Therapeutic and diagnostic methods for cancer

Abstract

To provide therapeutic and diagnostic methods and compositions for cancer, such as lung cancer, bladder cancer, kidney cancer, breast cancer or melanoma.SOLUTION: A method of identifying an individual having a cancer who may benefit from treatment including a PD-L1 axis binding antagonist comprises determining a tissue tumor mutational burden (tTMB) score from a tumor sample from the individual, where a tTMB score from the tumor sample that is at or above a reference tTMB score identifies the individual as one who may benefit from treatment including a PD-L1 axis binding antagonist. A method of treating an individual having a cancer comprises: (a) determining a tTMB score from a tumor sample from the individual, where the tTMB score from the tumor sample is at or above a reference tTMB score, and (b) administering an effective amount of a PD-L1 axis binding antagonist to the individual.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Provided herein are therapeutic and diagnostic methods and compositions for pathological conditions such as cancer (e.g., lung cancer (e.g., non-small cell lung cancer (NSCLC)), bladder cancer (e.g., urothelial carcinoma (UC)), kidney cancer (e.g., renal cell carcinoma (RCC)), breast cancer (e.g., triple-negative breast cancer (TNBC)), or melanoma), and methods of using PD-L1 axis-binding antagonists. Specifically, the present invention provides methods for patient selection and diagnosis, methods of treatment, articles of manufacture, diagnostic kits, and detection methods. [Background technology]

[0002] 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 is the second leading cause of death after heart disease, with approximately 1.3 million new cases occurring each year, accounting for approximately one in four deaths. For example, lung cancer is the most common form of cancer and is the leading cause of cancer-related mortality among American women. In particular, metastatic non-small cell lung cancer (NSCLC) is associated with a very poor outcome, with a five-year survival rate of approximately 1%.

[0003] Programmed death-ligand 1 (PD-L1) is a protein implicated in suppressing immune system responses in chronic infections, 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 the PD-L1 / PD-1 complex and PD-L1 / B7-1 complex negatively regulates T cell receptor signaling, subsequently downregulating T cell activation and suppressing antitumor immune activity.

[0004] Despite significant advances in the treatment of cancer (e.g., lung cancer (e.g., non-small cell lung cancer (NSCLC)), bladder cancer (e.g., urothelial carcinoma (UC)), kidney cancer (e.g., renal cell carcinoma (RCC)), breast cancer (e.g., triple-negative breast cancer (TNBC)), or melanoma), improved therapeutic and diagnostic methods remain needed. Summary of the Invention

[0005] The present invention provides therapeutic and diagnostic methods and compositions for cancer, such as lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma.

[0006] In one aspect, the invention features a method of identifying an individual having cancer who may benefit from a treatment comprising a PD-L1 axis-binding antagonist, the method including determining a tissue tumor mutational burden (tTMB) score from a tumor sample from the individual, wherein a tTMB score from the tumor sample that is equal to or greater than a reference tTMB score identifies the individual as one who may benefit from a treatment comprising a PD-L1 axis-binding antagonist.

[0007] In another aspect, the invention features a method for selecting a therapy for an individual having cancer, the method including determining a tTMB score from a tumor sample from the individual, wherein a tTMB score from the tumor sample that is equal to or greater than a reference tTMB score identifies the individual as one who may benefit from a therapy that includes a PD-L1 axis binding antagonist.

[0008] In some embodiments of any one of the preceding aspects, the tTMB score determined from the tumor sample is greater than or equal to a reference tTMB score, and the method further comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist.

[0009] In another aspect, the invention features a method of treating an individual having cancer, the method including: (a) determining a tTMB score from a tumor sample from the individual, wherein the tTMB score from the tumor sample is equal to or greater than a reference tTMB score; and (b) administering to the individual an effective amount of a PD-L1 axis binding antagonist.

[0010] In another aspect, the invention features a method of treating an individual having cancer, the method including administering to the individual an effective amount of a PD-L1 axis-binding antagonist, wherein prior to the administration, a tTMB score equal to or greater than a reference tTMB score has been determined from a tumor sample from the individual.

[0011] In some embodiments of any one of the preceding aspects, the reference tTMB score is a tTMB score within a reference population of individuals with cancer, the population consisting of a first subset of individuals being treated with a PD-L1 axis binding antagonist therapy and a second subset of individuals being treated with a non-PD-L1 axis binding antagonist therapy, where the non-PD-L1 axis binding antagonist therapy does not include a PD-L1 axis binding antagonist. In some embodiments, the reference tTMB score significantly separates each of the first and second subsets of individuals based on a significant difference in responsiveness to treatment with the PD-L1 axis binding antagonist therapy compared to responsiveness to treatment with the non-PD-L1 axis binding antagonist therapy. In some embodiments, the non-PD-L1 axis binding antagonist therapy comprises a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, or a combination thereof. In some embodiments, responsiveness to treatment is an increase in progression-free survival (PFS). In some embodiments, responsiveness to treatment is an increase in overall survival (OS). In some embodiments, responsiveness to treatment is an increase in overall response rate (ORR).

[0012] In some embodiments of any one of the preceding aspects, the tumor sample is determined to have an increased level of somatic mutations in at least one gene specified in Table 1 compared to a reference level of somatic mutations in at least one gene specified in Table 1. In some embodiments, the tumor sample from the individual is determined to have an increased level of somatic mutations in at least one-third of the genes specified in Table 1 compared to a reference level of somatic mutations in at least one-third of the genes specified in Table 1. In some embodiments, the tumor sample from the individual is determined to have an increased level of somatic mutations in at least one-half of the genes specified in Table 1 compared to a reference level of somatic mutations in at least one-half of the genes specified in Table 1. In some embodiments, the tumor sample from the individual is determined to have an increased level of somatic mutations in at least two-thirds of the genes specified in Table 1 compared to a reference level of somatic mutations in at least two-thirds of the genes specified in Table 1. In some embodiments, a tumor sample from the individual is determined to have an increased level of somatic mutations in at least three-quarters of the genes specified in Table 1 compared to a baseline level of somatic mutations in at least three-quarters of the genes specified in Table 1. In some embodiments, a tumor sample from the individual is determined to have an increased level of somatic mutations in the genes specified in Table 1 compared to a baseline level of somatic mutations in the genes specified in Table 1.

[0013] In some embodiments of any one of the preceding aspects, the somatic mutation is a substitution, deletion, and / or insertion. In some embodiments, the somatic mutation of at least one gene specified in Table 1 is a protein-modifying somatic mutation. In some embodiments, the substitution, deletion, and / or insertion is within a coding region. In some embodiments, the deletion and / or insertion is an indel. In some embodiments, the tumor sample from the individual has a higher genome-wide mutation load than a baseline level genome-wide mutation load. In some embodiments, the median genome-wide mutation load is at least about 10 mutations per megabase (mutations / Mb).

[0014] In some embodiments of any one of the preceding aspects, the reference tTMB score is a pre-assigned tTMB score. In some embodiments of any one of the preceding aspects, the reference tTMB score is about 5 to about 50 mutations per megabase (mutations / Mb). In some embodiments, the reference tTMB score is about 8 to about 30 mutations / Mb. In some embodiments, the reference tTMB score is about 10 to about 20 mutations / Mb. In some embodiments, the reference tTMB score is about 10 mutations / Mb. In some embodiments, the reference tTMB score is about 16 mutations / Mb. In some embodiments, the reference tTMB score is about 20 mutations / Mb.

[0015] In some embodiments of any one of the preceding aspects, the tTMB score from the tumor sample is about 5 or more mutations / Mb. In some embodiments, the tTMB score from the tumor sample is about 5 to about 100 mutations / Mb. In some embodiments, the tTMB score from the tumor sample is about 10 or more mutations / Mb. In some embodiments, the tTMB score from the tumor sample is about 10 to about 100 mutations / Mb. In some embodiments, the tTMB score from the tumor sample is about 16 or more mutations / Mb. In some embodiments, the tTMB score from the tumor sample is about 16 or more mutations / Mb, and the reference tTMB score is about 16 mutations / Mb. In some embodiments, the tTMB score from the tumor sample is about 20 or more mutations / Mb. In some embodiments, the tTMB score from the tumor sample is about 20 or more mutations / Mb, and the reference tTMB score is about 20 mutations / Mb.

[0016] In other embodiments of any one of the preceding aspects, the tTMB score determined from the tumor sample is less than a reference tTMB score. In some embodiments, the tTMB score determined from the tumor sample is less than a reference tTMB score, and the method further comprises administering to the individual a therapeutic agent other than, or in addition to, a PD-L1 axis-binding antagonist. In some embodiments, the tumor sample from the individual has a reduced level of somatic mutations in at least one gene specified in Table 1 compared to a reference level of somatic mutations in at least one gene specified in Table 1. In some embodiments, the tumor sample has been determined to have a reduced level of somatic mutations in at least one-third of the genes specified in Table 1 compared to a reference level of somatic mutations in at least one-third of the genes specified in Table 1. In some embodiments, the tumor sample has been determined to have a reduced level of somatic mutations in at least one-half of the genes specified in Table 1 compared to a reference level of somatic mutations in at least one-half of the genes specified in Table 1. In some embodiments, the tumor sample is determined to have a reduced level of somatic mutations in at least two-thirds of the genes specified in Table 1 compared to a reference level of somatic mutations in at least two-thirds of the genes specified in Table 1. In some embodiments, the tumor sample is determined to have a reduced level of somatic mutations in at least three-quarters of the genes specified in Table 1 compared to a reference level of somatic mutations in at least three-quarters of the genes specified in Table 1. In some embodiments, the tumor sample is determined to have a reduced level of somatic mutations in the genes specified in Table 1 compared to a reference level of somatic mutations in the genes specified in Table 1. In some embodiments, the tTMB score from the tumor sample is less than about 16 mutations / Mb. In some embodiments, the tTMB score from the tumor sample is less than about 16 mutations / Mb, and the reference tTMB score is about 16 mutations / Mb. In some embodiments, the tTMB score from the tumor sample is less than about 20 mutations / Mb.In some embodiments, the tTMB score from the tumor sample is less than about 20 mutations / Mb, and the reference tTMB score is about 20 mutations / Mb.

[0017] In some embodiments of any one of the preceding aspects, the tTMB score or reference tTMB score is expressed as the number of somatic mutations counted per defined number of sequenced bases. In some embodiments, the defined number of sequenced bases is about 100 kb to about 10 Mb. In some embodiments, the defined number of sequenced bases is about 1.1 Mb.

[0018] In some embodiments of any one of the preceding aspects, the tTMB score or reference tTMB score is an equivalent TMB value. In some embodiments, the equivalent TMB value is determined by whole exome sequencing (WES).

[0019] In some embodiments of any one of the preceding aspects, the tumor sample is determined to have a detectable expression level of PD-L1 in 1% or more of the tumor cells in the tumor sample. In some embodiments, the tumor sample is determined to have a detectable expression level of PD-L1 in 5% or more of the tumor cells in the tumor sample. In some embodiments, the tumor sample is determined to have a detectable expression level of PD-L1 in 10% or more of the tumor cells in the tumor sample. In some embodiments, the tumor sample is determined to have a detectable expression level of PD-L1 in 20% or more of the tumor cells in the tumor sample. In some embodiments, the tumor sample is determined to have a detectable expression level of PD-L1 in 50% or more of the tumor cells in the tumor sample.

[0020] In other embodiments of any one of the preceding aspects, the tumor sample has been determined to have an undetectable expression level of PD-L1 in tumor cells in the tumor sample.

[0021] In some embodiments of any one of the preceding aspects, the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells in the tumor sample. In some embodiments, the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise 1% or more of the tumor sample. In some embodiments, the tumor sample 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. In some embodiments, the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise 10% or more of the tumor sample.

[0022] In other embodiments of any one of the preceding aspects, the tumor sample has been determined to have an undetectable expression level of PD-L1 in tumor-infiltrating immune cells.

[0023] In some embodiments of any one of the preceding aspects, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist, a PD-1 binding antagonist, or 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 the binding of PD-L1 to one or more of its ligand binding partners. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is atezolizumab (MPDL3280A), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), or 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: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the PD-L1 axis-binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2.In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the antibody is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108. In some embodiments, the PD-1 binding antagonist is an Fc fusion protein. In some embodiments, the Fc fusion protein is AMP-224.

[0024] In some embodiments of any one of the preceding aspects, the method further comprises administering to the individual an effective amount of a second therapeutic agent, hi some embodiments, the second therapeutic agent is a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, or a combination thereof.

[0025] In another aspect, the invention features a PD-L1 axis-binding antagonist (e.g., for use in treating an individual afflicted with cancer), wherein a tumor sample from the individual has been determined to have a tTMB score that is equal to or greater than a reference tTMB score, and optionally, the tumor sample has been determined to have an increased level of somatic mutations in at least one gene specified in Table 1 compared to the reference level of somatic mutations in at least one gene specified in Table 1, a detectable level of expression of PD-L1 in tumor-infiltrating immune cells that comprise about 1% or more of the tumor sample, and / or a detectable level of expression of PD-L1 in tumor-infiltrating immune cells that comprise 1% or more of the tumor sample.

[0026] In another 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 an individual suffering from cancer, wherein a tumor sample from the individual has been determined to have a tTMB score that is equal to or greater than the reference tTMB score, and optionally, the tumor sample has been determined to have an increased level of somatic mutations in at least one gene specified in Table 1 compared to the reference level of somatic mutations in at least one gene specified in Table 1, a detectable level of expression of PD-L1 in tumor-infiltrating immune cells that comprise about 1% or more of the tumor sample, and / or a detectable level of expression of PD-L1 in tumor-infiltrating immune cells that comprise 1% or more of the tumor sample.

[0027] In another aspect, the invention features a composition comprising an effective amount of a PD-L1 axis-binding antagonist for use in a method of treating an individual afflicted with cancer, wherein a tumor sample from the individual has been determined to have a tTMB score that is equal to or greater than a reference tTMB score, and optionally, the tumor sample has been determined to have an increased level of somatic mutations in at least one gene specified in Table 1 compared to the reference level of somatic mutations in at least one gene specified in Table 1, a detectable level of expression of PD-L1 in tumor-infiltrating immune cells that comprise about 1% or more of the tumor sample, and / or a detectable level of expression of PD-L1 in tumor-infiltrating immune cells that comprise 1% or more of the tumor sample.

[0028] In some embodiments of any one of the preceding aspects, the individual has a high level of microsatellite instability (MSI), e.g., a tumor sample from the individual has an MSI-high (MSI-H) phenotype.

[0029] In some embodiments of any one of the preceding aspects, the cancer is lung cancer, kidney cancer, bladder cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, endometrial cancer, skin cancer, or hematological malignancy. In some embodiments, the cancer is lung cancer, bladder cancer, melanoma, kidney cancer, colorectal cancer, or head and neck cancer. In some embodiments, the cancer is lung cancer.

[0030] In some embodiments of any one of the preceding aspects, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is metastatic NSCLC. In other embodiments, the NSCLC is locally advanced NSCLC. In some embodiments, the NSCLC is stage IIIB NSCLC. In some embodiments, the NSCLC is stage IV NSCLC. In some embodiments, the NSCLC is recurrent NSCLC. In some embodiments, the individual has received at least one prior treatment for NSCLC with a platinum-containing regimen. In some embodiments, the individual has received at least two prior treatments for NSCLC with a platinum-containing regimen. In some embodiments, the individual has not received prior treatment for NSCLC with a platinum-containing regimen. In some embodiments, the bladder cancer is locally advanced or metastatic urothelial carcinoma. In some embodiments, the bladder cancer is metastatic urothelial carcinoma. In some embodiments, the individual (i) has not received any prior treatment for bladder cancer, or (ii) the individual has received at least one prior treatment (e.g., one or two prior treatments) for bladder cancer.

[0031] In some embodiments of any one of the preceding aspects, 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.

[0032] In some embodiments of any one of the preceding aspects, 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 some embodiments, the protein expression level of PD-L1 is detected using an anti-PD-L1 antibody.

[0033] In some embodiments of any one of the preceding aspects, the PD-L1 expression level is an mRNA expression level. In some embodiments, the PD-L1 mRNA expression level 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). [Brief explanation of the drawings]

[0034] [Figure 1]Figure 6 shows the overall survival (OS) benefit of atezolizumab treatment compared with docetaxel treatment in the POPLAR (clinical trial ID number NCT01903993) patient population. The OS benefit correlates with increased PD-L1 expression. However, patients with low or no PD-L1 expression on tumor cells (TC) and tumor-infiltrating immune cells (IC) can also derive clinical benefit from atezolizumab treatment. TC0 and IC0 indicate a TC score and IC IHC score of 0, respectively. TC1 / 2 / 3 and IC1 / 2 / 3 indicate a TC score and IC IHC score of 1, 2, or 3, respectively. TC2 / 3 and IC2 / 3 indicate a TC score and IC IHC score of 2 or 3, respectively. TC3 and IC3 indicate a TC score and IC IHC score of 3, respectively. Table 6 provides a summary of the TC and IC IHC scoring criteria. ITT, intention to treat analysis; NE, not estimable; a, unstratified hazard ratios (HRs) for subgroups and ITT stratification; mo, months. [Figure 2A] 1 is a graph showing that the level of somatic mutations, also referred to as mutation burden or tumor mutation burden (TMB), is associated with improved clinical response to atezolizumab as determined by RECIST v1.1 in the BIRCH (clinical trial ID number NCT02031458) and FIR (clinical trial ID number NCT01846416) patient populations. CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. [Figure 2B] (b) A pair of graphs showing that TMB is associated with improved progression-free survival (PFS) in the BIRCH and FIR patient populations. TMB quartile cutoffs (e.g., 25%, 50%, and 75% TMB quartiles) and their associated hazard ratios are presented in the left panel. The right panel is a Kaplan-Meier plot showing the PFS probability for patients below (<75%) and above (≥75%) the 75% TMB quartile cutoff. (c) The objective response rate (ORR) was 19% for both the biomarker-evaluable population (BEP) (n=371) and the intention-to-treat (ITT) (n=613) populations. [Figure 2C](a) and (b) are a pair of graphs showing that TMB is associated with improved overall survival (OS) in the BIRCH and FIR patient populations. The TMB quartile cutoffs (e.g., 25%, 50%, and 75% TMB quartiles) and their associated hazard ratios are presented in the left panel. The right panel is a Kaplan-Meier plot showing the OS probability for patients below (<75%) and above (≥75%) the 75% TMB quartile cutoff. (b) The ORR was 19% for both the BEP (n=371) and ITT (n=613) populations. [Figure 3A] This pair of graphs (upper panel) shows the association between the efficacy of TMB and atezolizumab treatment compared with the efficacy of docetaxel treatment in 2L+ NSCLC PD-L1-unselected patients from the randomized POPLAR study (clinical trial ID number NCT01903993). The lower left panel is a Kaplan-Meier plot showing the PFS probability for patients below the 75% TMB quartile cutoff (<75%, low docetaxel) and above the 75% TMB quartile cutoff (≥75%, high docetaxel). The lower right panel is a Kaplan-Meier plot showing the PFS probability for patients below the 75% TMB quartile cutoff (<75%, low atezolizumab) and above the 75% TMB quartile cutoff (≥75%, high atezolizumab). a, Unadjusted and unstratified HRs of atezolizumab compared with docetaxel at each TMB quantile cutoff. [Figure 3B]A pair of graphs (upper panel) show the association between TMB and atezolizumab treatment efficacy compared with docetaxel treatment efficacy in 2L+ NSCLC PD-L1-unselected patients from the randomized POPLAR study. The lower left panel is a Kaplan-Meier plot showing OS probability below the 75% TMB quartile cutoff (<75%, low-docetaxel) and above the 75% TMB quartile cutoff (≥75%, high-docetaxel). The lower right panel is a Kaplan-Meier plot showing OS probability for patients below the 75% TMB quartile cutoff (<75%, low-atezolizumab) and above the 75% TMB quartile cutoff (≥75%, high-atezolizumab). a, Unadjusted and unstratified HRs for atezolizumab compared with docetaxel at each TMB quartile cutoff. [Figure 4] Graph showing the association between TMB and PD-L1 expression on TC or IC in the combined BIRCH, FIR, and POPLAR patient populations. [Figure 5] Graph showing that response to atezolizumab treatment increased with increasing TMB and with increasing levels of PD-L1 expression on TCs or ICs. [Figure 6] Graph showing that the level of somatic mutations, also referred to as mutational burden or tumor mutation burden (TMB), is associated with improved clinical response to atezolizumab as determined by RECIST v1.1 in the BIRCH (clinical trial ID number NCT02031458) and FIR (clinical trial ID number NCT01846416) patient populations. BCOR, best confirmed overall response. [Figure 7A]Figure 7A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the entire patient population of the BIRCH and FIR studies. Figure 7A is a Kaplan-Meier plot showing the PFS probability for patients with less than the 16 mutations / Mb cutoff (<16 mutations / Mb, corresponding to the <75th quartile) and patients with ≥16 mutations / Mb cutoff (≥16 mutations / Mb, corresponding to the ≥75th quartile). Figure 7B is a Kaplan-Meier plot showing the PFS probability for patients with less than the 20 mutations / Mb cutoff (<20 mutations / Mb, corresponding to the <85th quartile) and patients with ≥20 mutations / Mb cutoff (≥20 mutations / Mb, corresponding to the ≥85th quartile). [Figure 7B] Figure 7A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the entire patient population of the BIRCH and FIR studies. Figure 7A is a Kaplan-Meier plot showing the PFS probability for patients with less than the 16 mutations / Mb cutoff (<16 mutations / Mb, corresponding to the <75th quartile) and patients with ≥16 mutations / Mb cutoff (≥16 mutations / Mb, corresponding to the ≥75th quartile). Figure 7B is a Kaplan-Meier plot showing the PFS probability for patients with less than the 20 mutations / Mb cutoff (<20 mutations / Mb, corresponding to the <85th quartile) and patients with ≥20 mutations / Mb cutoff (≥20 mutations / Mb, corresponding to the ≥85th quartile). [Figure 7C] Figure 7C is a pair of graphs showing the association between TMB and atezolizumab treatment efficacy in first-line (1L) patients from the BIRCH and FIR studies. Figure 7C is a Kaplan-Meier plot showing the PFS probability for patients with less than the 9 mutations / Mb cutoff (corresponding to the <50% quantile) and patients with 9 mutations / Mb or more (corresponding to the ≥50% quantile). Figure 7D is a Kaplan-Meier plot showing the PFS probability for patients with less than the 16 mutations / Mb cutoff (corresponding to the <85% quantile) and patients with 16 mutations / Mb or more (corresponding to the ≥85% quantile). [Figure 7D]Figure 7C is a pair of graphs showing the association between TMB and atezolizumab treatment efficacy in first-line (1L) patients from the BIRCH and FIR studies. Figure 7C is a Kaplan-Meier plot showing the PFS probability for patients with less than the 9 mutations / Mb cutoff (corresponding to the <50% quantile) and patients with 9 mutations / Mb or more (corresponding to the ≥50% quantile). Figure 7D is a Kaplan-Meier plot showing the PFS probability for patients with less than the 16 mutations / Mb cutoff (corresponding to the <85% quantile) and patients with 16 mutations / Mb or more (corresponding to the ≥85% quantile). [Figure 7E] Figure 7A shows a pair of graphs depicting the association between TMB and atezolizumab treatment efficacy in second- or third-line (2L+) patients from the BIRCH and FIR studies. Figure 7E is a Kaplan-Meier plot showing the PFS probability for patients below the 75% quantile cutoff (<75% quantile) and for patients above the 75% quantile cutoff (≥75% quantile). Figure 7F is a Kaplan-Meier plot showing the PFS probability for patients below the 85% quantile cutoff (<85% quantile) and for patients above the 85% quantile cutoff (≥85% quantile). [Figure 7F] Figure 7A shows a pair of graphs depicting the association between TMB and atezolizumab treatment efficacy in second- or third-line (2L+) patients from the BIRCH and FIR studies. Figure 7E is a Kaplan-Meier plot showing the PFS probability for patients below the 75% quantile cutoff (<75% quantile) and for patients above the 75% quantile cutoff (≥75% quantile). Figure 7F is a Kaplan-Meier plot showing the PFS probability for patients below the 85% quantile cutoff (<85% quantile) and for patients above the 85% quantile cutoff (≥85% quantile). [Figure 8A]Figure 8A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the entire patient population of the BIRCH and FIR studies. Figure 8A is a Kaplan-Meier plot showing the OS probability for patients with less than the 16 mutations / Mb cutoff (<16 mutations / Mb, corresponding to the <75th quartile) and patients with 16 mutations / Mb or more (≥16 mutations / Mb, corresponding to the ≥75th quartile). Figure 8B is a Kaplan-Meier plot showing the OS probability for patients with less than the 20 mutations / Mb cutoff (<20 mutations / Mb, corresponding to the <85th quartile) and patients with 20 mutations / Mb or more (≥20 mutations / Mb, corresponding to the ≥85th quartile). [Figure 8B] Figure 8A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the entire patient population of the BIRCH and FIR studies. Figure 8A is a Kaplan-Meier plot showing the OS probability for patients with less than the 16 mutations / Mb cutoff (<16 mutations / Mb, corresponding to the <75th quartile) and patients with 16 mutations / Mb or more (≥16 mutations / Mb, corresponding to the ≥75th quartile). Figure 8B is a Kaplan-Meier plot showing the OS probability for patients with less than the 20 mutations / Mb cutoff (<20 mutations / Mb, corresponding to the <85th quartile) and patients with 20 mutations / Mb or more (≥20 mutations / Mb, corresponding to the ≥85th quartile). [Figure 8C] Figure 8C is a pair of graphs showing the association between TMB and atezolizumab treatment efficacy in 1L patients from the BIRCH and FIR studies. Figure 8C is a Kaplan-Meier plot showing the OS probability for patients with less than the 9 mutations / Mb cutoff (corresponding to the <50% quantile) and patients with 9 mutations / Mb or more (corresponding to the ≥50% quantile). Figure 8D is a Kaplan-Meier plot showing the OS probability for patients with less than the 16 mutations / Mb cutoff (corresponding to the <85% quantile) and patients with 16 mutations / Mb or more (corresponding to the ≥85% quantile). [Figure 8D]Figure 8C is a pair of graphs showing the association between TMB and atezolizumab treatment efficacy in 1L patients from the BIRCH and FIR studies. Figure 8C is a Kaplan-Meier plot showing the OS probability for patients with less than the 9 mutations / Mb cutoff (corresponding to the <50% quantile) and patients with 9 mutations / Mb or more (corresponding to the ≥50% quantile). Figure 8D is a Kaplan-Meier plot showing the OS probability for patients with less than the 16 mutations / Mb cutoff (corresponding to the <85% quantile) and patients with 16 mutations / Mb or more (corresponding to the ≥85% quantile). [Figure 8E] Figure 8E is a pair of graphs showing the association between TMB and atezolizumab treatment efficacy in 2L+ patients from the BIRCH and FIR studies. Figure 8E is a Kaplan-Meier plot showing the OS probability for patients below the 75% quantile cutoff (<75% quantile) and for patients above the 75% quantile cutoff (≥75% quantile). Figure 8F is a Kaplan-Meier plot showing the OS probability for patients below the 85% quantile cutoff (<85% quantile) and for patients above the 85% quantile cutoff (≥85% quantile). [Figure 8F] Figure 8E is a pair of graphs showing the association between TMB and atezolizumab treatment efficacy in 2L+ patients from the BIRCH and FIR studies. Figure 8E is a Kaplan-Meier plot showing the OS probability for patients below the 75% quantile cutoff (<75% quantile) and for patients above the 75% quantile cutoff (≥75% quantile). Figure 8F is a Kaplan-Meier plot showing the OS probability for patients below the 85% quantile cutoff (<85% quantile) and for patients above the 85% quantile cutoff (≥85% quantile). [Figure 9A]

[0033] Figure 9A is a set of graphs showing the association between TMB and efficacy of atezolizumab treatment in the OAK (Figure 9A) and POPLAR (Figure 9B) studies. The graphs show the response rates in the biomarker-evaluable subgroups, the <16 mutations / Mb subgroup, the ≥16 mutations / Mb subgroup, and the ≥20 mutations / Mb subgroup. [Figure 9B]

[0033] Figure 9A is a set of graphs showing the association between TMB and efficacy of atezolizumab treatment in the OAK (Figure 9A) and POPLAR (Figure 9B) studies. The graphs show the response rates in the biomarker-evaluable subgroups, the <16 mutations / Mb subgroup, the ≥16 mutations / Mb subgroup, and the ≥20 mutations / Mb subgroup. [Figure 10A] Figure 10A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the POPLAR study. Figure 10A is a Kaplan-Meier plot showing the PFS probability for patients treated with atezolizumab (MPDL3280A) or docetaxel who had less than the 16 mutations / Mb cutoff (<16 mutations / Mb) and those who had more than or equal to the 16 mutations / Mb cutoff (≥16 mutations / Mb). Figure 10B is a Kaplan-Meier plot showing the PFS probability for patients treated with atezolizumab or docetaxel who had less than the 20 mutations / Mb cutoff (<20 mutations / Mb) and those who had more than or equal to the 20 mutations / Mb cutoff (≥20 mutations / Mb). [Figure 10B] Figure 10A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the POPLAR study. Figure 10A is a Kaplan-Meier plot showing the PFS probability for patients treated with atezolizumab (MPDL3280A) or docetaxel who had less than the 16 mutations / Mb cutoff (<16 mutations / Mb) and those who had more than or equal to the 16 mutations / Mb cutoff (≥16 mutations / Mb). Figure 10B is a Kaplan-Meier plot showing the PFS probability for patients treated with atezolizumab or docetaxel who had less than the 20 mutations / Mb cutoff (<20 mutations / Mb) and those who had more than or equal to the 20 mutations / Mb cutoff (≥20 mutations / Mb). [Figure 11A]Figure 11A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the POPLAR study. Figure 11A is a Kaplan-Meier plot showing the OS probability for patients with less than the 16 mutations / Mb cutoff (<16 mutations / Mb) and patients with more than or equal to the 16 mutations / Mb cutoff (≥16 mutations / Mb) treated with atezolizumab (MPDL3280A) or docetaxel. Figure 11B is a Kaplan-Meier plot showing the OS probability for patients with less than the 20 mutations / Mb cutoff (<20 mutations / Mb) and patients with more than or equal to the 20 mutations / Mb cutoff (≥20 mutations / Mb) treated with atezolizumab or docetaxel. [Figure 11B] Figure 11A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the POPLAR study. Figure 11A is a Kaplan-Meier plot showing the OS probability for patients with less than the 16 mutations / Mb cutoff (<16 mutations / Mb) and patients with more than or equal to the 16 mutations / Mb cutoff (≥16 mutations / Mb) treated with atezolizumab (MPDL3280A) or docetaxel. Figure 11B is a Kaplan-Meier plot showing the OS probability for patients with less than the 20 mutations / Mb cutoff (<20 mutations / Mb) and patients with more than or equal to the 20 mutations / Mb cutoff (≥20 mutations / Mb) treated with atezolizumab or docetaxel. [Figure 12A] Figure 12A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the OAK study. Figure 12A is a Kaplan-Meier plot showing the PFS probability for patients with less than the 16 mutations / Mb cutoff (<16 mutations / Mb) and patients with more than or equal to the 16 mutations / Mb cutoff (≥16 mutations / Mb) treated with atezolizumab or docetaxel. Figure 12B is a Kaplan-Meier plot showing the PFS probability for patients with less than the 20 mutations / Mb cutoff (<20 mutations / Mb) and patients with more than or equal to the 20 mutations / Mb cutoff (≥20 mutations / Mb) treated with atezolizumab or docetaxel. [Figure 12B]Figure 12A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the OAK study. Figure 12A is a Kaplan-Meier plot showing the PFS probability for patients with less than the 16 mutations / Mb cutoff (<16 mutations / Mb) and patients with more than or equal to the 16 mutations / Mb cutoff (≥16 mutations / Mb) treated with atezolizumab or docetaxel. Figure 12B is a Kaplan-Meier plot showing the PFS probability for patients with less than the 20 mutations / Mb cutoff (<20 mutations / Mb) and patients with more than or equal to the 20 mutations / Mb cutoff (≥20 mutations / Mb) treated with atezolizumab or docetaxel. [Figure 13A] Figure 13A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the OAK study. Figure 13A is a Kaplan-Meier plot showing the OS probability for patients with less than the 16 mutations / Mb cutoff (<16 mutations / Mb) and patients with more than or equal to the 16 mutations / Mb cutoff (≥16 mutations / Mb) treated with atezolizumab (MPDL3280A) or docetaxel. Figure 13B is a Kaplan-Meier plot showing the OS probability for patients with less than the 20 mutations / Mb cutoff (<20 mutations / Mb) and patients with more than or equal to the 20 mutations / Mb cutoff (≥20 mutations / Mb) treated with atezolizumab or docetaxel. [Figure 13B] Figure 13A is a pair of graphs showing the association between TMB and the efficacy of atezolizumab treatment in the OAK study. Figure 13A is a Kaplan-Meier plot showing the OS probability for patients with less than the 16 mutations / Mb cutoff (<16 mutations / Mb) and patients with more than or equal to the 16 mutations / Mb cutoff (≥16 mutations / Mb) treated with atezolizumab (MPDL3280A) or docetaxel. Figure 13B is a Kaplan-Meier plot showing the OS probability for patients with less than the 20 mutations / Mb cutoff (<20 mutations / Mb) and patients with more than or equal to the 20 mutations / Mb cutoff (≥20 mutations / Mb) treated with atezolizumab or docetaxel. [Figure 14A]Figure 14A is a pair of graphs showing the association between TMB and duration of response of atezolizumab treatment in the OAK study at cutoffs of 16 or more and 20 or more mutations / Mb. Figure 14A shows a forest plot, while Figures 14B and 14C show Kaplan-Meier plots showing duration of response (DoR) at cutoffs of 16 or more and 20 or more mutations / Mb, respectively. [Figure 14B] Figure 14A is a pair of graphs showing the association between TMB and duration of response of atezolizumab treatment in the OAK study at cutoffs of 16 or more and 20 or more mutations / Mb. Figure 14A shows a forest plot, while Figures 14B and 14C show Kaplan-Meier plots showing duration of response (DoR) at cutoffs of 16 or more and 20 or more mutations / Mb, respectively. [Figure 14C] Figure 14A is a pair of graphs showing the association between TMB and duration of response of atezolizumab treatment in the OAK study at cutoffs of 16 or more and 20 or more mutations / Mb. Figure 14A shows a forest plot, while Figures 14B and 14C show Kaplan-Meier plots showing duration of response (DoR) at cutoffs of 16 or more and 20 or more mutations / Mb, respectively. [Figure 15-1] Figures 15A-15C are a pair of graphs showing that increased TMB correlates with outcome in 1L metastatic urothelial carcinoma (mUC) patients (Cohort 1) in the IMvigor210 study. Figure 15A shows the mutational burden per megabase plotted against response in 1L cisplatin-ineligible mUC patients. Figures 15B and 15C show PFS and OS, respectively, at the indicated TMB quartiles. Figures 15D and 15E are a pair of graphs showing that high TMB is associated with increased OS in 1L cisplatin-ineligible mUC patients in the IMvigor210 trial, with TMB cutoffs of 16 or more mutations / Mb (Figure 15D) and 20 or more mutations / Mb (Figure 15E). [Figure 15-2]Figures 15A-15C are a pair of graphs showing that increased TMB correlates with outcome in 1L metastatic urothelial carcinoma (mUC) patients (Cohort 1) in the IMvigor210 study. Figure 15A shows the mutational burden per megabase plotted against response in 1L cisplatin-ineligible mUC patients. Figures 15B and 15C show PFS and OS, respectively, at the indicated TMB quartiles. Figures 15D and 15E are a pair of graphs showing that high TMB is associated with increased OS in 1L cisplatin-ineligible mUC patients in the IMvigor210 trial, with TMB cutoffs of 16 or more mutations / Mb (Figure 15D) and 20 or more mutations / Mb (Figure 15E). [Figure 15-3] Figures 15A-15C are a pair of graphs showing that increased TMB correlates with outcome in 1L metastatic urothelial carcinoma (mUC) patients (Cohort 1) in the IMvigor210 study. Figure 15A shows the mutational burden per megabase plotted against response in 1L cisplatin-ineligible mUC patients. Figures 15B and 15C show PFS and OS, respectively, at the indicated TMB quartiles. Figures 15D and 15E are a pair of graphs showing that high TMB is associated with increased OS in 1L cisplatin-ineligible mUC patients in the IMvigor210 trial, with TMB cutoffs of 16 or more mutations / Mb (Figure 15D) and 20 or more mutations / Mb (Figure 15E). [Figure 15-4] Figures 15A-15C are a pair of graphs showing that increased TMB correlates with outcome in 1L metastatic urothelial carcinoma (mUC) patients (Cohort 1) in the IMvigor210 study. Figure 15A shows the mutational burden per megabase plotted against response in 1L cisplatin-ineligible mUC patients. Figures 15B and 15C show PFS and OS, respectively, at the indicated TMB quartiles. Figures 15D and 15E are a pair of graphs showing that high TMB is associated with increased OS in 1L cisplatin-ineligible mUC patients in the IMvigor210 trial, with TMB cutoffs of 16 or more mutations / Mb (Figure 15D) and 20 or more mutations / Mb (Figure 15E). [Figure 16-1]Figures 16A-16C are a pair of graphs showing that increasing TMB correlates with outcome in 2L+ mUC patients (Cohort 2) in the IMvigor210 study. Figure 16A shows the mutational burden per megabase plotted against response in 2L+ mUC patients. Figures 16B and 16C show PFS and OS, respectively, at the indicated TMB quartiles. Figures 16D and 16E are a pair of graphs showing that high TMB (TMB-H) is associated with increased OS in 2L+ mUC patients in the IMvigor210 trial, with cutoffs of 16 or more and 20 or more mutations / Mb. [Figure 16-2] Figures 16A-16C are a pair of graphs showing that increasing TMB correlates with outcome in 2L+ mUC patients (Cohort 2) in the IMvigor210 study. Figure 16A shows the mutational burden per megabase plotted against response in 2L+ mUC patients. Figures 16B and 16C show PFS and OS, respectively, at the indicated TMB quartiles. Figures 16D and 16E are a pair of graphs showing that high TMB (TMB-H) is associated with increased OS in 2L+ mUC patients in the IMvigor210 trial, with cutoffs of 16 or more and 20 or more mutations / Mb. [Figure 16-3] Figures 16A-16C are a pair of graphs showing that increasing TMB correlates with outcome in 2L+ mUC patients (Cohort 2) in the IMvigor210 study. Figure 16A shows the mutational burden per megabase plotted against response in 2L+ mUC patients. Figures 16B and 16C show PFS and OS, respectively, at the indicated TMB quartiles. Figures 16D and 16E are a pair of graphs showing that high TMB (TMB-H) is associated with increased OS in 2L+ mUC patients in the IMvigor210 trial, with cutoffs of 16 or more and 20 or more mutations / Mb. [Figure 16-4]Figures 16A-16C are a pair of graphs showing that increasing TMB correlates with outcome in 2L+ mUC patients (Cohort 2) in the IMvigor210 study. Figure 16A shows the mutational burden per megabase plotted against response in 2L+ mUC patients. Figures 16B and 16C show PFS and OS, respectively, at the indicated TMB quartiles. Figures 16D and 16E are a pair of graphs showing that high TMB (TMB-H) is associated with increased OS in 2L+ mUC patients in the IMvigor210 trial, with cutoffs of 16 or more and 20 or more mutations / Mb. [Figure 17] Graph showing the response rates of 1L or 2L+ mUC patients at TMB cutoffs of 16 or more and 20 or more mutations / Mb in the IMvigor210 study. [Figure 18] Graph showing that high TMB has been identified across common and rare cancer indications, and therefore numerous indications are expected to derive benefit from immunotherapy with, for example, PD-L1 axis-binding antagonists (such as, for example, anti-PD-L1 antibodies (e.g., atezolizumab)). [Figure 19] Venn diagram showing the overlap between microsatellite instability-high (MSI-H) and high TMB, and response to atezolizumab monotherapy in the IMvigor210 study. DETAILED DESCRIPTION OF THE INVENTION

[0035] I. Introduction The present invention provides therapeutic and diagnostic methods and compositions for cancer, lung cancer (eg, non-small cell lung cancer (NSCLC)). The present invention is based, at least in part, on the discovery that determining the level of somatic mutations in a tumor tissue sample obtained from a patient and deriving a tissue tumor mutational burden (tTMB) score can be used as a biomarker (e.g., a predictive biomarker) in the treatment of patients with cancer for diagnosing a patient with cancer, for determining whether a patient with cancer is likely to respond to treatment with an anti-cancer therapy comprising an immune checkpoint inhibitor such as 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 comprising an immune checkpoint inhibitor such as a PD-L1 axis-binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atezolizumab), and / or for selecting patients for anti-cancer therapy comprising an immune checkpoint inhibitor such as a PD-L1 axis-binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atezolizumab).

[0036] II. Definition It is understood that 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.

[0037] As used herein, the term "about" refers to a normal error range for the respective value, which is readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, a description of "about X" includes a description of "X". In some embodiments, a "about" value refers to a value of up to ±10% of the cited value, e.g., ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%.

[0038] As used herein, the terms "mutational burden," "mutational load," "mutational burden," "tumor mutational burden score," "TMB score," "tissue tumor mutational burden score," and "tTMB score" can each be used interchangeably and refer to the level (e.g., number) of alterations (e.g., one or more alterations, e.g., one or more somatic alterations) per preselected unit (e.g., per megabase) in a predetermined set of genes (e.g., in the coding regions of a predetermined set of genes) detected in a tumor tissue sample (e.g., a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample). The tTMB score can be measured, for example, based on the whole genome or exome, or based on a subset of the genome or exome. In certain embodiments, the tTMB score measured based on a subset of the genome or exome can be extrapolated to determine the whole genome or exome mutational burden. In some embodiments, the tTMB score refers to the level of cumulative somatic mutations within an individual (e.g., an animal (e.g., a human)). The tTMB score may refer to the cumulative somatic mutations in a patient with cancer (e.g., lung cancer, e.g., NSCLC). In some embodiments, the tTMB score refers to the cumulative mutations in an individual's entire genome. In some embodiments, the tTMB score refers to the cumulative mutations in a particular tissue sample collected from an individual (e.g., a tumor tissue sample biopsy, e.g., a lung cancer tumor sample, e.g., an NSCLC tumor sample).

[0039] The term "somatic mutation" or "somatic modification" refers to a genetic modification that occurs in somatic tissue (e.g., a cell outside the germline). Examples of genetic modifications include, but are not limited to, point mutations (e.g., exchange of a single nucleotide for another (e.g., silent mutations, missense mutations, and nonsense mutations)), insertions and deletions (e.g., addition and / or removal of one or more nucleotides (e.g., indels)), amplifications, gene duplications, copy number alterations (CNAs), rearrangements, and splice variants. The presence of certain mutations can be associated with a disease state (e.g., cancer, e.g., lung cancer, e.g., NSCLC).

[0040] In certain embodiments, the somatic modification is a silent mutation (e.g., a synonymous modification). In other embodiments, the somatic modification is a nonsynonymous single nucleotide variant (SNV). In other embodiments, the somatic modification is a passenger mutation (e.g., a modification that has no detectable effect on the fitness of the clone). In certain embodiments, the somatic modification is a variant of unknown significance (VUS), e.g., a modification whose pathogenicity cannot be confirmed or excluded. In certain embodiments, the somatic modification has not been identified as being associated with a cancer phenotype.

[0041] In certain embodiments, the somatic modification is not associated with, or is not known to be associated with, an effect on cell division, growth, or survival, hi other embodiments, the somatic modification is associated with an effect on cell division, growth, or survival.

[0042] In certain embodiments, the number of somatic modifications excludes functional modifications within the subgenomic interval.

[0043] In some embodiments, a functional modification is a modification that has an effect on cell division, growth, or survival (e.g., promotes cell division, growth, or survival) compared to a reference sequence (e.g., a wild-type sequence or an unmutated sequence). In certain embodiments, a functional modification is identified by inclusion in a database of functional modifications, such as the COSMIC database (see Forbes et al. Nucl. Acids Res. 43(D1):D805-D811, 2015, incorporated herein by reference in its entirety). In other embodiments, a functional modification is a modification with a known functional status (e.g., occurring as a known somatic modification in the COSMIC database). In certain embodiments, a functional modification is a modification with a potential functional status (e.g., a truncation in a tumor suppressor gene). In certain embodiments, a functional modification is a driver mutation (e.g., a modification that confers a selective advantage to a clone, e.g., within its microenvironment, by increasing cell survival or replication). In other embodiments, a functional modification is a modification that can cause clonal expansion. In certain embodiments, the functional alteration is one that can cause one, two, three, four, five, or all six of the following: (a) self-sufficiency in growth signals; (b) decreased insensitivity, e.g., to anti-growth signals; (c) decreased apoptosis; (d) increased replicative capacity; (e) sustained angiogenesis; or (f) tissue invasion or metastasis.

[0044] In certain embodiments, the functional modification is not a passenger mutation (e.g., a modification that has no detectable effect on the fitness of a clone of cells). In certain embodiments, the functional modification is not a variant of unknown significance (VUS) (e.g., a modification whose pathogenicity cannot be confirmed or excluded).

[0045] In certain embodiments, a plurality (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) of functional variations in preselected oncogenes in the predetermined set of genes are excluded. In certain embodiments, all functional variations in preselected genes (e.g., oncogenes) in the predetermined set of genes are excluded. In certain embodiments, a plurality of functional variations in multiple preselected genes (e.g., oncogenes) in the predetermined set of genes are excluded. In certain embodiments, all functional variations in all genes (e.g., oncogenes) in the predetermined set of genes are excluded.

[0046] In certain embodiments, the number of somatic modifications excludes germline mutations within the subgenomic interval.

[0047] In certain embodiments, the germline modification is a SNP, a base substitution, an insertion, a deletion, an indel, or a silent mutation (e.g., a synonymous mutation).

[0048] In certain embodiments, germline modifications are excluded by using a method that does not use comparison with a matched normal sequence. In other embodiments, germline modifications are excluded by a method that includes the use of an algorithm. In certain embodiments, germline modifications are identified by inclusion in a germline modification database, such as the dbSNP database (see Sherry et al. Nucleic Acids Res. 29(1):308-311, 2001, incorporated herein by reference in its entirety). In other embodiments, germline modifications are identified by inclusion in two or more counts of the ExAC database (see Exome Aggregation Consortium et al. bioRxiv preprint, October 30, 2015, incorporated herein by reference in its entirety). In some embodiments, germline modifications are identified by inclusion in the 1000Genome Project database (McVean et al. Nature 491, 56-65, 2012, incorporated herein by reference in its entirety). In some embodiments, germline modifications are identified as such by inclusion in the ESP database (Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA).

[0049] As used herein, the term "immune checkpoint inhibitor" refers to a therapeutic agent that targets at least one immune checkpoint protein to alter the regulation of the immune response, e.g., downregulate or inhibit the immune response. Immune checkpoint proteins are known in the art and include, but are not limited to, cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), programmed cell death-ligand 2 (PD-L2), V-domain Ig suppressor of T-cell activation (VISTA), B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, OX40, and A2aR. In some cases, immune checkpoint proteins may be expressed on the surface of activated T cells. Therapeutic agents that may function as immune checkpoint inhibitors useful in the methods of the present invention include, but are not limited to, therapeutic agents that target one or more of CTLA-4, PD-1, PD-L1, PD-L2, VISTA, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, OX40, and A2aR. In some cases, immune checkpoint inhibitors enhance or suppress the function of one or more targeted immune checkpoint proteins. In some cases, the immune checkpoint inhibitor is a PD-L1 axis binding antagonist described herein.

[0050] 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, so as to eliminate T cell dysfunction resulting from signaling on the PD-1 signaling axis, 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 interfere with the interaction between PD-L1 and PD-1 (e.g., PD-L2-Fc fusions).

[0051] The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. This term includes the common elements of both "exhaustion" and / or "anergy," in which antigen recognition can occur but the subsequent immune response is ineffective in controlling infection or tumor growth.

[0052] As used herein, the term "dysfunctional" also includes refractoriness or unresponsiveness to antigen recognition, specifically, an impaired ability to translate antigen recognition into downstream T cell effector functions such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing.

[0053] The term "anergy" refers to the incomplete or insufficient signal delivered through the T cell receptor (e.g., intracellular Ca in the absence of Ras activation). 2+ T cell anergy refers to a state of unresponsiveness to antigenic stimulation resulting from an increase in T cell proliferation (increase in T cell proliferation). T cell anergy can also occur upon stimulation by antigen in the absence of costimulation, resulting in 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 function.

[0054] The term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction resulting from persistent TCR signaling, which occurs in many chronic infections and cancers. It is distinct from anergy in that it results from persistent signaling rather than through defective or insufficient signaling. It is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state that differs from that of 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.).

[0055] "Enhancing T cell function" means inducing, causing, or stimulating T cells to have sustained or amplified biological function, or regenerating 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. Manners for measuring this enhancement are known to those skilled in the art.

[0056] "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.

[0057] "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 a PD-L1 axis-binding antagonist.

[0058] As used herein, a "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-L1 with any 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 particular 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, abrogate, or interfere with 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 through 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 one particular embodiment, the anti-PD-L1 antibody is YW243.55S70, as described herein. In another particular embodiment, the anti-PD-L1 antibody is MDX-1105, as described herein. In yet another particular embodiment, the anti-PD-L1 antibody is atezolizumab (MPDL3280A), as described herein. In yet another particular embodiment, the anti-PD-L1 antibody is MEDI4736 (dorvalumab), as described herein. In yet another particular embodiment, the anti-PD-L1 antibody is MSB0010718C (avelumab), as described herein.

[0059] As used herein, a "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, abrogates, or interferes with 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 a particular aspect, 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, abrogate, or interfere with 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 PD-L1 or cell surface proteins expressed on T lymphocytes and other cells through 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 one particular aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab), as described herein. In another particular aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab), as described herein. In another particular aspect, the PD-1 binding antagonist is CT-011 (pidilizumab), as described herein. In another particular aspect, the PD-1 binding antagonist is MEDI-0680 (AMP-514), as described herein. In another particular aspect, 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.

[0060] 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.

[0061] 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.

[0062] By "PD-L1 polypeptide variant" or variations thereof is meant a PD-L1 polypeptide, generally an active PD-L1 polypeptide, as defined herein, that has at least about 80% amino acid sequence identity to any 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- 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, PD-L1 variant polypeptides are at least about 10 amino acids in length, or 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, the PD-L1 variant polypeptide has no more than one conservative amino acid substitution compared to a native PD-L1 polypeptide sequence, or no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to a native PD-L1 polypeptide sequence.

[0063] As used interchangeably herein, "polynucleotide" or "nucleic acid" 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, a polynucleotide as defined herein includes, but is not limited to, single- and double-stranded DNA, DNA containing single- and double-stranded regions, single- and double-stranded RNA, and RNA containing single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded, or contain single- and double-stranded regions. Additionally, as used herein, the term "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The strands within such regions may be from the same molecule or different molecules. These regions can include all of one or more of the molecules, but more typically involve only a region of some of the molecules. Often, one of the molecules in a triple-helical region is an oligonucleotide. The term "polynucleotide" includes cDNA.

[0064] 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 "caps," which replace one or more of the naturally occurring nucleotides with an analog; internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoamidate, carbamate, etc.) and 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 with intercalating agents (e.g., acridine, psoralen, etc.); those with 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); and unmodified forms of polynucleotide(s). Additionally, any of the hydroxyl groups normally present on the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated for additional linkage to additional nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' terminal OH groups may be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized with standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, 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 the 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 substituted or unsubstituted alkyl (1-20C) (optionally containing ether (-O-) linkages), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages within a polynucleotide need be identical. A polynucleotide may contain one or more 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.

[0065] As used herein, "oligonucleotide" generally refers to a short, single-stranded polynucleotide that is less than about 250 nucleotides in length, but not necessarily. An oligonucleotide may be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and completely applicable to oligonucleotides.

[0066] The term "primer" refers to a single-stranded polynucleotide that can hybridize to a nucleic acid, generally by providing a free 3'-OH group, to allow polymerization of a complementary nucleic acid.

[0067] The term "small molecule" refers to any molecule having a molecular weight below about 2000 daltons, preferably below about 500 daltons.

[0068] 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 without regard to 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.

[0069] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The term includes vectors as self-replicating nucleic acid structures and vectors that have integrated into the genome of a host cell into which they have been 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."

[0070] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in 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.

[0071] The term "antibody" as used herein is used 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.

[0072] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that would interfere with research, diagnostic, and / or therapeutic uses of the antibody, and may include 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, as determined, for example, by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, for example, 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.

[0073] "Native antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although 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. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain.

[0074] 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.

[0075] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CH1, CH2, and CH3 domains (collectively CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0076] 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 variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding site.

[0077] The term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, 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-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light-chain variable domains each contain four FR regions that primarily 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 of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes 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.

[0078] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Antibodies generally contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). In natural antibodies, H3 and L3 represent the greatest diversity among these six HVRs, and H3 in particular is thought to play a unique role in conferring superior specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in 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, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0079] Several HVR delineations are used herein and are encompassed herein. 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)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are listed below. TIFF2025028833000001.tif83170

[0080] HVRs may comprise the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al. (supra) for each of these definitions.

[0081] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0082] 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. (supra). 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 FRs or HVRs 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 according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment of the regions of homology of the antibody's sequence with the "standard" Kabat numbering sequence.

[0083] The Kabat numbering system is commonly used when referring to residues within the variable domain (residues approximately 1-107 of the light chain and 1-113 of 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 within the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "The EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0084] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to 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 containing an Fc region.

[0085] 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.

[0086] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0087] An "Fv" is the minimum antibody fragment containing a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that in a 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 HVRs specific for an antigen) has the ability to recognize and bind to antigen, albeit with lower affinity than the complete binding site.

[0088] Fab fragments contain 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 conjugations of antibody fragments are also known.

[0089] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of 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 review of scFvs, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0090] The term "diabody" refers to an antibody fragment having two antigen-binding sites; these fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains can pair with complementary domains in another chain, creating two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are described in detail, for example, in EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0091] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively.

[0092] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, e.g., the individual antibodies comprising the population are identical except for possible minor mutations (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 comprise an antibody comprising a polypeptide sequence that binds to a target, where the target-binding polypeptide sequence has been obtained by a process that includes selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be 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 for the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, or to create a multispecific antibody, and that an antibody comprising 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.

[0093] 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 by, for example, hybridoma techniques (e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hongo et al., Hybridoma 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA techniques (see, for example, U.S. Pat. No. 4,816,567), phage display technology (e.g., Clackson et al., Nature 352:624-628 (1991); Marks et al. 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.USA101(34):12467-12472(2004), and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), as well as 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. These antibodies can be produced by a variety of techniques, including immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG1, IgG1, IgG2, IgG4, IgG5, IgG6, IgG1, IgG1, IgG2, IgG4 ...

[0094] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (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, the antigen-binding region of which is derived from, for example, an antibody produced by immunizing macaque monkeys with an antigen of interest.

[0095] A "human antibody" is one possessing an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0096] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human framework regions (FRs). In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of its HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of its FRs correspond 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.

[0097] 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 an unrelated, non-PD-L1 protein is less than about 10% of the binding of the antibody to PD-L1, as measured, for example, by radioimmunoassay (RIA). In a specific embodiment, the anti-PD-L1 antibody binds to an epitope of PD-L1 that is conserved among PD-L1 from different species.

[0098] 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 so 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 an unrelated, non-PD-1 protein 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.

[0099] A "blocking" or "antagonist" antibody is an antibody 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.

[0100] "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 represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0101] As used herein, the terms "binds to," "specifically binds 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 greater affinity, avidity, more readily, and / or for a longer period of time 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, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. 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.

[0102] An "affinity matured" antibody refers to an antibody that possesses one or more modifications in one or more hypervariable regions (HVRs), which modifications improve the affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0103] 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, a reference antibody that blocks the binding of an antibody to its antigen by 50% or more in a competitive assay.

[0104] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.

[0105] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous protein ("adhesin") with the effector functions of immunoglobulin constant domains. Structurally, immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity, which is other than the antigen recognition and binding site of an antibody (i.e., "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 can be derived 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 involve 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. See also U.S. Patent No. 5,428,130 for information on producing immunoglobulin fusions. For example, useful immunoadhesins as pharmaceuticals useful in the therapeutic methods 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 (such as PD-L1 ECD-Fc, PD-L2 ECD-Fc, and PD-1 ECD-Fc, respectively) fused to a constant domain of an immunoglobulin sequence. Immunoadhesins combining the Ig Fc and ECD of a cell surface receptor are sometimes referred to as soluble receptors.

[0106] "Fusion protein" and "fusion polypeptide" refer to a polypeptide having two portions covalently linked together, each of which is a polypeptide with a distinct property. The property can be a biological property, such as in vitro or in vivo activity. 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 portions can be linked directly by a single peptide bond or through a peptide linker, but are in reading frame with each other.

[0107] "Percent (%) amino acid sequence identity" with respect to a polypeptide sequence 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 being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment to determine 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 appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes of this specification, 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 submitted to the U.S. Copyright Office (Washington, DC, 20559) and is registered under U.S. Copyright 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.

[0108] 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 relative to a given amino acid sequence B (alternatively, it may be expressed as a given amino acid sequence A having or containing a particular % amino acid sequence identity to, with, or relative to 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 that program's 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 will not equal 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.

[0109] The term "detection" includes any method of detection, including direct and indirect detection.

[0110] As used herein, the term "biomarker" refers to an indicator, e.g., a predictive, diagnostic, and / or prognostic indicator, that can be detected in a sample, such as a particular gene, or a protein encoded by the gene, or one or more somatic mutations in the particular gene. A biomarker can serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by specific molecular, pathological, histological, and / or clinical characteristics (e.g., responsiveness to a therapy including a PD-L1 axis-binding antagonist). In some embodiments, a biomarker is a collection of genes or a collection number of mutations / alterations (e.g., somatic mutations) in a collection of genes. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), polynucleotide modifications (e.g., polynucleotide copy number modifications (e.g., DNA copy number modifications)), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrate, and / or glycolipid-based molecular markers.

[0111] The "amount" or "level" of somatic mutations 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 disclosed herein. The expression level or amount of the assessed somatic mutation can be used to determine response to treatment.

[0112] The term "level" refers to the amount of somatic mutation in a biological sample.

[0113] An "increased level," "increased levels," or "elevated level" of somatic mutation refers to an increased level of somatic mutation 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 reference gene). In some embodiments, the increased level of somatic mutation is present throughout the individual's entire genome. In other embodiments, the increased level of somatic mutation is present in a sample (e.g., a tissue sample) collected from the individual. In some embodiments, the individual has cancer (e.g., cancer, e.g., lung cancer, e.g., non-small cell lung cancer (NSCLC)).

[0114] A "reduced level," "reduced levels," "reduced level," or "reduced levels" of somatic mutations refers to a decrease in the level of somatic mutations 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 reference level). In some embodiments, the decreased level of somatic mutations is present throughout the individual's entire genome. In other embodiments, the decreased level of somatic mutations is present in a sample (e.g., a tissue sample) collected from the individual. In some embodiments, the individual has cancer (e.g., cancer, e.g., lung cancer, e.g., non-small cell lung cancer (NSCLC)).

[0115] As used herein, the term "reference tTMB score" refers to a tTMB score to which another tTMB score is compared, e.g., to make a diagnostic, predictive, and / or therapeutic decision. For example, a reference tTMB score can be a tTMB score in a reference sample, a reference population, and / or a predetermined value. In some cases, the reference tTMB score is a cutoff value that significantly separates a first subset of individuals (e.g., patients) within a reference population who are being treated with an immune checkpoint inhibitor (e.g., a PD-L1 axis-binding antagonist therapy) from a second subset of individuals (e.g., patients) within the same reference population who are being treated with a non-PD-L1 axis-binding antagonist therapy that does not include an immune checkpoint inhibitor (e.g., a PD-L1 axis-binding antagonist), based on a significant difference between the individual's responsiveness to treatment with an immune checkpoint inhibitor (e.g., a PD-L1 axis-binding antagonist therapy) and the individual's responsiveness to treatment with a non-PD-L1 axis-binding antagonist therapy at or above and / or below the cutoff value. In some cases, the individual's responsiveness to treatment with an immune checkpoint inhibitor (e.g., a PD-L1 axis-binding antagonist therapy) is significantly improved compared to the individual's responsiveness to treatment with a non-PD-L1 axis-binding antagonist therapy at or above the cutoff value. In some cases, the individual's responsiveness to treatment with the non-PD-L1 axis-binding antagonist therapy is significantly improved compared to the individual's responsiveness to treatment with an immune checkpoint inhibitor (e.g., a PD-L1 axis-binding antagonist therapy) below the cutoff value.

[0116] Those skilled in the art will appreciate that the numerical value of the reference tTMB score may be used to assess the relevance of a cancer type (e.g., lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer), kidney cancer (e.g., renal urothelial carcinoma or renal cell carcinoma (RCC)), bladder cancer (e.g., bladder urothelial (transitional cell) carcinoma (e.g., locally advanced or metastatic urothelial carcinoma, including first-line (1L) or second-line or higher (2L+) locally advanced or metastatic urothelial carcinoma)), breast cancer (e.g., human epidermal growth factor receptor-2 (HER2)+ breast cancer or hormone receptor positive (HR+) breast cancer), colorectal cancer (e.g., colon adenocarcinoma), ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, melanoma (e.g., cutaneous melanoma), skin cancer ( For example, cutaneous squamous cell carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), thyroid cancer, sarcoma (e.g., soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, osteosarcoma, chondrosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, leiomyosarcoma, or rhabdomyosarcoma), prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), ML), chronic eosinophilic leukemia, or chronic lymphocytic leukemia (CLL), lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma (NHL)), myeloma (e.g., multiple myeloma (MM)), mycosis fungoides, Merkel cell carcinoma, hematological malignancies, cancer of the blood tissue, B-cell cancer, bronchial cancer, gastric cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, testicular cancer, biliary tract cancer, small intestine cancer, or appendix cancer , salivary gland cancer, adrenal gland cancer, adenocarcinoma, inflammatory myofibroblastic tumor, gastrointestinal stromal tumor (GIST), colon cancer, myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), polycythemia vera, chordoma, synovium, Ewing's tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder cancercarcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinoma, thyroid carcinoma, small cell carcinoma, essential thrombocythemia, primary myelofibrosis, hypereosinophilic syndrome, systemic mastocytosis, familial hypereosinophilia, neuroendocrine carcinoma, or carcinoid tumor, it will be understood that the tTMB score may depend on the methodology used to measure it and / or the statistical methods used to generate it.

[0117] The term "equivalent TMB value" refers to a numerical value corresponding to a tTMB score, which can be calculated by dividing the count of somatic variants (e.g., somatic mutations) by the number of sequenced bases (e.g., about 1.1 Mb (e.g., about 1.125 Mb) as assessed by, e.g., a FOUNDATIONONE® panel). It is generally understood that the tTMB score is linearly related to the size of the genomic region being sequenced. Such an equivalent tTMB value indicates an equivalent degree of tumor mutation burden compared to the tTMB score and can be used interchangeably in the methods described herein, for example, to predict a cancer patient's response to an immune checkpoint inhibitor (e.g., an anti-PD-L1 antibody, e.g., atezolizumab). As an example, in some embodiments, the equivalent tTMB value is a normalized tTMB value, which can be calculated by dividing the count of somatic variants (e.g., somatic mutations) by the number of sequenced bases. For example, an equivalent tTMB value can be expressed as the number of somatic mutations counted over a defined number of sequenced bases (e.g., about 1.1 Mb (e.g., about 1.125 Mb) as assessed by, e.g., a FOUNDATIONONE® panel). For example, a tTMB score of about 25 (determined as the number of somatic mutations counted over about 1.1 Mb) corresponds to an equivalent tTMB value of about 23 mutations / Mb. It is understood that the tTMB scores described herein (e.g., a TMB score expressed as the number of somatic mutations counted over a defined number of sequenced bases (e.g., about 1.1 Mb (e.g., about 1.125 Mb) as assessed by a FOUNDATIONONE® panel) encompass equivalent tTMB values ​​obtained using different methodologies (e.g., whole-exome sequencing or whole-genome sequencing). As an example, for a whole-exome panel, the target region may be about 50 Mb, and a sample in which about 500 somatic mutations are detected would have a tTMB value equivalent to a tTMB score of 10 mutations / Mb.In some embodiments, the tTMB score, determined as the number of somatic mutations counted across a defined number of sequenced bases (e.g., about 1.1 Mb (e.g., about 1.125 Mb)) in a subset of the genome or exome (e.g., a predetermined genotype), deviates by less than about 30% (e.g., about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, or less) from the tTMB score determined by whole-exome sequencing. See, e.g., Chalmers et al., Genome Medicine 9:34, 2017.

[0118] 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 a structure present and functioning in a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or further polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of a transcribed polynucleotide, a translated polypeptide, or a polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered expressed, regardless of whether they are derived from a transcript generated by alternative splicing or a degraded transcript, or from post-translational processing of a polypeptide, for example, by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer and ribosomal RNA).

[0119] "Increased expression," "increased expression level," "increased level," "elevated expression," "elevated expression level," or "elevated level" refers to increased expression or increased 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).

[0120] "Decreased expression," "decreased expression level," "decreased level," "reduced expression," "reduced expression level," or "reduced level" refers to decreased expression or decreased 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).

[0121] As used herein, "amplification" generally refers to the process of producing multiple copies of a desired sequence. "Multiple copies" means at least two copies. A "copy" does not necessarily imply perfect sequence complementarity or identity to the template sequence. For example, a copy may contain nucleotide analogs such as deoxyinosine, intentional sequence modifications (such as those introduced through primers containing sequences that are hybridizable to, but not complementary to, the template), and / or sequence errors that occur during amplification.

[0122] The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acids obtained from a single source (e.g., an individual) using two or more primer sets to amplify two or more DNA sequences in a single reaction.

[0123] As used herein, the technique of "polymerase chain reaction" or "PCR" 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 these primers are identical to or similar to opposite strands of the template to be amplified. The 5' terminal nucleotides of these two primers can match the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences derived from total genomic DNA, cDNA transcribed from total cellular RNA, bacteriophage, 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.

[0124] "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, including, for example, Cronin et al., Am. J. Pathol. 164(1):35-42 (2004) and Ma et al., Cancer Cell 5:607-616 (2004).

[0125] The term "microarray" refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.

[0126] 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., particular genes or proteins encoded by said genes)).

[0127] The term "aiding in diagnosis" is used herein to refer to a method that helps make a clinical decision 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 that aids in the diagnosis of a disease or condition (e.g., cancer) may include measuring a particular somatic mutation in a biological sample from an individual.

[0128] As used herein, the term "sample" 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, e.g., based on physical, biochemical, chemical, and / or physiological characteristics. 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, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts (such as homogenized tissue, tumor tissue, cell extracts), and combinations thereof.

[0129] A "tissue sample" or "cell sample" refers to a collection of similar cells obtained from the tissue of a subject or individual. The source of a tissue or cell sample may be solid tissue, such as from fresh, frozen, and / or preserved organs, tissue samples, biopsies, and / or aspirates; blood or any blood constituent (e.g., plasma); bodily fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage in a subject's pregnancy or development. A tissue sample may also be primary or cultured cells or cell lines. Optionally, a 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 tissue sample may contain a population of mixed cell types (e.g., tumor and non-tumor cells, cancerous and non-cancerous cells). A tissue sample may contain compounds not naturally mixed with natural tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, or antibiotics. In some cases, the tissue sample or tumor tissue sample is not a blood sample or sample or blood constituent (such as plasma).

[0130] 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 (e.g., 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.

[0131] 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 of the body (e.g., tissue or cells) 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 of the body (e.g., tissue or cells) of an individual other than 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 who is not the subject or individual.

[0132] For purposes of this specification, a "section" of a tissue sample refers to a single portion or piece of a tissue sample, e.g., a thin slice 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 for polypeptides (e.g., by immunohistochemistry) and / or polynucleotides (e.g., by in situ hybridization).

[0133] "Correlate" or "correlating" means comparing, 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 may be used in performing a second protocol and / or may be used to determine whether a second analysis or protocol should be performed. With respect to polypeptide analysis or protocol embodiments, the results of a polypeptide expression analysis or protocol may be used to determine whether a particular therapeutic regimen should be performed. With respect to polynucleotide analysis or protocol embodiments, the results of a polynucleotide expression analysis or protocol may be used to determine whether a particular therapeutic regimen should be performed.

[0134] "Individual response" or "response" can be assessed using any endpoint that indicates benefit to an individual, including, but not limited to, (1) some degree of inhibition of disease progression (e.g., cancer progression), including slowing or complete cessation; (2) reduction in tumor size; (3) inhibition (i.e., reduction, slowing, or complete cessation) of cancer cell invasion into adjacent peripheral organs and / or tissues; (4) inhibition (i.e., reduction, slowing, or complete cessation) of metastasis; (5) some degree of alleviation of one or more symptoms associated with a disease or disorder (e.g., cancer); (6) increased or prolonged survival, including overall survival and progression-free survival; and / or (7) a reduction in mortality at a given time point after treatment.

[0135] A patient's "effective response" or patient "responsiveness" to treatment with a pharmaceutical agent, and similar terms, refers 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 includes 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, the level of somatic mutations in tumor cells (e.g., tumor mutational burden (TMB)), determined, for example, using the methods disclosed herein, is used to identify patients who are predicted to have an increased likelihood of being responsive to pharmaceutical treatment (e.g., treatment comprising a PD-L1 axis-binding antagonist, e.g., an anti-PD-L1 antibody) compared to patients who do not have the same level of somatic mutations. In one embodiment, elevated levels of somatic mutations in tumor cells, e.g., as determined using the methods disclosed herein, are used to identify patients who are predicted to have an increased likelihood of being responsive to pharmaceutical treatment (e.g., anti-PD-L1 antibodies) compared to patients who do not have elevated levels of somatic mutations. In another embodiment, a biomarker (e.g., PD-L1 expression, e.g., as determined using IHC) is used to identify patients who are predicted to have an increased likelihood of being responsive to pharmaceutical treatment (e.g., 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, e.g., as determined using IHC) is used to identify patients who are predicted to have an increased likelihood of being responsive to pharmaceutical treatment (e.g., anti-PD-L1 antibodies) compared to patients who do not express the biomarker at the same level. In one embodiment, the presence of a biomarker is used to identify patients who are more likely to respond to pharmaceutical treatment compared to patients who do not have the presence of the biomarker. In another embodiment, the presence of a biomarker is used to determine that a patient has an increased likelihood of benefiting from treatment with a pharmaceutical agent compared to a patient who does not have the presence of the biomarker.

[0136] "Objective response" refers to a measurable response, including a complete response (CR) or a partial response (PR). In some embodiments, "objective response rate (ORR)" refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0137] 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.

[0138] "Sustained response" refers to a sustained effect on tumor growth reduction after cessation of treatment. For example, tumor size may be the same size or smaller compared to the size at the beginning of the pharmaceutical administration period. In some embodiments, the sustained response has a duration at least equal to the duration of treatment, or at least 1.5, 2.0, 2.5, or 3.0 times the duration of treatment, or longer.

[0139] 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.

[0140] As used herein, "partial response" or "PR" refers to a decrease in the size of one or more tumors or lesions or the extent of cancer in the body in response to treatment. For example, in some embodiments, PR refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions relative to the baseline SLD.

[0141] As used herein, "stable disease" or "SD" refers to neither sufficient shrinkage of target lesions to qualify for PR nor sufficient increase to qualify for PD, based on the smallest SLD since treatment initiation.

[0142] As used herein, "progressive disease" or "PD" refers to at least a 20% increase in the SLD of a target lesion based on the smallest SLD recorded since the start of treatment or the presence of one or more new lesions.

[0143] The term "survival" refers to the survival of a patient, and includes overall survival and progression-free survival.

[0144] As used herein, "progression-free survival" or "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 can 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. As used herein, "overall survival" or "OS" refers to the percentage of individuals in a group who are likely to be alive after a particular period of time.

[0145] "Prolonged survival" means an increase in overall survival or progression-free survival in treated patients compared to untreated patients (i.e., compared to patients not treated with a pharmaceutical agent), or compared to patients who do not have somatic mutations at a specified level, and / or compared to patients treated with an anti-tumor agent.

[0146] As used herein, the term "substantially the same" refers to a sufficiently high degree of similarity between two numerical values ​​such 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 characteristic measured by the values ​​(e.g., Kd value or mutation 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% difference between the two values ​​as a function of the reference / comparator value.

[0147] The phrase "substantially different," as used herein, refers to a difference between two numerical values ​​that is sufficiently great 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 characteristic measured by the values ​​(e.g., Kd value or mutation 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 of a reference / comparator molecule.

[0148] As used herein, the word "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent, such as a polynucleotide probe, or to an 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 chemical modification of a substrate compound or composition that is detectable. The term is intended to encompass direct labeling of a probe or antibody by conjugating (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 with fluorescently labeled streptavidin.

[0149] A "therapeutically effective amount" refers to the amount of a therapeutic agent used to treat or prevent a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a therapeutic agent may reduce the number of cancer cells, reduce primary tumor size, inhibit (i.e., slow to some extent and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent a drug can prevent the growth of and / or kill existing cancer cells, the drug may be cytostatic and / or cytotoxic. With respect to cancer therapy, in vivo efficacy 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.

[0150] 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 disease in question.

[0151] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes benign and malignant cancers. "Early-stage cancer" or "early-stage tumor" means 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. Examples of cancer include lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer (e.g., renal urothelial carcinoma or RCC), bladder cancer (e.g., bladder urothelial (transitional cell) carcinoma (e.g., locally advanced or metastatic urothelial carcinoma, including 1L or 2L+ locally advanced or metastatic urothelial carcinoma), breast cancer, colorectal cancer (e.g., colon adenocarcinoma), ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, black lung cancer, and the like. melanoma (e.g., cutaneous melanoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), thyroid cancer, sarcoma (e.g., soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, osteosarcoma, chondrosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, leiomyosarcoma, or rhabdomyosarcoma), prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia (e.g., acute lymphocytic leukemia), (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic eosinophilic leukemia, or chronic lymphocytic leukemia (CLL)), lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma (NHL)), myeloma (e.g., multiple myeloma (MM)), mycosis fungoides, Merkel cell carcinoma, hematological malignancies, cancers of the blood tissue, B-cell cancer, bronchial cancer, gastric cancer, brain cancer, or are central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, adrenal gland cancer, adenocarcinoma, inflammatory myofibroblastic tumor, gastrointestinal stromal tumor (GIST), colon cancer, myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), polycythemia vera, chordoma, synovium, Ewing's tumor, squamous cell carcinoma, basal cell carcinoma,Also included are, but are not limited to, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinoma, thyroid carcinoma, small cell carcinoma, essential thrombocythemia, primary myelofibrosis, hypereosinophilic syndrome, systemic mastocytosis, familial hypereosinophilia, neuroendocrine carcinoma, or carcinoid tumor. More specific examples of such cancers include lung cancer (including NSCLC), squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer (SCLC)), and lung adenocarcinoma and lung squamous cell carcinoma. In specific examples, the lung cancer is NSCLC, for example, locally advanced or metastatic NSCLC (e.g., stage IIIB NSCLC, stage IV NSCLC, or recurrent NSCLC). In some embodiments, the lung cancer (e.g., NSCLC) is unresectable / inoperable lung cancer (e.g., unresectable NSCLC). Other examples include cancer of the peritoneum, hepatocellular carcinoma, bladder cancer (e.g., urothelial bladder cancer (e.g., transitional cell carcinoma or urothelial carcinoma, non-muscle invasive bladder cancer, muscle invasive bladder cancer, and metastatic bladder cancer) and non-urothelial bladder cancer), gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, Merkel cell carcinoma, mycosis fungoides, testicular cancer, esophageal cancer, tumors of the bile duct, and head and neck cancer, and hematological malignancies. In some embodiments, the cancer is a triple negative (ER-, PR-, ER+, PR+) tumor with locally recurrent or metastatic disease (where locally recurrent disease is not amenable to resection with curative intent), optionally histologically confirmed.Triple-negative metastatic breast cancer, including HER2- (HER2-) breast adenocarcinoma.

[0152] As used herein, the term "tumor" 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.

[0153] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient contained therein is in a form such that it is effective, and which does not contain additional components that have unacceptable toxicity to the subject to which the formulation is administered.

[0154] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0155] 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 prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, remission or palliation of disease symptoms, and remission or improved prognosis. In some embodiments, antibodies (e.g., anti-PD-L1 antibodies and / or anti-PD-1 antibodies) are used to delay the onset of disease or slow disease progression.

[0156] The term "anti-cancer therapy" refers to a therapy useful in treating cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, 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, and other bioactive agents and organic chemical agents. Combinations thereof are also encompassed by the present invention.

[0157] As used herein, the term "cytotoxic agent" 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 (such as nucleases), antibiotics, and toxins (including fragments and / or variants thereof) such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, as well as various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. Tumoricidal agents cause the destruction of tumor cells.

[0158] 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, metuledopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including the synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryophenone; Statins; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, colofosfamidis nitrogen mustards such as estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, fenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin γ1I and calicheamicin ω1I (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994))); dynemicins, including dynemicin A; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclanomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxycyano-doxorubicin). including cidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); denopterin, methotrexate, pteropterin, trimetrexate, etc. Folic acid analogues; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calucelone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglatone; and aldophosphamide Glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraelin; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and taxanes, including TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum or platinum-based chemotherapy agents and platinum analogs (such as cisplatin, carboplatin, oxaliplatin (ELOXATIN™), satraplatin, picoplatin, nedaplatin, triplatin, and lipoplatin); vinblastine (VELBAN®); platinum; etoposide (VP-16); ibuprofen (Vegas); phosphamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovorin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically 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 the treatment regimen of oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin). Additional chemotherapeutic agents include, for example, maytansinoids (e.g., DM1) and cytotoxic agents available as antibody-drug conjugates, such as the auristatins MMAE and MMAF.

[0159] "Chemotherapeutic agents" also include "antihormonal agents" or "endocrine therapeutic agents," which act to regulate, reduce, block, or inhibit the action of hormones that can promote cancer growth, often in the form of systemic, or whole-body, treatment. 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-hydroxytamoxifen, trioxifene, 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, acetaminophen, and acetaminophen. Luteinizing hormone-releasing hormone (LHRH) agonists, such as buserelin acid, 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 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, holmestein, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole.Additionally, such 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; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those targeting, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptors. those that inhibit the expression of genes in signal transduction pathways implicated in aberrant cell proliferation, such as EGFR; THERATOPE® vaccines and gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® 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.

[0160] 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 with therapeutic potential as drugs in combination with the compounds of the invention include apolizumab, acelizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, cetolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, nat ... tuzumab, nimotuzumab, norobizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tuxituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and interleukin-12 and anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), a recombinant human sequence-specific full-length IgG1 lambda antibody genetically modified to recognize the p40 protein.

[0161] Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise directly interact with EGFR and prevent or reduce its signaling activity, and are alternatively 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 MAb579 (ATCC CRL HB8506), MAb455 (ATCC CRL HB8507), MAb225 (ATCC CRL8508), MAb528 (ATCC CRL8509) (see U.S. Pat. No. 4,943,533 to Mendelsohn et al.), as well as variants thereof, such as chimerized 225 (C225 or cetuximab, ERBUTIX®) and reshaped human 225 (H225) (Imclone Systems, Inc.). Inc., WO 96 / 40210); 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 to Abgenix / Amgen); EMD55900 (Stragliotto et al. al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) (EMD / Merck), a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-alpha for EGFR binding; 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 Pat. No. 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 agents, thereby generating immunoconjugates (see, eg, EP 659,439 A2 to Merck Patent GmbH).EGFR antagonists include those described in U.S. Patent Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,800,000, and the like. 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and the following PCT publications: WO98 / 14451, WO98 / 50038, WO99 / 09016, and WO99 / 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-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer), AG1571 (SU5271, 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).

[0162] Chemotherapeutic agents include "tyrosine kinase inhibitors" such as the EGFR-targeted drugs mentioned in the previous paragraph; small molecule HER2 tyrosine kinase inhibitors such as TAK165 available from Takeda; CP-724,714 (Pfizer and OSI), an oral selective inhibitor of ErbB2 receptor tyrosine kinase; dual HER inhibitors such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2- and EGFR-overexpressing cells; lapatinib (GSK572016, available from Glaxo-SmithKline); oral HER2 and EGFR tyrosine kinase inhibitors; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033, Pharmacia); Raf-1 inhibitors such as ISIS-5132, an antisense drug available from ISIS Pharmaceuticals that inhibits Raf-1 signaling; imatinib mesylate (GLEEVEC®, Glaxo-SmithKline); non-HER-targeted TK inhibitors such as sunitinib (SUTENT®, available from Pfizer); 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 PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP59326, CGP60261, and CGP62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyro bro[2,3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); 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 (ISIS3521, Isis / Lilly); imatinib mesylate (GLEEVEC®); PKI166 (Novartis); GW2016 (GlaxoSmithKline); 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 and those described in any of WO1999 / 06378 (Warner Lambert), WO1999 / 06396 (Warner Lambert), WO1996 / 30347 (Pfizer, Inc.), WO1996 / 33978 (Zeneca), WO1996 / 3397 (Zeneca), and WO1996 / 33980 (Zeneca).

[0163] Chemotherapeutic agents include dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG (raw), bevacuzimab, 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.

[0164] 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, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate; phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN) Immunoselective anti-inflammatory peptides (ImSAIDs) such as those from BioTherapeutics, LLC; azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers (etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), cetolithumab pegol (CIMZIA®), golimumab (SIMPONI®), etc.), interleukin-1 (IL-1 ... antirheumatic drugs such as nakinra (e.g., KINERET®), T-cell costimulation blockers (e.g., abatacept (ORENCIA®)), and interleukin-6 (IL-6) blockers (e.g., tocilizumab (ACTEMERA®)); interleukin-13 (IL-13) blockers such as lebrikizumab; interferon alpha (IFN) blockers such as rontalizumab; beta-7 integrin blockers such as rhuMAb beta-7; 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 drugs such as thioplatin, PS-341, phenyl butyrate, ET-18-OCH3, and farnesyltransferase inhibitors (L-739749, L-744832); polyphenols and their derivatives such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin, flavanols, procyanidins, and betulinic acid; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MA) RINOL®; beta-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopoletin, and 9-aminocamptothecin; podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronic acid Also included are bisphosphonates such as ledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPE® 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 (SCH6636, SARASAR™); and pharmaceutically acceptable salts or derivatives of any of the above; and combinations of two or more of the above.

[0165] As used herein, the term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that has reduced cytotoxicity 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). 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- or 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 listed above.

[0166] As used herein, a "growth inhibitory agent" 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, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, also extend to S-phase arrest. Further information can be found in "The Molecular Basis of Cancer," Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and anticancer drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs, both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules derived from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.

[0167] "Radiation therapy" refers to the use of directed gamma or beta radiation to induce sufficient damage to cells so as 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 the dose and duration of treatment. A typical treatment is given as a single administration, with typical doses ranging from 10 to 200 units (Grays) per day.

[0168] As used herein, the terms "individual," "patient," and "subject" are used interchangeably and refer to any single animal, more preferably a mammal (including, e.g., 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, an individual or patient herein is a human.

[0169] As used herein, "administering" refers to a method of providing a subject (e.g., a patient) with a dosage of a compound (e.g., an antagonist) or pharmaceutical composition (e.g., a pharmaceutical composition comprising an antagonist). Administration can be by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusions 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 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.

[0170] 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 in which the administration of one or more agent(s) continues after the administration of one or more other agent(s) is discontinued.

[0171] "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, for example, to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.

[0172] "Package insert" is used to refer to instructions typically included in commercial 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 product.

[0173] A "sterile" preparation is aseptic or free of all viable microorganisms and their spores.

[0174] An "article of manufacture" is any product (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 product or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0175] As used herein, the phrase "based on" means that information about one or more biomarkers is used to inform, for example, treatment decisions, information provided on package inserts, or marketing / promotional guidelines.

[0176] III. Method A. Diagnostic methods based on tissue tumor mutation burden (tTMB) scores that reflect the level of somatic mutations in cancer-related genes Provided herein are methods for determining whether a patient suffering from cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) is likely to respond to a treatment comprising an immune checkpoint inhibitor, such as a PD-L1 axis-binding antagonist. Also provided herein are methods for predicting the responsiveness of a patient suffering from cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) to a treatment comprising an immune checkpoint inhibitor, such as a PD-L1 axis-binding antagonist. Further provided herein are methods for selecting a therapy for a patient suffering from cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma). Any of the foregoing methods may be based on a determined or predetermined tissue tumor mutational burden (tTMB) score determined from a tumor tissue sample from the patient. Accordingly, any of the foregoing methods may be based on the level of somatic mutations in any of the genes described herein (e.g., in Table 1 and / or Table 2) in the tumor sample. Any of the foregoing methods may also be based on the expression level of a biomarker, e.g., PD-L1 expression in the tumor sample (e.g., in tumor-infiltrating immune cells and / or tumor cells). Any of these methods may further comprise administering to the patient an immune checkpoint inhibitor, such as 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.

[0177] The present invention provides a method for identifying an individual having a cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) that may benefit from a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining a tTMB score from a tumor sample from the individual, wherein a tTMB score from the tumor sample that is equal to or greater than a reference tTMB score identifies the individual as one that may benefit from a treatment comprising a PD-L1 axis-binding antagonist.

[0178] The method also provides a method for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma), the method comprising determining a tTMB score from a tumor sample from the individual, wherein a tTMB score from the tumor sample equal to or greater than a reference tTMB score identifies the individual as one who may benefit from therapy comprising a PD-L1 axis-binding antagonist.

[0179] In any of the foregoing methods, the tTMB score determined from the tumor sample may be greater than or equal to a reference tTMB score, and the method may further comprise administering to the individual an effective amount of a PD-L1 axis binding antagonist.

[0180] In other embodiments of any one of the preceding aspects, the tTMB score determined from the tumor sample may be less than the reference tTMB score. In some embodiments, the tTMB score determined from the tumor sample is less than the reference tTMB score, and the method further comprises administering to the individual a therapeutic agent other than or in addition to a PD-L1 axis-binding antagonist.

[0181] In any of the foregoing methods, in some instances, the reference tTMB score is a tTMB score within a reference population of individuals with cancer, the population consisting of a first subset of individuals being treated with a PD-L1 axis binding antagonist therapy and a second subset of individuals being treated with a non-PD-L1 axis binding antagonist therapy that does not include a PD-L1 axis binding antagonist. In some instances, the reference tTMB score significantly separates each of the first and second subsets of individuals based on a significant difference in responsiveness to treatment with a PD-L1 axis binding antagonist therapy compared to responsiveness to treatment with a non-PD-L1 axis binding antagonist therapy. In some instances, responsiveness to treatment is an increase in progression-free survival (PFS). In some instances, responsiveness to treatment is an increase in overall survival (OS). In some instances, responsiveness to treatment is an increase in overall response rate (ORR).

[0182] In any of the foregoing methods, in some cases, the reference tTMB score is a pre-assigned tTMB score. In some cases, the reference tTMB score is between about 5 and about 100 mutations per Mb (mutations / Mb), e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 mutations / Mb. For example, in some cases, the reference tTMB score is about 8 to about 30 mutations / Mb (e.g., about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 mutations / Mb). In some cases, the reference tTMB score is about 10 to about 20 mutations / Mb (e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations / Mb). In particular cases, the reference tTMB score may be 10 mutations / Mb, 16 mutations / Mb, or 20 mutations / Mb.

[0183] In some cases of any of the aforementioned methods, the tumor sample from the patient has a tTMB score of about 5 or more mutations / Mb. For example, in some cases, the tTMB score from the tumor sample is between about 5 and about 100 mutations / Mb (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 mutations / Mb). In some cases, a tumor sample from a patient has a tTMB score of about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 or more mutations / Mb. For example, in some cases, a tumor sample from a patient has a tTMB score of about 10 or more mutations / Mb. In some embodiments, the baseline tTMB score is 10 mutations / Mb. In some cases, the tTMB score from a tumor sample is about 10-100 mutations / Mb. In some cases, the tTMB score from a tumor sample is about 10-20 mutations / Mb. In some cases, a tumor sample from a patient has a tTMB score of about 16 or more mutations / Mb.In some cases, the tumor sample from the patient has a tTMB score of about 16 or more mutations / Mb, and the reference tTMB score is 16 mutations / Mb. In other cases, the tumor sample from the patient has a tTMB score of about 20 or more mutations / Mb. In some cases, the tumor sample from the patient has a tTMB score of about 20 or more mutations / Mb, and the reference tTMB score is about 20 mutations / Mb.

[0184] In some cases of any of the aforementioned methods, the tTMB score or reference tTMB score is expressed as the number of somatic mutations counted per defined number of sequenced bases. For example, in some cases, the defined number of sequenced bases is about 100 kb to about 10 Mb. In some cases, the defined number of sequenced bases is about 1.1 Mb (e.g., about 1.125 Mb), e.g., as assessed by a FOUNDATIONONE® panel. In some cases, the tTMB score or reference tTMB score is an equivalent TMB value. In some cases, the equivalent TMB value is determined by whole exome sequencing (WES).

[0185] In any of the foregoing methods, the determined tTMB score may reflect the level of somatic mutations and / or rearrangements detected within the genes listed in Table 1 and / or Table 2. In some cases, the tTMB score is determined (or is determined) to be at least about 5 or more mutations / Mb (e.g., about 5 or more mutations / Mb, about 6 or more mutations / Mb, about 7 or more mutations / Mb, about 8 or more mutations / Mb, about 9 or more mutations / Mb, about 10 or more mutations / Mb, about 11 or more mutations / Mb, about 12 or more mutations / Mb, about 13 or more mutations / Mb, about 14 or more mutations / Mb, about 15 or more mutations / Mb, about 16 or more mutations / Mb, about 17 or more mutations / Mb, about 18 or more mutations / Mb, about 19 or more mutations / Mb, about 20 or more mutations / Mb, about 25 or more mutations / Mb, about 30 or more mutations / Mb, about 35 or more mutations / Mb, about 40 or more mutations / Mb, and about 50 or more mutations / Mb). This predicts responsiveness to a treatment (e.g., a treatment comprising a PD-L1 axis-binding antagonist). In some cases, a tTMB score predictive of responsiveness to a treatment (e.g., a treatment comprising a PD-L1 axis-binding antagonist) can be about 7 mutations / Mb to about 20 mutations / Mb. In some cases, a tTMB score predictive of responsiveness to a treatment can be about 10 mutations / Mb to about 15 mutations / Mb. In some cases, a tTMB score predictive of responsiveness to a treatment can be about 11 mutations / Mb to about 13 mutations / Mb. In some cases, a tTMB score predictive of responsiveness to a treatment can be about 12.5 mutations / Mb. In other cases, the tTMB score predictive of responsiveness to treatment may be about 10 or more mutations / Mb, e.g., about 10 or more mutations / Mb, about 11 or more mutations / Mb, about 12 or more mutations / Mb, about 13 or more mutations / Mb, about 14 or more mutations / Mb, about 15 or more mutations / Mb, about 16 or more mutations / Mb, about 17 or more mutations / Mb, about 18 or more mutations / Mb, about 19 or more mutations / Mb, or about 20 or more mutations / Mb.

[0186] In some cases of any of the aforementioned methods, the tumor sample obtained from the patient is determined to have an increased level of somatic mutations in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes specified in Table 1 compared to a baseline level of somatic mutations in at least one gene specified in Table 1. In other cases, 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 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 genes identified in Table 1 have been determined to have increased somatic mutations. For example, in some cases, a tumor sample obtained from a patient is determined to have increased levels of somatic mutations in at least one-half or about 50% of the genes specified in Table 1. In some cases, a tumor sample obtained from a patient is determined to have increased levels of somatic mutations in at least two-thirds or about 67% of the genes specified in Table 1. In some cases, a tumor sample obtained from a patient is determined to have increased levels of somatic mutations in at least three-quarters or about 75% of the genes specified in Table 1. TIFF2025028833000002.tif253170TIFF2025028833000003.tif181170

[0187] The presence and / or level (amount) of somatic mutations can be qualitatively and / or quantitatively determined based on any suitable criteria known in the art, including, but not limited to, measurements of DNA, mRNA, cDNA, protein, protein fragment, and / or gene copy number levels in an individual. In some cases, a comprehensive genomic profile of the individual is determined. In some cases, a comprehensive genomic profile of a sample collected from the individual (e.g., a tissue sample, a formalin-fixed, paraffin-embedded (FFPE) tissue sample, a core or fine needle biopsy) is determined. In some cases, determining the genomic profile involves applying next-generation sequencing methods known in the art or described herein to identify genomic alterations (e.g., somatic mutations (e.g., base substitutions, insertions and deletions (indels), copy number alterations (CNAs), and rearrangements)) that are known to be distinct drivers of cancer (e.g., solid tumors).

[0188] In some cases, the test involves about 300 genes (e.g., at least about 300 to about 400 genes, e.g., about 300 to about 400 genes, e.g., about 300 to about 400 genes) covering at least about 0.05 Mb to about 10 Mb (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Mb). Simultaneously sequence the coding regions of 100 genes (a diverse set of 00, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 genes) to a typical median exon coverage depth of at least about 500x (e.g., 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, or 1,000x). In other cases, the test simultaneously sequences the coding regions of about 400 genes, about 425 genes, about 450 genes, about 475 genes, about 500 genes, about 525 genes, about 550 genes, about 575 genes, about 600 genes, about 625 genes, about 650 genes, about 675 genes, about 700 genes, about 725 genes, about 750 genes, about 775 genes, about 800 genes, about 825 genes, about 850 genes, about 875 genes, about 900 genes, about 925 genes, about 950 genes, about 975 genes, about 1000 genes, or more than 1000 genes. In some cases, the set of genes includes one or more genes (e.g., cancer-associated genes) specified in Table 1. In some cases, the set of genes is a set of genes from a FOUNDATIONONE® panel (see, e.g., Frampton et al. Nat. Biotechnol. 31:1023-31, 2013, which is incorporated by reference in its entirety). In some cases, the set of genes is a set of genes from a FOUNDATIONONE® CDx panel.In some embodiments, the test involves analyzing more than about 10 Mb of the individual's genome, e.g., more than about 10 Mb, more than about 15 Mb, more than about 20 Mb, more than about 25 Mb, more than about 30 Mb, more than about 35 Mb, more than about 40 Mb, more than about 45 Mb, more than about 50 Mb, more than about 55 Mb, more than about 60 Mb, more than about 65 Mb, more than about 70 Mb, more than about 75 Mb The test sequences more than about 80 Mb, more than about 85 Mb, more than about 90 Mb, more than about 95 Mb, more than about 100 Mb, more than about 200 Mb, more than about 300 Mb, more than about 400 Mb, more than about 500 Mb, more than about 600 Mb, more than about 700 Mb, more than about 800 Mb, more than about 900 Mb, more than about 1 Gb, more than about 2 Gb, more than about 3 Gb, or more than about 3.3 Gb. In some cases, the test simultaneously sequences the coding regions of 315 cancer-related genes and introns from 28 genes that are frequently rearranged or altered in cancer to a typical median coverage depth of more than 500x. In some cases, each comprehensive sequencing read represents a unique DNA cross-section, enabling highly sensitive and specific detection of genomic alterations that occur at low frequencies due to tumor heterogeneity, low tumor purity, and small tissue samples. In other cases, the presence and / or level of somatic mutations is determined by whole exome sequencing. In some cases, the presence and / or level of somatic mutations is determined by whole genome sequencing.

[0189] In some cases where the tTMB score determined from the tumor sample is less than a reference tTMB score, the tumor sample from the individual may also have a reduced level of somatic mutations in at least one gene specified in Table 1 compared to a reference level of somatic mutations in at least one gene specified in Table 1. In some cases, for example, in some embodiments, the tumor sample has been determined to have a reduced level of somatic mutations in at least one-third of the genes specified in Table 1 compared to a reference level of somatic mutations in at least one-third of the genes specified in Table 1. In some cases, the tumor sample has been determined to have a reduced level of somatic mutations in at least one-half of the genes specified in Table 1 compared to a reference level of somatic mutations in at least one-half of the genes specified in Table 1. In some cases, the tumor sample has been determined to have a reduced level of somatic mutations in at least two-thirds of the genes specified in Table 1 compared to a reference level of somatic mutations in at least two-thirds of the genes specified in Table 1. In some cases, the tumor sample is determined to have a reduced level of somatic mutations in at least three-quarters of the genes specified in Table 1 compared to a reference level of somatic mutations in at least three-quarters of the genes specified in Table 1. In some cases, the tumor sample is determined to have a reduced level of somatic mutations in the genes specified in Table 1 compared to a reference level of somatic mutations in the genes specified in Table 1. In some cases, the tTMB score from the tumor sample is less than about 16 mutations / Mb. In some cases, the tTMB score from the tumor sample is about 16 mutations / Mb and the reference tTMB score is about 16 mutations / Mb. In some cases, the tTMB score from the tumor sample is less than about 20 mutations / Mb. In some cases, the tTMB score from the tumor sample is less than about 20 mutations / Mb and the reference tTMB score is about 20 mutations / Mb.In any of these cases where the tTMB score determined from the tumor sample is less than the reference tTMB score, and the individual has a decreased level of somatic mutations in at least one gene specified in Table 1 compared to the reference level of somatic mutations in at least one gene specified in Table 1, the method may further comprise administering to the individual a therapeutic agent other than or in addition to a PD-L1 axis-binding antagonist.

[0190] The present invention further provides a method for determining whether a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining the level of somatic mutations in a tumor sample obtained from the patient, wherein increased levels of somatic mutations within at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes specified in Table 1 in the tumor sample indicate that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. For example, in some instances, an increased level of somatic mutations in at least about one-third of the genes specified in Table 1 in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, an increased level of somatic mutations in at least about two-thirds of the genes specified in Table 1 in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, an increased level of somatic mutations in at least about three-quarters of the genes specified in Table 1 in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, 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 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 genes identified in Table 1 have been determined to have increased somatic mutations. For example, in some cases, tumor samples obtained from patients have been determined to have elevated levels of somatic mutations in at least half or about 50% of the genes specified in Table 1.In some cases, a tumor sample obtained from a patient is determined to have an increased level of somatic mutations in at least two-thirds, or about 67%, of the genes specified in Table 1. In some cases, a tumor sample obtained from a patient is determined to have an increased level of somatic mutations in at least three-quarters, or about 75%, of the genes specified in Table 1.

[0191] The present invention further provides methods for predicting the responsiveness of a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining the level of somatic mutations in a tumor sample obtained from the patient, wherein an increased level of somatic mutations within at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes specified in Table 1 in 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 instances, an increased level of somatic mutations in at least about one-third of the genes specified in Table 1 in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, an increased level of somatic mutations in at least about two-thirds of the genes specified in Table 1 in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, an increased level of somatic mutations in at least about three-quarters of the genes specified in Table 1 in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, 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 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 genes identified in Table 1 have been determined to have increased somatic mutations. For example, in some cases, tumor samples obtained from patients have been determined to have elevated levels of somatic mutations in at least half or about 50% of the genes specified in Table 1.In some cases, a tumor sample obtained from a patient is determined to have an increased level of somatic mutations in at least two-thirds, or about 67%, of the genes specified in Table 1. In some cases, a tumor sample obtained from a patient is determined to have an increased level of somatic mutations in at least three-quarters, or about 75%, of the genes specified in Table 1.

[0192] The present invention further provides a method for selecting a therapy for a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma), the method comprising determining the level of somatic mutations in a tumor sample obtained from the patient, wherein an increased level of somatic mutations within at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes specified in Table 1 in the tumor sample indicates that the patient is likely to respond to therapy comprising a PD-L1 axis-binding antagonist. For example, in some instances, an increased level of somatic mutations in at least about one-third of the genes specified in Table 1 in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, an increased level of somatic mutations in at least about two-thirds of the genes specified in Table 1 in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, an increased level of somatic mutations in at least about three-quarters of the genes specified in Table 1 in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, 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 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 genes identified in Table 1 have been determined to have increased somatic mutations. For example, in some cases, tumor samples obtained from patients have been determined to have elevated levels of somatic mutations in at least half or about 50% of the genes specified in Table 1.In some cases, a tumor sample obtained from a patient is determined to have an increased level of somatic mutations in at least two-thirds, or about 67%, of the genes specified in Table 1. In some cases, a tumor sample obtained from a patient is determined to have an increased level of somatic mutations in at least three-quarters, or about 75%, of the genes specified in Table 1.

[0193] In any of the foregoing methods, somatic mutations in a gene identified in Table 1 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, 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, or about 95% or more) compared to a baseline level of somatic mutations in a gene identified in Table 1. For example, in some instances, the level of one or more somatic mutations is determined to be increased by about 1% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 5% or more. In other cases, the level of one or more somatic mutations is determined to be increased by about 10% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 15% or more. In still other cases, the level of one or more somatic mutations is determined to be increased by about 20% or more. In still further cases, the level of one or more somatic mutations is determined to be increased by about 25% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 30% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 35% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 40% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 50% or more.In other cases, 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 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 genes identified in Table 1 have been determined to have increased somatic mutations. For example, in some cases, a tumor sample obtained from a patient is determined to have increased levels of somatic mutations in at least one-half or about 50% of the genes specified in Table 1. In some cases, a tumor sample obtained from a patient is determined to have increased levels of somatic mutations in at least two-thirds or about 67% of the genes specified in Table 1. In some cases, a tumor sample obtained from a patient is determined to have increased levels of somatic mutations in at least three-quarters or about 75% of the genes specified in Table 1.

[0194] In any of the foregoing methods, the method may further comprise determining the expression level of PD-L1 in tumor cells in a tumor sample obtained from the patient. In some embodiments, a detectable expression level of PD-L1 in about 1% or more (e.g., about 1%, about 2%, about 3%, about 4%, about 5% or more, about 10% or more, about 15%, about 20% or more, about 25% or more, about 30% or more, about 35%, about 40% or more, about 45% or more, about 50% or more, about 55%, 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 cells in 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 1% or more of the tumor cells in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, a detectable expression level of PD-L1 in 5% or more of the tumor cells in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, a detectable expression level of PD-L1 in 10% or more of the tumor cells in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, a detectable expression level of PD-L1 in 20% or more of the tumor cells in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, a detectable expression level of PD-L1 in 30% or more of the tumor cells in a tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, detectable expression levels of PD-L1 in 50% or more of the tumor cells in a tumor sample indicate that the patient is likely to respond to treatment that includes a PD-L1 axis-binding antagonist.

[0195] In some embodiments of any of the aforementioned methods, about 1% to about 99% (e.g., about 1% to about 99%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, A detectable level of PD-L1 expression of about 1% to about 5%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, or about 50% to about 55%) indicates that the patient is likely to respond to treatment with a PD-L1 binding antagonist.

[0196] In some embodiments, a tumor sample obtained from a patient has been determined to have an undetectable expression level of PD-L1 in tumor cells in the tumor sample.

[0197] In any of the foregoing methods, the method may further comprise determining the expression level of PD-L1 in tumor-infiltrating immune cells in a tumor sample (tumor area) obtained from the patient. In some embodiments, the tumor sample obtained from the patient has a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise more than 1% of the tumor sample (tumor area) (e.g., more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50%). For example, in some cases, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 1% or more of the tumor area in a section of the tumor sample, as determined by immunohistochemistry, e.g., using an anti-PD-L1 antibody, indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. In other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 5% or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 20% ​​or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 30% or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In yet other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 50% or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist.

[0198] In some embodiments of any of the aforementioned methods, about 1% to about 50% (e.g., about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 50%, about 5% A detectable expression level of PD-L1 in tumor-infiltrating immune cells of about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, or about 10% to about 15% indicates that the patient is likely to respond to treatment with a PD-L1 binding antagonist.

[0199] In some embodiments, a tumor sample obtained from a patient has been determined to have undetectable expression levels of PD-L1 within tumor-infiltrating immune cells in the tumor sample (tumor area).

[0200] In any of the aforementioned methods, the patient may have a high level of microsatellite instability. For example, the patient may have an MSI-high (MSI-H) phenotype, as assessed by a PCR-based approach such as the MSI Analysis System (Promega, Madison, WI), which is composed of five pseudomonomorphic mononucleotide repeat sequences (BAT-25, BAT-26, NR-21, NR-24, and MONO-27) to detect MSI and two pentanucleotide loci (PentaC and PendaD) to confirm the identity of normal and tumor samples. The base size of each microsatellite locus can be determined, for example, by gel electrophoresis, and a tumor can be designated as MSI-H if the length of two or more mononucleotide loci varies compared to germline DNA. See, e.g., Le et al. NEJM 372:2509-2520, 2015. In other embodiments, the patient may have low levels of microsatellite instability (e.g., MSI-low (MSI-L)).

[0201] In any of the foregoing methods, the method may further comprise administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist based on the level of somatic mutations in the tumor sample. The PD-L1 axis-binding antagonist may be any PD-L1 axis-binding antagonist known in the art or described herein, for example, in Section D, below.

[0202] For example, in some cases, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist, a PD-1 binding antagonist, or 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 MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), or 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: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0203] 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 still 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 MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or 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. In some cases, the PD-L1 axis binding antagonist is administered as monotherapy.

[0204] 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 a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, or a combination thereof.

[0205] In any of the foregoing methods, the individual is diagnosed with, for example, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer), kidney cancer (e.g., renal urothelial carcinoma or RCC), bladder cancer (e.g., bladder urothelial (transitional cell) carcinoma (e.g., locally advanced or metastatic urothelial carcinoma, including 1L or 2L+ locally advanced or metastatic carcinoma), breast cancer (e.g., TNBC, HER2+ breast cancer, or HR+ breast cancer), endometrial cancer, skin cancer (e.g., cutaneous squamous cell carcinoma), colorectal cancer ( Cancers include, but are not limited to, cancers of the ovary, pancreas, stomach, esophagus, mesothelioma, melanoma (e.g., cutaneous melanoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), thyroid cancer, sarcoma (e.g., soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, osteosarcoma, chondrosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, leiomyosarcoma, or rhabdomyosarcoma), prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia (AR), acute myeloid leukemia (AML), acute myeloid leukemia (AR), acute myeloid leukemia (AM), acute myeloid leukemia (AL ... leukemia (AML), chronic myeloid leukemia (CML), chronic eosinophilic leukemia, or chronic lymphocytic leukemia (CLL)), lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma (NHL)), myeloma (e.g., multiple myeloma (MM)), mycosis fungoides, Merkel cell carcinoma, hematological malignancies, cancer of the blood tissue, B-cell cancer, bronchial cancer, gastric cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, testicular cancer, biliary tract cancer, Small intestine or appendix cancer, salivary gland cancer, adrenal gland cancer, adenocarcinoma, inflammatory myofibroblastic tumor, gastrointestinal stromal tumor (GIST), colon cancer, myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), polycythemia vera, chordoma, synovium, Ewing's tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder cancer carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinomacarcinoma), thyroid cancer, small cell carcinoma, essential thrombocythemia, primary myelofibrosis, hypereosinophilic syndrome, systemic mastocytosis, familial hypereosinophilia, neuroendocrine carcinoma, or carcinoid tumor.

[0206] In some cases of any of the aforementioned methods, the individual has progressed after treatment with a platinum-containing regimen for the cancer (e.g., a regimen including a platinum-based chemotherapy agent, e.g., a regimen including cisplatin-based chemotherapy). In other cases, the individual may be ineligible for treatment with a platinum-containing regimen (e.g., a regimen including a platinum-based chemotherapy agent, e.g., a regimen including cisplatin-based chemotherapy) and / or has not received prior treatment for the cancer. In some cases, the individual has not received prior treatment with an immune checkpoint inhibitor, e.g., a PD-L1 axis-binding antagonist.

[0207] For example, in any of the above cases, the lung cancer may be NSCLC, including but not limited to locally advanced or metastatic (e.g., stage IIIB, stage IV, or recurrent) NSCLC. In some cases, the lung cancer (e.g., NSCLC) is unresectable / inoperable lung cancer (e.g., NSCLC). In other cases, the lung cancer (e.g., NSCLC) may have progressed during or after prior treatment with a platinum-containing regimen.

[0208] The presence and / or level (amount) of a somatic mutation can 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 fragment, and / or gene copy number in an individual.

[0209] In any of the foregoing cases, the somatic mutations can be substitutions, deletions, and / or insertions.

[0210] 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.

[0211] In certain cases, the presence and / or level (amount) of somatic mutations in a first sample is increased or elevated compared to the presence / absence and / or level (amount) of such somatic mutations in a second sample. In certain cases, the presence / absence and / or level (amount) of somatic mutations in a first sample is decreased or reduced compared to the presence and / or level (amount) of such somatic mutations in a second sample. In certain cases, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. Additional disclosures for determining the presence / absence and / or level (amount) of somatic mutations are provided herein.

[0212] 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, and is obtained at one or more time points 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 point 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 the initial diagnosis of cancer, and test sample is obtained later when cancer becomes metastatic.

[0213] In certain cases, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more healthy individuals who are not patients. In certain cases, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the subject or individual. In certain cases, the reference sample, reference cell, reference tissue, control sample, control cell, or 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 cases, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from tumor tissue, or a pooled plasma or serum sample from one or more individuals with a disease or disorder (e.g., cancer) who are not patients.

[0214] In some cases of any of the methods described herein, an elevated or increased level refers to an overall increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of somatic mutations compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods, such as those described herein. In certain cases, an elevated level refers to an increase in the level / amount of somatic mutations in a sample, which increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the level / amount of somatic mutations in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, respectively. In some cases, elevated levels refer to an overall increase of about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or more than about 3.25-fold compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some cases, elevated or increased levels of somatic mutations refer to an overall increase in the level of one or more classes of somatic mutations (e.g., point mutations, insertions and deletions (e.g., indels), amplifications, gene duplications, copy number alterations (CNAs), and rearrangements) and / or an overall increase in the level of a particular somatic mutation in a sample compared to a reference level.

[0215] In some cases of any of the methods described herein, a reduced level refers to an overall reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of somatic mutations compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods, such as those described herein. In certain cases, a reduced level refers to a decrease in the level / amount of somatic mutations 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 level / amount of somatic mutations in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, respectively. In some cases, a reduced or decreased level of somatic mutations refers to an overall decrease in the level of one or more classes of somatic mutations (e.g., point mutations, insertions and deletions (e.g., indels), amplifications, gene duplications, copy number alterations (CNAs), and rearrangements) and / or an overall decrease in the level of a particular somatic mutation in a sample compared to a reference level.

[0216] B. Diagnostic Methods Based on the Level of Gene Rearrangements in Cancer Provided herein are methods, which can be used individually or in combination with any of the aforementioned methods presented in Section A above, for determining whether a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist based on the level of rearrangement of any one of the genes listed in Table 2. For example, rearrangement of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) of the genes listed in Table 2 can determine whether a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. In other cases, for example, an increase in the level of rearrangements of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) of the genes listed in Table 2, combined with an increase in somatic mutations within one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes listed in Table 1 can determine whether a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist.

[0217] Also provided herein are methods, which can be used individually or in combination with any of the aforementioned methods presented in Section A above, for predicting the responsiveness of a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) to a treatment comprising a PD-L1 axis-binding antagonist based on the level of rearrangement of any one of the genes listed in Table 2. For example, rearrangement of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) of the genes listed in Table 2 can predict whether a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. In other cases, for example, an increase in the level of rearrangements of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) of the genes listed in Table 2, in combination with an increase in somatic mutations within one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes listed in Table 1, can predict whether a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. Any of these methods may further comprise administering to the patient a PD-L1 axis-binding antagonist (such as 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.

[0218] The invention provides methods for treating a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma), 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 an increased level of rearrangements in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) genes specified in Table 2 compared to a reference level of rearrangements in at least one gene specified in Table 2. In other cases, 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 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 genes identified in Table 2 were determined to have increased levels of rearrangement. For example, in some cases, a tumor sample obtained from a patient has been determined to have an increased level of rearrangement in at least one-half or about 50% of the genes specified in Table 2. In some cases, a tumor sample obtained from a patient has been determined to have an increased level of rearrangement in at least two-thirds or about 67% of the genes specified in Table 2. In some cases, a tumor sample obtained from a patient has been determined to have an increased level of rearrangement in at least three-quarters or about 75% of the genes specified in Table 2.In some cases, at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes specified in Table 1 have been determined to have an increased level of somatic mutations compared to a baseline level of somatic mutations in at least one gene specified in Table 1, in combination with an elevated level of rearrangements within any gene listed in Table 2. TIFF2025028833000004.tif29170

[0219] The presence and / or level (amount) of somatic mutations can be qualitatively and / or quantitatively determined based on any suitable criteria known in the art, including, but not limited to, measurements of DNA, mRNA, cDNA, protein, protein fragment, and / or gene copy number levels in an individual. In some cases, a comprehensive genomic profile of the individual is determined. In some cases, a comprehensive genomic profile of a sample collected from the individual (e.g., a tissue sample, a formalin-fixed, paraffin-embedded (FFPE) tissue sample, a core or fine needle biopsy) is determined. In some cases, determining the genomic profile involves applying next-generation sequencing methods known in the art or described herein to identify genomic alterations (e.g., somatic mutations (e.g., base substitutions, insertions and deletions (indels), copy number alterations (CNAs), and rearrangements)) that are known to be distinct drivers of cancer (e.g., solid tumors). In some cases, this test simultaneously sequences the coding regions of 315 cancer-associated genes and introns from 28 genes that are frequently rearranged in cancer to a typical median coverage depth of over 500. In some cases, each comprehensive sequencing read represents a unique cross-section of DNA, enabling highly sensitive and specific detection of genomic alterations that occur at low frequency due to tumor heterogeneity, low tumor purity, and small tissue samples.

[0220] The invention provides methods for determining whether a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist, the method comprising determining the level of rearrangements in a tumor sample obtained from the patient, wherein an increased level of rearrangements within at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) of the genes specified in Table 2 in 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, an increased level of rearrangements in at least about one-third of the genes specified in Table 2 in the tumor sample indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. In other cases, an increased level of rearrangements in at least about two-thirds of the genes specified in Table 2 in the tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, an increased level of rearrangements in at least about three-quarters of the genes specified in Table 2 in the tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, 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 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 genes specified in Table 2 were determined to have increased rearrangements. For example, in some cases, tumor samples obtained from patients have been determined to have increased levels of rearrangements in at least half or about 50% of the genes specified in Table 2.In some cases, a tumor sample obtained from a patient is determined to have an increased level of rearrangements in at least two-thirds, or about 67%, of the genes specified in Table 2. In some cases, a tumor sample obtained from a patient is determined to have an increased level of rearrangements in at least three-quarters, or about 75%, of the genes specified in Table 2. In some cases, in combination with an elevated level of somatic mutations in any gene listed in Table 2, at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes specified in Table 1 are determined to have an increased level of somatic mutations compared to a baseline level of somatic mutations in at least one gene specified in Table 1.

[0221] The present invention further provides methods for predicting the responsiveness of a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) to a therapy comprising a PD-L1 axis-binding antagonist, the method comprising determining the level of rearrangements in a tumor sample obtained from the patient, wherein an increased level of rearrangements within at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) of the genes specified in Table 2 in the tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. For example, in some cases, an increased level of rearrangements in one-third of the genes specified in Table 2 in the tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, increased levels of rearrangements in two-thirds of the genes specified in Table 2 in a tumor sample indicate that the patient is likely to respond to a therapy that includes a PD-L1 axis-binding antagonist. In other cases, increased levels of rearrangements in three-quarters of the genes specified in Table 2 in a tumor sample indicate that the patient is likely to respond to a therapy that includes a PD-L1 axis-binding antagonist. In other cases, 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 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 genes identified in Table 2 were determined to have increased rearrangements. For example, in some cases, tumor samples obtained from patients have been determined to have increased levels of rearrangements in at least one-half, or about 50%, of the genes specified in Table 2. In some cases, tumor samples obtained from patients have been determined to have increased levels of rearrangements in at least two-thirds, or about 67%, of the genes specified in Table 2.In some cases, a tumor sample obtained from a patient has been determined to have an elevated level of rearrangements in at least three-quarters or about 75% of the genes specified in Table 2. In some cases, in combination with an elevated level of rearrangements in any gene listed in Table 2, at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes specified in Table 1 have been determined to have an elevated level of somatic mutations compared to a baseline level of somatic mutations in at least one gene specified in Table 1.

[0222] The present invention further provides a method for selecting a therapy for a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma), the method comprising determining the level of rearrangements in a tumor sample obtained from the patient, wherein an increased level of rearrangements within at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) genes specified in Table 2 in the tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. For example, in some cases, an increased level of rearrangements in one-third of the genes specified in Table 2 in the tumor sample indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other cases, increased levels of rearrangements in two-thirds of the genes specified in Table 2 in a tumor sample indicate that the patient is likely to respond to a therapy that includes a PD-L1 axis-binding antagonist. In other cases, increased levels of rearrangements in three-quarters of the genes specified in Table 2 in a tumor sample indicate that the patient is likely to respond to a therapy that includes a PD-L1 axis-binding antagonist. In other cases, 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 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 genes identified in Table 2 were determined to have increased rearrangements. For example, in some cases, tumor samples obtained from patients have been determined to have increased levels of rearrangements in at least one-half, or about 50%, of the genes specified in Table 2. In some cases, tumor samples obtained from patients have been determined to have increased levels of rearrangements in at least two-thirds, or about 67%, of the genes specified in Table 2.In some cases, a tumor sample obtained from a patient has been determined to have an elevated level of rearrangements in at least three-quarters or about 75% of the genes specified in Table 2. In some cases, in combination with an elevated level of rearrangements in any gene listed in Table 2, at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes specified in Table 1 have been determined to have an elevated level of somatic mutations compared to a baseline level of somatic mutations in at least one gene specified in Table 1.

[0223] In any of the foregoing methods, rearrangements within a gene specified in Table 2 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, 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, or about 95% or more) compared to a reference level of rearrangements within a gene specified in Table 2. For example, in some cases, the level of one or more rearrangements is determined to be increased by about 1% or more. In some cases, the level of one or more rearrangements is determined to be increased by about 5% or more. In other cases, the level of one or more rearrangements is determined to be increased by about 10% or more. In some cases, the level of one or more rearrangements is determined to be increased by about 15% or more. In still other cases, the level of one or more rearrangements is determined to be increased by about 20% or more. In still further cases, the level of one or more rearrangements is determined to be increased by about 25% or more. In some cases, the level of one or more rearrangements is determined to be increased by about 30% or more. In some cases, the level of one or more rearrangements is determined to be increased by about 35% or more. In some cases, the level of one or more rearrangements is determined to be increased by about 40% or more. In some cases, the level of one or more rearrangements is determined to be increased by about 50% or more. In other cases, 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 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 genes identified in Table 2 were determined to have increased rearrangements.For example, in some cases, a tumor sample obtained from a patient has been determined to have an increased level of rearrangement in at least one-half or about 50% of the genes specified in Table 2. In some cases, a tumor sample obtained from a patient has been determined to have an increased level of rearrangement in at least two-thirds or about 67% of the genes specified in Table 2. In some cases, a tumor sample obtained from a patient has been determined to have an increased level of rearrangement in at least three-quarters or about 75% of the genes specified in Table 2. In some cases, at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) genes specified in Table 1 have been determined to have an increased level of somatic mutations compared to a baseline level of somatic mutations in at least one gene specified in Table 1, in combination with an elevated level of rearrangements within any gene listed in Table 2.

[0224] In some embodiments of any of the aforementioned methods, about 1% to about 99% (e.g., about 1% to about 99%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, A detectable level of PD-L1 expression of about 1% to about 5%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, or about 50% to about 55%) indicates that the patient is likely to respond to treatment with a PD-L1 binding antagonist.

[0225] In some embodiments, a tumor sample obtained from a patient has been determined to have an undetectable expression level of PD-L1 in tumor cells in the tumor sample.

[0226] In any of the foregoing methods, the method may further comprise determining the expression level of PD-L1 in tumor-infiltrating immune cells in a tumor sample (tumor area) obtained from the patient. In some embodiments, the tumor sample obtained from the patient has a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise more than 1% of the tumor sample (tumor area) (e.g., more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50%). For example, in some cases, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 1% or more of the tumor area in a section of the tumor sample, as determined by immunohistochemistry, e.g., using an anti-PD-L1 antibody, indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. In other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 5% or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 20% ​​or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 30% or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In yet other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 50% or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist.

[0227] In some embodiments of any of the aforementioned methods, about 1% to about 50% (e.g., about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 50%, about 5% A detectable expression level of PD-L1 in tumor-infiltrating immune cells of about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, or about 10% to about 15% indicates that the patient is likely to respond to treatment with a PD-L1 binding antagonist.

[0228] In some embodiments, a tumor sample obtained from a patient has been determined to have undetectable expression levels of PD-L1 within tumor-infiltrating immune cells in the tumor sample (tumor area).

[0229] In any of the aforementioned methods, the patient may have a high level of microsatellite instability. For example, the patient may have an MSI-high (MSI-H) phenotype, as assessed by a PCR-based approach such as the MSI Analysis System (Promega, Madison, WI), which is composed of five pseudomonomorphic mononucleotide repeat sequences (BAT-25, BAT-26, NR-21, NR-24, and MONO-27) to detect MSI and two pentanucleotide loci (PentaC and PendaD) to confirm the identity of normal and tumor samples. The base size of each microsatellite locus can be determined, for example, by gel electrophoresis, and a tumor can be designated as MSI-H if the length of two or more mononucleotide loci varies compared to germline DNA. See, e.g., Le et al. NEJM 372:2509-2520, 2015. In other embodiments, the patient may have low levels of microsatellite instability (e.g., MSI-low (MSI-L)).

[0230] In any of the foregoing methods, the method may further comprise administering to the patient a therapeutically effective amount of a PD-L1 axis-binding antagonist based on the level of somatic mutations in the tumor sample. The PD-L1 axis-binding antagonist may be any PD-L1 axis-binding antagonist known in the art or described herein, for example, in Section D, below.

[0231] 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 MPDL3280A (atezolizumab), YW243.55.S70, 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: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0232] 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 still 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. In some cases, the PD-L1 axis binding antagonist is administered as monotherapy.

[0233] 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.

[0234] In any of the foregoing methods, the individual is diagnosed with, for example, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer), kidney cancer (e.g., renal urothelial carcinoma or RCC), bladder cancer (e.g., bladder urothelial (transitional cell) carcinoma (e.g., locally advanced or metastatic urothelial carcinoma, including 1L or 2L+ locally advanced or metastatic carcinoma), breast cancer (e.g., TNBC, HER2+ breast cancer, or HR+ breast cancer), endometrial cancer, skin cancer (e.g., cutaneous squamous cell carcinoma), colorectal cancer ( Cancers include, but are not limited to, cancers of the ovary, pancreas, stomach, esophagus, mesothelioma, melanoma (e.g., cutaneous melanoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), thyroid cancer, sarcoma (e.g., soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, osteosarcoma, chondrosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, leiomyosarcoma, or rhabdomyosarcoma), prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia (AR), acute myeloid leukemia (AML), acute myeloid leukemia (AR), acute myeloid leukemia (AM), acute myeloid leukemia (AL ... leukemia (AML), chronic myeloid leukemia (CML), chronic eosinophilic leukemia, or chronic lymphocytic leukemia (CLL)), lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma (NHL)), myeloma (e.g., multiple myeloma (MM)), mycosis fungoides, Merkel cell carcinoma, hematological malignancies, cancer of the blood tissue, B-cell cancer, bronchial cancer, gastric cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, testicular cancer, biliary tract cancer, Small intestine or appendix cancer, salivary gland cancer, adrenal gland cancer, adenocarcinoma, inflammatory myofibroblastic tumor, gastrointestinal stromal tumor (GIST), colon cancer, myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), polycythemia vera, chordoma, synovium, Ewing's tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder cancer carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinomacarcinoma), thyroid cancer, small cell carcinoma, essential thrombocythemia, primary myelofibrosis, hypereosinophilic syndrome, systemic mastocytosis, familial hypereosinophilia, neuroendocrine carcinoma, or carcinoid tumor.

[0235] In some cases of any of the aforementioned methods, the individual has progressed after treatment with a platinum-containing regimen for the cancer (e.g., a regimen including a platinum-based chemotherapy agent, e.g., a regimen including cisplatin-based chemotherapy). In other cases, the individual may be ineligible for treatment with a platinum-containing regimen (e.g., a regimen including a platinum-based chemotherapy agent, e.g., a regimen including cisplatin-based chemotherapy) and / or has not received prior treatment for the cancer. In some cases, the individual has not received prior treatment with an immune checkpoint inhibitor, e.g., a PD-L1 axis-binding antagonist.

[0236] For example, in any of the above cases, the lung cancer may be NSCLC, including but not limited to locally advanced or metastatic (e.g., stage IIIB, stage IV, or recurrent) NSCLC. In some cases, the lung cancer (e.g., NSCLC) may be unresectable / inoperable lung cancer (e.g., NSCLC). In other cases, the lung cancer (e.g., NSCLC) may have progressed during or after prior treatment with a platinum-containing regimen.

[0237] In any of the foregoing cases, the presence and / or level (amount) of somatic mutations can 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 fragment, and / or gene copy number in an individual.

[0238] In any of the foregoing cases, the somatic mutations may be substitutions, deletions, and / or insertions. For example, in some cases, the somatic mutations may be copy number alterations and / or rearrangements.

[0239] 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.

[0240] In certain cases, the presence and / or level (amount) of a somatic mutation in a first sample is increased or elevated compared to the presence / absence and / or level (amount) of such a somatic mutation in a second sample. In certain cases, the presence / absence and / or level (amount) of a somatic mutation in a first sample is decreased or reduced compared to the presence and / or level (amount) of such a somatic mutation in a second sample. In certain cases, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. Additional disclosures for determining the presence / absence and / or level (amount) of a somatic mutation are provided herein.

[0241] 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, and is obtained at one or more time points 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 point 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 the initial diagnosis of cancer, and test sample is obtained later when cancer becomes metastatic.

[0242] In certain cases, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more healthy individuals who are not patients. In certain cases, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the subject or individual. In certain cases, the reference sample, reference cell, reference tissue, control sample, control cell, or 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 cases, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from tumor tissue, or a pooled plasma or serum sample from one or more individuals with a disease or disorder (e.g., cancer) who are not patients.

[0243] In some cases of any of the methods described herein, an elevated or increased level refers to an overall increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of somatic mutations compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods, such as those described herein. In certain cases, an elevated level refers to an increase in the level / amount of somatic mutations in a sample, which increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the level / amount of somatic mutations in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, respectively. In some cases, elevated levels refer to an overall increase of about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or more than about 3.25-fold compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some cases, elevated or increased levels of somatic mutations refer to an overall increase in the level of one or more classes of somatic mutations (e.g., point mutations, insertions and deletions (e.g., indels), amplifications, gene duplications, copy number alterations (CNAs), and rearrangements) and / or an overall increase in the level of a particular somatic mutation in a sample compared to a reference level.

[0244] In some cases of any of the methods described herein, a reduced level refers to an overall reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of somatic mutations compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods, such as those described herein. In certain cases, a reduced level refers to a decrease in the level / amount of somatic mutations 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 level / amount of somatic mutations in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, respectively. In some cases, a reduced or decreased level of somatic mutations refers to an overall decrease in the level of one or more classes of somatic mutations (e.g., point mutations, insertions and deletions (e.g., indels), amplifications, gene duplications, copy number alterations (CNAs), and rearrangements) and / or an overall decrease in the level of a particular somatic mutation in a sample compared to a reference level.

[0245] C. Treatment method The present invention provides methods for treating patients afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma). In some cases, the patient has progressed after treatment for the cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma) with a platinum-containing regimen (e.g., a regimen including a platinum-based chemotherapy agent, e.g., a regimen including cisplatin-based chemotherapy). In other cases, the patient may be ineligible for treatment with a platinum-containing regimen (e.g., a regimen including a platinum-based chemotherapy agent, e.g., a regimen including cisplatin-based chemotherapy) and / or has not received prior treatment for the cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma). In some instances, the methods of the invention include administering to a patient an anti-cancer therapy comprising a PD-L1 axis-binding antagonist. Any PD-L1 axis-binding antagonist described herein (see, e.g., Section D, below) or known in the art can be used in the methods. In some instances, the methods involve determining a tTMB score from a tumor sample from the patient, where the tTMB score from the tumor sample is equal to or greater than a reference tTMB score, and administering to the patient an anti-cancer therapy comprising a PD-L1 axis-binding antagonist based on an elevated tTMB score compared to the reference tTMB score. Such methods can include, for example, determining the presence and / or expression of somatic mutations in the sample using any of the methods described herein (e.g., those described in Sections A and B or in the Examples, below) or methods known in the art.

[0246] The invention provides methods of treating a patient suffering from cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma), the method comprising determining a tTMB score from a tumor sample from the patient, wherein the tTMB score from the tumor sample is equal to or greater than a reference tTMB score, and administering to the patient an effective amount of a PD-L1 axis-binding antagonist.

[0247] The method also provides a method of treating a patient having cancer, the method comprising administering to the patient an effective amount of a PD-L1 axis binding antagonist, wherein prior to the administration, a tTMB score equal to or greater than a reference tTMB score has been determined from a tumor sample from the patient.

[0248] In some cases, the tumor sample obtained from the patient is also determined to have an increased level of somatic mutations in at least one gene specified in Table 1 and / or Table 2 compared to a baseline level of somatic mutations in at least one gene specified in Table 1 and / or Table 2.

[0249] In any of the foregoing methods, somatic mutations in genes specified in Table 1 and / or Table 2 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, 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, or about 95% or more) relative to a baseline level of somatic mutations in genes specified in Table 1 and / or Table 2. For example, in some cases, the level of one or more somatic mutations (e.g., the level of one or more somatic mutations from different classes (e.g., insertions, deletions, and / or rearrangements), the level of a particular class of somatic mutations, and / or the level of a particular somatic mutation) is determined to be increased by about 1% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 5% or more. In other cases, the level of one or more somatic mutations is determined to be increased by about 10% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 15% or more. In still other cases, the level of one or more somatic mutations is determined to be increased by about 20% or more. In still further cases, the level of one or more somatic mutations is determined to be increased by about 25% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 30% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 35% or more. In some cases, the level of one or more somatic mutations is determined to be increased by about 40% or more. In some cases, the level of one or more somatic mutations was determined to be increased by about 50% or more.

[0250] In any of the foregoing methods, 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 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 genes identified in Table 1 and / or Table 2 were determined to have increased somatic mutations. For example, in some cases, a tumor sample obtained from a patient is determined to have increased levels of somatic mutations in at least one-half or about 50% of the genes shown in Table 1 and / or Table 2. In some cases, a tumor sample obtained from a patient is determined to have increased levels of somatic mutations in at least two-thirds or about 67% of the genes shown in Table 1 and / or Table 2. In some cases, a tumor sample obtained from a patient is determined to have increased levels of somatic mutations in at least three-quarters or about 75% of the genes shown in Table 1 and / or Table 2.

[0251] In some cases, the test involves about 300 genes (e.g., at least about 300 to about 400 genes, e.g., about 300 to about 400 genes, e.g., about 300 to about 400 genes) covering at least about 0.05 Mb to about 10 Mb (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Mb). Simultaneously sequence the coding regions of 100 genes (a diverse set of 00, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 genes) to a typical median exon coverage depth of at least about 500x (e.g., 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, or 1,000x). In other cases, the test simultaneously sequences the coding regions of about 400 genes, about 425 genes, about 450 genes, about 475 genes, about 500 genes, about 525 genes, about 550 genes, about 575 genes, about 600 genes, about 625 genes, about 650 genes, about 675 genes, about 700 genes, about 725 genes, about 750 genes, about 775 genes, about 800 genes, about 825 genes, about 850 genes, about 875 genes, about 900 genes, about 925 genes, about 950 genes, about 975 genes, about 1000 genes, or more than 1000 genes. In some cases, the set of genes includes one or more genes (e.g., cancer-associated genes) specified in Table 1. In some cases, the set of genes is a set of genes from a FOUNDATIONONE® panel (e.g., Frampton et al. Nat. Biotechnol. 31:1023-31, 2013, which is incorporated by reference in its entirety). In some cases, the set of genes is a set of genes from a FOUNDATIONONE® CDx panel.In some embodiments, the test involves analyzing more than about 10 Mb of the individual's genome, e.g., more than about 10 Mb, more than about 15 Mb, more than about 20 Mb, more than about 25 Mb, more than about 30 Mb, more than about 35 Mb, more than about 40 Mb, more than about 45 Mb, more than about 50 Mb, more than about 55 Mb, more than about 60 Mb, more than about 65 Mb, more than about 70 Mb, more than about 75 Mb The test sequences more than about 80 Mb, more than about 85 Mb, more than about 90 Mb, more than about 95 Mb, more than about 100 Mb, more than about 200 Mb, more than about 300 Mb, more than about 400 Mb, more than about 500 Mb, more than about 600 Mb, more than about 700 Mb, more than about 800 Mb, more than about 900 Mb, more than about 1 Gb, more than about 2 Gb, more than about 3 Gb, or more than about 3.3 Gb. In some cases, the test simultaneously sequences the coding regions of 315 cancer-related genes and introns from 28 genes that are frequently rearranged or altered in cancer to a typical median coverage depth of more than 500x. In some cases, each comprehensive sequencing read represents a unique DNA cross-section, enabling highly sensitive and specific detection of genomic alterations that occur at low frequencies due to tumor heterogeneity, low tumor purity, and small tissue samples. In other cases, the presence and / or level of somatic mutations is determined by whole exome sequencing. In some cases, the presence and / or level of somatic mutations is determined by whole genome sequencing.

[0252] In any of the foregoing methods, the reference tTMB score is a tTMB score within a reference population of individuals having cancer, the population consisting of a first subset of individuals being treated with a PD-L1 axis binding antagonist therapy and a second subset of individuals being treated with a non-PD-L1 axis binding antagonist therapy, where the non-PD-L1 axis binding antagonist therapy does not include a PD-L1 axis binding antagonist. In some instances, the reference tTMB score significantly separates each of the first and second subsets of individuals based on a significant difference in responsiveness to treatment with a PD-L1 axis binding antagonist therapy compared to responsiveness to treatment with a non-PD-L1 axis binding antagonist therapy. In some instances, responsiveness to treatment is an increase in progression-free survival (PFS), overall survival, and / or overall response rate (ORR).

[0253] In any of the foregoing methods, in some cases, the reference tTMB score is a pre-assigned tTMB score. In some cases, the reference tTMB score is between about 5 and about 100 mutations per Mb (mutations / Mb), e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 mutations / Mb. For example, in some cases, the reference tTMB score is about 8 to about 30 mutations / Mb (e.g., about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 mutations / Mb). In some cases, the reference tTMB score is about 10 to about 20 mutations / Mb (e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations / Mb). In particular cases, the reference tTMB score may be 10 mutations / Mb, 16 mutations / Mb, or 20 mutations / Mb.

[0254] In some cases of any of the aforementioned methods, the tumor sample from the patient has a tTMB score of about 5 or more mutations / Mb. For example, in some cases, the tTMB score from the tumor sample is between about 5 and about 100 mutations / Mb (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 mutations / Mb). In some cases, a tumor sample from a patient has a tTMB score of about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 or more mutations / Mb. For example, in some cases, a tumor sample from a patient has a tTMB score of about 10 or more mutations / Mb. In some embodiments, the baseline tTMB score is 10 mutations / Mb. In some cases, the tTMB score from a tumor sample is about 10-100 mutations / Mb. In some cases, the tTMB score from a tumor sample is about 10-20 mutations / Mb. In some cases, a tumor sample from a patient has a tTMB score of about 16 or more mutations / Mb.In some cases, the tumor sample from the patient has a tTMB score of about 16 or more mutations / Mb, and the reference tTMB score is 16 mutations / Mb. In other cases, the tumor sample from the patient has a tTMB score of about 20 or more mutations / Mb. In some cases, the tumor sample from the patient has a tTMB score of about 20 or more mutations / Mb, and the reference tTMB score is about 20 mutations / Mb.

[0255] In other cases, the invention provides methods of treating a patient afflicted with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UC), kidney cancer (e.g., RCC), breast cancer (e.g., TNBC), or melanoma), the method comprising determining a tTMB score from a tumor sample from the patient, wherein the tTMB score from the tumor sample is less than a reference tTMB score, and the method further comprises administering to the individual a therapeutic agent other than or in addition to a PD-L1 axis-binding antagonist. In some cases of any of the foregoing methods, the tTMB score or the reference tTMB score is expressed as the number of somatic mutations counted per defined number of sequenced bases. For example, in some cases, the defined number of sequenced bases is about 100 kb to about 10 Mb. In some cases, the defined number of sequenced bases is about 1.1 Mb (e.g., about 1.125 Mb), e.g., as assessed by a FOUNDATIONONE® panel). In some cases, the tTMB score or reference tTMB score is an equivalent TMB value. In some cases, the equivalent TMB value is determined by whole exome sequencing (WES). In any of the above methods, the determined tTMB score can reflect the level of somatic mutations and / or rearrangements detected in the genes listed in Table 1 and / or Table 2. In some cases, the tTMB score is determined (or is determined) to be at least about 5 or more mutations / Mb (e.g., about 5 or more mutations / Mb, about 6 or more mutations / Mb, about 7 or more mutations / Mb, about 8 or more mutations / Mb, about 9 or more mutations / Mb, about 10 or more mutations / Mb, about 11 or more mutations / Mb, about 12 or more mutations / Mb, about 13 or more mutations / Mb, about 14 or more mutations / Mb, about 15 or more mutations / Mb, about 16 or more mutations / Mb, about 17 or more mutations / Mb, about 18 or more mutations / Mb, about 19 or more mutations / Mb, about 20 or more mutations / Mb, about 25 or more mutations / Mb, about 30 or more mutations / Mb, about 35 or more mutations / Mb, about 40 or more mutations / Mb, and about 50 or more mutations / Mb). This predicts responsiveness to treatment (e.g., treatment including a PD-L1 axis-binding antagonist).In some cases, the mutational load predictive of responsiveness to a treatment (e.g., a treatment comprising a PD-L1 axis-binding antagonist) may be about 7 mutations / Mb to about 20 mutations / Mb. In some cases, the mutational load predictive of responsiveness to a treatment may be about 10 mutations / Mb to about 15 mutations / Mb. In some cases, the tTMB score predictive of responsiveness to a treatment may be about 11 mutations / Mb to about 13 mutations / Mb. In some cases, the tTMB score predictive of responsiveness to a treatment may be about 12.5 mutations / Mb. In other cases, the tTMB score predictive of responsiveness to treatment may be about 10 or more mutations / Mb, e.g., about 10 or more mutations / Mb, about 11 or more mutations / Mb, about 12 or more mutations / Mb, about 13 or more mutations / Mb, about 14 or more mutations / Mb, about 15 or more mutations / Mb, about 16 or more mutations / Mb, about 17 or more mutations / Mb, about 18 or more mutations / Mb, about 19 or more mutations / Mb, or about 20 or more mutations / Mb.

[0256] In some embodiments of any of the aforementioned methods, about 1% to about 99% (e.g., about 1% to about 99%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, A detectable level of PD-L1 expression of about 1% to about 5%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, or about 50% to about 55%) indicates that the patient is likely to respond to treatment with a PD-L1 binding antagonist.

[0257] In some embodiments, a tumor sample obtained from a patient has been determined to have an undetectable expression level of PD-L1 in tumor cells in the tumor sample.

[0258] In any of the foregoing methods, the method may further comprise determining the expression level of PD-L1 in tumor-infiltrating immune cells in a tumor sample (tumor area) obtained from the patient. In some embodiments, the tumor sample obtained from the patient has a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise more than 1% of the tumor sample (tumor area) (e.g., more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50%). For example, in some cases, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 1% or more of the tumor area in a section of the tumor sample, as determined by immunohistochemistry, e.g., using an anti-PD-L1 antibody, indicates that the patient is likely to respond to a treatment comprising a PD-L1 axis-binding antagonist. In other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 5% or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 20% ​​or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 30% or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist. In yet other instances, a detectable expression level of PD-L1 in tumor-infiltrating immune cells covering 50% or more of the tumor area in a tumor sample section indicates that the patient is likely to respond to a therapy comprising a PD-L1 axis-binding antagonist.

[0259] In some embodiments of any of the aforementioned methods, about 1% to about 50% (e.g., about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 50%, about 5% A detectable expression level of PD-L1 in tumor-infiltrating immune cells of about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, or about 10% to about 15% indicates that the patient is likely to respond to treatment with a PD-L1 binding antagonist.

[0260] In some embodiments, a tumor sample obtained from a patient has been determined to have undetectable expression levels of PD-L1 within tumor-infiltrating immune cells in the tumor sample (tumor area).

[0261] In any of the aforementioned methods, the patient may have a high level of microsatellite instability. For example, the patient may have an MSI-high (MSI-H) phenotype, as assessed by a PCR-based approach such as the MSI Analysis System (Promega, Madison, WI), which is composed of five pseudomonomorphic mononucleotide repeat sequences (BAT-25, BAT-26, NR-21, NR-24, and MONO-27) to detect MSI and two pentanucleotide loci (PentaC and PendaD) to confirm the identity of normal and tumor samples. The base size of each microsatellite locus can be determined, for example, by gel electrophoresis, and a tumor can be designated as MSI-H if the length of two or more mononucleotide loci varies compared to germline DNA. See, e.g., Le et al. NEJM 372:2509-2520, 2015. In other embodiments, the patient may have low levels of microsatellite instability (e.g., MSI-low (MSI-L)).

[0262] In any of the foregoing methods, the PD-L1 axis binding antagonist may be any PD-L1 axis binding antagonist known in the art or described herein, for example, in Section D, below.

[0263] For example, in some cases, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist, a PD-1 binding antagonist, or 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 MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), or 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: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0264] 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 still 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 MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or 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.

[0265] In any of the foregoing methods, the individual is diagnosed with, for example, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer), kidney cancer (e.g., renal urothelial carcinoma or RCC), bladder cancer (e.g., bladder urothelial (transitional cell) carcinoma (e.g., locally advanced or metastatic urothelial carcinoma, including 1L or 2L+ locally advanced or metastatic carcinoma), breast cancer (e.g., TNBC, HER2+ breast cancer, or HR+ breast cancer), endometrial cancer, skin cancer (e.g., cutaneous squamous cell carcinoma), colorectal cancer ( Cancers include, but are not limited to, cancers of the ovary, pancreas, stomach, esophagus, mesothelioma, melanoma (e.g., cutaneous melanoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), thyroid cancer, sarcoma (e.g., soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, osteosarcoma, chondrosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, leiomyosarcoma, or rhabdomyosarcoma), prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia (AR), acute myeloid leukemia (AML), acute myeloid leukemia (AR), acute myeloid leukemia (AM), acute myeloid leukemia (AL ... leukemia (AML), chronic myeloid leukemia (CML), chronic eosinophilic leukemia, or chronic lymphocytic leukemia (CLL)), lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma (NHL)), myeloma (e.g., multiple myeloma (MM)), mycosis fungoides, Merkel cell carcinoma, hematological malignancies, cancer of the blood tissue, B-cell cancer, bronchial cancer, gastric cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, testicular cancer, biliary tract cancer, Small intestine or appendix cancer, salivary gland cancer, adrenal gland cancer, adenocarcinoma, inflammatory myofibroblastic tumor, gastrointestinal stromal tumor (GIST), colon cancer, myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), polycythemia vera, chordoma, synovium, Ewing's tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder cancer carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinomacarcinoma), thyroid cancer, small cell carcinoma, essential thrombocythemia, primary myelofibrosis, hypereosinophilic syndrome, systemic mastocytosis, familial hypereosinophilia, neuroendocrine carcinoma, or carcinoid tumor.

[0266] In some cases of any of the aforementioned methods, the individual has progressed after treatment with a platinum-containing regimen for the cancer (e.g., a regimen including a platinum-based chemotherapy agent, e.g., a regimen including cisplatin-based chemotherapy). In other cases, the individual may be ineligible for treatment with a platinum-containing regimen (e.g., a regimen including a platinum-based chemotherapy agent, e.g., a regimen including cisplatin-based chemotherapy) and / or has not received prior treatment for the cancer. In some cases, the individual has not received prior treatment with an immune checkpoint inhibitor, e.g., a PD-L1 axis-binding antagonist.

[0267] For example, in any of the above cases, the lung cancer may be NSCLC, including but not limited to locally advanced or metastatic (e.g., stage IIIB, stage IV, or recurrent) NSCLC. In some cases, the lung cancer (e.g., NSCLC) may be unresectable / inoperable lung cancer (e.g., NSCLC). In other cases, the lung cancer (e.g., NSCLC) may have progressed during or after prior treatment with a platinum-containing regimen.

[0268] In a further aspect, the invention provides use of a PD-L1 axis-binding antagonist in the manufacture or preparation of a medicament. In one example, the medicament is for the treatment of cancer. In a further example, the medicament is for use in a method for treating cancer, comprising administering an effective amount of the medicament to a patient suffering from cancer (e.g., lung cancer, e.g., NSCLC). In one such example, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below.

[0269] The compositions (e.g., PD-L1 axis-binding antagonists) utilized in the methods described herein can be administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, transdermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, 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 local perfusion directly bathing target cells, by catheter, by lavage, in a cream, or in a lipid composition. The compositions utilized in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various 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 administration is short-term 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.

[0270] The PD-L1 axis binding antagonists (e.g., antibodies, binding polypeptides, and / or small molecules) described herein (and any additional therapeutic agents) may be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context 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 drug delivery, the method of administration, the administration schedule, and other factors known to medical professionals. The PD-L1 axis binding antagonists are optionally, but not necessarily, formulated with and / or co-administered 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 by the routes of administration described herein, or about 1-99% of the dosages described herein, or at any dosage and by any route empirically / clinically determined to be appropriate.

[0271] The appropriate dosage of a PD-L1 axis binding antagonist 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., lung cancer, e.g., NSCLC) depends 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, previous therapy, 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. A typical daily dosage can 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 longer, depending on the condition, treatment is generally 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., so that the patient receives, for example, from about 2 to about 20, or for example, about 6 doses of the PD-L1 axis-binding antagonist). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0272] For example, as a general suggestion, 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 the patient's body weight, whether administered 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 instances, the antibody is administered at 15 mg / kg. However, other dosage regimens may be useful. In one instance, an 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 instances, the anti-PD-L1 antibody atezolizumab (MPDL3280A) is administered intravenously at 1200 mg every 3 weeks (q3w). The dose may be administered as a single dose, such as by injection, or as multiple doses (for example, two or three doses). The dose of the antibody administered in combination therapy may be reduced compared to that in monotherapy. The progress of this therapy is easily monitored by conventional techniques.

[0273] 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 a combination thereof. In some cases, the PD-L1 axis-binding antagonist may be administered in conjunction with chemotherapy or a 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 a targeted therapy agent. In some cases, the PD-L1 axis-binding antagonist may be administered in conjunction with an immunotherapy or an 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. In some cases, the PD-L1 axis-binding antagonist is administered as monotherapy.

[0274] Such combination therapy as described above encompasses combined administration (wherein the two or more therapeutic agents are in the same formulation or separate formulations) as well as separate administration, where administration of the PD-L1 axis binding antagonist can occur before, concurrently with, and / or after administration of the additional therapeutic agent(s). In one case, administration of the PD-L1 axis binding antagonist and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0275] Without wishing 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 ...

Claims

1. 1. A method for identifying an individual having cancer who may benefit from a treatment comprising a PD-L1 axis binding antagonist, comprising determining a tissue tumor mutational burden (tTMB) score from a tumor sample from the individual, wherein a tTMB score from the tumor sample that is equal to or greater than a reference tTMB score identifies the individual as an individual who may benefit from a treatment comprising a PD-L1 axis binding antagonist.

2. 1. A method for selecting a therapy for an individual having cancer, comprising determining a tTMB score from a tumor sample from said individual, wherein a tTMB score from said tumor sample that is equal to or greater than a reference tTMB score identifies said individual as one who may benefit from a therapy comprising a PD-L1 axis binding antagonist.

3. 3. The method of claim 1 or 2, wherein the tTMB score determined from the tumor sample is equal to or greater than the reference tTMB score, and the method further comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist.

4. 3. The method of claim 1 or 2, wherein the tTMB score determined from the tumor sample is less than the reference tTMB score.

5. 1. A method of treating an individual having cancer, comprising: (a) determining a tTMB score from a tumor sample from the individual, wherein the tTMB score from the tumor sample is equal to or greater than a reference tTMB score; (b) administering to the individual an effective amount of a PD-L1 axis binding antagonist.

6. 1. A method of treating an individual having cancer, comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist, wherein prior to said administration, a tTMB score that is equal to or greater than a reference tTMB score has been determined from a tumor sample from the individual.

7. 7. The method of any one of claims 1 to 6, wherein the reference tTMB score is a tTMB score within a reference population of individuals having the cancer, the population of individuals consisting of a first subset of individuals being treated with a PD-L1 axis binding antagonist therapy and a second subset of individuals being treated with a non-PD-L1 axis binding antagonist therapy, and the non-PD-L1 axis binding antagonist therapy does not include a PD-L1 axis binding antagonist.

8. 8. The method of claim 7, wherein the baseline tTMB score significantly separates each of the first and second subsets of individuals based on a significant difference in responsiveness to treatment with the PD-L1 axis binding antagonist therapy compared to responsiveness to treatment with the non-PD-L1 axis binding antagonist therapy.

9. 9. The method of claim 8, wherein responsiveness to treatment is an increase in progression-free survival (PFS).

10. 9. The method of claim 8, wherein responsiveness to treatment is an increase in overall survival (OS).

11. 9. The method of claim 8, wherein responsiveness to treatment is an increase in overall response rate (ORR).

12. 12. The method of any one of claims 1-3 and 5-11, wherein the tumor sample has been determined to have an increased level of somatic mutations in at least one gene identified in Table 1 compared to a reference level of somatic mutations in at least one gene identified in Table 1.

13. 13. The method of claim 12, wherein the tumor sample has been determined to have an increased level of somatic mutations in at least one-third of the genes set forth in Table 1 compared to a reference level of somatic mutations in at least one-third of the genes set forth in Table 1.

14. 14. The method of claim 13, wherein the tumor sample has been determined to have an increased level of somatic mutations in at least one half of the genes set forth in Table 1 compared to a reference level of somatic mutations in at least one half of the genes set forth in Table 1.

15. 15. The method of claim 14, wherein the tumor sample has been determined to have an increased level of somatic mutations in at least two-thirds of the genes specified in Table 1 compared to a reference level of somatic mutations in at least two-thirds of the genes specified in Table 1.

16. 16. The method of claim 15, wherein the tumor sample has been determined to have an increased level of somatic mutations in at least three-quarters of the genes set forth in Table 1 compared to a reference level of somatic mutations in at least three-quarters of the genes set forth in Table 1.

17. 17. The method of claim 16, wherein the tumor sample has been determined to have an increased level of somatic mutations in the genes specified in Table 1 compared to a reference level of somatic mutations in the genes specified in Table 1.

18. The method according to any one of claims 12 to 17, wherein the somatic mutation is a substitution, a deletion, and / or an insertion.

19. 18. The method of any one of claims 12 to 17, wherein said somatic mutation in said at least one gene specified in Table 1 is a protein-modifying somatic mutation.

20. 20. The method of claim 18 or 19, wherein the substitutions, deletions and / or insertions are in a coding region.

21. The method according to any one of claims 18 to 20, wherein the deletion and / or insertion is an indel.

22. The method of any one of claims 1 to 21, wherein the reference tTMB score is a pre-assigned tTMB score.

23. 23. The method of any one of claims 1-22, wherein the baseline tTMB score is from about 5 to about 50 mutations per megabase (mutations / Mb).

24. 24. The method of claim 23, wherein the baseline tTMB score is from about 8 to about 30 mutations / Mb.

25. 25. The method of claim 24, wherein the baseline tTMB score is about 10 to about 20 mutations / Mb.

26. 26. The method of claim 25, wherein the baseline tTMB score is about 10 mutations / Mb.

27. 26. The method of claim 25, wherein the baseline tTMB score is about 16 mutations / Mb.

28. 26. The method of claim 25, wherein the baseline tTMB score is about 20 mutations / Mb.

29. The method of any one of claims 1 to 28, wherein the tTMB score from the tumor sample is about 5 mutations / Mb or more.

30. 30. The method of claim 29, wherein the tTMB score from the tumor sample is from about 5 to about 100 mutations / Mb.

31. 30. The method of claim 29, wherein the tTMB score from the tumor sample is about 10 mutations / Mb or more.

32. 32. The method of claim 31, wherein the tTMB score from the tumor sample is from about 10 to about 100 mutations / Mb.

33. The method of any one of claims 29 to 32, wherein the tTMB score from the tumor sample is about 16 mutations / Mb or more.

34. 34. The method of claim 33, wherein the baseline tTMB score is about 16 mutations / Mb.

35. The method of any one of claims 29 to 32, wherein the tTMB score from the tumor sample is about 20 mutations / Mb or more.

36. 36. The method of claim 35, wherein the baseline tTMB score is about 20 mutations / Mb.

37. 37. The method of any one of claims 1 to 36, wherein the tTMB score or the reference tTMB score is expressed as the number of somatic mutations counted per defined number of sequenced bases.

38. 38. The method of claim 37, wherein the defined number of sequenced bases is from about 100 kb to about 10 Mb.

39. 39. The method of claim 38, wherein the defined number of sequenced bases is about 1.1 Mb.

40. 40. The method of any one of claims 1 to 39, wherein the tTMB score or the reference tTMB score is an equivalent TMB value.

41. 41. The method of claim 40, wherein the equivalent TMB value is determined by whole exome sequencing (WES).

42. 42. The method of any one of claims 1-41, wherein the tumor sample has been determined to have a detectable expression level of PD-L1 in 1% or more of the tumor cells in the tumor sample.

43. 43. The method of claim 42, wherein the tumor sample has been determined to have a detectable expression level of PD-L1 in greater than or equal to 5% of the tumor cells in the tumor sample.

44. 44. The method of claim 43, wherein the tumor sample has been determined to have a detectable expression level of PD-L1 in greater than or equal to 10% of the tumor cells in the tumor sample.

45. 45. The method of claim 44, wherein the tumor sample has been determined to have a detectable expression level of PD-L1 in greater than or equal to 20% of the tumor cells in the tumor sample.

46. 46. ​​The method of claim 45, wherein the tumor sample has been determined to have a detectable expression level of PD-L1 in greater than or equal to 50% of the tumor cells in the tumor sample.

47. 42. The method of any one of claims 1 to 41, wherein the tumor sample has been determined to have an undetectable expression level of PD-L1 in the tumor cells in the tumor sample.

48. 48. The method of any one of claims 1 to 47, wherein the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells in the tumor sample.

49. 49. The method of claim 48, wherein the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise 1% or more of the tumor sample.

50. 50. The method of claim 49, wherein the tumor sample 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.

51. 51. The method of claim 50, wherein the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise 10% or more of the tumor sample.

52. 48. The method of any one of claims 1 to 47, wherein the tumor sample has been determined to have an undetectable expression level of PD-L1 in the tumor-infiltrating immune cells.

53. 53. The method of any one of claims 1 to 52, wherein the PD-L1 axis binding antagonist is a PD-L1 binding antagonist, a PD-1 binding antagonist, or a PD-L2 binding antagonist.

54. 54. The method of claim 53, wherein the PD-L1 axis binding antagonist is a PD-L1 binding antagonist.

55. 55. The method of claim 54, wherein the PD-L1 binding antagonist inhibits binding of PD-L1 to one or more of its ligand binding partners.

56. 56. The method of claim 55, wherein the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1.

57. 56. The method of claim 55, wherein the PD-L1 binding antagonist inhibits binding of PD-L1 to B7-1.

58. The method of any one of claims 55 to 57, wherein the PD-L1 binding antagonist inhibits binding of PD-L1 to both PD-1 and B7-1.

59. The method of any one of claims 54 to 58, wherein the PD-L1 binding antagonist is an antibody.

60. 60. The method of claim 59, wherein the antibody is atezolizumab (MPDL3280A), YW243.55S70, MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab).

61. 60. The method of claim 59, 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.

62. 60. The method of claim 59, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

4.

63. 54. The method of claim 53, wherein the PD-L1 axis binding antagonist is a PD-1 binding antagonist.

64. 64. The method of claim 63, wherein the PD-1 binding antagonist inhibits binding of PD-1 to one or more of its ligand binding partners.

65. 64. The method of claim 63, wherein the PD-1 binding antagonist inhibits binding of PD-1 to PD-L1.

66. 64. The method of claim 63, wherein the PD-1 binding antagonist inhibits binding of PD-1 to PD-L2.

67. The method of any one of claims 64 to 66, wherein the PD-1 binding antagonist inhibits binding of PD-1 to both PD-L1 and PD-L2.

68. 68. The method of any one of claims 63-67, wherein the PD-1 binding antagonist is an antibody.

69. 69. The method of claim 68, wherein the antibody is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108.

70. 68. The method of any one of claims 63-67, wherein the PD-1 binding antagonist is an Fc fusion protein.

71. 71. The method of claim 70, wherein the Fc fusion protein is AMP-224.

72. 72. The method of any one of claims 3 and 5-71, further comprising administering to the individual an effective amount of a second therapeutic agent.

73. 73. The method of claim 72, wherein the second therapeutic agent is a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, or a combination thereof.

74. 5. The method of claim 4, further comprising administering to the individual a therapeutic agent other than or in addition to a PD-L1 axis binding antagonist.

75. The method of any one of claims 7 to 74, wherein the non-PD-L1 axis binding antagonist therapy comprises a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, or a combination thereof.

76. 76. The method of any one of claims 1 to 75, wherein the cancer is lung cancer, kidney cancer, bladder cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, endometrial cancer, skin cancer, or hematological malignancies.

77. 77. The method of claim 76, wherein the cancer is lung cancer, bladder cancer, melanoma, renal cancer, colorectal cancer, or head and neck cancer.

78. 78. The method of claim 77, wherein the cancer is lung cancer.

79. The method of any one of claims 76 to 78, wherein the lung cancer is non-small cell lung cancer (NSCLC).

80. 80. The method of claim 79, wherein the NSCLC is metastatic NSCLC.

81. 81. The method of claim 79 or 80, wherein the NSCLC is locally advanced NSCLC.

82. The method of any one of claims 79 to 81, wherein the NSCLC is stage IIIB NSCLC.

83. The method of any one of claims 79 to 81, wherein the NSCLC is stage IV NSCLC.

84. The method of any one of claims 79 to 81, wherein the NSCLC is recurrent NSCLC.

85. The method of any one of claims 79-84, wherein said individual has received at least one prior treatment with a platinum-containing regimen for said NSCLC.

86. 86. The method of claim 85, wherein the individual has received at least two prior treatments with platinum-containing regimens for the NSCLC.

87. The method of any one of claims 1-84, wherein said individual has not been previously treated with a platinum-containing regimen for said NSCLC.

88. 78. The method of claim 77, wherein the bladder cancer is locally advanced or metastatic urothelial carcinoma.

89. 89. The method of claim 88, wherein the bladder cancer is metastatic urothelial carcinoma.

90. 90. The method of claim 88 or 89, wherein the individual (i) has not had a prior treatment for the bladder cancer, or (ii) the individual has had at least one prior treatment for the bladder cancer.

91. 91. The method of any one of claims 1 to 90, 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.

92. The method of any one of claims 42 to 91, wherein the expression level of PD-L1 is a protein expression level.

93. 93. The method of claim 92, 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.

94. 94. The method of claim 93, wherein the protein expression level of PD-L1 is determined using IHC.

95. The method of any one of claims 92 to 94, wherein the protein expression level of PD-L1 is detected using an anti-PD-L1 antibody.

96. The method according to any one of claims 42 to 91, wherein the expression level of PD-L1 is an mRNA expression level.

97. 97. The method of claim 96, wherein the PD-L1 mRNA expression level 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).

98. 13. A PD-L1 axis binding antagonist for use in treating an individual suffering from cancer, wherein a tumor sample from said individual has been determined to have a tTMB score that is equal to or greater than a reference tTMB score, and optionally, wherein said tumor sample has been determined to have an increased level of somatic mutations in at least one gene set forth in Table 1 compared to a reference level of somatic mutations in at least one gene set forth in Table 1, a detectable level of expression of PD-L1 in tumor infiltrating immune cells that constitute about 1% or more of said tumor sample, and / or a detectable level of expression of PD-L1 in tumor infiltrating immune cells that constitute 1% or more of said tumor sample.

99. 16. Use of an effective amount of a PD-L1 axis binding antagonist in the manufacture of a medicament for use in treating an individual suffering from cancer, wherein a tumor sample from said individual has been determined to have a tTMB score that is equal to or greater than a reference tTMB score, and optionally, wherein said tumor sample has been determined to have an increased level of somatic mutations in at least one gene set forth in Table 1 compared to a reference level of somatic mutations in at least one gene set forth in Table 1, a detectable level of expression of PD-L1 in tumor infiltrating immune cells that constitute about 1% or more of said tumor sample, and / or a detectable level of expression of PD-L1 in tumor infiltrating immune cells that constitute 1% or more of said tumor sample.

100. 13. A composition comprising an effective amount of a PD-L1 axis binding antagonist for use in a method of treating an individual suffering from cancer, wherein a tumor sample from said individual has been determined to have a tTMB score that is equal to or greater than a reference tTMB score, and optionally, wherein said tumor sample has been determined to have an increased level of somatic mutations in at least one gene set forth in Table 1 compared to a reference level of somatic mutations in at least one gene set forth in Table 1, a detectable level of expression of PD-L1 in tumor infiltrating immune cells that constitute about 1% or more of said tumor sample, and / or a detectable level of expression of PD-L1 in tumor infiltrating immune cells that constitute 1% or more of said tumor sample.

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