Therapeutic and diagnostic methods for cancer

Abstract

To provide therapeutic and diagnostic methods and compositions for cancer, for example, bladder cancer.SOLUTION: The invention provides methods of treating bladder cancer, methods of determining whether a patient suffering from bladder cancer is likely to respond to treatment comprising a PD-L1 axis binding antagonist, methods of predicting responsiveness of a patient suffering from bladder cancer to treatment comprising a PD-L1 axis binding antagonist, and methods of selecting a therapy for a patient suffering from bladder cancer based on expression levels of a biomarker of the invention (e.g., PD-L1 expression levels in tumor-infiltrating immune cells in tumor samples obtained from patients) and / or on tumor sample subtype determination.SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy, created on May 20, 2016, is named 50474-116WO3_Sequence_Listing_5_20_16_ST25 and is 23,626 bytes in size.

[0002] Provided herein are therapeutic and diagnostic methods and compositions for conditions such as cancer (e.g., bladder cancer (e.g., urothelial bladder cancer)) and methods using PD-L1 axis binding antagonists. In particular, provided herein are biomarkers, therapeutic methods, products, diagnostic kits, and detection methods for patient selection and diagnosis. [Background technology]

[0003] Cancer remains one of the most deadly threats to human health. Cancer or malignant tumors metastasize and grow rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult. In the United States, cancer affects approximately 1.3 million new patients each year and is the second leading cause of death after heart disease, accounting for approximately one in four deaths. Solid tumors are responsible for the majority of such deaths. Bladder cancer is the fifth most common malignancy worldwide, with nearly 400,000 newly diagnosed cases and approximately 150,000 associated deaths reported per year. In particular, metastatic urothelial bladder cancer is associated with poor prognosis, represents a major unmet medical need, and has few effective treatments to date.

[0004] Programmed death-ligand 1 (PD-L1) is a protein that has been implicated in suppressing immune system responses in chronic infection, pregnancy, tissue allografts, autoimmune diseases, and cancer. PD-L1 regulates immune responses by binding to an inhibitory receptor known as programmed death 1 (PD-1), which is expressed on the surface of T cells, B cells, and monocytes. PD-L1 also negatively regulates T cell function through its interaction with another receptor, B7-1. Formation of PD-L1 / PD-1 and PD-L1 / B7-1 complexes negatively regulates T cell receptor signaling, subsequently resulting in downregulation of T cell activation and suppression of antitumor immune activity.

[0005] Despite significant advances in the treatment of cancer, such as bladder cancer (eg, urothelial bladder cancer), there remains a need for improved therapeutic and diagnostic methods. Summary of the Invention

[0006] The present invention provides therapeutic and diagnostic methods and compositions for cancer, eg, bladder cancer (eg, urothelial bladder cancer).

[0007] In a first aspect, the invention features a method of treating a patient suffering from bladder cancer, the method comprising administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that make up about 1% or more (e.g., about 1%, about 2%, about 3%, or about 4% or more) of the tumor sample. In some embodiments, a tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that make up about 1% to about 65% or more (e.g., about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 65%) of the tumor sample. In some embodiments, the median overall survival of patients administered a therapeutically effective amount of a PD-L1 axis-binding antagonist is at least about 8 months. In some embodiments, the median overall survival of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 8.8 months. In other embodiments, the median overall survival of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 7 to about 11 months. In another embodiment, the response rate of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is about 10% to about 35% (e.g., about 10% to about 20%, about 20% to about 30%, about 30% to about 35%). In yet another embodiment, the response rate of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is about 13% to about 24%. In other embodiments, the response rate of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 12%. In other embodiments, the response rate of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 21%. In some embodiments, the response rate in patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is at least about 18%.

[0008] In some embodiments, a tumor sample obtained from a patient is determined to have a detectable expression level of PD-L1 in tumor infiltrating immune cells that comprise about 5% or more (e.g., about 5%, about 6%, about 7%, about 8%, or about 9% or more) of the tumor sample. In some embodiments, the median overall survival of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 9 months. In some embodiments, the median overall survival of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 11 months. In some embodiments, the median progression-free survival of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 4 months. In some embodiments, the median progression-free survival of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 3 to about 6 months. In another embodiment, the response rate of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is about 10% to about 45%. In some embodiments, the response rate of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is about 35% to about 45%. In yet another embodiment, the response rate of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is about 18% to about 37%. In other embodiments, the response rate of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 14%. In other embodiments, the response rate of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 25%. In some embodiments, the response rate of patients administered a therapeutically effective amount of a PD-L1 axis binding antagonist is at least about 27%.

[0009] In some embodiments, the tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise about 10% or more (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, or about 60% or more) of the tumor sample. In other embodiments, the tumor sample obtained from the patient has been determined to be a luminal subtype tumor.

[0010] In a second aspect, the invention features a method of treating a patient suffering from bladder cancer, the method comprising administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to be a luminal subtype tumor.

[0011] In some embodiments of the first and second aspects, the expression level of at least one of CDKN2A, GATA3, FOXA1, and ERBB2 in a tumor sample obtained from a patient is determined to be increased compared to a reference level of the at least one gene, and / or the expression level of at least one of FGFR3, KRT5, KRT14, and EGFR in a tumor sample obtained from a patient is determined to be decreased compared to a reference level of the at least one gene. In some embodiments, the expression level of CDKN2A, GATA3, FOXA1, and ERBB2 in a tumor sample obtained from a patient is determined to be increased compared to a reference level of the gene, and / or the expression level of FGFR3, KRT5, KRT14, and EGFR in a tumor sample obtained from a patient is determined to be decreased compared to a reference level of the gene. In some embodiments, the expression levels of CDKN2A, GATA3, FOXA1, and ERBB2 in the tumor sample obtained from the patient are determined to be increased compared to the reference level of the genes, and the expression levels of FGFR3, KRT5, KRT14, and EGFR in the tumor sample obtained from the patient are determined to be decreased compared to the reference level of the genes. In other embodiments, the expression levels of miR-99a-5p or miR100-5p in the tumor sample obtained from the patient are determined to be increased compared to the reference level of microRNA (miRNA). In some embodiments, the expression levels of miR-99a-5p and miR100-5p in the tumor sample obtained from the patient are determined to be increased compared to the reference level of miRNA. In yet other embodiments, the expression level of at least one of CD8A, GZMA, GZMB, IFNG, CXCL9, CXCL10, PRF1, and TBX21 in the tumor sample obtained from the patient is determined to be increased compared to the reference level of the at least one gene. In some embodiments, the expression levels of at least CXCL9 and CXCL10 in a tumor sample obtained from a patient are determined to be increased relative to reference levels of the genes, hi other embodiments, the luminal subtype tumor is a luminal cluster II subtype tumor.

[0012] In a third aspect, the invention features a method of determining whether a patient suffering from bladder cancer is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining the expression level of PD-L1 in tumor-infiltrating immune cells in a tumor sample obtained from the patient, wherein a detectable expression level of PD-L1 in tumor-infiltrating immune cells comprising about 1% or more of the tumor sample indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist.

[0013] In a fourth aspect, the invention features a method of predicting responsiveness of a patient suffering from bladder cancer to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining the expression level of PD-L1 in tumor-infiltrating immune cells in a tumor sample obtained from the patient, wherein a detectable expression level of PD-L1 in tumor-infiltrating immune cells constituting about 1% or more of the tumor sample indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist.

[0014] In a fifth aspect, the invention features a method of selecting a therapy comprising a PD-L1 axis-binding antagonist for a patient suffering from bladder cancer, the method comprising determining the expression level of PD-L1 in tumor-infiltrating immune cells in a tumor sample obtained from the patient, and selecting a therapy comprising a PD-L1 axis-binding antagonist for the patient based on the detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 1% or more of the tumor sample.

[0015] In some embodiments of the third, fourth, and fifth aspects, a tumor sample obtained from a patient has been determined to have a detectable expression level of PD-L1 in tumor infiltrating immune cells that comprise 5% or more of the tumor sample, hi some embodiments, a tumor sample obtained from a patient has been determined to have a detectable expression level of PD-L1 in tumor infiltrating immune cells that comprise at least about 10% of the tumor sample.

[0016] In a sixth aspect, the invention features a method of determining whether a patient suffering from bladder cancer is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining a tumor subtype from a tumor sample obtained from the patient, wherein a luminal subtype tumor indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist.

[0017] In a seventh aspect, the invention features a method of predicting responsiveness of a patient suffering from bladder cancer to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining a tumor subtype from a tumor sample obtained from the patient, wherein a luminal subtype tumor indicates that the patient is more likely to respond to a treatment comprising a PD-L1 axis-binding antagonist.

[0018] In an eighth aspect, the invention features a method of selecting a therapy for a patient suffering from bladder cancer, the method includes determining a tumor subtype from a tumor sample obtained from the patient, and selecting a therapy comprising a PD-L1 axis binding antagonist for the patient based on a determination that the tumor is a luminal subtype tumor.

[0019] In some embodiments of the third through eighth aspects, the method further comprises administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist based on the expression level of PD-L1 on tumor cells or tumor-infiltrating immune cells in the tumor sample.

[0020] In some embodiments of any of the foregoing aspects, the PD-L1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist. In some embodiments, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to one or more of its ligand binding partners. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to B7-1. In other embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to both PD-1 and B7-1. In yet other embodiments, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is selected from the group consisting of atezolizumab (MPDL3280A), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). In some embodiments, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 19, the HVR-H2 sequence of SEQ ID NO: 20, and the HVR-H3 sequence of SEQ ID NO: 21, and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 22, the HVR-L2 sequence of SEQ ID NO: 23, and the HVR-L3 sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. In other embodiments, the PD-L1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to one or more of its ligand binding partners. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to PD-L2. In other embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to both PD-L1 and PD-L2. In yet other embodiments, the PD-1 binding antagonist is an antibody.In some embodiments, the antibody is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. In other embodiments, the PD-1 binding antagonist is an Fc fusion protein. In some embodiments, the Fc fusion protein is AMP-224. In still other embodiments, the method further comprises administering to the patient an effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, and combinations thereof. In other embodiments, the bladder cancer is urothelial bladder cancer (UBC). In some embodiments, the UBC is metastatic urothelial bladder cancer. In other embodiments, the UBC is locally advanced urothelial bladder cancer. In some embodiments, the patient has progressed following treatment with a platinum-based chemotherapy agent (i.e., the patient's disease (e.g., UBC) has progressed following prior treatment with a platinum-based chemotherapy agent). In still other embodiments, the tumor sample is a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample. In other embodiments, the expression level of PD-L1 is a protein expression level. In some embodiments, the protein expression level of PD-L1 is determined using a method selected from the group consisting of immunohistochemistry (IHC), immunofluorescence, flow cytometry, and Western blot. In some embodiments, the protein expression level of PD-L1 is determined using IHC. In other embodiments, the protein expression level of PD-L1 is detected using an anti-PD-L1 antibody. In still other embodiments, the expression level of PD-L1 is an mRNA expression level. In some embodiments, the mRNA expression level of PD-L1 is determined using a method selected from the group consisting of quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), RNA sequencing, microarray analysis, in situ hybridization, and serial analysis of gene expression (SAGE).

[0021] In a ninth aspect, the invention features a PD-L1 antagonist for use in treating a patient suffering from bladder cancer, wherein a tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 1% or more of the tumor sample.

[0022] In a tenth aspect, the invention features the use of an effective amount of a PD-L1 axis-binding antagonist in the manufacture of a medicament for use in treating a patient suffering from bladder cancer, wherein a tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 1% or more of the pre-tumor sample.

[0023] In an eleventh aspect, the invention features a composition comprising an effective amount of a PD-L1 axis-binding antagonist for use in treating a patient suffering from bladder cancer, wherein a tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 1% or more of the tumor sample. [Brief description of the drawings]

[0024] [Figure 1A] 1 is a table showing the prevalence of PD-L1 expression in UBC with the indicated IC scores. Results are based on staining of archival tumor tissue from patients prescreened in an ongoing Phase Ia clinical trial (see Example 2). [Figure 1B] Images showing PD-L1 expression in tumor-infiltrating immune cells (IC) as assessed by immunohistochemistry using a rabbit monoclonal anti-PD-L1 antibody. PD-L1 staining is shown in dark brown. [Diagram 2]1 is a table showing that PD-L1 expression in IC is associated with response of UBC patients to treatment with atezolizumab (MPDL3280A). Objective response rate (ORR), complete response (CR), and partial response (PR) are shown for patients with the indicated IC scores. Efficacy evaluable patients with measurable disease at baseline by RECIST v1.1. Four IC2 / 3 patients and seven IC0 / 1 patients were lost or unevaluable. [Diagram 3] Graph showing UBC patient response to treatment with atezolizumab (MPDL3280A). Patient IC scores are shown. SLD, longest diameter of target lesion. Seven patients without post-baseline tumor assessments were not included. Asterisks indicate nine CR patients who were not all confirmed by the data cut-off date, seven of whom had less than 100% reduction due to lymph node target lesions. All lymph nodes returned to normal size by RECIST v1.1. aSLD change >100%. [Figure 4] Graph showing duration of treatment and duration of response in UBC patients treated with atezolizumab (MPDL3280A). Markers for discontinuation and ongoing response status do not imply timing. [Figure 5A] 1 is a table showing the association between PD-L1 expression in IC and survival in UBC patients treated with atezolizumab (MPDL3280A). Graphs show median and 1-year progression-free survival (PFS) and overall survival (OS) for IC2 / 3 and IC0 / 1 UBC patients treated with atezolizumab (MPDL3280A). [Figure 5B] Graph showing OS for IC2 / 3 and IC0 / 1 UBC patients treated with atezolizumab (MPDL3280A). [Figure 6] A series of graphs showing the association between expression levels of immune blocker gene signatures (CTLA4, BTLA, LAG3, HAVCR2, PD1) or CTLA4 in peripheral blood mononuclear cells (PBMCs) and response during treatment of UBC patients with atezolizumab. C, cycles; D, days. [Figure 7] Schematic diagram of the overall design of the Phase II study. Evaluable tumor tissues for PD-L1 testing were prospectively evaluated by a central laboratory. Patients and investigators were blinded to PD-L1 IHC status. [Figure 8] Overview of the cohorts participating in the phase II study. The excluded group includes patients who were rescreened. The treatment group consisted of 311 patients and the efficacy-evaluable group consisted of 310 patients. One patient was removed from the treatment group because the site from which his tumor sample originated was unknown. [Figure 9A] Graph showing change over time in sum of longest tumor diameters from baseline in IC2 / 3 patients experiencing a partial or complete response to atezolizumab (MPDL3280A). [Figure 9B] FIG. 1 is a graph showing the change from baseline in the sum of the longest tumor diameters over time in IC2 / 3 patients who remained stable on atezolizumab (MPDL3280A). [Figure 9C] FIG. 13 is a graph showing the change from baseline in the sum of the longest tumor diameters over time in IC2 / 3 patients with progressive disease on atezolizumab (MPDL3280A). [Figure 9D] Graph showing overall survival of IC0, IC1, and IC2 / 3 patients. [Figure 10A] Graph showing sum of longest tumor diameter from baseline in IC0 patients with a response to treatment with atezolizumab. Green dashed line = PR / CR (n=8). [Figure 10B] Graph showing sum of longest tumor diameter from baseline in IC0 patients with stable disease treated with atezolizumab. Blue dashed line = SD (n=25). [Figure 10C] Graph showing sum of longest tumor diameter from baseline in IC0 patients with progressive disease treated with atezolizumab. Red line = PD (n=52). [Figure 10D]Graph showing sum of longest tumor diameter from baseline in IC1 patients with a response to treatment with atezolizumab. Green dashed line = PR / CR (n=11). [Figure 10E] Graph showing sum of longest tumor diameter from baseline in IC1 patients with stable disease treated with atezolizumab. Blue dashed line = SD (n=18). [Figure 10F] Graph showing sum of longest tumor diameter from baseline in IC1 patients with progressive disease treated with atezolizumab. Red line = PD (n=61). [Figure 11A] Graph showing change over time in sum of longest tumor diameters by best response in IC0 patients treated with atezolizumab after progression. Middle grey line = -30 or less (n=2), black line = -30 or more and 20 or less (n=8), light grey line = >20 (n=17). [Figure 11B] Graph showing change over time in sum of longest tumor diameters by best response in IC1 patients treated with atezolizumab after progression. Middle grey line = -30 or less (n=8), black line = -30 or more and 20 or less (n=10), light grey line = >20 (n=14). [Figure 11C] Graph showing change over time in sum of longest tumor diameters by best response in IC2 / 3 patients treated with atezolizumab after progression. Middle grey line = -30 or less (n=10), black line = -30 or more and 20 or less (n=15), light grey line = >20 (n=11). [Figure 12A] Graph showing the association between PD-L1 immunohistochemical expression (e.g., IC score) and genes in the CD8 effector set (e.g., CXCL9 and CXCL10). [Figure 12B] Graph showing the association between PD-L1 immunohistochemical expression (e.g., IC score) and genes in the CD8 effector set (e.g., CXCL9 and CXCL10). [Figure 12C] Graph showing the association between CD8 infiltration and PD-L1 immunohistochemical expression (e.g., IC score). [Figure 12D] Graph showing the association between CD8 infiltration and response. [Figure 12E] Graph showing the association between PD-L1 immunohistochemical expression on tumor-infiltrating immune cell (IC) tumor subtypes. [Figure 12F] Graph showing the association between PD-L1 immunohistochemical expression on tumor cells (TC) with tumor subtypes. [Figure 12G] 1 is a graph showing the association between tumor subtype and response. [Figure 13A] Graph showing association of the complete CD8 T effector gene set (e.g., CD8A, GZMA, GZMB, IFNG, CXCL9, CXCL10, PRF1, TBX21) with PD-L1 immunohistochemistry IC status. [Figure 13B] Graph showing association of the complete CD8 T effector gene set (e.g., CD8A, GZMA, GZMB, IFNG, CXCL9, CXCL10, PRF1, TBX21) with patient response. [Figure 14] 13 is a heatmap showing the relationship between putative molecular subtype, response, IC and TC scores, and gene expression of two gene sets: (i) genes used to assign TCGA subtypes, and (ii) genes commonly associated with CD8 T effector activity. [Figure 15] FIG. 1 shows the relationship of logistic regression fitted to response (CR / PR vs. SD / PD) on one or more biomarkers: PD-L1 IHC IC score (IC0 / 1 vs. IC2 / 3) and TCGA gene expression subtype. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] I. Introduction The present invention provides therapeutic and diagnostic methods and compositions for cancer, e.g., bladder cancer (e.g., urothelial bladder cancer, UBC).The present invention is based, at least in part, on the discovery that determining the expression level of a biomarker of the invention, e.g., PD-L1 and / or a tumor subtype, in a sample obtained from a patient is useful in treating a patient suffering from cancer, for diagnosing a patient suffering from cancer, for determining whether a patient having cancer is likely to respond to treatment with an anti-cancer therapy that includes a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atezolizumab (MPDL3280A)), for optimizing the therapeutic efficacy of an anti-cancer therapy that includes a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atezolizumab), and / or for selecting patients for an anti-cancer therapy that includes a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atezolizumab).

[0026] II. Definition It is understood that the aspects and embodiments of the invention described herein include aspects and embodiments "comprising," "consisting of," and "consisting essentially of." As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0027] The term "about" as used herein refers to the normal error range of the respective value, which is readily understood by those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) an embodiment that is directed to the value or parameter itself. For example, a statement that refers to "about X" includes a statement of "X".

[0028] The term "tumor subtype" or "tumor sample subtype" refers to the intrinsic molecular characteristics of a tumor or cancer (e.g., DNA, RNA, and / or protein expression levels (e.g., genomic profile)). A particular subtype of a tumor or cancer (e.g., urothelial bladder cancer (UBC tumor)) can be determined by histopathological criteria or subtype-associated molecular characteristics (e.g., expression of one or more biomarkers (e.g., specific genes, RNA (e.g., mRNA, microRNA), or proteins encoded by the genes)) (see, e.g., Cancer Genome Atlas Research Network Nature 507:315-22,2014; Jiang et al. Bioinformatics 23:306-13,2007; Dong et al. Nat. Med. 8:793-800,2002).

[0029] The term "PD-L1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-L1 axis binding partner with one or more of its binding partners, such that T cell dysfunction resulting from signaling on the PD-1 signaling axis is ablated, resulting in 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 disrupt the interaction between PD-L1 and PD-1 (e.g., PD-L2-Fc fusions).

[0030] The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. This term includes common elements of both "exhaustion" and / or "anergy," in which antigen recognition can occur but the subsequent immune response is ineffective in controlling infection or tumor growth.

[0031] As used herein, the term "dysfunctional" also includes unresponsiveness or non-responsiveness to antigen recognition, specifically, impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing.

[0032] The term "anergy" refers to defective or insufficient signals delivered via the T cell receptor (e.g., intracellular Ca in the absence of Ras activation). 2+ Anergy refers to a state of unresponsiveness to antigenic stimulation resulting from an increase in T cell proliferation (increase in T cell proliferation and proliferation) in response to T cell stimulation. T cell anergy can also occur upon stimulation with antigen in the absence of costimulation, resulting in the cells becoming refractory to subsequent activation by antigen, even in the context of costimulation. The unresponsive state can often be reversed by the presence of interleukin 2. Anergic T cells do not undergo clonal expansion and / or acquire effector functions.

[0033] The term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction due to persistent TCR signaling that occurs during many chronic infections and cancer development. It is distinct from anergy in that it is due to persistent signaling rather than defective or insufficient signaling. It is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state that differs from functional effector or memory T cells. Exhaustion prevents optimal control of infections and tumors. Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunoregulatory cytokines) and cell-intrinsic negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).

[0034] "Enhancing T cell function" means inducing, causing, or stimulating T cells to have sustained or amplified biological function, or renewing or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include increased secretion of gamma-interferon from CD8+ T cells, increased proliferation, and increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance) compared to such levels before the intervention. In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200% enhancement. The manner of measuring this enhancement is known to those skilled in the art.

[0035] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated and tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0036] "Immunogenicity" refers to the ability of a particular substance to induce an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps the immune response to clear tumor cells. An example of enhancing tumor immunogenicity is treatment with PD-L1 axis binding antagonists.

[0037] As used herein, a "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, neutralizes, or prevents signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules that reduce, block, inhibit, neutralize, or prevent signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In one embodiment, the PD-L1 binding antagonist reduces negative signals mediated by or through PD-L1 or cell surface proteins expressed on T lymphocytes and other cells via PD-1, thereby alleviating the dysfunctional state of dysfunctional T cells. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific embodiment, the anti-PD-L1 antibody is YW243.55.S70, as described herein. In another specific embodiment, the anti-PD-L1 antibody is MDX-1105, as described herein. In yet another specific embodiment, the anti-PD-L1 antibody is atezolizumab (MPDL3280A), as described herein. In yet another specific embodiment, the anti-PD-L1 antibody is MEDI4736 (druvalumab), as described herein. In yet another specific embodiment, the anti-PD-L1 antibody is MSB0010718C (avelumab), as described herein.

[0038] As used herein, a "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, neutralizes, or prevents signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and / or PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partners. In specific aspects, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules that reduce, block, inhibit, neutralize, or prevent signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative signals mediated by or through cell surface proteins expressed on T lymphocytes and other cells via PD-L1 or PD-1, thereby alleviating the dysfunctional state of dysfunctional T cells. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific embodiment, the PD-1 binding antagonist is MDX-1106 (nivolumab), as described herein. In another specific embodiment, the PD-1 binding antagonist is MK-3475 (pembrolizumab), as described herein. In another specific embodiment, the PD-1 binding antagonist is CT-011 (pidilizumab), as described herein. In another specific embodiment, the PD-1 binding antagonist is MEDI-0680 (AMP-514), as described herein. In another specific embodiment, the PD-1 binding antagonist is PDR001, as described herein. In another specific embodiment, the PD-1 binding antagonist is REGN2810, as described herein. In another specific embodiment, the PD-1 binding antagonist is BGB-108, as described herein. In another specific embodiment, the PD-1 binding antagonist is AMP-224, as described herein.

[0039] The terms "programmed death ligand 1" and "PD-L1," as used herein, refer to native sequence PD-L1 polypeptides, polypeptide variants, and fragments of native sequence polypeptides and polypeptide variants, as further defined herein. The PD-L1 polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.

[0040] A "native sequence PD-L1 polypeptide" includes a polypeptide that has the same amino acid sequence as a corresponding PD-L1 polypeptide derived from nature.

[0041] By "PD-L1 polypeptide variant" or variations thereof is meant that a PD-L1 polypeptide, typically an active PD-L1 polypeptide, as defined herein, has at least about 80% amino acid sequence identity to any of the native sequence PD-L1 polypeptide sequences disclosed herein. Such PD-L1 polypeptide variants include, for example, PD-L1 polypeptides having one or more amino acid residues added or deleted at the N-terminus 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 disclosed herein. Typically, a PD-L1 variant polypeptide is at least about 10 amino acids in length, alternatively 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, or 289 amino acids in length or more. Optionally, a PD-L1 variant polypeptide has no more than one conservative amino acid substitution compared to a native PD-L1 polypeptide sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to a native PD-L1 polypeptide sequence.

[0042] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. The 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. Thus, for example, polynucleotides as defined herein include, but are not limited to, single-stranded and double-stranded DNA, DNA containing single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA containing single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or may contain single-stranded and double-stranded regions. In addition, the term "polynucleotide" as used herein refers to triple-stranded regions that contain RNA or DNA, or both RNA and DNA. The strands in such regions can be from the same molecule or from different molecules. These regions can include all of one or more of these molecules, but more typically include only some regions of these molecules. One of the molecules of the triple helix region is often an oligonucleotide. The term "polynucleotide" specifically includes cDNA.

[0043] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps" which replace one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those containing intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH may be phosphorylated or replaced with amines or organic capping group moieties of 1-20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars that are commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, analogs of carbocyclic sugars, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages within a polynucleotide need be identical. A polynucleotide may contain one or more different types of modifications described herein and / or multiple modifications of the same type. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0044] As used herein, "oligonucleotide" refers to a short, single-stranded polynucleotide that is generally, but not necessarily, less than about 250 nucleotides in length. Oligonucleotides may be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and completely applicable to oligonucleotides.

[0045] The term "primer" refers to a single-stranded polynucleotide that can hybridize to a nucleic acid and permit polymerization of a complementary nucleic acid, generally by providing a free 3'-OH group.

[0046] The term "small molecule" refers to any molecule having a molecular weight below about 2000 daltons, preferably below about 500 daltons.

[0047] 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 its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0048] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0049] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained within a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0050] The term "antibody" is used herein in the broadest sense and encompasses a variety of 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.

[0051] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with the research, diagnostic, and / or therapeutic uses of the antibody, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95%, and in some embodiments, greater than 99%, by weight of the antibody, e.g., as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, e.g., using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, e.g., using Coomassie blue or silver staining. Isolated antibodies include antibodies in situ, within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

[0052] A "native antibody" is usually a heterotetrameric glycoprotein of about 150,000 daltons consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain and the variable domain of the light chain aligned with the variable domain of the heavy chain. It is believed that certain amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain.

[0053] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0054] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portions of the immunoglobulin, the variable domains that contain the antigen binding sites. The constant domains contain the CH1, CH2, and CH3 domains of the heavy chain (collectively, CH) and the CHL (or CL) domain of the light chain.

[0055] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The heavy chain variable domain may be referred to as "VH". The light chain variable domain may be referred to as "VL". These domains are generally the most variable parts of an antibody and contain the antigen binding sites.

[0056] The term "variable" refers to the fact that the sequence of certain parts of the variable domains differs widely among antibodies and is used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration connected by three HVRs that form loops that connect and, in some cases, form part of the beta-sheet structure. The HVRs within each chain are held together in close proximity by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular toxicity.

[0057] As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain that is sequence hypervariable and / or forms structurally defined loops. Generally, an antibody contains six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the highest diversity of the six HVRs, and H3 in particular is thought to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0058] Several HVR delineations are used and encompassed herein. The Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs are a compromise between the Kabat HVRs and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of available complex crystal structures. The residues of each of these HVRs are described below. TIFF2025003997000001.tif100170

[0059] HVRs may include the following "extended HVRs": in the VL, 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3), and in the VH, 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3). The variable domain residues are numbered according to Kabat et al. (see above) for each of these definitions.

[0060] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0061] The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al. (see above). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody sequence with the "standard" Kabat numbered sequence at the regions of homology.

[0062] The Kabat numbering system is commonly used when referring to residues in the variable domain (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is commonly used when referring to residues in immunoglobulin heavy chain constant regions (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0063] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody in its substantially intact form, rather than an antibody fragment, as defined below. These terms specifically refer to an antibody having a heavy chain that contains an Fc region.

[0064] An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen-binding region thereof. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0065] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and an "Fc" fragment, the name reflecting its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0066] "Fv" is the smallest antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight non-covalent association. In a single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that in the two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0067] Fab fragments contain the heavy and light chain variable domains, and also contain the light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0068] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0069] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between these two domains on the same chain, these domains are forced to pair with complementary domains of another chain, creating two antigen-binding sites. Diabodies can be bivalent or bispecific. For diabodies, 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). For triabodies and tetrabodies, see also Hudson et al., Nat. Med. 9:129-134 (2003).

[0070] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively.

[0071] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogenous antibodies, e.g., the individual antibodies that make up the population are identical except for possible mutations that may be present in minor amounts, e.g., naturally occurring mutations. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of separate antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies that include a polypeptide sequence that binds to a target, and the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It is understood that the selected target-binding sequence may be further modified, e.g., to improve affinity to the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, and create a multispecific antibody, and an antibody that includes a modified target-binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0072] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be produced using, for example, hybridoma methods (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., Nature, 352:624-628 (1991)), and the like. al., J.Mol.Biol.222:581-597(1992), Sidhu et al., J.Mol.Biol.338(2):299-310(2004), Lee et al. al., J.Mol.Biol.340(5):1073-1093(2004), Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004), and Lee et al.,J.Immunol.Methods 284(1-2):119-132 (2004)), and techniques for producing human antibodies or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO1998 / 24893, WO1996 / 34096, WO1996 / 33735, WO1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993), Jakobovits et al., Nature 362:255-258 (1993), Bruggemann et al., Year in Immunol. 7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992), Lonberg et al., Nature 368:856-859 (1994), Morrison, Nature 368:812-813 (1994), Fishwild et al., Nature Biotechnol. 14:845-851 (1996), Neuberger, Nature Biotechnol. 14:826 (1996), and Lonberg et al. al., Intern. Rev. Immunol. 13:65-93 (1995)).

[0073] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, so long as the desired biological activity is exhibited (see, e.g., U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies, whose antigen-binding regions are derived from, for example, antibodies produced by immunizing macaque monkeys with an antigen of interest.

[0074] A "human antibody" is one that possesses an amino acid sequence that corresponds to the amino acid sequence of an antibody or other human antibody coding sequence produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0075] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from a non-human hypervariable region (HVR) and amino acid residues from a human framework region (FR). In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of its HVRs (e.g., CDRs) corresponding to those of a non-human antibody and all or substantially all of its FRs corresponding to those of a human antibody. A humanized antibody may optionally 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.

[0076] The terms "anti-PD-L1 antibody" and "antibody that binds to PD-L1" refer to an antibody that can bind to PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting PD-L1. In one embodiment, the extent of binding of the anti-PD-L1 antibody 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 an epitope of PD-L1 that is conserved among PD-L1 from different species.

[0077] The terms "anti-PD-1 antibody" and "antibody that binds to PD-1" refer to an antibody that can bind to PD-1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting PD-1. In one embodiment, the extent of binding of the anti-PD-1 antibody to unrelated, non-PD-1 proteins is less than about 10% of the binding of the antibody to PD-1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the anti-PD-1 antibody binds to an epitope of PD-1 that is conserved among PD-1 from different species.

[0078] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0079] "Affinity" refers to the strength of the sum of 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 as a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0080] As used herein, the terms "bind", "specifically bind to", or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for longer than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0081] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody that does not possess such alterations, which alterations improve the affinity of the antibody for antigen.

[0082] 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 competitive assay, and conversely, an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay.

[0083] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.

[0084] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity that is not the antigen recognition and binding site of an antibody (i.e., is "heterologous") with an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence that includes at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin may be taken from any immunoglobulin, such as IgG1, IgG2 (including IgG2A and IgG2B), IgG3, or IgG4 subtypes, IgA (including IgA1 and IgA2), IgE, IgD, or IgM. Ig fusions preferably include the substitution of a polypeptide or antibody domain as described herein in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2, and CH3, or the hinge, CH1, CH2, and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions, see U.S. Patent No. 5,428,130. For example, immunoadhesins useful as therapeutic medicaments herein include polypeptides comprising the extracellular domain (ECD) or PD-1 binding portion of PD-L1 or PD-L2, or the extracellular or PD-L1 or PD-L2 binding portion of PD-1, e.g., PD-L1 ECD-Fc, PD-L2 ECD-Fc, and PD-1 ECD-Fc, respectively, fused to a constant domain of an immunoglobulin sequence. The combination of an immunoadhesin with the Ig Fc and ECD of a cell surface receptor is sometimes referred to as a soluble receptor.

[0085] "Fusion protein" and "fusion polypeptide" refer to a polypeptide having two moieties covalently linked together, where each of these moieties is a polypeptide with a different property. The property can be a biological property, such as activity in vitro or in vivo. The property can also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two moieties can be linked directly by a single peptide bond or through a peptide linker, but are in reading frame with each other.

[0086] "Percent amino acid sequence identity" with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide to be compared, after aligning the sequences and introducing gaps as necessary to obtain the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved 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. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent 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, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0087] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, it may be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It is understood 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 is not equal to the % amino acid sequence identity of B to A. Unless specifically indicated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0088] The term "detection" includes any method of detection, including direct and indirect detection.

[0089] The term "biomarker" as used herein refers to an indicator that can be detected in a sample, such as a predictive, diagnostic, and / or prognostic indicator, such as PD-L1, FGFR3, miR-99a-5p, miR-100-5p, CDKN2A, KRT5, KRT6A, KRT14, EGFR, GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, E-cadhherin, ERBB2, or ESR2. A biomarker can serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and / or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotide (e.g., DNA and / or RNA), polynucleotide copy number alterations (e.g., DNA copy number), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and / or glycolipid-based molecular markers.

[0090] The "amount" or "level" of a biomarker associated with increased clinical benefit to an individual is the level detectable in a biological sample. These can be measured by methods known to those skilled in the art and also disclosed herein. The expression level or amount of the biomarker evaluated can be used to determine response to treatment.

[0091] 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., genetic coding information and / or epigenetic information) is converted into structures present and functional in a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide) should also be considered as expressed, regardless of whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of a polypeptide, e.g., by proteolysis. "Expressed genes" include genes that are transcribed into polynucleotides as mRNA and then translated into a polypeptide, and also include genes that are transcribed into RNA but not translated into a polypeptide (e.g., transfer RNA and ribosomal RNA).

[0092] "Increased expression," "increased expression level," "increased level," "elevated expression," "elevated expression level," or "elevated level" refers to an increase in expression or an increase in the level of a biomarker in an individual compared to a control, such as an individual(s) not afflicted with a disease or disorder (e.g., cancer) or an internal control (e.g., a housekeeping biomarker).

[0093] "Decreased expression," "decreased expression level," "decreased level," "reduced expression," "reduced expression level," or "reduced level" refers to reduced expression or reduced levels of a biomarker in an individual compared to a control, such as an individual(s) not afflicted with a disease or disorder (e.g., cancer) or an internal control (e.g., a housekeeping biomarker). In some embodiments, reduced expression is little or no expression.

[0094] The term "housekeeping biomarker" refers to a biomarker or group of biomarkers (e.g., polynucleotides and / or polypeptides) that are typically present similarly in all cell types. In some embodiments, a housekeeping biomarker is a "housekeeping gene." "Housekeeping gene," as used herein, refers to a gene or group of genes that encode a protein whose activity is essential for maintaining cellular function and that are typically present similarly in all cell types.

[0095] As used herein, "amplification" generally refers to the process of generating multiple copies of a desired sequence. "Multiple copies" means at least two copies. "Copy" does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies may contain nucleotide analogs such as deoxyinosine, deliberate sequence modifications (such as those introduced by primers that contain sequences that are hybridizable to but not complementary to the template), and / or sequence errors that occur during amplification.

[0096] The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acid obtained from a single source (e.g., an individual) using more than one primer set in order to amplify two or more DNA sequences in a single reaction.

[0097] As used herein, the term "polymerase chain reaction" or "PCR" technique generally refers to a procedure in which minute specific pieces of nucleic acid, RNA, and / or DNA are amplified, as described, for example, in U.S. Pat. No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond must be available so that oligonucleotide primers can be designed, the sequences of which are identical or similar to opposite strands of the template to be amplified. The 5' terminal nucleotides of these two primers may coincide with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, cDNA transcribed from total cellular RNA, bacteriophage sequences, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987) and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). As used herein, PCR is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample that involves the use of known nucleic acids (DNA or RNA) as primers and utilizes a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid or a specific piece of nucleic acid complementary to a specific nucleic acid.

[0098] "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 various publications, such as Cronin et al., Am. J. Pathol. 164(1):35-42 (2004), and Ma et al., Cancer Cell 5:607-616 (2004).

[0099] The term "microarray" refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.

[0100] The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease, or condition (e.g., 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 subtype of cancer, for example, by histopathological criteria or by molecular features (e.g., a subtype characterized by the expression of one or a combination of biomarkers (e.g., a particular gene, or the protein encoded by the gene)).

[0101] The term "aiding in diagnosis" is used herein to refer to a method that helps make a clinical determination regarding the presence or nature of a particular type of symptom or condition of a disease or disorder (e.g., cancer). For example, a method of aiding in the diagnosis of a disease or condition (e.g., cancer) can include measuring a certain biomarker (e.g., PD-L1) in a biological sample from an individual.

[0102] The term "sample" as used herein refers to a composition obtained or derived from a subject and / or individual of interest that contains cells and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological properties. For example, the phrase "disease sample" and variations thereof refer to any sample obtained from a subject of interest that is expected to contain or is known to contain the cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, cells from blood, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts, e.g., homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0103] "Tissue sample" or "cell sample" refers to a collection of similar cells obtained from the tissue of a subject or individual. The source of the tissue or cell sample can be solid tissue from fresh, frozen, and / or preserved organs, tissue samples, biopsies, and / or aspirates, or any blood component, e.g., plasma; bodily fluids, e.g., cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells at 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. For example, a "tumor sample" is a tissue sample obtained from a tumor or other cancerous tissue. A tumor sample can contain a population of mixed cell types (e.g., tumor and non-tumor cells, cancerous and non-cancerous cells). A tissue sample can contain compounds that are not naturally mixed with tissue in nature, e.g., preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0104] "Tumor-infiltrating immune cells," as used herein, refer to any immune cells present in a tumor or a sample thereof. Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, other tumor stromal cells (e.g., fibroblasts), or any combination thereof. Such tumor-infiltrating immune cells may be, for example, T lymphocytes (such as CD8+ T lymphocytes and / or CD4+ T lymphocytes), B lymphocytes, or other myeloid cells, including granulocytes (e.g., neutrophils, eosinophils, and basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer cells.

[0105] As used herein, "tumor cell" refers to any tumor cell present in a tumor or a sample thereof. Tumor cells can be distinguished from other cells, such as stromal cells and tumor-infiltrating immune cells, that may be present in a tumor sample using methods known in the art and / or described herein.

[0106] As used herein, a "reference sample", "reference cell", "reference tissue", "control sample", "control cell", or "control tissue" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of the same subject or individual. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue can be a healthy and / or non-diseased cell or tissue adjacent to a diseased cell or tissue (e.g., a cell or tissue adjacent to a tumor). In another embodiment, the reference sample is obtained from an untreated tissue and / or cell of the body of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of an individual that is not the subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from untreated tissues and / or cells from the body of an individual that is not the subject or individual.

[0107] For purposes herein, a "section" of a tissue sample refers to a single portion or piece of a tissue sample, e.g., a thin section of tissue or cells cut from a tissue sample (e.g., a tumor sample). It is understood that multiple sections of a tissue sample can be taken and subjected to analysis, provided that the same section of a tissue sample can be analyzed at both the morphological and molecular levels, or analyzed for polypeptides (e.g., by immunohistochemistry) and / or polynucleotides (e.g., by in situ hybridization).

[0108] "Correlate" or "correlating" means to compare, in any manner, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, the results of a first analysis or protocol can be used in performing a second protocol and / or can be used to determine whether a second analysis or protocol should be performed. With respect to embodiments of a polypeptide analysis or protocol, the results of a polypeptide expression analysis or protocol can be used to determine whether a particular treatment regimen should be performed. With respect to embodiments of a polynucleotide analysis or protocol, the results of a polynucleotide expression analysis or protocol can be used to determine whether a particular treatment regimen should be performed.

[0109] "Individual response" or "response" may be assessed using any endpoint that indicates benefit to an individual, including, without limitation, (1) inhibition to some extent of disease progression (e.g., cancer progression), including slowing or complete halt; (2) reduction in tumor size; (3) inhibition (i.e., reduction, slowing, or complete halt) of cancer cell invasion into adjacent peripheral organs and / or tissues; (4) inhibition (i.e., reduction, slowing, or complete halt) of metastasis; (5) alleviation to some extent of one or more symptoms associated with a disease or disorder (e.g., cancer); (6) increase or prolongation in length of survival, including overall survival and progression-free survival; and / or (7) reduced mortality at a given time point following treatment.

[0110] A patient's "effective response" to a pharmaceutical treatment or patient "responsiveness" and similar words refer to a clinical or therapeutic benefit conferred on a patient at risk for or suffering from a disease or disorder, such as cancer. In one embodiment, such benefit may include any one or more of extending survival (including overall survival and / or progression-free survival), producing an objective response (including a complete or partial response), or ameliorating the signs or symptoms of cancer. In one embodiment, a biomarker (e.g., PD-L1 expression on tumor-infiltrating immune cells, e.g., as determined using IHC) is used to identify patients who are predicted to have an increased likelihood of being responsive to a pharmaceutical treatment (e.g., a treatment comprising a PD-L1 axis-binding antagonist, e.g., an anti-PD-L1 antibody) compared to patients who do not express the biomarker. In one embodiment, a biomarker (e.g., PD-L1 expression on tumor infiltrating immune cells, e.g., as determined using IHC) is used to identify patients who are predicted to have an increased likelihood of being responsive to treatment with a pharmaceutical agent (e.g., an anti-PD-L1 antibody) relative to patients who do not express the biomarker at the same level. In one embodiment, the presence of the biomarker is used to identify patients who are more likely to respond to treatment with a pharmaceutical agent relative to patients who do not have the presence of the biomarker. In another embodiment, the presence of the biomarker is used to determine that a patient has an increased likelihood of benefiting from treatment with a pharmaceutical agent relative to patients who do not have the presence of the biomarker.

[0111] "Response" refers to a measurable response, including complete response (CR) or partial response (PR). In some embodiments, "response rate (ORR)" refers to the combined complete response (CR) rate and partial response (PR) rate.

[0112] By "complete response" or "CR" is intended the disappearance of all signs of cancer (e.g., disappearance of all target lesions) in response to treatment. This does not necessarily mean that the cancer has been cured.

[0113] "Sustained response" refers to the sustained effect of reducing tumor growth after the cessation of treatment.For example, tumor size can remain the same size or become smaller compared to the size at the beginning of the drug administration period.In some embodiments, the sustained response has a duration of at least the same as the treatment period, or at least 1.5 times, 2.0 times, 2.5 times, or 3.0 times the length of the treatment period, or more.

[0114] As used herein, "reducing or inhibiting cancer recurrence" means reducing or inhibiting the recurrence of a tumor or cancer, or the progression of a tumor or cancer. As disclosed herein, cancer recurrence and / or progression includes, but is not limited to, cancer metastasis.

[0115] As used herein, "partial response" or "PR" refers to a reduction in the size of one or more tumors or lesions, or the extent of cancer in the body, in response to treatment. For example, in some embodiments, PR refers to at least a 30% reduction in the sum of the longest diameters (SLD) of target lesions, with reference to the baseline SLD.

[0116] As used herein, "stable disease" or "SD" refers to neither sufficient shrinkage of target lesions to qualify as PR nor sufficient increase to qualify as PD, with reference to the smallest SLD since the start of treatment.

[0117] As used herein, "progressive disease" or "PD" refers to at least a 20% increase in the SLD of a target lesion, referenced to the smallest SLD recorded since the start of treatment, or the presence of one or more new lesions.

[0118] The term "survival" refers to the survival of a patient, and includes overall survival, as well as progression-free survival.

[0119] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not worsen. Progression-free survival may include the amount of time a patient experiences a complete or partial response, as well as the amount of time a patient experiences stable disease.

[0120] As used herein, "overall survival" (OS) refers to the proportion of individuals in a group who are likely to be alive after a particular period of time.

[0121] By "prolonged survival" is meant an increase in overall survival or progression-free survival in a treated patient relative to untreated patients (i.e., relative to patients not treated with a pharmaceutical agent), or relative to patients who do not express the biomarker at a specified level, and / or relative to patients treated with an anti-tumor agent.

[0122] As used herein, the term "substantially the same" refers to a sufficiently high degree of similarity between two numerical values ​​that one of skill in the art would consider the difference between the two values ​​to have little or no biological and / or statistical significance in the context of the biological attribute measured by the value (e.g., Kd value or expression level), e.g., less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10% as a function of the reference / comparison value.

[0123] The phrase "substantially different" as used herein refers to a sufficiently high degree of difference between two numerical values ​​that one of skill in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological trait measured by the values ​​(e.g., Kd value or expression level), e.g., 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 value for the reference / comparator molecule.

[0124] The word "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent such as a polynucleotide probe or antibody, facilitating detection of the reagent to which it is conjugated or fused. The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a substrate compound or composition that is detectable. The term is intended to encompass direct labeling of a probe or antibody by binding (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of a probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling of a DNA probe with biotin such that it can be detected using fluorescently labeled streptavidin.

[0125] "Therapeutically effective amount" refers to the amount of a therapeutic agent to treat or prevent a disease or disorder 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 a primary tumor, inhibit (i.e., slow down to some extent and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more of the symptoms associated with the disorder. To the extent that a drug can prevent the growth of existing cancer cells and / or kill them, the drug can be cytostatic and / or cytotoxic. With respect to cancer treatment, efficacy in vivo can be measured, for example, by assessing survival time, time to disease progression (TTP), response rate (e.g., CR and PR), duration of response, and / or quality of life.

[0126] A "disorder" is any condition that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0127] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth. This definition includes benign and malignant cancers. "Early stage cancer" or "early stage tumor" refers to a cancer that is not invasive or metastatic or is classified as stage 0, 1, or 2 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 tumor, gastrinoma, and islet cell carcinoma), mesothelioma, Schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid tumors. More specific examples of such cancers include bladder cancer (e.g., urothelial bladder cancer (e.g., transitional cell or urothelial carcinoma, non-muscle invasive bladder cancer, muscle invasive bladder cancer, and metastatic bladder cancer) and non-urothelial bladder cancer), 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, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer, ) or stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, Merkel cell carcinoma, mycosis fungoides, testicular cancer, esophageal cancer, bile duct tumors, as well as head and neck cancer and multiple myeloma. In some embodiments, the cancer is triple-negative metastatic breast cancer, including any histologically confirmed triple-negative (ER-, PR-, HER2-) breast adenocarcinoma with locally recurrent or metastatic disease (locally recurrent disease is not amenable to resection with curative intent). In some embodiments, the cancer is bladder cancer. In certain embodiments, the bladder cancer is urothelial bladder cancer.

[0128] The term "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive as referred to herein.

[0129] The term "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained therein is effective, and that does not contain any additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0130] "Pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical formulation that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0131] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention aimed at altering the natural history of the individual being treated and may be performed either for prophylaxis or during the clinical pathology process. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or remission of disease, and remission or improvement of prognosis. In some embodiments, antibodies (e.g., anti-PD-L1 antibodies and / or anti-PD-1 antibodies) are used to delay the development of disease or slow the progression of disease.

[0132] The term "anti-cancer therapy" refers to a therapy useful in the treatment of cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as 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(s), TRAIL / Apo2, other biologically active and organic chemical agents, and the like. Combinations of these are also included in the present invention.

[0133] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. This term includes radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , and radioactive isotopes of Lu), chemotherapeutic agents such as methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents, enzymes and fragments thereof, e.g., nucleases, 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), as well as various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. Tumoricidal agents cause the destruction of tumor cells.

[0134] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. ethylenimines and methylamelamines, including olomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and including 9-aminocamptothecin;bryostatin;kallistatin;CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs);podophyllotoxin;podophyllic acid;teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatins;duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1);eleutherobin;pancratistatin;sarcodictiin;spongistatin;chlorambucil, chlornaphtham nitrogen mustards such as chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin γ1I and calicheamicin ω1I (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); the dynemicins, including dynemicin A; the esperamicins; and the neocarzinostatin chromophore and related enediyne antibiotic chromophores), antibiotics such as aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, doxorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, chelamycin, rodorubicin, Streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxacin, Pyrimidine analogues such as uridine;androgens such as calcineurone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone;antiadrenal agents such as aminoglutethimide, mitotane, and trilostane;folic acid supplements such as floric acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabulil;bisantrene;edatrexate;defofamine;demecolcine;diaziquone;elfornithine;elliptinium acetate;epothilones;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidanmol;Nitraerin;Pentostatin;Fenameth;Pirarubicin;Rosoxantrone;2-Ethylhydrazide;Procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethanes; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as taxanes including TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ cremophor-free, albumin engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum or platinum-based chemotherapy agents and platinum analogues such as cisplatin, carboplatin, oxaliplatin (ELOXATIN™), satraplatin, picoplatin, nedaplatin, triplatin, and lipoplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate;include the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharma- ceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP (an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone), and FOLFOX (an abbreviation for a treatment regimen that uses oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin). Additional chemotherapeutic agents include, for example, maytansinoids (e.g., DM1), and cytotoxic agents useful as antibody drug conjugates, such as the auristatins MMAE and MMAF;

[0135] "Chemotherapeutic agents" also include "anti-hormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit hormonal action that may promote the growth of cancer, often in the form of systemic or whole body treatments. They may themselves be hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4-droxytamoxifene, trioxyfene, ketoxifene, LY117018, onapristone, and FARESTON® toremifene; antiprogesterones; estrogen receptor downregulators (ERDs); agents that function to suppress or shut down the ovaries, such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, acetate These include luteinizing hormone releasing hormone (LHRH) agonists, such as buserelin, and tripterelin; other antiandrogens, such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which block the enzyme aromatase, which controls estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formstein, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole.Additionally, this definition of chemotherapeutic agent includes bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid / zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor. These include those that inhibit the expression of genes in signaling pathways implicated in abherant cell proliferation, such as receptors (EGFR); THERATOOPE® vaccines and gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitors; ABARLIX® rmRH; lapatinib ditosylate (also known as GW572016, an ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor); and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0136] Chemotherapeutic agents also include antibodies, such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech), cetuximab (ERBITUX®, Imclone), panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Additional humanized monoclonal antibodies that have therapeutic potential as drugs in combination with the compounds of the present invention include apolizumab, acelizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, and cefotaxime.mertansine, cantuzumab mertansine, cedelizumab, cetrizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizu mab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan These include tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), an exclusively human sequence recombinant full-length IgG1 lambda antibody genetically modified to recognize the interleukin-12 p40 protein.

[0137] Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise directly interact with EGFR and block or reduce its signaling activity, also referred to as "EGFR antagonists." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. Pat. No. 4,943,533 (Mendelsohn et al.)), as well as mutant forms thereof, such as chimeric 225 (C225 or cetuximab, ERBUTIX®) and reshaped human 225 (H225) (see WO 96 / 40210 (Imclone Systems, Inc., U.S. Pat. No. 6,225-6,325). Inc.), IMC-11F8, a fully human EGFR targeting antibody (Imclone); antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind to EGFR as described in U.S. Pat. No. 5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98 / 50433 (Abgenix / Amgen)); EMD55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996); the humanized EGFR antibody EMD7200 (matuzumab) directed against EGFR which competes with both EGF and TGF-alpha for EGFR binding (EMD / Merck); the human EGFR antibody HuMax-EGFR (GenMab); the fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3 and described in US 6,235,883; MDX-447 (Medarex Inc); and mAb806 or humanized mAb806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)).Anti-EGFR antibodies can be conjugated to cytotoxic drugs to produce immunoconjugates (see, e.g., EP 659,439A2, Merck Patent GmbH). EGFR antagonists include those described in U.S. Patents 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,86 Nos. 6,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and the following PCT publications: WO 98 / 14451, WO 98 / 50038, WO 99 / 09016, and WO 99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech / OSI Pharmaceuticals), PD183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.), ZD1839, gefitinib (IRESSA®) 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca), ZM105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca), BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim), PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol), (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine), CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide), EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinazolinyl]-4-(dimethylamino)-2-butynamide) (Wyeth), AG1478 (Pfizer), AG1571 (SU 5271, Pfizer), and dual EGFR / HER2 tyrosine kinase inhibitors, such as lapatinib (TYKERB®, GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

[0138] Chemotherapeutic agents include "tyrosine kinase inhibitors" including the EGFR targeted drugs described in the previous paragraph; small molecule HER2 tyrosine kinase inhibitors, e.g., TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors, e.g., EKB-569, which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells (available from Wyeth); lapatinib (GSK572016, available from Glaxo-SmithKline); oral HER2 and EGFR tyrosine kinase inhibitors; PKI-166 (available from Novartis); pan-HER inhibitors, e.g., canertinib (CI-1033, Pharmacia); Raf-1 inhibitors, e.g., ISIS, which inhibits Raf-1 signaling. antisense drug ISIS-5132 available from Pharmaceuticals; non-HER targeted TK inhibitors such as imatinib mesylate (GLEEVEC® available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT® available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584 available from Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261, and CGP 62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. Patent No. 5,804,396); tryphostins (U.S. Patent No. 5,804,396); ZD6474 (Astra Zeneca);PTK-787 (Novartis / Schering AG); pan-HER inhibitors, such as CI-1033 (Pfizer); Affinitac (ISIS 3521, Isis / Lilly); imatinib mesylate (GLEEVEC®); PKI166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®); or the following patent publications: U.S. Pat. No. 5,804,396, WO1999 / 09016 (American Cyanamid), WO1998 / 43960 (American Cyanamid), WO1997 / 38983 (Warner Lambert), WO1999 / 06378 (Warner Lambert), WO1999 / 06396 (Warner Lambert), WO1996 / 30347 (Pfizer, Inc), WO1996 / 33978 (Zeneca), WO1996 / 3397 (Zeneca), and WO1996 / 33980 (Zeneca).

[0139] Chemotherapeutic agents include dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, and interferon alfa- Also included are 2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.

[0140] Chemotherapeutic agents include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate ... thizone-17-valerate, aclomethasone propionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasone-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-form (feG) (IMULAN BioTherapeutics, LLC; antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide minocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers such as etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), cetrizumab pegol (CIMZIA®), golimumab ( SIMPONI®), interleukin 1 (IL-1) blockers such as anakinra (KINERET®), T cell costimulation blockers such as abatacept (ORENCIA®), interleukin 6 (IL-6) blockers, tocilizumab (ACTEMERA®); interleukin 13 (IL-13) blockers such as lebrikizumab; interferon alpha (IFN) blockers such as rontalizumab; beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers such as anti-lymphotoxin alpha (LTa);Various investigational agents, such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, and farnesyltransferase inhibitors (L-739749, L-744832); polyphenols, such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid and its derivatives; autophagy inhibitors, such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin; podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®). (registered trademark), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); as well as epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOOPE® vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium (GENASENSE®); pixantrone; farnesyltransferase inhibitors, such as lonafarnib (SCH 6636, SARASAR™); and pharma- ceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0141] The term "prodrug" as used herein refers to a precursor or derivative form of a pharmacologic active substance that is less cytotoxic to tumor cells compared to the parent drug and can be activated or converted by enzymes into a more active parent 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-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs, which can be converted to more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms for use in the present invention include, but are not limited to, the chemotherapeutic agents mentioned above.

[0142] A "growth inhibitory agent," as used herein, refers to a compound or composition that inhibits the growth and / or proliferation of cells (e.g., cells whose growth is dependent on PD-L1 expression) either in vitro or in vivo. Thus, a growth inhibitory agent may be one that significantly reduces the percentage of cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as the anthracycline antibiotic doxorubicin ((8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione), epirubicin, daunorubicin, etoposide, and bleomycin. Agents that arrest G1 also extend to S-phase arrest, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in "The Molecular Basis of Cancer," by Murakami et al., Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antitineoplastic drugs" (WB Saunders: Philadelphia, 1995), especially page 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs, both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew tree, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules derived from tubulin dimers, stabilize microtubules by preventing depolymerization, and inhibit mitosis in cells.

[0143] "Radiation therapy" refers to the use of directed gamma or beta radiation to induce sufficient damage to cells to limit their ability to function normally or to destroy them completely. It is understood that there are many methods known in the art for determining dosage and duration of treatment. A typical therapeutic agent is given as a single dose, with typical dosages ranging from 10 to 200 units (grays) per day.

[0144] As used herein, the terms "patient" or "subject" are used interchangeably and refer to any single animal, more preferably a mammal (including, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human animals such as non-human primates), for which treatment is desired. In certain embodiments, a patient herein is a human.

[0145] As used herein, "administering" refers to a method of providing a dosage of a compound (e.g., an antagonist) or pharmaceutical composition (e.g., a pharmaceutical composition comprising an antagonist) to a subject (e.g., a patient). Administration can be by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if localized treatment is desired. Parenteral injections include, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0146] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents, where the administration at least partially overlaps in time. Thus, concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after the cessation of administration of one or more other agent(s).

[0147] "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. Reduction or inhibition may refer to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.

[0148] The term "package insert" is used to refer to instructions typically included in the packaging of a therapeutic product that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic agent.

[0149] A "sterile" preparation is aseptic or free of all viable microorganisms and their spores.

[0150] An "article of manufacture" is any article of manufacture (e.g., package or container) or kit that includes at least one reagent, e.g., a pharmaceutical agent for the treatment of a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker (e.g., PD-L1) described herein. In certain embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0151] The phrase "based on," as used herein, means that information about one or more biomarkers is used to inform, for example, treatment decisions, information provided on a package insert, or marketing / promotional guidelines.

[0152] III. Method A. Diagnostic Methods Based on PD-L1 Expression Levels Provided herein is a method of determining whether a patient suffering from cancer (e.g., bladder cancer, e.g., urothelial bladder cancer (UBC)) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. Also provided herein is a method of predicting the responsiveness of a patient suffering from cancer (e.g., UBC) to a treatment comprising a PD-L1 axis-binding antagonist. Also provided herein is a method of selecting a therapy for a patient suffering from cancer (e.g., UBC). Any of the foregoing methods may be based on the expression level of a biomarker provided herein, e.g., PD-L1 expression in a tumor sample, e.g., in tumor-infiltrating immune cells. Any of the methods may further be based on determining a tumor sample subtype. Any of the methods may further comprise administering to the patient a PD-L1 axis-binding antagonist (e.g., as described in Section D below). Any of the methods may further comprise administering to the patient an effective amount of a second therapeutic agent.

[0153] The invention provides a method of determining whether a patient suffering from bladder cancer is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining the expression level of PD-L1 in tumor-infiltrating immune cells in a tumor sample obtained from the patient, wherein a detectable expression level of PD-L1 in tumor-infiltrating immune cells making up about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) of the tumor sample indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. For example, in some cases, a detectable expression level of PD-L1 in tumor infiltrating immune cells that comprise about 1% or more of a tumor sample indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis-binding antagonist. In some cases, a detectable expression level of PD-L1 in tumor infiltrating immune cells that comprise about 5% or more of a tumor sample indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis-binding antagonist. In other cases, a detectable expression level of PD-L1 in tumor infiltrating immune cells that comprise about 10% or more of a tumor sample indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis-binding antagonist.

[0154] The invention further provides a method of predicting a patient suffering from bladder cancer's responsiveness to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining the expression level of PD-L1 in tumor-infiltrating immune cells in a tumor sample obtained from the patient, wherein a detectable expression level of PD-L1 in tumor-infiltrating immune cells making up about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) of the tumor sample indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. For example, in some cases, a detectable expression level of PD-L1 in tumor infiltrating immune cells that comprise about 1% or more of a tumor sample indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis-binding antagonist. In some cases, a detectable expression level of PD-L1 in tumor infiltrating immune cells that comprise about 5% or more of a tumor sample indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis-binding antagonist. In other cases, a detectable expression level of PD-L1 in tumor infiltrating immune cells that comprise about 10% or more of a tumor sample indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis-binding antagonist.

[0155] The invention further provides a method of selecting a therapy for a patient suffering from bladder cancer, the method comprising determining the expression level of PD-L1 in tumor-infiltrating immune cells in a tumor sample obtained from the patient, and selecting a therapy comprising a PD-L1 axis-binding antagonist for the patient based on the detectable expression level of PD-L1 in tumor-infiltrating immune cells that make up about 1% or more of the tumor sample (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more). For example, in some cases, the method includes selecting a therapy that includes a PD-L1 axis-binding antagonist based on a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise about 1% or more of the tumor sample. In some cases, a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise about 5% or more of the tumor sample indicates that the patient is likely to respond to a therapy that includes a PD-L1 axis-binding antagonist. In other cases, the method includes selecting a therapy that includes a PD-L1 axis-binding antagonist based on a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise about 10% or more of the tumor sample.

[0156] In any of the above methods, the tumor-infiltrating immune cells may cover about 1% or more of the tumor area in a section of a tumor sample obtained from a patient (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, or about 90% or more). For example, in some cases, the tumor-infiltrating immune cells may cover about 1% or more of the tumor area in a section of a tumor sample. In some cases, the tumor infiltrating immune cells may cover about 5% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 10% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 15% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 20% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 25% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 30% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 35% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 40% or more of the tumor area in the section of the tumor sample. In some cases, tumor infiltrating immune cells may cover about 50% or more of the tumor area in a section of a tumor sample.

[0157] In any of the foregoing methods, about 1% (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more) of the tumor-infiltrating immune cells in a tumor sample may express a detectable expression level of PD-L1.

[0158] In any of the foregoing methods, the method further comprises administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist based on the expression level of PD-L1 on tumor-infiltrating immune cells in the tumor sample. The PD-L1 axis-binding antagonist can be any PD-L1 axis-binding antagonist known in the art or described herein, for example, in Section D below.

[0159] For example, in some cases, the PD-L1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist. In some cases, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In still other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some cases, the PD-L1 binding antagonist is an antibody. In some instances, the antibody is selected from the group consisting of YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). In some instances, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 19, the HVR-H2 sequence of SEQ ID NO: 20, and the HVR-H3 sequence of SEQ ID NO: 21, and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 22, the HVR-L2 sequence of SEQ ID NO: 23, and the HVR-L3 sequence of SEQ ID NO: 24. In some instances, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0160] In some cases, the PD-L1 axis binding antagonist is a PD-1 binding antagonist. For example, in some cases, the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners. In some cases, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In other cases, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In yet other cases, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some cases, the PD-1 binding antagonist is an antibody. In some cases, the antibody is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. In some cases, the PD-1 binding antagonist is an Fc fusion protein. For example, in some cases, the Fc fusion protein is AMP-224.

[0161] In some cases, the method further comprises administering to the patient an effective amount of a second therapeutic agent, hi some cases, the second therapeutic agent is selected from the group consisting of a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, and combinations thereof.

[0162] In any of the foregoing cases, the bladder cancer can be urothelial bladder cancer, including, but not limited to, non-muscle invasive urothelial bladder cancer, muscle invasive urothelial bladder cancer, or metastatic urothelial bladder cancer. In some cases, the urothelial bladder cancer is metastatic urothelial bladder cancer.

[0163] The presence and / or expression level / amount of a biomarker (e.g., PD-L1) may be determined qualitatively and / or quantitatively based on any suitable criteria known in the art, including but not limited to DNA, mRNA, cDNA, protein, protein fragments, and / or gene copy number.

[0164] In any of the foregoing methods, the sample obtained from the patient is selected from the group consisting of tissue, whole blood, plasma, serum, and combinations thereof. In some cases, the sample is a tissue sample. In some cases, the tissue sample is a tumor sample. In some cases, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, or any combination thereof. In any of the foregoing cases, the tumor sample can be a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0165] In any of the foregoing methods, the method can include determining the presence and / or expression level of an additional biomarker. In some cases, the additional biomarker is a biomarker described in WO2014 / 151006, the disclosure of which is incorporated herein by reference in its entirety. In some cases, the additional biomarker is selected from circulating Ki-67+CD8+ T cells, interferon gamma, MCP-1, or a myeloid cell-associated gene. In some cases, the myeloid cell-associated gene is selected from IL18, CCL2, and IL1B.

[0166] The presence and / or expression levels / amounts of the various biomarkers described herein in a sample can be analyzed by a number of methodologies, many of which are known in the art and understood by those of skill in the art, including immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, quantitative blood-based assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, fluorescent in situ hybridization (FISH), semi-transfected ELISA, and the like. Examples of assays that can be used to assess the status of genes and gene products include, but are not limited to, any one of a wide variety of assays that can be performed by gene analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (PCR) (including quantitative real-time PCR (qRT-PCR) and other amplification-based detection methods (e.g., branched DNA, SISBA, TMA, etc.), RNA-Seq, microarray analysis, gene expression profiling, and / or serial analysis of gene expression ("SAGE"), as well as protein, gene, and / or tissue array analysis. Exemplary protocols for assessing the status of genes and gene products are found, for example, in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern blotting), 4 (Southern blotting), 15 (immunoblotting), and 18 (PCR analysis). Multiplexed immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD"), may also be used.

[0167] In any of the foregoing methods, the presence and / or expression level / amount of a biomarker (e.g., PD-L1) is measured by determining the protein expression level of the biomarker. In certain cases, the method includes contacting the biological sample with an antibody (e.g., an anti-PD-L1 antibody) that specifically binds to the biomarker described herein 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 some cases, the antibody is used to select a biomarker for selection of a subject, e.g., an individual, eligible for treatment with a PD-L1 axis-binding antagonist. Any method of measuring protein expression levels known in the art or described herein may be used. For example, in some cases, the protein expression level of a biomarker (e.g., PD-L1) is determined using a method selected from the group consisting of flow cytometry (e.g., fluorescence activated cell sorting (FACS™)), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry (IHC), immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC. In some cases, the protein expression level of a biomarker (e.g., PD-L1) is determined in tumor infiltrating immune cells. In some cases, the protein expression level of a biomarker (e.g., PD-L1) is determined in tumor cells. In some cases, the protein expression level of a biomarker (e.g., PD-L1) is determined in tumor infiltrating immune cells and / or tumor cells.

[0168] In certain cases, the presence and / or expression level / amount of a biomarker protein (e.g., PD-L1) is tested using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method of determining or detecting the presence of a protein in a sample. In some cases of any of these methods, assays, and / or kits, the biomarker is PD-L1. In one case, the expression level of the biomarker is determined using a method that includes (a) performing an IHC analysis of a sample (e.g., a tumor sample obtained from a patient) using an antibody, and (b) determining the expression level of the biomarker in the sample. In some cases, the IHC staining intensity is determined relative to a reference. In some cases, the reference is a reference value. In some cases, the reference is a reference sample (e.g., a control cell line stained sample, a tissue sample from a non-cancer patient, or a PD-L1 negative tumor sample).

[0169] IHC may be performed in combination with additional techniques such as morphological staining and / or in situ hybridization (e.g., FISH). Two general IHC methods are available: direct and indirect assays. According to the first assay, the binding of the antibody to the target antigen is directly determined. This direct assay uses a labeling reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction. In a typical indirect assay, an uncomplexed primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. If the secondary antibody is conjugated to an enzyme label, a chromogenic or fluorogenic substrate is added, resulting in the visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.

[0170] The primary and / or secondary antibodies used in IHC will typically be labeled with a detectable moiety. Numerous labels are available, which can generally be grouped into the following categories: (a) radioisotopes, e.g. 35 S, 14 C. 125 1. 3 H, and131 (b) colloidal gold particles; (c) fluorescent labels including, but not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycocrytherin, phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7, and / or derivatives of any one or more of the above; (d) a variety of enzyme-substrate labels are available, and U.S. Pat. No. 4,275,149 provides a review of some of these. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase, see, e.g., U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.

[0171] Examples of enzyme and substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, alkaline phosphatase (AP) with para-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, for example, U.S. Patent Nos. 4,275,149 and 4,318,980.

[0172] Specimens can be prepared, for example, manually or using an automated staining device (e.g., Ventana Benchmark XT or Benchmark ULTRA device) (see, for example, Example 1 below). The specimens thus prepared can be mounted and coverslipped. Slide evaluation can then be determined, for example, using a microscope, and staining intensity criteria routinely used in the art can be utilized. In one instance, when cells and / or tissues from a tumor are examined using IHC, it is understood that staining is generally determined or assessed in tumor cell(s) and / or tissue (as opposed to stroma or surrounding tissue that may be present in the sample). In some instances, when cells and / or tissues from a tumor are examined using IHC, it is understood that staining includes determining or assessing in tumor-infiltrating immune cells, including intratumoral or peritumoral immune cells. In some cases, the presence of a biomarker (e.g., PD-L1) is detected by IHC in more than 0% of the samples, in at least 1% of the samples, in at least 5% of the samples, in at least 10% of the samples, in at least 15% of the samples, in at least 15% of the samples, in at least 20% of the samples, in at least 25% of the samples, in at least 30% of the samples, in at least 35% of the samples, in at least 40% of the samples, in at least 45% of the samples, in at least 50% of the samples, in at least 55% of the samples, in at least 60% of the samples, in at least 65% of the samples, in at least 70% of the samples, in at least 75% of the samples, in at least 80% of the samples, in at least 85% of the samples, in at least 90% of the samples, in at least 95% of the samples, or more. Samples can be scored, for example, by a pathologist or automated image analysis, using any of the criteria described herein (see, e.g., Table 2).

[0173] In some cases of any of the methods described herein, PD-L1 is detected by immunohistochemistry using an anti-PD-L1 diagnostic antibody (i.e., a primary antibody). In some cases, the PD-L1 diagnostic antibody specifically binds to human PD-L1. In some cases, the PD-L1 diagnostic antibody is a non-human antibody. In some cases, the PD-L1 diagnostic antibody is a rat, mouse, or rabbit antibody. In some cases, the PD-L1 diagnostic antibody is a rabbit antibody. In some cases, the PD-L1 diagnostic antibody is a monoclonal antibody. In some cases, the PD-L1 diagnostic antibody is directly labeled. In some cases, the PD-L1 diagnostic antibody is indirectly labeled.

[0174] In some cases of any of the aforementioned methods, the expression level of PD-L1 is detected using IHC in tumor-infiltrating immune cells, tumor 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, and other tumor stromal cells (e.g., fibroblasts). Such tumor-infiltrating immune cells may be T lymphocytes (such as CD8+ T lymphocytes and / or CD4+ T lymphocytes), B lymphocytes, or other myeloid cells including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer cells. In some cases, staining for PD-L1 is detected as membrane staining, cytoplasmic staining, and a combination thereof. In other cases, the absence of PD-L1 is detected as absence or no staining in the sample.

[0175] In any of the above methods, the expression level of a biomarker (e.g., PD-L1) can be a nucleic acid expression level. In some cases, the nucleic acid expression level is determined using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, or in situ hybridization (e.g., FISH). In some cases, the expression level of a biomarker (e.g., PD-L1) is determined in tumor cells, tumor-infiltrating immune cells, stromal cells, or a combination thereof. In some cases, the expression level of a biomarker (e.g., PD-L1) is determined in tumor-infiltrating immune cells. In some cases, the expression level of a biomarker (e.g., PD-L1) is determined in tumor cells.

[0176] Methods for evaluating mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization, Northern blots, and related techniques using labeled riboprobes specific for one or more genes) and various nucleic acid amplification assays (RT-PCR using complementary primers specific for one or more genes, and other amplification-based detection methods, such as branched DNA, SISBA, TMA, etc.). In addition, such methods may include one or more steps that allow the level of target mRNA to be determined in a biological sample (for example, by simultaneously testing the level of a comparative control mRNA sequence of a "housekeeping" gene, such as an actin family member). Optionally, the sequence of the amplified target cDNA may be determined. Optional methods include protocols for testing or detecting mRNA, such as target mRNA, in tissue or cell samples by microarray technology. Using nucleic acid microarrays, 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 an array of nucleic acids immobilized on a solid support. The array is constructed such that the sequence and location of each member of the array is known. For example, a selection of genes whose expression correlates with increased or decreased clinical benefit of a therapy that includes a PD-L1 axis binding antagonist can be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.

[0177] In certain cases, the presence and / or expression level / amount of the biomarker in the first sample is increased or elevated compared to the presence / absence and / or expression level / amount in the second sample. In certain cases, the presence / absence and / or expression level / amount of the biomarker in the first sample is decreased or reduced compared to the presence and / or expression level / amount in the second sample. In certain cases, the second sample is a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue. Further disclosures for determining the presence / absence and / or expression level / amount of a gene are described herein.

[0178] In certain cases, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a single sample or a combination of multiple samples from the same subject or individual that are obtained at one or more times different from when test sample is obtained.For example, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from the same subject or individual at an earlier time than when test sample is obtained.Such reference sample, reference cell, reference tissue, control sample, control cell or control tissue can be useful when reference sample is obtained during early diagnosis of cancer and test sample is obtained when cancer becomes metastatic cancer.

[0179] In certain cases, the reference sample, the reference cell, the reference tissue, the control sample, the control cell, or the control tissue is a combination of multiple samples from one or more healthy individuals who are not patients. In certain cases, the reference sample, the reference cell, the reference tissue, the control sample, the control cell, or the control tissue is a combination of multiple samples from one or more individuals who are not subjects or individuals and have a disease or disorder (e.g., cancer). In certain embodiments, the reference sample, the reference cell, the reference tissue, the control sample, the control cell, or the control tissue is a pooled RNA sample from normal tissue, or a pooled plasma or serum sample from one or more individuals who are not patients. In certain embodiments, the reference sample, the reference cell, the reference tissue, the control sample, the control cell, or the control tissue is a pooled RNA sample from tumor tissue, or a pooled plasma or serum sample from one or more individuals who are not patients and have a disease or disorder (e.g., cancer).

[0180] In some embodiments of any of these methods, elevated or increased expression refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of a biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)) as detected by standard art-known methods, such as those described herein, compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, elevated expression refers to an increase in the expression level / amount of a biomarker in a sample, which increase is about any of 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 of the expression level / amount of the respective 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 greater than 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, reference cell, reference tissue, control sample, control cell, control tissue, or an internal control (e.g., a housekeeping gene).

[0181] In some embodiments of any of these methods, reduced expression refers to an overall reduction of any 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., protein or nucleic acid (e.g., gene or mRNA)) as detected by standard art-known methods, such as those described herein, compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression refers to a decrease in the expression level / amount of a biomarker in a sample, which 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 of the expression level / amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0182] B. Diagnostic Methods Based on Assessment of Tumor Subtype Provided herein is a method for determining whether a patient suffering from cancer (e.g., bladder cancer (e.g., UBC)) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist based on the assessment of tumor subtype, which can be used independently or in combination with any of the aforementioned methods presented in section A above. For example, the present invention provides a method for determining whether a patient suffering from bladder cancer is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising: determining a tumor subtype from a tumor sample obtained from the patient, and a luminal subtype tumor indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. In some cases, determining that the tumor sample is a luminal subtype II tumor indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. In some cases, the level of one or more of the biomarkers listed in Table 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) compared to a reference level of the biomarker can be used to determine the tumor subtype. In some cases, the level of one or more of the biomarkers listed in Table 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) compared to a reference level of the biomarker can be used to determine a luminal subtype tumor. In some cases, the level of one or more of the biomarkers listed in Table 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) compared to a reference level of the biomarker can be used to determine a luminal subtype II tumor. In some cases, the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of the biomarkers listed in Table 1 relative to a reference level of the biomarker can be used to determine whether a patient suffering from cancer (e.g., bladder cancer (e.g., UBC)) is likely to respond to a treatment that includes a PD-L1 axis binding antagonist.In other cases, e.g., an increase and / or decrease in the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 16) of the biomarkers listed in Table 1 relative to a reference level of the biomarker is detected in about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, In combination with detectable expression levels of PD-L1 in tumor infiltrating immune cells that constitute about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more), the expression levels of PD-L1 in tumor infiltrating immune cells can be used to determine whether a patient suffering from cancer (bladder cancer (e.g., UBC)) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. Any of these methods may further comprise administering to the patient a PD-L1 axis-binding antagonist (e.g., those described in Section D below). Any of these methods may further comprise administering to the patient an effective amount of a second therapeutic agent. TIFF2025003997000002.tif114170

[0183] The methods of predicting the responsiveness of a patient suffering from cancer (e.g., bladder cancer (e.g., UBC) to a treatment comprising a PD-L1 axis-binding antagonist based on an assessment of the tumor subtype may be used independently or in combination with any of the aforementioned methods presented in section A above. For example, the present invention provides a method of predicting whether a patient suffering from bladder cancer is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining the tumor subtype from a tumor sample obtained from the patient, the method comprising: In some cases, a determination that a tumor sample is a luminal subtype II tumor indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis binding antagonist. In some cases, a determination that a tumor sample is a luminal subtype II tumor indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis binding antagonist. In some cases, a determination that a tumor sample is a luminal subtype II tumor indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis binding antagonist. In some cases, a determination that a tumor sample is a luminal subtype II tumor indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis binding antagonist. In some cases, the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of the biomarkers listed in Table 1 compared to a reference level of the biomarker can be used to determine a luminal subtype tumor. In some cases, the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of the biomarkers listed in Table 1 compared to a reference level of the biomarker can be used to determine a luminal subtype tumor. , 13, 14, 15, or 16) of the biomarkers listed in Table 1 relative to a reference level of the biomarker can be used to determine whether a patient suffering from cancer (e.g., bladder cancer (e.g., UBC)) is likely to respond to a treatment that includes a PD-L1 axis binding antagonist.In other cases, for example, an increase and / or decrease in the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 16) of the biomarkers listed in Table 1 relative to a reference level of the biomarker is detected in about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more) of tumor samples. In combination with detectable expression levels of PD-L1 in tumor infiltrating immune cells that constitute about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more of the tumor, it can be possible to predict whether a patient suffering from cancer (bladder cancer (e.g., UBC)) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. Any of the methods may further comprise administering to the patient a PD-L1 axis-binding antagonist (e.g., those described in Section D below). Any of the methods may further comprise administering to the patient an effective amount of a second therapeutic agent.

[0184] The method of selecting a therapy for a patient suffering from cancer (e.g., bladder cancer (e.g., UBC)) comprising selecting a PD-L1 axis-binding antagonist based on an assessment of tumor subtype can be used independently or in combination with any of the aforementioned methods presented in section A above. In some cases, the method comprises determining a tumor subtype from a tumor sample obtained from the patient, and the PD-L1 axis-binding antagonist is selected based on a determination that the tumor is a luminal subtype tumor. In some cases, the PD-L1 axis-binding antagonist is selected based on a determination that the tumor is a luminal subtype II tumor. In some cases, the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of the biomarkers listed in Table 1 relative to a reference level of the biomarker can be used to determine the tumor subtype. In some cases, the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of the biomarkers listed in Table 1 compared to a reference level of the biomarker can be used to determine a luminal subtype tumor. In some cases, the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of the biomarkers listed in Table 1 compared to a reference level of the biomarker can be used to determine a luminal subtype II tumor. In some cases, the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of the biomarkers listed in Table 1 relative to a reference level of the biomarker can be used to select a PD-L1 axis binding antagonist as an appropriate therapy for a patient suffering from cancer (e.g., bladder cancer (e.g., UBC)).In other cases, for example, an increase and / or decrease in the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 16) of the biomarkers listed in Table 1 relative to a reference level of the biomarker is detected in about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, In combination with detectable expression levels of PD-L1 in tumor infiltrating immune cells that constitute about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more), can inform the selection of a PD-L1 axis-binding antagonist for a patient suffering from cancer (e.g., bladder cancer (e.g., UBC)). Any of the methods may further comprise administering to the patient a PD-L1 axis-binding antagonist (e.g., those described in Section D below). Any of the methods may further comprise administering to the patient an effective amount of a second therapeutic agent.

[0185] In any of the foregoing methods, the biomarkers set forth in Table 1 are determined to be increased and / or decreased by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, or about 90% or more) relative to a reference level of the biomarker set forth in Table 1. For example, in some instances, the level of one or more biomarkers is determined to be increased and / or decreased by about 1% or more. In some cases, the level of one or more biomarkers is determined to be increased and / or decreased by about 5% or more. In other cases, the level of one or more biomarkers is determined to be increased and / or decreased by about 10% or more. In some cases, the level of one or more biomarkers is determined to be increased and / or decreased by about 15% or more. In still other cases, the level of one or more biomarkers is determined to be increased and / or decreased by about 20% or more. In further cases, the level of one or more biomarkers is determined to be increased and / or decreased by about 25% or more. In some cases, the level of one or more biomarkers is determined to be increased and / or decreased by about 30% or more. In some cases, the level of one or more biomarkers is determined to be increased and / or decreased by about 35% or more. In some cases, the level of one or more biomarkers is determined to be increased and / or decreased by about 40% or more. In some cases, the level of one or more biomarkers is determined to be increased and / or decreased by about 50% or more.

[0186] In any of the above cases, the tumor sample obtained from the patient is determined to be a luminal subtype tumor (e.g., a UBC luminal subtype tumor). In some cases, the tumor is determined to be a luminal subtype II tumor. In some cases, the expression levels of at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group A of Table 1 (e.g., FGFR3, miR-99a-5p, miR-100-5p, CDKN2A) and at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group B of Table 1 (e.g., KRT5, KRT6A, KRT14, EGFR) can be used to determine the luminal subtype II classification. In some cases, the expression levels of at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group A of Table 1 (e.g., FGFR3, miR-99a-5p, miR-100-5p, CDKN2A) and at least one or more (e.g., 1, 2, 3, 4, 5, or 6) biomarkers selected from Group C of Table 1 (e.g., GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, E-cadherin) can be used to determine the luminal subtype II classification. In some cases, the expression levels of at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group A of Table 1 (e.g., FGFR3, miR-99a-5p, miR-100-5p, CDKN2A) and at least one or more (e.g., 1 or 2) biomarkers selected from Group D of Table 1 (e.g., ERBB2, ESR2) can be used to determine the luminal subtype II classification.In some cases, the expression levels of at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group A of Table 1 (e.g., FGFR3, miR-99a-5p, miR-100-5p, CDKN2A), at least one or more (e.g., 1, 2, 3, 4, 5, or 6) biomarkers selected from Group C of Table 1 (e.g., GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, E-cadherin), and at least one or more (e.g., 1 or 2) biomarkers selected from Group D of Table 1 (e.g., ERBB2, ESR2) can be used to determine the luminal subtype II classification. In some cases, the expression levels of at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group A of Table 1 (e.g., FGFR3, miR-99a-5p, miR-100-5p, CDKN2A), at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group B of Table 1 (e.g., KRT5, KRT6A, KRT14, EGFR), at least one or more (e.g., 1, 2, 3, 4, 5, or 6) biomarkers selected from Group C of Table 1 (e.g., GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, E-cadherin), and at least one or more (e.g., 1 or 2) biomarkers selected from Group D of Table 1 (e.g., ERBB2, ESR2) can be used to determine the luminal subtype II classification. In any of the foregoing cases, the biomarker level is an mRNA level, a protein level, and / or a microRNA (e.g., miRNA) level.

[0187] In some cases, an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3 in combination with a decrease in the expression level of at least one of KRT5, KRT6A, KRT14, and EGFR, compared to a reference level of the biomarker, can be used to determine the luminal subtype II classification. In some cases, an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3 in combination with an increase in the level of at least one of GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, and E-cadherin, compared to a reference level of the biomarker, can be used to determine the luminal subtype II classification. In some cases, a combination of an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3 and an increase in the level of ERBB2 and / or ESR2 compared to a reference level of the biomarkers can be used to determine the luminal subtype II classification. In some cases, an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3, an increase in the level of at least one of GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, and E-cadherin, and an increase in the level of ERBB2 and / or ESR2 compared to a reference level of the biomarkers can be used to determine the luminal subtype II classification.

[0188] In some cases, an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3; a decrease in the level of at least one of KRT5, KRT6A, KRT14, and EGFR; and an increase in the level of ERBB2 and / or ESR2 compared to a reference level of the biomarkers can be used to determine the luminal subtype II classification. In some cases, the luminal subtype II classification can be determined using an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3; a decrease in the level of at least one of KRT5, KRT6A, KRT14, and EGFR; an increase in the level of at least one of GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, and E-cadherin; and an increase in the level of ERBB2 and / or ESR2, compared to a reference level of the biomarker. In any of the above cases, the level of the biomarker is an mRNA level, a protein level, and / or a microRNA (e.g., miRNA) level.

[0189] In some cases, the expression level of at least one of CDKN2A, GATA3, FOXA1, ERBB2, FGFR3, KRT5, KRT14, EGFR, CD8A, GZMA, GZMB, IFNG, CXCL9, CXCL10, PRF1, and TBX21 in a tumor sample obtained from a patient is determined to be altered by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) compared to a reference level of the at least one gene.

[0190] In some cases, the expression level of at least one of CDKN2A, GATA3, FOXA1, and ERBB2 in a tumor sample obtained from a patient is determined to be increased by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) relative to a reference level of the at least one gene. and / or the expression level of at least one of FGFR3, KRT5, KRT14, and EGFR in a tumor sample obtained from the patient has been determined to be decreased by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) compared to a reference level of the at least one gene.

[0191] In some cases, the expression levels of CDKN2A, GATA3, FOXA1, and ERBB2 in a tumor sample obtained from a patient are determined to be increased by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) relative to a reference level of the gene. and / or the expression levels of FGFR3, KRT5, KRT14, and EGFR in a tumor sample obtained from a patient have been determined to be decreased by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) compared to a reference level of the gene.

[0192] In some cases, the expression level of CDKN2A, GATA3, FOXA1, and ERBB2 in a tumor sample obtained from a patient is determined to be increased by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) relative to a reference level of the gene. and the expression levels of FGFR3, KRT5, KRT14, and EGFR in a tumor sample obtained from a patient have been determined to be decreased by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) compared to the reference level of the gene.

[0193] In other cases, the expression level of miR-99a-5p or miR100-5p in the tumor sample obtained from the patient is determined to be changed by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) compared to the reference level of the miRNA. In other cases, the expression level of miR-99a-5p or miR100-5p in the tumor sample obtained from the patient is determined to be increased compared to the reference level of the miRNA. In other cases, the expression level of miR-99a-5p or miR100-5p in a tumor sample obtained from a patient is determined to be increased relative to a reference level of the miRNA. In some cases, the expression level of miR-99a-5p and miR100-5p in a tumor sample obtained from a patient is determined to be increased by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) relative to a reference level of the miRNA.

[0194] In still other cases, the expression level of at least one of CD8A, GZMA, GZMB, IFNG, CXCL9, CXCL10, PRF1, and TBX21 in a tumor sample obtained from a patient is determined to be increased by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) compared to a reference level of the at least one gene. In some cases, the expression levels of at least CXCL9 and CXCL10 in a tumor sample obtained from a patient are determined to be increased by about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) compared to a reference level of the genes. In other cases, the luminal subtype tumor is a luminal cluster II subtype tumor.

[0195] In any of the foregoing methods, the method further comprises administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist based on the expression level of PD-L1 on tumor-infiltrating immune cells in the tumor sample. The PD-L1 axis-binding antagonist can be any PD-L1 axis-binding antagonist known in the art or described herein, for example, in Section D below.

[0196] For example, in some cases, the PD-L1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist. In some cases, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In still other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some cases, the PD-L1 binding antagonist is an antibody. In some instances, the antibody is selected from the group consisting of YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). In some instances, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 19, the HVR-H2 sequence of SEQ ID NO: 20, and the HVR-H3 sequence of SEQ ID NO: 21, and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 22, the HVR-L2 sequence of SEQ ID NO: 23, and the HVR-L3 sequence of SEQ ID NO: 24. In some instances, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0197] In some cases, the PD-L1 axis binding antagonist is a PD-1 binding antagonist. For example, in some cases, the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners. In some cases, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In other cases, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In yet other cases, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some cases, the PD-1 binding antagonist is an antibody. In some cases, the antibody is selected from the group consisting of MDX1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. In some cases, the PD-1 binding antagonist is an Fc fusion protein. For example, in some cases, the Fc fusion protein is AMP-224.

[0198] In some cases, the method further comprises administering to the patient an effective amount of a second therapeutic agent, hi some cases, the second therapeutic agent is selected from the group consisting of a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, and combinations thereof.

[0199] In any of the foregoing cases, the bladder cancer may be urothelial bladder cancer (UBC), including, but not limited to, non-muscle invasive urothelial bladder cancer, muscle invasive urothelial bladder cancer, or metastatic urothelial bladder cancer. In some cases, the urothelial bladder cancer is metastatic urothelial bladder cancer.

[0200] The presence and / or expression level / amount of a biomarker (e.g., PD-L1) may be determined qualitatively and / or quantitatively based on any suitable criteria known in the art, including but not limited to DNA, mRNA, cDNA, protein, protein fragments, and / or gene copy number.

[0201] In any of the foregoing methods, the sample obtained from the patient is selected from the group consisting of tissue, whole blood, plasma, serum, and combinations thereof. In some cases, the sample is a tissue sample. In some cases, the tissue sample is a tumor sample. In some cases, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, or any combination thereof. In any of the foregoing cases, the tumor sample can be a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0202] In any of the foregoing methods, the method can include determining the presence and / or expression level of an additional biomarker. In some cases, the additional biomarker is a biomarker described in WO2014 / 151006, the disclosure of which is incorporated herein by reference in its entirety. In some cases, the additional biomarker is selected from circulating Ki-67+CD8+ T cells, interferon gamma, MCP-1, or a myeloid cell-associated gene. In some cases, the myeloid cell-associated gene is selected from IL18, CCL2, and IL1B.

[0203] The presence and / or expression levels / amounts of the various biomarkers described herein in a sample may be analyzed by a number of methodologies, many of which are known in the art and understood by those of skill in the art, including, but not limited to, any one of a wide variety of assays that may be performed by immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, quantitative blood-based assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, fluorescent in situ hybridization (FISH), Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (PCR) (including quantitative real-time PCR (qRT-PCR) and other amplified detection methods, e.g., branched DNA, SISBA, TMA, etc.), RNA-Seq, microarray analysis, gene expression profiling, and / or serial analysis of gene expression ("SAGE"), as well as protein, gene, and / or tissue array analysis. Exemplary protocols for assessing the status of genes and gene products are found, for example, in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern blotting), 4 (Southern blotting), 15 (immunoblotting), and 18 (PCR analysis). Multiplexed immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD"), can also be used.

[0204] In any of the above methods, the presence and / or expression level / amount of a biomarker (e.g., PD-L1) is measured by determining the protein expression level of the biomarker. In certain cases, the method includes contacting the biological sample with an antibody (e.g., an anti-PD-L1 antibody) that specifically binds to a biomarker described herein under conditions that allow binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such a method can be an in vitro method or an in vivo method. In some cases, the antibody is used to select subjects (e.g., a biomarker for the selection of individuals) eligible for treatment with a PD-L1 axis-binding antagonist. Any method of measuring protein expression levels known in the art or described herein may be used. For example, in some cases, the protein expression level of a biomarker (e.g., PD-L1) is determined using a method selected from the group consisting of flow cytometry (e.g., fluorescence activated cell sorting (FACS™)), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry (IHC), immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC. In some cases, the protein expression level of a biomarker (e.g., PD-L1) is determined in tumor infiltrating immune cells. In some cases, the protein expression level of a biomarker (e.g., PD-L1) is determined in tumor cells. In some cases, the protein expression level of a biomarker (e.g., PD-L1) is determined in tumor infiltrating immune cells and / or tumor cells.

[0205] In certain cases, the presence and / or expression level / amount of a biomarker protein (e.g., PD-L1) is tested using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method of determining or detecting the presence of a protein in a sample. In some cases of any of these methods, assays, and / or kits, the biomarker is PD-L1. In one case, the expression level of the biomarker is determined using a method that includes (a) performing an IHC analysis of a sample (e.g., a tumor sample obtained from a patient) using an antibody, and (b) determining the expression level of the biomarker in the sample. In some cases, the IHC staining intensity is determined relative to a reference. In some cases, the reference is a reference value. In some cases, the reference is a reference sample (e.g., a control cell line stained sample, a tissue sample from a non-cancer patient, or a PD-L1 negative tumor sample).

[0206] IHC may be performed in combination with additional techniques such as morphological staining and / or in situ hybridization (e.g., FISH). Two general IHC methods are available: direct and indirect assays. According to the first assay, the binding of the antibody to the target antigen is directly determined. This direct assay uses a labeling reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction. In a typical indirect assay, an uncomplexed primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. If the secondary antibody is conjugated to an enzyme label, a chromogenic or fluorogenic substrate is added, resulting in the visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.

[0207] The primary and / or secondary antibodies used in IHC will typically be labeled with a detectable moiety. Numerous labels are available, which can generally be grouped into the following categories: (a) radioisotopes, e.g. 35 S, 14 C. 125 1. 3 H, and131 (b) colloidal gold particles; (c) fluorescent labels including, but not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycocrytherin, phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7, and / or derivatives of any one or more of the above; (d) a variety of enzyme-substrate labels are available, and U.S. Pat. No. 4,275,149 provides a review of some of these. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase, see, e.g., U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.

[0208] Examples of enzyme and substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, alkaline phosphatase (AP) with para-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, for example, U.S. Patent Nos. 4,275,149 and 4,318,980.

[0209] Specimens can be prepared, for example, manually or using an automated staining device (e.g., Ventana Benchmark XT or Benchmark ULTRA device) (see, for example, Example 1 below). The specimens thus prepared can be mounted and coverslipped. Slide evaluation can then be determined, for example, using a microscope, and staining intensity criteria routinely used in the art can be utilized. In one instance, when cells and / or tissues from a tumor are examined using IHC, it is understood that staining is generally determined or assessed in tumor cell(s) and / or tissue (as opposed to stroma or surrounding tissue that may be present in the sample). In some instances, when cells and / or tissues from a tumor are examined using IHC, it is understood that staining includes determining or assessing in tumor-infiltrating immune cells, including intratumoral or peritumoral immune cells. In some cases, the presence of a biomarker (e.g., PD-L1) is detected by IHC in more than 0% of the samples, in at least 1% of the samples, in at least 5% of the samples, in at least 10% of the samples, in at least 15% of the samples, in at least 15% of the samples, in at least 20% of the samples, in at least 25% of the samples, in at least 30% of the samples, in at least 35% of the samples, in at least 40% of the samples, in at least 45% of the samples, in at least 50% of the samples, in at least 55% of the samples, in at least 60% of the samples, in at least 65% of the samples, in at least 70% of the samples, in at least 75% of the samples, in at least 80% of the samples, in at least 85% of the samples, in at least 90% of the samples, in at least 95% of the samples, or more. Samples can be scored, for example, by a pathologist or automated image analysis, using any of the criteria described herein (see, e.g., Table 2).

[0210] In some cases of any of the methods described herein, PD-L1 is detected by immunohistochemistry using an anti-PD-L1 diagnostic antibody. In some cases, the PD-L1 diagnostic antibody specifically binds to human PD-L1. In some cases, the PD-L1 diagnostic antibody is a non-human antibody. In some cases, the PD-L1 diagnostic antibody is a rat, mouse, or rabbit antibody. In some cases, the PD-L1 diagnostic antibody is a rabbit antibody. In some cases, the PD-L1 diagnostic antibody is a monoclonal antibody. In some cases, the PD-L1 diagnostic antibody is directly labeled. In some cases, the PD-L1 diagnostic antibody is indirectly labeled.

[0211] In some cases of any of the aforementioned methods, the expression level of PD-L1 is detected using IHC in tumor-infiltrating immune cells, tumor 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, and other tumor stromal cells (e.g., fibroblasts). Such tumor-infiltrating immune cells may be T lymphocytes (such as CD8+ T lymphocytes and / or CD4+ T lymphocytes), B lymphocytes, or other myeloid cells including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer cells. In some cases, staining for PD-L1 is detected as membrane staining, cytoplasmic staining, and a combination thereof. In other cases, the absence of PD-L1 is detected as absence or no staining in the sample.

[0212] In any of the above methods, the expression level of a biomarker (e.g., PD-L1) can be a nucleic acid expression level. In some cases, the nucleic acid expression level is determined using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, or in situ hybridization (e.g., FISH). In some cases, the expression level of a biomarker (e.g., PD-L1) is determined in tumor cells, tumor-infiltrating immune cells, stromal cells, or a combination thereof. In some cases, the expression level of a biomarker (e.g., PD-L1) is determined in tumor-infiltrating immune cells. In some cases, the expression level of a biomarker (e.g., PD-L1) is determined in tumor cells.

[0213] Methods for evaluating mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization, Northern blots, and related techniques using labeled riboprobes specific for one or more genes) and various nucleic acid amplification assays (RT-PCR using complementary primers specific for one or more genes, and other amplification-based detection methods, such as branched DNA, SISBA, TMA, etc.). In addition, such methods may include one or more steps that allow the level of target mRNA to be determined in a biological sample (for example, by simultaneously testing the level of a comparative control mRNA sequence of a "housekeeping" gene, such as an actin family member). Optionally, the sequence of the amplified target cDNA may be determined. Optional methods include protocols for testing or detecting mRNA, such as target mRNA, in tissue or cell samples by microarray technology. Using nucleic acid microarrays, 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 an array of nucleic acids immobilized on a solid support. The array is constructed such that the sequence and location of each member of the array is known. For example, a selection of genes whose expression correlates with increased or decreased clinical benefit of a therapy that includes a PD-L1 axis binding antagonist can be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.

[0214] In certain cases, the presence and / or expression level / amount of the biomarker in the first sample is increased or elevated compared to the presence / absence and / or expression level / amount in the second sample. In certain cases, the presence / absence and / or expression level / amount of the biomarker in the first sample is decreased or reduced compared to the presence and / or expression level / amount in the second sample. In certain cases, the second sample is a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue. Further disclosures for determining the presence / absence and / or expression level / amount of a gene are described herein.

[0215] In certain cases, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a single sample or a combination of multiple samples from the same subject or individual that are obtained at one or more times different from when test sample is obtained.For example, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from the same subject or individual at an earlier time than when test sample is obtained.Such reference sample, reference cell, reference tissue, control sample, control cell or control tissue can be useful when reference sample is obtained during early diagnosis of cancer and test sample is obtained when cancer becomes metastatic cancer.

[0216] In certain cases, the reference sample, the reference cell, the reference tissue, the control sample, the control cell, or the control tissue is a combination of multiple samples from one or more healthy individuals who are not patients. In certain cases, the reference sample, the reference cell, the reference tissue, the control sample, the control cell, or the control tissue is a combination of multiple samples from one or more individuals who are not subjects or individuals and have a disease or disorder (e.g., cancer). In certain embodiments, the reference sample, the reference cell, the reference tissue, the control sample, the control cell, or the control tissue is a pooled RNA sample from normal tissue, or a pooled plasma or serum sample from one or more individuals who are not patients. In certain embodiments, the reference sample, the reference cell, the reference tissue, the control sample, the control cell, or the control tissue is a pooled RNA sample from tumor tissue, or a pooled plasma or serum sample from one or more individuals who are not patients and have a disease or disorder (e.g., cancer).

[0217] In some embodiments of any of these methods, elevated or increased expression refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of a biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)) as detected by standard art-known methods, such as those described herein, compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, elevated expression refers to an increase in the expression level / amount of a biomarker in a sample, which increase is about any of 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 of the expression level / amount of the respective 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 greater than 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, reference cell, reference tissue, control sample, control cell, control tissue, or an internal control (e.g., a housekeeping gene).

[0218] In some embodiments of any of these methods, reduced expression refers to an overall reduction of any 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., protein or nucleic acid (e.g., gene or mRNA)) as detected by standard art-known methods, such as those described herein, compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression refers to a decrease in the expression level / amount of a biomarker in a sample, which 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 of the expression level / amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0219] C. Treatment method The invention provides methods of treating a patient suffering from cancer, e.g., bladder cancer (e.g., urothelial bladder cancer). In some cases, the methods of the invention involve administering to the patient an anti-cancer therapy, including a PD-L1 axis-binding antagonist. Any of the PD-L1 axis-binding antagonists described herein (see, e.g., Section D, below) or known in the art can be used in the methods. In some cases, the methods involve determining the presence and / or expression level of PD-L1 in a sample obtained from the patient (e.g., in tumor-infiltrating immune cells in a tumor sample) and administering to the patient an anti-cancer therapy based on the presence and / or expression level of PD-L1 in the sample, e.g., using a method described herein (e.g., Sections A, B, or in the Examples, below) or known in the art.

[0220] The invention provides a method of treating a patient suffering from bladder cancer, the method comprising administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute 1% or more of the tumor sample (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more).

[0221] The invention further provides a method of treating a patient suffering from bladder cancer, the method comprising administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to be a luminal subtype tumor. In some cases, the tumor has been determined to be a luminal subtype II tumor.

[0222] In any of the above methods, the tumor-infiltrating immune cells may cover about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) of the tumor area in the section of the tumor sample obtained from the patient. For example, in some cases, the tumor-infiltrating immune cells may cover about 1% or more of the tumor area in the section of the tumor sample. In some cases, the tumor-infiltrating immune cells may cover about 5% or more of the tumor area in the section of the tumor sample. In some cases, the tumor-infiltrating immune cells may cover about 10% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 15% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 20% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 25% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 30% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 35% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 40% or more of the tumor area in the section of the tumor sample. In some cases, the tumor infiltrating immune cells may cover about 50% or more of the tumor area in the section of the tumor sample.

[0223] In any of the foregoing methods, about 1% (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more) of the tumor-infiltrating immune cells in a tumor sample may express a detectable expression level of PD-L1.

[0224] The invention provides a method of treating a patient suffering from bladder cancer, the method comprising administering a therapeutically effective amount of a PD-L1 axis-binding antagonist to the patient, wherein a tumor sample obtained from the patient is determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 1% or more (e.g., about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more) of the tumor sample, indicating that the patient is likely to respond to treatment comprising a PD-L1 axis-binding antagonist. For example, in some cases, a detectable expression level of PD-L1 in tumor infiltrating immune cells that constitute about 1% or more of a tumor sample indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis-binding antagonist. In some cases, a detectable expression level of PD-L1 in tumor infiltrating immune cells that constitute about 5% or more of a tumor sample indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis-binding antagonist. In other cases, a detectable expression level of PD-L1 in tumor infiltrating immune cells that constitute about 10% or more of a tumor sample indicates that the patient is likely to respond to a treatment that includes a PD-L1 axis-binding antagonist. In some cases, changes in the levels of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of the biomarkers listed in Table 1 may be used to aid in determining the tumor subtype. In some cases, the tumor sample (e.g., a UBC tumor sample) is a luminal subtype tumor (e.g., a luminal subtype II tumor). The present invention provides a method of treating a patient suffering from bladder cancer, the method comprising administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist, wherein the tumor sample obtained from the patient is determined to be a luminal subtype tumor (e.g., a UBC luminal subtype tumor). In some cases, the tumor is determined to be a luminal subtype II tumor.In some cases, the expression levels of at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group A of Table 1 (e.g., FGFR3, miR-99a-5p, miR-100-5p, CDKN2A) and at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group B of Table 1 (e.g., KRT5, KRT6A, KRT14, EGFR) can be used to determine the luminal subtype II classification. In some cases, the expression levels of at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group A of Table 1 (e.g., FGFR3, miR-99a-5p, miR-100-5p, CDKN2A) and at least one or more (e.g., 1, 2, 3, 4, 5, or 6) biomarkers selected from Group C of Table 1 (e.g., GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, E-cadherin) can be used to determine the luminal subtype II classification. In some cases, the expression levels of at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group A of Table 1 (e.g., FGFR3, miR-99a-5p, miR-100-5p, CDKN2A) and at least one or more (e.g., 1 or 2) biomarkers selected from Group D of Table 1 (e.g., ERBB2, ESR2) can be used to determine the luminal subtype II classification. In some cases, the expression levels of at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group A of Table 1 (e.g., FGFR3, miR-99a-5p, miR-100-5p, CDKN2A), at least one or more (e.g., 1, 2, 3, 4, 5, or 6) biomarkers selected from Group C of Table 1 (e.g., GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, E-cadherin), and at least one or more (e.g., 1 or 2) biomarkers selected from Group D of Table 1 (e.g., ERBB2, ESR2) can be used to determine the luminal subtype II classification.In some cases, the expression levels of at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group A of Table 1 (e.g., FGFR3, miR-99a-5p, miR-100-5p, CDKN2A), at least one or more (e.g., 1, 2, 3, or 4) biomarkers selected from Group B of Table 1 (e.g., KRT5, KRT6A, KRT14, EGFR), at least one or more (e.g., 1, 2, 3, 4, 5, or 6) biomarkers selected from Group C of Table 1 (e.g., GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, E-cadherin), and at least one or more (e.g., 1 or 2) biomarkers selected from Group D of Table 1 (e.g., ERBB2, ESR2) can be used to determine the luminal subtype II classification. In any of the foregoing cases, the biomarker level is an mRNA level, a protein level, and / or a microRNA (e.g., miRNA) level.

[0225] In some cases, an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3 in combination with a decrease in the expression level of at least one of KRT5, KRT6A, KRT14, and EGFR, compared to a reference level of the biomarker, can be used to determine the luminal subtype II classification. In some cases, an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3 in combination with an increase in the level of at least one of GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, and E-cadherin, compared to a reference level of the biomarker, can be used to determine the luminal subtype II classification. In some cases, a combination of an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3 and an increase in the level of ERBB2 and / or ESR2 compared to a reference level of the biomarkers can be used to determine the luminal subtype II classification. In some cases, an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3, an increase in the level of at least one of GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, and E-cadherin, and an increase in the level of ERBB2 and / or ESR2 compared to a reference level of the biomarkers can be used to determine the luminal subtype II classification. In some cases, an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3; a decrease in the level of at least one of KRT5, KRT6A, KRT14, and EGFR; and an increase in the level of ERBB2 and / or ESR2 compared to a reference level of the biomarkers can be used to determine the luminal subtype II classification.In some cases, an increase in the expression level of at least one of miR-99a-5p, miR-100-5p, and CDKN2A, and / or a decrease in the expression level of FGFR3; a decrease in the level of at least one of KRT5, KRT6A, KRT14, and EGFR; an increase in the level of at least one of GATA3, FOXA1, UPK3A, miR-200a-3p, miR-200b-3p, and E-cadherin; and an increase in the level of ERBB2 and / or ESR2, compared to a reference level of the biomarker, can be used to determine the luminal subtype II classification. In any of the above cases, the level of the biomarker is an mRNA level, a protein level, and / or a miRNA level.

[0226] In any of the foregoing methods, the PD-L1 axis binding antagonist can be any PD-L1 axis binding antagonist known in the art or described herein, for example, in Section D below.

[0227] For example, in some cases, the PD-L1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist. In some cases, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In still other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some cases, the PD-L1 binding antagonist is an antibody. In some instances, the antibody is selected from the group consisting of YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). In some instances, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 19, the HVR-H2 sequence of SEQ ID NO: 20, and the HVR-H3 sequence of SEQ ID NO: 21, and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 22, the HVR-L2 sequence of SEQ ID NO: 23, and the HVR-L3 sequence of SEQ ID NO: 24. In some instances, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0228] In some cases, the PD-L1 axis binding antagonist is a PD-1 binding antagonist. For example, in some cases, the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners. In some cases, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In other cases, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In yet other cases, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some cases, the PD-1 binding antagonist is an antibody. In some cases, the antibody is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. In some cases, the PD-1 binding antagonist is an Fc fusion protein. For example, in some cases, the Fc fusion protein is AMP-224.

[0229] In some cases, the method further comprises administering to the patient an effective amount of a second therapeutic agent. In some cases, the second therapeutic agent is selected from the group consisting of a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, and combinations thereof. In some cases, the second therapeutic agent is an agonist directed against an activating costimulatory molecule. In some cases, the second therapeutic agent is an antagonist directed against an inhibitory costimulatory molecule.

[0230] In any of the foregoing cases, the urothelial bladder cancer can be, for example, non-muscle invasive urothelial bladder cancer, muscle invasive urothelial bladder cancer, or metastatic urothelial bladder cancer.

[0231] In a further aspect, the present invention provides the use of a PD-L1 axis binding antagonist in the manufacture or preparation of a medicament. In one instance, the medicament is for the treatment of cancer. In a further instance, the medicament is for use in a method of treating cancer, comprising administering an effective amount of the medicament to a patient suffering from cancer (e.g., bladder cancer (e.g., urothelial bladder cancer)). In one such instance, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below.

[0232] The compositions (e.g., PD-L1 axis binding antagonists) used in the methods described herein may be administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, transdermally, intraarterially, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, topically, transdermally, intravitreally (e.g., by intravitreal injection), by eye drops, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in a cream, or in a lipid composition. The compositions used in the methods described herein may also be administered systemically or locally. Methods of administration can vary depending on a variety of factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). In some cases, the PD-L1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. A variety of dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time points, bolus doses, and pulse infusions.

[0233] The PD-L1 axis binding antagonists (e.g., antibodies, binding polypeptides, and / or small molecules) described herein (any additional therapeutic agents) may be formulated, dosed, and administered in a manner consistent with the principles of good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to the physician. The PD-L1 axis binding antagonists need not be, but are optionally, formulated with or administered simultaneously with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of PD-L1 axis binding antagonist present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and routes of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0234] The appropriate dosage of the PD-L1 axis binding antagonists described herein (when used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of cancer (e.g., bladder cancer (e.g., urothelial bladder cancer)) will depend on the type of disease being treated, the severity and course of the disease, whether the PD-L1 axis binding antagonist is administered for prophylactic or therapeutic purposes, treatment experience, 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 suitably administered to the patient at one time or over a series of treatments. One typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or more depending on the pathology, treatment will generally be sustained until a desired suppression of disease symptoms occurs. Such doses may be administered intermittently, for example every week or every three weeks (e.g., such that the patient receives from about 2 to about 20, or for example about 6, doses of the PD-L1 axis-binding antagonist). A higher initial loading dose, followed by one or more lower doses, may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0235] For example, as a general proposition, a therapeutically effective amount of a PD-L1 axis-binding antagonist antibody administered to a human will be in the range of about 0.01 to about 50 mg / kg of patient body weight, whether in one or more doses. In some cases, the antibody used will be about 0.01 mg / kg to about 45 mg / kg, about 0.01 mg / kg to about 40 mg / kg, about 0.01 mg / kg to about 35 mg / kg, about 0.01 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 25 mg / kg, about 0.01 mg / kg to about 20 mg / kg, about 0.01 mg / kg to about 15 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 5 mg / kg, or about 0.01 mg / kg to about 1 mg / kg, administered, for example, daily, weekly, every two weeks, every three weeks, or monthly. In some cases, the antibody is administered at 15 mg / kg. However, other dosage regimens may be useful. In one case, the anti-PD-L1 antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, or about 1800 mg on day 1 of a 21-day cycle (every 3 weeks, q3w). In some cases, the anti-PD-L1 antibody MPDL3280A is administered at 1200 mg intravenously every 3 weeks (q3w). The dose can be administered in a single dose or multiple doses (e.g., 2 or 3 doses), such as by injection. The dose of the antibody administered in combination therapy can be reduced compared to monotherapy. The progress of this therapy is easily monitored by conventional techniques.

[0236] In some cases, the method further involves administering to the patient an effective amount of a second therapeutic agent. In some cases, the second therapeutic agent is selected from the group consisting of a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, and combinations thereof. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with a chemotherapy or chemotherapeutic agent. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with a radiotherapeutic agent. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with a targeted therapy or targeted therapy agent. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with an immunotherapy or immunotherapeutic agent, such as a monoclonal antibody. In some cases, the second therapeutic agent is an agonist directed against an activating costimulatory molecule. In some cases, the second therapeutic agent is an antagonist directed against an inhibitory costimulatory molecule.

[0237] Such combination therapy as described above encompasses combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of the PD-L1 axis binding antagonist can occur prior to, concurrently with, and / or subsequent to administration of the additional therapeutic agent(s). In one instance, administration of the PD-L1 axis binding antagonist and administration of the additional therapeutic agent occur within about one month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0238] Without intending to be bound by theory, it is believed that enhancing T cell stimulation by promoting activating costimulatory molecules or inhibiting negative costimulatory molecules can promote tumor cell death, thereby treating or slowing the progression of cancer. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with an agonist directed against an activating costimulatory molecule. In some cases, the activating costimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some cases, the agonist directed against an activating costimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with an antagonist directed against an inhibitory costimulatory molecule. In some cases, the inhibitory costimulatory molecule can include CTLA-4 (also known as CD152), TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase. In some cases, the antagonist directed against the inhibitory costimulatory molecule is an antagonist antibody that binds to CTLA-4, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase.

[0239] In some cases, the PD-L1 axis binding antagonist may be administered in combination with an antagonist directed against CTLA-4 (aka CD152), such as a blocking antibody. In some cases, the PD-L1 axis binding antagonist may be administered in combination with ipilimumab (aka MDX-010, MDX-101, or YERVOY®). In some cases, the PD-L1 axis binding antagonist may be administered in combination with tremelimumab (aka ticilimumab or CP-675,206). In some cases, the PD-L1 axis binding antagonist may be administered in combination with an antagonist directed against B7-H3 (aka CD276), such as a blocking antibody. In some cases, the PD-L1 axis binding antagonist may be administered in combination with MGA271. In some cases, the PD-L1 axis-binding antagonist may be administered in combination with an antagonist directed against TGF-beta, such as methemoglobin (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.

[0240] In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with a treatment comprising adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR). In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with a treatment comprising adoptive transfer of T cells comprising a dominant negative TGF beta receptor, e.g., a dominant negative TGF beta type II receptor. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with a treatment comprising the HERCREEM protocol (see, e.g., ClinicalTrials.gov Identifier NCT00889954).

[0241] In some cases, the PD-L1 axis binding antagonist may be administered in combination with an agonist, such as an activating antibody, directed against CD137 (aka TNFRSF9, 4-1BB, or ILA). In some cases, the PD-L1 axis binding antagonist may be administered in combination with urelumab (aka BMS-663513). In some cases, the PD-L1 axis binding antagonist may be administered in combination with an agonist, such as an activating antibody, directed against CD40. In some cases, the PD-L1 axis binding antagonist may be administered in combination with CP-870893. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an agonist, such as an activating antibody, directed against OX40 (aka CD134). In some cases, the PD-L1 axis binding antagonist may be administered in combination with an anti-OX40 antibody (e.g., AgonOX). In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with an agonist directed against CD27, such as an activating antibody. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with CDX-1127. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with an antagonist directed against indoleamine-2,3-dioxygenase (IDO). In some cases, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).

[0242] In some cases, the PD-L1 axis binding antagonist may be administered in combination with an antibody-drug conjugate. In some cases, the antibody-drug conjugate includes mertansine or monomethyl auristatin E (MMAE). In some cases, the PD-L1 axis binding antagonist may be administered in combination with an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599). In some cases, the PD-L1 axis binding antagonist may be administered in combination with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or KADCYLA®, Genentech). In some cases, the PD-L1 axis binding antagonist may be administered in combination with DMUC5754A. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an antibody-drug conjugate targeting the endothelin B receptor (EDNBR), such as an antibody directed against EDNBR conjugated with MMAE.

[0243] In some cases, the PD-L1 axis binding antagonist may be administered in combination with an anti-angiogenic agent. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an antibody directed against VEGF, such as VEGF-A. In some cases, the PD-L1 axis binding antagonist may be administered in combination with bevacizumab (aka AVASTIN®, Genentech). In some cases, the PD-L1 axis binding antagonist may be administered in combination with an antibody directed against angiopoietin 2 (aka Ang2). In some cases, the PD-L1 axis binding antagonist may be administered in combination with MEDI3617. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an anti-tumor agent. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an agent that targets CSF-1R (aka M-CSFR or CD115). In some cases, the PD-L1 axis binding antagonist may be administered in combination with anti-CSF-1R (aka IMC-CS4). In some cases, the PD-L1 axis binding antagonist may be administered in combination with an interferon, such as interferon alpha or interferon gamma. In some cases, the PD-L1 axis binding antagonist may be administered in combination with Roferon-A (aka recombinant interferon alpha-2a). In some cases, the PD-L1 axis binding antagonist may be administered in combination with GM-CSF (aka recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or LEUKINE®). In some cases, the PD-L1 axis binding antagonist may be administered in combination with IL-2 (aka aldesleukin or PROLEUKIN®). In some cases, the PD-L1 axis binding antagonist may be administered in combination with IL-12. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with an antibody that targets CD20. In some cases, the antibody that targets CD20 is obinutuzumab (also known as GA101 or GAZYVA®) or rituximab.In some cases, the PD-L1 axis binding antagonist may be administered in combination with an antibody that targets GITR. In some cases, the antibody that targets GITR is TRX518.

[0244] In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with a cancer vaccine. In some cases, the cancer vaccine is a peptide cancer vaccine, which in some cases is a personalized peptide vaccine. In some cases, the peptide cancer vaccine is a multivalent long peptide, multi-peptide, peptide cocktail, hybrid peptide, or peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci. 104:14-21, 2013). In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with an adjuvant. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with a therapeutic agent, including a TLR agonist, for example, Poly-ICLC (also known as HILTONOL®), LPS, MPL, or CpG ODN. In some cases, the PD-L1 axis binding antagonist may be administered in conjunction with tumor necrosis factor (TNF) alpha. In some cases, the PD-L1 axis binding antagonist may be administered in combination with IL-1. In some cases, the PD-L1 axis binding antagonist may be administered in combination with HMGB1. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an IL-10 antagonist. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an IL-4 antagonist. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an IL-13 antagonist. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an HVEM antagonist. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an ICOS agonist, for example, by administration of ICOS-L, or in combination with an agonistic antibody directed against ICOS. In some cases, the PD-L1 axis binding antagonist may be administered in combination with a therapeutic agent that targets CX3CL1. In some cases, the PD-L1 axis binding antagonist may be administered in combination with a therapeutic agent that targets CXCL9. In some cases, the PD-L1 axis binding antagonist may be administered in combination with a therapeutic agent that targets CXCL10.In some cases, the PD-L1 axis binding antagonist may be administered in combination with a therapeutic agent that targets CCL5. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an LFA-1 or ICAM1 agonist. In some cases, the PD-L1 axis binding antagonist may be administered in combination with a selectin agonist.

[0245] In some cases, the PD-L1 axis binding antagonist may be administered in combination with a targeted therapy. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an inhibitor of B-Raf. In some cases, the PD-L1 axis binding antagonist may be administered in combination with vemurafenib (aka ZELBORAF®). In some cases, the PD-L1 axis binding antagonist may be administered in combination with dabrafenib (aka TAFINLAR®). In some cases, the PD-L1 axis binding antagonist may be administered in combination with erlotinib (aka TARCEVA®). In some cases, the PD-L1 axis binding antagonist may be administered in combination with an inhibitor of MEK, such as MEK1 (aka MAP2K1) or MEK2 (aka MAP2K2). In some cases, the PD-L1 axis binding antagonist may be administered in combination with cobimetinib (aka GDC-0973 or XL-518). In some cases, the PD-L1 axis binding antagonist may be administered in combination with trametinib (aka MEKINIST®). In some cases, the PD-L1 axis binding antagonist may be administered in combination with an inhibitor of K-Ras. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an inhibitor of c-Met. In some cases, the PD-L1 axis binding antagonist may be administered in combination with onartuzumab (aka MetMAb). In some cases, the PD-L1 axis binding antagonist may be administered in combination with an inhibitor of Alk. In some cases, the PD-L1 axis binding antagonist may be administered in combination with AF802 (aka CH5424802 or alectinib). In some cases, the PD-L1 axis binding antagonist may be administered in combination with an inhibitor of phosphatidylinositol 3-kinase (PI3K). In some cases, the PD-L1 axis binding antagonist may be administered in combination with BKM120. In some cases, the PD-L1 axis binding antagonist may be administered in combination with idelalisib (also known as GS-1101 or CAL-101).In some embodiments, the PD-L1 axis binding antagonist may be administered in combination with perifosine (aka KRX-0401). In some embodiments, the PD-L1 axis binding antagonist may be administered in combination with an inhibitor of Akt. In some embodiments, the PD-L1 axis binding antagonist may be administered in combination with MK2206. In some cases, the PD-L1 axis binding antagonist may be administered in combination with GSK690693. In some cases, the PD-L1 axis binding antagonist may be administered in combination with GDC-0941. In some cases, the PD-L1 axis binding antagonist may be administered in combination with an inhibitor of mTOR. In some cases, the PD-L1 axis binding antagonist may be administered in combination with sirolimus (aka rapamycin). In some cases, the PD-L1 axis binding antagonist may be administered in combination with temsirolimus (aka CCI-779 or TORISEL®). In some cases, the PD-L1 axis binding antagonist may be administered in combination with everolimus (aka RAD001). In some cases, the PD-L1 axis binding antagonist may be administered in combination with ridaforolimus (aka AP-23573, MK-8669, or deforolimus). In some cases, the PD-L1 axis binding antagonist may be administered in combination with OSI-027. In some cases, the PD-L1 axis binding antagonist may be administered in combination with AZD8055. In some cases, the PD-L1 axis binding antagonist may be administered in combination with INK128. In some cases, the PD-L1 axis binding antagonist may be administered in combination with a dual PI3K / mTOR inhibitor. In some cases, the PD-L1 axis binding antagonist may be administered in combination with XL765. In some cases, the PD-L1 axis binding antagonist may be administered in combination with GDC-0980. In some cases, the PD-L1 axis binding antagonist may be administered in combination with BEZ235 (also known as NVP-BEZ235). In some cases, the PD-L1 axis binding antagonist may be administered in combination with BGT226.In some cases, the PD-L1 axis binding antagonist may be administered in combination with GSK2126458. In some cases, the PD-L1 axis binding antagonist may be administered in combination with PF-04691502. In some cases, the PD-L1 axis binding antagonist may be administered in combination with PF-05212384 (also known as PKI-587).

[0246] D. PD-L1 Axis Binding Antagonists for Use in the Methods of the Invention Provided herein is a method of treating or delaying the progression of cancer in a patient, e.g., bladder cancer (e.g., urothelial bladder cancer), comprising administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist. Provided herein is a method of determining whether a patient suffering from cancer (e.g., bladder cancer, (e.g., urothelial bladder cancer)) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. Provided herein is a method of predicting the responsiveness of a patient suffering from cancer (e.g., bladder cancer, (e.g., urothelial bladder cancer)) to a treatment comprising a PD-L1 axis-binding antagonist. Provided herein is a method of selecting a therapy for a patient suffering from cancer (e.g., bladder cancer (e.g., urothelial bladder cancer)). Any of the foregoing methods may be based on the expression level of a biomarker provided herein, e.g., PD-L1 expression in a tumor sample, e.g., in tumor-infiltrating immune cells.

[0247] For example, PD-L1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1", "PDCD1", "CD279", and "SLEB2". An exemplary PD-1 is set forth in UniProtKB / Swiss-Prot Accession No. Q15116. PD-L1 (programmed death ligand 1) is also referred to in the art as "programmed cell death 1 ligand 1", "PDCD1LG1", "CD274", "B7-H", and "PDL1". An exemplary human PD-L1 is set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7.1. PD-L2 (Programmed Death Ligand 2) is also referred to in the art as "Programmed Cell Death 1 Ligand 2", "PDCD1LG2", "CD273", "B7-DC", "Btdc", and "PDL2". An exemplary human PD-L2 is set forth in UniProtKB / Swiss-Prot Accession No. Q9BQ51. In some instances, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[0248] In some cases, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific embodiment, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another case, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding ligand. In a specific embodiment, the PD-L1 binding partner is PD-1 and / or B7-1. In another case, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its ligand binding partner. In a specific embodiment, the PD-L2 binding ligand partner is PD-1. The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0249] In some cases, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), e.g., as described below. In some cases, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in WO2006 / 121168. MK-3475, also known as pembrolizumab or lambrolizumab, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT, hBAT-1, or pidilizumab, is an anti-PD-1 antibody described in WO2009 / 101611. In some cases, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin that includes an extracellular or PD-1 binding portion of PD-L1 or PD-L2 in a constant region (e.g., an Fc region of an immunoglobulin sequence). In some cases, the PD-1 binding antagonist is AMP-224. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0250] In some instances, the anti-PD-1 antibody is MDX-1106. Alternative names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558, and nivolumab. In some instances, the anti-PD-1 antibody is nivolumab (CAS Registry Number 946414-94-4). In still further instances, an isolated anti-PD-1 antibody is provided that includes a heavy chain variable region that includes a heavy chain variable region amino acid sequence from SEQ ID NO: 1, and / or a light chain variable region that includes a light chain variable region amino acid sequence from SEQ ID NO: 2. In still further instances, an isolated anti-PD-1 antibody is provided that includes a heavy chain and / or light chain sequence, (a) The heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to TIFF2025003997000003.tif43170, and (b) the light chain sequence TIFF2025003997000004.tif27170 has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to TIFF2025003997000004.tif27170.

[0251] In some cases, the PD-L1 axis binding antagonist is a PD-L2 binding antagonist. In some cases, the PD-L2 binding antagonist is an anti-PD-L2 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some cases, the PD-L2 binding antagonist is an immunoadhesin.

[0252] In some cases, the PD-L1 binding antagonist is an anti-PD-L1 antibody, such as those described below. In some cases, the anti-PD-L1 antibody can inhibit the binding between PD-L1 and PD-1 and / or between PD-L1 and B7-1. In some cases, the anti-PD-L1 antibody is a monoclonal antibody. In some cases, 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 cases, the anti-PD-L1 antibody is a humanized antibody. In some cases, the anti-PD-L1 antibody is a human antibody. In some instances, the anti-PD-L1 antibody is selected from the group consisting of YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, and MEDI4736 (durvalumab), and MSB0010718C (avelumab). Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO2010 / 077634. ​​MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007 / 005874. MEDI4736 (durvalumab) is an anti-PD-L1 monoclonal antibody described in WO2011 / 066389 and US2013 / 034559. Examples of anti-PD-L1 antibodies useful for use in the invention, and methods for making them, are described in PCT Patent Application Nos. WO2010 / 077634, WO2007 / 005874, WO2011 / 066389, U.S. Patent No. 8,217,149, and US2013 / 034559, which are hereby incorporated by reference.

[0253] The anti-PD-L1 antibodies described in WO2010 / 077634A1 and US8,217,149 may be used in the methods described herein. In some cases, the anti-PD-L1 antibody comprises a heavy chain variable region sequence of SEQ ID NO: 3 and / or a light chain variable region sequence of SEQ ID NO: 4. In still further cases, an isolated anti-PD-L1 antibody is provided that comprises a heavy chain variable region and / or a light chain variable region sequence, (a) The heavy chain sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 3), and (b) the light chain sequence It has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

[0254] In one example, the anti-PD-L1 antibody comprises a heavy chain variable region comprising the sequences HVR-H1, HVR-H2, and HVR-H3; TIFF2025003997000005.tif38170 further wherein X1 is D or G, X2 is S or L, and X3 is T or S. In a specific embodiment, X1 is D, X2 is S, and X3 is T. In another embodiment, the polypeptide further comprises a variable region heavy chain framework sequence juxtaposed between the HVRs according to the following formula: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4). In yet another embodiment, the framework sequence is derived from a human consensus framework sequence. In a further embodiment, the framework sequence is a VH subgroup III consensus framework. In yet a further embodiment, at least one of the framework sequences is the following: TIFF2025003997000006.tif50170

[0255] In yet a further embodiment, the heavy chain polypeptide is further combined with a variable region light chain comprising HVR-L1, HVR-L2, and HVR-L3, In the formula, 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, and X 10 is Y or A, and X 11 is Y, G, F, or S, and X 12 is L, Y, F, or W, and X 13 is Y, N, A, T, G, F, or I, and X 14 is H, V, P, T, or I, and X 15 is A, W, R, P, or T. In still further embodiments, X4 is D, X5 is V, X6 is S, X7 is A, X8 is V, X9 is F, and X 10 is Y and X 11 is Y and X 12 is L and X 13 is Y and X 14 is H and X 15 is A.

[0256] In yet a further embodiment, the light chain further comprises a variable region light chain framework sequence juxtaposed between the HVRs according to the following formula: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4). In yet a further embodiment, the framework sequence is derived from a human consensus framework sequence. In yet a further embodiment, the framework sequence is a VL kappa I consensus framework. In yet a further embodiment, at least one of the framework sequences is the following: TIFF2025003997000008.tif50170

[0257] In another aspect, an isolated anti-PD-L1 antibody or antigen-binding fragment is provided that comprises a heavy chain and a light chain variable region sequence, (a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, and further (b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, and further comprises In the formula, 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, and X 10 is Y or A, and X 11 is Y, G, F, or S, and X 12 is L, Y, F, or W, and X 13 is Y, N, A, T, G, F, or I, and X 14 is H, V, P, T, or I, and X 15 is A, W, R, P, or T. In a specific embodiment, X1 is D, X2 is S, and X3 is T. In another embodiment, X4 is D, X5 is V, X6 is S, X7 is A, X8 is V, X9 is F, and X 10 is Y and X 11 is Y and X 12 is L and X 13 is Y and X 14 is H and X 15 In yet another embodiment, X1 is D, X2 is S, X3 is T, X4 is D, X5 is V, X6 is S, X7 is A, X8 is V, X9 is F, and X 10 is Y and X 11 is Y and X 12 is L and X 13 is Y and X14 is H and X 15 is A.

[0258] In a further embodiment, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4). In yet a further embodiment, the framework sequences are derived from a human consensus framework sequence. In yet a further embodiment, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In yet a further embodiment, the heavy chain framework sequences are VH subgroup III consensus framework. In yet a further embodiment, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs: 8, 9, 10, and 11. In yet further embodiments, the light chain framework sequences are derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet further embodiments, the light chain framework sequences are VL kappa I consensus frameworks. In yet further embodiments, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17, and 18.

[0259] In yet further specific embodiments, the antibody further comprises a human or mouse constant region. In still further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In still further specific embodiments, the human constant region is IgG1. In still further embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In still further embodiments, the mouse constant region is in IgG2A. In still further specific embodiments, the antibody has reduced or minimal effector function. In still further specific embodiments, the minimal effector function is due to an "effector-less Fc mutation" or aglycosylation. In still further cases, the effector-less Fc mutation is a N297A or D265A / N297A substitution in the constant region.

[0260] In yet another aspect, an anti-PD-L1 antibody is provided that comprises a heavy chain and a light chain variable region sequence, (a) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20), and RHWPGGFDY (SEQ ID NO: 21), respectively; or (b) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity to RASQDVSTAVA (sequence number 22), SASFLYS (sequence number 23), and QQYLYHPAT (sequence number 24), respectively.

[0261] In specific embodiments, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0262] In another embodiment, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4). In yet another embodiment, the framework sequences are derived from a human consensus framework sequence. In yet a further embodiment, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet a further embodiment, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet a further embodiment, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs: 8, 9, 10, and 11. In yet further embodiments, the light chain framework sequences are derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet further embodiments, the light chain framework sequences are VL kappa I consensus frameworks. In yet further embodiments, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17, and 18.

[0263] In yet further specific embodiments, the antibody further comprises a human or mouse constant region. In still further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In still further specific embodiments, the human constant region is IgG1. In still further embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In still further embodiments, the mouse constant region is in IgG2A. In still further specific embodiments, the antibody has reduced or minimal effector function. In still further specific embodiments, the minimal effector function is due to an "effector-less Fc mutation" or aglycosylation. In still further cases, the effector-less Fc mutation is a N297A or D265A / N297A substitution in the constant region.

[0264] In another further aspect, an isolated anti-PD-L1 antibody is provided, comprising a heavy chain and a light chain variable region sequence: (a) The heavy chain sequence and / or has at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 25); and / or (b) the light chain sequence It has at least 85% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

[0265] In specific embodiments, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In another embodiment, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4). In yet another embodiment, the framework sequences are derived from human consensus framework sequences. In further embodiments, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In still further embodiments, the heavy chain framework sequences are VH subgroup III consensus frameworks. In still further embodiments, one or more of the heavy chain framework sequences are set forth as SEQ ID NO:8, 9, 10, and WGQGTLVTVSS (SEQ ID NO:27).

[0266] In yet further embodiments, the light chain framework sequences are derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet further embodiments, the light chain framework sequences are VL kappa I consensus frameworks. In yet further embodiments, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17, and 18.

[0267] In yet further specific embodiments, the antibody further comprises a human or mouse constant region. In still further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In still further specific embodiments, the human constant region is IgG1. In still further embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In still further embodiments, the mouse constant region is in IgG2A. In still further specific embodiments, the antibody has reduced or minimal effector function. In still further specific embodiments, the minimal effector function results from production in a prokaryotic cell. In still further specific embodiments, the minimal effector function results from an "effector-less Fc mutation" or aglycosylation. In still further cases, the effector-less Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0268] In a further embodiment, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4). In yet a further embodiment, the framework sequences are derived from a human consensus framework sequence. In yet a further embodiment, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In yet a further embodiment, the heavy chain framework sequences are VH subgroup III consensus frameworks. In yet a further embodiment, one or more of the heavy chain framework sequences are the following: TIFF2025003997000011.tif38170

[0269] In still further embodiments, the light chain framework sequences are derived from a Kabat kappa I, II, II, or IV subgroup sequence. In still further embodiments, the light chain framework sequences are VL kappa I consensus frameworks. In still further embodiments, one or more of the light chain framework sequences are the following: TIFF2025003997000012.tif38170

[0270] In yet further specific embodiments, the antibody further comprises a human or mouse constant region. In still further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In still further specific embodiments, the human constant region is IgG1. In still further embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In still further embodiments, the mouse constant region is in IgG2A. In still further specific embodiments, the antibody has reduced or minimal effector function. In still further specific embodiments, the minimal effector function is due to an "effector-less Fc mutation" or aglycosylation. In still further cases, the effector-less Fc mutation is a N297A or D265A / N297A substitution in the constant region.

[0271] In yet another aspect, an anti-PD-L1 antibody is provided that comprises a heavy chain and a light chain variable region sequence, (c) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20), and RHWPGGFDY (SEQ ID NO: 21), respectively; and / or (d) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity to RASQDVSTAVA (sequence number 22), SASFLYS (sequence number 23), and QQYLYHPAT (sequence number 24), respectively.

[0272] In specific embodiments, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0273] In another embodiment, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4). In yet another embodiment, the framework sequences are derived from a human consensus framework sequence. In yet a further embodiment, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet a further embodiment, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet a further embodiment, one or more of the heavy chain framework sequences are set forth as SEQ ID NO:8, 9, 10, and WGQGTLVTVSSASTK (SEQ ID NO:31).

[0274] In still further embodiments, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In still further embodiments, the light chain framework sequence is a VL kappa I consensus framework. In still further embodiments, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17, and 18. In still further specific embodiments, the antibody further comprises a human or mouse constant region. In still further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In still further specific embodiments, the human constant region is IgG1. In still further embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In still further embodiments, the mouse constant region in IgG2A. In still further specific embodiments, the antibody has reduced or minimal effector function. In still further specific embodiments, the minimal effector function is due to an "effector-less Fc mutation" or aglycosylation. In still further cases, the effector-less Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0275] In still further instances, there is provided an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, (a) The heavy chain sequence or has at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 26); (b) the light chain sequence It has at least 85% sequence identity with DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

[0276] In some cases, an isolated anti-PD-L1 antibody is provided that comprises heavy and light chain variable region sequences, where the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some cases, an isolated anti-PD-L1 antibody is provided that comprises heavy and light chain variable region sequences, where the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:26. In some instances, an isolated anti-PD-L1 antibody is provided that comprises heavy and light chain variable region sequences, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4, and the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:26. In some cases, 1, 2, 3, 4, or 5 amino acid residues at the N-terminus of the heavy and / or light chain may be deleted, substituted or modified.

[0277] In still further instances, there is provided an isolated anti-PD-L1 antibody comprising heavy and light chain sequences, (a) The heavy chain sequence has at least 85% sequence identity to TIFF2025003997000013.tif42170, and / or (b) the light chain sequence It has at least 85% sequence identity to TIFF2025003997000014.tif27170.

[0278] In some cases, an isolated anti-PD-L1 antibody is provided comprising heavy and light chain sequences, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 33. In some cases, an isolated anti-PD-L1 antibody is provided comprising heavy and light chain sequences, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 32. In some cases, an isolated anti-PD-L1 antibody is provided comprising heavy and light chain sequences, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:33, and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:32.

[0279] In some cases, the isolated anti-PD-L1 antibody is aglycosylated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of the sugars N-aceylgalactosamine, galactose, or xylose to one hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Removal of glycosylation sites from an antibody is conveniently accomplished by modifying the amino acid sequence to remove one of the above tripeptide sequences (for N-linked glycosylation sites). This can be done by substituting an asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

[0280] In any of the cases herein, the isolated anti-PD-L1 antibodies may bind to human PD-L1, for example human PD-L1 as set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof.

[0281] In still further instances, an isolated nucleic acid is provided that encodes any of the antibodies described herein. In some instances, the nucleic acid further comprises a vector suitable for expressing the nucleic acid encoding any of the aforementioned anti-PD-L1 antibodies. In still further specific embodiments, the vector is in a host cell suitable for expressing the nucleic acid. In still further specific embodiments, the host cell is a eukaryotic or prokaryotic cell. In still further specific embodiments, the eukaryotic cell is a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell.

[0282] Antibodies or antigen-binding fragments thereof can be produced using methods known in the art under conditions suitable for producing such antibodies or fragments, for example by a process which includes culturing a host cell containing nucleic acid encoding any of the above-mentioned anti-PD-L1 antibodies or antigen-binding fragments in a form suitable for expression, and recovering the antibody or fragment.

[0283] It is expressly contemplated that such PD-L1 antibodies (e.g. anti-PD-L1 antibodies, anti-PD-1 antibodies, and anti-PD-L2 antibodies) or other antibodies described herein (e.g. anti-PD-L1 antibodies for detecting PD-L1 expression levels) for use in any of the cases listed above may have any of the features described in sections 1-7 below, either alone or in combination.

[0284] 1. Antibody affinity In certain cases, an antibody provided herein (e.g., an anti-PD-L1 antibody or an anti-PD-1 antibody) may have a concentration of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0285] In one instance, Kd is measured by radiolabeled antigen binding assay (RIA). In one instance, an RIA is performed using an antibody of interest and a Fab version of its antigen. For example, the solution binding affinity of a Fab for an antibody is determined by binding the Fab to the lowest concentration ( 125I) equilibration with labeled antigen and then measurement of bound antibody with anti-Fab antibody-coated plates (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multi-well plates are coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 I] The antigen is mixed with serial dilutions of the Fab of interest (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, although this incubation may be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium has been achieved. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., for 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μL / well of scintillant (MICROSCINT-20™, Packard) is added and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that results in 20% or less of maximum binding is selected for use in the competitive binding assay.

[0286] In another example, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25° C. with an immobilized antigen CM5 chip at about 10 response units (RU). In one example, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / mL (about 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 injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μL / min in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). off / k on The on-rate is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). 6 M -1 s -1If the on-rate exceeds 100%, it can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm, emission=340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS at 25° C. in the presence of increasing antigen concentrations as measured with a spectrometer such as a spectrophotometer equipped with a stopped flow (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer equipped with a stirred cuvette (ThermoSpectronic).

[0287] 2. Antibody fragment In certain cases, the antibodies provided herein (e.g., anti-PD-L1 antibodies or anti-PD-1 antibodies) are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as 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, e.g., Pluckthun, 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. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments which contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0288] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., 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). See also, Hudson et al. Nat. Med. 9:129-134 (2003), for triabodies and tetrabodies.

[0289] A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain cases, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0290] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as described herein, as well as production by recombinant host cells (e.g., E. coli or phage).

[0291] 3. Chimeric and humanized antibodies In certain cases, the antibodies provided herein (e.g., anti-PD-L1 or anti-PD-1 antibodies) 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 a non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of a parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0292] In certain cases, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains, the HVRs, e.g., CDRs (or portions thereof), of which are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody will also optionally comprise at least a portion of a human constant region. In some cases, some FR residues in the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0293] Humanized antibodies and methods for their production are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are also described, e.g., in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Natl. 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 specificity determining region (SDR) grafting), Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"), Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"), as well as Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0294] Human framework regions that may be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992), and Presta et al. J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions obtained from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0295] 4. Human antibodies In certain cases, the antibodies provided herein (e.g., anti-PD-L1 antibodies or anti-PD-1 antibodies) are human antibodies. Human antibodies may be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0296] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or are extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, 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). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE™ technology), 5,770,429 (describing HUMAB® technology), 7,041,870 (describing KM MOUSE® technology), and U.S. Patent Application Publication No. US2007 / 0061900 (describing VELOCIMOUSE® technology). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0297] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147:86 (1991)). Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):937-2005(27) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 185(927):91-2005(3).

[0298] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0299] 5. Library-derived Antibodies The antibodies of the invention (e.g., anti-PD-L1 antibodies and anti-PD-1 antibodies) may be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and 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,Methods in Molecular Biology 248:161-175(Lo,ed.,Human Press,Totowa,NJ,2003), Sidhu et al.,J.Mol.Biol.338(2):299-310(2004),Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0300] In one particular phage display method, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined into a phage library, which can then be screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunized sources provide high affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide single-source antibodies against a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J. 12:725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V gene fragments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve in vitro rearrangement, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. 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.

[0301] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0302] 6. Multispecific antibodies In any one of the above embodiments, the antibodies provided herein (e.g., anti-PD-L1 antibodies or anti-PD-1 antibodies) may be multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain cases, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. In certain cases, one of the binding specificities is for PD-L1 and the other is for any other antigen. In certain cases, bispecific antibodies may 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.

[0303] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-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 "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by engineering electrostatic steering effects to create antibody Fc heterodimeric molecules (see, e.g., WO 2009 / 089004 A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol. 1999, 20:111-112 (1999)), and / or using glycerol-containing cleavage sites (see, e.g., Gruber et al., J. Immunol. 1999, 20:111-112 (1999)). al., J. Immunol. 152:5368 (1994)), as well as by preparing trispecific antibodies (e.g., as described in Tutt et al. J. Immunol. 147:60 (1991)).

[0304] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US 2006 / 0025576 A1).

[0305] The antibodies or fragments herein also include "dual action Fabs" or "DAFs" that contain antigen binding sites that bind to PD-L1 as well as another distinct antigen.

[0306] 7. Antibody variants In certain cases, amino acid sequence variants of the antibodies of the invention (e.g., anti-PD-L1 antibodies and anti-PD-1 antibodies) are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding.

[0307] I. Substitution, insertion, and deletion mutations In certain cases, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions." More substantial changes are shown in Table 2 under the heading of "exemplary substitutions," and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products can be screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). TIFF2025003997000015.tif166170

[0308] Amino acids can be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu (4) Basic: His, Lys, Arg (5) Residues that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe.

[0309] Non-conservative substitutions will involve the exchange of a member of one of these classes for another class.

[0310] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have an alteration (e.g., improvement) in a particular biological property (e.g., increased affinity and / or reduced immunogenicity) compared to the parent antibody and / or will have a particular biological property of the parent antibody substantially retained. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0311] Modifications (e.g., substitutions) may be made within the HVRs, for example, to improve antibody affinity. Such modifications may be made in HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in residues that contact the antigen, and the resulting mutant VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some cases of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed 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. CDR-H3 and CDR-L3 in particular are often targeted.

[0312] In certain cases, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made within the HVRs. Such modifications may, for example, be outside of the antigen contact residues within the HVRs. In the particular case of the above-mentioned variant VH and VL sequences, each HVR is either unmodified or has no more than one, two, or three amino acid substitutions.

[0313] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" and is described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of 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 of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted as candidates for substitution or can be eliminated. The mutants can be screened to determine whether they have the desired properties.

[0314] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of an antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0315] II. Glycosylation variants In certain cases, an antibody of the invention can be modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody of the invention can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0316] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides, generally linked to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may comprise a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some cases, modifications of the oligosaccharides in the antibodies of the present invention may be made to generate antibody variants with improved specific properties.

[0317] In one case, antibody variants are provided that have carbohydrate structures attached (directly or indirectly) to the Fc region that lack fucose. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (EU numbering of Fc region residues) in the Fc region, although Asn297 may also be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294-300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. US2003 / 0157108 and US2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 013214, and US2005 / 014214. 0, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 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, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-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 WO2003 / 085107).

[0318] Further provided are antibody variants with 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, for example, in WO2003 / 011878, U.S. Patent No. 6,602,684, and US2005 / 0123546. Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087, WO1998 / 58964, and WO1999 / 22764.

[0319] III. Fc Region Mutations In certain cases, one or more amino acid modifications may be introduced into the Fc region of an antibody of the invention, thereby generating an Fc region variant. The Fc region variant may com...

Claims

1. 1. A method of treating a patient suffering from bladder cancer, comprising administering to said patient a therapeutically effective amount of a PD-L1 axis binding antagonist, wherein a tumor sample obtained from said patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 1% or more of said tumor sample.

2. 2. The method of claim 1, wherein the median overall survival of patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is at least 8 months.

3. 3. The method of claim 1 or 2, wherein the median overall survival of patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is at least about 8.8 months.

4. The method of any one of claims 1 to 3, wherein the response rate of patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is at least about 12%.

5. The method of any one of claims 1 to 4, wherein the response rate of patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is at least about 21%.

6. The method of any one of claims 1 to 4, wherein the response rate of patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is about 18%.

7. 2. The method of claim 1, wherein the tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise about 5% or more of the tumor sample.

8. The method of claim 7, wherein the median overall survival of patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is at least 9 months.

9. The method of claim 7 or 8, wherein the median overall survival of patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is at least about 11 months.

10. The method of any one of claims 7 to 9, wherein the response rate of patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is at least about 14%.

11. The method of any one of claims 7 to 10, wherein the response rate of patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is at least about 25%.

12. The method of any one of claims 7 to 11, wherein the response rate in patients administered a therapeutically effective amount of the PD-L1 axis binding antagonist is about 27%.

13. 8. The method of claim 7, wherein the tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells comprising about 10% or more of the tumor sample.

14. The method of any one of claims 1 to 13, wherein the tumor sample obtained from the patient has been determined to be a luminal subtype tumor.

15. 1. A method of treating a patient suffering from bladder cancer, comprising administering to said patient a therapeutically effective amount of a PD-L1 axis binding antagonist, wherein a tumor sample obtained from said patient has been determined to be a luminal subtype tumor.

16. (a) the expression level of at least one of CDKN2A, GATA3, FOXA1, and ERBB2 in the tumor sample obtained from the patient is determined to be increased compared to a reference level of the at least one gene; and / or (b) the expression level of at least one of FGFR3, KRT5, KRT14, and EGFR in the tumor sample obtained from the patient is determined to be decreased compared to a reference level of the at least one gene.

17. (a) the expression levels of CDKN2A, GATA3, FOXA1, and ERBB2 in the tumor sample obtained from the patient are determined to be increased compared to reference levels of the genes; and / or 17. The method of claim 16, wherein (b) the expression levels of FGFR3, KRT5, KRT14, and EGFR in the tumor sample obtained from the patient are determined to be decreased compared to reference levels of the genes.

18. (a) determining that expression levels of CDKN2A, GATA3, FOXA1, and ERBB2 in the tumor sample obtained from the patient are increased relative to reference levels of the genes; 18. The method of claim 17, wherein (b) the expression levels of FGFR3, KRT5, KRT14, and EGFR in the tumor sample obtained from the patient are determined to be decreased compared to reference levels of the genes.

19. The method of any one of claims 14 to 18, wherein the expression level of miR-99a-5p or miR100-5p in the tumor sample obtained from the patient is determined to be increased compared to a reference level of said microRNA (miRNA).

20. The method of claim 19, wherein expression levels of miR-99a-5p and miR100-5p in the tumor sample obtained from the patient are determined to be increased compared to reference levels of the miRNAs.

21. 21. The method of any one of claims 14 to 20, wherein the expression level of at least one of CD8A, GZMA, GZMB, IFNG, CXCL9, CXCL10, PRF1, and TBX21 in the tumor sample obtained from the patient is determined to be increased compared to a reference level of the at least one gene.

22. 22. The method of claim 21, wherein the expression levels of at least CXCL9 and CXCL10 in the tumor sample obtained from the patient are determined to be increased compared to reference levels of the genes.

23. The method according to any one of claims 14 to 22, wherein the luminal subtype tumor is a luminal cluster II subtype tumor.

24. 1. A method for determining whether a patient suffering from bladder cancer is likely to respond to a treatment comprising a PD-L1 axis binding antagonist, comprising: determining the expression level of PD-L1 on tumor-infiltrating immune cells in a tumor sample obtained from said patient; The method, wherein a detectable expression level of PD-L1 in tumor-infiltrating immune cells comprising about 1% or more of the tumor sample indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist.

25. 1. A method for predicting the responsiveness of a patient suffering from bladder cancer to a treatment comprising a PD-L1 axis binding antagonist, comprising: determining the expression level of PD-L1 on tumor-infiltrating immune cells in a tumor sample obtained from said patient; The method, wherein a detectable expression level of PD-L1 in tumor-infiltrating immune cells comprising about 1% or more of the tumor sample indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist.

26. 1. A method for selecting a therapy for a patient suffering from bladder cancer, comprising: determining the expression level of PD-L1 in tumor-infiltrating immune cells in a tumor sample obtained from the patient; and selecting a therapy comprising a PD-L1 axis binding antagonist for the patient based on a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 1% or more of the tumor sample.

27. 27. The method of any one of claims 24-26, wherein the tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 5% or more of the tumor sample.

28. 28. The method of claim 27, wherein the tumor sample obtained from the patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise at least about 10% of the tumor sample.

29. 1. A method for determining whether a patient suffering from bladder cancer is likely to respond to a treatment comprising a PD-L1 axis binding antagonist, comprising: determining a subtype of the tumor from a tumor sample obtained from the patient, indicating that a luminal subtype tumor is more likely to respond to a treatment comprising a PD-L1 axis binding antagonist.

30. 1. A method for predicting the responsiveness of a patient suffering from bladder cancer to a treatment comprising a PD-L1 axis binding antagonist, comprising: determining the subtype of the tumor from a tumor sample obtained from the patient; The method indicates that a luminal subtype tumor is likely to respond to a treatment comprising a PD-L1 axis binding antagonist.

31. 1. A method for selecting a therapy for a patient suffering from bladder cancer, comprising: determining the subtype of the tumor from a tumor sample obtained from the patient; selecting a therapy comprising a PD-L1 axis binding antagonist for the patient based on the determination that the tumor is a luminal subtype tumor.

32. The method of any one of claims 24-31, further comprising administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist based on the expression level of PD-L1 in tumor-infiltrating immune cells in the tumor sample.

33. 33. The method of any one of claims 1 to 32, wherein the PD-L1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist.

34. 34. The method of claim 33, wherein the PD-L1 axis binding antagonist is a PD-L1 binding antagonist.

35. 35. The method of claim 34, wherein the PD-L1 binding antagonist inhibits binding of PD-L1 to one or more of its ligand binding partners.

36. 36. The method of claim 35, wherein the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1.

37. 36. The method of claim 35, wherein the PD-L1 binding antagonist inhibits binding of PD-L1 to B7-1.

38. The method of any one of claims 35 to 37, wherein the PD-L1 binding antagonist inhibits binding of PD-L1 to both PD-1 and B7-1.

39. The method of any one of claims 34 to 38, wherein the PD-L1 binding antagonist is an antibody.

40. 40. The method of claim 39, wherein the antibody is selected from the group consisting of atezolizumab ((MPDL3280A), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab).

41. 40. The method of claim 39, wherein the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 19, the HVR-H2 sequence of SEQ ID NO: 20, and the HVR-H3 sequence of SEQ ID NO: 21, and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 22, the HVR-L2 sequence of SEQ ID NO: 23, and the HVR-L3 sequence of SEQ ID NO:

24.

42. 40. The method of claim 39, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

4.

43. 34. The method of claim 33, wherein the PD-L1 axis binding antagonist is a PD-1 binding antagonist.

44. 44. The method of claim 43, wherein the PD-1 binding antagonist inhibits binding of PD-1 to one or more of its ligand binding partners.

45. 45. The method of claim 44, wherein the PD-1 binding antagonist inhibits binding of PD-1 to PD-L1.

46. 45. The method of claim 44, wherein the PD-1 binding antagonist inhibits binding of PD-1 to PD-L2.

47. The method of any one of claims 44 to 46, wherein the PD-1 binding antagonist inhibits binding of PD-1 to both PD-L1 and PD-L2.

48. 48. The method of any one of claims 43-47, wherein the PD-1 binding antagonist is an antibody.

49. 49. The method of claim 48, wherein the antibody is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108.

50. 48. The method of any one of claims 33-47, wherein the PD-1 binding antagonist is an Fc fusion protein.

51. The method of claim 50, wherein the Fc fusion protein is AMP-224.

52. The method of any one of claims 1-23 and 32-51, further comprising administering to the patient an effective amount of a second therapeutic agent.

53. 53. The method of claim 52, wherein the second therapeutic agent is selected from the group consisting of a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, and combinations thereof.

54. The method of any one of claims 1 to 53, wherein the bladder cancer is urothelial bladder cancer.

55. 55. The method of claim 54, wherein the urothelial bladder cancer is metastatic urothelial bladder cancer.

56. 55. The method of claim 54, wherein the urothelial bladder cancer is locally advanced urothelial bladder cancer.

57. The method of any one of claims 1 to 56, wherein the patient is progressing following treatment with a platinum-based chemotherapy agent.

58. 58. The method of any one of claims 1 to 57, wherein the tumor sample is a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample.

59. The method of any one of claims 1 to 14, 16 to 28, and 52 to 58, wherein the expression level of PD-L1 is a protein expression level.

60. 60. The method of claim 59, wherein the protein expression level of PD-L1 is determined using a method selected from the group consisting of immunohistochemistry (IHC), immunofluorescence, flow cytometry, and Western blot.

61. 61. The method of claim 60, wherein the protein expression level of PD-L1 is determined using IHC.

62. The method of claim 60 or 61, wherein the protein expression level of PD-L1 is detected using an anti-PD-L1 antibody.

63. The method according to any one of claims 1 to 14, 16 to 28, and 52 to 58, wherein the expression level of PD-L1 is an mRNA expression level.

64. 64. The method of claim 63, wherein the mRNA expression level of PD-L1 is determined using a method selected from the group consisting of quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), RNA sequencing, microarray analysis, in situ hybridization, and serial analysis of gene expression (SAGE).

65. 1. A PD-L1 axis binding antagonist for use in treating a patient suffering from bladder cancer, wherein a tumor sample obtained from said patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 1% or more of said tumor sample.

66. 1. Use of an effective amount of a PD-L1 axis binding antagonist in the manufacture of a medicament for use in treating a patient suffering from bladder cancer, wherein a tumor sample obtained from said patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 1% or more of said tumor sample.

67. 1. A composition comprising an effective amount of a PD-L1 axis binding antagonist for use in treating a patient suffering from bladder cancer, wherein a tumor sample obtained from said patient has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that constitute about 1% or more of said tumor sample.

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