆孢菌素(roridin)A和蛇行菌素(anguidine))；乌拉坦(urethan)；长春地辛(vindesine)达卡巴嗪(dacarbazine)；甘露醇氮芥(mannomustine)；二溴甘露醇(mitobronitol)；二溴卫矛醇(mitolactol)；哌泊溴烷(pipobroman)；gacytosine；阿糖胞苷(arabinoside)(“Ara-C”)；塞替派(thiotepa)；类紫杉醇(taxoid)，例如帕利他塞(paclitaxel)清蛋白改造的纳米颗粒剂型帕利他塞(ABRAXANETM)和多西他塞(docetaxel)苯丁酸氮芥(chloranbucil)；6-硫鸟嘌呤(thioguanine)；巯基嘌呤(mercaptopurine)；甲氨蝶呤(methotrexate)；铂剂，诸如顺铂(cisplatin)、奥沙利铂(oxaliplatin)(例如)和卡铂(carboplatin)；长春药类(vincas)，其阻止微管蛋白聚合形成微管，包括长春碱(vinblastine)长春新碱(vincristine)长春地辛(vindesine)和长春瑞滨(vinorelbine)依托泊苷(etoposide)(VP-16)；异环磷酰胺(ifosfamide)；米托蒽醌(mitoxantrone)；亚叶酸(leucovorin)；能灭瘤(novantrone)；依达曲沙(edatrexate)；道诺霉素(daunomycin)；氨基蝶呤(aminopterin)；伊本膦酸盐(ibandronate)；拓扑异构酶抑制剂RFS 2000；二氟甲基鸟氨酸(DMFO)；类维A酸(retinoids)，诸如维A酸(retinoic acid)，包括贝沙罗汀(bexarotene)二膦酸盐类(bisphosphonates)，诸如氯膦酸盐(clodronate)(例如或)、依替膦酸钠(etidronate)NE-58095、唑来膦酸/唑来膦酸盐(zoledronic acid/zoledronate)阿伦膦酸盐(alendronate)帕米膦酸盐(pamidronate)替鲁膦酸盐(tiludronate)或利塞膦酸盐(risedronate)以及曲沙他滨(troxacitabine)(1,3-二氧戊环核苷胞嘧啶类似物)；反义寡核苷酸，特别是抑制牵涉异常细胞增殖的信号途经中的基因表达的反义寡核苷酸，诸如例如PKC-α、Raf、H-Ras和表皮生长因子受体(EGF-R)；疫苗，诸如疫苗和基因疗法疫苗，例如疫苗、疫苗和疫苗；拓扑异构酶1抑制剂(例如)；rmRH(例如)；BAY439006(sorafenib；Bayer)；SU-11248(sunitinib，Pfizer)；哌立福辛(perifosine)，COX-2抑制剂(如塞来考昔(celecoxib)或艾托考昔(etoricoxib))，蛋白体抑制剂(例如PS341)；bortezomibCCI-779；tipifarnib(R11577)；orafenib，ABT510；Bcl-2抑制剂，诸如oblimersen sodiumpixantrone；EGFR抑制剂(见下文定义)；酪氨酸激酶抑制剂(见下文定义)；丝氨酸-苏氨酸激酶抑制剂，诸如雷帕霉素(rapamycin)(sirolimus，)；法尼基转移酶抑制剂，诸如lonafarnib(SCH 6636，SARASARTM)；及任何上述各项的药学可接受盐、酸或衍生物；以及两种或更多种上述各项的组合，诸如CHOP(环磷酰胺、多柔比星、长春新碱和泼尼松龙联合疗法的缩写)和FOLFOX(奥沙利铂(ELOXATINTM)联合5-FU和亚叶酸的治疗方案的缩写)。"Chemotherapeutic agent" refers to a chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide, and alkyl sulfonates. sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; acetogenins (especially bullatacin and butyrosine); bullatacinone); delta-9-tetrahydrocannabinol (dronabinol); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecins (including the synthetic analogs topotecan CPT-11 (irinotecan), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, 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 the enediynes (e.g., calicheamicin, especially calicheamicin gamma and calicheamicin omega (see, e.g., Nicolaou et al., Angew. Chem. Am. Am., 1996; 1996; 1997; 1998; 2001; 2003; 2004; 2005; 2006; 2007; 2008; 2009; 2010; 2011; 2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022; 2023; 2024; 2025; 2026; 2027; 2028; 2029; 2030; 2031 Intl. Ed. Engl., 33:183-186 (1994); CDP323, an oral α-4 integrin inhibitor; anthracycline antibiotics (dynemicin), including dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolidodoxorubicin, doxorubicin hydrochloride liposomal injection, liposomal doxorubicin TLC D-99 PEGylated liposomal doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfibrinocin, iromycin), puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine, tegafur ur) capecitabine, epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine Pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, and doxycycline; propionate, epitiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, OR; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");thiotepa; taxoids, such as paclitaxel; albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANE™) . ) and docetaxel; chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin (for example), and carboplatin; vincas, which prevent tubulin from polymerizing to form microtubules, including vinblastine, vincristine, and vinblastine. tine; vindesine and vinorelbine; etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid, including bexarotene; bisphosphonates, such as clodronate (e.g., clodronate or vinorelbine), etidronate NE-58095, zoledronic acid/zoledronic acid. acid/zoledronate) alendronate, pamidronate, tiludronate or risedronate, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly antisense oligonucleotides that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as vaccines and gene therapy vaccines, e.g., vaccines, vaccines, and vaccines vaccines; topoisomerase 1 inhibitors (e.g., rmRH); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors, such as rapamycin (sirolimus,); farnesyl transferase inhibitors, such as lonafarnib (SCH 6636, SARASAR™ ); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP (an abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy) and FOLFOX (an abbreviation for the treatment regimen of oxaliplatin (ELOXATIN™ ) combined with 5-FU and folinic acid).
如本文中定义的化疗剂包括起调节、降低、阻断或抑制可促进癌生长的激素效果作用的“抗激素剂”或“内分泌治疗剂”类。它们自身可以是激素，包括但不限于：具有混合的激动剂/拮抗剂特性的抗雌激素类，包括他莫昔芬(tamoxifen)4-羟基他莫昔芬、托瑞米芬(toremifene)艾多昔芬(idoxifene)、屈洛昔芬(droloxifene)、雷洛昔芬(raloxifene)曲沃昔芬(trioxifene)、那洛昔芬(keoxifene)，和选择性雌激素受体调节剂类(SERM)，诸如SERM3；没有激动剂特性的纯的抗雌激素类，诸如氟维司群(fulvestrant)和EM800(此类药剂可阻断雌激素受体(ER)二聚化、抑制DNA结合、提高ER周转、和/或遏制ER水平)；芳香酶抑制剂类，包括类固醇芳香酶抑制剂类，诸如福美坦(formestane)和依西美坦(exemestane)和非类固醇芳香酶抑制剂类，诸如阿那曲唑(anastrazole)来曲唑(letrozole)和氨鲁米特(aminoglutethimide)，和其它芳香酶抑制剂类，包括伏氯唑(vorozole)醋酸甲地孕酮(megestrol acetate)法倔唑(fadrozole)和4(5)-咪唑；促黄体生成激素释放激素激动剂类，包括亮丙瑞林(leuprolide)(和)、戈舍瑞林(goserelin)、布舍瑞林(buserelin)和曲普瑞林(tripterelin)；性类固醇类(sex steroids)，包括妊娠素类(progestine)，诸如醋酸甲地孕酮(megestrol acetate)和醋酸甲羟孕酮(medroxyprogesterone acetate)，雌激素类，诸如二乙基己烯雌酚(diethylstilbestrol)和普雷马林(premarin)，和雄激素类/类视黄酸类，诸如氟甲睾酮(fluoxymesterone)、所有反式视黄酸(transretionic acid)和芬维A胺(fenretinide)；奥那司酮(onapristone)；抗孕酮类；雌激素受体下调剂类(ERD)；抗雄激素类，诸如氟他米特(flutamide)、尼鲁米特(nilutamide)和比卡米特(bicalutamide)；及任何上述物质的药剂学可接受的盐、酸或衍生物；以及两种或多种上述物质的组合。Chemotherapeutic agents as defined herein include "antihormonal agents" or "endocrine therapeutic agents" that act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth. They can themselves be hormones, including but not limited to: antiestrogens with mixed agonist/antagonist properties, including tamoxifen, 4-hydroxytamoxifen, toremifene, idoxifene, droloxifene, raloxifene, trioxifene, naloxifene, and selective estrogen receptor modulators (SERMs), such as SERM3; pure antiestrogens without agonist properties, such as fulvestrant; nt) and EM800 (such agents can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane and nonsteroidal aromatase inhibitors such as anastrazole, letrozole, and aminoglutethimide, and other aromatase inhibitors including vorozole, megestrol acetate, and steroids. acetate), fadrozole, and 4(5)-imidazole; luteinizing hormone-releasing hormone agonists, including leuprolide (and), goserelin, buserelin, and tripterelin; sex steroids, including progestins, such as megestrol acetate and medroxyprogesterone acetate, estrogens, such as diethylstilbestrol and premarin, and androgens/retinoids, such as fluoxymesterone, all transretinoic acid, and dapoxetine. acid) and fenretinide; onapristone; antiprogestins; estrogen receptor downregulators (ERDs); antiandrogens, such as flutamide, nilutamide, and bicalutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing; and combinations of two or more of the foregoing.
术语“前药”在用于本申请时指与母药(parent drug)相比对肿瘤细胞的细胞毒性较小并能够酶促活化或转变为更具活性母药形式的药用活性物质的前体或衍生物形式。参见例如Wilman,“Prodrugs in Cancer Chemotherapy”Biochemical SocietyTransactions,14,pp.375-382,615th Meeting Belfast(1986)和Stella et al.,“Prodrugs:A Chemical Approach to Targeted Drug Delivery,”Directed DrugDelivery,Borchardt et al.,(ed.),pp.247-267,Humana Press(1985)。本发明的前药包括但不限于含磷酸盐/酯前药、含硫代磷酸盐/酯前药、含硫酸盐/酯前药、含肽前药、D-氨基酸修饰前药、糖基化前药、含β-内酰胺前药、含任选取代苯氧基乙酰胺的前药或含任选取代苯乙酰胺的前药、可转化为更具活性而无细胞毒性的药物的5-氟胞嘧啶和其它5-氟尿苷前药。可衍生为本发明使用的前药形式的细胞毒性药物的例子包括但不限于上文描述的那些化疗剂。The term "prodrug" as used herein refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and can be enzymatically activated or converted into the more active parent drug form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy," Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs of the present invention include, but are not limited to, phosphate/ester prodrugs, thiophosphate/ester prodrugs, sulfate/ester prodrugs, peptide prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam prodrugs, optionally substituted phenoxyacetamide prodrugs, or optionally substituted phenylacetamide prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted into more active, non-cytotoxic drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms for use in the present invention include, but are not limited to, those chemotherapeutic agents described above.
“生长抑制剂”在用于本文时指抑制细胞(例如在体外或在体内其生长依赖于PD-L1表达的细胞)生长的化合物或组合物。生长抑制剂的例子包括阻断细胞周期行进(处于S期以外的位置)的药剂，诸如诱导G1停滞和M期停滞的药剂。经典的M期阻断剂包括长春药类(vincas)(长春新碱(vincristine)和长春碱(vinblastine))、紫杉烷类(taxanes)、和拓扑异构酶II抑制剂诸如多柔比星(doxorubicin)、表柔比星(epirubicin)、柔红霉素(daunorubicin)、依托泊苷(etoposide)和博来霉素(bleomycin)。那些阻滞G1的药剂也溢出进入S期停滞，例如DNA烷化剂类诸如他莫昔芬(tamoxifen)、泼尼松(prednisone)、达卡巴嗪(dacarbazine)、双氯乙基甲胺(mechlorethamine)、顺铂(cisplatin)、甲氨蝶呤(methotrexate)、5-氟尿嘧啶(5-fluorouracil)和ara-C。更多信息可参见TheMolecularBasis of Cancer,Mendelsohn and Israel,eds.,Chapter 1,entitled"Cell cycleregulation,oncogenes,and antineoplastic drugs"by Murakami et al.(WB Saunders:Philadelphia,1995)，尤其是第13页。紫杉烷类(帕利他赛(paclitaxel)和多西他赛(docetaxel))是衍生自紫杉树的抗癌药。衍生自欧洲紫杉的多西他赛(Rhone-Poulenc Rorer)是帕利他赛(Bristol-Myers Squibb)的半合成类似物。帕利他赛和多西他赛促进由微管蛋白二聚体装配成微管并通过防止解聚使微管稳定，导致对细胞中有丝分裂的抑制。"Growth inhibitory agent" as used herein refers to a compound or composition that inhibits the growth of cells (e.g., cells whose growth depends on PD-L1 expression in vitro or in vivo). Examples of growth inhibitory agents include agents that block cell cycle progression (at a position other than the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Drugs that arrest G1 also spill over into S phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. For more information, seeTheMolecularBasis of Cancer , Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel (Rhone-Poulenc Rorer), derived from the European yew tree, is a semisynthetic analog of paclitaxel (Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, leading to the inhibition of mitosis in cells.
“放射疗法”指使用定向伽马射线或贝塔射线来诱发对细胞的足够损伤，以限制细胞正常发挥功能的能力或全然破坏细胞。应当领会，本领域知道许多方式来确定治疗的剂量和持续时间。典型的治疗作为一次施用来给予，而典型的剂量范围为每天10-200个单位(戈瑞(Gray))。"Radiotherapy" refers to the use of directed gamma or beta rays to induce sufficient damage to cells to limit the ability of the cells to function normally or to destroy the cells altogether. It will be appreciated that many ways are known in the art to determine the dosage and duration of treatment. Typical treatments are given as a single administration, and typical dosages range from 10 to 200 units (Gray) per day.
“个体”或“受试者”指哺乳动物。哺乳动物包括但不限于驯养动物(例如牛、绵羊、猫、犬、和马)、灵长类(例如人和非人灵长类诸如猴)、兔、和啮齿类(例如小鼠和大鼠)。在某些实施方案中，个体或受试者是人。"Individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
术语“并行”在本文中用于指施用两种或更多种治疗剂，其中至少部分施用在时间上交叠。因而，并行施用包括如下的剂量给药方案，一种或多种药剂的施用中断后继续施用一种或多种其它药剂。The term "concurrent" is used herein to refer to the administration of two or more therapeutic agents wherein at least some of the administration overlaps in time. Thus, concurrent administration includes dosing regimens in which the administration of one or more agents is interrupted followed by the continued administration of one or more other agents.
“降低或抑制”指引起20％、30％、40％、50％、60％、70％、75％、80％、85％、90％、95％、或更大的总体降低的能力。降低或抑制可以指所治疗的病症的症状、转移的存在或大小、或原发性肿瘤的大小。"Reduce or inhibit" refers to the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more. Reduce or inhibit can refer to the symptoms of the condition being treated, the presence or size of metastases, or the size of the primary tumor.
术语“包装插页”用于指通常包括在治疗用产品的商业包装中的说明书，它们包含有关涉及此类治疗用产品应用的适应征、用法、剂量、施用、组合疗法、禁忌症和/或警告的信息。The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
“制品”是包含至少一种试剂，例如用于治疗疾病或病症(例如癌症)的药物或用于特异性检测本文所述生物标志物的探针的任何制造物(例如包装或容器)或试剂盒。在某些实施方案中，制造物或试剂盒以用于实施本文所述方法的单位推销、分销或销售。An "article of manufacture" is any article of manufacture (e.g., a package or container) or kit comprising at least one agent, such as a drug for treating a disease or condition (e.g., cancer) or a probe for specifically detecting a biomarker described herein. In certain embodiments, the article of manufacture or kit is marketed, distributed, or sold as a unit for practicing the methods described herein.
“目标受众”指接受如通过推销或做广告(尤其为了特定用途、治疗、或适应症)进行的特定药物宣传或意图进行的特定药物宣传的人群或机构，诸如个体，群体，报纸、医学文献、和杂志读者，电视或因特网观众，无线电或因特网听众，内科医生，药品公司等。"Target audience" refers to the group of people or institutions to whom a particular drug is promoted or intended to be promoted, such as through marketing or advertising (especially for a specific use, treatment, or indication), such as individuals, groups, readers of newspapers, medical literature, and magazines, television or Internet viewers, radio or Internet listeners, physicians, pharmaceutical companies, etc.
在本文中使用时，短语“基于”意味着关于一种或多种生物标志物的信息用于告知治疗决定、包装插页上提供的信息、或营销/促销指导、等。As used herein, the phrase "based on" means that information about one or more biomarkers is used to inform treatment decisions, information provided on a package insert, or marketing/promotional guidance, etc.
如本领域技术人员理解的，本文中提述“约”某值或参数包括(且描述)针对该值或参数本身的实施方案。例如，提述“约X”的描述包括对“X”的描述。As understood by those skilled in the art, references herein to "about" a value or parameter include (and describe) embodiments directed to that value or parameter itself. For example, a description referring to "about X" includes a description of "X."
应当理解，本文中描述的方面和实施方案包括由和/或基本上由各方面和实施方案“组成”。如本文中使用的，单数形式“一个”、“一种”和“所述/该”包括复数提及物，除非另外指示。It should be understood that the aspects and embodiments described herein include “consisting of” and/or consisting essentially of the various aspects and embodiments. As used herein, the singular forms “a,” “an,” and “the” include plural references unless otherwise indicated.
I.方法和用途I. Methods and Uses
本文中提供的是利用PD-L1生物标志物的方法。特别是，提供利用PD-L1轴结合拮抗剂和PD-L1生物标志物的方法。Provided herein are methods utilizing the PD-L1 biomarker. In particular, methods utilizing a PD-L1 axis binding antagonist and a PD-L1 biomarker are provided.
诊断方法Diagnostic methods
本文中提供的是用于鉴定更有可能响应PD-L1轴结合拮抗剂治疗的具有疾病或病症的个体的方法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，其中该样品中存在PD-L1生物标志物指示该个体更有可能响应该PD-L1轴结合拮抗剂治疗，并提供该个体会更有可能响应PD-L1轴结合拮抗剂治疗的推荐。Provided herein are methods for identifying an individual having a disease or condition who is more likely to respond to treatment with a PD-L1 axis binding antagonist, the method comprising: determining the presence of a PD-L1 biomarker in a sample from the individual, wherein the presence of the PD-L1 biomarker in the sample indicates that the individual is more likely to respond to treatment with the PD-L1 axis binding antagonist, and providing a recommendation that the individual will be more likely to respond to treatment with the PD-L1 axis binding antagonist.
本文中进一步提供的是用于预测具有疾病或病症的个体对PD-L1轴结合拮抗剂治疗的响应性的方法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，其中该样品中存在PD-L1生物标志物指示该个体更有可能响应该PD-L1轴结合拮抗剂治疗，并提供该个体会具有升高的可能性响应PD-L1轴结合拮抗剂治疗的推荐。Further provided herein are methods for predicting responsiveness of an individual having a disease or condition to treatment with a PD-L1 axis binding antagonist, the method comprising: determining the presence of a PD-L1 biomarker in a sample from the individual, wherein the presence of the PD-L1 biomarker in the sample indicates that the individual is more likely to respond to treatment with the PD-L1 axis binding antagonist, and providing a recommendation that the individual will have an increased likelihood of responding to treatment with the PD-L1 axis binding antagonist.
本文中进一步提供的是用于确定具有疾病或病症的个体会展现受益于PD-L1轴结合拮抗剂治疗的可能性的方法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，其中该样品中存在PD-L1生物标志物指示该个体具有升高的可能性受益于该PD-L1轴结合拮抗剂治疗，并提供该个体会具有升高的可能性受益于PD-L1轴结合拮抗剂治疗的推荐。Further provided herein are methods for determining a likelihood that an individual having a disease or condition will exhibit benefit from treatment with a PD-L1 axis binding antagonist, the method comprising: assaying a sample from the individual for the presence of a PD-L1 biomarker, wherein the presence of the PD-L1 biomarker in the sample indicates that the individual has an increased likelihood of benefiting from treatment with the PD-L1 axis binding antagonist, and providing a recommendation that the individual will have an increased likelihood of benefiting from treatment with a PD-L1 axis binding antagonist.
本文中进一步提供的是用于为具有疾病或病症的个体选择疗法的方法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，并基于该样品中PD-L1生物标志物的存在提供为该个体选择的疗法包含PD-L1轴结合拮抗剂治疗的推荐。Further provided herein are methods for selecting a therapy for an individual having a disease or condition, the method comprising: determining the presence of a PD-L1 biomarker in a sample from the individual, and providing a recommendation that the therapy selected for the individual comprises treatment with a PD-L1 axis binding antagonist based on the presence of the PD-L1 biomarker in the sample.
在一些实施方案中，该方法进一步包括将有效量的PD-L1轴结合拮抗剂施用于该个体。In some embodiments, the method further comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist.
在一些实施方案中，PD-L1生物标志物选自下组：PD-L1，PD-1，PD-L2及其任意组合。In some embodiments, the PD-L1 biomarker is selected from the group consisting of PD-L1, PD-1, PD-L2, and any combination thereof.
在一些实施方案中，PD-L1生物标志物是免疫相关标志物。免疫相关标志物指由免疫细胞或其它细胞(例如肿瘤细胞，内皮细胞，成纤维细胞或其它基质细胞)表达的标志物。如果由免疫细胞以外的细胞表达的话，标志物可以涉及调节免疫细胞生物学或功能，诸如活化，引发，抗原识别和呈递，细胞因子和趋化因子生成，增殖，迁移，存活，抗体生成等等。在一些实施方案中，免疫相关标志物是T细胞相关标志物。在一些实施方案中，T细胞相关标志物选自下组：CD8A，IFN-g，EOMES，粒酶-A，CXCL9及其任意组合。在一些实施方案中，免疫相关标志物选自下组：CX3CL1，CD45RO，IDO1，半乳凝素9，MIC-A，MIC-B，CTLA-4及其任意组合。In some embodiments, the PD-L1 biomarker is an immune-related marker. Immune-related markers refer to markers expressed by immune cells or other cells (e.g., tumor cells, endothelial cells, fibroblasts or other stromal cells). If expressed by cells other than immune cells, the marker may be involved in regulating immune cell biology or function, such as activation, priming, antigen recognition and presentation, cytokine and chemokine production, proliferation, migration, survival, antibody production, and the like. In some embodiments, the immune-related marker is a T cell-related marker. In some embodiments, the T cell-related marker is selected from the group consisting of CD8A, IFN-g, EOMES, granzyme-A, CXCL9, and any combination thereof. In some embodiments, the immune-related marker is selected from the group consisting of CX3CL1, CD45RO, IDO1, galectin 9, MIC-A, MIC-B, CTLA-4, and any combination thereof.
在一些实施方案中，PD-L1生物标志物的存在指示当用PD-L1轴结合拮抗剂治疗个体时该个体有可能具有增大的临床受益。在一些实施方案中，增大的临床受益包括下述一项或多项的相对增大：总体存活(OS)，无进展存活(PFS)，完全响应(CR)，部分响应(PR)及其组合。In some embodiments, the presence of a PD-L1 biomarker indicates that the individual is likely to have an increased clinical benefit when treated with a PD-L1 axis binding antagonist. In some embodiments, the increased clinical benefit comprises a relative increase in one or more of the following: overall survival (OS), progression-free survival (PFS), complete response (CR), partial response (PR), and combinations thereof.
在一些实施方案中，当0％的样品包含PD-L1生物标志物时，它是样品中缺失的。在一些实施方案中，当超过0％的样品包含PD-L1生物标志物时，它是样品中存在的。在一些实施方案中，至少1％的样品中存在PD-L1生物标志物。在一些实施方案中，至少5％的样品中存在PD-L1生物标志物。在一些实施方案中，至少10％的样品中存在PD-L1生物标志物。In some embodiments, the PD-L1 biomarker is absent from the sample when 0% of the sample comprises it. In some embodiments, the PD-L1 biomarker is present in the sample when greater than 0% of the sample comprises it. In some embodiments, the PD-L1 biomarker is present in at least 1% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 5% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 10% of the sample.
在一些实施方案中，使用选自下组的方法检测样品中的PD-L1生物标志物：FACS，Western印迹，ELISA，免疫沉淀，免疫组织化学，免疫荧光，放射免疫测定法，点印迹，免疫检测方法，HPLC，表面等离振子共振，光谱术，质谱术，HPLC，qPCR，RT-qPCR，多重qPCR或RT-qPCR，RNA-seq，微阵列分析，SAGE，MassARRAY技术，和FISH，及其组合。在一些实施方案中，通过蛋白质表达检测样品中的PD-L1生物标志物。在一些实施方案中，通过免疫组织化学(IHC)测定蛋白质表达。在一些实施方案中，使用抗PD-L1抗体检测PD-L1生物标志物。In some embodiments, the PD-L1 biomarker in the sample is detected using a method selected from the group consisting of FACS, Western blotting, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection method, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof. In some embodiments, the PD-L1 biomarker in the sample is detected by protein expression. In some embodiments, protein expression is determined by immunohistochemistry (IHC). In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 antibody.
在一些实施方案中，通过IHC以弱染色强度检测PD-L1生物标志物。在一些实施方案中，通过IHC以中等染色强度检测PD-L1生物标志物。在一些实施方案中，通过IHC以强染色强度检测PD-L1生物标志物。In some embodiments, the PD-L1 biomarker is detected by IHC with weak staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC with moderate staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC with strong staining intensity.
在一些实施方案中，使用蛋白质表达分析诸如IHC分析在肿瘤细胞，肿瘤浸润性免疫细胞或其组合上检测PD-L1生物标志物。肿瘤浸润性免疫细胞包括但不限于肿瘤内免疫细胞，肿瘤周围免疫细胞或其任意组合，其它肿瘤基质细胞(例如成纤维细胞)。此类肿瘤浸润性免疫细胞可以是T淋巴细胞(诸如CD8+T淋巴细胞和/或CD4+T淋巴细胞)，B淋巴细胞，或其它骨髓谱系细胞，包括粒细胞(嗜中性细胞，嗜曙红细胞，嗜碱性细胞)，单核细胞，巨噬细胞，树突细胞(即指状树突细胞)，组织细胞，和天然杀伤细胞。In some embodiments, protein expression analysis such as IHC analysis is used to detect PD-L1 biomarkers on tumor cells, tumor-infiltrating immune cells, or a combination thereof. Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, or any combination thereof, other tumor stromal cells (e.g., fibroblasts). Such tumor-infiltrating immune cells can be T lymphocytes (such as CD8+ T lymphocytes and/or CD4+ T lymphocytes), B lymphocytes, or other bone marrow lineage cells, including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (i.e., interdigitating dendritic cells), tissue cells, and natural killer cells.
在一些实施方案中，以膜染色，胞质染色及其组合检测PD-L1生物标志物的染色。在其它实施方案中，以样品中没有或无染色检测PD-L1生物标志物的缺失。In some embodiments, staining of the PD-L1 biomarker is detected by membrane staining, cytoplasmic staining, and combinations thereof. In other embodiments, absence of the PD-L1 biomarker is detected by the absence or lack of staining in the sample.
在一些实施方案中，通过核酸表达检测样品中的PD-L1生物标志物。在一些实施方案中，使用qPCR，rtPCR，RNA-seq，多重qPCR或RT-qPCR，微阵列分析，SAGE，MassARRAY技术，或FISH测定核酸表达。In some embodiments, the PD-L1 biomarker is detected in a sample by nucleic acid expression. In some embodiments, nucleic acid expression is measured using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, or FISH.
在一些实施方案中，使用核酸表达诸如qPCR分析在肿瘤细胞，肿瘤浸润性免疫细胞，基质细胞及其任意组合上检测PD-L1生物标志物。In some embodiments, the PD-L1 biomarker is detected on tumor cells, tumor-infiltrating immune cells, stromal cells, and any combination thereof using nucleic acid expression assays such as qPCR analysis.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1轴结合拮抗剂选自下组：PD-L1结合拮抗剂和PD-1结合拮抗剂。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist and a PD-1 binding antagonist.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1轴结合拮抗剂是PD-L1结合拮抗剂。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合它的配体结合配偶。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合PD-1。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合B7-1。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合PD-1和B7-1二者。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to its ligand binding partner. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to PD-1. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to B7-1. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to both PD-1 and B7-1.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂是抗体。在任何方法、测定法和/或试剂盒的一些实施方案中，抗体是单克隆抗体。在任何方法、测定法和/或试剂盒的一些实施方案中，抗体是人抗体，人源化抗体或嵌合抗体。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist is an antibody. In some embodiments of any of the methods, assays, and/or kits, the antibody is a monoclonal antibody. In some embodiments of any of the methods, assays, and/or kits, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1轴结合拮抗剂是PD-1结合拮抗剂。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合它的配体结合配偶。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L1。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L2。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L1和PD-L2二者。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to its ligand binding partner. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to PD-L1. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to PD-L2. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to both PD-L1 and PD-L2.
在一些实施方案中，自个体获得的样品选自下组：组织，全血，血浆，血清及其组合。在一些实施方案中，样品是组织样品。在一些实施方案中，样品是肿瘤组织样品。在一些实施方案中，肿瘤组织样品包含肿瘤细胞，肿瘤浸润性免疫细胞，基质细胞或其任意组合。In some embodiments, the sample obtained from the individual is selected from the group consisting of tissue, whole blood, plasma, serum, and combinations thereof. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a tumor tissue sample. In some embodiments, the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, or any combination thereof.
在一些实施方案中，样品是在PD-L1轴结合拮抗剂治疗之前获得的。在一些实施方案中，组织样品是福尔马林固定和石蜡包埋的，存档的，新鲜的或冷冻的。In some embodiments, the sample is obtained prior to treatment with a PD-L1 axis binding antagonist. In some embodiments, the tissue sample is formalin-fixed and paraffin-embedded, archived, fresh, or frozen.
在一些实施方案中，样品是全血。在一些实施方案中，全血包含免疫细胞，循环肿瘤细胞及其任意组合。In some embodiments, the sample is whole blood. In some embodiments, the whole blood comprises immune cells, circulating tumor cells, and any combination thereof.
在一些实施方案中，疾病或病症为增殖性疾病或病症。在一些实施方案中，疾病或病症为免疫相关疾病或病症。在一些实施方案中，疾病或病症为癌症。在一些实施方案中，癌症为非小细胞肺癌，小细胞肺癌，肾细胞癌，结肠直肠癌，卵巢癌，乳腺癌，胰腺癌，胃癌，膀胱癌，食道癌，间皮瘤，黑素瘤，头和颈癌，甲状腺癌，肉瘤，前列腺癌，成胶质细胞瘤，宫颈癌，胸腺癌，白血病，淋巴瘤，骨髓瘤，蕈样肉芽肿，梅克尔细胞癌，和其它血液学恶性肿瘤。In some embodiments, the disease or condition is a proliferative disease or condition. In some embodiments, the disease or condition is an immune-related disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies.
可以基于本领域中已知的任何适宜标准定性和/或定量地测定生物标志物(例如PD-L1)的存在和/或表达水平/量，所述标准包括但不限于DNA、mRNA、cDNA、蛋白质、蛋白质片段和/或基因拷贝数。在某些实施方案中，第一样品中生物标志物的存在和/或表达水平/量相比于第二样品中的存在/缺失和/或表达水平/量增加或升高。在某些实施方案中，第一样品中生物标志物的存在/缺失和/或表达水平/量相比于第二样品中的存在和/或表达水平/量减少或降低。在某些实施方案中，第二样品是参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织。本文中描述了用于测定基因的存在/缺失和/或表达水平/量的其他公开内容。The presence and/or expression level/amount of a biomarker (e.g., PD-L1) can be determined qualitatively and/or quantitatively based on any suitable standard known in the art, including but not limited to DNA, mRNA, cDNA, protein, protein fragment, and/or gene copy number. In certain embodiments, the presence and/or expression level/amount of a biomarker in a first sample is increased or increased compared to the presence/absence and/or expression level/amount in a second sample. In certain embodiments, the presence/absence and/or expression level/amount of a biomarker in a first sample is decreased or reduced compared to the presence and/or expression level/amount in a second sample. In certain embodiments, the second sample is a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue. Other disclosures for determining the presence/absence and/or expression level/amount of a gene are described herein.
在任何方法的一些实施方案中，升高的表达指生物标志物(例如蛋白质或核酸(例如基因或mRNA))水平中相比于参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织的任意的约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的总体增加，其通过标准的本领域已知方法诸如本文中描述的那些检测。在某些实施方案中，升高的表达指样品中生物标志物的表达水平/量的增加，其中所述增加是参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织中相应生物标志物表达水平/量的任意的至少约1.5倍、1.75倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、25倍、50倍、75倍或100倍。在一些实施方案中，升高的表达指相比参照样品、参照细胞、参照组织、对照样品、对照细胞、对照组织或内部对照(例如持家基因)高约1.5倍、约1.75倍、约2倍、约2.25倍、约2.5倍、约2.75倍、约3.0倍或约3.25倍的总体增加。In some embodiments of any of the methods, elevated expression refers to an overall increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) as compared to any of a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods such as those described herein. In certain embodiments, elevated expression refers to an increase in the expression level/amount of a biomarker in a sample, wherein the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the expression level/amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression refers to an overall increase of about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to a reference sample, a reference cell, a reference tissue, a control sample, a control cell, a control tissue, or an internal control (e.g., a housekeeping gene).
在任何方法的一些实施方案中，降低的表达指生物标志物(例如蛋白质或核酸(例如基因或mRNA))水平中相比于参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织的任意的约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的总体降低，其通过标准的本领域已知方法诸如本文中描述的那些检测。在某些实施方案中，降低的表达指样品中生物标志物的表达水平/量的降低，其中所述降低是参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织中相应生物标志物表达水平/量的任意的至少约0.9倍、0.8倍、0.7倍、0.6倍、0.5倍、0.4倍、0.3倍、0.2倍、0.1倍、0.05倍或0.01倍。In some embodiments of any of the methods, decreased expression refers to an overall decrease in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue as detected by standard art-known methods such as those described herein. In certain embodiments, decreased expression refers to a decrease in the expression level/amount of a biomarker in a sample, wherein the decrease is at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold greater than the expression level/amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
可通过许多方法学来分析样品中各种生物标志物的存在和/或表达水平/量，其中许多是本领域中已知且熟练技术人员理解的，包括但不限于免疫组织化学(“IHC”)、Western印迹分析、免疫沉淀、分子结合测定法、ELISA、ELIFA、荧光激活细胞分选(“FACS”)、MassARRAY、蛋白组学、基于血液的定量测定法(如例如血清ELISA)、生化酶活性测定法、原位杂交、Southern分析、Northern分析、全基因组测序、聚合酶链式反应(“PCR”)(包括定量实时PCR(“qRT-PCR”)和其他扩增类型检测方法，如例如分支的DNA、SISBA、TMA等)、RNA-Seq、FISH、微阵列分析、基因表达概况分析、和/或基因表达系列分析(“SAGE”)，以及可通过蛋白质、基因和/或组织阵列分析实施的许多种测定法中的任一种。用于评估基因和基因产物状态的典型方案见于例如Ausubel et al.,eds.,1995,Current Protocols InMolecular Biology，Units 2(Northern Blotting),4(Southern Blotting),15(Immunoblotting)和18(PCR Analysis)。还可使用多重免疫测定法如那些可从RulesBased Medicine或Meso Scale Discovery(“MSD”)获得的。The presence and/or expression levels/amounts of various biomarkers in a sample can be analyzed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including but not limited to immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, blood-based quantitative assays (such as, for example, serum ELISA), biochemical enzyme activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction ("PCR") (including quantitative real-time PCR ("qRT-PCR") and other amplification-type detection methods, such as, for example, branched DNA, SISBA, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling, and/or serial analysis of gene expression ("SAGE"), as well as any of a variety of assays that can be performed by protein, gene and/or tissue array analysis. Typical protocols for assessing the status of genes and gene products are found in, for example, Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting), and 18 (PCR Analysis). Multiplex immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD") can also be used.
在一些实施方案中，使用包括以下的方法测定生物标志物的存在和/或表达水平/量：(a)在样品(如患者癌症样品)上实施基因表达概况分析、PCR(诸如rtPCR或qRT-PCR)、RNA-seq、微阵列分析、SAGE、MassARRAY技术、或FISH；并(b)测定生物标志物在样品中的存在和/或表达水平/量。在一些实施方案中，微阵列方法包括使用微阵列芯片，其具有一种或多种能在严格条件下与编码上文所述基因的核酸分子杂交的核酸分子或具有一种或多种能与一种或多种由上文所述基因编码的蛋白质结合的多肽(诸如肽或抗体)。在一个实施方案中，PCR方法是qRT-PCR。在一个实施方案中，PCR方法是多重PCR。在一些实施方案中，通过微阵列测量基因表达。在一些实施方案中，通过qRT-PCR测量基因表达。在一些实施方案中，通过多重PCR来测量表达。In some embodiments, the presence and/or expression level/amount of a biomarker is determined using a method comprising: (a) performing gene expression profiling, PCR (such as rtPCR or qRT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technology, or FISH on a sample (such as a patient cancer sample); and (b) determining the presence and/or expression level/amount of a biomarker in a sample. In some embodiments, the microarray method comprises using a microarray chip having one or more nucleic acid molecules that can hybridize under stringent conditions with nucleic acid molecules encoding the genes described above or having one or more polypeptides (such as peptides or antibodies) that can bind to one or more proteins encoded by the genes described above. In one embodiment, the PCR method is qRT-PCR. In one embodiment, the PCR method is multiplex PCR. In some embodiments, gene expression is measured by microarray. In some embodiments, gene expression is measured by qRT-PCR. In some embodiments, expression is measured by multiplex PCR.
用于评估细胞中mRNA的方法是众所周知的，包括例如使用互补DNA探针的杂交测定法(诸如使用经标记的特异于一种或多种基因的核糖核酸探针的原位杂交、Northern印迹和相关技术)和各种核酸扩增测定法(诸如使用特异于一种或多种基因的互补引物的RT-PCR，及其它扩增型检测方法，诸如例如分支DNA、SISBA、TMA等)。Methods for assessing mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes specific for one or more genes, Northern blotting, and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for one or more genes, and other amplification-based detection methods such as, for example, branched DNA, SISBA, TMA, etc.).
可以使用Northern、点印迹或PCR分析方便地对来自哺乳动物的样品测定mRNA。另外，此类方法可以包括一个或多个如下步骤，其容许测定生物学样品中靶mRNA的水平(例如通过同时检查“持家”基因诸如肌动蛋白家族成员的比较性对照mRNA序列的水平)。任选的是，可以测定所扩增靶cDNA的序列。mRNA can be conveniently assayed for samples from mammals using Northern, dot blot, or PCR analysis. In addition, such methods can include one or more steps that allow the determination of target mRNA levels in a biological sample (e.g., by simultaneously examining the levels of comparative control mRNA sequences of "housekeeping" genes such as members of the actin family). Optionally, the sequence of the amplified target cDNA can be determined.
任选方法包括通过微阵列技术在组织或细胞样品中检查或检测mRNA如靶mRNA的方案。使用核酸微阵列，将来自测试和对照组织样品的测试和对照mRNA样品逆转录并标记以生成cDNA探针。然后，将探针与固定化于固体支持物上的核酸阵列杂交。阵列配置为使得阵列每个成员的序列和位置是已知的。例如，可将其表达与抗血管生成疗法的增加或降低的临床益处相关的基因选择集在固体支持物上呈阵列。经标记探针与特定阵列成员的杂交指示得到该探针的样品表达该基因。Optional methods include protocols for examining or detecting mRNA, such as target mRNA, in tissue or cell samples using microarray technology. Using a nucleic acid microarray, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to a nucleic acid array immobilized on a solid support. The array is configured so that the sequence and position of each member of the array are known. For example, a gene selection set whose expression is associated with an increase or decrease in the clinical benefit of anti-angiogenic therapy can be arrayed on a solid support. Hybridization of the labeled probe with a specific array member indicates that the sample expressing the probe expresses the gene.
依照一些实施方案，通过观察前述基因的蛋白质表达水平来测量存在和/或表达水平/量。在某些实施方案中，所述方法包括使生物学样品与针对本文所述生物标志物的抗体(例如抗PD-L1抗体)在允许生物标志物结合的条件下接触，并检测抗体与生物标志物之间是否形成复合物。这类方法可以是体外或体内方法。在一个实施方案中，使用抗体来选择符合使用PD-L1轴结合拮抗剂疗法的资格的受试者，例如用于选择患者的生物标志物。According to some embodiments, the presence and/or expression level/amount is measured by observing the protein expression level of the aforementioned genes. In certain embodiments, the method comprises contacting a biological sample with an antibody to a biomarker described herein (e.g., an anti-PD-L1 antibody) under conditions that allow binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such methods can be in vitro or in vivo methods. In one embodiment, an antibody is used to select subjects who are eligible for treatment with a PD-L1 axis binding antagonist, such as a biomarker for selecting patients.
在某些实施方案中，使用IHC和染色方案来检查样品中生物标志物蛋白质的存在和/或表达水平/量。已显示对组织切片的IHC染色是一种测定或检测样品中蛋白质的存在的可靠方法。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1生物标志物为PD-L1。在一些实施方案中，通过免疫组织化学来检测PD-L1。在一些实施方案中，来自个体的样品中升高的PD-L1生物标志物表达为升高的蛋白质表达，而且在又一些实施方案中，是使用IHC测定的。在一个实施方案中，使用包括下述的方法来测定生物标志物的表达水平：(a)用抗体对样品(诸如受试者癌症样品)实施IHC分析；和(b)测定样品中生物标志物的表达水平。在一些实施方案中，IHC染色强度是相对于参照确定的。在一些实施方案中，参照为参照值。在一些实施方案中，参照为参照样品(例如对照细胞系染色样品或来自非癌症患者的组织样品)。In certain embodiments, IHC and staining protocols are used to examine the presence and/or expression level/amount of biomarker proteins in a sample. IHC staining of tissue sections has been shown to be a reliable method for determining or detecting the presence of proteins in a sample. In some embodiments of any method, assay, and/or kit, the PD-L1 biomarker is PD-L1. In some embodiments, PD-L1 is detected by immunohistochemistry. In some embodiments, elevated PD-L1 biomarker expression in a sample from an individual is elevated protein expression, and in yet other embodiments, is determined using IHC. In one embodiment, a method comprising the following is used to determine the expression level of a biomarker: (a) performing IHC analysis on a sample (such as a subject's cancer sample) using an antibody; and (b) determining the expression level of the biomarker in the sample. In some embodiments, the IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control cell line stained sample or a tissue sample from a non-cancer patient).
IHC可与另外的技术如形态学染色和/或荧光原位杂交组合实施。IHC有两种一般性方法；直接和间接测定法。依照第一种测定法，直接测定抗体对靶抗原的结合。该直接测定法使用经标记的试剂，如荧光标签或酶标记的一抗，其可以在没有别的抗体相互作用的情况下可视化。在典型的间接测定法中，未缀合的一抗结合抗原，然后经标记的二抗结合该一抗。在二抗缀合于酶标记物的情况下，添加生色或产荧光底物以提供抗原的可视化。发生信号扩增，因为几个二抗可以与一抗上的不同表位反应。IHC can be performed in combination with other techniques such as morphological staining and/or fluorescence in situ hybridization. There are two general methods for IHC: direct and indirect assays. According to the first assay, the binding of the antibody to the target antigen is directly measured. The direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without the interaction of other antibodies. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. In the case where the secondary antibody is conjugated to an enzyme label, a chromogenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody.
用于IHC的一抗和/或二抗通常用可检测模块标记。存在大量标记，其一般可分组成以下类别：(a)放射性同位素，如35S、14C、125I、3H和131I；(b)胶体金颗粒；(c)荧光标记物，包括但不限于稀土螯合物(铕螯合物)、Texas Red、罗丹明、荧光素、丹酰、丽丝胺(Lissamine)、伞形酮(umbelliferone)、藻红蛋白(phycocrytherin)、藻蓝蛋白、或商品化的荧光团如SPECTRUM ORANGE7和SPECTRUM GREEN7和/或上述任意一种或多种的衍生物；(d)存在各种酶-底物标记物，且美国专利No.4,275,149提供了对其中一些的综述。酶标记物的例子包括萤光素酶(例如萤火虫萤光素酶和细菌萤光素酶；美国专利No.4,737,456)、萤光素、2,3-二氢酞嗪二酮(dihydrophthalazinedione)、苹果酸脱氢酶、脲酶、过氧化物酶如辣根过氧化物酶(HRPO)、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖氧化酶(例如葡萄糖氧化酶、半乳糖氧化酶、和葡萄糖-6-磷酸脱氢酶)、杂环氧化酶(如尿酸酶和黄嘌呤氧化酶)、乳过氧化物酶(lactoperoxidase)、微过氧化物酶(microperoxidase)等。Primary and/or secondary antibodies used in IHC are typically labeled with a detectable moiety. A large number of labels exist, which can generally be grouped into the following categories: (a) radioisotopes, such as35 S,14 C,125 I,3 H, and131 I; (b) colloidal gold particles; (c) fluorescent labels, including but not limited to rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycoerythrin, phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more of the foregoing; (d) various enzyme-substrate labels exist, and U.S. Patent No. 4,275,149 provides a review of some of them. Examples of enzyme labels include luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
酶-底物组合的例子包括，例如辣根过氧化物酶(HRPO)和作为底物的过氧化氢酶；碱性磷酸酶(AP)和作为生色底物的对硝基苯基磷酸；和β-D-半乳糖苷酶(β-D-Gal)与生色底物(例如对硝基苯基-β-D-半乳糖苷酶)或产荧光底物(例如4-甲基伞形基(methylumbelliferyl)-β-D-半乳糖苷酶)。对于这些的一般性综述，参见美国专利No.4,275,149和4,318,980。Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) and catalase as a substrate; alkaline phosphatase (AP) and p-nitrophenyl phosphate as a chromogenic substrate; and β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate (e.g., 4-methylumbelliferyl-β-D-galactosidase). For a general review of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980.
在任何方法的一些实施方案中，使用抗PD-L1诊断性抗体(即一抗)通过免疫组织化学来检测PD-L1。在一些实施方案中，PD-L1诊断性抗体特异性结合人PD-L1。在一些实施方案中，PD-L1诊断性抗体为非人抗体。在一些实施方案中，PD-L1诊断性抗体为大鼠、小鼠、或家兔抗体。在一些实施方案中，PD-L1诊断性抗体为单克隆抗体。在一些实施方案中，PD-L1诊断性抗体是直接标记的。In some embodiments of any of the methods, PD-L1 is detected by immunohistochemistry using an anti-PD-L1 diagnostic antibody (i.e., a primary antibody). In some embodiments, the PD-L1 diagnostic antibody specifically binds to human PD-L1. In some embodiments, the PD-L1 diagnostic antibody is a non-human antibody. In some embodiments, the PD-L1 diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the PD-L1 diagnostic antibody is a monoclonal antibody. In some embodiments, the PD-L1 diagnostic antibody is directly labeled.
可将如此制备的标本封固并盖上盖玻片。然后确定载玻片评估，例如使用显微镜，并可采用本领域中常规使用的染色强度标准。在一个实施方案中，理解当使用IHC检查来自肿瘤的细胞和/或组织时，通常在肿瘤细胞和/或组织中测定或评估染色(相对于可能存在于样品中的基质或周围组织)。在一些实施方案中，理解的是，当使用IHC检查来自肿瘤的细胞和/或组织时，染色包括肿瘤浸润性免疫细胞，包括肿瘤内或肿瘤周围的免疫细胞中的测定或评估。在一些实施方案中，在>0％的样品中，在至少1％的样品中，在至少5％的样品中，在至少10％的样品中通过IHC检测PD-L1生物标志物的存在。The specimen thus prepared can be sealed and covered with a coverslip. The slide assessment is then determined, for example using a microscope, and staining intensity standards routinely used in the art can be used. In one embodiment, it is understood that when IHC is used to examine cells and/or tissues from a tumor, staining is typically measured or assessed in tumor cells and/or tissues (relative to the matrix or surrounding tissue that may be present in the sample). In some embodiments, it is understood that when IHC is used to examine cells and/or tissues from a tumor, staining includes determination or assessment of tumor-infiltrating immune cells, including immune cells within or around the tumor. In some embodiments, the presence of the PD-L1 biomarker is detected by IHC in>0% of the samples, in at least 1% of the samples, in at least 5% of the samples, and in at least 10% of the samples.
在任何方法、测定法和/或试剂盒的一些实施方案中，通过IHC以任何强度的PD-L1染色检测PD-L1生物标志物的存在。在一些实施方案中，通过IHC以弱染色强度检测PD-L1生物标志物。在一些实施方案中，通过IHC以中等染色强度检测PD-L1生物标志物。在一些实施方案中，通过IHC以强染色强度检测PD-L1生物标志物。In some embodiments of any of the methods, assays, and/or kits, the presence of the PD-L1 biomarker is detected by IHC at any intensity of PD-L1 staining. In some embodiments, the PD-L1 biomarker is detected by IHC at a weak staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC at a moderate staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC at a strong staining intensity.
在一些实施方案中，通过IHC在肿瘤细胞，肿瘤浸润性免疫细胞及其组合中检测PD-L1生物标志物。In some embodiments, the PD-L1 biomarker is detected by IHC in tumor cells, tumor-infiltrating immune cells, and combinations thereof.
适合在IHC中使用的抗PD-L1抗体是本领域公知的。普通技术人员理解，可以使用例如本文中公开的IHC方案通过与抗PD-L1抗体比较来鉴定和表征别的合适的抗PD-L1抗体。Anti-PD-L1 antibodies suitable for use in IHC are well known in the art. One of ordinary skill will appreciate that additional suitable anti-PD-L1 antibodies can be identified and characterized by comparison with anti-PD-L1 antibodies using, for example, the IHC protocol disclosed herein.
使用胎盘和扁桃体组织(强PD-L1染色强度)；经重组人PD-L1转染的HEK-293细胞(不同程度的PD-L1染色强度，弱，中等和强强度)例示阳性组织对照。对于例示性的PD-L1IHC标准，可以见下文。Positive tissue controls were exemplified using placenta and tonsil tissue (strong PD-L1 staining intensity); HEK-293 cells transfected with recombinant human PD-L1 (different degrees of PD-L1 staining intensity, weak, moderate, and strong). For exemplary PD-L1 IHC criteria, see below.
在一些实施方案中，下文提供PD-L1IHC诊断性评估的标准：In some embodiments, the criteria for PD-L1 IHC diagnostic assessment are provided below:
在备选的方法中，可使样品与特异于所述生物标志物的抗体在足以形成抗体-生物标志物复合物的条件下接触，然后检测该复合物。可以许多途径来检测生物标志物的存在，如通过Western印迹和ELISA规程，其用于测定很多种组织和样品，包括血浆或血清。有大量使用这类测定形式的免疫测定技术，参见例如美国专利No.4,016,043、4,424,279和4,018,653。这些包括非竞争型的单位点和双位点二者或“三明治式”测定法，以及传统的竞争性结合测定法。这些测定法还包括经标记抗体对靶生物标志物的直接结合。In an alternative approach, the sample can be contacted with an antibody specific for the biomarker under conditions sufficient to form an antibody-biomarker complex, and the complex is then detected. The presence of biomarkers can be detected in many ways, such as by Western blotting and ELISA procedures, which are used to assay a wide variety of tissues and samples, including plasma or serum. There are a large number of immunoassay techniques using this type of assay format, see, for example, U.S. Patent Nos. 4,016,043, 4,424,279, and 4,018,653. These include both non-competitive single-site and two-site or "sandwich" assays, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to target biomarkers.
选定的生物标志物在组织或细胞样品中的存在和/或表达水平/量还可经由功能性或基于活性的测定法来检查。例如，如果生物标志物是酶，可以进行本领域中已知的测定法来测定或检测给定的酶活性在组织或细胞样品中的存在。The presence and/or expression level/amount of a selected biomarker in a tissue or cell sample can also be examined via functional or activity-based assays. For example, if the biomarker is an enzyme, assays known in the art can be performed to determine or detect the presence of a given enzyme activity in a tissue or cell sample.
在某些实施方案中，针对测定的生物标志物的量中的差异和使用的样品质量中的可变性，以及测定轮数之间的变异性两者将样品标准化。这类标准化可通过检测并纳入特定标准化生物标志物(包括公知的看家基因)表达来实现。或者，标准化可基于所有测定基因或其较大子集的均值或中值信号(全局标准化办法)。在一个基因接一个基因的基础上，将受试者肿瘤mRNA或蛋白质的测量的经标准化的量与在参照集中发现的量比较。每种mRNA或蛋白质每份测试肿瘤每位受试者的经标准化表达水平可表示为在参照集中测量的表达水平的百分数。在要分析的特定受试者样品中测量的存在和/或表达水平/量将落在该范围内的某个百分数处，这可通过本领域中公知的方法来测定。In certain embodiments, the sample is standardized for the difference in the amount of the biomarker measured and the variability in the sample quality used, as well as the variability between the number of rounds of measurement. This type of standardization can be achieved by detecting and incorporating specific standardized biomarker (including well-known housekeeping genes) expression. Alternatively, standardization can be based on the mean or median signal of all measured genes or a larger subset thereof (global standardization approach). On a gene-by-gene basis, the standardized amount of the measurement of the subject's tumor mRNA or protein is compared with the amount found in the reference set. The standardized expression level of each mRNA or protein per test tumor per subject can be expressed as a percentage of the expression level measured in the reference set. The presence and/or expression level/amount measured in the specific subject sample to be analyzed will fall within a certain percentage within this range, which can be determined by methods well known in the art.
在一个实施方案中，所述样品是临床样品。在另一个实施方案中，所述样品用在诊断性测定法中。在一些实施方案中，所述样品从原发性或转移性肿瘤获得。经常使用组织活组织检查(biopsy)来获得肿瘤组织的代表性的片/块。或者，可以以已知或认为含有感兴趣肿瘤细胞的组织或流体的形式间接获得肿瘤细胞。例如，可通过切除、支气管镜检、细针抽吸、支气管刷检、或从痰、胸膜液或血液获得肺癌损伤的样品。可从癌症或肿瘤组织或从其他身体样品如尿液、痰、血清或血浆检测基因或基因产物。上文论述的用于检测癌性样品中靶基因或基因产物的相同技术可应用于其他身体样品。癌细胞可能从癌损伤脱落并出现在这类身体样品中。通过筛选这类身体样品，可实现对这些癌症的简单的早期诊断。另外，通过测试这类身体样品中的靶基因或基因产物能更容易地监测治疗的进展。In one embodiment, the sample is a clinical sample. In another embodiment, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a primary or metastatic tumor. Biopsy is often used to obtain representative pieces/pieces of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissues or fluids known or thought to contain tumor cells of interest. For example, a sample of lung cancer damage can be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushings, or from sputum, pleural fluid, or blood. Genes or gene products can be detected from cancer or tumor tissue or from other body samples such as urine, sputum, serum, or plasma. The same technology discussed above for detecting target genes or gene products in cancerous samples can be applied to other body samples. Cancer cells may fall off from cancer damage and appear in such body samples. By screening such body samples, simple early diagnosis of these cancers can be achieved. In addition, the progress of treatment can be more easily monitored by testing the target genes or gene products in such body samples.
在某些实施方案中，参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织是来自同一受试者或个体的单一样品或组合的多重样品，其在不同于获得测试样品时的一个或多个时间点获得。例如，参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织在早于获得测试样品时的时间点从同一受试者或个体获得。如果参照样品在癌症的初始诊断期间获得而测试样品在癌症变成转移性时的更晚时候获得，那么这类参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织可以是有用的。In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined multiple sample from the same subject or individual that is obtained at one or more time points different from when the test sample is obtained. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual at a time point earlier than when the test sample is obtained. Such reference samples, reference cells, reference tissues, control samples, control cells, or control tissues can be useful if the reference sample is obtained during the initial diagnosis of the cancer and the test sample is obtained later when the cancer becomes metastatic.
在某些实施方案中，参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织是来自一个或多个并非该受试者或个体的健康个体的组合的多重样品。在某些实施方案中，参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织是来自患有疾病或病症(例如癌症)的、并非该受试者或个体的一个或多个个体的组合的多重样品。在某些实施方案中，参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织是来自并非该受试者或个体的一个或多个个体的正常组织的合并RNA样品或合并的血浆或血清样品。在某些实施方案中，参照样品、参照细胞、参照组织、对照样品、对照细胞或对照组织是来自患有疾病或病症(例如癌症)的、并非该受试者或个体的一个或多个个体的肿瘤组织的合并RNA样品或合并的血浆或血清样品。In certain embodiments, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a multiple sample of a combination of one or more healthy individuals that are not the subject or individual. In certain embodiments, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a multiple sample of a combination of one or more individuals that are not the subject or individual suffering from a disease or condition (e.g., cancer). In certain embodiments, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a combined RNA sample or the plasma or serum sample of a normal tissue that is not the subject or individual. In certain embodiments, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a combined RNA sample or the plasma or serum sample of a tumor tissue that is not the subject or individual suffering from a disease or condition (e.g., cancer).
在一些实施方案中，样品为来自个体的组织样品。在一些实施方案中，组织样品为肿瘤组织样品(例如活检组织)。在一些实施方案中，组织样品为肺组织。在一些实施方案中，组织样品为肾组织。在一些实施方案中，组织样品为皮肤组织。在一些实施方案中，组织样品为胰腺组织。在一些实施方案中，组织样品为胃组织。在一些实施方案中，组织样品为膀胱组织。在一些实施方案中，组织样品为食道组织。在一些实施方案中，组织样品为间皮组织。在一些实施方案中，组织样品为乳腺组织。在一些实施方案中，组织样品为甲状腺组织。在一些实施方案中，组织样品为结肠直肠组织。在一些实施方案中，组织样品为头和颈组织。在一些实施方案中，组织样品为骨肉瘤组织。在一些实施方案中，组织样品为前列腺组织。在一些实施方案中，组织样品为卵巢组织，HCC(肝)，血细胞，淋巴结，骨/骨髓。In some embodiments, the sample is a tissue sample from an individual. In some embodiments, the tissue sample is a tumor tissue sample (e.g., a biopsy). In some embodiments, the tissue sample is lung tissue. In some embodiments, the tissue sample is kidney tissue. In some embodiments, the tissue sample is skin tissue. In some embodiments, the tissue sample is pancreatic tissue. In some embodiments, the tissue sample is stomach tissue. In some embodiments, the tissue sample is bladder tissue. In some embodiments, the tissue sample is esophageal tissue. In some embodiments, the tissue sample is mesothelial tissue. In some embodiments, the tissue sample is breast tissue. In some embodiments, the tissue sample is thyroid tissue. In some embodiments, the tissue sample is colorectal tissue. In some embodiments, the tissue sample is head and neck tissue. In some embodiments, the tissue sample is osteosarcoma tissue. In some embodiments, the tissue sample is prostate tissue. In some embodiments, the tissue sample is ovarian tissue, HCC (liver), blood cells, lymph nodes, bone/bone marrow.
在任何方法的一些实施方案中，疾病或病症为肿瘤。在一些实施方案中，肿瘤为恶性癌性肿瘤(即癌症)。在一些实施方案中，肿瘤和/或癌症为实体瘤或非实体或软组织肿瘤。软组织肿瘤的例子包括白血病(例如慢性髓性白血病、急性髓性白血病、成人急性成淋巴细胞性白血病、急性髓性白血病、成熟B-细胞急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、多形细胞性(polymphocytic)白血病、或毛细胞性白血病)或淋巴瘤(例如非何杰金(Hodgkin)氏淋巴瘤、皮肤T-细胞淋巴瘤、或何杰金氏病)。实体瘤包括除血液、骨髓、或淋巴系统以外的身体组织的任何癌症。实体瘤可进一步分成上皮细胞起源的和非上皮细胞起源的。上皮细胞实体瘤的例子包括胃肠道、结肠、结直肠(例如基样(basaloid)结直肠癌)、乳腺、前列腺、肺、肾、肝、胰腺、卵巢(例如子宫内膜样(endometrioid)卵巢癌)、头和颈、口腔、胃、十二指肠、小肠、大肠、肛门、胆囊、阴唇、鼻咽、皮肤、子宫、男性生殖器官、泌尿器官(例如尿路上皮癌、发育异常尿路上皮癌、移行细胞癌)、膀胱、和皮肤的肿瘤。非上皮起源的实体瘤包括肉瘤、脑瘤、和骨瘤。在一些实施方案中，癌症为非小细胞肺癌(NSCLC)。在一些实施方案中，癌症为二线或三线局部晚期或转移性非小细胞肺癌。在一些实施方案中，癌症为腺癌。在一些实施方案中，癌症为鳞状细胞癌。In some embodiments of any method, disease or illness is tumor.In some embodiments, tumor is malignant cancerous tumor (i.e. cancer).In some embodiments, tumor and/or cancer is solid tumor or non-solid or soft tissue tumor.The example of soft tissue tumor includes leukemia (such as chronic myeloid leukemia, acute myeloid leukemia, adult acute lymphoblastic leukemia, acute myeloid leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, polymorphic cell (polymphocytic) leukemia or hairy cell leukemia) or lymphoma (such as non-Hodgkin (Hodgkin) lymphoma, cutaneous T-cell lymphoma or Hodgkin's disease).Solid tumor includes any cancer of body tissue except blood, bone marrow or lymphatic system.Solid tumor can be further divided into epithelial cell origin and non-epithelial cell origin. Examples of epithelial cell solid tumors include tumors of the gastrointestinal tract, colon, colorectum (e.g., basaloid colorectal cancer), breast, prostate, lung, kidney, liver, pancreas, ovary (e.g., endometrioid ovarian cancer), head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, labia, nasopharynx, skin, uterus, male reproductive organs, urinary organs (e.g., urothelial carcinoma, dysplastic urothelial carcinoma, transitional cell carcinoma), bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is second-line or third-line locally advanced or metastatic non-small cell lung cancer. In some embodiments, the cancer is adenocarcinoma. In some embodiments, the cancer is squamous cell carcinoma.
在一些实施方案中，使用选自下组的方法在样品中检测PD-L1生物标志物：FACS，Western印迹，ELISA，免疫沉淀，免疫组织化学，免疫荧光，放射免疫测定法，点印迹，免疫检测方法，HPLC，表面等离振子共振，光谱术，质谱术，HPLC，qPCR，RT-qPCR，多重qPCR或RT-qPCR，RNA-seq，微阵列分析，SAGE，MassARRAY技术，和FISH，及其组合。在一些实施方案中，使用FACS分析检测PD-L1生物标志物。在一些实施方案中，PD-L1生物标志物为PD-L1。在一些实施方案中，在血液样品中检测PD-L1表达。在一些实施方案中，在血液样品中的循环免疫细胞上检测PD-L1表达。在一些实施方案中，循环免疫细胞为CD3+/CD8+T细胞。在一些实施方案中，在分析之前，自血液样品分离免疫细胞。可以使用分离/富集此类细胞群体的任何合适方法，包括但不限于细胞分选。在一些实施方案中，来自个体的样品中的PD-L1表达响应PD-L1/PD-1轴途径的抑制剂(诸如抗PD-L1抗体)治疗而升高。在一些实施方案中，血液样品中的循环免疫细胞(诸如CD3+/CD8+T细胞)上的PD-L1表达升高。In some embodiments, the PD-L1 biomarker is detected in a sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection method, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof. In some embodiments, the PD-L1 biomarker is detected using FACS analysis. In some embodiments, the PD-L1 biomarker is PD-L1. In some embodiments, PD-L1 expression is detected in a blood sample. In some embodiments, PD-L1 expression is detected on circulating immune cells in a blood sample. In some embodiments, the circulating immune cells are CD3+/CD8+ T cells. In some embodiments, immune cells are isolated from a blood sample prior to analysis. Any suitable method for isolating/enriching such cell populations may be used, including but not limited to cell sorting. In some embodiments, PD-L1 expression in a sample from an individual is elevated in response to treatment with an inhibitor of the PD-L1/PD-1 axis pathway, such as an anti-PD-L1 antibody. In some embodiments, PD-L1 expression is elevated on circulating immune cells, such as CD3+/CD8+ T cells, in a blood sample.
治疗方法Treatment
提供的是用于治疗个体中疾病或病症的方法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，并将有效量的PD-L1轴结合拮抗剂施用于该个体。Provided are methods for treating a disease or condition in an individual, the method comprising: determining the presence of a PD-L1 biomarker in a sample from the individual, and administering to the individual an effective amount of a PD-L1 axis binding antagonist.
本文中进一步提供的是用于治疗个体中疾病或病症的方法，其包括对该个体施用有效量的PD-L1轴结合拮抗剂，其中治疗基于来自该个体的样品中PD-L1生物标志物的存在。Further provided herein are methods for treating a disease or condition in an individual comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist, wherein the treatment is based on the presence of a PD-L1 biomarker in a sample from the individual.
在一些实施方案中，PD-L1生物标志物选自下组：PD-L1，PD-1，PD-L2及其任意组合。In some embodiments, the PD-L1 biomarker is selected from the group consisting of PD-L1, PD-1, PD-L2, and any combination thereof.
在一些实施方案中，PD-L1生物标志物为免疫相关标志物。免疫相关标志物指由免疫细胞或其它细胞(例如肿瘤细胞，内皮细胞，成纤维细胞或其它基质细胞)表达的标志物。如果由免疫细胞以外的细胞表达的话，标志物可以涉及调节免疫细胞生物学或功能，诸如活化，引发，抗原识别和呈递，细胞因子和趋化因子生成，增殖，迁移，存活，抗体生成等等。在一些实施方案中，免疫相关标志物为T细胞相关标志物。在一些实施方案中，T细胞相关标志物选自下组：CD8A，IFN-g，EOMES，粒酶-A，CXCL9及其任意组合。在一些实施方案中，免疫相关标志物选自下组：CX3CL1，CD45RO，IDO1，半乳凝素9，MIC-A，MIC-B，CTLA-4及其任意组合。In some embodiments, the PD-L1 biomarker is an immune-related marker. Immune-related markers refer to markers expressed by immune cells or other cells (e.g., tumor cells, endothelial cells, fibroblasts or other stromal cells). If expressed by cells other than immune cells, the marker may be involved in regulating immune cell biology or function, such as activation, priming, antigen recognition and presentation, cytokine and chemokine production, proliferation, migration, survival, antibody production, and the like. In some embodiments, the immune-related marker is a T cell-related marker. In some embodiments, the T cell-related marker is selected from the group consisting of CD8A, IFN-g, EOMES, granzyme-A, CXCL9, and any combination thereof. In some embodiments, the immune-related marker is selected from the group consisting of CX3CL1, CD45RO, IDO1, galectin 9, MIC-A, MIC-B, CTLA-4, and any combination thereof.
在一些实施方案中，PD-L1生物标志物的存在指示当用PD-L1轴结合拮抗剂治疗个体时该个体有可能具有增大的临床受益。在一些实施方案中，增大的临床受益包括下述一项或多项的相对增大：总体存活(OS)，无进展存活(PFS)，完全响应(CR)，部分响应(PR)及其组合。In some embodiments, the presence of a PD-L1 biomarker indicates that the individual is likely to have an increased clinical benefit when treated with a PD-L1 axis binding antagonist. In some embodiments, the increased clinical benefit comprises a relative increase in one or more of the following: overall survival (OS), progression-free survival (PFS), complete response (CR), partial response (PR), and combinations thereof.
在一些实施方案中，当0％的样品包含PD-L1生物标志物时，它是样品中缺失的。在一些实施方案中，当超过0％的样品包含PD-L1生物标志物时，它是样品中存在的。在一些实施方案中，至少1％的样品中存在PD-L1生物标志物。在一些实施方案中，至少5％的样品中存在PD-L1生物标志物。在一些实施方案中，至少10％的样品中存在PD-L1生物标志物。In some embodiments, the PD-L1 biomarker is absent from the sample when 0% of the sample comprises it. In some embodiments, the PD-L1 biomarker is present in the sample when greater than 0% of the sample comprises it. In some embodiments, the PD-L1 biomarker is present in at least 1% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 5% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 10% of the sample.
在一些实施方案中，使用选自下组的方法检测样品中的PD-L1生物标志物：FACS，Western印迹，ELISA，免疫沉淀，免疫组织化学，免疫荧光，放射免疫测定法，点印迹，免疫检测方法，HPLC，表面等离振子共振，光谱术，质谱术，HPLC，qPCR，RT-qPCR，多重qPCR或RT-qPCR，RNA-seq，微阵列分析，SAGE，MassARRAY技术，和FISH，及其组合。In some embodiments, the PD-L1 biomarker is detected in the sample using a method selected from the group consisting of FACS, Western blotting, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof.
在一些实施方案中，通过蛋白质表达检测样品中的PD-L1生物标志物。在一些实施方案中，通过免疫组织化学(IHC)测定蛋白质表达。在一些实施方案中，使用抗PD-L1抗体检测PD-L1生物标志物。In some embodiments, the PD-L1 biomarker is detected in a sample by protein expression. In some embodiments, protein expression is determined by immunohistochemistry (IHC). In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 antibody.
在一些实施方案中，通过IHC以弱染色强度检测PD-L1生物标志物。在一些实施方案中，通过IHC以中等染色强度检测PD-L1生物标志物。在一些实施方案中，通过IHC以强染色强度检测PD-L1生物标志物。In some embodiments, the PD-L1 biomarker is detected by IHC with weak staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC with moderate staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC with strong staining intensity.
在一些实施方案中，使用蛋白质表达分析诸如IHC分析在肿瘤细胞，肿瘤浸润性免疫细胞或其组合上检测PD-L1生物标志物。肿瘤浸润性免疫细胞包括但不限于肿瘤内免疫细胞，肿瘤周围免疫细胞或其任意组合，其它肿瘤基质细胞(例如成纤维细胞)。此类肿瘤浸润性免疫细胞可以是T淋巴细胞(诸如CD8+T淋巴细胞和/或CD4+T淋巴细胞)，B淋巴细胞，或其它骨髓谱系细胞，包括粒细胞(嗜中性细胞，嗜曙红细胞，嗜碱性细胞)，单核细胞，巨噬细胞，树突细胞(即指状树突细胞)，组织细胞，和天然杀伤细胞。In some embodiments, protein expression analysis such as IHC analysis is used to detect PD-L1 biomarkers on tumor cells, tumor-infiltrating immune cells, or a combination thereof. Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, or any combination thereof, other tumor stromal cells (e.g., fibroblasts). Such tumor-infiltrating immune cells can be T lymphocytes (such as CD8+ T lymphocytes and/or CD4+ T lymphocytes), B lymphocytes, or other bone marrow lineage cells, including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (i.e., interdigitating dendritic cells), tissue cells, and natural killer cells.
在一些实施方案中，以膜染色，胞质染色及其组合检测PD-L1生物标志物的染色。在其它实施方案中，以样品中没有或无染色检测PD-L1生物标志物的缺失。In some embodiments, staining of the PD-L1 biomarker is detected by membrane staining, cytoplasmic staining, and combinations thereof. In other embodiments, absence of the PD-L1 biomarker is detected by the absence or lack of staining in the sample.
在一些实施方案中，通过核酸表达检测样品中的PD-L1生物标志物。在一些实施方案中，使用qPCR，rtPCR，RNA-seq，多重qPCR或RT-qPCR，微阵列分析，SAGE，MassARRAY技术，或FISH测定核酸表达。In some embodiments, the PD-L1 biomarker is detected in a sample by nucleic acid expression. In some embodiments, nucleic acid expression is measured using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, or FISH.
在一些实施方案中，使用核酸表达诸如qPCR分析在肿瘤细胞，肿瘤浸润性免疫细胞，基质细胞及其组合上检测PD-L1生物标志物。In some embodiments, the PD-L1 biomarker is detected on tumor cells, tumor-infiltrating immune cells, stromal cells, and combinations thereof using nucleic acid expression assays such as qPCR analysis.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1轴结合拮抗剂选自下组：PD-L1结合拮抗剂和PD-1结合拮抗剂。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist and a PD-1 binding antagonist.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1轴结合拮抗剂为PD-L1结合拮抗剂。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合它的配体结合配偶。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合PD-1。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合B7-1。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合PD-1和B7-1二者。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to its ligand binding partner. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to PD-1. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to B7-1. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist inhibits PD-L1 from binding to both PD-1 and B7-1.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1结合拮抗剂为抗体。在任何方法、测定法和/或试剂盒的一些实施方案中，抗体为单克隆抗体。在任何方法、测定法和/或试剂盒的一些实施方案中，抗体为人抗体，人源化抗体或嵌合抗体。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 binding antagonist is an antibody. In some embodiments of any of the methods, assays, and/or kits, the antibody is a monoclonal antibody. In some embodiments of any of the methods, assays, and/or kits, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
在任何方法、测定法和/或试剂盒的一些实施方案中，PD-L1轴结合拮抗剂为PD-1结合拮抗剂。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合它的配体结合配偶。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L1。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L2。在任何方法、测定法和/或试剂盒的一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L1和PD-L2二者。In some embodiments of any of the methods, assays, and/or kits, the PD-L1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to its ligand binding partner. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to PD-L1. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to PD-L2. In some embodiments of any of the methods, assays, and/or kits, the PD-1 binding antagonist inhibits PD-1 from binding to both PD-L1 and PD-L2.
对本发明的方法有用的抗PD-L1抗体及其制备方法记载于PCT专利申请WO 2010/077634 A1，通过援引将其收入本文。Anti-PD-L1 antibodies useful for the methods of the present invention and methods for their preparation are described in PCT patent application WO 2010/077634 A1, which is incorporated herein by reference.
在一些实施方案中，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，抗PD-L1抗体为单克隆抗体。在一些实施方案中，抗PD-L1抗体为选自下组的抗体片段：Fab，Fab'-SH，Fv，scFv，和(Fab')2片段。在一些实施方案中，抗PD-L1抗体为人源化抗体。在一些实施方案中，抗PD-L1抗体为人抗体。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 the group consisting of: 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.
在一个实施方案中，抗PD-L1抗体含有包含HVR-H1，HVR-H2和HVR-H3序列的重链可变区多肽，其中：In one embodiment, the anti-PD-L1 antibody comprises a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:
(a)HVR-H1序列为GFTFSX1SWIH(SEQ ID NO:1)；(a) HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO: 1);
(b)HVR-H2序列为AWIX2PYGGSX3YYADSVKG(SEQ ID NO:2)；(b) HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 2);
(c)HVR-H3序列为RHWPGGFDY(SEQ ID NO:3)；(c) HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 3);
且其中：X1为D或G；X2为S或L；X3为T或S。And wherein: X1 is D or G; X2 is S or L; X3 is T or S.
在一个特定方面，X1为D；X2为S和X3为T。在另一个方面，该多肽进一步包含依照下式并置HVR之间的可变区重链框架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)。在还有另一个方面，该框架序列是自人共有框架序列衍生的。在又一个方面，该框架序列为VH亚组III共有框架。在仍有又一个方面，该框架序列中的至少一个为下述：In one specific aspect, X1 is D; X2 is S and X3 is T. 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 sequence is derived from a human consensus framework sequence. In yet another aspect, the framework sequence is a VH subgroup III consensus framework. In yet another aspect, at least one of the framework sequences is as follows:
HC-FR1为EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:4)
HC-FR2为WVRQAPGKGLEWV(SEQ ID NO:5)HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 5)
HC-FR3为RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 6)
HC-FR4为WGQGTLVTVSA(SEQ ID NO:7)。HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 7).
在仍有又一个方面，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 still 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)HVR-L1序列为RASQX4X5X6TX7X8A(SEQ ID NO:8)；(a) HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 8);
(b)HVR-L2序列为SASX9LX10S(SEQ ID NO:9)；(b) HVR-L2 sequence is SASX9LX10S (SEQ ID NO: 9);
(c)HVR-L3序列为QQX11X12X13X14PX15T(SEQ ID NO:10)；(c) HVR-L3 sequence is QQX11X12X13X14PX15T (SEQ ID NO: 10);
且其中：X4为D或V；X5为V或I；X6为S或N；X7为A或F；X8为V或L；X9为F或T；X10为Y或A；X11为Y，G，F，或S；X12为L，Y，F或W；X13为Y，N，A，T，G，F或I；X14为H，V，P，T或I；X15为A，W，R，P或T。And 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.
在仍有又一个方面，X4为D；X5为V；X6为S；X7为A；X8为V；X9为F；X10为Y；X11为Y；X12为L；X13为Y；X14为H；X15为A。在仍有又一个方面，该轻链进一步包含依照下式并置HVR之间的可变区轻链框架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。在仍有又一个方面，该框架序列是自人共有框架序列衍生的。在仍有又一个方面，该框架序列为VL卡帕I共有框架。在仍有又一个方面，该框架序列中的至少一个为下述：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; X15 is A. In yet another aspect, the light chain further comprises a variable region light chain framework sequence juxtaposed between the HVRs according to the following 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 VL kappa I consensus framework. In yet another aspect, at least one of the framework sequences is as follows:
LC-FR1为DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:11)LC-FR1 is DIQMTQSPSSSLSASVGDRVTITC (SEQ ID NO:11)
LC-FR2为WYQQKPGKAPKLLIY(SEQ ID NO:12)LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO:12)
LC-FR3为GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:13)
LC-FR4为FGQGTKVEIKR(SEQ ID NO:14)。LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).
在另一个实施方案中，提供的是分离的抗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, provided is an isolated anti-PD-L1 antibody or antigen-binding fragment comprising heavy and light chain variable region sequences, wherein:
(a)该重链包含HVR-H1，HVR-H2和HVR-H3，且其中：(a) the heavy chain comprises HVR-H1, HVR-H2 and HVR-H3, and wherein:
(i)HVR-H1序列为GFTFSX1SWIH(SEQ ID NO:1)(i) HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO: 1)
(ii)HVR-H2序列为AWIX2PYGGSX3YYADSVKG(SEQ ID NO:2)(ii) HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 2)
(iii)HVR-H3序列为RHWPGGFDY(SEQ ID NO:3)，且(iii) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 3), and
(b)该轻链包含HVR-L1，HVR-L2和HVR-L3，且其中：(b) the light chain comprises HVR-L1, HVR-L2 and HVR-L3, and wherein:
(i)HVR-L1序列为RASQX4X5X6TX7X8A(SEQ ID NO:8)(i) HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 8)
(ii)HVR-L2序列为SASX9LX10S(SEQ ID NO:9)(ii) HVR-L2 sequence is SASX9LX10S (SEQ ID NO: 9)
(iii)HVR-L3序列为QQX11X12X13X14PX15T(SEQ ID NO:10)(iii) HVR-L3 sequence is QQX11X12X13X14PX15T (SEQ ID NO: 10)
且其中：X1为D或G；X2为S或L；X3为T或S；X4为D或V；X5为V或I；X6为S或N；X7为A或F；X8为V或L；X9为F或T；X10为Y或A；X11为Y，G，F，或S；X12为L，Y，F或W；X13为Y，N，A，T，G，F或I；X14为H，V，P，T或I；X15为A，W，R，P或T。and 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; and X15 is A, W, R, P, or T.
在一个特定方面，X1为D；X2为S和X3为T。在另一个方面，X4为D；X5为V；X6为S；X7为A；X8为V；X9为F；X10为Y；X11为Y；X12为L；X13为Y；X14为H；X15为A。在还有另一个方面，X1为D；X2为S和X3为T，X4为D；X5为V；X6为S；X7为A；X8为V；X9为F；X10为Y；X11为Y；X12为L；X13为Y；X14为H和X15为A。In one specific aspect, 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; X15 is A. In yet another aspect, X1 is D; X2 is S and 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 and X15 is A.
在又一个方面，该重链可变区包含如下并置HVR之间的一种或多种框架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)，且该轻链可变区包含如下并置HVR之间的一种或多种框架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。在仍有又一个方面，该框架序列是自人共有框架序列衍生的。在仍有又一个方面，该重链框架序列是自Kabat亚组I，II，或III序列衍生的。在仍有又一个方面，该重链框架序列为VH亚组III共有框架。在仍有又一个方面，该重链框架序列中的一种或多种为下述：In yet another aspect, the heavy chain variable region comprises one or more framework sequences between the following juxtaposed HVRs: (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 between the following juxtaposed HVRs: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In still another aspect, the framework sequences are derived from human consensus framework sequences. In still another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In still another aspect, the heavy chain framework sequences are VH subgroup III consensus frameworks. In still another aspect, one or more of the heavy chain framework sequences are as follows:
HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)
HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)
HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)
HC-FR4 WGQGTLVTVSA(SEQ ID NO:7)。HC-FR4 WGQGTLVTVSA (SEQ ID NO:7).
在仍有又一个方面，该轻链框架序列是自Kabat卡帕I，II，II或IV亚组序列衍生的。在仍有又一个方面，该轻链框架序列为VL卡帕I共有框架。在仍有又一个方面，该轻链框架序列中的一种或多种为如下：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 are as follows:
LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:11)LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:11)
LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)
LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)
LC-FR4 FGQGTKVEIKR(SEQ ID NO:14)。LC-FR4 FGQGTKVEIKR (SEQ ID NO: 14).
在仍有又一个特定方面，该抗体进一步包含人或鼠恒定区。在仍有又一个方面，该人恒定区选自下组：IgG1，IgG2，IgG2，IgG3，IgG4。在仍有又一个特定方面，该人恒定区为IgG1。在仍有又一个方面，该鼠恒定区选自下组：IgG1，IgG2A，IgG2B，IgG3。在仍有又一个方面，该鼠恒定区为IgG2A。在仍有又一个特定方面，该抗体具有降低的或最小程度的效应器功能。在仍有又一个特定方面，该最小程度的效应器功能源自“效应器减小性Fc突变”或无糖基化。在仍有又一个实施方案中，该效应器减小性Fc突变为恒定区中的N297A或D265A/N297A替代。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 the group consisting of 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 the group consisting of 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-reducing Fc mutation" or aglycosylation. In yet another embodiment, the effector-reducing Fc mutation is an N297A or D265A/N297A substitution in the constant region.
在仍有另一个实施方案中，提供的是包含重链和轻链可变区序列的抗PD-L1抗体，其中：In yet another embodiment, provided is an anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:
该重链进一步包含分别与GFTFSDSWIH(SEQ ID NO:15)，AWISPYGGSTYYADSVKG(SEQID NO:16)和RHWPGGFDY(SEQ ID NO:3)具有至少85％序列同一性的HVR-H1，HVR-H2和HVR-H3序列，或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: 15), AWISPYGGSTYYADSVKG (SEQ ID NO: 16) and RHWPGGFDY (SEQ ID NO: 3), respectively, or
该轻链进一步包含分别与RASQDVSTAVA(SEQ ID NO:17)，SASFLYS(SEQ ID NO:18)和QQYLYHPAT(SEQ ID NO:19)具有至少85％序列同一性的HVR-L1，HVR-L2和HVR-L3序列。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: 17), SASFLYS (SEQ ID NO: 18) and QQYLYHPAT (SEQ ID NO: 19), respectively.
在一个特定方面，该序列同一性为86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％。在另一个方面，该重链可变区包含如下并置HVR之间的一种或多种框架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)，且该轻链可变区包含如下并置HVR之间的一种或多种框架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。在还有另一个方面，该框架序列是自人共有框架序列衍生的。在仍有又一个方面，该重链框架序列是自Kabat亚组I，II，或III序列衍生的。在仍有又一个方面，该重链框架序列为VH亚组III共有框架。在仍有又一个方面，该重链框架序列中的一种或多种为下述：In a specific aspect, 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 between the following juxtaposed HVRs: (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 between the following juxtaposed HVRs: (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 sequence is a VH subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences are:
HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)
HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)
HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)
HC-FR4 WGQGTLVTVSA(SEQ ID NO:7)。HC-FR4 WGQGTLVTVSA (SEQ ID NO:7).
在仍有又一个方面，该轻链框架序列是自Kabat卡帕I，II，II或IV亚组序列衍生的。在仍有又一个方面，该轻链框架序列为VL卡帕I共有框架。在仍有又一个方面，该轻链框架序列中的一种或多种为下述：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 as follows:
LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:11)LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:11)
LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)
LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)
LC-FR4 FGQGTKVEIKR(SEQ ID NO:14)。LC-FR4 FGQGTKVEIKR (SEQ ID NO: 14).
在仍有又一个特定方面，该抗体进一步包含人或鼠恒定区。在仍有又一个方面，该人恒定区选自下组：IgG1，IgG2，IgG2，IgG3，IgG4。在仍有又一个特定方面，该人恒定区为IgG1。在仍有又一个方面，该鼠恒定区选自下组：IgG1，IgG2A，IgG2B，IgG3。在仍有又一个方面，该鼠恒定区为IgG2A。在仍有又一个特定方面，该抗体具有降低的或最小程度的效应器功能。在仍有又一个特定方面，该最小程度的效应器功能源自“效应器减小性Fc突变”或无糖基化。在仍有又一个实施方案中，该效应器减小性Fc突变为恒定区中的N297A或D265A/N297A替代。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 the group consisting of 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 the group consisting of 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-reducing Fc mutation" or aglycosylation. In yet another embodiment, the effector-reducing Fc mutation is an N297A or D265A/N297A substitution in the constant region.
在仍有又一个实施方案中，提供的是分离的包含重链和轻链可变区序列的抗PD-L1抗体，其中：In yet another embodiment, provided is an isolated anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:
(a)重链序列与重链序列EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA(SEQID NO:20)具有至少85％序列同一性，或(a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 20), or
(b)轻链序列与轻链序列DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:21)具有至少85％序列同一性。(b) the light chain sequence has at least 85% sequence identity to the light chain sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 21).
在一个特定方面，该序列同一性为86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％。在另一个方面，该重链可变区包含如下并置HVR之间的一种或多种框架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)，且该轻链可变区包含如下并置HVR之间的一种或多种框架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。在还有另一个方面，该框架序列是自人共有框架序列衍生的。在又一个方面，该重链框架序列是自Kabat亚组I，II，或III序列衍生的。在仍有又一个方面，该重链框架序列为VH亚组III共有框架。在仍有又一个方面，该重链框架序列中的一种或多种为下述：In a specific aspect, 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 between the following juxtaposed HVRs: (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 between the following juxtaposed HVRs: (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 sequence is a VH subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences are as follows:
HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)
HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)
HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)
HC-FR4 WGQGTLVTVSA(SEQ ID NO:7)。HC-FR4 WGQGTLVTVSA (SEQ ID NO:7).
在仍有又一个方面，该轻链框架序列是自Kabat卡帕I，II，II或IV亚组序列衍生的。在仍有又一个方面，该轻链框架序列为VL卡帕I共有框架。在仍有又一个方面，该轻链框架序列中的一种或多种为下述：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 as follows:
LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:11)LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:11)
LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)
LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)
LC-FR4 FGQGTKVEIKR(SEQ ID NO:14)。LC-FR4 FGQGTKVEIKR (SEQ ID NO: 14).
在仍有又一个特定方面，该抗体进一步包含人或鼠恒定区。在仍有又一个方面，该人恒定区选自下组：IgG1，IgG2，IgG2，IgG3，IgG4。在仍有又一个特定方面，该人恒定区为IgG1。在仍有又一个方面，该鼠恒定区选自下组：IgG1，IgG2A，IgG2B，IgG3。在仍有又一个方面，该鼠恒定区为IgG2A。在仍有又一个特定方面，该抗体具有降低的或最小程度的效应器功能。在仍有又一个特定方面，该最小程度的效应器功能源自在原核细胞中生成。在仍有又一个特定方面，该最小程度的效应器功能源自“效应器减小性Fc突变”或无糖基化。在仍有又一个实施方案中，该效应器减小性Fc突变为恒定区中的N297A或D265A/N297A替代。In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another specific aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another specific aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In yet another specific 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 results from production in prokaryotic cells. In yet another specific aspect, the minimal effector function results from an "effector-reducing Fc mutation" or aglycosylation. In yet another embodiment, the effector-reducing Fc mutation is an N297A or D265A/N297A substitution in the constant region.
在仍有又一个实施方案中，本发明提供组合物，其包含与至少一种药学可接受载剂组合的任何上文所述抗PD-L1抗体。In still another embodiment, the present invention provides a composition comprising any of the anti-PD-L1 antibodies described above in combination with at least one pharmaceutically acceptable carrier.
在仍有又一个实施方案中，提供的是分离的编码抗PD-L1抗体的轻链或重链可变区序列的核酸，其中：In yet another embodiment, provided is an isolated nucleic acid encoding a light chain or heavy chain variable region sequence of an anti-PD-L1 antibody, wherein:
(a)重链进一步包含分别与GFTFSDSWIH(SEQ ID NO:15)，AWISPYGGSTYYADSVKG(SEQ ID NO:16)和RHWPGGFDY(SEQ ID NO:3)具有至少85％序列同一性的HVR-H1，HVR-H2和HVR-H3序列，且(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: 15), AWISPYGGSTYYADSVKG (SEQ ID NO: 16) and RHWPGGFDY (SEQ ID NO: 3), respectively, and
(b)轻链进一步包含分别与RASQDVSTAVA(SEQ ID NO:17)，SASFLYS(SEQ ID NO:18)和QQYLYHPAT(SEQ ID NO:19)的具有至少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: 17), SASFLYS (SEQ ID NO: 18) and QQYLYHPAT (SEQ ID NO: 19), respectively.
在一个特定方面，该序列同一性为86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％。在一个方面，该重链可变区包含如下并置HVR之间的一种或多种框架序列：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)，且该轻链可变区包含如下并置HVR之间的一种或多种框架序列：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)。在还有另一个方面，该框架序列是自人共有框架序列衍生的。在又一个方面，该重链框架序列是自Kabat亚组I，II，或III序列衍生的。在仍有又一个方面，该重链框架序列为VH亚组III共有框架。在仍有又一个方面，该重链框架序列中的一种或多种为下述：In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In one aspect, the heavy chain variable region comprises one or more framework sequences between the following juxtaposed HVRs: (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 between the following juxtaposed HVRs: (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 sequence is a VH subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences are:
HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)
HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)
HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)
HC-FR4 WGQGTLVTVSA(SEQ ID NO:7)。HC-FR4 WGQGTLVTVSA (SEQ ID NO:7).
在仍有又一个方面，该轻链框架序列是自Kabat卡帕I，II，II或IV亚组序列衍生的。在仍有又一个方面，该轻链框架序列为VL卡帕I共有框架。在仍有又一个方面，该轻链框架序列中的一种或多种为下述：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 as follows:
LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:11)LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:11)
LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)
LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)
LC-FR4 FGQGTKVEIKR(SEQ ID NO:14)。LC-FR4 FGQGTKVEIKR (SEQ ID NO: 14).
在仍有又一个特定方面，该抗体进一步包含人或鼠恒定区。在仍有又一个方面，该人恒定区选自下组：IgG1，IgG2，IgG2，IgG3，IgG4。在仍有又一个特定方面，该人恒定区为IgG1。在仍有又一个方面，该鼠恒定区选自下组：IgG1，IgG2A，IgG2B，IgG3。在仍有又一个方面，该鼠恒定区为IgG2A。在仍有又一个特定方面，该抗体具有降低的或最小程度的效应器功能。在仍有又一个特定方面，该最小程度的效应器功能源自在原核细胞中生成。在仍有又一个特定方面，该最小程度的效应器功能源自“效应器减小性Fc突变”或无糖基化。在仍有又一个方面，该效应器减小性Fc突变为恒定区中的N297A或D265A/N297A替代。In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another specific aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another specific aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In yet another specific 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 results from production in prokaryotic cells. In yet another specific aspect, the minimal effector function results from an "effector-reducing Fc mutation" or aglycosylation. In yet another specific aspect, the effector-reducing Fc mutation is an N297A or D265A/N297A substitution in the constant region.
在仍有又一个方面，该核酸进一步包含在适合于表达编码任何先前所述抗PD-L1抗体的核酸的载体中。在仍有又一个特定方面，该载体进一步包含在适合于表达该核酸的宿主细胞中。在仍有又一个特定方面，该宿主细胞为真核细胞或原核细胞。在仍有又一个特定方面，该真核细胞为哺乳动物细胞，诸如中国仓鼠卵巢(CHO)。In yet another aspect, the nucleic acid is further comprised in a vector suitable for expressing the nucleic acid encoding any of the previously described anti-PD-L1 antibodies. In yet another specific aspect, the vector is further comprised 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PD-L1抗体或其抗原结合片段可以使用本领域已知方法来制备，例如通过包括下述的工艺：在适合于生成此类抗体或片段的条件下培养以适合于表达的形式含有编码任何先前所述抗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。在仍有又一个实施方案中，本发明提供组合物，其包含本文中提供的抗PD-L1抗体或其抗原结合片段和至少一种药学可接受载剂。Anti-PD-L1 antibodies or antigen-binding fragments thereof can be prepared using methods known in the art, for example, by a process 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 the production of such antibodies or fragments, and recovering the antibody or fragment. In yet another embodiment, the present invention provides a composition comprising an anti-PD-L1 antibody or antigen-binding fragment thereof provided herein and at least one pharmaceutically acceptable carrier.
A.抗体A. Antibodies
1.抗体亲和力1. Antibody affinity
在某些实施方案中，本文中提供的抗体具有≤1μM的解离常数。在一个实施方案中，Kd是通过如下述测定法所述用Fab型式的感兴趣抗体及其抗原实施的放射性标记抗原结合测定法(RIA)来测量的。通过在存在未标记抗原的滴定系列的情况中用最小浓度的(125I)标记抗原平衡Fab，然后用抗Fab抗体包被板捕捉结合的抗原来测量Fab对抗原的溶液结合亲和力(见例如Chen等,J.Mol.Biol.293:865-881(1999))。为了建立测定法的条件，将多孔板(Thermo Scientific)用50mM碳酸钠(pH 9.6)中的5μg/ml捕捉用抗Fab抗体(Cappel Labs)包被过夜，随后用PBS中的2％(w/v)牛血清清蛋白于室温(约23℃)封闭2-5小时。在非吸附板(Nunc#269620)中，将100pM或26pM[125I]-抗原与连续稀释的感兴趣Fab(例如与Presta等,Cancer Res.57:4593-4599(1997)中抗VEGF抗体，Fab-12的评估一致)混合。然后将感兴趣的Fab温育过夜；然而，温育可持续更长时间(例如约65小时)以确保达到平衡。此后，将混合物转移至捕捉板，于室温温育(例如1小时)。然后除去溶液，并用PBS中的0.1％聚山梨酯20洗板8次。平板干燥后，加入150μl/孔闪烁液(MICROSCINT-20TM；Packard)，然后在TOPCOUNTTM伽马计数器(Packard)上对平板计数10分钟。选择各Fab给出小于或等于最大结合之20％的浓度用于竞争性结合测定法。In certain embodiments, the antibodies provided herein have a dissociation constant of ≤1 μM. In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with a Fab version of the antibody of interest and its antigen as described in the following assay. The 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 establish the conditions for the assay, multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w/v) bovine serum albumin in PBS at room temperature (about 23° C.) for 2-5 hours. In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [125 I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). 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. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 in PBS. After the plate has dried, 150 μl/well scintillation fluid (MICROSCINT-20™ ; Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). Concentrations of each Fab that give less than or equal to 20% of maximal binding are selected for use in competitive binding assays.
依照另一个实施方案，Kd是使用表面等离振子共振测定法使用或(BIAcore,Inc.,Piscataway,NJ)于25℃使用固定化抗原CM5芯片在约10个响应单位(RU)测量的。简言之，依照供应商的用法说明书用盐酸N-乙基-N’-(3-二甲氨基丙基)-碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)活化羧甲基化右旋糖苷生物传感器芯片(CM5,BIACORE,Inc.)。将抗原用10mM乙酸钠pH 4.8稀释至5μg/ml(约0.2μM)，然后以5μl/分钟的流速注射以获得约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基团。为了进行动力学测量，于25℃以约25μl/分钟的流速注入在含0.05％聚山梨酯20(TWEEN-20TM)表面活性剂的PBS(PBST)中两倍连续稀释的Fab(0.78nM至500nM)。使用简单一对一朗格缪尔(Langmuir)结合模型(Evaluation Software version 3.2)通过同时拟合结合和解离传感图计算结合速率(kon)和解离速率(koff)。平衡解离常数(Kd)以比率koff/kon计算。见例如Chen等,J.Mol.Biol.293:865-881(1999)。如果根据上文表面等离振子共振测定法，结合速率超过106M-1S-1，那么结合速率可使用荧光淬灭技术来测定，即根据分光计诸如配备了断流装置的分光光度计(AvivInstruments)或8000系列SLM-AMINCOTM分光光度计(ThermoSpectronic)中用搅拌比色杯的测量，在存在浓度渐增的抗原的情况中，测量PBS pH 7.2中20nM抗抗原抗体(Fab形式)于25℃的荧光发射强度(激发＝295nm；发射＝340nm，16nm带通)的升高或降低。According to another embodiment, Kd is measured using a surface plasmon resonance assay using a BIAcore® or a BIAcore® (BIAcore, Inc., Piscataway, NJ) at 25°C using an immobilized antigen CM5 chip at approximately 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was 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 obtain approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine was 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 (TWEEN-20™ ) surfactant (PBST) were injected at 25° C. at a flow rate of approximately 25 μl/min. Association rates (kon ) and dissociation rates (koff ) were calculated by simultaneous fitting of the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio koff /kon . See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M-1 S-1 according to the surface plasmon resonance assay above, the on-rate can be determined using a fluorescence quenching technique, i.e., measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured in a spectrometer such as a spectrophotometer equipped with a cut-off device (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.
2.抗体片段2. Antibody fragments
在某些实施方案中，本文中提供的抗体是抗体片段。抗体片段包括但不限于Fab、Fab’、Fab’-SH、F(ab’)2、Fv、和scFv片段，及下文所描述的其它片段。关于某些抗体片段的综述，见Hudson等Nat.Med.9:129-134(2003)。关于scFv片段的综述，见例如Pluckthün,于The Pharmacology of Monoclonal Antibodies,第113卷,Rosenburg和Moore编,(Springer-Verlag,New York),第269-315页(1994)；还可见WO 93/16185；及美国专利No.5,571,894和5,587,458。关于包含补救受体结合表位残基，并且具有延长的体内半衰期的Fab和F(ab’)2片段的讨论，见美国专利No.5,869,046。In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2 , Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have extended in vivo half-lives, see U.S. Pat. No. 5,869,046.
双抗体是具有两个抗原结合位点的抗体片段，其可以是二价的或双特异性的。见例如EP 404,097；WO 1993/01161；Hudson等,Nat.Med.9:129-134(2003)；及Hollinger等,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)。三抗体和四抗体也记载于Hudson等,Nat.Med.9:129-134(2003)。Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; 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).
单域抗体是包含抗体的整个或部分重链可变域或整个或部分轻链可变域的抗体片段。在某些实施方案中，单域抗体是人单域抗体(Domantis,Inc.,Waltham,MA；见例如美国专利No.6,248,516B1)。Single-domain antibodies are antibody fragments that contain 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. 6,248,516 Bl).
可以通过多种技术，包括但不限于对完整抗体的蛋白水解消化及重组宿主细胞(例如大肠杆菌或噬菌体)的生成来生成抗体片段，如本文中所描述的。Antibody fragments can be produced by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production in recombinant host cells (eg, E. coli or phage), as described herein.
3.嵌合的和人源化的抗体3. Chimeric and humanized antibodies
在某些实施方案中，本文中提供的抗体是嵌合抗体。某些嵌合抗体记载于例如美国专利No.4,816,567；及Morrison等,Proc.Natl.Acad.Sci.USA,81:6851-6855(1984))。在一个例子中，嵌合抗体包含非人可变区(例如，自小鼠、大鼠、仓鼠、家兔、或非人灵长类，诸如猴衍生的可变区)和人恒定区。在又一个例子中，嵌合抗体是“类转换的”抗体，其中类或亚类已经自亲本抗体的类或亚类改变。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, for example, in 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，将非人抗体人源化以降低对人的免疫原性，同时保留亲本非人抗体的特异性和亲和力。一般地，人源化抗体包含一个或多个可变域，其中HVR，例如CDR(或其部分)自非人抗体衍生，而FR(或其部分)自人抗体序列衍生。任选地，人源化抗体还会至少包含人恒定区的一部分。在一些实施方案中，将人源化抗体中的一些FR残基用来自非人抗体(例如衍生HVR残基的抗体)的相应残基替代，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, non-human antibodies are humanized to reduce immunogenicity to people 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) are derived from non-human antibodies, and FR (or part thereof) are derived from human antibody sequences. Optionally, humanized antibodies also include at least a portion of human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (such as antibodies derived from HVR residues), for example, to restore or improve antibody specificity or affinity.
人源化抗体及其生成方法综述于例如Almagro和Fransson,Front.Biosci.13:1619-1633(2008)，并且进一步记载于例如Riechmann等,Nature 332:323-329(1988)；Queen等,Proc.Nat’l Acad.Sci.USA 86:10029-10033(1989)；美国专利No.5,821,337,7,527,791,6,982,321和7,087,409；Kashmiri等,Methods 36:25-34(2005)(描述了SDR(a-CDR)嫁接)；Padlan,Mol.Immunol.28:489-498(1991)(描述了“重修表面”)；Dall’Acqua等,Methods 36:43-60(2005)(描述了“FR改组”)；及Osbourn等,Methods 36:61-68(2005)和Klimka等,Br.J.Cancer,83:252-260(2000)(描述了FR改组的“引导选择”方法)。Humanized antibodies and methods for their production are reviewed in, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, 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 “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” method of FR shuffling).
可以用于人源化的人框架区包括但不限于：使用“最佳拟合(best-fit)”方法选择的框架区(见例如Sims等J.Immunol.151:2296(1993))；自轻或重链可变区的特定亚组的人抗体的共有序列衍生的框架区(见例如Carter等Proc.Natl.Acad.Sci.USA,89:4285(1992)；及Presta等J.Immunol.,151:2623(1993))；人成熟的(体细胞突变的)框架区或人种系框架区(见例如Almagro和Fransson,Front.Biosci.13:1619-1633(2008))；和通过筛选FR文库衍生的框架区(见例如Baca等,J.Biol.Chem.272:10678-10684(1997)及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 "best-fit" 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)). 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 by 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)).
4.人抗体4. Human Antibodies
在某些实施方案中，本文中提供的抗体是人抗体。可以使用本领域中已知的多种技术来生成人抗体。一般地，人抗体记载于van Dijk和van de Winkel,Curr.Opin.Pharmacol.5:368-74(2001)及Lonberg,Curr.Opin.Immunol.20:450-459(2008)。In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Generally, human antibodies are 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，所述转基因动物已经修饰为响应抗原性攻击而生成完整人抗体或具有人可变区的完整抗体。此类动物通常含有所有或部分人免疫球蛋白基因座，其替换内源免疫球蛋白基因座，或者其在染色体外存在或随机整合入动物的染色体中。在此类转基因小鼠中，一般已经将内源免疫球蛋白基因座灭活。关于自转基因动物获得人抗体的方法的综述，见Lonberg,Nat.Biotech.23:1117-1125(2005)。还可见例如美国专利No.6,075,181和6,150,584，其描述了XENOMOUSETM技术；美国专利No.5,770,429，其描述了技术；美国专利No.7,041,870，其描述了K-M技术，和美国专利申请公开文本No.US 2007/0061900，其描述了技术)。可以例如通过与不同人恒定区组合进一步修饰来自由此类动物生成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 generate 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 loci, which replace the endogenous immunoglobulin loci, or they exist extrachromosomally or are randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). Also visible are, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes technology; U.S. Patent No. 7,041,870, which describes KM technology, and U.S. Patent Application Publication No. US 2007/0061900, which describes technology). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with a different human constant region.
也可以通过基于杂交瘤的方法生成人抗体。已经描述了用于生成人单克隆抗体的人骨髓瘤和小鼠-人异骨髓瘤细胞系(见例如Kozbor J.Immunol.,133:3001(1984)；Brodeur等,Monoclonal Antibody Production Techniques and Applications,第51-63页(Marcel Dekker,Inc.,New York,1987)；及Boerner等,J.Immunol.,147:86(1991))。经由人B细胞杂交瘤技术生成的人抗体也记载于Li等,Proc.Natl.Acad.Sci.USA,103:3557-3562(2006)。其它方法包括那些例如记载于美国专利No.7,189,826(其描述了自杂交瘤细胞系生成单克隆人IgM抗体)和Ni,Xiandai Mianyixue,26(4):265-268(2006)(其描述了人-人杂交瘤)的。人杂交瘤技术(Trioma技术)也记载于Vollmers和Brandlein,Histologyand Histopathology,20(3):927-937(2005)及Vollmers和Brandlein,Methods andFindings in Experimental and Clinical Pharmacology,27(3):185-91(2005)。Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for generating 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 generated via 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, for example, in U.S. Pat. No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (Trioma 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).
也可以通过分离自人衍生的噬菌体展示文库选择的Fv克隆可变域序列生成人抗体。然后，可以将此类可变域序列与期望的人恒定域组合。下文描述了自抗体文库选择人抗体的技术。It is also possible to generate human antibodies by separating the Fv clone variable domain sequence selected from the phage display library derived from humans.Then, such variable domain sequence can be combined with the desired human constant domain.The technology of selecting human antibodies from the antibody library is described below.
5.文库衍生的抗体5. Library-derived Antibodies
可以通过对组合文库筛选具有期望的一种或多种活性的抗体来分离抗体。例如，用于生成噬菌体展示文库并对此类文库筛选拥有期望结合特征的抗体的多种方法是本领域中已知的。此类方法综述于例如Hoogenboom等于Methods in Molecular Biology 178:1-37(O’Brien等编,Human Press,Totowa,NJ,2001)，并且进一步记载于例如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,于Methods in Molecular Biology248:161-175(Lo编,Human Press,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.Methods284(1-2):119-132(2004)。Antibodies can be isolated by screening combinatorial libraries for antibodies with the desired one or more activities. For example, a variety of methods for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics are known in the art. Such methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., 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 et al., eds., 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).
在某些噬菌体展示方法中，将VH和VL基因的全集分别通过聚合酶链式反应(PCR)克隆，并在噬菌体文库中随机重组，然后可以对所述噬菌体文库筛选抗原结合噬菌体，如记载于Winter等,Ann.Rev.Immunol.,12:433-455(1994)的。噬菌体通常以单链Fv(scFv)片段或以Fab片段展示抗体片段。来自经免疫的来源的文库提供针对免疫原的高亲和力抗体，而不需要构建杂交瘤。或者，可以(例如自人)克隆未免疫全集以在没有任何免疫的情况中提供针对一大批非自身和还有自身抗原的抗体的单一来源，如由Griffiths等,EMBO J,12:725-734(1993)描述的。最后，也可以通过自干细胞克隆未重排的V基因区段，并使用含有随机序列的PCR引物编码高度可变的CDR3区并在体外实现重排来合成生成未免疫文库，如由Hoogenboom和Winter,J.Mol.Biol.,227:381-388(1992)所描述的。描述人抗体噬菌体文库的专利公开文本包括例如：美国专利No.5,750,373、和美国专利公开文本No.2005/0079574,2005/0119455,2005/0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936和2009/0002360。In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be 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 as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, non-immunized repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a large number of non-self and also self-antigens in the absence of any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, unimmunized libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications 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.
认为自人抗体文库分离的抗体或抗体片段是本文中的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.
6.多特异性抗体6. Multispecific Antibodies
在某些实施方案中，本文中提供的抗体是多特异性抗体，例如双特异性抗体。多特异性抗体是对至少两个不同位点具有结合特异性的单克隆抗体。在某些实施方案中，结合特异性之一针对PD-L1，而另一种针对任何其它抗原。在某些实施方案中，双特异性抗体可以结合PD-L1的两个不同表位。也可以使用双特异性抗体来将细胞毒剂定位于表达PD-L1的细胞。双特异性抗体可以以全长抗体或抗体片段制备。In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for PD-L1, and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of PD-L1. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing PD-L1. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.
用于生成多特异性抗体的技术包括但不限于具有不同特异性的两对免疫球蛋白重链-轻链对的重组共表达(见Milstein和Cuello,Nature 305:537(1983))、WO 93/08829、和Traunecker等,EMBO J.10:3655(1991))、和“突起-入-空穴”工程化(见例如美国专利No.5,731,168)。也可以通过用于生成抗体Fc-异二聚体分子的工程化静电操纵效应(WO2009/089004A1)；交联两个或更多个抗体或片段(见例如美国专利No.4,676,980,及Brennan等,Science,229:81(1985))；使用亮氨酸拉链来生成双特异性抗体(见例如Kostelny等,J.Immunol.,148(5):1547-1553(1992))；使用用于生成双特异性抗体片段的“双抗体”技术(见例如Hollinger等,Proc.Natl.Acad.Sci.USA,90:6444-6448(1993))；及使用单链Fv(sFv)二聚体(见例如Gruber等,J.Immunol.,152:5368(1994))；及如例如Tutt等J.Immunol.147:60(1991)中所描述的，制备三特异性抗体来生成多特异性抗体。Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305:537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-into-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). It is also possible to generate antibody Fc-heterodimer molecules by engineering electrostatic manipulation effects (WO2009/089004A1); cross-linking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992 ... Multispecific antibodies can be generated by using the "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by preparing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).
本文中还包括具有三个或更多个功能性抗原结合位点的工程化改造抗体，包括“章鱼抗体”(见例如US 2006/0025576A1)。Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576A1).
本文中的抗体或片段还包括包含结合PD-L1及另一种不同抗原的抗原结合位点的“双重作用FAb”或“DAF”。The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs" that contain antigen binding sites that bind to both PD-L1 and another, different antigen.
7.抗体变体7. Antibody variants
a)糖基化变体a) Glycosylation variants
在某些实施方案中，改变本文中提供的抗体以提高或降低抗体糖基化的程度。可以通过改变氨基酸序列，使得创建或消除一个或多个糖基化位点来方便地实现对抗体的糖基化位点的添加或删除。In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.
在抗体包含Fc区的情况中，可以改变其附着的碳水化合物。由哺乳动物细胞生成的天然抗体通常包含分支的、双触角寡糖，其一般通过N连接附着于Fc区的CH2域的Asn297。见例如Wright等TIBTECH 15:26-32(1997)。寡糖可以包括各种碳水化合物，例如，甘露糖、N-乙酰葡糖胺(GlcNAc)、半乳糖、和唾液酸，以及附着于双触角寡糖结构“主干”中的GlcNAc的岩藻糖。在一些实施方案中，可以对抗体中的寡糖进行修饰以创建具有某些改善的特性的抗体变体。In the case where the antibody comprises an Fc district, the carbohydrate to which it is attached can be changed. The natural antibody produced by mammalian cells generally comprises branched, biantennary oligosaccharides, which are generally attached to the Asn297 in the CH2 domain of the Fc district by N connection. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose attached to the GlcNAc in the biantennary oligosaccharide structure " backbone ". In some embodiments, the oligosaccharides in the antibody can be modified to create antibody variants with some improved characteristics.
在一个实施方案中，提供了抗体变体，其具有缺乏附着(直接或间接)于Fc区的岩藻糖的碳水化合物结构。例如，此类抗体中的岩藻糖量可以是1％至80％、1％至65％、5％至65％或20％至40％。通过相对于附着于Asn297的所有糖结构(例如，复合的、杂合的和高甘露糖的结构)的总和，计算Asn297处糖链内岩藻糖的平均量来测定岩藻糖量，如通过MALDI-TOF质谱术测量的，例如如记载于WO 2008/077546的。Asn297指位于Fc区中的约第297位(Fc区残基的Eu编号方式)的天冬酰胺残基；然而，Asn297也可以由于抗体中的微小序列变异而位于第297位上游或下游约±3个氨基酸，即在第294位和第300位之间。此类岩藻糖基化变体可以具有改善的ADCC功能。见例如美国专利公开文本No.US 2003/0157108(Presta,L.)；US 2004/0093621(Kyowa Hakko Kogyo Co.,Ltd)。涉及“脱岩藻糖基化的”或“岩藻糖缺乏的”抗体变体的出版物的例子包括：US 2003/0157108；WO 2000/61739；WO 2001/29246；US2003/0115614；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等J.Mol.Biol.336:1239-1249(2004)；Yamane-Ohnuki等Biotech.Bioeng.87:614(2004)。能够生成脱岩藻糖基化抗体的细胞系的例子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No US 2003/0157108 A1,Presta,L；及WO 2004/056312 A1,Adams等，尤其在实施例11)，和敲除细胞系，诸如α-1,6-岩藻糖基转移酶基因FUT8敲除CHO细胞(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, antibody variants are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. 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 Asn297, as measured by MALDI-TOF mass spectrometry, for example as described in 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, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants can 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, Presta, L; and WO 2004/056312 A1, Adams et al., particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (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).
进一步提供了具有两分型寡糖的抗体变体，例如其中附着于抗体Fc区的双触角寡糖是通过GlcNAc两分的。此类抗体变体可以具有降低的岩藻糖基化和/或改善的ADCC功能。此类抗体变体的例子记载于例如WO 2003/011878(Jean-Mairet等)；美国专利No.6,602,684(Umana等)；及US 2005/0123546(Umana等)。还提供了在附着于Fc区的寡糖中具有至少一个半乳糖残基的抗体变体。此类抗体变体可以具有改善的CDC功能。此类抗体变体记载于例如WO 1997/30087(Patel等)；WO 1998/58964(Raju,S.)；及WO 1999/22764(Raju,S.)。Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). 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 in, for example, WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
b)Fc区变体b) Fc region variants
在某些实施方案中，可以将一处或多处氨基酸修饰引入本文中提供的抗体的Fc区中，由此生成Fc区变体。Fc区变体可以包含在一个或多个氨基酸位置包含氨基酸修饰(例如替代)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 be comprised of a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.
在某些实施方案中，本发明涵盖拥有一些但不是所有效应器功能的抗体变体，所述效应器功能使其成为如下应用的期望候选物，其中抗体的体内半衰期是重要的，而某些效应器功能(诸如补体和ADCC)是不必要的或有害的。可以进行体外和/或体内细胞毒性测定法以确认CDC和/或ADCC活性的降低/消减。例如，可以进行Fc受体(FcR)结合测定法以确保抗体缺乏FcγR结合(因此有可能缺乏ADCC活性)，但是保留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。在Ravetch和Kinet,Annu.Rev.Immunol.9:457-492(1991)的第464页上的表3中汇总了造血细胞上的FcR表达。评估感兴趣分子的ADCC活性的体外测定法的非限制性例子记载于美国专利No.5,500,362(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(见Bruggemann,M.等,J.Exp.Med.166:1351-1361(1987))。或者，可以采用非放射性测定方法(见例如用于流式细胞术的ACTITM非放射性细胞毒性测定法(CellTechnology,Inc.Mountain View,CA；和CytoTox非放射性细胞毒性测定法(Promega,Madison,WI))。对于此类测定法有用的效应细胞包括外周血单个核细胞(PBMC)和天然杀伤(NK)细胞。或者/另外，可以在体内评估感兴趣分子的ADCC活性，例如在动物模型中，诸如披露于Clynes等Proc.Nat’l Acad.Sci.USA 95:652-656(1998)的。也可以实施C1q结合测定法以确认抗体不能结合C1q，并且因此缺乏CDC活性。见例如WO 2006/029879和WO 2005/100402中的C1q和C3c结合ELISA。为了评估补体激活，可以实施CDC测定法(见例如Gazzano-Santoro等,J.Immunol.Methods 202:163(1996)；Cragg,M.S.等,Blood 101:1045-1052(2003)；及Cragg,M.S.和M.J.Glennie,Blood 103:2738-2743(2004))。也可以使用本领域中已知的方法来实施FcRn结合和体内清除/半衰期测定(见例如Petkova,S.B.等,Int’l.Immunol.18(12):1759-1769(2006))。In certain embodiments, the present invention encompasses antibody variants that possess some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important and certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/reduction 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 primary 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 assessing 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 Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and the CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in a 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, e.g., 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, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, 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, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).
具有降低的效应器功能的抗体包括那些具有Fc区残基238,265,269,270,297,327和329中的一个或多个的替代的(美国专利No.6,737,056)。此类Fc突变体包括在氨基酸位置265、269、270、297和327中的两处或更多处具有替代的Fc突变体，包括残基265和297替代成丙氨酸的所谓的“DANA”Fc突变体(美国专利No.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 those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine ( U.S. Patent No. 7,332,581 ).
描述了具有改善的或降低的对FcR的结合的某些抗体变体(见例如美国专利No.6,737,056；WO 2004/056312，及Shields等,J.Biol.Chem.9(2):6591-6604(2001))。在某些实施方案中，抗体变体包含具有改善ADCC的一处或多处氨基酸替代，例如Fc区的位置298、333、和/或334(残基的EU编号方式)的替代的Fc区。在一些实施方案中，对Fc区做出改变，其导致改变的(即，改善的或降低的)C1q结合和/或补体依赖性细胞毒性(CDC)，例如，如记载于美国专利No.6,194,551、WO 99/51642、及Idusogie等J.Immunol.164:4178-4184(2000)的。Certain antibody variants with improved or decreased binding to FcRs have been described (see, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 (EU numbering of residues) of the Fc region. In some embodiments, alterations are made to the Fc region that result in altered (i.e., improved or decreased) C1q binding and/or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).
具有延长的半衰期和改善的对新生儿Fc受体(FcRn)的结合的抗体记载于US2005/0014934A1(Hinton等)，新生儿Fc受体(FcRn)负责将母体IgG转移至胎儿(Guyer等,J.Immunol.117:587(1976)及Kim等,J.Immunol.24:249(1994))。那些抗体包含其中具有改善Fc区对FcRn结合的一处或多处替代的Fc区。此类Fc变体包括那些在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中的一处或多处具有替代，例如，Fc区残基434的替代的(美国专利No.7,371,826)。还可见Duncan和Winter,Nature 322:738-40(1988)；美国专利No.5,648,260；美国专利No.5,624,821；及WO 94/29351，其关注Fc区变体的其它例子。Antibodies with extended 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 with one or more substitutions that improve Fc region binding to FcRn. Such Fc variants include those having 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, for example, substitution of Fc region residue 434 ( U.S. Patent No. 7,371,826 ). See also Duncan and Winter, Nature 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 for other examples of Fc region variants.
c)经半胱氨酸工程化改造的抗体变体c) Cysteine-engineered antibody variants
在某些实施方案中，可以期望创建经半胱氨酸工程化改造的抗体，例如，“thioMAb”，其中抗体的一个或多个残基用半胱氨酸残基替代。在具体的实施方案中，替代的残基存在于抗体的可接近位点。通过用半胱氨酸替代那些残基，反应性硫醇基团由此定位于抗体的可接近位点，并且可以用于将抗体与其它模块，诸如药物模块或接头-药物模块缀合，以创建免疫缀合物，如本文中进一步描述的。在某些实施方案中，可以用半胱氨酸替代下列残基之任一个或多个：轻链的V205(Kabat编号方式)；重链的A118(EU编号方式)；和重链Fc区的S400(EU编号方式)。可以如例如美国专利No.7,521,541所述生成经半胱氨酸工程化改造的抗体。In certain embodiments, it may be desirable to create an antibody engineered with cysteine, e.g., a "thioMAb," wherein one or more residues of an antibody are replaced with cysteine residues. In a specific embodiment, the substituted residues are present at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thus located at accessible sites of the antibody and can be used to conjugate the antibody to other modules, such as drug modules or linker-drug modules, to create immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine-engineered antibodies may be generated as described in, for example, U.S. Patent No. 7,521,541.
d)免疫缀合物d) Immunoconjugates
本发明还提供包含本文中抗PD-L1抗体的免疫缀合物，该抗体与一种或多种细胞毒剂诸如化疗剂或药物、生长抑制剂、毒素(例如蛋白质毒素、细菌、真菌、植物、或动物起源的酶活性毒素、或其片段)、或放射性同位素缀合。The invention also provides immunoconjugates comprising an anti-PD-L1 antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
在一个实施方案中，免疫缀合物是抗体-药物缀合物(ADC)，其中抗体与一种或多种药物缀合，包括但不限于美登木生物碱(maytansinoid)(参见美国专利No.5,208,020、No.5,416,064和欧洲专利EP 0 425 235 B1)；auristatin诸如单甲基auristatin药物模块DE和DF(MMAE和MMAF)(参见美国专利No.5,635,483和No.5,780,588，及No.7,498,298)；多拉司他汀(dolastatin)；加利车霉素(calicheamicin)或其衍生物(参见美国专利No.5,712,374、No.5,714,586、No.5,739,116、No.5,767,285、No.5,770,701、No.5,770,710、No.5,773,001、和No.5,877,296；Hinman等人，Cancer Res.53:3336-3342(1993)；及Lode等人，Cancer Res.58:2925-2928(1998))；蒽环类抗生素诸如道诺霉素(daunomycin)或多柔比星(doxorubicin)(参见Kratz等人，Current Med.Chem.13:477-523(2006)；Jeffrey等人，Bioorganic&Med.Chem.Letters 16:358-362(2006)；Torgov等人，Bioconj.Chem.16:717-721(2005)；Nagy等人，Proc.Natl.Acad.Sci.USA 97:829-834(2000)；Dubowchik等人，Bioorg.&Med.Chem.Letters 12:1529-1532(2002)；King等人，J.Med.Chem.45:4336-4343(2002)；及美国专利No.6,630,579)；甲氨蝶呤；长春地辛(vindesine)；紫杉烷诸如多西他赛(docetaxel)、帕利他赛(paclitaxel)、larotaxel、tesetaxel、和ortataxel；单端孢菌素(trichothecene)；和CC1065。In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235). B1); auristatin such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.
在另一个实施方案中，免疫缀合物包含本文所述抗体，该抗体与酶活性毒素或其片段缀合，包括但不限于白喉毒素A链、白喉毒素的非结合活性片段、外毒素A链(来自铜绿假单胞菌Pseudomonas aeruginosa)、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、蒴莲根毒蛋白(modeccin)A链、α-帚曲霉素(sarcin)、油桐(Aleurites fordii)毒蛋白、香石竹(dianthin)毒蛋白、美洲商陆(Phytolaca americana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)、局限曲菌素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)和单端孢菌素(tricothecenes)。In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria toxin A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis toxin, scutellaria baicalensis toxin, scutellaria serrata ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
在另一个实施方案中，免疫缀合物包含本文所述抗体，该抗体与放射性原子缀合以形成放射缀合物。多种放射性同位素可用于生成放射缀合物。实例包括At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32、Pb212和Lu的放射性同位素。在将放射缀合物用于检测时，它可包含放射性原子用于闪烁照相研究，例如tc99或I123，或自旋标记物用于核磁共振(NMR)成像(也称为磁共振成像，mri)，诸如碘-123、碘-131、铟-111、氟-19、碳-13、氮-15、氧-17、钆、锰或铁。In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to generate radioconjugates. Examples include At211 , I131 , I125 , Y90 , Re186 , Re188 , Sm153 , Bi212 , P32 , Pb212 and radioisotopes of Lu. When the radioconjugate is used for detection, it can contain radioactive atoms for scintigraphic studies, such as tc99 or I123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
可使用多种双功能蛋白质偶联剂来制备抗体和细胞毒剂的缀合物，诸如N-琥珀酰亚氨基-3-(2-吡啶基二硫代)丙酸酯(SPDP)、琥珀酰亚氨基-4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯(SMCC)、亚氨基硫烷(IT)、亚氨酸酯(诸如盐酸己二酰亚氨酸二甲酯)、活性酯类(诸如辛二酸二琥珀酰亚氨基酯)、醛类(诸如戊二醛)、双叠氮化合物(诸如双(对-叠氮苯甲酰基)己二胺)、双重氮衍生物(诸如双(对-重氮苯甲酰基)乙二胺)、二异氰酸酯(诸如甲苯2,6-二异氰酸酯)、和双活性氟化合物(诸如1,5-二氟-2,4-二硝基苯)的双功能衍生物。例如，可如Vitetta等,Science 238:1098(1987)中所述制备蓖麻毒蛋白免疫毒素。碳-14标记的1-异硫氰酸苯甲基-3-甲基二亚乙基三胺五乙酸(MX-DTPA)是用于将放射性核苷酸与抗体缀合的例示性螯合剂。参见WO 94/11026。接头可以是便于在细胞中释放细胞毒药物的“可切割接头”。例如，可使用酸不稳定接头、肽酶敏感接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Res.52:127-131(1992)；美国专利No.5,208,020)。Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94/11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020).
本文中的免疫缀合物或ADC明确涵盖但不限于用下列交联剂制备的此类缀合物，包括但不限于：商品化(如购自Pierce Biotechnology Inc.,Rockford,IL,U.S.A)的BMPS、EMCS、GMBS、HBVS、LC-SMCC、MBS、MPBH、SBAP、SIA、SIAB、SMCC、SMPB、SMPH、sulfo-EMCS、sulfo-GMBS、sulfo-KMUS、sulfo-MBS、sulfo-SIAB、sulfo-SMCC、和sulfo-SMPB、和SVSB(琥珀酰亚胺基-(4-乙烯基砜)苯甲酸酯)。The immunoconjugates or ADCs herein specifically encompass, but are not limited to, conjugates prepared with the following cross-linkers, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology Inc., Rockford, IL, U.S.A.).
C.结合多肽C. Binding peptide
结合多肽是如下的多肽，其结合，优选特异性结合如本文所述的PD-L1。在一些实施方案中，结合多肽为PD-L1轴结合拮抗剂。结合多肽可以使用已知的多肽合成方法学化学合成，或者可使用重组技术制备和纯化。结合多肽的长度通常是至少约5个氨基酸，或者长度为至少约6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100个氨基酸或更长，其中此类结合多肽能够结合，优选特异性结合本文所述的靶物，即PD-L1。结合多肽无需过多试验就可使用公知技术来鉴定。在这点上，注意到用于对多肽文库筛选能够特异性结合多肽靶物的结合多肽的技术是本领域公知的(参见例如美国专利5,556,762,5,750,373,4,708,871,4,833,092,5,223,409,5,403,484,5,571,689,5,663,143；PCT公开号WO84/03506和WO 84/03564；Geysen等,Proc.Natl.Acad.Sci.U.S.A.81:3998-4002(1984)；Geysen等,Proc.Natl.Acad.Sci.U.S.A.82:178-182(1985)；Geysen等,in SyntheticPeptides as Antigens,130-149(1986)；Geysen等,J.Immunol.Meth.102:259-274(1987)；Schoofs等,J.Immunol.140:611-616(1988)；Cwirla,S.E.等,(1990)Proc.Natl.Acad.Sci.USA 87:6378；Lowman,H.B.等,(1991)Biochemistry 30:10832；Clackson,T.等,(1991)Nature 352:624；Marks,J.D.等,(1991),J.Mol.Biol.222:581；Kang,A.S.等,(1991)Proc.Natl.Acad.Sci.USA 88:8363；Smith,G.P.,(1991)CurrentOpin.Biotechnol.2:668)。Binding polypeptides are polypeptides that bind, preferably specifically bind, to PD-L1 as described herein. In some embodiments, the binding polypeptide is a PD-L1 axis binding antagonist. The binding polypeptide can be chemically synthesized using known polypeptide synthesis methodologies, or can be prepared and purified using recombinant technology. The binding polypeptide is typically at least about 5 amino acids in length, or at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 , 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 amino acids or longer, wherein such binding polypeptides are capable of binding, preferably specifically binding, to a target as described herein, i.e., PD-L1. Binding polypeptides can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for screening polypeptide libraries for binding polypeptides capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Patents 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO 84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A. 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A. 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth. 102: 259-274 (1987); Schoofs et al., J. Immunol. 140: 611-616 (1988); Cwirla, S. E. et al., (1990) Proc. Natl. Acad. Sci. USA 87:6378; Lowman, H.B. et al., (1991) Biochemistry 30:10832; Clackson, T. et al., (1991) Nature 352:624; Marks, J.D. et al., (1991), J.Mol.Biol.222:581; Kang, A.S. et al., (1991) Proc.Natl.Acad.Sci.USA 88:8363; Smith, G.P., (1991) Current Opin. Biotechnol. 2:668).
在这点上，噬菌体展示是一种可以筛选大型多肽文库来鉴定那些文库中能够特异性结合靶多肽，即PD-L1的成员的公知技术。噬菌体展示是将变体多肽作为与外壳蛋白的融合蛋白展示在噬菌体颗粒表面的一种技术(Scott,J.K.and Smith,G.P.(1990)Science249:386)。噬菌体展示的效用在于，可以对选择性随机化蛋白质变体(或随机克隆cDNA)的大型文库迅速且有效地分选那些以高亲和力结合靶分子的序列的事实。在噬菌体上展示肽(Cwirla,S.E.等,(1990)Proc.Natl.Acad.Sci.USA 87:6378)或蛋白质(Lowman,H.B.等,(1991)Biochemistry 30:10832；Clackson,T.等,(1991)Nature 352:624；Marks,J.D.等,(1991)J.MoI.Biol.222:581；Kang,A.S.等,(1991)Proc.Natl.Acad.Sci.USA 88:8363)文库已经用于对数百万多肽或寡肽筛选具有特异结合特性的那些多肽或寡肽(Smith,G.P.(1991)Current Opin.Biotechnol.2:668)。分选随机突变体的噬菌体文库需要构建和扩充大量变体的策略，使用靶受体进行亲和纯化的流程，及评估结合富集的结果的手段。参见美国专利5,223,409,5,403,484,5,571,689和5,663,143。In this regard, phage display is a well-known technology that can screen large polypeptide libraries to identify those members that can specifically bind to the target polypeptide, i.e., PD-L1. Phage display is a technology that displays variant polypeptides as fusion proteins with coat proteins on the surface of phage particles (Scott, J.K. and Smith, G.P. (1990) Science 249:386). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be quickly and efficiently sorted for sequences that bind to the target molecule with high affinity. Display of peptide (Cwirla, S.E. et al., (1990) Proc. Natl. Acad. Sci. USA 87:6378) or protein (Lowman, H.B. et al., (1991) Biochemistry 30:10832; Clackson, T. et al., (1991) Nature 352:624; Marks, J.D. et al., (1991) J. Mol. Biol. 222:581; Kang, A.S. et al., (1991) Proc. Natl. Acad. Sci. USA 88:8363) libraries on phage has been used to screen millions of polypeptides or oligopeptides for those with specific binding properties (Smith, G.P. (1991) Current Opin. Biotechnol. 2:668). Sorting phage libraries of random mutants requires strategies for constructing and expanding large numbers of variants, protocols for affinity purification using target receptors, and means for evaluating the results of binding enrichment. See U.S. Patents 5,223,409, 5,403,484, 5,571,689, and 5,663,143.
尽管大多数噬菌体展示方法已经使用丝状噬菌体，λ类(lambdoid)噬菌体展示系统(WO 95/34683；US 5,627,024)、T4噬菌体展示系统(Ren等,Gene 215:439(1998)；Zhu等,Cancer Research 58(15):3209-3214(1998)；Jiang等,Infection&Immunity 65(11):4770-4777(1997)；Ren等,Gene 195(2):303-311(1997)；Ren,Protein Sci.5:1833(1996)；Efimov等,Virus Genes 10:173(1995))和T7噬菌体展示系统(Smith and Scott,Methodsin Enzymology 217:228-257(1993)；US 5,766,905)也是已知的。Although most phage display methods have used filamentous phage, the lambdoid phage display system (WO 95/34683; US 5,627,024), the T4 phage display system (Ren et al., Gene 215:439 (1998); Zhu et al., Cancer Research 58(15):3209-3214 (1998); Jiang et al., Infection & Immunity 65(11):4770-4777 (1997); Ren et al., Gene 195(2):303-311 (1997); Ren, Protein Sci. 5:1833 (1996); Efimov et al., Virus Genes 10:173 (1995)) and the T7 phage display system (Smith and Scott, Methods in Enzymology 217:228-257 (1993); US 5,766,905) are also known.
别的改进增强了展示系统对肽文库筛选与选定靶分子的结合及展示功能性蛋白质的能力，所述功能性蛋白质具有对这些蛋白质筛选期望特性的潜力。已经开发了用于噬菌体展示反应的组合反应装置(WO 98/14277)，而且噬菌体展示文库已经用于分析和控制生物分子相互作用(WO 98/20169；WO 98/20159)和受约束(constrained)螺旋肽的特性(WO98/20036)。WO 97/35196描述了分离亲和配体的方法，其中使噬菌体展示文库接触第一种溶液和第二种溶液以选择性分离结合的配体，在第一种溶液中配体将结合靶分子，而在第二种溶液中亲和配体将不会结合靶分子。WO 97/46251描述了这样一种方法，即用亲和纯化的抗体生物淘选随机噬菌体展示库，然后分离结合的噬菌体，随后使用微量板的孔进行淘选过程以分离高亲和力结合的噬菌体。已经报道了金黄色葡萄球菌(Staphylococcusaureus)蛋白A作为亲和标签的使用(Li等,(1998)Mol.Biotech.9:187)。WO 97/47314描述了底物扣除文库用于区别酶特异性的用途，其中使用可以是噬菌体展示库的组合文库。WO97/09446描述了使用噬菌体展示选择适用于洗涤剂的酶的方法。美国专利5,498,538,5,432,018和WO 98/15833中描述了选择特异性结合的蛋白质的其它方法。Other improvements enhance the ability of display systems to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential to screen these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO 98/14277), and phage display libraries have been used to analyze and control biomolecular interactions (WO 98/20169; WO 98/20159) and properties of constrained helical peptides (WO 98/20036). WO 97/35196 describes a method for separating affinity ligands, wherein a phage display library is contacted with a first solution and a second solution to selectively separate the bound ligand, in which the ligand will bind to the target molecule, while in the second solution the affinity ligand will not bind to the target molecule. WO 97/46251 describes such a method, i.e., using affinity-purified antibody biopanning random phage display libraries, then separating the bound phage, and subsequently using the wells of a microplate to perform a panning process to separate the phage bound by high affinity. The use of Staphylococcus aureus protein A as an affinity tag has been reported (Li et al., (1998) Mol.Biotech.9:187). WO 97/47314 describes the use of substrate subtraction libraries for distinguishing enzyme specificity, wherein the use may be a combinatorial library of phage display libraries. WO 97/09446 describes a method for selecting enzymes suitable for detergents using phage display. Other methods for selecting specifically bound proteins are described in U.S. Patents 5,498,538,5,432,018 and WO 98/15833.
产生肽文库和筛选这些文库的方法还公开于美国专利5,723,286,5,432,018,5,580,717,5,427,908,5,498,530,5,770,434,5,734,018,5,698,426,5,763,192,和5,723,323。Methods for generating peptide libraries and screening these libraries are also disclosed in U.S. Patents 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and 5,723,323.
D.结合小分子D. Binding small molecules
本文中提供了作为PD-L1小分子拮抗剂使用的结合小分子。Provided herein are binding small molecules for use as small molecule antagonists of PD-L1.
优选地，结合小分子指本文所定义的结合多肽或抗体以外，结合、优选特异性结合本文所述PD-L1的有机分子。结合有机小分子可以使用已知方法学来鉴定和化学合成(参见例如PCT公开号WO 00/00823和WO 00/39585)。结合有机小分子的大小通常小于约2000道尔顿，或者其大小小于约1500、750、500、250或200道尔顿，其中此类能够结合、优选特异性结合本文所述多肽的有机小分子无需过多实验就可使用公知技术来鉴定。在这点上，注意到用于对有机小分子文库筛选能够结合多肽靶物的分子的技术是本领域公知的(参见例如PCT公开号WO 00/00823和WO 00/39585)。结合有机小分子可以是例如醛、酮、肟、腙、缩氨基脲(semicarbazone)、卡巴肼(carbazide)、伯胺、仲胺、叔胺、N-取代的肼、酰肼、醇、醚、硫醇、硫醚、二硫化物、羧酸、酯、酰胺、脲、氨基甲酸酯(carbamate)、碳酸酯(carbonate)、缩酮、硫缩酮(thioketal)、缩醛、硫缩醛、芳基卤、芳基磺酸酯(aryl sulfonate)、烃基卤、烃基磺酸酯(alkyl sulfonate)、芳香族化合物、杂环化合物、苯胺、烯烃、炔烃、二醇、氨基醇、口恶唑烷、口恶唑啉、噻唑烷、噻唑啉、烯胺、磺酰胺(sulfonamide)、环氧化物、吖丙啶(aziridine)、异氰酸酯(isocyanate)、磺酰氯、重氮化合物、酰基氯(acid chloride)等。Preferably, the binding small molecule refers to an organic molecule that binds to, preferably specifically binds to, the PD-L1 described herein, in addition to the binding polypeptide or antibody defined herein. Binding organic small molecules can be identified and chemically synthesized using known methodologies (see, for example, PCT Publication Nos. WO 00/00823 and WO 00/39585). The size of the binding organic small molecule is generally less than about 2000 Daltons, or its size is less than about 1500, 750, 500, 250 or 200 Daltons, wherein such organic small molecules that can bind, preferably specifically bind to, the polypeptide described herein can be identified using known techniques without too much experimentation. In this regard, it is noted that the techniques for screening organic small molecule libraries for molecules that can bind to polypeptide targets are well known in the art (see, for example, PCT Publication Nos. WO 00/00823 and WO 00/39585). The bound organic small molecule can be, for example, an aldehyde, a ketone, an oxime, a hydrazone, a semicarbazone, a carbazide, a primary amine, a secondary amine, a tertiary amine, an N-substituted hydrazine, a hydrazide, an alcohol, an ether, a thiol, a thioether, a disulfide, a carboxylic acid, an ester, an amide, a urea, a carbamate, a carbonate, a ketal, a thioketal, an acetal, a thioacetal, an aryl halide, an aryl sulfonate, an alkyl halide, an alkyl sulfonate, an aromatic compound, a heterocyclic compound, an aniline, an alkene, an alkyne, a diol, an amino alcohol, an oxazolidine, an oxazoline, a thiazolidine, a thiazoline, an enamine, a sulfonamide, an epoxide, an aziridine, an isocyanate, a sulfonyl chloride, a diazo compound, an acid chloride, and the like.
E.拮抗剂多核苷酸E. Antagonist Polynucleotides
本文中提供了多核苷酸拮抗剂。多核苷酸可以是反义核酸和/或核酶。反义核酸包含至少与PD-L1基因的RNA转录物的一部分互补的序列。然而，尽管优选绝对互补性，但是其不是必要的。Provided herein are polynucleotide antagonists. The polynucleotides may be antisense nucleic acids and/or ribozymes. The antisense nucleic acids comprise a sequence that is complementary to at least a portion of the RNA transcript of the PD-L1 gene. However, although absolute complementarity is preferred, it is not essential.
本文中提及的“至少与RNA的一部分互补的”序列意指具有足够的互补性以能够与RNA杂交，形成稳定的双链体的序列；在双链PD-L1反义核酸的情况中，如此可以测试双链体DNA的单链，或者可以测定三链体形成。杂交能力会取决于互补性程度和反义核酸的长度两者。一般地，杂交的核酸越大，与PD-L1RNA的碱基错配越多，它可以含有且仍形成稳定的双链体(或三链体，情况也可以如此)。本领域技术人员可以通过使用标准规程测定杂交复合物的熔点来确认可容许的错配程度。As used herein, a sequence that is "complementary to at least a portion of an RNA" means a sequence that has sufficient complementarity to be able to hybridize with the RNA to form a stable duplex; in the case of a double-stranded PD-L1 antisense nucleic acid, this can be done by testing a single strand of the duplex DNA, or by determining triplex formation. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with the PD-L1 RNA it can contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can determine the degree of mismatch tolerance by determining the melting point of the hybrid complex using standard procedures.
与信息5’端(例如5’非翻译序列直至AUG起始密码子且包括AUG起始密码子)互补的多核苷酸在抑制翻译方面应当最有效起作用。然而，已经显示了与mRNA的3’非翻译序列互补的序列也有效抑制mRNA的翻译。一般见Wagner,R.,1994,Nature 372:333-335。如此，与PD-L1基因的5’-或3’-非翻译、非编码区互补的寡核苷酸可以在反义方法中使用以抑制内源PD-L1mRNA的翻译。与mRNA的5’非翻译区互补的多核苷酸应当包括AUG起始密码子的互补物。与mRNA编码区互补的反义多核苷酸是不太有效的翻译抑制剂，但是可以依照本发明使用。不论设计为与PD-L1mRNA的5’、3’或编码区杂交，反义核酸的长度应当是至少6个核苷酸，并且优选是长度范围为6至约50个核苷酸的寡核苷酸。在具体的实施方案中，寡核苷酸是至少10个核苷酸、至少17个核苷酸、至少25个核苷酸或至少50个核苷酸。Polynucleotides complementary to the 5' end of the message (e.g., the 5' untranslated sequence up to and including the AUG start codon) should work most effectively in inhibiting translation. However, sequences complementary to the 3' untranslated sequence of the mRNA have been shown to also effectively inhibit the translation of the mRNA. See generally Wagner, R., 1994, Nature 372: 333-335. Thus, oligonucleotides complementary to the 5'- or 3'-untranslated, non-coding regions of the PD-L1 gene can be used in antisense methods to inhibit the translation of endogenous PD-L1 mRNA. Polynucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon. Antisense polynucleotides complementary to the mRNA coding region are less effective translation inhibitors, but can be used in accordance with the present invention. Regardless of whether they are designed to hybridize to the 5', 3' or coding region of the PD-L1 mRNA, the antisense nucleic acid should be at least 6 nucleotides in length, and preferably are oligonucleotides ranging in length from 6 to about 50 nucleotides. In specific embodiments, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.
在一个实施方案中，PD-L1反义核酸通过自外源序列转录在细胞内生成。例如，转录载体或其部分，生成PD-L1基因的反义核酸(RNA)。此类载体会含有编码PD-L1反义核酸的序列。此类载体可以仍然是附加体的或者变为染色体整合的，只要它可以被转录以生成期望的反义RNA。可以通过本领域中标准的重组DNA技术方法构建此类载体。载体可以是用于在脊椎动物细胞中复制和表达的质粒、病毒、或本领域中已知的其它载体。可以通过本领域中已知在脊椎动物(优选人细胞)中起作用的任何启动子表达编码PD-L1或其片段的序列。此类启动子可以是诱导型或组成性的。此类启动子包括但不限于SV40早期启动子区(Bernoist and Chambon,Nature 29:304-310(1981)、劳氏肉瘤病毒的3’长末端重复中含有的启动子(Yamamoto等,Cell 22:787-797(1980)、疱疹胸苷启动子(Wagner等,Proc.Natl.Acad.Sci.U.S.A.78:1441-1445(1981)、金属硫蛋白基因的调节序列(Brinster等,Nature 296:39-42(1982))，等等。In one embodiment, the PD-L1 antisense nucleic acid is generated in the cell by transcription from an exogenous sequence. For example, a vector or a portion thereof is transcribed to generate an antisense nucleic acid (RNA) of the PD-L1 gene. Such a vector will contain a sequence encoding the PD-L1 antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to generate the desired antisense RNA. Such vectors can be constructed by standard recombinant DNA technology methods in the art. The vector can be a plasmid, virus, or other vector known in the art for replication and expression in vertebrate cells. The sequence encoding PD-L1 or its fragment can be expressed by any promoter known in the art to function in vertebrates (preferably human cells). Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatory sequence of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)), and the like.
F.抗体和结合多肽变体F. Antibody and Binding Polypeptide Variants
在某些实施方案中，涵盖本文中提供的抗体和/或结合多肽的氨基酸序列变体。例如，可以期望改善抗体和/或结合多肽的结合亲和力和/或其它生物学特性。可以通过将合适的修饰引入编码抗体和/或结合多肽的核苷酸序列中，或者通过肽合成来制备抗体和/或结合多肽的氨基酸序列变体。此类修饰包括例如对抗体和/或结合多肽的氨基酸序列内的残基的删除、和/或插入和/或替代。可以进行删除、插入、和替代的任何组合以得到最终的构建体，只要最终的构建体拥有期望的特征，例如，靶物结合。In certain embodiments, the amino acid sequence variants of the antibodies and/or binding polypeptides provided herein are encompassed. For example, it is desirable to improve the binding affinity and/or other biological properties of the antibodies and/or binding polypeptides. The amino acid sequence variants of the antibodies and/or binding polypeptides can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibodies and/or binding polypeptides, or by peptide synthesis. Such modifications include, for example, deletion and/or insertion and/or substitution of residues in the amino acid sequence of the antibodies and/or binding polypeptides. Any combination of deletion, insertion, and substitution can be performed to obtain the final construct, as long as the final construct has the desired characteristics, for example, the target binds.
在某些实施方案中，提供了具有一处或多处氨基酸替代的抗体变体和/或结合多肽变体。替代诱变感兴趣的位点包括HVR和FR。保守替代在表1中在“保守替代”的标题下显示。更实质的变化在表1中在“例示性替代”的标题下提供，并且如下文参照氨基酸侧链类别进一步描述。可以将氨基酸替代引入感兴趣的抗体和/或结合多肽中，并且对产物筛选期望的活性，例如保留/改善的抗原结合、降低的免疫原性、或改善的ADCC或CDC。In certain embodiments, antibody variants and/or binding polypeptide variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Conservative Substitutions." More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions," and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody and/or binding polypeptide of interest, and the product screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
表1Table 1
依照共同的侧链特性，氨基酸可以如下分组：According to common side chain properties, amino acids can be grouped as follows:
(1)疏水性的：正亮氨酸,Met,Ala,Val,Leu,Ile；(1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
(2)中性、亲水性的：Cys,Ser,Thr,Asn,Gln；(2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3)酸性的：Asp,Glu；(3) Acidic: Asp, Glu;
(4)碱性的：His,Lys,Arg；(4) Basic: His, Lys, Arg;
(5)影响链取向的残基：Gly,Pro；(5) Residues that affect chain orientation: Gly, Pro;
(6)芳香族的：Trp,Tyr,Phe。(6) Aromatic: Trp, Tyr, Phe.
非保守替代会需要用这些类别之一的成员替换另一个类别的。Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.
一类替代变体牵涉替代亲本抗体(例如人源化或人抗体)的一个或多个高变区残基。一般地，为进一步研究选择的所得变体相对于亲本抗体会具有某些生物学特性的改变(例如改善)(例如升高的亲和力、降低的免疫原性)和/或会基本上保留亲本抗体的某些生物学特性。例示性的替代变体是亲和力成熟的抗体，其可以例如使用基于噬菌体展示的亲和力成熟技术诸如本文中所描述的那些技术来方便地生成。简言之，将一个或多个HVR残基突变，并将变体抗体在噬菌体上展示，并对其筛选特定的生物学活性(例如结合亲和力)。A class of substitution variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have changes (e.g., improvements) in certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and/or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display, such as those described herein. In short, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).
可以对HVR做出变化(例如，替代)，例如以改善抗体亲和力。可以对HVR“热点”，即由在体细胞成熟过程期间以高频率经历突变的密码子编码的残基(见例如Chowdhury,Methods Mol.Biol.207:179-196(2008))，和/或SDR(a-CDR)做出此类变化，其中对所得的变体VH或VL测试结合亲和力。通过次级文库的构建和再选择进行的亲和力成熟已经记载于例如Hoogenboom等于Methods in Molecular Biology 178:1-37(O’Brien等编,HumanPress,Totowa,NJ,(2001))。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、或寡核苷酸指导的诱变)中的任何方法将多样性引入为成熟选择的可变基因。然后，创建次级文库。然后，筛选文库以鉴定具有期望的亲和力的任何抗体变体。另一种引入多样性的方法牵涉HVR指导的方法，其中将几个HVR残基(例如，一次4-6个残基)随机化。可以例如使用丙氨酸扫描诱变或建模来特异性鉴定牵涉抗原结合的HVR残基。特别地，经常靶向CDR-H3和CDR-L3。HVR can be changed (e.g., replaced), for example to improve antibody affinity. Such changes can be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutations at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol.Biol.207: 179-196 (2008)), and/or SDR (a-CDR), where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and reselection of secondary libraries has been described in, for example, Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., 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-guided mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves an HVR-guided approach 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. In particular, CDR-H3 and CDR-L3 are often targeted.
在某些实施方案中，可以在一个或多个HVR内发生替代、插入、或删除，只要此类变化不实质性降低抗体结合抗原的能力。例如，可以对HVR做出保守变化(例如，保守替代，如本文中提供的)，其不实质性降低结合亲和力。此类变化可以在HVR“热点”或SDR外部。在上文提供的变体VH和VL序列的某些实施方案中，每个HVR或是未改变的，或是含有不超过1、2或3处氨基酸替代。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 antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. 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 either unchanged or contains no more than 1, 2, or 3 amino acid substitutions.
一种可用于鉴定抗体和/或结合多肽中可以作为诱变靶位的残基或区域的方法称作“丙氨酸扫描诱变”，如由Cunningham和Wells(1989)Science,244:1081-1085所描述的。在此方法中，将残基或靶残基的组(例如，带电荷的残基诸如arg、asp、his、lys、和glu)鉴定，并用中性或带负电荷的氨基酸(例如，丙氨酸或多丙氨酸)替换以测定抗体与抗原的相互作用是否受到影响。可以在对初始替代表明功能敏感性的氨基酸位置引入进一步的替代。或者/另外，利用抗原-抗体复合物的晶体结构来鉴定抗体与抗原间的接触点。作为替代的候选，可以靶向或消除此类接触残基和邻近残基。可以筛选变体以确定它们是否含有期望的特性。A method for identifying residues or regions that can be used as mutagenesis targets in antibodies and/or binding polypeptides is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, groups of residues or target residues (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 between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitutions. Alternatively or in addition, the crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and the antigen. As candidates for substitution, such contact residues and adjacent residues can be targeted or eliminated. Variants can be screened to determine whether they contain desired properties.
氨基酸序列插入包括长度范围为1个残基至含有100或更多个残基的多肽的氨基和/或羧基端融合，及单个或多个氨基酸残基的序列内插入。末端插入的例子包括具有N端甲硫氨酰基残基的抗体。抗体分子的其它插入变体包括抗体的N或C端与酶(例如对于ADEPT)或延长抗体的血清半衰期的多肽的融合物。Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from 1 residue to polypeptides containing 100 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 the antibody molecule include fusions of the N- or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.
G.抗体和结合多肽衍生物G. Antibodies and Binding Polypeptide Derivatives
在某些实施方案中，可进一步修饰本文中提供的抗体和/或结合多肽以包含本领域中已知的且容易获得的别的非蛋白质性质模块。适合于抗体和/或结合多肽衍生化的模块包括但不限于水溶性聚合物。水溶性聚合物的非限制性例子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、右旋糖苷(dextran)、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二氧杂环戊烷、聚-1,3,6-三氧杂环戊烷、乙烯/马来酸酐共聚物、聚氨基酸(或是同聚物或是随机共聚物)，右旋糖苷或聚(n-乙烯吡咯烷酮)聚乙二醇、聚丙二醇(propropylene glycol)同聚物、聚环氧丙烷(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。聚乙二醇丙醛由于其在水中的稳定性而有利于制备。聚合物可以是任何分子量的，且可以是分支的或不分支的。附着于抗体和/或结合多肽的聚合物数目是变化的，且若附着超过一个聚合物，则它们可以是相同的或不同的分子。通常，可基于以下考虑确定用于衍生化的聚合物的数目和/或类型，所述考虑包括但不限于，待改进之抗体和/或结合多肽的具体特性或功能，抗体衍生物和/或结合多肽衍生物是否会在限定条件下用于治疗，等等。In certain embodiments, the antibodies and/or binding polypeptides provided herein can be further modified to include other non-proteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of antibodies and/or binding polypeptides include, but are not limited to, water-soluble polymers. 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-trioxolane, ethylene/maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde is advantageous for preparation due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody and/or binding polypeptide can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody and/or binding polypeptide to be improved, whether the antibody derivative and/or binding polypeptide derivative will be used therapeutically under defined conditions, etc.
在另一个实施方案中，提供了抗体和/或结合多肽与可通过暴露于辐射而选择性加热的非蛋白质性质模块的缀合物。在一个实施方案中，非蛋白质性质模块是碳纳米管(Kam等，Proc.Natl.Acad.Sci.USA 102:11600-11605(2005))。辐射可以是任何波长的，且包括但不限于不会伤害普通细胞但将非蛋白质性质模块加热至邻近抗体和/或结合多肽-非蛋白质性质模块的细胞被杀死的温度的波长。In another embodiment, a conjugate of an antibody and/or binding polypeptide with a non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-proteinaceous moiety to a temperature at which cells adjacent to the antibody and/or binding polypeptide-non-proteinaceous moiety are killed.
在一些实施方案中，样品为组织样品。在一些实施方案中，样品为肿瘤组织样品。在一些实施方案中，肿瘤组织样品包含肿瘤细胞，肿瘤浸润性免疫细胞，肿瘤内免疫细胞，肿瘤周围免疫细胞或其任意组合，肿瘤基质细胞(例如成纤维细胞)。在一些实施方案中，样品是患者的癌的。在一些实施方案中，样品是在PD-L1轴结合拮抗剂治疗之前获得的。在一些实施方案中，样品是福尔马林固定和石蜡包埋的。In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a tumor tissue sample. In some embodiments, the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, intratumoral immune cells, peritumoral immune cells, or any combination thereof, tumor stromal cells (e.g., fibroblasts). In some embodiments, the sample is a patient's cancer. In some embodiments, the sample is obtained prior to treatment with a PD-L1 axis binding antagonist. In some embodiments, the sample is formalin-fixed and paraffin-embedded.
在一些实施方案中，疾病或病症为增殖性疾病或病症。在一些实施方案中，疾病或病症为免疫相关疾病或病症。在一些实施方案中，疾病或病症为癌症。在一些实施方案中，癌症为非小细胞肺癌，肾细胞癌，卵巢癌，胰腺癌，胃癌，膀胱癌，食道癌，间皮瘤，黑素瘤，乳腺癌，甲状腺癌，结肠直肠癌，头和颈癌，骨肉瘤，前列腺癌，或成胶质细胞瘤。在一些实施方案中，癌症为非小细胞肺癌(NSCLC)。在一些实施方案中，NSCLC为二线或三线局部晚期或转移性NSCLC。在一些实施方案中，NSCLC为腺癌。在一些实施方案中，NSCLC为鳞状细胞癌。In some embodiments, the disease or condition is a proliferative disease or condition. In some embodiments, the disease or condition is an immune-related disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is non-small cell lung cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, breast cancer, thyroid cancer, colorectal cancer, head and neck cancer, osteosarcoma, prostate cancer, or glioblastoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, NSCLC is second-line or third-line locally advanced or metastatic NSCLC. In some embodiments, NSCLC is adenocarcinoma. In some embodiments, NSCLC is squamous cell carcinoma.
在任何方法的一些实施方案中，依照任何上述实施方案的个体可以是人。In some embodiments of any of the methods, the individual according to any of the above embodiments can be a human.
在又一个实施方案中，本文中提供的是用于治疗癌症的方法。在一个实施方案中，该方法包括对具有此类癌症的个体施用有效量的PD-L1轴结合拮抗剂。在一个此类实施方案中，该方法进一步包括对该个体施用有效量的至少一种别的治疗剂。在一些实施方案中，个体可以是人。In yet another embodiment, provided herein are methods for treating cancer. In one embodiment, the method comprises administering to an individual having such cancer an effective amount of a PD-L1 axis binding antagonist. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In some embodiments, the individual can be human.
可以在疗法中单独或与其它药剂组合使用本文中描述的PD-L1轴结合拮抗剂。例如，可以与至少一种别的治疗剂共施用本文中描述的PD-L1轴结合拮抗剂。在某些实施方案中，别的治疗剂是化疗剂。The PD-L1 axis binding antagonists described herein can be used alone or in combination with other agents in therapy. For example, the PD-L1 axis binding antagonists described herein can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent.
上文记载的此类组合疗法涵盖组合施用(其中两种或更多种治疗剂包含在同一配制剂或分开的配制剂中)，和分开施用(在该情况中拮抗剂可以在别的治疗剂和/或佐剂施用之前、同时、和/或之后施用)。本文中描述的PD-L1轴结合拮抗剂还可以与放射疗法组合使用。Such combination therapies described above encompass combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration (in which case the antagonist can be administered before, simultaneously with, and/or after the administration of the other therapeutic agent and/or adjuvant). The PD-L1 axis binding antagonists described herein can also be used in combination with radiation therapy.
可以通过任何合适的手段，包括胃肠外、肺内、和鼻内，及若期望用于局部治疗的话，损伤内施用来施用本文中描述的PD-L1轴结合拮抗剂(例如抗体、结合多肽、和/或小分子)(及任何别的治疗剂)。胃肠外输注包括肌肉内、静脉内、动脉内、腹膜内、或皮下施用。部分根据施用是短暂的还是长期的，给药可以通过任何合适的路径，例如通过注射，诸如静脉内或皮下注射进行。本文中涵盖各种给药日程表，包括但不限于单次施用或在多个时间点里的多次施用、推注施用、和脉冲输注。The PD-L1 axis binding antagonists (e.g., antibodies, binding polypeptides, and/or small molecules) described herein (and any other therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse infusions.
本文中描述的PD-L1轴结合拮抗剂(例如抗体、结合多肽、和/或小分子)可以一种符合良好的医学实践的方式配制、确定剂量及施用。在此背景中考虑的因素包括在治疗的特定病症、在治疗的特定哺乳动物、个体患者的临床状态、病症原因、药剂递送部位、施用方法、施用日程以及其它为从业医生所知的因素。PD-L1轴结合拮抗剂无需但可任选地与一种或多种目前用于预防或治疗所述病症的药剂一起配制。这类其它药剂的有效量取决于配方中所存在的PD-L1轴结合拮抗剂的量、病症或治疗的类型、以及其它上述讨论的因素。这些药剂通常以相同的剂量使用并具有本文中所描述的施用途径，或以约1-99％的本文所描述的剂量使用，或以任何剂量并通过任何途径使用，所述剂量和途径是凭经验确定的/经临床测定合适的。The PD-L1 axis binding antagonists described herein (e.g., antibodies, binding polypeptides, and/or small molecules) can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical status of the individual patient, the cause of the condition, the site of agent delivery, the method of administration, the schedule of administration, and other factors known to practitioners. The PD-L1 axis binding antagonists need not be but may optionally be formulated with one or more agents currently used to prevent or treat the condition. The effective amount of such other agents depends on the amount of PD-L1 axis binding antagonist present in the formulation, the type of condition or treatment, and other factors discussed above. These agents are generally used in the same doses and with the routes of administration described herein, or at about 1-99% of the doses described herein, or at any dose and by any route, the dose and route being empirically determined/clinically appropriate.
为了预防或治疗疾病，本文中描述的PD-L1轴拮抗剂(当单独或与一种或多种其它别的治疗剂联合使用时)的合适剂量应取决于所要治疗的疾病的类型、疾病的严重性和病程、施用PD-L1轴结合拮抗剂是出于预防还是治疗目的、之前的治疗、患者的临床史和对PD-L1轴结合拮抗剂的响应、以及主治医师的斟酌决定。PD-L1轴结合拮抗剂适合于在一次或一系列的治疗中给予患者。一个典型的日剂量可在约1μg/kg-100mg/kg或更多的范围内，取决于上文所述因素。对于在数天或更长时间内的重复施用，根据状况，治疗一般将持续直至发生期望的对疾病症状的抑制。这类剂量可间歇施用，如每周或每三周施用，例如使得患者接受约2-约20剂，或例如约6剂的PD-L1轴结合拮抗剂。可施用初始较高的负荷剂量，接着施用一个或多个较低的剂量。例示性的给药方案包括施用。然而，可使用其它给药方案。通过常规技术和测定法易于监测该治疗的进展。For the prevention or treatment of disease, the appropriate dosage of the PD-L1 axis antagonists described herein (when used alone or in combination with one or more other therapeutic agents) should depend on the type of disease to be treated, the severity and course of the disease, whether the PD-L1 axis binding antagonist is administered for preventive or therapeutic purposes, previous treatment, the patient's clinical history and response to the PD-L1 axis binding antagonist, and the discretion of the attending physician. The PD-L1 axis binding antagonist is suitable for administration to the patient in one or a series of treatments. A typical daily dose may be in the range of about 1 μg/kg-100 mg/kg or more, depending on the factors described above. For repeated administration over several days or longer, depending on the situation, treatment will generally continue until the desired suppression of disease symptoms occurs. Such doses may be administered intermittently, such as weekly or every three weeks, for example, so that the patient receives about 2 to about 20 doses, or for example about 6 doses of the PD-L1 axis binding antagonist. An initial higher loading dose may be administered, followed by one or more lower doses. Exemplary dosing regimens include administration. However, other dosing regimens may be used.The progress of this treatment is easily monitored by conventional techniques and assays.
在任何方法的一些实施方案中，以约0.3-30mg/kg的剂量施用PD-L1轴结合拮抗剂(例如抗PD-L1抗体)。在一些实施方案中，以约0.3mg/kg，0.5mg/kg，1mg/kg，2mg/kg，4mg/kg，8mg/kg，15mg/kg，20mg/kg，或30mg/kg任一的剂量施用PD-L1轴结合拮抗剂(例如抗PD-L1抗体)。在一些实施方案中，在21天周期中以约2mg/kg，4mg/kg，8mg/kg，15mg/kg，或30mg/kg任一的剂量施用PD-L1轴结合拮抗剂(例如抗PD-L1抗体)。理解的是，任何上述配制剂或治疗方法可使用免疫缀合物代替或补充PD-L1轴结合拮抗剂来进行。In some embodiments of any method, the PD-L1 axis binding antagonist (e.g., anti-PD-L1 antibody) is administered at a dose of about 0.3-30 mg/kg. In some embodiments, the PD-L1 axis binding antagonist (e.g., anti-PD-L1 antibody) is administered at a dose of about 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 4 mg/kg, 8 mg/kg, 15 mg/kg, 20 mg/kg, or 30 mg/kg. In some embodiments, the PD-L1 axis binding antagonist (e.g., anti-PD-L1 antibody) is administered at a dose of about 2 mg/kg, 4 mg/kg, 8 mg/kg, 15 mg/kg, or 30 mg/kg in a 21-day cycle. It is understood that any of the above formulations or treatment methods can be performed using an immunoconjugate instead of or in addition to the PD-L1 axis binding antagonist.
通过将具有期望纯度的此类抗体与一种或多种任选的药学可接受载体(Remington’s Pharmaceutical Sciences第16版,Osol,A.编(1980))混合以冻干配制剂或水性溶液形式制备如本文中所描述的PD-L1轴结合拮抗剂的药物配制剂。在一些实施方案中，PD-L1轴结合拮抗剂是结合小分子、抗体、结合多肽、和/或多核苷酸。一般地，药学可接受载体在所采用的剂量和浓度对接受者是无毒的，而且包括但不限于缓冲剂，诸如磷酸盐、柠檬酸盐、和其它有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(诸如氯化十八烷基二甲基苄基铵；氯化己烷双胺；苯扎氯铵、苄索氯铵；酚、丁醇或苯甲醇；对羟基苯甲酸烃基酯，诸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，诸如血清清蛋白、明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯烷酮；氨基酸，诸如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其它碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，诸如EDTA；糖类，诸如蔗糖、甘露醇、海藻糖或山梨醇；成盐相反离子，诸如钠；金属复合物(例如Zn-蛋白质复合物)；和/或非离子表面活性剂，诸如聚乙二醇(PEG)。本文中的例示性的药学可接受载体进一步包含间质药物分散剂诸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，诸如rHuPH20(Baxter International,Inc.)。某些例示性的sHASEGP和使用方法，包括rHuPH20记载于美国专利公开文本No.2005/0260186和2006/0104968。在一个实施方案中，将sHASEGP与一种或多种别的糖胺聚糖酶诸如软骨素酶组合。Pharmaceutical formulations of the PD-L1 axis binding antagonists as described herein are prepared by mixing such antibodies having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of lyophilized formulations or aqueous solutions. In some embodiments, the PD-L1 axis binding antagonist is a binding small molecule, antibody, binding polypeptide, and/or polynucleotide. Generally, pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as 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); Polypeptides; 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 carbohydrates 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), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.
例示性的冻干配制剂记载于美国专利No.6,267,958。水性抗体配制剂包括那些记载于美国专利No.6,171,586和WO2006/044908的，后一种配制剂包含组氨酸-乙酸盐缓冲液。Exemplary lyophilized 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.
本文中的配制剂还可含有超过一种所治疗具体适应症所必需的活性组分，优选那些活性互补且彼此没有不利影响的组分。此类活性组分适于以有效用于所需目的的量而组合存在。The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those whose activities complement each other and do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the desired purpose.
活性成分可包载于例如通过凝聚技术或通过界面聚合制备的微胶囊中(例如分别是羟甲基纤维素或明胶微胶囊和聚(甲基丙烯酸甲酯)微胶囊)，在胶状药物投递系统中(例如脂质体、清蛋白微球体、微乳剂、纳米颗粒和纳米胶囊)，或在粗滴乳状液中。此类技术披露于Remington’s Pharmaceutical Sciences,第16版,Osol,A.编(1980)。The active ingredient can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), 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).
可以制备持续释放制剂。持续释放制剂的合适的例子包括含有PD-L1轴结合拮抗剂的固体疏水性聚合物的半透性基质，该基质为成形商品形式，例如膜，或微胶囊。Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the PD-L1 axis binding antagonist, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
用于体内施用的配制剂一般是无菌的。无菌性可容易地实现，例如通过穿过无菌滤膜过滤。Formulations for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.
理解的是，任何上述制品可包括本文所述免疫缀合物代替或补充PD-L1拮抗剂。It is understood that any of the above-described articles of manufacture may include an immunoconjugate described herein in place of or in addition to a PD-L1 antagonist.
鉴定和使用生物标志物的方法Methods for identifying and using biomarkers
本文中提供的是用于鉴定治疗响应性生物标志物的方法。Provided herein are methods for identifying biomarkers of treatment responsiveness.
在一些实施方案中，可以基于分析物表达水平和受试者的响应相对于一种或多种治疗前基线响应的积极或消极变化之间的限定相关性或关联来鉴定药效学生物标志物。在一些实施方案中，可以在治疗或治疗干预之前，期间和之后自受试者收集的样品中测量分析物表达水平。In some embodiments, a pharmacodynamic biomarker can be identified based on a defined correlation or association between the analyte expression level and a positive or negative change in the subject's response relative to one or more pre-treatment baseline responses. In some embodiments, the analyte expression level can be measured in samples collected from the subject before, during, and after treatment or therapeutic intervention.
药效学生物标志物可用于但不限于治疗监测和评估治疗有效性。例如，可以给临床医师提供药效学生物标志物水平用于建立或改变用于受试者的治疗过程。当选择治疗和治疗开始时，可以周期性监测受试者，即在两个或更多个间隔收集生物学样品，测定与治疗之前，期间和之后的给定时间间隔对应的临床响应，并随时间比较临床响应。基于这些响应和就提高，降低或稳定临床响应而言观察到的任何趋势或药效学生物标志物水平中的变化，临床医生，治疗学家，或其他健康护理专业人员可以选择继续当前的治疗，中止治疗，或调节治疗计划，目的是随时间看到改善。Pharmacodynamic biomarkers can be used for, but are not limited to, treatment monitoring and assessing treatment effectiveness. For example, a pharmacodynamic biomarker level can be provided to a clinician for use in establishing or changing a treatment course for a subject. When a treatment is selected and treatment begins, the subject can be monitored periodically, i.e., biological samples are collected at two or more intervals, and the clinical response corresponding to a given time interval before, during, and after treatment is determined, and the clinical response is compared over time. Based on these responses and any trends or changes in pharmacodynamic biomarker levels observed with respect to improvement, reduction, or stabilization of the clinical response, a clinician, therapist, or other health care professional can choose to continue the current treatment, discontinue treatment, or adjust the treatment plan with the goal of seeing improvement over time.
因而，本文中提供的是用于评估个体对PD-L1轴结合拮抗剂的治疗响应的方法，该方法包括：(a)测定在施用PD-L1轴结合拮抗剂期间或之后的一个时间点自个体衍生的生物学样品中一种或多种生物标志物的水平；并(b)基于生物学样品中一种或多种生物标志物的水平与参照水平的比较维持，调节，或停止个体的治疗，其中生物学样品中一种或多种生物标志物的水平与参照水平相比的变化指示对PD-L1轴结合拮抗剂治疗的响应。Thus, provided herein are methods for assessing a subject's response to treatment with a PD-L1 axis binding antagonist, the methods comprising: (a) determining the level of one or more biomarkers in a biological sample derived from the subject at a time point during or after administration of the PD-L1 axis binding antagonist; and (b) maintaining, adjusting, or discontinuing treatment of the subject based on a comparison of the level of the one or more biomarkers in the biological sample to a reference level, wherein a change in the level of the one or more biomarkers in the biological sample compared to the reference level indicates a response to treatment with the PD-L1 axis binding antagonist.
本文中进一步提供的是用于监测用PD-L1轴结合拮抗剂治疗的个体的响应的方法，该方法包括：(a)测定在施用PD-L1轴结合拮抗剂期间或之后的一个时间点自个体衍生的生物学样品中一种或多种生物标志物的水平；并(b)将生物学样品中一种或多种生物标志物的水平与参照水平比较以监测经历PD-L1轴结合拮抗剂治疗的个体的响应。Further provided herein are methods for monitoring the response of an individual treated with a PD-L1 axis binding antagonist, the method comprising: (a) determining the level of one or more biomarkers in a biological sample derived from the individual at a time point during or after administration of the PD-L1 axis binding antagonist; and (b) comparing the level of the one or more biomarkers in the biological sample to a reference level to monitor the response of the individual undergoing treatment with the PD-L1 axis binding antagonist.
在一些实施方案中，一种或多种生物标志物的参照水平选自下组：(1)施用PD-L1轴结合拮抗剂之前来自个体的一种或多种生物标志物的水平；(2)来自参照群体的一种或多种生物标志物的水平；(3)一种或多种生物标志物的预指派水平；和(4)在第一时间点之前的第二时间点来自个体的一种或多种生物标志物的水平。In some embodiments, the reference level of one or more biomarkers is selected from the group consisting of: (1) the level of one or more biomarkers from the individual before administration of the PD-L1 axis binding antagonist; (2) the level of one or more biomarkers from a reference population; (3) a pre-assigned level of one or more biomarkers; and (4) the level of one or more biomarkers from the individual at a second time point prior to the first time point.
为了关联和比较个体的生物学样品与参照群体，有必要获得关于由接受治疗的个体的群体(即临床群体)在PD-L1轴结合拮抗剂治疗之前和/或之后展现的临床响应的数据。这种临床数据可以通过临床试验结果的回顾性分析来获得。或者，可以通过设计和进行一项或多项新的临床试验来获得临床数据。临床群体数据的分析对于定义标准参照群体是有用的，标准参照群体继而对于将受试者分类以选择治疗性处理和/或将受试者分类为对PD-L1轴结合拮抗剂治疗展现积极响应是有用的。In order to associate and compare individual biological samples with reference populations, it is necessary to obtain data on the clinical response exhibited by a population of treated individuals (i.e., a clinical population) before and/or after treatment with a PD-L1 axis binding antagonist. Such clinical data can be obtained by retrospective analysis of clinical trial results. Alternatively, clinical data can be obtained by designing and conducting one or more new clinical trials. Analysis of clinical population data is useful for defining a standard reference population, which in turn is useful for classifying subjects to select therapeutic treatments and/or classifying subjects as exhibiting a positive response to treatment with a PD-L1 axis binding antagonist.
在一些实施方案中，生物学样品中一种或多种生物标志物的水平与参照水平相比的变化为水平的升高。In some embodiments, a change in the level of one or more biomarkers in a biological sample compared to a reference level is an increase in the level.
在一些实施方案中，生物学样品中一种或多种生物标志物的水平与参照水平相比的变化为水平的降低。In some embodiments, a change in the level of one or more biomarkers in a biological sample compared to a reference level is a decrease in the level.
在一些实施方案中，一种或多种生物标志物选自下组：PD-L1，PD-1，PD-L2及其任意组合。在一些实施方案中，选自生物学样品中PD-L1，PD-1，PD-L2及其任意组合的一种或多种生物标志物与参照水平相比升高指示对治疗的积极响应。In some embodiments, the one or more biomarkers are selected from the group consisting of PD-L1, PD-1, PD-L2, and any combination thereof. In some embodiments, an increase in one or more biomarkers selected from PD-L1, PD-1, PD-L2, and any combination thereof in a biological sample compared to a reference level indicates a positive response to treatment.
在一些实施方案中，一种或多种生物标志物为免疫相关标志物。In some embodiments, the one or more biomarkers are immune-related markers.
在一些实施方案中，一种或多种生物标志物为T细胞相关标志物。In some embodiments, the one or more biomarkers are T cell-associated markers.
在一些实施方案中，一种或多种生物标志物为T细胞活化标志物。In some embodiments, the one or more biomarkers are T cell activation markers.
在一些实施方案中，生物学样品中的T细胞活化标志物与参照水平相比升高。In some embodiments, the T cell activation marker is elevated in the biological sample compared to a reference level.
在一些实施方案中，T细胞活化标志物选自下组：CD8，IFN-g，粒酶-A，TNF-a，穿孔蛋白及其任意组合。在一些实施方案中，生物学样品中选自CD8，IFN-g，粒酶-A，TNF-a，穿孔蛋白及其任意组合的T细胞活化标志物与参照水平相比升高指示对治疗的积极响应。In some embodiments, the T cell activation marker is selected from the group consisting of CD8, IFN-g, granzyme-A, TNF-a, perforin, and any combination thereof. In some embodiments, an increase in the T cell activation marker selected from the group consisting of CD8, IFN-g, granzyme-A, TNF-a, perforin, and any combination thereof in the biological sample compared to a reference level indicates a positive response to treatment.
在一些实施方案中，一种或多种生物标志物为活化的增殖T细胞。In some embodiments, the one or more biomarkers is activated, proliferating T cells.
在一些实施方案中，生物学样品中的活化的增殖T细胞与参照水平相比增多。In some embodiments, activated, proliferating T cells are increased in the biological sample compared to a reference level.
在一些实施方案中，活化的增殖T细胞为CD8+/Ki67+细胞，CD8+/HLA-DR+/Ki67+细胞及其任意组合。In some embodiments, the activated proliferating T cells are CD8+/Ki67+ cells, CD8+/HLA-DR+/Ki67+ cells, and any combination thereof.
在一些实施方案中，一种或多种生物标志物为IL-6。In some embodiments, the one or more biomarkers is IL-6.
在一些实施方案中，生物学样品中的IL-6水平与参照水平相比降低。在一些实施方案中，生物学样品中的IL-6水平与参照水平相比降低指示对治疗的积极响应。在一些实施方案中，生物学样品中的IL-6水平与参照水平相比升高。在一些实施方案中，生物学样品中的IL-6水平与参照水平相比升高指示对治疗无响应。In some embodiments, the IL-6 level in the biological sample is reduced compared to the reference level. In some embodiments, a reduction in the IL-6 level in the biological sample compared to the reference level indicates a positive response to treatment. In some embodiments, the IL-6 level in the biological sample is increased compared to the reference level. In some embodiments, an increase in the IL-6 level in the biological sample compared to the reference level indicates no response to treatment.
在一些实施方案中，自个体衍生的生物学样品选自下组：细胞，组织，组织培养物，肿瘤，生物学流体及其组合。In some embodiments, the biological sample derived from the individual is selected from the group consisting of a cell, a tissue, a tissue culture, a tumor, a biological fluid, and combinations thereof.
在一些实施方案中，生物学流体选自下组：血浆，血清，全血，PBMC及其组合。In some embodiments, the biological fluid is selected from the group consisting of plasma, serum, whole blood, PBMCs, and combinations thereof.
在一些实施方案中，组织为肿瘤组织。In some embodiments, the tissue is tumor tissue.
在一些实施方案中，肿瘤组织选自下组：肿瘤细胞，肿瘤浸润性细胞，基质细胞及其任意组合。In some embodiments, the tumor tissue is selected from the group consisting of tumor cells, tumor-infiltrating cells, stromal cells, and any combination thereof.
在一些实施方案中，细胞为循环肿瘤细胞(CTC)。In some embodiments, the cells are circulating tumor cells (CTCs).
在一些实施方案中，个体罹患增殖性疾病或病症。In some embodiments, the subject suffers from a proliferative disease or disorder.
在一些实施方案中，个体罹患癌症或恶性肿瘤。In some embodiments, the individual suffers from cancer or a malignancy.
在一些实施方案中，癌症或恶性肿瘤选自非小细胞肺癌，小细胞肺癌，肾细胞癌，结肠直肠癌，卵巢癌，乳腺癌，胰腺癌，胃癌，膀胱癌，食道癌，间皮瘤，黑素瘤，头和颈癌，甲状腺癌，肉瘤，前列腺癌，成胶质细胞瘤，宫颈癌，胸腺癌，白血病，淋巴瘤，骨髓瘤，蕈样肉芽肿，梅克尔细胞癌，和其它血液学恶性肿瘤。In some embodiments, the cancer or malignancy is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematologic malignancies.
在一些实施方案中，个体罹患免疫相关疾病或病症。In some embodiments, the individual suffers from an immune-related disease or disorder.
在一些实施方案中，PD-L1轴结合拮抗剂为PD-L1结合拮抗剂。In some embodiments, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist.
在一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合它的配体结合配偶。In some embodiments, a PD-L1 binding antagonist inhibits the binding of PD-L1 to its ligand binding partner.
在一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合PD-1。In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1.
在一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合B7-1。In some embodiments, the PD-L1 binding antagonist inhibits PD-L1 binding to B7-1.
在一些实施方案中，PD-L1结合拮抗剂抑制PD-L1结合PD-1和B7-1二者。In some embodiments, the PD-L1 binding antagonist inhibits PD-L1 binding to both PD-1 and B7-1.
在一些实施方案中，PD-L1结合拮抗剂为抗体。In some embodiments, the PD-L1 binding antagonist is an antibody.
在一些实施方案中，抗体为单克隆抗体。In some embodiments, the antibody is a monoclonal antibody.
在一些实施方案中，抗体为人抗体，人源化抗体或嵌合抗体。In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
在一些实施方案中，PD-L1轴结合拮抗剂为PD-1结合拮抗剂。In some embodiments, the PD-L1 axis binding antagonist is a PD-1 binding antagonist.
在一些实施方案中，PD-1结合拮抗剂抑制PD-1结合它的配体结合配偶。In some embodiments, a PD-1 binding antagonist inhibits PD-1 from binding to its ligand binding partner.
在一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L1。In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1.
在一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L2。In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2.
在一些实施方案中，PD-1结合拮抗剂抑制PD-1结合PD-L1和PD-L2二者。In some embodiments, the PD-1 binding antagonist inhibits PD-1 binding to both PD-L1 and PD-L2.
在一些实施方案中，PD-1结合拮抗剂为抗体。In some embodiments, the PD-1 binding antagonist is an antibody.
在一些实施方案中，抗体为单克隆抗体。In some embodiments, the antibody is a monoclonal antibody.
在一些实施方案中，抗体为人抗体，人源化抗体或嵌合抗体。In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
做广告的方法How to advertise
本文中进一步提供的是用于为PD-L1轴结合拮抗剂做广告的方法，其包括给目标受众宣传PD-L1轴结合拮抗剂用于基于PD-L1生物标志物的存在和/或水平来治疗具有疾病或病症的个体的用途。在一些实施方案中，PD-L1轴结合拮抗剂的用途基于升高水平的PD-L1生物标志物。Further provided herein are methods for advertising a PD-L1 axis binding antagonist, comprising advertising to a target audience the use of the PD-L1 axis binding antagonist for treating an individual having a disease or condition based on the presence and/or level of a PD-L1 biomarker. In some embodiments, the use of the PD-L1 axis binding antagonist is based on elevated levels of a PD-L1 biomarker.
做广告为经由非个人媒体进行的、通常付费的通讯，其中发起人受到鉴别且信息受到控制。为本文目的的做广告包括宣传、公共关系、产品布置、赞助、保险(underwriting)、和促销。该术语还包括出现在任何印刷传播媒体中的赞助的信息公告，其设计用于引起大众的兴趣以劝说、通知、宣传、激发、或以其它方式改变行为，向购买、支持、或认可本文中发明的有利方式发展。Advertising is communication, usually paid, via non-personal media in which the originator is identified and the message is controlled. For purposes of this document, advertising includes publicity, public relations, product placement, sponsorship, underwriting, and sales promotion. The term also includes sponsored information announcements appearing in any printed communication media designed to arouse public interest in order to persuade, inform, publicize, inspire, or otherwise change behavior in a manner that leads to the purchase, support, or endorsement of the inventions herein.
本文中诊断方法的广告和宣传可通过任何手段实现。用于传递这些信息的广告媒体的例子包括电视、电台、电影、杂志、报纸、因特网、和告示板，包括商业性的，即出现在广播媒体中的信息。广告还包括那些在食品货车座位上、机场通道墙壁上、和公共汽车侧面上的广告、或电话等候信息或店内布告(PA)系统中听到的广告、或可放置视觉或听觉通讯的任何地方的广告。The advertising and publicity of diagnostic methods herein can be achieved by any means. Examples of advertising media for delivering these messages include television, radio, movies, magazines, newspapers, the Internet, and notice boards, including commercial, i.e., information that appears in broadcast media. Advertisements also include those on food truck seats, on airport aisle walls, and on the sides of buses, or heard on telephone waiting information or in-store notice (PA) systems, or anywhere else where visual or auditory communication can be placed.
宣传或做广告手段的更特定的例子包括电视、电台、电影、因特网(诸如网络传播和网络研讨会(webinar))、意图到达同步用户的交互式计算机网络、固定的或电子的告示板和其它公共标牌、海报、传统的或电子的文献(诸如杂志和报纸)、其它媒体渠道、讲座或个体接触，例如通过电子邮件、电话、即时信息、邮寄、快递、大众(mass)、或承运人邮件(carrier mail)、亲自访问等进行。More specific examples of promotional or advertising means include television, radio, motion pictures, the Internet (such as webcasts and webinars), interactive computer networks intended to reach simultaneous users, fixed or electronic billboards and other public signage, posters, traditional or electronic literature (such as magazines and newspapers), other media channels, lectures, or personal contact, for example, by email, telephone, instant messaging, postal mail, courier, mass or carrier mail, personal visits, etc.
所使用的做广告的类型会取决于许多因素，例如待传达的目标受众的性质，例如医院、保险公司、诊所、医生、护士、和患者，以及成本考虑和管理药物和诊断剂做广告的相关管辖权法律和条例。可根据由服务相互作用和/或其它数据(诸如用户人口统计状况和地理学定位)限定的用户表征使做广告个性化或用户化。The type of advertising used will depend on many factors, such as the nature of the target audience to be communicated, e.g., hospitals, insurance companies, clinics, doctors, nurses, and patients, as well as cost considerations and relevant jurisdictional laws and regulations governing the advertising of pharmaceuticals and diagnostics. Advertising can be personalized or customized based on user profiles defined by service interactions and/or other data, such as user demographics and geographic location.
诊断试剂盒，测定法和制品Diagnostic kits, assays and products
本文中提供的是诊断试剂盒，其包含用于测定来自具有疾病或病症的个体的样品中PD-L1生物标志物的存在的一种或多种试剂，其中PD-L1生物标志物的存在意味着用PD-L1轴结合拮抗剂治疗个体时更高可能性的功效，且其中PD-L1生物标志物的缺失意味着用PD-L1轴结合拮抗剂治疗具有疾病的个体时更少可能性的功效。任选地，该试剂盒进一步包含使用该试剂盒来选择药物(例如PD-L1轴结合拮抗剂，诸如抗PD-L1抗体)以治疗疾病或病症的说明书，如果该个体表达PD-L1生物标志物的话。在另一个实施方案中，说明书是使用该试剂盒来选择除PD-L1轴结合拮抗剂以外的药物，如果该个体不表达PD-L1生物标志物的话。Provided herein are diagnostic kits comprising one or more reagents for determining the presence of a PD-L1 biomarker in a sample from an individual with a disease or condition, wherein the presence of a PD-L1 biomarker means a higher likelihood of efficacy when treating an individual with a PD-L1 axis binding antagonist, and wherein the absence of a PD-L1 biomarker means a lower likelihood of efficacy when treating an individual with a disease with a PD-L1 axis binding antagonist. Optionally, the kit further comprises instructions for using the kit to select a drug (e.g., a PD-L1 axis binding antagonist, such as an anti-PD-L1 antibody) to treat a disease or condition if the individual expresses the PD-L1 biomarker. In another embodiment, the instructions are for using the kit to select a drug other than a PD-L1 axis binding antagonist if the individual does not express the PD-L1 biomarker.
本文中还提供的是用于鉴定具有疾病或病症的个体来接受PD-L1轴结合拮抗剂的测定法，该方法包括：测定来自该个体的样品中PD-L1生物标志物的存在，并基于PD-L1生物标志物的存在推荐PD-L1轴结合拮抗剂。Also provided herein are assays for identifying an individual with a disease or condition to receive a PD-L1 axis binding antagonist, the method comprising: determining the presence of a PD-L1 biomarker in a sample from the individual, and recommending a PD-L1 axis binding antagonist based on the presence of the PD-L1 biomarker.
本文中还提供的是制品，其包含包装在一起的PD-L1轴结合拮抗剂(例如抗PD-L1抗体)和包装插页，该PD-L1轴结合拮抗剂在药学可接受载体中，该包装插页指示该PD-L1轴结合拮抗剂(例如抗PD-L1抗体)用于基于PD-L1生物标志物的表达来治疗具有疾病或病症的患者。治疗方法包括本文中公开的任何治疗方法。本发明进一步提供的是用于制造制品的方法，包括在包装中组合药物组合物和包装插页，该药物组合物包含PD-L1轴结合拮抗剂(例如抗PD-L1抗体)，该包装插页指示该药物组合物用于基于PD-L1生物标志物的表达治疗具有疾病或病症的患者。Also provided herein are articles of manufacture comprising a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody) packaged together and a package insert, the PD-L1 axis binding antagonist being in a pharmaceutically acceptable carrier, the package insert indicating that the PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody) is used to treat a patient having a disease or condition based on the expression of a PD-L1 biomarker. The methods of treatment include any of the methods disclosed herein. Further provided herein are methods for making an article of manufacture comprising combining in a package a pharmaceutical composition comprising a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody) and a package insert indicating that the pharmaceutical composition is used to treat a patient having a disease or condition based on the expression of a PD-L1 biomarker.
所述制品包括容器和容器上或伴随容器的标签或包装插页。合适的容器包括例如瓶(bottles)、管形瓶(vials)、注射器(syringes)、等。所述容器可以用多种材料诸如玻璃或塑料制成。所述容器容纳或装有包含癌症药物作为活性剂的组合物，并可以具有无菌出入口(例如该容器可以是具有可被皮下注射针头刺穿的塞子的静脉内溶液袋或管形瓶)。The article of manufacture includes a container and a label or package insert on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be made of a variety of materials, such as glass or plastic. The container holds or contains a composition comprising a cancer drug as an active agent and can have sterile access ports (for example, the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle).
所述制品可以进一步包括第二容器，该容器装有药学可接受的稀释缓冲剂，诸如抑菌性注射用水(BWFI)、磷酸盐缓冲盐水、林格氏(Ringer)溶液、和右旋糖溶液。制品还可包括从商业和使用者观点看有需要的其它材料，包括其它缓冲剂、稀释剂、滤器、针头、和注射器。The article of manufacture may further include a second container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
本发明的制品还包括信息，例如以包装插页的形式，指示所述组合物用于基于本文中生物标志物的表达水平，治疗癌症。所述插页或标签可采取任何形式，诸如纸或在电子介质上，诸如磁记录介质(例如软盘)或CD-ROM。所述标签或插页还可包括关于所述试剂盒或制品中药物组合物和剂量形式的其它信息。The articles of manufacture of the present invention may also include information, such as in the form of a package insert, indicating that the composition is used to treat cancer based on the expression levels of the biomarkers described herein. The insert or label may take any form, such as paper or on an electronic medium, such as a magnetic recording medium (e.g., a floppy disk) or a CD-ROM. The label or insert may also include other information regarding the pharmaceutical composition and dosage form in the kit or article.
本发明还涉及用于制造制品的方法，包括在包装中组合药物组合物和包装插页，该药物组合物包含PD-L1轴结合拮抗剂(例如抗PD-L1抗体)，该包装插页指示该药物组合物用于基于PD-L1生物标志物的表达治疗癌症(诸如NSCLC)患者。The present invention also relates to a method for making an article of manufacture comprising combining in a package a pharmaceutical composition comprising a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody) and a package insert indicating that the pharmaceutical composition is used to treat a patient with cancer (such as NSCLC) based on expression of the PD-L1 biomarker.
所述制品可以进一步包括别的容器，该容器装有药学可接受的稀释缓冲剂，诸如抑菌性注射用水(BWFI)、磷酸盐缓冲盐水、林格氏(Ringer)溶液、和/或右旋糖溶液。制品还可包括从商业和使用者观点看有需要的其它材料，包括其它缓冲剂、稀释剂、滤器、针头、和注射器。The article of manufacture may further include another container containing a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution. The article of manufacture may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.
实施例Example
以下是方法和组合物的实施例。应当理解，鉴于上文提供的一般描述，可以实施各种其它实施方案。The following are examples of methods and compositions.It is understood that various other embodiments may be practiced, given the general description provided above.
用于实施例的材料和方法Materials and Methods Used in the Examples
样品：分析肿瘤样品或癌细胞系的福尔马林固定、石蜡包埋(FFPE)切片。Samples: Formalin-fixed, paraffin-embedded (FFPE) sections of tumor samples or cancer cell lines were analyzed.
免疫组织化学(IHC)：在抗原修复之前，将福尔马林固定、石蜡包埋的组织切片脱石蜡，封闭，并与抗PD-L1抗体一起温育。与二抗一起温育和酶促显色之后，将切片复染色并在系列醇和二甲苯中脱水，之后盖上盖玻片。Immunohistochemistry (IHC): Formalin-fixed, paraffin-embedded tissue sections were deparaffinized, blocked, and incubated with anti-PD-L1 antibody before antigen retrieval. After incubation with secondary antibody and enzymatic development, sections were counterstained and dehydrated in a series of alcohols and xylene before coverslipping.
使用下述方案进行IHC。使用下述试剂和材料，使用Ventana Benchmark XT或Benchmark Ultra系统实施PD-L1IHC染色：IHC was performed using the following protocol. PD-L1 IHC staining was performed using the Ventana Benchmark XT or Benchmark Ultra systems using the following reagents and materials:
一抗：抗PD-L1家兔单克隆一抗Primary antibody: anti-PD-L1 rabbit monoclonal primary antibody
标本类型：不同染色强度的组织样品和对照细胞团粒的福尔马林固定、石蜡包埋(FFPE)切片Specimen type: Formalin-fixed, paraffin-embedded (FFPE) sections of tissue samples with varying staining intensities and control cell pellets
规程物种：人Species of Regulation: Human
仪器：BenchMark XT或Benchmark UltraInstrument: BenchMark XT or Benchmark Ultra
表位恢复条件：细胞条件化，标准1(CC1，Ventana，产品目录#950-124)Epitope retrieval conditions: Cell conditioning, Standard 1 (CC1, Ventana, catalog #950-124)
一抗条件：1/100，6.5μg/ml/于36℃ 16分钟Primary antibody conditions: 1/100, 6.5 μg/ml/at 36°C for 16 minutes
稀释剂：抗体稀释缓冲液(含有载体蛋白和Brig-35的Tris缓冲盐水)Diluent: Antibody dilution buffer (Tris-buffered saline containing carrier protein and Brig-35)
阴性对照：未免疫家兔IgG，6.5μg/ml(Cell Signaling)或单独的稀释剂Negative control: non-immunized rabbit IgG, 6.5 μg/ml (Cell Signaling) or diluent alone
检测：依照制造商的说明书(Ventana)使用Optiview或Ultraview通用DAB检测试剂盒(Ventana)和扩增试剂盒(如果适用的话)。Detection: Optiview or Ultraview Universal DAB Detection Kit (Ventana) and amplification kit (if applicable) were used according to the manufacturer's instructions (Ventana).
复染色：Ventana苏木精II(产品目录#790-2208)/带发蓝试剂(产品目录#760-2037)(分别为4分钟和4分钟)Counterstain: Ventana Hematoxylin II (Catalog #790-2208)/Bluing Reagent (Catalog #760-2037) (4 minutes and 4 minutes, respectively)
Benchmark方案如下：The Benchmark solution is as follows:
1.石蜡(选择)1. Paraffin (optional)
2.脱石蜡(选择)2. Deparaffinization (optional)
3.细胞条件化(选择)3. Cell Conditioning (Selection)
4.调节器#1(选择)4. Regulator #1 (select)
5.标准CC1(选择)5. Standard CC1 (selection)
6.抗体温育温度(选择)6. Antibody incubation temperature (selection)
7.36℃抗体温育(选择)7.36℃ Antibody Incubation (Selection)
8.滴定(选择)8. Titration (selection)
9.自动分发(一抗)，并温育(16分钟)9. Automatically distribute (primary antibody) and incubate (16 minutes)
10.复染色(选择)10. Counterstaining (optional)
11.应用一滴(苏木精II)(复染色)，应用盖玻片，并温育(4分钟)11. Apply one drop of (Hematoxylin II) (counter-stain), apply a coverslip, and incubate (4 minutes)
12.后复染色(选择)12. Post-counter-staining (optional)
13.应用一滴(发蓝试剂)(后复染色)，应用盖玻片，并温育(4分钟)13. Apply one drop of (blueing reagent) (post-counter-staining), apply a coverslip, and incubate (4 minutes)
14.在肥皂水中清洗载玻片以去除油14. Wash the slides in soapy water to remove oil
15.用水漂洗载玻片15. Rinse the slides with water
16.经由95％乙醇、100％乙醇至二甲苯使载玻片脱水(Leica自动染色机程序#9)16. Dehydrate the slides through 95% ethanol, 100% ethanol, and then xylene (Leica autostainer program #9)
17.盖上盖玻片。17. Cover with a coverslip.
实施例1--通过IHC对PD-L1表达打分Example 1 - Scoring PD-L1 expression by IHC
通过IHC在人福尔马林固定、石蜡包埋(FFPE)组织中使用能检测PD-L1的抗PD-L1特异性抗体评估肿瘤标本中PD-L1表达的存在或缺失。为了测量和量化肿瘤样品中PD-L1的相对表达，开发了一种PD-L1IHC打分系统来测量肿瘤细胞和肿瘤浸润性免疫细胞中的PD-L1特异性信号。免疫细胞定义为具有淋巴样和/或巨噬细胞/组织细胞形态的细胞。The presence or absence of PD-L1 expression in tumor specimens is assessed by IHC in human formalin-fixed, paraffin-embedded (FFPE) tissue using an anti-PD-L1-specific antibody that detects PD-L1. To measure and quantify the relative expression of PD-L1 in tumor samples, a PD-L1 IHC scoring system was developed to measure PD-L1-specific signals in tumor cells and tumor-infiltrating immune cells. Immune cells are defined as cells with lymphoid and/or macrophage/histiocyte morphology.
肿瘤细胞染色表述为显示任何强度的膜染色的所有肿瘤细胞的百分比。浸润性免疫细胞染色定义为被显示任何强度的染色的免疫细胞占据的总肿瘤面积的百分比。总肿瘤面积涵盖恶性细胞以及肿瘤相关基质，包括紧邻和毗邻主要肿瘤块的免疫浸润物的面积。另外，浸润性免疫细胞染色定义为所有肿瘤浸润性免疫细胞的百分比。Tumor cell staining was expressed as the percentage of all tumor cells that showed membrane staining of any intensity. Infiltrating immune cell staining was defined as the percentage of the total tumor area occupied by immune cells that showed staining of any intensity. The total tumor area encompassed malignant cells as well as tumor-associated stroma, including the area of immune infiltrates immediately adjacent to and adjacent to the main tumor mass. Additionally, infiltrating immune cell staining was defined as the percentage of all tumor-infiltrating immune cells.
PD-L1染色强度在肿瘤组织中有较宽的动态范围。不管亚细胞定位，还将信号分类为强，中等，弱，或阴性染色。PD-L1 staining intensity showed a wide dynamic range in tumor tissues. Regardless of subcellular localization, the signal was categorized as strong, moderate, weak, or negative staining.
如图1所示，阴性信号强度特征在于缺失任何可检测信号，如使用HEK-293细胞例示的。比较而言，阳性信号强度特征在于金色至深褐色膜染色，如使用用重组人PD-L1转染的HEK-293细胞例示的。最后，阳性信号强度还通过胎盘滋养层的染色和扁桃体隐窝区域中的强染色来例示，而且常常是膜样式，特征为金色至深褐色染色。在肿瘤组织中，PD-L1阴性样品限定为使用20倍物镜评估时没有可检测信号或只有弱胞质背景染色。比较而言，PD-L1阳性样品主要呈现肿瘤细胞和/或浸润性免疫细胞中的膜染色。以不同强度观察PD-L1染色，从弱(纤细，浅褐色膜)到强(深褐色厚膜，在低放大率轻易认出)。如图2所示，显示三种代表性PD-L1阳性肿瘤样品：(A)三重阴性乳腺癌，其中大多数肿瘤细胞是PD-L1强阳性的，显示膜和胞质染色的组合(100倍放大率)；(B)恶性黑素瘤，其中显示一簇免疫细胞，一些具有PD-L1膜染色；罕见肿瘤细胞(箭)具有PD-L1膜染色(400倍放大率)；(C)NSCLC，腺癌，其中显示一簇免疫细胞具有强PD-L1染色；数个肿瘤细胞(箭)具有膜和/或胞质PD-L1染色(400倍放大率)。As shown in Figure 1, negative signal intensity is characterized by the absence of any detectable signal, as exemplified using HEK-293 cells. In comparison, positive signal intensity is characterized by golden to dark brown membrane staining, as exemplified using HEK-293 cells transfected with recombinant human PD-L1. Finally, positive signal intensity is also exemplified by staining of the placental trophoblast and strong staining in the tonsil crypt region, and is often a membrane pattern characterized by golden to dark brown staining. In tumor tissue, PD-L1 negative samples are defined as having no detectable signal or only weak cytoplasmic background staining when evaluated using a 20x objective. In comparison, PD-L1 positive samples primarily present membrane staining in tumor cells and/or infiltrating immune cells. PD-L1 staining is observed at varying intensities, from weak (slender, light brown membrane) to strong (dark brown thick membrane, easily recognized at low magnification). As shown in Figure 2, three representative PD-L1-positive tumor samples are shown: (A) triple-negative breast cancer, in which the majority of tumor cells are strongly positive for PD-L1, showing a combination of membrane and cytoplasmic staining (100x magnification); (B) malignant melanoma, in which a cluster of immune cells is shown, some with PD-L1 membrane staining; rare tumor cells (arrows) have PD-L1 membrane staining (400x magnification); (C) NSCLC, adenocarcinoma, in which a cluster of immune cells is shown with strong PD-L1 staining; several tumor cells (arrows) have membrane and/or cytoplasmic PD-L1 staining (400x magnification).
阳性案例中的染色趋向于就空间分布和强度而言集中。目测估算显示任何强度的染色的肿瘤或免疫细胞的百分比并用于确定PD-L1状态。使用同种型阴性对照来评估测试样品中背景的存在情况。Staining in positive cases tended to be concentrated in terms of spatial distribution and intensity. The percentage of tumor or immune cells showing staining of any intensity was estimated visually and used to determine PD-L1 status. Isotype negative controls were used to assess the presence of background in test samples.
染色要求一系列组织切片用于H&E、第二系列组织切片用于抗PD-L1、和第三系列组织切片用于同种型阴性对照抗体。使用经PD-L1转染的HEK-293细胞系对照或扁桃体载玻片作为测定法特异性的参照和运行对照。Staining requires one series of tissue sections for H&E, a second series of tissue sections for anti-PD-L1, and a third series of tissue sections for an isotype negative control antibody. HEK-293 cell line control or tonsil slides transfected with PD-L1 were used as a reference and running control for assay specificity.
PDL-1状态标准PDL-1 Status Standards
在一些情况中，PD-L1阳性状态可包含肿瘤细胞或肿瘤浸润性免疫细胞中存在任何强度的可辨别的PD-L1染色，直至50％的被肿瘤细胞，相关肿瘤内，和连续肿瘤周围促结缔组织生成性基质占据的肿瘤面积。如此，PD-L1阳性染色包括高至50％的肿瘤细胞或肿瘤浸润性免疫细胞显示任何强度的染色。In some instances, PD-L1 positive status can include the presence of discernible PD-L1 staining of any intensity in tumor cells or tumor-infiltrating immune cells up to 50% of the tumor area occupied by tumor cells, associated intratumoral, and continuous peritumoral desmoplastic stroma. Thus, PD-L1 positive staining includes up to 50% of tumor cells or tumor-infiltrating immune cells showing staining of any intensity.
如上所述评估用抗PD-L1染色的可评估载玻片。阴性染色强度特征在于缺失任何可检测信号或表现为浅灰色至蓝色(而非褐色或棕色)的信号且缺失膜增强。如果没有(例如缺失)膜染色，那么该案例为阴性。Evaluable slides stained with anti-PD-L1 were evaluated as described above. Negative staining intensity was characterized by the absence of any detectable signal or a signal that appeared as light gray to blue (rather than brown or tan) and the absence of membrane enhancement. If there was no (e.g., absent) membrane staining, the case was negative.
实施例2--使用抗PD-L1抗体治疗Example 2 - Treatment with anti-PD-L1 antibodies
一项I期研究设计具体评估PD-L1肿瘤状态(如下评估：(a)抗PD-L1 IHC试剂，(b)PD-L1基因表达，如通过PD-L1 qPCR试剂测量的，(c)免疫基因签名，如通过多重qPCR“免疫芯片”测量的)和抑制PD-L1/PD-1途径的单药疗法的临床好处(如下测量：(i)基于RECIST1.1的响应，(ii)免疫相关响应标准，(iii)PFS，(iv)OS，(v)完全响应率，(vi)响应持久性，(vii)6周时的PD)之间的关联。要求扩展组中的患者提供肿瘤组织来评估PD-L1肿瘤状态，并登记入不管PD-L1肿瘤状态的扩展组或登记入基于通过针对PD-L1的IHC测定法测量的PD-L1肿瘤状态前瞻性选择患者的扩展组。登记的肿瘤类型具体包括NSCLC(鳞状和非鳞状组织学)，黑素瘤，RCC，CRC，胃癌，乳腺癌，SCCHN，胰腺癌，膀胱癌和血液学恶性肿瘤。另外，还(已经)登记具有淋巴瘤，骨髓瘤，肉瘤，卵巢癌，前列腺癌，食道癌，小细胞肺癌，蕈样肉芽肿，梅克尔细胞癌，宫颈癌，HPV或EBV+SCCHN，和胸腺癌的患者。A Phase I study was designed to specifically evaluate the association between PD-L1 tumor status (assessed as follows: (a) anti-PD-L1 IHC reagents, (b) PD-L1 gene expression, as measured by a PD-L1 qPCR reagent, and (c) immune gene signature, as measured by a multiplex qPCR "immunochip") and the clinical benefit of monotherapy that inhibits the PD-L1/PD-1 pathway (measured as follows: (i) response based on RECIST 1.1, (ii) immune-related response criteria, (iii) PFS, (iv) OS, (v) complete response rate, (vi) durability of response, and (vii) PD at 6 weeks). Patients in the expansion cohort were required to provide tumor tissue for assessment of PD-L1 tumor status and were enrolled in the expansion cohort regardless of PD-L1 tumor status or in an expansion cohort in which patients were prospectively selected based on PD-L1 tumor status as measured by an IHC assay specific for PD-L1. Tumor types enrolled specifically include NSCLC (squamous and non-squamous histology), melanoma, RCC, CRC, gastric cancer, breast cancer, SCCHN, pancreatic cancer, bladder cancer, and hematologic malignancies. In addition, patients with lymphoma, myeloma, sarcoma, ovarian cancer, prostate cancer, esophageal cancer, small cell lung cancer, mycosis fungoides, Merkel cell carcinoma, cervical cancer, HPV or EBV+ SCCHN, and thymic carcinoma are also enrolled.
在评估基线PD-L1肿瘤状态与来自抑制PD-L1/PD-1途径的单药疗法的临床好处的关联以外，该研究还评估下述好处：(a)在存档的肿瘤样品较之新鲜的或最近的肿瘤活检样品中测量PD-L1状态；(b)用抗CD8IHC试剂评估肿瘤中的CD8+T细胞浸润；(c)评估不同细胞类型％，隔室中的PD-L1染色或染色强度；(d)肿瘤周围较之肿瘤内PD-L1染色或CD8的影响；(e)PD-L1染色放大的影响；(f)qPCR或免疫芯片评估之前肿瘤宏观解剖的影响；(g)组织样品年龄和固定对PD-L1状态评估的影响和与好处的关联；(h)治疗中肿瘤活检用于使用上文所述肿瘤表征方法评估临床好处或毒性的价值；(i)FDG PET成像和CT反差增强用于评估治疗中好处或患者选择的价值；(j)肿瘤突变/癌基因状态(例如KRAS，bRAF，PI3K途径突变状态，Met状态，Her2neu状态，PTEN状态)在预测上述治疗的好处中的价值；(k)CTC数目和PD-L1表征；(l)循环细胞类型，子集和数目；(m)循环血浆/血清生物标志物；(n)种族差异；(o)吸烟状态；(p)FcgRIII多态性状态；(q)免疫相关多态性状态。In addition to assessing the association of baseline PD-L1 tumor status with clinical benefit from monotherapy inhibiting the PD-L1/PD-1 pathway, this study also evaluated the benefits of: (a) measuring PD-L1 status in archived tumor samples compared with fresh or recent tumor biopsies; (b) assessing CD8+ T cell infiltration in tumors using anti-CD8 IHC reagents; (c) assessing PD-L1 staining or staining intensity in different cell types and compartments; (d) the impact of PD-L1 staining or CD8 in the tumor periphery compared with within the tumor; (e) the impact of PD-L1 staining amplification; (f) the impact of tumor macrodissection prior to qPCR or immunomicroarray assessment; (g) the impact of tissue sample age and fixation on the assessment of PD-L1 status and association with benefit; (h) the value of on-treatment tumor biopsies for assessing clinical benefit or toxicity using the tumor characterization methods described above; (i) FDG The value of PET imaging and contrast-enhanced CT for assessing benefit from treatment or patient selection; (j) the value of tumor mutation/oncogene status (e.g., KRAS, bRAF, PI3K pathway mutation status, Met status, Her2neu status, PTEN status) in predicting benefit from the above treatments; (k) CTC number and PD-L1 characterization; (l) circulating cell types, subsets, and numbers; (m) circulating plasma/serum biomarkers; (n) racial differences; (o) smoking status; (p) FcgRIII polymorphism status; (q) immune-related polymorphism status.
研究设计。此研究是一项I期多中心试验，设计用于评估实体和液体肿瘤中使用抗PD-L1抗体(MPDL3280A)的PD-L1/PD-1途径抑制治疗的初步活性和安全性。在超过17个多国地点间登记超过250名患者。根据患者的临床状态，继续MPDL3280A治疗直至疾病进展，不可接受的毒性(即，容许有疾病进展的证据的患者继续进行研究治疗，如果他们维持他们的ECOG PS且潜在存在临床好处的话，如由调查人员评估的)。在启动研究后的多个时间对来自此研究的数据实施中期分析，包括在2013年1月10日对在研究中登记的患者，包括2012年7月1日之前登记的患者(n＝122)。此数据提示在研究中，其肿瘤表达更低水平的PD-L1的患者确实获得最小程度的来自PD-L1/PD-1途径抑制的好处，但是在其肿瘤中具有更高水平的PD-L1(特别是在肿瘤免疫浸润性细胞上测量的)的患者获得重大好处，如通过持久响应测量的。Study design. This study is a Phase I multicenter trial designed to evaluate the preliminary activity and safety of PD-L1/PD-1 pathway inhibition therapy using an anti-PD-L1 antibody (MPDL3280A) in solid and liquid tumors. More than 250 patients were enrolled across more than 17 multinational sites. Depending on the patient's clinical status, MPDL3280A treatment was continued until disease progression and unacceptable toxicity (i.e., patients with evidence of disease progression were allowed to continue study treatment if they maintained their ECOG PS and potential clinical benefit was present, as assessed by the investigator). Interim analyses of data from this study were performed at multiple times after the start of the study, including on January 10, 2013, for patients enrolled in the study, including patients enrolled before July 1, 2012 (n=122). This data suggests that in the study, patients whose tumors expressed lower levels of PD-L1 did derive minimal benefit from PD-L1/PD-1 pathway inhibition, but patients with higher levels of PD-L1 in their tumors (particularly as measured on tumor immune-infiltrating cells) derive significant benefit, as measured by durable responses.
在研究期间，如上所述收集肿瘤测量和存活状态方面的数据来评估PFS，总体存活(OS)和总体响应率(ORR)和其它测量。在基线时和大约每6周获得CT扫描。一些患者中的成像包括FDG-PET成像。关于基于血液的和基于细胞子集的生物标志物，在基线时和在研究中评估血液生物标志物。将这些和其它肿瘤生物标志物与临床结局关联起来会有助于鉴定预测性生物标志物，例如可反映药物活性或对疗法的响应的循环中的标志物。在预先规定的时间自同意的患者采集血液用于获得血清和血浆，并评估这些探索性标志物的水平。During the study, data on tumor measurements and survival status were collected as described above to assess PFS, overall survival (OS) and overall response rate (ORR) and other measurements. CT scans were obtained at baseline and approximately every 6 weeks. Imaging in some patients included FDG-PET imaging. With regard to blood-based and cell subset-based biomarkers, blood biomarkers were assessed at baseline and during the study. Correlating these and other tumor biomarkers with clinical outcomes can help identify predictive biomarkers, such as markers in the circulation that can reflect drug activity or response to therapy. Blood was collected from consenting patients at pre-specified times to obtain serum and plasma, and the levels of these exploratory markers were assessed.
实施例3--来自用抗PD-L1抗体治疗的个体的样品通过IHC的打分显示PD-L1表达Example 3 - Scoring of samples from individuals treated with anti-PD-L1 antibodies for PD-L1 expression by IHC与对治疗的响应之间的关联Association with response to treatment
如图3A所示，对来自用抗PD-L1抗体MPDL3280A治疗的I期患者的肿瘤样品分析PD-L1表达。数据集包括2012年7月1日之前登记的患者。使用上文所述IHC方案实施肿瘤样品中PD-L1状态的染色。As shown in Figure 3A, tumor samples from phase I patients treated with the anti-PD-L1 antibody MPDL3280A were analyzed for PD-L1 expression. The dataset includes patients enrolled before July 1, 2012. Staining for PD-L1 status in tumor samples was performed using the IHC protocol described above.
初步结果显示肿瘤浸润性细胞(IC)中的PD-L1表达和患者对抗PD-L1治疗的临床响应之间存在关联。特别地，对抗PD-L1治疗展示部分响应(PR)或完全响应(CR)的患者与肿瘤样品区域内PD-L1表达性肿瘤浸润性细胞的染色有关，如通过IHC检测的。肿瘤样品区域涵盖恶性细胞以及肿瘤相关基质，包括与主要肿瘤块紧邻和毗邻的免疫浸润物的区域。比较而言，对抗PD-L1治疗不展示临床响应的患者(例如展现进行性疾病(PD))的肿瘤在肿瘤样品区域内的肿瘤浸润性免疫细胞中展现更低的PD-L1表达。p<0.0001。Preliminary results show that there is a correlation between PD-L1 expression in tumor-infiltrating cells (ICs) and the patient's clinical response to anti-PD-L1 treatment. In particular, patients who showed a partial response (PR) or complete response (CR) to anti-PD-L1 treatment were associated with staining of PD-L1-expressing tumor-infiltrating cells within the tumor sample area, as detected by IHC. The tumor sample area covers malignant cells as well as tumor-associated stroma, including areas of immune infiltrates immediately adjacent to and adjacent to the main tumor mass. In comparison, tumors of patients who did not show a clinical response to anti-PD-L1 treatment (e.g., showing progressive disease (PD)) showed lower PD-L1 expression in tumor-infiltrating immune cells within the tumor sample area. p<0.0001.
还观察到患者对抗PD-L1治疗的临床响应和总免疫细胞内PD-L1表达性肿瘤浸润性免疫细胞(IC)的染色之间的关联。如图3B所示，对用抗PD-L1治疗进行的治疗展现响应性的患者与肿瘤样品内总免疫浸润物内PD-L1表达性肿瘤浸润性细胞的染色有关。通过H&E染色测定肿瘤样品内免疫浸润物的总数目。对抗PD-L1治疗展现部分响应(PR)或完全响应(CR)的患者与总免疫浸润物内PD-L1表达性肿瘤浸润性细胞的染色有关。比较而言，对抗PD-L1治疗不展示临床响应的患者(例如展现进行性疾病(PD))的肿瘤在肿瘤样品区域内肿瘤浸润性免疫细胞中展现更低的PD-L1表达。p<0.0005。The association between the patient's clinical response to anti-PD-L1 treatment and the staining of PD-L1-expressing tumor-infiltrating immune cells (IC) in total immune cells was also observed. As shown in Figure 3B, patients who showed responsiveness to treatment with anti-PD-L1 treatment were associated with the staining of PD-L1-expressing tumor-infiltrating cells in total immune infiltrates in tumor samples. The total number of immune infiltrates in tumor samples was determined by H&E staining. Patients who showed a partial response (PR) or a complete response (CR) to anti-PD-L1 treatment were associated with the staining of PD-L1-expressing tumor-infiltrating cells in total immune infiltrates. In comparison, tumors of patients who did not show a clinical response to anti-PD-L1 treatment (e.g., showing progressive disease (PD)) showed lower PD-L1 expression in tumor-infiltrating immune cells within the tumor sample area. p<0.0005.
初步数据提示PD-L1肿瘤状态可能是用于鉴定更有可能响应涉及使用抗PD-L1抗体治疗抑制PD-L1/PD-1途径的癌症疗法的患者的预测标志物。至今观察到的初始临床好处包括PR和/或CR，但是继续监测可反映别的好处，包括响应持久性，评估PFS，总体存活(OS)和总体响应率(ORR)。此初步数据为肿瘤样品中的PD-L1表达(包括肿瘤浸润性免疫细胞(IC)上的表达)可预测患者对涉及使用抗PD-L1抗体治疗抑制PD-L1/PD-1途径的癌症疗法的响应性提供支持。该数据进一步支持PD-L1肿瘤状态可确定患者会展现受益于抗PD-L1抗体治疗的可能性。Preliminary data suggest that PD-L1 tumor status may be a predictive marker for identifying patients who are more likely to respond to cancer therapies involving the inhibition of the PD-L1/PD-1 pathway using anti-PD-L1 antibody treatment. Initial clinical benefits observed to date include PR and/or CR, but continued monitoring may reflect additional benefits, including durability of response, assessment of PFS, overall survival (OS), and overall response rate (ORR). This preliminary data provides support for the ability of PD-L1 expression in tumor samples, including expression on tumor-infiltrating immune cells (ICs), to predict patient responsiveness to cancer therapies involving the inhibition of the PD-L1/PD-1 pathway using anti-PD-L1 antibody treatment. This data further supports that PD-L1 tumor status can determine the likelihood that a patient will show benefit from anti-PD-L1 antibody treatment.
实施例4--来自用抗PD-L1抗体治疗的个体的样品通过qPCR的打分显示PD-L1表达Example 4 - Scoring of samples from individuals treated with anti-PD-L1 antibodies for PD-L1 expression by qPCR与对治疗的响应之间的关联Association with response to treatment
为了评估PD-L1基因表达状态是否与响应抗PD-L1治疗的患者有关，通过qPCR测定肿瘤样品中PD-L1的基因表达水平。宏观解剖来自1期患者的组织以富集肿瘤内容。自FFPE切片分离RNA并使用基于PCR的方法学(Fluidigm)测量PD-L1基因表达。将PD-L1表达针对持家基因(GusB)标准化。To assess whether PD-L1 gene expression status is associated with patients responding to anti-PD-L1 therapy, PD-L1 gene expression levels in tumor samples were determined by qPCR. Tissue from stage 1 patients was macroscopically dissected to enrich for tumor content. RNA was isolated from FFPE sections and PD-L1 gene expression was measured using a PCR-based methodology (Fluidigm). PD-L1 expression was normalized to the housekeeping gene (GusB).
FFPE RNA分离FFPE RNA isolation
由病理学家对来自FFPE肿瘤标本的H&E载玻片验证组织诊断和肿瘤内容评估。如果总体肿瘤内容小于70-75％的话，自宏观解剖组织分离RNA以富集肿瘤内容。Histological diagnosis and tumor content assessment were verified by a pathologist on H&E slides from FFPE tumor specimens. RNA was isolated from macrodissected tissue to enrich for tumor content if overall tumor content was less than 70-75%.
使用Envirene试剂(Hardy Diagnostics，Santa Maria，CA，USA)将FFPE组织切片脱石蜡，之后制备组织裂解物。使用LC Pertuzumab FFPET RNA试剂盒(Roche Diagnosticpart#06474 969 001)实施RNA分离。用ND-2000/8000 UV-Vis分光光度计测定RNA浓度和260/280比。对于每份样品，用20ng-200ng RNA(2μL体积)使用BioMark实时PCR平台(Immune Fluid igm panel)进行基因表达分析。使用110ng-115ng RNA进行PDL1 qPCR测定法。FFPE tissue sections were deparaffinized using Envirene reagent (Hardy Diagnostics, Santa Maria, CA, USA) and tissue lysates were prepared thereafter. RNA was isolated using the LC Pertuzumab FFPET RNA kit (Roche Diagnostic part # 06474 969 001). RNA concentration and 260/280 ratio were determined using an ND-2000/8000 UV-Vis spectrophotometer. For each sample, 20 ng-200 ng RNA (2 μL volume) was used to perform gene expression analysis using a BioMark real-time PCR platform (Immune Fluid igm panel). PDL1 qPCR assay was performed using 110 ng-115 ng RNA.
PD-L1 qPCR测定法PD-L1 qPCR assay
使用由Roche Molecular Science(RMS)开发的PDL1 mRNA qRT-PCR测定法实施PD-L1 qPCR。使用由RMS提供的反应混合物和寡聚物混合物并依照制造商的说明书逆转录，扩增并检测PDL1和参照基因(GusB或TMEM55B)mRNA。热循环条件如下：1个周期的50℃5min，1个周期的95℃1min，1个周期的61℃ 30min，然后是2个周期的95℃ 15sec和61℃30sec，然后是53个周期的92℃ 15sec和61℃ 30sec，接着是1个周期的40℃ 30sec和25℃10sec。在Cobas z480分析仪(Roche)中实施反应。如下使用ΔCt(dCt)方法测定PD-L1表达水平：Ct(PD-L1)-Ct(参照基因)。数据集包括在2012年11月1日之前有样品可得的患者。PD-L1 qPCR was performed using the PDL1 mRNA qRT-PCR assay developed by Roche Molecular Science (RMS). PDL1 and reference gene (GusB or TMEM55B) mRNA were reverse transcribed, amplified, and detected using the reaction mixture and oligomer mixture provided by RMS and according to the manufacturer's instructions. The thermal cycling conditions were as follows: 1 cycle of 50°C for 5 min, 1 cycle of 95°C for 1 min, 1 cycle of 61°C for 30 min, followed by 2 cycles of 95°C for 15 sec and 61°C for 30 sec, then 53 cycles of 92°C for 15 sec and 61°C for 30 sec, followed by 1 cycle of 40°C for 30 sec and 25°C for 10 sec. The reaction was performed in a Cobas z480 analyzer (Roche). PD-L1 expression levels were determined using the ΔCt (dCt) method as follows: Ct(PD-L1)-Ct(reference gene). The dataset includes patients with samples available before November 1, 2012.
如图4所示，初步结果显示肿瘤样品中升高的PD-L1基因表达和患者对抗PD-L1治疗的临床响应之间存在关联。对抗PD-L1治疗展示部分响应(PR)或完全响应(CR)的患者与肿瘤样品内的PD-L1基因表达有关。比较而言，对抗PD-L1治疗不展示临床响应(例如展现进行性疾病(PD))的患者的肿瘤在肿瘤样品内展现更低的PD-L1基因表达。p＝0.0037。As shown in Figure 4, preliminary results show that there is an association between elevated PD-L1 gene expression in tumor samples and the patient's clinical response to anti-PD-L1 treatment. Patients who showed a partial response (PR) or complete response (CR) to anti-PD-L1 treatment were associated with PD-L1 gene expression within tumor samples. In comparison, tumors of patients who did not show a clinical response to anti-PD-L1 treatment (e.g., showed progressive disease (PD)) showed lower PD-L1 gene expression within tumor samples. p = 0.0037.
此初步数据提示PD-L1肿瘤基因表达状态可能是一种有用的生物标志物，用于预测患者对涉及使用抗PD-L1抗体抑制PD-L1/PD-1途径的癌症疗法的响应性。PD-L1肿瘤基因表达概况可以衍生自肿瘤细胞，肿瘤浸润性细胞或二者组合。These preliminary data suggest that PD-L1 tumor gene expression status may be a useful biomarker for predicting patient responsiveness to cancer therapies involving inhibition of the PD-L1/PD-1 pathway with anti-PD-L1 antibodies. The PD-L1 tumor gene expression profile can be derived from tumor cells, tumor-infiltrating cells, or a combination of both.
实施例5--通过qPCR对来自用抗PD-L1抗体治疗的个体的样品的打分显示PD-1表Example 5 - Scoring of samples from individuals treated with anti-PD-L1 antibodies to reveal PD-1 expression by qPCR达与对治疗的响应之间的关联Association between expression and response to treatment
在肿瘤样品中的PD-L1基因表达和患者临床响应之间观察到的关联以外，PD-1基因表达状态还显示与临床响应有关。如图5所示，观察到肿瘤样品中的PD-1基因表达和患者对抗PD-L1治疗的临床响应之间的关联。对抗PD-L1治疗展示部分响应(PR)的患者与肿瘤样品内的PD-1基因表达有关。比较而言，PD-1基因表达状态与对抗PD-L1治疗不展示临床响应(例如PD)的患者的关联较少。p＝0.0206。数据集包括在2012年11月1日之前有样品可得的患者。In addition to the association observed between PD-L1 gene expression in tumor samples and patient clinical responses, PD-1 gene expression status was also shown to be associated with clinical response. As shown in Figure 5, an association was observed between PD-1 gene expression in tumor samples and the patient's clinical response to anti-PD-L1 treatment. Patients who showed a partial response (PR) to anti-PD-L1 treatment were associated with PD-1 gene expression within tumor samples. In comparison, PD-1 gene expression status was less associated with patients who did not show a clinical response (e.g., PD) to anti-PD-L1 treatment. p=0.0206. The data set included patients whose samples were available before November 1, 2012.
此初步数据提示PD-1肿瘤状态可能是另一种预测标志物，用于鉴定更有可能响应涉及使用抗PD-L1抗体治疗抑制PD-L1/PD-1途径的癌症疗法的患者。肿瘤样品中的PD-1基因表达(包括肿瘤浸润性免疫细胞(IC)，肿瘤细胞或二者组合中的表达)可预测患者对涉及使用抗PD-L1抗体治疗抑制PD-L1/PD-1途径的癌症疗法的响应性。This preliminary data suggests that PD-1 tumor status may be another predictive marker for identifying patients who are more likely to respond to cancer therapies involving inhibition of the PD-L1/PD-1 pathway with anti-PD-L1 antibody treatment. PD-1 gene expression in tumor samples (including expression in tumor-infiltrating immune cells (ICs), tumor cells, or a combination of both) may predict patient responsiveness to cancer therapies involving inhibition of the PD-L1/PD-1 pathway with anti-PD-L1 antibody treatment.
此初步数据提示PD-1肿瘤状态可能是另一种预测标志物，用于鉴定更有可能响应使用抗PD-L1抗体治疗涉及抑制PD-L1/PD-1途径的癌症疗法的患者。肿瘤样品中的PD-1基因表达(包括肿瘤浸润性免疫细胞(IC)，肿瘤细胞或二者组合中的表达)可预测患者对涉及使用抗PD-L1抗体治疗抑制PD-L1/PD-1途径的癌症疗法的响应性。This preliminary data suggests that PD-1 tumor status may be another predictive marker for identifying patients who are more likely to respond to cancer therapies involving inhibition of the PD-L1/PD-1 pathway using anti-PD-L1 antibodies. PD-1 gene expression in tumor samples (including expression in tumor-infiltrating immune cells (ICs), tumor cells, or a combination of both) may predict patient responsiveness to cancer therapies involving inhibition of the PD-L1/PD-1 pathway using anti-PD-L1 antibodies.
实施例6--来自用抗PD-L1抗体治疗的个体的样品的肿瘤免疫基因签名显示与对Example 6 - Tumor immune gene signatures from samples from individuals treated with anti-PD-L1 antibodies show similarity to治疗的响应的关联Correlation with treatment response
为了测定某些免疫基因签名和响应抗PD-L1抗体治疗的患者之间是否存在关联，实施下述方案。To determine whether there is an association between certain immune gene signatures and patient response to anti-PD-L1 antibody treatment, the following protocol was performed.
Fluidigm基因表达分析Fluidigm gene expression analysis
使用BioMark实时PCR平台(Immune Fluidigm)实施基因表达分析。将2μl总RNA逆转录成cDNA，并使用Superscript III/Platinum Taq和2X反应混合物(Invitrogen)在一份反应中预扩增。预扩增反应中以0.2X Taqman测定法浓度(Applied Biosystems)的最终稀释度包括96 Taqman引物/探针套组。热循环条件如下：1个循环的50℃ 15min，1个循环的70℃ 2min，然后18个循环的95℃ 15sec和60℃ 4min。Gene expression analysis was performed using the BioMark real-time PCR platform (Immune Fluidigm). 2 μl of total RNA was reverse transcribed into cDNA and preamplified in one reaction using Superscript III/Platinum Taq and 2X reaction mix (Invitrogen). The preamplification reaction included a 96-well Taqman primer/probe set at a final dilution of 0.2X Taqman assay concentration (Applied Biosystems). Thermal cycling conditions were as follows: 1 cycle of 50°C for 15 min, 1 cycle of 70°C for 2 min, followed by 18 cycles of 95°C for 15 sec and 60°C for 4 min.
将预扩增cDNA稀释1.94倍，然后依照制造商的说明书在BioMark BMK-M-96.96平台(Fluidigm)上使用Taqman通用PCR大师级混合物(Applied Biosystems)扩增。一式三份测定所有样品。表达组中的所有Taqman测定法为FAM-MGB，而且是经Life Technologies定购的，或是订单生产的或是客户设计的，包括五种参照基因(GusB，SDHA，SP2，TMEM55B和VPS-33B)。为每份样品计算参照基因的Ct值的中值，并如下使用△Ct(dCt)方法确定表达水平：Ct(靶基因)–中值Ct(参照基因)。或者，在指明的任何时候，在将每种靶基因的Ct值针对所有基因的中值Ct值标准化之后，确定表达水平。The pre-amplified cDNA was diluted 1.94-fold and then amplified using Taqman Universal PCR Master Mix (Applied Biosystems) on a BioMark BMK-M-96.96 platform (Fluidigm) according to the manufacturer's instructions. All samples were assayed in triplicate. All Taqman assays in the expression panel were FAM-MGB and were ordered from Life Technologies, either made to order or custom-designed, and included five reference genes (GusB, SDHA, SP2, TMEM55B, and VPS-33B). The median Ct value of the reference gene was calculated for each sample, and the expression level was determined using the ΔCt (dCt) method as follows: Ct (target gene) - median Ct (reference gene). Alternatively, at any time indicated, the expression level was determined after normalizing the Ct value of each target gene to the median Ct value of all genes.
如图6所示，某些免疫基因签名和患者对抗PD-L1抗体治疗的响应之间存在关联。结果显示某些免疫基因的表达与响应抗PD-L1抗体治疗的患者有关。例如，发现T细胞活化免疫基因(包括IFN-g，CD8A，EOMES，粒酶A和CXCL9)与患者部分响应抗PD-L1治疗的有关。数据集包括在2012年11月1日之前有样品可得的患者。As shown in Figure 6, there is an association between certain immune gene signatures and patient response to anti-PD-L1 antibody treatment. The results show that the expression of certain immune genes is associated with patients who respond to anti-PD-L1 antibody treatment. For example, T cell activation immune genes (including IFN-g, CD8A, EOMES, granzyme A and CXCL9) were found to be associated with patients' partial response to anti-PD-L1 treatment. The dataset includes patients whose samples were available before November 1, 2012.
此初步数据提示已经鉴定出别的预测生物标志物，可有助于鉴定更有可能响应涉及使用抗PD-L1抗体治疗抑制PD-L1/PD-1途径的癌症疗法的患者。免疫基因签名包括但不限于IFN-g，CD8A，EOMES，粒酶A和CXCL9，而且与免疫细胞活化有关。This preliminary data suggests that additional predictive biomarkers have been identified that may help identify patients who are more likely to respond to cancer therapies involving inhibition of the PD-L1/PD-1 pathway using anti-PD-L1 antibody treatment. The immune gene signature includes, but is not limited to, IFN-g, CD8A, EOMES, granzyme A, and CXCL9, and is associated with immune cell activation.
实施例7--PD-L1和PD-L2表达与对抗PD-L1抗体治疗的响应的关联Example 7 - Correlation of PD-L1 and PD-L2 Expression with Response to Anti-PD-L1 Antibody Treatment
用PD-L1轴结合拮抗剂(诸如抗PD-L1抗体)治疗的具有局部晚期或转移性肿瘤的患者的临床活性，安全性和生物标志物。Clinical activity, safety, and biomarkers in patients with locally advanced or metastatic tumors treated with PD-L1 axis binding antagonists, such as anti-PD-L1 antibodies.
已经报告了PD-L1和PD-L2调节Th1和Th2免疫应答。肿瘤表达的PD-L1在结合活化的T细胞上的PD-1或B7.1时能介导癌症免疫逃避。抑制PD-L1结合它的受体代表一种有吸引力的策略来恢复肿瘤特异性T细胞免疫。然而，肿瘤微环境中表达的PD-L2也可结合PD-1表达性T细胞，消除它们的功能。这里记载MPDL3280A(抗PD-L1抗体，一种人单克隆抗体，其含有设计用于促进Th1驱动的应答的工程化改造的Fc域以优化功效和安全性)连同I期结果。PD-L1 and PD-L2 have been reported to regulate Th1 and Th2 immune responses. Tumor-expressed PD-L1 can mediate cancer immune evasion when it binds to PD-1 or B7.1 on activated T cells. Inhibiting PD-L1 from binding to its receptor represents an attractive strategy to restore tumor-specific T cell immunity. However, PD-L2 expressed in the tumor microenvironment can also bind to PD-1-expressing T cells, abrogating their function. Here, MPDL3280A (anti-PD-L1 antibody, a human monoclonal antibody containing an engineered Fc domain designed to promote a Th1-driven response to optimize efficacy and safety) is described, along with Phase I results.
材料和方法：在具有局部晚期或转移性实体瘤的患者中用IV q3w施用的MPDL3280A进行了一项研究，包括3+3剂量扩大和扩展组。通过RECIST v1.1评估ORR，包括u/cCR和u/cPR。在存档的肿瘤标本中，通过IHC测量PD-L1(阳性对阴性)，并通过qPCR测量PD-L2(高对低)。Materials and Methods: A study was conducted in patients with locally advanced or metastatic solid tumors using MPDL3280A administered IV every 3 weeks, including 3+3 dose escalation and expansion cohorts. ORR, including u/cCR and u/cPR, was assessed by RECIST v1.1. PD-L1 (positive vs. negative) was measured by IHC, and PD-L2 (high vs. low) was measured by qPCR in archival tumor specimens.
结果：到2013年2月1日，171名患者可评估安全性。施用的剂量包括≤1(n＝9)，3(n＝3)，10(n＝35)，15(n＝57)和20mg/kg(n＝67)。剂量扩大组中的患者没有经历剂量限制性毒性(DLT)。没有鉴定最大耐受剂量(MTD)。患者接受MPDL3280A的中值持续时间为147天(范围1-450)。41％的患者报告G3/4AE，不管归因。没有观察到急性肺炎。122名2012年7月1日之前登记的患者可评估功效。在多种肿瘤类型中观察到RECIST响应，包括NSCLC(9/37)，RCC(5/39)，黑素瘤(9/35)，CRC(1/4)和胃癌(1/1)。在非选择实体瘤中观察到21％(25/122)的ORR，响应持续时间范围1+至253+天。其他患者在明显放射线照相术进展(没有包括在ORR中)之后具有延迟的响应。24周PFS为42％。94名患者具有可评估PD-L1状态的肿瘤，且81名患者具有可评估PD-L2的肿瘤。PD-L1阳性肿瘤中的中值PD-L2表达比PD-L1阴性肿瘤要高约2倍。具有PD-L1阳性肿瘤的患者的ORR为39％(13/33)，比较而言，具有PD-L1阴性肿瘤的患者为13％(8/61)。具有PD-L2高肿瘤的患者显示27％(11/41)的ORR，比较而言，具有PD-L2低肿瘤的患者为13％(5/40)。Results: As of February 1, 2013, 171 patients were evaluable for safety. Doses administered included ≤1 (n=9), 3 (n=3), 10 (n=35), 15 (n=57), and 20 mg/kg (n=67). No patients in the dose-expansion group experienced dose-limiting toxicity (DLT). No maximum tolerated dose (MTD) was identified. The median duration of MPDL3280A treatment was 147 days (range 1-450). 41% of patients reported G3/4 AEs, regardless of attribution. No acute pneumonitis was observed. 122 patients enrolled before July 1, 2012, were evaluable for efficacy. RECIST responses were observed in multiple tumor types, including NSCLC (9/37), RCC (5/39), melanoma (9/35), CRC (1/4), and gastric cancer (1/1). An ORR of 21% (25/122) was observed in non-selected solid tumors, with a duration of response ranging from 1+ to 253+ days. Other patients had delayed responses after significant radiographic progression (not included in the ORR). The 24-week PFS was 42%. 94 patients had tumors that could be evaluated for PD-L1 status, and 81 patients had tumors that could be evaluated for PD-L2. The median PD-L2 expression in PD-L1-positive tumors was approximately 2 times higher than that in PD-L1-negative tumors. The ORR for patients with PD-L1-positive tumors was 39% (13/33), compared to 13% (8/61) for patients with PD-L1-negative tumors. Patients with PD-L2-high tumors showed an ORR of 27% (11/41), compared to 13% (5/40) for patients with PD-L2-low tumors.
MPDL3280A得到较好耐受，没有肺炎相关死亡。在多种肿瘤中观察到持久响应。PD-L1和PD-L2肿瘤状态表现出与对MPDL3280A的响应有关。此初步数据为肿瘤样品中的PD-L1以及PD-L2表达(包括肿瘤细胞，肿瘤浸润性免疫细胞(IC)上的和/或肿瘤微环境诸如基质细胞内的表达及其任意组合)可预测患者对涉及抑制PD-L1/PD-1途径的癌症疗法的响应性提供另外的支持。该数据进一步支持PD-L1和PD-L2肿瘤状态可确定患者会展现受益于PD-L1轴结合拮抗剂治疗(诸如抗PD-L1抗体)的可能性。MPDL3280A was well tolerated, with no pneumonia-related deaths. Durable responses were observed in a variety of tumors. PD-L1 and PD-L2 tumor status appeared to be associated with the response to MPDL3280A. This preliminary data provides additional support for the responsiveness of patients to cancer therapies involving inhibition of the PD-L1/PD-1 pathway by PD-L1 and PD-L2 expression in tumor samples (including expression on tumor cells, tumor-infiltrating immune cells (IC) and/or in the tumor microenvironment, such as stromal cells, and any combination thereof). The data further support the possibility that PD-L1 and PD-L2 tumor status can determine whether a patient will benefit from treatment with a PD-L1 axis-binding antagonist (such as an anti-PD-L1 antibody).
实施例8--抗PD-L1抗体治疗在响应治疗的PD-L1+患者中导致升高的T细胞活化Example 8 - Anti-PD-L1 Antibody Treatment Leads to Increased T Cell Activation in PD-L1+ Patients Responding to Treatment
评估治疗中肿瘤活检用于评估对响应抗PD-L1抗体的患者的临床好处的价值和与治疗有效性有关的药效学(PD)生物标志物的鉴定。To evaluate the value of on-treatment tumor biopsies for assessing clinical benefit in patients responding to anti-PD-L1 antibodies and to identify pharmacodynamic (PD) biomarkers associated with treatment effectiveness.
如图7所示，评估来自进行中的I期研究的来自用抗PD-L1抗体治疗的患者的系列治疗前/中肿瘤活检。使用抗CD8IHC试剂以及药效学生物标志物经基因表达分析对来自用抗PD-L1抗体治疗的罹患多种适应症(包括黑素瘤，RCC，NSCLC，H&N，CRC，胃，和乳腺癌)的患者(n＝26)的成对基线(包括治疗前的或存档的肿瘤组织)和治疗中肿瘤活检分析CD8+T细胞浸润。Serial pre-treatment/on-treatment tumor biopsies from patients treated with anti-PD-L1 antibodies from an ongoing Phase I study were evaluated as shown in Figure 7. Paired baseline (including pre-treatment or archived tumor tissue) and on-treatment tumor biopsies from patients (n=26) with various indications (including melanoma, RCC, NSCLC, H&N, CRC, gastric, and breast cancer) treated with anti-PD-L1 antibodies were analyzed for CD8+ T cell infiltration by gene expression analysis using anti-CD8 IHC reagents as well as pharmacodynamic biomarkers.
如图8(a)所示，来自响应抗PD-L1抗体治疗的患者的肿瘤样品中CD8+T细胞浸润的升高与PD-L1表达升高有关。在基线条件下，T细胞和PD-L1+肿瘤细胞可共定位，而且focalPD-L1表达可代表癌细胞和免疫细胞之间的界面(抗肿瘤T细胞攻击可以通过肿瘤或免疫细胞PD-L1表达来控制)。在第1周期第1天(C1D1)抗PD-L1抗体治疗后第4周，检测到肿瘤样品内PD-L1表达的升高连同密集的淋巴细胞浸润，特别是CD8+T细胞浸润。此CD8+T细胞增多可导致T细胞介导的肿瘤细胞杀伤，其继而可导致T细胞增殖和活化。此类活化的T细胞可释放IFN-g且还在邻近肿瘤细胞和/或免疫细胞中可诱导PD-L1表达。As shown in Figure 8(a), the increase in CD8+T cell infiltration in tumor samples from patients who responded to anti-PD-L1 antibody treatment was associated with increased PD-L1 expression. Under baseline conditions, T cells and PD-L1+ tumor cells can be co-localized, and focal PD-L1 expression can represent the interface between cancer cells and immune cells (anti-tumor T cell attack can be controlled by tumor or immune cell PD-L1 expression). At week 4 after treatment with anti-PD-L1 antibodies on day 1 of cycle 1 (C1D1), an increase in PD-L1 expression in tumor samples was detected together with dense lymphocyte infiltration, particularly CD8+T cell infiltration. This increase in CD8+T cells can lead to T cell-mediated tumor cell killing, which in turn can lead to T cell proliferation and activation. Such activated T cells can release IFN-g and can also induce PD-L1 expression in adjacent tumor cells and/or immune cells.
如图8(b)所示，发现T细胞活化标志物的数目在响应抗PD-L1抗体治疗的患者中增多。发现T细胞活化标志物(包括粒酶A，穿孔蛋白，IFN-g，TNFa和CD8)的基因表达水平在响应抗PD-L1抗体治疗的患者中在抗PD-L1抗体治疗之后与治疗前的基线水平相比升高。As shown in Figure 8(b), the number of T cell activation markers was found to be increased in patients who responded to anti-PD-L1 antibody treatment. The gene expression levels of T cell activation markers (including granzyme A, perforin, IFN-g, TNFa, and CD8) were found to be increased in patients who responded to anti-PD-L1 antibody treatment after anti-PD-L1 antibody treatment compared to the baseline levels before treatment.
此数据提示来自响应抗PD-L1抗体治疗的患者的肿瘤样品中CD8+T细胞浸润的升高与PDL-1表达的升高有关。而且，发现多种T细胞活化标志物(包括但不限于粒酶A，穿孔蛋白，IFN-g，TNFa和CD8)的表达在来自响应抗PD-L1抗体治疗的患者的肿瘤样品中升高。这些标志物可能是有用的药效学生物标志物，用于评估涉及抑制PD-L1/PD-1途径的疗法(包括使用抗PD-L1抗体)的临床好处和功效。This data suggests that increased CD8+ T cell infiltration in tumor samples from patients responding to anti-PD-L1 antibody treatment is associated with increased PDL-1 expression. Moreover, the expression of multiple T cell activation markers (including but not limited to granzyme A, perforin, IFN-g, TNFa and CD8) was found to be elevated in tumor samples from patients responding to anti-PD-L1 antibody treatment. These markers may be useful pharmacodynamic biomarkers for evaluating the clinical benefits and efficacy of therapies involving inhibition of the PD-L1/PD-1 pathway, including the use of anti-PD-L1 antibodies.
实施例9--PD-L1表达的适应性升高在响应治疗的患者中是突出的Example 9 - Adaptive increase in PD-L1 expression is prominent in patients responding to treatment
在来自响应抗PD-L1抗体治疗的患者的肿瘤样品中CD8+T细胞浸润和T细胞活化标志物表达的升高以外，在响应抗PD-L1抗体治疗的患者中还观察到肿瘤PD-L1表达的升高。In addition to increased CD8+ T cell infiltration and expression of T cell activation markers in tumor samples from patients who responded to anti-PD-L1 antibody treatment, increased tumor PD-L1 expression was also observed in patients who responded to anti-PD-L1 antibody treatment.
如图9所示，呈现成对肿瘤活检中对抗PD-L1抗体的响应的汇总。在响应抗PD-L1抗体治疗的患者中靶病变最长直径总和(SLD)有>30％降低的所有情况中(4/4名患者；100％)，肿瘤PD-L1表达也有升高，如通过PD-L1 IHC测量的。尽管响应抗PD-L1抗体治疗的患者中SLD有0-30％降低，33％的患者(2/6名患者)在治疗之后展示肿瘤PD-L1表达升高。As shown in Figure 9, a summary of the response to anti-PD-L1 antibodies in paired tumor biopsies is presented. In all cases where there was a >30% decrease in the sum of the longest diameters of target lesions (SLD) in patients responding to anti-PD-L1 antibody treatment (4/4 patients; 100%), tumor PD-L1 expression also increased, as measured by PD-L1 IHC. Despite a 0-30% decrease in SLD in patients responding to anti-PD-L1 antibody treatment, 33% of patients (2/6 patients) showed an increase in tumor PD-L1 expression after treatment.
比较而言，在不响应抗PD-L1抗体治疗且展示SLD升高0-20％的患者中，只有1/10名患者展现肿瘤PD-L1表达升高。而且，对于展示>20％SLD升高的患者，无一(0/4名患者)展示任何可测量的肿瘤PD-L1表达升高。In comparison, among patients who did not respond to anti-PD-L1 antibody treatment and showed a 0-20% increase in SLD, only 1 in 10 patients showed increased tumor PD-L1 expression. Moreover, for patients who showed a >20% increase in SLD, none (0 of 4 patients) showed any measurable increase in tumor PD-L1 expression.
此初步数据提示肿瘤中的PD-L1表达可能在响应抗PD-L1抗体治疗的患者中升高，而且此类升高可能是适应性升高，其可充当药效学生物标志物(局部肿瘤浸润性白细胞(TIL)攻击肿瘤的指示剂)且还可充当评估涉及抑制PD-L1/PD-1途径的疗法(包括使用抗PD-L1抗体)的临床好处和功效的标志物。These preliminary data suggest that PD-L1 expression in tumors may be increased in patients who respond to anti-PD-L1 antibody treatment and that such increases may be adaptive, serving as a pharmacodynamic biomarker (an indicator of tumor attack by local tumor-infiltrating leukocytes (TILs)) and also as a marker for assessing the clinical benefit and efficacy of therapies involving inhibition of the PD-L1/PD-1 pathway, including the use of anti-PD-L1 antibodies.
实施例10--抗PD-L1抗体治疗导致血液中活化的T细胞的频率升高Example 10 - Anti-PD-L1 antibody treatment leads to an increase in the frequency of activated T cells in the blood
还评估血液中抗PD-L1抗体治疗的药效学(PD)生物标志物的鉴定。Identification of pharmacodynamic (PD) biomarkers of anti-PD-L1 antibody treatment in blood was also evaluated.
流式细胞术(FACS)分析：Flow cytometry (FACS) analysis:
在肝素钠(NaHep)血液收集管中收集全血。通过缓慢倒置收集管将血液混合。用适宜抗体组合对细胞染色，并在黑暗中于室温温育30分钟。温育后，将FACSLyse添加至所有管。将管漩涡震荡，并在黑暗中于室温温育10分钟。用含1％BSA的PBS清洗细胞。清洗后，将FACSPerm2添加至所有管，并在黑暗中于室温温育10分钟。温育后，用含1％BSA的PBS清洗细胞，并在适用时与抗体一起温育。温育后，用含1％BSA的PBS清洗细胞，并在1％低聚甲醛中重悬浮。将管保存于2-8℃直至在FACSCantoII流式细胞仪上捕获。Whole blood is collected in sodium heparin (NaHep) blood collection tubes. The blood is mixed by slowly inverting the collection tube. The cells are stained with an appropriate antibody combination and incubated in the dark at room temperature for 30 minutes. After incubation, FACSLyse is added to all tubes. The tubes are vortexed and incubated in the dark at room temperature for 10 minutes. The cells are washed with PBS containing 1% BSA. After washing, FACSPerm2 is added to all tubes and incubated in the dark at room temperature for 10 minutes. After incubation, the cells are washed with PBS containing 1% BSA and incubated with antibodies when applicable. After incubation, the cells are washed with PBS containing 1% BSA and resuspended in 1% paraformaldehyde. The tubes are stored at 2-8°C until captured on a FACSCantoII flow cytometer.
如图10(a)所示，血液中的增殖T细胞(作为CD8+/Ki67+鉴定)在抗PD-L1抗体治疗过程期间增多，C2D1(第2周期第1天)时与C1D1(第1周期第1天)时相比增多约2倍。而且，如图10(b)所示，血液中同样活化的增殖T细胞(作为CD8+/HLA-DR+/Ki67+鉴定)在抗PD-L1治疗过程期间增多，而且C2D1时更加常见(增多约2倍)。As shown in Figure 10(a), proliferating T cells (identified as CD8+/Ki67+) in the blood increased during anti-PD-L1 antibody treatment, increasing approximately 2-fold on C2D1 (Day 1 of Cycle 2) compared to C1D1 (Day 1 of Cycle 1). Furthermore, as shown in Figure 10(b), similarly activated proliferating T cells (identified as CD8+/HLA-DR+/Ki67+) in the blood increased during anti-PD-L1 treatment and were more common on C2D1 (approximately 2-fold increase).
此初步数据提示血液中的活化的T细胞增殖可能在抗PD-L1抗体治疗过程期间升高，而且可充当涉及抑制PD-L1/PD-1途径的疗法(包括使用抗PD-L1抗体)的药效学生物标志物。These preliminary data suggest that activated T cell proliferation in the blood may be elevated during the course of anti-PD-L1 antibody treatment and may serve as a pharmacodynamic biomarker for therapies involving inhibition of the PD-L1/PD-1 pathway, including the use of anti-PD-L1 antibodies.
实施例11--血浆中的IL-6表达降低可能与响应治疗的患者有关Example 11 - Reduced IL-6 expression in plasma may be associated with patients responding to treatment
还评估血浆中抗PD-L1抗体治疗的药效学(PD)生物标志物的鉴定。Identification of pharmacodynamic (PD) biomarkers of anti-PD-L1 antibody treatment in plasma was also evaluated.
血浆分析Plasma analysis
将血液收集入肝素钠收集管中。通过缓慢倒置收集管将管彻底混合。随后，将收集管在冷冻离心机中以最小1500-2000x g离心15分钟。将血浆转移至聚丙烯冷冻管并保持冷冻直至分析。依照制造商的推荐使用修改的ELISA对血浆分析IL-6和其它细胞因子。Blood was collected into sodium heparin collection tubes. Thoroughly mix the tubes by gently inverting them. Subsequently, the tubes were centrifuged in a refrigerated centrifuge at a minimum of 1500-2000 x g for 15 minutes. Plasma was transferred to polypropylene cryovials and kept frozen until analysis. Plasma was analyzed for IL-6 and other cytokines using a modified ELISA according to the manufacturer's recommendations.
如图11所示，血浆中的IL-6水平降低与响应抗PD-L1抗体治疗的患者有关。具体地，在治疗过程上展现有益PR/CR响应(部分响应/完全响应)的患者还展现可测量的IL-6水平降低。As shown in Figure 11, reduced IL-6 levels in plasma are associated with patients who respond to anti-PD-L1 antibody treatment. Specifically, patients who exhibit a beneficial PR/CR response (partial response/complete response) over the course of treatment also exhibit measurable reductions in IL-6 levels.
比较而言，在治疗过程上没有受益于抗PD-L1抗体治疗的患者与血浆中的IL-6水平升高有关。如图11所示，在治疗过程上展现PD响应(进行性疾病)的患者展现可测量的IL-6水平升高。In contrast, patients who did not benefit from anti-PD-L1 antibody treatment over the course of treatment were associated with elevated IL-6 levels in plasma. As shown in Figure 11, patients who exhibited a PD response (progressive disease) over the course of treatment exhibited measurable increases in IL-6 levels.
此初步数据提示血浆中的IL-6水平可充当药效学生物标志物来评估涉及抑制PD-L1/PD-1途径的疗法(包括使用抗PD-L1抗体)的临床好处和功效。These preliminary data suggest that plasma IL-6 levels may serve as a pharmacodynamic biomarker to assess the clinical benefit and efficacy of therapies involving inhibition of the PD-L1/PD-1 pathway, including the use of anti-PD-L1 antibodies.
实施例12--来自用抗PD-L1抗体治疗的个体的样品中的肿瘤免疫基因表达显示与Example 12 - Tumor immune gene expression in samples from individuals treated with anti-PD-L1 antibodies shows对治疗的响应的关联Correlation with response to treatment
为了进一步评估别的免疫基因签名和患者对依照方案的治疗的响应性，如先前实施例6所述使用Fluidigm基因表达分析实施基因表达分析。To further assess additional immune gene signatures and patient responsiveness to per-protocol treatment, gene expression analysis was performed using the Fluidigm gene expression assay as previously described in Example 6.
如图12所示，多种别的免疫相关基因和患者对抗PD-L1抗体治疗的响应之间存在关联。具体而言，与具有进行性疾病(PD)的患者相比，观察到CTLA4和CD45RO的基因表达水平在抗PD-L1抗体治疗之后展示部分响应(PR)或完全响应(CR)的患者中更高。图12还显示，与具有PD的患者相比，观察到CX3CL1(一种趋化因子)，LGLS9(半乳凝素-9)，MIC-A和MIC-B的基因表达水平在响应抗PD-L1抗体治疗的患者(PR/CR)中更低。HK。此数据代表来自自罹患下述癌症适应症的患者收集的样品的合并基因表达水平：黑素瘤，RCC，NSCLC，CRC，胃癌，膀胱癌，卵巢癌，乳腺癌，头和颈癌，胰腺癌，肉瘤，食道癌，SCLC，多发性骨髓瘤，NHL，和子宫内膜癌。As shown in Figure 12, there is a correlation between a variety of other immune-related genes and the patient's response to anti-PD-L1 antibody treatment. Specifically, compared with patients with progressive disease (PD), it was observed that the gene expression levels of CTLA4 and CD45RO were higher in patients who showed a partial response (PR) or complete response (CR) after anti-PD-L1 antibody treatment. Figure 12 also shows that compared with patients with PD, the gene expression levels of CX3CL1 (a chemokine), LGLS9 (galectin-9), MIC-A and MIC-B were observed to be lower in patients (PR/CR) who responded to anti-PD-L1 antibody treatment. HK. This data represents the combined gene expression levels of samples collected from patients with the following cancer indications: melanoma, RCC, NSCLC, CRC, gastric cancer, bladder cancer, ovarian cancer, breast cancer, head and neck cancer, pancreatic cancer, sarcoma, esophageal cancer, SCLC, multiple myeloma, NHL, and endometrial cancer.
有趣的是，某些免疫相关基因根据疾病适应症展示不同的与患者对抗PD-L1抗体治疗的响应的关联样式。如图13所示，与具有进行性疾病(PD)的患者相比，IDO1(吲哚胺-吡咯2,3-双加氧酶)的基因表达水平在抗PD-L1抗体治疗之后展示部分响应(PR)或完全响应(CR)的黑素瘤患者中更高。然而，在NSCLC患者中，与具有PD的患者相比，IDO1的基因表达水平在抗PD-L1抗治疗体之后展示PR或CR的患者中更低。如此，有可能的是这些生物标志物可能根据疾病适应症显示不同关联概况。Interestingly, certain immune-related genes show different association patterns with patients' responses to anti-PD-L1 antibody treatment according to disease indications. As shown in Figure 13, compared with patients with progressive disease (PD), the gene expression level of IDO1 (indoleamine-pyrrole 2,3-dioxygenase) is higher in melanoma patients who show a partial response (PR) or a complete response (CR) after anti-PD-L1 antibody treatment. However, in NSCLC patients, compared with patients with PD, the gene expression level of IDO1 is lower in patients who show a PR or CR after anti-PD-L1 anti-treatment. Thus, it is possible that these biomarkers may show different association profiles according to disease indications.
这些结果提示已经鉴定出别的预测生物标志物，可有助于鉴定更有可能响应涉及诸如使用抗PD-L1抗体抑制PD-L1/PD-1途径的癌症疗法的患者。These results suggest that additional predictive biomarkers have been identified that may help identify patients who are more likely to respond to cancer therapies that involve inhibition of the PD-L1/PD-1 pathway, such as with anti-PD-L1 antibodies.
实施例13--血液中的循环T细胞上的PD-L1表达与对抗PD-L1抗体治疗的响应有关Example 13—PD-L1 expression on circulating T cells in blood is associated with response to anti-PD-L1 antibody therapy
为了评估循环T细胞上的PD-L1表达是否与患者对抗PD-L1抗体治疗的响应有关，在治疗之前60天内收集血液样品并实施FACs分析以测定样品中的T细胞上的PD-L1表达水平。To assess whether PD-L1 expression on circulating T cells is associated with patient response to anti-PD-L1 antibody treatment, blood samples were collected within 60 days before treatment and subjected to FACs analysis to measure PD-L1 expression levels on T cells in the samples.
简言之，在含有抗凝剂(例如肝素钠，EDTA，或柠檬酸盐)的管中收集来自治疗前患者的血液样品。通过缓慢倒置收集管至少3次，在搜集管中彻底混合收集的血液。将大约100μL抗凝全血移液入适当标记的测试管中。用下述一抗对血液染色，并在黑暗中于室温温育30分钟：抗CD3抗体，抗CD8抗体和抗PD-L1抗体。一抗染色后，然后使用例如氯化铵溶液裂解红细胞，然后用2mL含1％BSA的PBS清洗细胞。然后在黑暗中于室温用二抗或适当量的链霉亲合素-(染料)(如果使用生物素化一抗的话)对血细胞染色20分钟。然后再次用含1％BSA的PBS清洗细胞，在1％低聚甲醛中重悬浮，并保存于2-8℃直至在流式细胞仪上捕获它们。Briefly, blood samples from pre-treatment patients are collected in tubes containing an anticoagulant (e.g., sodium heparin, EDTA, or citrate). The collected blood is thoroughly mixed in the collection tube by slowly inverting the collection tube at least 3 times. Approximately 100 μL of anticoagulated whole blood is pipetted into appropriately labeled test tubes. The blood is stained with the following primary antibodies and incubated in the dark at room temperature for 30 minutes: anti-CD3 antibody, anti-CD8 antibody, and anti-PD-L1 antibody. After primary antibody staining, the red blood cells are then lysed using, for example, ammonium chloride solution, and the cells are then washed with 2 mL of PBS containing 1% BSA. The blood cells are then stained with a secondary antibody or an appropriate amount of streptavidin-(dye) (if a biotinylated primary antibody is used) at room temperature for 20 minutes in the dark. The cells are then washed again with PBS containing 1% BSA, resuspended in 1% paraformaldehyde, and stored at 2-8°C until they are captured on a flow cytometer.
如图14所示，循环T细胞(CD3+/CD8+)上升高的PD-L1表达和患者对抗PD-L1治疗的临床响应之间有关联。在抗PD-L1治疗之后展示部分响应(PR)或完全响应(CR)的患者与他们的循环T细胞上升高的PD-L1表达水平有关。比较而言，对抗PD-L1治疗不展示临床响应的患者(例如具有进行性疾病(PD))在他们的循环T细胞上展现更低的PD-L1表达。As shown in Figure 14, there is a correlation between elevated PD-L1 expression on circulating T cells (CD3+/CD8+) and the patient's clinical response to anti-PD-L1 treatment. Patients who showed a partial response (PR) or complete response (CR) after anti-PD-L1 treatment were associated with elevated PD-L1 expression levels on their circulating T cells. In comparison, patients who did not show a clinical response to anti-PD-L1 treatment (e.g., with progressive disease (PD)) exhibited lower PD-L1 expression on their circulating T cells.
这些结果提示循环T细胞上的PD-L1表达可能是有价值和有用的生物标志物，用于预测患者对涉及使用例如抗PD-L1抗体抑制PD-L1/PD-1途径的癌症疗法的响应性。These results suggest that PD-L1 expression on circulating T cells may be a valuable and useful biomarker for predicting patient responsiveness to cancer therapies involving inhibition of the PD-L1/PD-1 pathway using, for example, anti-PD-L1 antibodies.
实施例14--用抗PD-L1抗体治疗的个体的1a期研究和诊断选择个体中与对治疗的Example 14 - Phase 1a Study of Individuals Treated with Anti-PD-L1 Antibodies and Diagnostic Selection of Individuals with Respondents to Treatment响应的关联Correlation of responses
研究PCD4989g是一项正在进行的具有晚期实体瘤和血液学恶性肿瘤的患者中的Ia期试验，用于评估通过每3周静脉内输注施用的抗PD-L1抗体(MPDL3280A)的安全性和耐受性。该研究含有一大组NSCLC患者(n＝79，包括具有最少6个月随访的53人)。Study PCD4989g is an ongoing Phase Ia trial in patients with advanced solid tumors and hematologic malignancies evaluating the safety and tolerability of an anti-PD-L1 antibody (MPDL3280A) administered by intravenous infusion every 3 weeks. The study includes a large group of NSCLC patients (n=79, including 53 with a minimum of 6 months of follow-up).
来自研究PCD4989g的这些初步结果提示肿瘤浸润性免疫细胞中的PD-L1表达与对MPDL3280A的响应有关。NSCLC中的PD-L1阳性定义为肿瘤浸润性免疫细胞中任何强度的可辨别的PD-L1染色，覆盖≥5％的被肿瘤细胞，相关肿瘤内，和连续肿瘤周围促结缔组织生成性基质占据的肿瘤面积。下文提供NSCLC中PD-L1诊断性评估的建议标准：These preliminary results from Study PCD4989g suggest that PD-L1 expression in tumor-infiltrating immune cells is associated with response to MPDL3280A. PD-L1 positivity in NSCLC is defined as discernible PD-L1 staining of any intensity in tumor-infiltrating immune cells covering ≥5% of the tumor area occupied by tumor cells, associated intratumoral, and contiguous peritumoral desmoplastic stroma. The following are suggested criteria for the diagnostic assessment of PD-L1 in NSCLC:
到2013年4月30日数据截止，有53名具有局部晚期或转移性NSCLC的患者在2012年10月1日之前服药，有最少6个月随访。这个组的中值年龄为62岁(范围为24-84岁)，而且该组代表大量在先治疗的患者群体：84.9％具有≥2种在先系统性疗法且52.8％具有≥4种在先系统性疗法。在NSCLC中观察到显著响应，包括在多种系统性疗法失败的和/或在开始治疗之前有症状的患者中。中值响应时间为11.7周，大约90％的响应观察了24周(6个月)。具有最少6个月随访的所有NSCLC患者中的客观响应率(ORR)为22.6％(95％CI：12.3％-35.1％)。As of the data cutoff of April 30, 2013, 53 patients with locally advanced or metastatic NSCLC who were treated before October 1, 2012, had a minimum of 6 months of follow-up. The median age of this group was 62 years (range, 24-84 years), and the group represented a heavily pre-treated patient population: 84.9% had ≥2 prior systemic therapies and 52.8% had ≥4 prior systemic therapies. Significant responses were observed in NSCLC, including in patients who had failed multiple systemic therapies and/or were symptomatic before starting treatment. The median duration of response was 11.7 weeks, with approximately 90% of responses observed by 24 weeks (6 months). The objective response rate (ORR) among all NSCLC patients with a minimum of 6 months of follow-up was 22.6% (95% CI: 12.3%-35.1%).
肿瘤浸润性免疫细胞的PD-L1阳性表现出预测用MPDL3280A治疗的NSCLC患者中更高的响应。具有≥5％PD-L1阳性肿瘤浸润性免疫细胞(IHC 2/3)的NSCLC患者具有46.2％(95％CI：22.4％-74.0％)的ORR，比较而言，具有IHC 0/1的诊断概况的患者为18.2％(95％CI：8.2％-33.8％)。当使用更高的诊断阈即≥10％PD-L1阳性肿瘤浸润性免疫细胞(IHC 3)时，PD-L1阳性患者具有83.3％(95％CI：40.2％-99.1％)的ORR，比较而言，具有IHC 0/1的诊断概况的患者为17.5％(95％CI：7.8％-31.5％)。初步实验显示诊断截留IHC 2/3与用MPDL3280A治疗的NSCLC患者显著的临床好处有关。响应的患者表现出已经形成持久的抗肿瘤免疫，所有NSCLC响应在数据截止时在研究上持续170至534天。PD-L1 positivity on tumor-infiltrating immune cells was shown to predict a higher response in NSCLC patients treated with MPDL3280A. NSCLC patients with ≥5% PD-L1-positive tumor-infiltrating immune cells (IHC 2/3) had an ORR of 46.2% (95% CI: 22.4%-74.0%), compared to 18.2% (95% CI: 8.2%-33.8%) for patients with an IHC 0/1 diagnostic profile. When a higher diagnostic threshold of ≥10% PD-L1-positive tumor-infiltrating immune cells (IHC 3) was used, PD-L1-positive patients had an ORR of 83.3% (95% CI: 40.2%-99.1%), compared to 17.5% (95% CI: 7.8%-31.5%) for patients with an IHC 0/1 diagnostic profile. Preliminary experiments showed that diagnostic IHC cutoff 2/3 was associated with significant clinical benefit in NSCLC patients treated with MPDL3280A. Responding patients demonstrated the development of durable anti-tumor immunity, with all NSCLC responses lasting 170 to 534 days on study at data cutoff.
实施例15--来自用抗PD-L1抗体治疗的个体的样品的肿瘤免疫基因签名显示与对Example 15 - Tumor immune gene signatures from samples from individuals treated with anti-PD-L1 antibodies show similarity to治疗的响应的关联Correlation with treatment response
在来自用MPDL3280A治疗的患者的肿瘤和血液中评估免疫学药效学作用。在治疗中，响应性肿瘤显示肿瘤细胞表达和肿瘤浸润性免疫细胞PD-L1表达，CD8+T细胞和Th1优势免疫浸润物浸润升高，为适应性PD-L1上调提供证据。非响应者显示最小程度的肿瘤CD8+T细胞浸润和T细胞活化缺失(通过粒酶，穿孔蛋白和EOMES表达测量)。Immunological pharmacodynamic effects were assessed in tumors and blood from patients treated with MPDL3280A. During treatment, responding tumors showed increased expression of PD-L1 on tumor cells and tumor-infiltrating immune cells, as well as elevated infiltration of CD8+ T cells and Th1-dominant immune infiltrates, providing evidence for adaptive PD-L1 upregulation. Non-responders showed minimal tumor CD8+ T cell infiltration and loss of T cell activation (measured by granzyme, perforin, and EOMES expression).
如图15所示，对MPDL3280A的抗肿瘤响应与T细胞生物学相关标志物有关。具体地，在基线时在肿瘤组织中检测到更高的细胞毒性Th1细胞，IFN-g和T细胞运输标志物的基因表达，而且这与MPDL3280A活性有关。例如，发现T细胞活化免疫基因(包括IFN-g，CD8A，粒酶B和CXCL9)与部分响应/完全响应MPDL3280A治疗的患者有关。数据集包括在2013年6月1日之前有样品可得的患者。As shown in Figure 15, the anti-tumor response to MPDL3280A was associated with markers related to T cell biology. Specifically, higher gene expression of cytotoxic Th1 cells, IFN-g, and T cell trafficking markers was detected in tumor tissue at baseline, and this was associated with MPDL3280A activity. For example, T cell activation immune genes (including IFN-g, CD8A, granzyme B, and CXCL9) were found to be associated with partial/complete responses in patients treated with MPDL3280A. The dataset includes patients for whom samples were available before June 1, 2013.
如图16所示，MPDL3280A导致具有响应治疗的黑素瘤的患者中升高的T细胞活化。具体地，发现一些T细胞活化标志物在响应MPDL3280A的患者中升高，包括粒酶A，粒酶B，穿孔蛋白，EOMES，IFN-g，TNF，CXCL9，CXCL10，CD8A和ICOS。As shown in Figure 16, MPDL3280A resulted in elevated T cell activation in patients with melanoma that responded to treatment. Specifically, several T cell activation markers were found to be elevated in patients who responded to MPDL3280A, including granzyme A, granzyme B, perforin, EOMES, IFN-g, TNF, CXCL9, CXCL10, CD8A, and ICOS.
比较而言，具有不响应MPDL3280A的黑素瘤的患者展现低频率的肿瘤内T细胞且缺乏T细胞活化，如图17所示。In comparison, patients with melanoma that did not respond to MPDL3280A exhibited low frequencies of intratumoral T cells and lacked T cell activation, as shown in FIG17 .
还对循环生物标志物评估它们与临床结局的联系。在所有患者中评估时，CD8+HLA-DR+Ki67+T细胞在血液中的频率在第一剂MPDL3280A之后不久升高并到周期2结束时返回基线水平，代表PD-L1抑制的瞬时药效学测量。如图18所示，MPDL3280A导致活化的T细胞在血液中的频率瞬时升高，而且提示CD8+HLA-DR+Ki67+T细胞可能是MPDL3280A治疗的一种潜在药效学生物标志物。Circulating biomarkers were also evaluated for their association with clinical outcomes. When assessed in all patients, the frequency of CD8+HLA-DR+Ki67+ T cells in the blood increased shortly after the first dose of MPDL3280A and returned to baseline levels by the end of Cycle 2, representing a transient pharmacodynamic measure of PD-L1 inhibition. As shown in Figure 18, MPDL3280A resulted in a transient increase in the frequency of activated T cells in the blood, suggesting that CD8+HLA-DR+Ki67+ T cells may be a potential pharmacodynamic biomarker of MPDL3280A treatment.
观察到CD4+/ICOS+T细胞中的显著波动，以及此T细胞群体中延迟的与响应有关的增多和与疾病进展有关的减少(周期3后发生)，如图19所示。CD4+/ICOS+T细胞的增多可能反映MPDL3280A治疗后发起强CD8+抗肿瘤T细胞响应的患者中T辅助细胞响应的辅助活化。A significant fluctuation in CD4+/ICOS+ T cells was observed, as well as a delayed response-associated increase and disease progression-associated decrease in this T cell population (occurring after cycle 3), as shown in Figure 19. The increase in CD4+/ICOS+ T cells likely reflects the auxiliary activation of T helper cell responses in patients who mounted strong CD8+ anti-tumor T cell responses after MPDL3280A treatment.
而且，PD-L1表达的适应性升高在响应MPDL3280A的患者中是突出的。如图20所示，呈现了配对肿瘤活检中对MPDL3280A的响应的汇总。在响应MPDL3280A的患者中，肿瘤细胞PD-L1表达以及肿瘤浸润性免疫细胞PDL-1表达二者中均存在升高，如通过PD-L1IHC测定法测量的。Furthermore, an adaptive increase in PD-L1 expression was prominent in patients responding to MPDL3280A. As shown in Figure 20, a summary of responses to MPDL3280A in paired tumor biopsies is presented. In patients responding to MPDL3280A, there was an increase in both tumor cell PD-L1 expression and tumor-infiltrating immune cell PDL-1 expression, as measured by the PD-L1 IHC assay.
实施例16--CTLA4和Fractalkine表达与抗PD-L1抗体治疗之后的响应或进展的关Example 16 - Correlation between CTLA4 and Fractalkine expression and response or progression after anti-PD-L1 antibody treatment联Alliance
在多种癌症类型间，展现Th1相关基因表达(CTLA4)存在和fractalkine/CX3CL1缺失的治疗前肿瘤与活性有关。具体地，CTLA4的表达与对MPDL3280A的响应强相关，而治疗前肿瘤中fractalkine/CX3CL1的表达与对MPDL3280A的进展强相关，如图21所示。Across multiple cancer types, pre-treatment tumors exhibiting expression of Th1-associated genes (CTLA4) and loss of fractalkine/CX3CL1 correlated with activity. Specifically, CTLA4 expression was strongly correlated with response to MPDL3280A, while fractalkine/CX3CL1 expression in pre-treatment tumors was strongly correlated with progression to MPDL3280A, as shown in Figure 21.
CTLA4的作用较好地确立为一种由T细胞表达，可导致抑制进一步T细胞活化的因子。不同肿瘤类型间响应MPDL3280A的患者中的更高治疗前CTLA4表达的关联提示CTLA4充当抗癌免疫响应中的一种重要反馈机制，而且代表活跃T细胞免疫和炎症的一种标志物。然而，在外周中，CTLA4作为阴性调节物的功能性作用看来不如PD-L1重要。The role of CTLA4 is well established as a factor expressed by T cells that can lead to inhibition of further T cell activation. The association of higher pre-treatment CTLA4 expression in patients who respond to MPDL3280A across different tumor types suggests that CTLA4 serves as an important feedback mechanism in the anti-cancer immune response and represents a marker of active T cell immunity and inflammation. However, in the periphery, the functional role of CTLA4 as a negative regulator appears to be less important than that of PD-L1.
对MPDL3280A经历疾病进展的患者中的更高治疗前fractalkine(CX3CL1)表达的关联也是出乎意料的，因为此趋化因子通常与驱动T细胞浸润有关。然而，以它的未切割形式，fractalkine诱导淋巴细胞粘附至内皮细胞并因此可实际约束T细胞进入肿瘤床中。对于用MPDL3280A治疗的患者，肿瘤中的fractalkine表达和进行性疾病的联系提示fractalkine表达也可能代表缺乏活跃免疫响应的肿瘤的一种反馈机制或代表一种活跃肿瘤免疫响应阻抑性因子。The association of higher pre-treatment fractalkine (CX3CL1) expression in patients with MPDL3280A experiencing disease progression was also unexpected, as this chemokine is typically associated with driving T cell infiltration. However, in its uncleaved form, fractalkine induces lymphocyte adhesion to endothelial cells and can therefore actually restrict T cells from entering the tumor bed. For patients treated with MPDL3280A, the association of fractalkine expression in tumors with progressive disease suggests that fractalkine expression may also represent a feedback mechanism for tumors lacking an active immune response or represent a suppressive factor for an active tumor immune response.
这些结果提示CTLA4和fractalkine可能是有价值的预测生物标志物，有助于鉴定更有可能响应涉及抑制PD-L1/PD-1途径的癌症疗法(诸如使用抗PD-L1抗体)的患者。These results suggest that CTLA4 and fractalkine may be valuable predictive biomarkers to help identify patients who are more likely to respond to cancer therapies that involve inhibition of the PD-L1/PD-1 pathway, such as the use of anti-PD-L1 antibodies.
实施例17--六种癌症类型间的肿瘤浸润性淋巴细胞签名及它们与疾病预后因子Example 17 - Tumor-infiltrating lymphocyte signatures across six cancer types and their association with disease prognostic factors的联系Contact
为了进一步评估和了解可调控或抑制抗肿瘤免疫并因此促成对免疫调控疗法的响应或抗性的因素的复杂性，使用多种高度灵敏的免疫基因表达测定法(iCHIP)(使用Fluidigm Biomark平台)来调查六种癌症适应症间免疫响应的质量，包括CRC(n＝48)，BC(n＝126)，NSCLC(n＝51)，黑素瘤(n＝35)，RCC(n＝48)，和膀胱癌(n＝42)。iCHIP平台由96种基因组成，它们代表与IFNg途径，细胞毒性T细胞，Th2细胞，T效应细胞，T效应细胞，T调节细胞，Th17细胞，髓样细胞，树突细胞，NK细胞，B细胞和免疫检查点标志物有关的签名。To further assess and understand the complexity of factors that can regulate or inhibit anti-tumor immunity and thus contribute to response or resistance to immunomodulatory therapies, a highly sensitive immune gene expression assay (iCHIP) (using the Fluidigm Biomark platform) was used to investigate the quality of immune responses across six cancer indications, including CRC (n=48), BC (n=126), NSCLC (n=51), melanoma (n=35), RCC (n=48), and bladder cancer (n=42). The iCHIP platform consists of 96 genes representing signatures related to the IFNg pathway, cytotoxic T cells, Th2 cells, T effector cells, T effector cells, T regulatory cells, Th17 cells, myeloid cells, dendritic cells, NK cells, B cells, and immune checkpoint markers.
自福尔马林固定、石蜡包埋的存档组织(衍生自临床收集或在进行中的MPDL3280A(抗PD-L1抗体)I期研究中收集)提取RNA。自伦理审查委员会获得关于生物标志物的探索性评估的适当的患者知情同意。RNA was extracted from formalin-fixed, paraffin-embedded archival tissues (derived from clinical collections or collected in the ongoing MPDL3280A (anti-PD-L1 antibody) Phase I study). Appropriate patient informed consent for the exploratory evaluation of biomarkers was obtained from the institutional review board.
如图22所示，显示了与Teff(T效应细胞)，Treg(T调节细胞)，和Th17有关的基因签名。Teff细胞通过下述基因簇来定义：CD8A，GZMB，IFNg，EOMES，GZMA，穿孔蛋白；Treg细胞通过下述基因簇来定义：FOXP3；而Th17细胞通过下述基因簇来定义：RORC，IL17F，IL17A。As shown in Figure 22, gene signatures associated with Teff (T effector cells), Treg (T regulatory cells), and Th17 are shown. Teff cells are defined by the following gene clusters: CD8A, GZMB, IFNg, EOMES, GZMA, perforin; Treg cells are defined by the following gene clusters: FOXP3; and Th17 cells are defined by the following gene clusters: RORC, IL17F, IL17A.
免疫基因表达分析显示适应症间免疫阻抑性和免疫响应性因子和细胞类型的独特样式。适应症(包括三重阴性乳腺癌(TNBC)，NSCLC和膀胱癌)代表IFN-g签名的最高流行性，CRC和激素受体阳性乳腺癌构成具有最低表达的疾病。在TNBC中的高IFN-g签名以外，与黑素瘤(代表最高的IFN-g:Treg基因表达比)相比时，这种亚型的乳腺癌还由高Treg签名组成。与所有其它适应症相比，Th17基因签名在CRC中最流行。这些基因签名与疾病阶段，结局(在可得时)和其它疾病特异性已知预后因子(包括分子亚型和KRAS，BRAF，PIK3CA和EGFR中的突变)的联系当前正在进行中。Immune gene expression analysis shows a unique pattern of immune suppression and immune response factors and cell types between indications. Indications (including triple negative breast cancer (TNBC), NSCLC and bladder cancer) represent the highest prevalence of IFN-g signatures, and CRC and hormone receptor positive breast cancer constitute the diseases with the lowest expression. In addition to the high IFN-g signature in TNBC, this subtype of breast cancer is also composed of a high Treg signature when compared with melanoma (representing the highest IFN-g:Treg gene expression ratio). Compared with all other indications, Th17 gene signatures are the most popular in CRC. The connection between these gene signatures and disease stage, outcome (when available) and other disease-specific known prognostic factors (including molecular subtypes and mutations in KRAS, BRAF, PIK3CA and EGFR) is currently underway.
特别地，在RCC患者的一个组中，在不响应抗PD-L1治疗的患者中观察到朝向更高的IL17F的肿瘤基因表达的趋势，如图23所示，尽管PD-L1(CD274)的肿瘤基因表达在抗PD-L1治疗的响应者中更高。In particular, in one group of RCC patients, a trend toward higher tumor gene expression of IL17F was observed in patients who did not respond to anti-PD-L1 treatment, as shown in Figure 23, although tumor gene expression of PD-L1 (CD274) was higher in responders to anti-PD-L1 treatment.
而且，在适应症(黑素瘤，肺癌，RCC)间，IL-17F的肿瘤基因表达在对抗PD-L1具有晚期响应(在6个月疗法后响应)的患者中更高，如图24所示。Furthermore, across indications (melanoma, lung cancer, RCC), tumor gene expression of IL-17F was higher in patients with late responses (responses after 6 months of therapy) to anti-PD-L1, as shown in FIG24 .
如此，有可能的是某些生物标志物可根据疾病阶段和涉及抑制PD-L1/PD-1途径的疗法的时机显示不同关联概况。Thus, it is possible that certain biomarkers may show different association profiles depending on the disease stage and the timing of therapies involving inhibition of the PD-L1/PD-1 pathway.
实施例18--通过MPDL3280A抑制PD-L1在具有转移性尿路上皮膀胱癌(UBC)的患者Example 18 - Inhibition of PD-L1 by MPDL3280A in Patients with Metastatic Urothelial Bladder Cancer (UBC)中导致临床活性Leading to clinical activity
转移性UBC与较差的预后和有限的治疗选项有关。PD-L1表达在这种疾病中流行，而且可通过结合它的受体PD-1和B7.1保护癌细胞免于免疫介导的破坏。Metastatic UBC is associated with a poor prognosis and limited treatment options. PD-L1 expression is prevalent in this disease and can protect cancer cells from immune-mediated destruction by binding to its receptors PD-1 and B7.1.
在I期研究中，UBC患者接受MPDL3280A 15mg/kg IV q3w，持续1年。通过RECISTv1.1评估客观响应率(ORR；包括未确认的响应)。平行地，评估肿瘤和循环生物标志物以研究MPDL3280A免疫关联。In a Phase I study, UBC patients received MPDL3280A 15 mg/kg IV every 3 weeks for 1 year. Objective response rate (ORR; including unconfirmed responses) was assessed by RECIST v1.1. In parallel, tumor and circulating biomarkers were assessed to investigate the immune association of MPDL3280A.
到2013年9月19日，用MPDL3280A治疗了31名PD-L1+UBC患者。患者为84％男性，中值年龄66岁(42-86)，57％为ECOG PS 1且68％具有内脏转移。71％接受≥2种在先疗法；97％接受在先基于铂的化疗。到数据截止时患者接受MPDL3280A的中值持续时间为43天(1-153)。在≥2名患者中发生的G1-4治疗相关AE为发热，贫血，食欲减退，疲劳和恶心。3.2％的患者中发生相关G3-4AE。没有免疫相关AE。20名患者在分析时可评估功效，中值随访为2.8个月(1.4-5)。ORR为50％(1例CR和9例PR)，中值响应时间为43天(39-82)，对应于第一次放射线照相术评估且包括在基线时具有CNS，肺和骨转移的患者。所有响应者在临床截止时仍然是响应的。As of September 19, 2013, 31 PD-L1+UBC patients had been treated with MPDL3280A. 84% of the patients were male, with a median age of 66 years (42-86), 57% were ECOG PS 1 and 68% had visceral metastases. 71% received ≥2 prior therapies; 97% received prior platinum-based chemotherapy. The median duration of MPDL3280A treatment in patients at the time of data cutoff was 43 days (1-153). G1-4 treatment-related AEs occurring in ≥2 patients were fever, anemia, decreased appetite, fatigue and nausea. Related G3-4 AEs occurred in 3.2% of patients. There were no immune-related AEs. 20 patients were evaluable for efficacy at the time of analysis, with a median follow-up of 2.8 months (1.4-5). The ORR was 50% (1 CR and 9 PRs), with a median duration of response of 43 days (range, 39-82), corresponding to the first radiographic assessment and including patients with CNS, lung, and bone metastases at baseline. All responders remained responsive at the time of clinical cutoff.
已经观察到肿瘤浸润性免疫细胞上的PD-L1状态和对抗PD-L1治疗的响应之间的联系，而且正在进行进一步评估以确定肿瘤细胞上的PD-L1状态和对抗PD-L1治疗的响应的联系。An association between PD-L1 status on tumor-infiltrating immune cells and response to anti-PD-L1 therapy has been observed, and further evaluation is underway to determine the association between PD-L1 status on tumor cells and response to anti-PD-L1 therapy.
治疗导致循环Ki-67+CD8+T细胞的瞬时增多，代表UBC患者中对PD-1/PD-L1途径抑制剂疗法的活性的一种潜在药效学(PD)生物标志物。如图25所示，循环Ki-67+CD8+T细胞在MPDL3280A治疗期间展现瞬时升高。Treatment resulted in a transient increase in circulating Ki-67+CD8+ T cells, representing a potential pharmacodynamic (PD) biomarker of activity against PD-1/PD-L1 pathway inhibitor therapy in UBC patients. As shown in Figure 25, circulating Ki-67+CD8+ T cells exhibited a transient increase during MPDL3280A treatment.
治疗还导致IFN-g信号传导上游的血浆蛋白质(诸如IL-18)的瞬时升高，代表活性的另一种PD生物标志物，如图26所示。而且，基线血浆MCP-1在具有部分响应/完全响应(PR/CR)的患者中更低。IL-18和MCP-1均在单核细胞(髓样细胞的一种成分)中优势表达(见图26)。Treatment also resulted in a transient increase in plasma proteins upstream of IFN-g signaling, such as IL-18, representing another PD biomarker of activity, as shown in Figure 26. Moreover, baseline plasma MCP-1 was lower in patients with partial response/complete response (PR/CR). Both IL-18 and MCP-1 are predominantly expressed in monocytes, a component of myeloid cells (see Figure 26).
来自治疗前肿瘤的基因表达数据显示进展的患者具有按比例更高的髓样基因签名。如图27所示，具有进行性疾病(PD)的患者展示升高水平的IL-8，CCL2，和IL1B，而且这些与在髓样类型细胞(例如单核细胞，树突细胞)中优势存在有关。Gene expression data from pre-treatment tumors showed that patients who progressed had a proportionally higher myeloid gene signature. As shown in Figure 27, patients with progressive disease (PD) displayed elevated levels of IL-8, CCL2, and IL1B, and these were associated with a predominant presence in myeloid cell types (e.g., monocytes, dendritic cells).
MPDL3280A在这个治疗前PD-L1+UBC群体中得到较好耐受。50％接受治疗的患者响应治疗。响应是快速的和进行中的。生物标志物分析揭示PD标志物，以及对疗法的抗性的潜在机制的标志物。MPDL3280A was well tolerated in this pre-treatment PD-L1+ UBC population. 50% of treated patients responded to treatment. Responses were rapid and ongoing. Biomarker analysis revealed markers of PD, as well as potential mechanisms of resistance to therapy.
实施例19--升高的可溶性PD-L1水平在响应治疗的患者中是突出的Example 19 - Elevated soluble PD-L1 levels are prominent in patients who respond to treatment
在来自在进行中的1期研究中响应抗PD-L1抗体治疗的患者的血液样品还观察到升高的可溶性PD-L1基线血浆水平。Elevated baseline plasma levels of soluble PD-L1 were also observed in blood samples from patients who responded to anti-PD-L1 antibody treatment in an ongoing Phase 1 study.
将血液收集入肝素钠收集管中。通过缓慢倒置收集管将管彻底混合。随后，将收集管在冷冻离心机中以最小1500-2000x g离心15分钟。将血浆转移至聚丙烯冷冻管并保持冷冻直至分析。依照制造商的推荐使用修改的ELISA对血浆分析IL-6和其它细胞因子。Blood was collected into sodium heparin collection tubes. Thoroughly mix the tubes by gently inverting them. Subsequently, the tubes were centrifuged in a refrigerated centrifuge at a minimum of 1500-2000 x g for 15 minutes. Plasma was transferred to polypropylene cryovials and kept frozen until analysis. Plasma was analyzed for IL-6 and other cytokines using a modified ELISA according to the manufacturer's recommendations.
如图28所示，发现靶病变最长直径总和(SLD)>＝30％降低响应抗PD-L1抗体治疗的RCC患者与他们血浆样品中与只展示>＝20％SLD降低的患者相比更高的可溶性PD-L1(sPDL1)水平有关。As shown in Figure 28, it was found that RCC patients who responded to anti-PD-L1 antibody treatment with a >= 30% reduction in the sum of the longest diameters of target lesions (SLD) were associated with higher soluble PD-L1 (sPDL1) levels in their plasma samples compared to patients who only showed a >= 20% reduction in SLD.
此初步数据提示对于预测患者对涉及抑制PD-L1/PD-1途径的癌症疗法的响应性，血浆中的可溶性PD-L1表达可能是有价值和有用的生物标志物。These preliminary data suggest that soluble PD-L1 expression in plasma may be a valuable and useful biomarker for predicting patient responsiveness to cancer therapies involving inhibition of the PD-L1/PD-1 pathway.
实施例20--肿瘤浸润性免疫细胞和肿瘤细胞上的PD-L1表达与对抗PD-L1治疗的Example 20 - PD-L1 expression on tumor-infiltrating immune cells and tumor cells and response to anti-PD-L1 therapy响应的联系Responsive Contact
在进行中的1期研究中，观察到对抗PD-L1治疗的响应与肿瘤浸润性免疫细胞(IC)和肿瘤细胞二者中的PD-L1表达的清楚联系。如图29所示，在具有NSCLC的患者中(图29(a))以及在所有患者中(图29(b))观察到肿瘤浸润性免疫细胞(IC)中的PD-L1表达和对抗PD-L1治疗的响应之间的联系。类似地，在具有NSCLC的患者中(图30(a))以及所有患者中(图30(b))观察到肿瘤细胞(TC)中的PD-L1表达和对抗PD-L1治疗的响应之间的联系。In the ongoing Phase 1 study, a clear connection was observed between the response to anti-PD-L1 treatment and the expression of PD-L1 in both tumor-infiltrating immune cells (IC) and tumor cells. As shown in Figure 29, the connection between PD-L1 expression in tumor-infiltrating immune cells (IC) and the response to anti-PD-L1 treatment was observed in patients with NSCLC (Figure 29 (a)) and in all patients (Figure 29 (b)). Similarly, the connection between PD-L1 expression in tumor cells (TC) and the response to anti-PD-L1 treatment was observed in patients with NSCLC (Figure 30 (a)) and in all patients (Figure 30 (b)).
尽管为了理解清楚的目的，上述发明已经通过例示和实施例较为详细地描述，描述和实施例不应解释为限制范围。通过提及明确完整收录本文中引用的所有专利和科学文献的公开内容。Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.
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