HK40019960A

HK40019960A - Diagnostic and therapeutic methods for cancer

Info

Publication number: HK40019960A
Application number: HK62020009769.4A
Authority: HK
Inventors: Marcin KOWANETZ; Mahrukh HUSENI; Wei Zou
Original assignee: 豪夫迈‧罗氏有限公司
Priority date: 2017-04-14
Filing date: 2018-04-13
Publication date: 2020-10-16

Description

Methods for diagnosis and treatment of cancer

Sequence listing

This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on 30/3/2018, entitled 50474-158WO3_ Sequence _ Listing _3.30.18_ ST25 and has a size of 96,571 bytes.

Technical Field

The present invention is directed to diagnostic and therapeutic methods for the treatment of cancer using PD-L1 axis binding antagonists. Also provided are related assays and kits.

Background

Cancer remains one of the most fatal threats to human health. In the united states, cancer affects almost 130 million new patients each year and is the second leading cause of death after a resident heart disease, accounting for approximately 1 of 4 deaths. It is also predicted that cancer may be the first cause of death in five years beyond cardiovascular disease. Solid tumors are responsible for most of those deaths.

Studies in humans with immune checkpoint inhibitors have shown promise in the steering immune system to control and eradicate tumor growth. The programmed death 1(PD-1) receptor and its ligand programmed death ligand 1(PD-L1) is an immune checkpoint protein that has been linked to the suppression of immune system responses during chronic infections, pregnancy, tissue allografts, autoimmune diseases, and cancer. PD-L1 modulates immune responses by binding to the inhibitory receptor PD-1 expressed on the surface of T cells, B cells, and monocytes. PD-L1 also down-regulates T cell function via interaction with another receptor, B7-1. The formation of the PD-L1/PD-1 and PD-L1/B7-1 complexes negatively regulates T cell receptor signaling, leading to subsequent T cell activation down-regulation and suppression of anti-tumor immune activity.

Although there have been significant advances in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved only about 10% over the past 20 years. In particular, malignant solid tumors rapidly metastasize and grow in an uncontrolled manner, making their timely detection and treatment extremely difficult.

Despite significant advances in cancer treatment, improved diagnostic methods and cancer therapies are still sought.

Summary of The Invention

The present invention provides therapeutic and diagnostic methods and compositions for treating an individual having cancer.

In one aspect, provided herein is a method of identifying an individual having cancer who is likely to benefit from treatment with a PD-L1 binding antagonist, the method comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment with a PD-L1 binding antagonist, wherein the reference immune score expression level is the immune score expression level of PD-L1, CXCL9, and IFNG in a reference population.

In another aspect, provided herein is a method for selecting a therapy for an individual having cancer, the method comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 binding antagonist, wherein the reference immune score expression level is the immune score expression level of PD-L1, CXCL9, and IFNG in a reference population.

In some embodiments, the immune score expression level of PD-L1, CXCL9, and IFNG in the sample is greater than the reference immune score expression level and the method further comprises administering to the individual an effective amount of a PD-L1 binding antagonist. In some embodiments, an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is lower than the reference immune score expression level identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 binding antagonist. In some embodiments, the immune score expression level of PD-L1, CXCL9, and IFNG in the sample is below the reference immune score expression level and the method further comprises administering to the individual an effective amount of an anti-cancer therapy that replaces or supplements a PD-L1 binding antagonist (e.g., an anti-cancer therapy that replaces or supplements a PD-L1 binding antagonist can include a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) alone or in addition to a PD-L1 axis binding antagonist and/or any additional therapeutic agent described herein such as a cytotoxic agent, growth inhibitory agent, radiation therapy, anti-angiogenic agent, or a combination thereof, as described herein).

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising (a) determining the expression level of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein the immune score expression level of PD-L1, CXCL9, and IFNG in the sample has been determined to be higher than a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, CXCL9, and IFNG in a reference population, and (b) administering to the individual an effective amount of a PD-L1 binding antagonist based on the immune score expression level of PD-L1, CXCL9, and IFNG determined in step (a).

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 binding antagonist, wherein the expression levels of PD-L1, CXCL9, and IFNG have been determined in a sample from the individual prior to treatment and the expression levels of the immune scores of PD-L1, CXCL9, and IFNG in the sample have been determined to be higher than the expression level of the immune score of a reference, wherein the expression level of the immune score of the reference is the expression level of the immune score of PD-L1, CXCL9, and IFNG in the reference population.

In some embodiments, the immune score expression levels of PD-L1, CXCL9, and IFNG in the sample are in the first 80 th percentile of the immune score expression levels of PD-L1, CXCL9, and IFNG in the reference population. In some embodiments, the immune score expression levels of PD-L1, CXCL9, and IFNG in the sample are in the first 50 th percentile of the immune score expression levels of PD-L1, CXCL9, and IFNG in the reference population. In some embodiments, the immune score expression levels of PD-L1, CXCL9, and IFNG in the sample are in the top 20 th percentile of the immune score expression levels of PD-L1, CXCL9, and IFNG in the reference population.

In some embodiments, the reference population is a population of individuals with cancer consisting of a first subset of individuals who have been treated with PD-L1 binding antagonist therapy and a second subset of individuals who have been treated with non-PD-L1 binding antagonist therapy, wherein the non-PD-L1 binding antagonist therapy does not comprise a PD-L1 binding antagonist.

In some embodiments, the immune score expression levels of PD-L1, CXCL9, and IFNG are the average of the expression levels of each of PD-L1, CXCL9, and IFNG. In some embodiments, the average of the expression levels of each of PD-L1, CXCL9, and IFNG is the average of the normalized expression levels of each of PD-L1, CXCL9, and IFNG. In some embodiments, the immune score expression level of PD-L1, CXCL9, and IFNG is the median of the expression levels of each of PD-L1, CXCL9, and IFNG. In some embodiments, the immune score expression level of PD-L1, CXCL9, and IFNG is the median of the normalized expression levels of each of PD-L1, CXCL9, and IFNG. In some embodiments, the normalized expression level for each of PD-L1, CXCL9, and IFNG is the expression level for each of PD-L1, CXCL9, and IFNG normalized to a reference gene. In some embodiments, the reference immune score expression level is a pre-assigned expression level of PD-L1, CXCL9, and IFNG.

In another aspect, provided herein is a method of identifying an individual having cancer who is likely to benefit from treatment with a PD-L1 binding antagonist, the method comprising determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the sample are higher than the expression level of the reference immune score identifies the individual as an individual who is likely to benefit from treatment with the PD-L1 binding antagonist, wherein the expression level of the reference immune score is the expression level of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the reference population.

In another aspect, provided herein is a method for selecting a therapy for an individual having cancer, the method comprising determining the expression level of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein the expression level of the immune score of PD-L1, IFNG, GZMB, and CD8A in the sample is higher than the expression level of a reference immune score identifies the individual as an individual who is likely to benefit from treatment with a PD-L1 binding antagonist, wherein the expression level of the reference immune score is the expression level of the immune score of PD-L1, IFNG, GZMB, and CD8A in the reference population.

In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample is greater than the reference immune score expression level and the method further comprises administering to the individual an effective amount of a PD-L1 binding antagonist.

In some embodiments, an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample that is lower than the reference immune score expression level identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 binding antagonist.

In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample is below the reference immune score expression level and the method further comprises administering to the individual an effective amount of an anti-cancer therapy that replaces or supplements the PD-L1 binding antagonist (e.g., an anti-cancer therapy that replaces or supplements the PD-L1 binding antagonist may include a cytotoxic agent, a growth inhibitory agent, a radiation therapy, an anti-angiogenic agent, or a combination thereof as described herein, alone or in addition to the PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and/or any additional therapeutic agent described herein.

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising (a) determining the expression level of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample has been determined to be higher than a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population, and (b) administering an effective amount of a PD-L1 binding antagonist to the individual based on the immune score expression level of PD-L1, IFNG, GZMB, and CD8A determined in step (a).

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 binding antagonist, wherein the expression levels of PD-L1, IFNG, GZMB, and CD8A have been determined in a sample from the individual prior to treatment and the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the sample have been determined to be higher than the expression levels of the reference immune scores, wherein the expression levels of the reference immune scores are the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the reference population.

In some embodiments, the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the sample are in the first 80 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the reference population. In some embodiments, the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the sample are in the first 50 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the reference population. In some embodiments, the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the sample are in the first 20 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the reference population.

In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, and CD8A is an average of the expression levels of each of PD-L1, IFNG, GZMB, and CD 8A. In some embodiments, the average expression level of each of PD-L1, IFNG, GZMB, and CD8A is the average of the normalized expression levels of each of PD-L1, IFNG, GZMB, and CD 8A. In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, and CD8A is the median of the expression levels of each of PD-L1, IFNG, GZMB, and CD 8A. In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, and CD8A is the median of the normalized expression levels of each of PD-L1, IFNG, GZMB, and CD 8A. In some embodiments, the normalized expression level of each of PD-L1, IFNG, GZMB, and CD8A is the expression level of each of PD-L1, IFNG, GZMB, and CD8A normalized to a reference gene. In some embodiments, the reference immune score expression level is a pre-assigned expression level of PD-L1, IFNG, GZMB, and CD 8A.

In another aspect, provided herein is a method of identifying an individual having cancer who is likely to benefit from treatment with a PD-L1 binding antagonist, the method comprising determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is higher than a reference immune score expression level identifies the individual as likely to benefit from treatment with the PD-L1 binding antagonist, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population.

In another aspect, provided herein is a method for selecting a therapy for an individual having cancer, the method comprising determining the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 binding antagonist, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample is greater than the reference immune score expression level and the method further comprises administering to the individual an effective amount of a PD-L1 binding antagonist. In some embodiments, an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 binding antagonist. In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample is below the reference immune score expression level and the method further comprises administering to the individual an effective amount of an anti-cancer therapy that replaces or supplements the PD-L1 binding antagonist (e.g., an anti-cancer therapy that replaces or supplements the PD-L1 binding antagonist may include a cytotoxic agent, a growth inhibitory agent, a radiation therapy, an anti-angiogenic agent, as described herein, alone or in addition to the PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) and/or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and/or any additional therapeutic agent described herein, or a combination thereof.

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising (a) determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample have been determined relative to a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population, and (b) administering an effective amount of a PD-L1 binding antagonist to the individual based on the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 determined in step (a).

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 binding antagonist, wherein the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample from the individual prior to treatment and the expression levels of the immune scores of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample have been determined to be higher than a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population.

In some embodiments, the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample are in the first 80 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population. In some embodiments, the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample are in the first 50 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population. In some embodiments, the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample are in the first 20 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 is an average of the expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1. In some embodiments, the average of the expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1 is the average of the normalized expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1.

In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 is the median of the expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1.

In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 is the median of the normalized expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1. In some embodiments, the normalized expression level of each of PD-L1, IFNG, GZMB, CD8A, and PD-1 is the expression level of each of PD-L1, IFNG, GZMB, CD8A, and PD-1 normalized to a reference gene. In some embodiments, the reference immune score expression level is a pre-assigned expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1.

In some embodiments of any of the above aspects, the reference population is a population of individuals with cancer consisting of a first subset of individuals who have been treated with PD-L1 binding antagonist therapy and a second subset of individuals who have been treated with non-PD-L1 binding antagonist therapy, wherein the non-PD-L1 binding antagonist therapy does not comprise a PD-L1 binding antagonist.

In some embodiments of any of the above aspects, the reference immune score expression level significantly separates each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy and the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy above the reference immune score expression level, wherein the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy.

In some embodiments of any of the above aspects, the reference immune score expression level significantly separates each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy and the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy below the reference immune score expression level, wherein the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy.

In some embodiments of any of the above aspects, responsiveness to treatment is prolongation of PFS.

In some embodiments of any of the above aspects, responsiveness to treatment is prolongation of OS.

In some embodiments of any of the above aspects, the reference gene is a housekeeping gene. In some embodiments, the housekeeping gene is TMEM 55B.

In some embodiments of any of the above aspects, the benefit from treatment comprising a PD-L1 binding antagonist is prolongation of OS.

In some embodiments of any of the above aspects, the benefit from treatment comprising a PD-L1 binding antagonist is prolongation of PFS.

In some embodiments of any of the above aspects, the benefit from treatment comprising a PD-L1 binding antagonist is prolongation of OS and PFS.

In some embodiments of any of the above aspects, the expression level is a nucleic acid expression level. In some embodiments, the nucleic acid expression level is an mRNA expression level. In some embodiments, mRNA expression levels are determined by RNA-seq, RT-qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or combinations thereof. In some embodiments, mRNA expression levels are detected using RNA-seq. In some embodiments, mRNA expression levels are detected using RT-qPCR. In some embodiments, the expression level is detected in a tumor cell, a tumor-infiltrating immune cell, a stromal cell, or a combination thereof.

In some embodiments of any of the above aspects, the sample is a tissue sample, a cell sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the tissue sample is a tumor tissue sample. In some embodiments, the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, or a combination thereof. In some embodiments, the tumor tissue sample is a formalin-fixed and paraffin-embedded (FFPE) sample, an archived sample, a fresh sample, or a frozen sample. In some embodiments, the tumor tissue sample is an FFPE sample.

In some embodiments of any of the above aspects, the cancer is selected from the group consisting of 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, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoides, merkel cell carcinoma, or hematological malignancy. In some embodiments, the cancer is lung cancer, kidney cancer, bladder cancer, or breast cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the renal cancer is Renal Cell Carcinoma (RCC). In some embodiments, the bladder cancer is Urothelial Bladder Cancer (UBC). In some embodiments, the breast cancer is Triple Negative Breast Cancer (TNBC).

In some embodiments of any of the above aspects, the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1, binding of PD-L1 to B7-1, or binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody.

In some embodiments of any of the above aspects, the anti-PD-L1 antibody is selected from the group consisting of atuzumab (MPDL3280A), yw243.55.s70, MSB0010718C, MDX-1105, and MEDI 4736. In some embodiments, the anti-PD-L1 antibody comprises the following hypervariable regions (a) the HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO: 9); (b) AWISPYGGSTYYADSVKG (SEQ ID NO:10) of HVR-H2 sequence; (c) RHWPGGFDY (SEQ ID NO:11) of HVR-H3 sequence; (d) RASQDVSTAVA (SEQ ID NO:12) of HVR-L1 sequence; (e) the HVR-L2 sequence of SASFLYS (SEQ ID NO: 13); and (f) the HVR-L3 sequence of QQYLYHPAT (SEQ ID NO: 14). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID No. 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO. 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO. 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO 16; (b) a VL domain comprising the amino acid sequence of SEQ ID NO 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments the anti-PD-L1 antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO 16; and (b) a VL domain comprising the amino acid sequence of SEQ ID NO 17. In some embodiments, the anti-PD-L1 antibody is atelizumab.

In some embodiments of any of the above aspects, the non-PD-L1 binding antagonist is an anti-neoplastic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, or a cytotoxic agent.

In some embodiments of any of the above aspects, the anti-cancer therapy is an anti-tumor agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, or a cytotoxic agent.

In some embodiments of any of the above aspects, the subject has not previously been treated for cancer. In some embodiments of any of the above aspects, the individual has not previously been administered a PD-L1 binding antagonist.

In some embodiments of any of the above aspects, the treatment comprising a PD-L1 binding antagonist is a monotherapy.

In some embodiments of any of the above aspects, the method further comprises administering to the individual an effective amount of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anti-neoplastic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, or a cytotoxic agent.

In some embodiments of any of the above aspects, the subject is a human.

In another aspect, provided herein is a kit for identifying an individual having cancer who may benefit from treatment with a PD-L1 binding antagonist, the kit comprising (a) reagents for determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual; and, optionally, (b) instructions for using the agent to identify an individual having cancer who is likely to benefit from treatment with the PD-L1 binding antagonist.

In another aspect, provided herein is a kit for identifying an individual having cancer who may benefit from treatment with a PD-L1 binding antagonist, the kit comprising (a) reagents for determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual; and, optionally, (b) instructions for using the agent to identify an individual having cancer who is likely to benefit from treatment with the PD-L1 binding antagonist.

In another aspect, provided herein is a kit for identifying an individual having cancer who is likely to benefit from treatment with a PD-L1 binding antagonist, the kit comprising reagents for determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual; and, optionally, instructions for using the agent to identify an individual having cancer who is likely to benefit from treatment with the agent comprising a PD-L1 binding antagonist.

In another aspect, provided herein is an assay for identifying an individual having cancer as a candidate for treatment comprising a PD-L1 binding antagonist, the assay comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 binding antagonist, and wherein the reference immune score expression level is the immune score expression level of PD-L1, CXCL9, and IFNG in the reference population.

In another aspect, provided herein is an assay for identifying an individual having cancer as a candidate for treatment comprising a PD-L1 binding antagonist, the assay comprising determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 binding antagonist, and wherein the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the reference population.

In another aspect, provided herein is an assay for identifying an individual having cancer as a candidate for treatment comprising a PD-L1 binding antagonist, the assay comprising determining the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 binding antagonist, and wherein the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

In another aspect, provided herein is a method of identifying an individual having cancer who may benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is higher than a reference immune score expression level identifies the individual as an individual who may benefit from treatment with a PD-L1 axis binding antagonist, wherein the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population.

In another aspect, provided herein is a method for selecting a therapy for an individual having cancer, the method comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, wherein the reference immune score expression level is the immune score expression level of PD-L1, CXCL9, and IFNG in the reference population.

In some embodiments, the immune score expression level of PD-L1, CXCL9, and IFNG in the sample is greater than the reference immune score expression level and the method further comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist. In some embodiments, an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is lower than the reference immune score expression level identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist. In some embodiments, the immune score expression level of PD-L1, CXCL9, and IFNG in the sample is below the reference immune score expression level and the method further comprises administering to the individual an effective amount of an anti-cancer therapy that replaces or supplements the PD-L1 axis binding antagonist (e.g., an anti-cancer therapy that replaces or supplements the PD-L1 axis binding antagonist can include a cytotoxic agent, growth inhibitory agent, radiation therapy, anti-angiogenic agent, or a combination thereof as described herein alone or in addition to the PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and/or any additional therapeutic agent described herein.

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising (a) determining the expression level of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein the immune score expression level of PD-L1, CXCL9, and IFNG in the sample has been determined to be higher than a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, CXCL9, and IFNG in a reference population, and (b) administering to the individual an effective amount of a PD-L1 axis binding antagonist based on the immune score expression level of PD-L1, CXCL9, and IFNG determined in step (a).

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist, wherein the expression levels of PD-L1, CXCL9, and IFNG have been determined in a sample from the individual prior to treatment and the expression levels of the immune scores of PD-L1, CXCL9, and IFNG in the sample have been determined to be higher than the expression level of the immune score of a reference, wherein the expression level of the immune score of the reference is the expression level of the immune score of PD-L1, CXCL9, and IFNG in the reference population.

In some embodiments, the reference population is a population of individuals with cancer consisting of a first subset of individuals who have been treated with PD-L1 axis binding antagonist therapy and a second subset of individuals who have been treated with non-PD-L1 axis binding antagonist therapy, wherein the non-PD-L1 axis binding antagonist therapy does not comprise a PD-L1 axis binding antagonist.

In another aspect, provided herein is a method of identifying an individual having cancer who is likely to benefit from treatment with a PD-L1 axis binding antagonist, the method comprising determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the sample are higher than the expression level of the reference immune score identifies the individual as an individual who is likely to benefit from treatment with the PD-L1 axis binding antagonist, wherein the expression level of the reference immune score is the expression level of the immune scores of PD-L1, IFNG, gb, and CD8A in the reference population.

In another aspect, provided herein is a method for selecting a therapy for an individual having cancer, the method comprising determining the expression level of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, wherein the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the reference population.

In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample is greater than the reference immune score expression level and the method further comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist.

In some embodiments, an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample that is lower than the reference immune score expression level identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist.

In some embodiments, the expression level of the immune score of PD-L1, IFNG, GZMB, and CD8A in the sample is below the reference immune score expression level and the method further comprises administering to the individual an effective amount of an anti-cancer therapy that replaces or supplements the PD-L1 axis binding antagonist (e.g., an anti-cancer therapy that replaces or supplements the PD-L1 axis binding antagonist may include a cytotoxic agent, a growth inhibitory agent, a radiation therapy, an anti-angiogenic agent, or a combination thereof as described herein, alone or in addition to the PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and/or any additional therapeutic agent described herein.

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising (a) determining the expression level of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample has been determined to be higher than a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population, and (b) administering an effective amount of a PD-L1 axis binding antagonist to the individual based on the immune score expression level of PD-L1, IFNG, GZMB, and CD8A determined in step (a).

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist, wherein the expression levels of PD-L1, IFNG, GZMB, and CD8A have been determined in a sample from the individual prior to treatment and the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the sample have been determined to be higher than the expression levels of the reference immune scores, wherein the expression levels of the reference immune scores are the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the reference population.

In another aspect, provided herein is a method of identifying an individual having cancer who is likely to benefit from treatment with a PD-L1 axis binding antagonist, the method comprising determining the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein an expression level of an immune score for PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment with the PD-L1 axis binding antagonist, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population.

In another aspect, provided herein is a method for selecting a therapy for an individual having cancer, the method comprising determining the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample is greater than the reference immune score expression level and the method further comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist. In some embodiments, an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist. In some embodiments, the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample is less than the reference immune score expression level and the method further comprises administering to the individual an effective amount of an anti-cancer therapy that replaces or supplements the PD-L1 axis binding antagonist (e.g., an anti-cancer therapy that replaces or supplements the PD-L1 axis binding antagonist may comprise a cytotoxic agent, growth inhibitory agent, radiation therapy, anti-angiogenic agent, or a combination thereof as described herein alone or in addition to the PD-L1 axis binding antagonist (e.g., a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL32 3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and/or any additional therapeutic agent described herein.

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising (a) determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample have been determined relative to a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population, and (b) administering an effective amount of a PD-L1 axis binding antagonist to the individual based on the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 determined in step (a).

In another aspect, provided herein is a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist, wherein the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample from the individual prior to treatment and the expression levels of the immune scores of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample have been determined to be higher than the expression levels of the reference immune scores, wherein the expression levels of the reference immune scores are the expression levels of the immune scores of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

In some embodiments of any of the above aspects, the reference population is a population of individuals with cancer consisting of a first subset of individuals who have been treated with PD-L1 axis binding antagonist therapy and a second subset of individuals who have been treated with non-PD-L1 axis binding antagonist therapy, wherein the non-PD-L1 axis binding antagonist therapy does not comprise a PD-L1 axis binding antagonist.

In some embodiments of any of the above aspects, the reference immune score expression level significantly separates each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy and the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy above the reference immune score expression level, wherein the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy.

In some embodiments of any of the above aspects, the reference immune score expression level significantly separates each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy and the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy below the reference immune score expression level, wherein the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy.

In some embodiments of any of the above aspects, the benefit from treatment comprising a PD-L1 axis binding antagonist is prolongation of OS.

In some embodiments of any of the above aspects, the benefit from treatment comprising a PD-L1 axis binding antagonist is prolongation of PFS.

In some embodiments of any of the above aspects, the benefit from treatment comprising a PD-L1 axis binding antagonist is prolongation of OS and PFS.

In some embodiments of any of the above aspects, the PD-L1 axis binding antagonist inhibits binding of PD-L1 to PD-1, PD-L1 to B7-1, or PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In other embodiments, the PD-L1 axis binding antagonist is a PD-1 binding antagonist.

In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody (e.g., atelizumab (atezolizumab) (MPDL3280A), yw243.55.s70, MSB0010718C (avilamab)), MDX-1105, or MEDI4736 (durvalumab)). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., MDX 1106 (nivolumab)), MK-3475 (pembrolizumab), CT-011 (pidilizumab)), MEDI-0680(AMP-514), PDR001, REGN2810, or BGB-108).

In some embodiments of any of the above aspects, the anti-PD-L1 antibody is selected from the group consisting of atuzumab (MPDL3280A), yw243.55.s70, MSB0010718C, MDX-1105, and MEDI 4736. In some embodiments, the anti-PD-L1 antibody comprises the following hypervariable regions (a) the HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO: 9); (b) AWISPYGGSTYYADSVKG (SEQ ID NO:10) of HVR-H2 sequence; (c) RHWPGGFDY (SEQ ID NO:11) of HVR-H3 sequence; (d) RASQDVSTAVA (SEQ ID NO:12) of HVR-L1 sequence; (e) the HVR-L2 sequence of SASFLYS (SEQ ID NO: 13); and (f) the HVR-L3 sequence of QQYLYHPAT (SEQ ID NO: 14). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID No. 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO. 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO. 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 16; (b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, an anti-PD-L1 antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO 16; (b) a VL domain comprising the amino acid sequence of SEQ ID NO 17; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments the anti-PD-L1 antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO 16; and (b) a VL domain comprising the amino acid sequence of SEQ ID NO 17. In some embodiments, the anti-PD-L1 antibody is atelizumab. In some embodiments, the anti-PD-L1 antibody is

In some embodiments, the PD-L1 axis binding antagonist is an anti-PD-1 antibody.

In some embodiments of any of the above aspects, the non-PD-L1 axis binding antagonist is an anti-neoplastic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, or a cytotoxic agent.

In some embodiments of any of the above aspects, the subject has not previously been treated for cancer. In some embodiments of any of the above aspects, the individual has not previously been administered a PD-L1 axis binding antagonist.

In some embodiments of any of the above aspects, the treatment comprising a PD-L1 axis binding antagonist is monotherapy.

In some embodiments of any of the above aspects, the treatment comprising a PD-L1 binding antagonist is a combination therapy.

In some embodiments of any of the above aspects, the method further comprises administering to the individual an effective amount of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anti-neoplastic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, a cytotoxic agent, or a combination thereof.

In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is carboplatin; -Parietai; or carboplatin and paclitaxel. In certain embodiments, the chemotherapeutic agent is carboplatin and paclitaxel.

In some embodiments, the additional therapeutic agent is an anti-angiogenic agent. In some embodiments, the anti-angiogenic agent is an anti-VEGF antibody (e.g., bevacizumab).

In some embodiments, the additional therapeutic agent is a combination of an anti-angiogenic agent and a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is carboplatin; -Parietai; or carboplatin and paclitaxel. In some embodiments, the chemotherapeutic agent is carboplatin and paclitaxel. In some embodiments, the anti-angiogenic agent is an anti-VEGF antibody (e.g., bevacizumab).

In some embodiments of any of the above aspects, the subject is a human.

In another aspect, provided herein is a kit for identifying an individual having cancer who may benefit from treatment with a PD-L1 axis binding antagonist, the kit comprising (a) reagents for determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual; and, optionally, (b) instructions for using the agent to identify an individual having cancer who is likely to benefit from treatment with the agent comprising a PD-L1 axis binding antagonist.

In another aspect, provided herein is a kit for identifying an individual having cancer who may benefit from treatment with a PD-L1 axis binding antagonist, the kit comprising (a) reagents for determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual; and, optionally, (b) instructions for using the agent to identify an individual having cancer who is likely to benefit from treatment with the agent comprising a PD-L1 axis binding antagonist.

In another aspect, provided herein is a kit for identifying an individual having cancer who may benefit from treatment with a PD-L1 axis binding antagonist, the kit comprising reagents for determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual; and, optionally, instructions for using the agent to identify an individual having cancer who is likely to benefit from treatment with the agent comprising a PD-L1 axis binding antagonist.

In another aspect, provided herein is an assay for identifying an individual having cancer as a candidate for treatment comprising a PD-L1 axis binding antagonist, the assay comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, and wherein the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population.

In another aspect, provided herein is an assay for identifying an individual having cancer as a candidate for treatment comprising a PD-L1 axis binding antagonist, the assay comprising determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising the PD-L1 axis binding antagonist, and wherein the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the reference population.

In another aspect, provided herein is an assay for identifying an individual having cancer as a candidate for treatment comprising a PD-L1 axis binding antagonist, the assay comprising determining the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising the PD-L1 axis binding antagonist, and wherein the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

Brief Description of Drawings

Figure 1 is a graph showing Kaplan-Meier curves of patients in the attrituximab (MPDL3280A) treated (black) arm and docetaxel control (grey) arm of the OAK trial (clinical trial ID No.: NCT02008227) evaluable population (BEP) (nBEP 753 patients) for Progression Free Survival (PFS), each arm stratified according to the immune score expression levels of PD-L1, CXCL9, and IFNG. Patients with higher expression levels of PD-L1, CXCL9, and the immune score of IFNG than about 50% of the total BEP (cut-off: mean normalized dCt ≧ 1.9) are indicated by solid lines while patients with lower expression levels of PD-L1, CXCL9, and the immune score of IFNG than about 50% of the total BEP (cut-off: mean normalized dCt < -1.9) are indicated by dashed lines. Also shown is a table listing the number of patients within each subgroup of BEPs that do not have a PFS event at a given point in time. The time point for each bar corresponds to the time displayed along the x-axis of the above-described graph. The mean normalized dCt is the mean of the normalized dCt values for each of PD-L1, CXCL9, and IFNG. dCt (target gene) ═ Ct (control gene) -Ct (target gene).

Figure 2 is a table and forest plot showing the Hazard Ratio (HR) of PFS in patients treated with atuzumab (MPDL3280A) compared to docetaxel (control) in an OAK trial (clinical trial ID No.: NCT 02008227). The HR between subgroups of patients as defined by different cut-off values (mean normalized dCt values at different percentile cut-offs of BEP) of the immune score expression levels of PD-L1, CXCL9, and IFNG are listed. The mean normalized dCt is the mean of the normalized dCt values for each of PD-L1, CXCL9, and IFNG. dCt (target gene) ═ Ct (control gene) -Ct (target gene).

FIG. 3 is a graph showing Kaplan-Meier curves of Overall Survival (OS) of BEP for patients in the Attributab (MPDL3280A) treated (black) arm and docetaxel control (gray) arm of the OAK test (clinical trial ID No.: NCT02008227), each arm stratified according to the immune score expression levels of PD-L1, CXCL9, and IFNG. Patients with higher expression levels of PD-L1, CXCL9, and the immune score of IFNG than about 50% of the total BEP (cut-off: mean normalized dCt ≧ 1.9) are indicated by solid lines while patients with lower expression levels of PD-L1, CXCL9, and the immune score of IFNG than about 50% of the total BEP (cut-off: mean normalized dCt < -1.9) are indicated by dashed lines. Also shown is a table listing the number of viable patients in each subgroup of the BEP at a given time point. The time point for each bar corresponds to the time displayed along the x-axis of the above-described graph. The mean normalized dCt is the mean of the normalized dCt values for each of PD-L1, CXCL9, and IFNG. dCt (target gene) ═ Ct (control gene) -Ct (target gene).

Figure 4 is a table and forest plot showing HR of OS in patients treated with atuzumab (MPDL3280A) compared to docetaxel (control) in an OAK trial. The HR between subgroups of patients as defined by different cut-off values (mean normalized dCt values at different percentile cut-offs of BEP) of the immune score expression levels of PD-L1, CXCL9, and IFNG are listed. The mean normalized dCt is the mean of the normalized dCt values for each of PD-L1, CXCL9, and IFNG. dCt (target gene) ═ Ct (control gene) -Ct (target gene).

Figure 5 is a table and forest plot showing HR of PFS in patients treated with atuzumab (MPDL3280A) compared to docetaxel (control) in an OAK trial. The HR between subgroups of patients defined by different cut-off values (mean normalized dCt values at different percentile cut-offs of BEP) of the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A are listed. The mean normalized dCt is the mean of the normalized dCt values for each of PD-L1, IFNG, GZMB, and CD 8A. dCt (target gene) ═ Ct (control gene) -Ct (target gene).

Figure 6 is a table and forest plot showing HR of OS in patients treated with atuzumab (MPDL3280A) compared to docetaxel (control) in an OAK trial. The HR between subgroups of patients defined by different cut-off values (mean normalized dCt values at different percentile cut-offs of BEP) of the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A are listed. The mean normalized dCt is the mean of the normalized dCt values for each of PD-L1, IFNG, GZMB, and CD 8A. dCt (target gene) ═ Ct (control gene) -Ct (target gene).

Figure 7 is a table showing prevalence in OAK trials in patients treated with atuzumab (MPDL3280A), HR of PFS, and HR of OS compared to docetaxel (control). Listed by (i) CXCL 9; (ii) IFNG; (ii) PD-L1(CD274) and PD-1; (iii) PD-L1(CD274) and IFNG; (iv) CD8A, GZMB, PD-L1(CD274), IFNG, and CXCL 9; and (v) HR between subgroups of patients defined by different cut-off values of immune score expression levels of GZMB, PD-L1(CD274), IFNG, CXCL9, and PD-1 (mean normalized dCt values at different quartile cut-offs of BEP). dCt (control gene) -Ct (target gene), wherein a higher dCt indicates a higher expression level of the target gene.

Figure 8A is a table and forest plot showing HR of PFS in patients treated with atuzumab (MPDL3280A) compared to docetaxel (control) in the POPLAR trial (clinical trial ID No.: NCT 01903993). The HR between subgroups of patients as defined by different cut-off values (mean normalized dCt values at different percentile cut-offs of BEP) of the immune score expression levels of PD-L1, CXCL9, and IFNG are listed. The mean normalized dCt is the mean of the normalized dCt values for each of PD-L1, CXCL9, and IFNG. dCt (target gene) ═ Ct (control gene) -Ct (target gene).

Fig. 8B is a table indicating Objective Response Rates (ORR) for the respective patient populations in fig. 8A.

Figure 9 is a table and forest plot showing HR of OS in patients treated with atuzumab (MPDL3280A) compared to docetaxel (control) in the POPLAR trial (clinical trial ID No.: NCT 01903993). The HR between subgroups of patients as defined by different cut-off values (mean normalized dCt values at different percentile cut-offs of BEP) of the immune score expression levels of PD-L1, CXCL9, and IFNG are listed. The mean normalized dCt is the mean of the normalized dCt values for each of PD-L1, CXCL9, and IFNG. dCt (target gene) ═ Ct (control gene) -Ct (target gene).

FIG. 10 is a graph showing the Kaplan-Meier curve of OS with BEP of urothelial bladder cancer patients treated with atzumab in cohort 2 of the IMvigor210 trial (clinical trial ID No.: NCT02108652), layered according to the immune score expression levels of PD-L1, CXCL9, and IFNG. Patients with higher expression levels of PD-L1, CXCL9, and the immune score of IFNG than about 66% of all BEPs (cut-off ≧ 66 th percentile cut-off of BEP) are indicated by solid lines while patients with lower expression levels of PD-L1, CXCL9, and the immune score of IFNG than about 66% of all BEPs (cut-off: < 66 th percentile cut-off of BEP) are indicated by dashed lines. Also shown is a table listing the number of viable patients in each subgroup of the BEP at a given time point. The time point for each bar corresponds to the time displayed along the x-axis of the above-described graph.

FIG. 11 is a graph showing the Kaplan-Meier curves of PFS of ATTRAUzumab (MPDL3280A) and bevacizumab combinations for IMmotion150 trials (clinical trial ID No.: NCT01984242) treating BEP of patients with renal cell carcinoma in the (black) arm and sunitinib (grey) arm, each arm stratified according to the expression levels of immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1. Patients with higher expression levels of the immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1 than about 50% of all BEPs (cut-off value ≧ 50 th percentile cut-off of BEP) are indicated by a solid line and patients with lower expression levels of the immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1 than about 50% of all BEPs (cut-off value: < 50 th percentile cut-off of BEP) are indicated by a dashed line. Also shown is a table listing the number of patients within each subgroup of BEPs that do not have a PFS event at a given point in time. The time point for each bar corresponds to the time displayed along the x-axis of the above-described graph.

FIG. 12 is a graph showing a Kaplan-Meier curve of OS for BEP of patients treated with atuzumab in the PCD4989g trial, layered according to the immune score expression levels of PD-L1, CXCL9, and IFNG. Patients with higher expression levels of PD-L1, CXCL9, and the immune score of IFNG than about 50% of all BEPs (cut-off:. gtoreq.50 th percentile cut-off of BEP) are indicated by solid lines while patients with lower expression levels of PD-L1, CXCL9, and the immune score of IFNG than about 50% of all BEPs (cut-off: < 50 th percentile cut-off of BEP) are indicated by dashed lines. Also shown is a table listing the number of viable patients in each subgroup of the BEP at a given time point. The time point for each bar corresponds to the time displayed along the x-axis of the above-described graph.

Figure 13 is a block diagram showing the correlation between mean normalized expression and full or partial response (CR/PR), Stable Disease (SD), and Progressive Disease (PD) of PD-L1(CD274), IFNG, and CXCL9 in TNBC-bearing patients treated with atuzumab (MPDL3280A) in the PCD4989g trial (clinical trial ID No.: NCT 01375842).

Figure 14 is a hierarchical graph showing the study design of the phase III IMpower150 trial (clinical trial ID No. nct02366143).

FIG. 15 is a CONSORT graph of the IMpower150 test.

FIG. 16 is a Kaplan-Meier curve of PFS in the Intent To Treat (ITT) -WT population for the IMpower150 tested Atlizumab, bevacizumab, carboplatin, and paclitaxel arm (ABCP; arm B) or bevacizumab, carboplatin, and paclitaxel arm (BCP, arm C). The layered (randomisation factor by ITT-WT) HR is given.

FIGS. 17A and 17B show the ITT-WT population (FIG. 17A) or ITT ISEL of ABCP arms (arm B) or BCP arms (arm C) tested by IMpower150^{Height of}Kaplan-Meier curves of PFS assessed by the independent examination agency (IRF) in WT (FIG. 17B). ITT-WT (FIG. 17A; layering of ITT-WT by randomization factor) and ISEL are given^{Height of}WT (FIG. 17B; ISEL)^{Height of}Stratification of WT by gender and liver metastasis).

FIGS. 18A and 18B show ISEL of the ABCP arm (arm B) or the BCP arm (arm C) for the IMpower150 assay^{Height of}WT population (FIG. 18A) and ISEL^{Is low in}Kaplan-Meier curves of PFS in WT population (FIG. 18B). ISEL^{Height of}Layered (ISEL) of WT^{Height of}-WT translocation through gender and liver) HR; ISEL^{Is low in}-non-stratified HR of WT.

Figure 19 is a table and forest plot showing HR of PFS in patients treated with ABCP (arm B) or BCP (arm C) in the IMpower150 trial. The HR between subgroups of patients as defined by different cut-off values (mean normalized dCt values at different percentile cut-offs of BEP) of the immune score expression levels of PD-L1, CXCL9, and IFNG are listed. The mean normalized dCt is the mean of the normalized dCt values for each of PD-L1, CXCL9, and IFNG. dCt (target gene) ═ Ct (control gene) -Ct (target gene).

Figure 20 is a Kaplan-Meier curve of PFS in patients with EGFR or ALK genomic alterations in the ABCP arm (arm B) or BCP arm (arm C) of the IMpower150 assay.

Figure 21 is a Kaplan-Meier curve of PFS in the ITT population (including patients with EGFR mutations or ALK translocations) in the ABCP arm (arm B) or BCP arm (arm C) of the IMpower150 assay. Layered (by randomization factor) HR.

FIG. 22 is a table and forest plot showing HR and 95% Confidence Intervals (CI) for PFS in the clinical subgroup of the ITT-WT population.

FIG. 23 is a Kaplan-Meier curve of mid-stage OS analysis in ITT-WT populations in either the ABCP arm (arm B) or the BCP arm (arm C) of the IMpower150 assay. Layered (following randomization factor) HR.

FIG. 24 is a table and forest chart showing the HR of PFS in patients treated with atuzumab, carboplatin, and paclitaxel (ACP; arm A) or BCP (arm C) in the IMpower150 trial. The HR between subgroups of patients as defined by different cut-off values (mean normalized dCt values at different percentile cut-offs of BEP) of the immune score expression levels of PD-L1, CXCL9, and IFNG are listed. The mean normalized dCt is the mean of the normalized dCt values for each of PD-L1, CXCL9, and IFNG. dCt (target gene) ═ Ct (control gene) -Ct (target gene).

FIGS. 25A and 25B show ISEL of ACP arm (arm A) or BCP arm (arm C) in IMpower150 assay^{Height of}WT population and ISEL^{Is low in}Kaplan-Meier curves of PFS at different immune score expression level cutoffs (approximately 44% prevalence (fig. 25A) and approximately 25% prevalence (fig. 25B)) in the WT population.

FIG. 26 is a Kaplan-Meier curve for OS in the intent-to-treat (ITT) population of the ATTRAUzumab arm or chemotherapy arm tested by IMvigor 211.

FIGS. 27A and 27B show ISEL of the ATTRAUzumab arm or chemotherapy arm of the IMvigor211 assay^{Height of}WT population (FIG. 27A) and ISEL^{Is low in}Kaplan-Meier curves for OS in WT population (FIG. 27B).

Detailed Description

The present invention provides diagnostic methods, therapeutic methods, and compositions for the treatment of cancer, such as lung cancer (e.g., non-small cell lung cancer (NSCLC)), bladder cancer (e.g., Urothelial Bladder Cancer (UBC)), kidney cancer (e.g., Renal Cell Carcinoma (RCC)), and breast cancer (e.g., Triple Negative Breast Cancer (TNBC)). The present invention is based, at least in part, on the expression level of an immune score in a sample obtained from an individual having cancer selected from at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4) selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, and PD-1) can be used to identify whether the individual is likely to respond to a treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritzuki 3224) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)); selecting a therapy for treating the subject; optimizing therapeutic efficacy of a treatment comprising a PD-L1 axis binding antagonist; and/or monitoring the discovery of a biomarker (e.g., a predictive biomarker) in a method of responding to treatment comprising a PD-L1 axis binding antagonist in the individual.

I. Definition of

As used herein, the term "about" refers to the usual error range for corresponding numerical values as would be readily understood by one of ordinary skill in the art. References herein to "about" a value or parameter include (and describe) embodiments that relate to that value or parameter itself.

As used herein, "administering" refers to a method of administering a dose of a compound (e.g., PD-L1 axis binding antagonist (e.g., PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising a PD-L1 axis binding antagonist) to a subject the compounds and/or compositions utilized in the methods described herein can be, e.g., intravenous (e.g., by intravenous infusion), subcutaneous, intramuscular, intradermal, transdermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topical, intratumoral, intraperitoneal, subconjunctival, intravesical, transmucosal, intrapericardial, intraumbilical, intraocular, oral, topical, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion bathing target cells directly, by catheter, by lavage, in cream, or in lipid compositions. The method of administration may vary depending on a variety of factors, such as the compound or composition being administered and the severity of the condition, disease, or disorder being treated.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an 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 the molecule X for its partner Y can generally be determined by the dissociation constant (K)_D) To represent. Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

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

As used herein, "amplification" generally refers to the process of generating multiple copies of a desired sequence. "multiple copies" means at least two copies. "copy" does not necessarily imply complete sequence complementarity or identity to the template sequence. For example, the copies may include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced via primers comprising sequences that are hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.

The term "antibody" 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.

An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ')₂(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. An exemplary competition assay is provided herein.

The terms "anti-PD-L1 antibody" and "antibody that binds to PD-L1" refer to an antibody that is capable of binding PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting PD-L1. In one embodiment, the extent to which the anti-PD-L1 antibody binds 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 certain embodiments, the anti-PD-L1 antibody binds to a PD-L1 epitope that is conserved among PD-L1 from different species. In certain embodiments, the anti-PD-L1 antibody is atuzumab (MPDL 3280A). PD-L1 (programmed death ligand 1) is also known in the art as "programmed cell death 1 ligand 1", "PDCD 1LG 1", "CD 274", "B7-H", and "PDL 1". An exemplary human PD-L1 is shown in UniProtKB/Swiss-Prot accession No. Q9NZQ7.1.

The term "anti-cancer therapy" refers to a therapy useful for treating cancer, such as lung cancer (e.g., non-small cell lung cancer (NSCLC)), bladder cancer (e.g., Urothelial Bladder Cancer (UBC)), kidney cancer (e.g., Renal Cell Carcinoma (RCC)), or breast cancer (e.g., Triple Negative Breast Cancer (TNBC)). Examples of anticancer therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents for use in radiotherapyAgents for use, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-CD 20 antibodies, platelet derived growth factor inhibitors (e.g., GLEEVEC)^TM(imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more targets PDGFR-beta, BlyS, APRIL, BCMA receptor, TRAIL/Apo2, other biologically active and organic chemicals, and the like. Combinations thereof are also included in the present invention.

An "article of manufacture" is any manufacture (e.g., a package or container) or kit comprising at least one agent, e.g., a drug for treating a disease or disorder (e.g., cancer, e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) or a probe for specifically detecting a biomarker described herein. In certain embodiments, the article of manufacture or kit is marketed, distributed, or sold by a unit for performing the methods described herein.

As used herein, the phrase "based on" means using information about one or more biomarkers to inform treatment decisions, information provided on package inserts, or marketing/promotional instructions, etc.

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

"binding domain" means a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Binding domains include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab'₂scFv antibodies, SMIPs, domain antibodies, diabodies, minibodies (minibodies), scFv-Fc, affibodies (affibodies), nanobodies (nanobodies), and VH and/or VL domains of antibodies), receptors, ligands, aptamers, and other molecules with identified binding partners.

As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, such as a predictive, diagnostic, and/or prognostic indicator (e.g., PD-L1, CXCL9, IFNG, GZMB, CD8A, PD-1, or a combination thereof, including, for example, PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; or PD-L1, IFNG, GZMB, CD8A, and PD-1). Biomarkers can serve as indicators of particular disease or disorder subtypes characterized by particular molecular, pathological, histological, and/or clinical characteristics (e.g., cancer, such as lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)). In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polynucleotide copy number alterations (e.g., DNA copy number), polypeptides and polynucleotide modifications (e.g., post-translational modifications), carbohydrate and/or glycolipid-based molecular markers.

The terms "biomarker signature," "biomarker expression signature," or "expression signature" are used interchangeably herein to refer to one or a group of biomarkers (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; or PD-L1, IFNG, GZMB, CD8A, and the level of immune score expression of PD-1) whose expression is an indicator, e.g., predictive, diagnostic, and/or prognostic indicator. Biomarker signatures can serve as indicators of specific disease or disorder subtypes characterized by certain molecular, pathological, histological, and/or clinical characteristics (e.g., cancer, such as lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)). In some embodiments, the biomarker signature is a "gene signature". The term "gene signature" is used interchangeably with "gene expression signature" and refers to a polynucleotide or set of polynucleotides whose expression is an indicator, e.g., a predictive, diagnostic and/or prognostic indicator. In some embodiments, the biomarker signature is a "protein signature". The terms "protein signature" and "protein expression signature" are used interchangeably to refer to a polypeptide or set of polypeptides whose expression is an indicator, e.g., a predictive, diagnostic and/or prognostic indicator.

As used herein, the term "CD 8A" refers to any native CD8A from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed CD8A, as well as any form of CD8A that results from processing in a cell. The term also encompasses naturally occurring variants of CD8A, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human CD8A is set forth in SEQ ID NO. 1. An exemplary amino acid sequence of the protein encoded by human CD8A is shown in SEQ ID NO 2.

As used herein, the term "GZMB" refers to any native GZMB (granzyme B) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed GZMB as well as any form of GZMB that results from processing in a cell. The term also encompasses naturally occurring variants of GZMB, such as splice variants or allelic variants. One exemplary nucleic acid sequence of human GZMB is set forth in SEQ ID NO 3. An exemplary amino acid sequence of a protein encoded by human GZMB is shown in SEQ ID NO 4.

As used herein, the term "IFNG" refers to any native IFNG (interferon, gamma) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed IFNG as well as any form of IFNG that results from processing in a cell. The term also encompasses naturally occurring variants of IFNG, such as splice variants or allelic variants. An exemplary nucleic acid sequence for human IFNG is set forth in SEQ ID NO. 5. An exemplary amino acid sequence of the protein encoded by human IFNG is shown in SEQ ID NO 6.

As used herein, the term "CXCL 9" refers to any native CXCL9 (chemokine (C-X-C motif) ligand 9) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CXCL9 as well as any form of CXCL9 that results from processing in a cell. The term also encompasses naturally occurring variants of CXCL9, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human CXCL9 is set forth in SEQ ID NO 7. An exemplary amino acid sequence of a protein encoded by human CXCL9 is shown in SEQ ID No. 8.

As used herein, the term "CD 27" refers to any native CD27 (also referred to in the art as CD27L receptor or TNFRSF7) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed CD27 as well as any form of CD27 that results from processing in a cell. The term also encompasses naturally occurring variants of CD27, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human CD27 is set forth in SEQ ID NO 21. An exemplary amino acid sequence of the protein encoded by human CD27 is shown in SEQ ID NO 22.

As used herein, the term "FOXP 3" refers to any native FOXP3 (prong box P3, also known in the art as scurfin) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed FOXP3 as well as any form of FOXP3 that results from processing in a cell. The term also encompasses naturally occurring variants of FOXP3, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human FOXP3 is set forth in SEQ ID NO. 23. An exemplary amino acid sequence of the protein encoded by human FOXP3 is shown in SEQ ID No. 24.

As used herein, the term "CTLA 4" refers to any native CTLA4 (cytotoxic T lymphocyte-associated protein 4, also known in the art as CD152) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CTLA4 as well as any form of CTLA4 that results from processing in a cell. The term also encompasses naturally occurring variants of CTLA4, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human CTLA4 is set forth in SEQ ID NO. 25. An exemplary amino acid sequence of a protein encoded by human CTLA4 is shown in SEQ ID No. 26.

As used herein, the term "TIGIT" refers to any native TIGIT (T cell immunoreceptor with Ig and ITIM domains) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed TIGIT as well as any form of TIGIT that results from processing in a cell. The term also encompasses naturally occurring variants of TIGIT, such as splice variants or allelic variants. The nucleic acid sequence of an exemplary human TIGIT is set forth in SEQ ID No. 27. An exemplary amino acid sequence of a protein encoded by human TIGIT is shown in SEQ ID No. 28.

As used herein, the term "IDO 1" refers to any native IDO1 (indoleamine 2, 3-dioxygenase 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed IDO1 as well as any form of IDO1 that results from processing in the cell. The term also encompasses naturally occurring variants of IDO1, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human IDO1 is set forth in SEQ ID NO. 29. An exemplary amino acid sequence of the protein encoded by human IDO1 is shown in SEQ ID No. 30.

As used herein, the term "CXCL 10" refers to any native CXCL10(C-X-C motif chemokine 10; also known in the art as interferon gamma-induced protein 10 or small inducible cytokine B10) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CXCL10 as well as any form of CXCL10 that results from processing in a cell. The term also encompasses naturally occurring variants of CXCL10, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human CXCL10 is set forth in SEQ ID NO 31. An exemplary amino acid sequence of a protein encoded by human CXCL10 is shown in SEQ ID No. 32.

As used herein, the term "CXCL 11" refers to any native CXCL11(C-X-C motif chemokine 11) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CXCL11 as well as any form of CXCL11 that results from processing in a cell. The term also encompasses naturally occurring variants of CXCL11, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human CXCL11 is set forth in SEQ ID NO 33. An exemplary amino acid sequence of a protein encoded by human CXCL11 is shown in SEQ ID No. 34.

As used herein, the term "PSMB 8" refers to any native PSMB8 (proteasomal subunit β 8 type) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed PSMB8, as well as any form of PSMB8 that results from processing in a cell. The term also encompasses naturally occurring variants of PSMB8, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human PSMB8 is set forth in SEQ ID NO. 35. An exemplary amino acid sequence of a protein encoded by human PSMB8 is shown in SEQ ID NO. 36.

As used herein, the term "PSMB 9" refers to any native PSMB9 (proteasomal subunit β 9 type) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed PSMB9, as well as any form of PSMB9 that results from processing in a cell. The term also encompasses naturally occurring variants of PSMB9, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human PSMB9 is set forth in SEQ ID NO. 37. An exemplary amino acid sequence of a protein encoded by human PSMB9 is shown in SEQ ID NO. 38.

As used herein, the term "TAP 1" refers to any native TAP1 (transporter 1 associated with antigen processing; also referred to in the art as antigenic peptide transporter 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed TAP1 as well as any form of TAP1 that results from processing in a cell. The term also encompasses naturally occurring variants of TAP1, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human TAP1 is set forth in SEQ ID NO. 39. An exemplary amino acid sequence of the protein encoded by human TAP1 is shown in SEQ ID NO 40.

As used herein, the term "TAP 2" refers to any native TAP2 (antigenic peptide transporter 2) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed TAP2 as well as any form of TAP2 that results from processing in a cell. The term also encompasses naturally occurring variants of TAP2, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human TAP2 is set forth in SEQ ID NO. 41. An exemplary amino acid sequence of the protein encoded by human TAP2 is shown in SEQ ID NO 42.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); bladder cancer (e.g., Urothelial Bladder Cancer (UBC), Muscle Invasive Bladder Cancer (MIBC), and BCG refractory non-muscle invasive bladder cancer (NMIBC)); kidney cancer or cancer of the kidney (e.g., Renal Cell Carcinoma (RCC)); cancer of the urethra; breast cancer (e.g., HER2+ breast cancer and estrogen receptor (ER-), progesterone receptor (PR-), and HER2(HER2-) negative Triple Negative Breast Cancer (TNBC)); prostate cancer, such as Castration Resistant Prostate Cancer (CRPC); peritoneal cancer; hepatocellular carcinoma; gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer); pancreatic cancer; a glioblastoma; cervical cancer; ovarian cancer; liver cancer; hepatoma; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; prostate cancer; vulvar cancer; thyroid cancer; cancer of the liver; anal cancer; penile cancer; melanomas, including superficial invasive melanoma, lentigo malignant melanoma, acromelasma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); Small Lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high immunoblastic NHL; high lymphoblastic NHL; high small non-nucleated NHL; storage disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); acute Myeloid Leukemia (AML); hairy cell leukemia; chronic Myeloblastic Leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndrome (MDS), as well as abnormal vascular proliferation associated with scarring nevus, edema (such as associated with brain tumors), Meigs' syndrome, brain cancer, head and neck cancer, and related metastases.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)). In another embodiment, the cell proliferative disorder is a tumor.

A "chemotherapeutic agent" is a chemical compound useful for treating cancer (e.g., cancer, such as lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)). Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and cyclophosphamide (cyclophosphamide)Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodopa), carboquone (carboquone), metoclopramide (meteredopa) and uretepa (uredpa); ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimetalmamine; annonaceous acetogenins (especially bullatacin and bullatacin); delta-9-tetrahydrocannabinol (dronabinol)abinol),) (ii) a Beta-lapachone (lapachone); lapachol (lapachol); colchicines (colchicines); betulinic acid (betulinic acid); camptothecin (camptothecin) (including the synthetic analogue topotecan)CPT-11 (irinotecan),) Acetyl camptothecin, scopolectin (scopolectin) and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin) and bizelesin (bizelesin) synthetic analogs); podophyllotoxin (podophylotoxin); podophyllinic acid (podophyllic acid); teniposide (teniposide); cryptophycins (especially cryptophycins 1 and 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin (spongistatin); nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (chlorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine (prednimustine), tramadol (trofosfamide), uracil mustard (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranirnustine); antibiotics, such as enediynes (enediynes) (e.g. calicheamicin, especially calicheamicin gamma)₁ ^IAnd calicheamicin ω I1 (see, e.g., Nicolaou et al, Angew. chem Intl. Ed. Engl.,33:183- "186 (1994)); CDP323, an oral alpha-4 integrinA white inhibitor; anthracyclines (kinemicin), including kinemicin a; epothilones (esperamicins); and neocarzinostatin (neocarzinostatin) chromophores and related chromoproteenediyne antibiotic chromophores, aclacinomycin (acrinomycin), actinomycin (actinomycin), anthranilic (authramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), carbacidin, carminomycin (caminomycin), carcinomycin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditorelbircin (desoubicin), 6-diaza-5-oxo-L-norleucine, doxorubicin (doxorubicin) (includingMorpholino doxorubicin, cyano morpholino doxorubicin, 2-pyrrolyl doxorubicin, doxorubicin hydrochloride liposome injectionLiposomal doxorubicin TLC D-99PEGylated liposomal doxorubicinAnd doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcelllomycin), mitomycins (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (peplomycin), pofiomycin (porfirlomycin), puromycin (puromycin), triiron doxorubicin (quelamycin), rodobicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozocin), tubercidin (tubicin), ubenimex (enumemex), stastin (zinostatin), zorubicin (zorubicin); antimetabolites, such as methotrexate, gemcitabine (gemcitabine)Tegafur (tegafur)Capecitabine (capecitabine)Epothilone (epothilone) and 5-fluorouracil (5-FU); combretastatin (combretastatin); folic acid analogs such as denopterin, methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (frilic acid); acetoglucurolactone (acegultone); an aldophosphamide glycoside (aldophosphamideglycoside); aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defofamine); dimecorsine (demecolcine); diazaquinone (diaziqutone); elformithine; ammonium etitanium acetate; an epothilone; etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidainine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidanmol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine);polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane (rizoxane); rhizomycin (rhizoxin); sisofilan (sizofuran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2, 2' -trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin (verrucin) A, bacillocin (roridin) A and snakes (anguidine); urethane (urethan); vindesine (vindesine)Dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); thiotepa (thiotepa); taxols (taxoid), e.g. paclitaxel (paclitaxel) ((R))Bristol-Myers Squibb Oncology, Princeton, N.J.), albumin engineered nanoparticle dosage form paclitaxel (ABRAXANE)^TM) And docetaxel (docetaxel) ((docetaxel))Rhome-Poulene Rorer, Antony, France); chlorambucil (chlorenbucil); 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum agents, such as cisplatin (cissplatin), oxaliplatin (oxaliplatin) (e.g. cisplatin)) And carboplatin (carboplatin); vinblastines (vincas), which prevent tubulin polymerization to form microtubules, include vinblastine (vinblastine)Vincristine (vincristine)Vindesine (vindesine)And vinorelbine (vinorelbine)Etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); leucovorin (leucovorin); oncostatin (novantrone); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); ibandronate (ibandronate); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as tretinoids, including bexarotene (bexarotene)Diphosphonates (bisphosphates), such as clodronate (e.g. clodronate)Or) Etidronate sodium (etidronate)NE-58095, zoledronic acid/zoledronateAlendronate (alendronate)Pamidronate (pamidronate)Tiludronate (tirudronate)Or risedronate (risedronate)And troxacitabine (a 1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, in particular antisense oligonucleotides that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC- α, Raf, H-Ras and epidermal growth factor receptor (EGF-R) (e.g., erlotinib (erlotinib) (TARCEVA)^TM) (ii) a And VEGF-A that reduces cell proliferation; vaccines, e.g.Vaccines and gene therapy vaccines, e.g.A vaccine is provided which comprises a vaccine,a vaccine anda vaccine; topoisomerase 1 inhibitors (e.g. topoisomerase 1 inhibitors)) (ii) a rmRH (e.g. rmRH)) (ii) a BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib),pfizer); perifosine (perifosine), COX-2 inhibitors (such as celecoxib (celecoxib) or etoricoxib (etoricoxib)), proteosome inhibitors (such as PS 341); bortezomib (bortezomib)CCI-779; tipifarnib (R11577); orafenaib, ABT 510; bcl-2 inhibitors, such asoblimersen sodiumPixantrone (pixantrone); an EGFR inhibitor; tyrosine kinase inhibitors; serine-threonine kinase inhibitors, such as rapamycin (rapamycin) (sirolimus),) (ii) a Farnesyl transferase inhibitors, such as lonafarnib (SCH 6636, SARASAR)^TM) (ii) a And pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy) and FOLFOX (oxaliplatin)^TM) Abbreviations for treatment regimens combining 5-FU and folinic acid), and pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the foregoing.

Chemotherapeutic agents as defined herein also include "anti-hormonal agents" or "endocrine therapeutic agents" which act to modulate, reduce, block, or inhibit the effects of hormones that promote the growth of cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)). They may themselves be hormones including, but not limited to, antiestrogens and Selective Estrogen Receptor Modulators (SERMs), including, for example, tamoxifen (includingTamoxifen), raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxytamoxifene, trioxifene (trioxifene), naloxifene (keoxifene), LY117018, onapristone (onapristone) andtoremifene (toremifene); aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal gland, such as, for example, 4(5) -imidazole, aminoglutethimide,megestrol acetate (megestrol acetate),exemestane (exemestane), formestane (formestanine), fadrozole (fadrozole),the compound is vorozole (vorozole),letrozole (letrozole) andanastrozole (anastrozole); anti-androgens such as flutamide/flutamide (flutamide), nilutamide (nilutamide), bicalutamide/bicalutamide (bicalutamide), leuprolide (leuprolide), and goserelin (goserelin); and troxacitabine (a 1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly antisense oligonucleotides that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC- α, Raf and H-Ras; ribozymes, such as VEGF expression inhibitors (e.g.Ribozymes) and inhibitors of HER2 expression; vaccines, such as gene therapy vaccines, e.g.A vaccine is provided which comprises a vaccine,a vaccine anda vaccine;rIL-2；a topoisomerase 1 inhibitor;rmRH; vinorelbine (Vinorelbine) and Esperamicins (Esperamicins) (see U.S. patent No.4,675,187); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are 5 major classes of antibodies, IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁,IgG₂,IgG₃,IgG₄,IgA₁And IgA₂. The constant domains of heavy chains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At)²¹¹,I¹³¹,I¹²⁵,Y⁹⁰,Re¹⁸⁶,Re¹⁸⁸,Sm¹⁵³,Bi²¹²,P³²,Pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate), doxorubicin (adriamicin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed hereinafter.

The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents, wherein at least some of the administrations overlap in time. Thus, concurrent administration includes a dosing regimen in which administration of one or more agents is discontinued and administration of one or more other agents continues.

As used herein, "delaying the progression of a condition or disease" means delaying, impeding, slowing, delaying, stabilizing, and/or delaying the formation of a disease or condition (e.g., cancer, such as lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)). This delay can be of varying lengths of time depending on the history of the disease and/or the subject being treated. As will be apparent to one of skill in the art, a sufficient or significant delay may essentially encompass prevention, as the subject does not develop disease.

The terms "determine", "determining", "detecting" and grammatical variations thereof include any determination/determination or detection means, including direct and indirect determination/determination or detection.

A "disorder" or "disease" is any condition that would benefit from management, including but not limited to chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder in question (e.g., cancer, such as lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)).

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, such as lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)). For example, "diagnosis" may refer to the identification of a particular type of cancer. "diagnosis" may also refer to the classification of a particular subtype of cancer, for example a subtype characterized by histopathological criteria or by molecular characteristics, for example by the expression of one or a set of biomarkers, for example a particular gene or a protein encoded by said gene.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include C1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., PD-L1); and B cell activation.

An "effective amount" of a compound (e.g., a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., an anti-PD 3280A) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) or composition thereof (e.g., a pharmaceutical composition) is at least the minimum amount required to achieve a desired therapeutic or prophylactic result, such as a measurable extension of Overall Survival (OS) or Progression Free Survival (PFS) for a particular disease or disorder (e.g., cancer, e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)). Beneficial or desired results include results such as elimination or reduction of risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during disease development. An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to effect prophylactic or therapeutic treatment, either directly or indirectly. As understood in the clinical setting, an effective amount of a drug, compound, or pharmaceutical composition can be achieved with or without another drug, compound, or pharmaceutical composition. As such, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the desired result can be achieved or achieved, along with one or more other agents. For example, an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) as a cancer treatment can reduce the number of cancer cells; reducing the size of the primary tumor; inhibit (i.e., slow to some extent, preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent, preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or to alleviate one or more symptoms associated with the condition to some extent. To the extent that the drug can prevent the growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy can be measured by, for example, assessing duration of survival, time to disease progression (TTP), response rate (PR), duration of response, and/or quality of life.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest,5th ed.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. In general, the FRs of the variable domains consist of 4 FR domains FR1, FR2, FR3, and FR 4. Thus, the HVR and FR sequences in VH (or VL) generally occur in the order FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using the repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.mol.biol.227:381 (1991); Marks et al, J.mol.biol.222:581 (1991)). Can also be used for preparingHuman Monoclonal Antibodies are described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); boerner et al, J.Immunol.147(1):86-95 (1991). See also van Dijk and van de Winkel, curr, opin, pharmacol, 5:368-74 (2001). Human antibodies can be made by administering an antigen to a transgenic animal, such as an immunized XENOMOUSE (xenomic), that has been modified to produce human antibodies in response to antigenic challenge, but whose endogenous loci have been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XeNOMOUS^TMA technique). See also, e.g., Li et al, Proc. Natl. Ascad. Sci. USA,103:3557-3562(2006), for human antibodies generated via human B-cell hybridoma technology.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a "humanized form" of a non-human antibody, refers to an antibody that has undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or which forms structurally defined loops ("hypervariable loops") and/or which contains antigen-contacting residues ("antigen contacts"). Typically, antibodies comprise 6 HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops present at amino acid residues 26-32(L1),50-52(L2),91-96(L3),26-32(H1),53-55(H2), and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987));

(b) CDRs present at amino acid residues 24-34(L1),50-56(L2),89-97(L3),31-35b (H1),50-65(H2), and 95-102(H3) (Kabat et al, Sequences of Proteins of immunological interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigen contacts, present at amino acid residues 27c-36(L1),46-55(L2),89-96(L3),30-35b (H1),47-58(H2), and 93-101(H3) (MacCallum et al, J.mol.biol.262:732-745 (1996)); and (d) combinations of (a), (b), and/or (c) comprising HVR amino acid residues 46-56(L2),47-56(L2),48-56(L2),49-56(L2),26-35(H1),26-35b (H1),49-65(H2),93-102(H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al, supra.

An "isolated" antibody refers to an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromager.b 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its native chromosomal location.

As used herein, the word "label" refers to a detectable compound or composition. The label is typically conjugated or fused, directly or indirectly, to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent conjugated or fused thereto. The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that produces a detectable product.

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., gene-encoded and/or epigenetic) is converted into structures present and operating in a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Transcribed polynucleotides, translated polypeptides, or fragments of a polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered expressed, whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of a polypeptide (e.g., by proteolysis). "expressed gene" includes genes that are transcribed into a polynucleotide (e.g., mRNA) and then translated into a polypeptide, as well as genes that are transcribed into RNA but not translated into a polypeptide (e.g., transport and ribosomal RNA). Expression levels can be measured by methods disclosed herein, as known to those skilled in the art, including, for example, RT-qPCR and RNA-seq. The assessed expression level can be used to determine a response to treatment.

The term "immune score expression level" refers to a numerical value that reflects the expression level of a single gene of interest (e.g., a normalized expression level), or the aggregate expression level of more than one gene of interest (e.g., at least two, at least three, at least four, at least five, or at least six genes of interest) that is associated with an immune response. The immune score expression level of more than one gene of interest can be determined by one skilled in the art, as well as the clustering methods disclosed herein, including, for example, by calculating a median or mean of the expression levels of all genes of interest. The expression level of each gene of interest can be normalized prior to staging, by using statistical methods known to those skilled in the art, as well as disclosed herein, including, for example, normalizing for the expression level of one or more housekeeping genes, or normalizing for the entire library volume, or normalizing for the median or mean expression level value measured across all genes. In some cases, the normalized expression level of each gene of interest can be normalized by using statistical methods known to those skilled in the art, as well as disclosed herein, prior to clustering among the multiple genes of interest, including, for example, by calculating a Z-score for the normalized expression level of each gene of interest. In some cases, each gene of interest may have an assigned weight score and the immune score expression level of a plurality of genes of interest may be calculated by incorporating the weight scores to determine a mean of the weighted expression levels of all genes of interest. For example, an immune score expression level can refer to a value reflecting a normalized expression level of a single gene selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. Alternatively, the immune score expression level may, for example, refer to a value reflecting the aggregate normalized expression level (e.g., median normalized expression level or mean normalized expression level) of at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4), and optionally further reflecting the expression level of other T-responsive cell related genes, including, for example, cxg, GZMB, ifmes, PRF1, PD-L CXCL 48, PD-L CXCL1, CXCL9, it 27, CTLA 638, tigo 638, tfo 1, one or more genes (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen genes) of the group consisting of PSMB8, PSMB9, TAP1, and TAP2, or a combination thereof, wherein the one or more biomarkers associated with T effector cells are different from one or more genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. In some cases, an immune score expression level can, for example, refer to a value that reflects the aggregate Z-score expression level (e.g., the mean of Z-score normalized expression levels or the median of Z-score normalized expression levels) of at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4), selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, IFNG, PD, preof 2, PD-1 5, PD-L1, CD 3884, FOXP 4642, CD-1, CD-4, and optionally further reflects the expression level of other T effector cell-associated genes, including, for example, CD A, GZMA, GZMB, IFNG, preof 2, PRF 685f 2, prq7325, CD 3884, FOXP-1, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, TAP1, and TAP2 (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen genes), or a combination thereof, wherein the one or more genes associated with T-effector cells are different from one or more genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

As used herein, the term "reference immune score expression level" refers to an immune score expression level, another immune score expression level (e.g., of at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)) against which it is compared, e.g., for making a diagnosis, prediction, prognosis, and/or treatment determination. For example, a reference immune score expression level can be derived from a reference sample, an expression level in a reference population (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4), and/or a pre-assigned value (e.g., a cutoff value previously determined based on a significant difference between the responsiveness of an individual to treatment with the PD-L1 axis binding antagonist therapy above and/or below the cutoff value and the responsiveness of an individual to treatment with the non-PD-L1 axis binding antagonist therapy (e.g., a statistically significant separation of the reference population) A first subset of individuals who have been treated with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein above the cut-off value the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy and/or below the cut-off value the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) relative to the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy Statistically significant) improvement). One skilled in the art will appreciate that the value of the reference immune score expression level can vary with the indication (e.g., cancer (e.g., breast, lung, kidney, or bladder cancer)), methodology used to detect the expression level (e.g., RNA-seq or RT-qPCR), statistical methods used to generate the immune score, and/or the particular combination of genes examined (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or combinations thereof (e.g., PD-L1, CXCL9, and ng; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, gb, CD8A, and PD-1; or any combination of genes listed in tables 1-4).

"elevated expression", "elevated expression level", or "elevated level" refers to an increased level of expression or any one of the listed combinations of genes in a subject or internal control (e.g., a housekeeping gene, e.g., TMEM55B), such as a control, such as one or more subjects not suffering from a disease or disorder (e.g., cancer, e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), or a reference level, such as a reference immune score expression level, a gene or combination of genes in a subject (e.g., selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1), or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 358; PD-L1, ifzmng, gc A, and PD-1; or a combination of genes listed in a table)).

"reduced expression", "reduced expression level", or "reduced level" refers to a reduced level or a reduced level of expression of any one of the listed genes or combinations of genes in a subject, such as one or more subjects not suffering from a disease or disorder (e.g., cancer, e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) or an internal control (e.g., a housekeeping gene, e.g., TMEM55B), or a reference level, such as a reference immune score expression level, a gene or combination of genes in a subject (e.g., selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1), or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 358; PD-L1, ifzmng, gcm 26, CD-1; or PD-1-4)). In some embodiments, the reduced expression is little or no expression.

As used herein, "reference gene" refers to a gene or set of genes (e.g., one, two, three, four, five, or six or more genes) for comparison purposes, such as a housekeeping gene. "housekeeping gene" refers herein to a gene or set of genes (e.g., one, two, three, four, five, or six or more genes) that encodes a protein whose activity is essential for the maintenance of cellular function and which typically occurs similarly in all cell types (e.g., TMEM 55B).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for generating monoclonal antibodies are described herein.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. The naked antibody may be present in a pharmaceutical formulation.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is an heterotetrameric glycan protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has one variable region (VH), also called variable or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a Constant Light (CL) domain. Antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences.

The term "oligonucleotide" refers to relatively short polynucleotides (e.g., less than about 250 nucleotides in length), including but not limited to single-stranded deoxyribonucleotides, single-or double-stranded ribonucleotides, RNA: DNA hybrids, and double-stranded DNA. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using commercially available automated oligonucleotide synthesizers. However, oligonucleotides can be prepared by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNA in cells and organisms.

The term "package insert" is used to refer to instructions for use typically contained in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings relating to the use of such therapeutic products.

The term "pharmaceutical formulation" refers to a preparation that is in a form such as to allow the biological activity of the active ingredient contained therein to be effective, and free of additional ingredients that have unacceptable toxicity to a subject that will receive administration of the formulation.

"pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation that is different from the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, the term "protein" refers to any native protein from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed protein as well as any form of protein that is processed from the cell. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without any conservative substitutions being considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech corporation and the source code has been submitted with the user document to the U.S. copyright office (Washington d.c.,20559) and registered with U.S. copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech corporation (South San Francisco, Calif.) or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of amino acid sequence comparisons using ALIGN-2, the% amino acid sequence identity of a given amino acid sequence A relative to (to), with (with), or against (against) a given amino acid sequence B (or can be stated as a given amino acid sequence A having or comprising some% amino acid sequence identity relative to, with, or against a given amino acid sequence B) is calculated as follows:

100 times fraction X/Y

Wherein X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a relative to B will not be equal to the% amino acid sequence identity of B relative to a. Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The terms "apoptosis-ligand 1" and "PD-L1" refer herein to native sequence PD-L1 polypeptides, polypeptide variants, and fragments of native sequence polypeptides and polypeptide variants (which are further defined herein). The PD-L1 polypeptides described herein may be isolated from a variety of sources, such as from a human tissue type or from another source, or prepared by recombinant or synthetic methods.

By "PD-L1 polypeptide variant" or variations thereof is meant a PD-L1 polypeptide, typically an active PD-L1 polypeptide, as defined herein, having at least about 80% amino acid sequence identity to any native sequence PD-L1 polypeptide sequence as disclosed herein. For example, such PD-L1 polypeptide variants include PD-L1 polypeptides in which one or more amino acid residues are added 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, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a native sequence PD-L1 polypeptide sequence as disclosed herein. Typically, a PD-L1 variant polypeptide is at least about 10 amino acids in length, alternatively at least about 20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,281,282,283,284,285,286,287,288,289 amino acids in length, or more. Optionally, the PD-L1 variant polypeptide will have no more than one conservative amino acid substitution as compared to the native PD-L1 polypeptide sequence, or no more than 2,3,4,5,6,7,8,9, or 10 conservative amino acid substitutions as compared to the native PD-L1 polypeptide sequence.

A "native sequence PD-L1 polypeptide" comprises a polypeptide having the same amino acid sequence as a corresponding PD-L1 polypeptide derived from nature.

The term "PD-L1 axis binding antagonist" refers to a molecule that inhibits the interaction of the PD-L1 axis binding partner with one or more of its binding partners, thereby removing T cell dysfunction resulting from signaling on the PD-1 signaling axis, one result being restoration or enhancement of T cell function. As used herein, PD-L1 axis binding antagonists include PD-L1 binding antagonists and PD-1 binding antagonists as well as molecules that interfere with the interaction between PD-L1 and PD-1 (e.g., PD-L2-Fc fusion).

The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 or B7-1. In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding its binding partner. In a particular aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonist includes an anti-PD-L1 antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, oligopeptide, and other molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 or B7-1. In one embodiment, the PD-L1 binding antagonist reduces negative costimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes that mediate signaling via PD-L1, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a particular aspect, the anti-PD-L1 antibody is atelizumab (CAS registry No.: 1422185-06-5), also known as MPDL3280A, described herein. In another specific aspect, the anti-PD-L1 antibody is yw243.55.s70 described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1105 as described herein. In yet another specific aspect, the anti-PD-L1 antibody is MEDI4736 (dulafumab) described herein. In yet another specific aspect, the anti-PD-L1 antibody is MSB0010718C (avizumab) described herein.

As used herein, a "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates 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 partner. In a particular aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies and antigen-binding fragments thereof that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules. In one embodiment, the PD-1 binding antagonist reduces negative signaling mediated by or via cell surface proteins expressed on T lymphocytes and other cells that mediate signaling via PD-1 or PD-L1, thereby rendering dysfunctional T cells less dysfunctional. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a particular aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In another specific aspect, the PD-1 binding antagonist is CT-011 (piditumumab). In another specific aspect, the PD-1 binding antagonist is MEDI-0680 (AMP-514). In another specific aspect, the PD-1 binding antagonist is PDR 001. In another specific aspect, the PD-1 binding antagonist is REGN 2810. In another specific aspect, the PD-1 binding antagonist is BGB-108. In another specific aspect, the PD-1 binding antagonist is AMP-224.

As used interchangeably herein, "multiple coresA nucleotide "or" nucleic acid "refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and/or analogs thereof, or any substrate that can be incorporated into the polymer by a DNA or RNA polymerase, or by a synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. If present, modifications to the nucleotide structure may be administered before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of the polynucleotide. Additionally, any hydroxyl groups typically present in sugars may be replaced with, for example, phosphonic acid (phosphonate) groups, phosphoric acid (phosphate) groups, protected with standard protecting groups, or activated in preparation for additional attachment to additional nucleotides, or a solid or semi-solid support may be conjugated. The 5 'and 3' terminal OH groups may be phosphorylated or substituted with amines or organic capping group modules of 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized to standard protecting groups. The polynucleotide may also contain forms of ribose or deoxyribose sugar analogs generally known in the art, including, for example, 2 '-O-methyl-, 2' -O-allyl-, 2 '-fluoro-, or 2' -azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and alkali-free sugarsNucleoside analogs such as methyl ribonucleosides. One or more phosphodiester linkages may be replaced with alternative linking groups. Such alternative linking groups include, but are not limited to, embodiments wherein the phosphate ester is substituted with P (O) S ("thioester"), P (S) S ("dithioate"), (O) NR₂("amide ester"), P (O) R, P (O) OR', CO OR CH₂(formal) in which R or R' are each independently H or a substituted or unsubstituted hydrocarbyl group (1-20C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aryl hydrocarbyl (araldyl). Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "polymerase chain reaction" or "PCR" techniques generally refer to procedures in which minute amounts of specific segments of nucleic acid, RNA and/or DNA are amplified as described in U.S. Pat. No.4,683,195, issued 7/28/1987. Generally, sequence information from the ends of the region of interest or beyond needs to be available to enable the design of oligonucleotide primers; these primers will be identical or similar in sequence to the opposite strand of the template to be amplified. The 5' terminal nucleotides of both primers may coincide with the ends of the amplified material. Specific RNA sequences, specific DNA sequences, can be amplified using PCR from total genomic DNA, and cDNA, phage, or plasmid sequences transcribed from total cellular RNA, and the like. See generally Mullis et al, Cold spring harbor Symp. Quant.biol.,51:263 (1987); erlich, ed., PCR Technology, (StocktonPress, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, including using a known nucleic acid (DNA or RNA) as a primer and utilizing a nucleic acid polymerase to amplify or generate a particular nucleic acid segment or to amplify or generate a particular nucleic acid segment that is complementary to a particular nucleic acid.

As used herein, the term "reverse transcriptase polymerase chain reaction" or "RT-PCR" refers to the replication and amplification of RNA sequences. In this method, reverse transcription is coupled to PCR, for example as described in U.S. Pat. No.5,322,770, which is incorporated herein by reference in its entirety. In RT-PCR, an RNA template is converted to cDNA by the reverse transcriptase activity of the enzyme and then amplified using the polymerization activity of the same or a different enzyme. Both thermostable and thermolabile reverse transcriptase and polymerase enzymes can be used. "reverse transcriptase" (RT) may include reverse transcriptases from retroviruses, other viruses, and DNA polymerases that exhibit reverse transcriptase activity.

As used herein, the term "reverse transcriptase quantitative polymerase chain reaction" or "RT-qPCR" is a form of PCR in which the nucleic acid to be amplified is RNA, the RNA is first reverse transcribed into cDNA, and the amount of PCR product is measured at each step of the PCR reaction.

"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 a number of publications including Cronin et al, am.j.pathol.164(1):35-42 (2004); and Ma et al, Cancer cell.5: 607-.

The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acids obtained from a single source (e.g., an individual) using more than one set of primers for the purpose of amplifying two or more DNA sequences in a single reaction.

The term "RNA-seq," also known as "Whole Transcriptome Shotgun Sequencing (WTSS)," refers to sequencing and/or quantifying cDNA using high-throughput sequencing techniques to obtain information about the RNA content of a sample. Publications describing RNA-Seq include Wanget, a reliable tool for transcriptitomics, Nature reviews genetics 10(1):57-63(January 2009); ryan et al, BioTechniques 45(1):81-94 (2008); and Maher et al, "transaction sequencing to detection gene fusions insincer", Nature 458(7234):97-101(January 2009).

The term "polynucleotide" when used in the singular or plural generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, polynucleotides as defined herein include, but are not limited to, single and double stranded DNA, DNA comprising single and double stranded regions, single and double stranded RNA, and RNA comprising single and double stranded regions, hybrid molecules comprising DNA and RNA, which may be single stranded, or more typically double stranded, or comprise single and double stranded regions. In addition, as used herein, the term "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chains in such regions may be from the same molecule or from different molecules. The region may comprise the entire molecule or molecules, but more typically is a region involving only some molecules. One of the molecules of the triple-helical region is often an oligonucleotide. The term "polynucleotide" specifically includes cDNA. The term includes DNA (including cDNA) and RNA that contain one or more modified bases. Thus, a DNA or RNA whose backbone is modified for stability or other reasons is also a "polynucleotide" for which the term is intended herein. In addition, DNA or RNA comprising rare bases such as inosine or modified bases such as tritiated bases are also included within the term "polynucleotide" as defined herein. In general, the term "polynucleotide" encompasses all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.

"response to treatment", "responsiveness to treatment", or "benefit from treatment" can be assessed using any endpoint that indicates a benefit to the individual, including, but not limited to, (1) inhibition of disease progression (e.g., cancer progression) to some extent, including slowing and complete arrest; (2) reducing the size of the tumor; (3) inhibit (i.e., reduce, slow, or completely stop) cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibit (i.e., reduce, slow, or completely stop) metastasis; (5) alleviating to some extent one or more symptoms associated with a disease or disorder (e.g., cancer); (6) prolongation or extension of the length of survival, including overall survival (OS HR <1) and progression free survival (PFS HR < 1); and/or (9) a reduction in mortality at a given time point following treatment (e.g., treatment comprising a PD-L1 axis binding antagonist, e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

As used herein, "progression-free survival" or "PFS" refers to the length of time during and after treatment that the treated disease (e.g., cancer, e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) has not progressed or deteriorated. Progression-free survival may include the amount of time an individual experiences a complete response or a partial response, as well as the amount of time an individual experiences stable disease.

As used herein, "overall survival" or "OS" refers to the percentage of subjects in a group that are likely to be alive after a particular duration (e.g., 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years, or more than 20 years from the time of self-diagnosis or treatment).

As used herein, "complete response" or "CR" refers to the disappearance of all signs of cancer in response to treatment. This does not necessarily mean that the cancer has already cured.

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.

As used herein, "hazard ratio" or "HR" is a statistical definition of the ratio of events. For the purposes of the present invention, hazard ratio is defined as representing the probability of an event (e.g. PFS or OS) in the test (e.g. treatment) group/arm divided by the probability of an event in the control group/arm at any particular point in time. HR with a value of 1 indicates that the relative risk of endpoint (e.g. death) is equal in both the "treatment" and "control" groups; a value greater than 1 indicates a greater risk in the treated group relative to the control group; whereas a value less than 1 indicates a greater risk in the control group relative to the treated group. The "hazard ratio" (i.e. PFS HR) in the progression free survival assay is a summary of the difference between the two progression free survival curves, representing a reduced risk of mortality for treatment compared to control over the follow-up period. The "hazard ratio" (i.e. OS HR) in the overall survival analysis is a summary of the difference between the two overall survival curves, representing a reduced risk of mortality for treatment compared to control over the follow-up period.

By "extended survival" is meant extending the overall survival or progression-free survival in the treated individual relative to an untreated individual (i.e., relative to an individual not treated with the drug), or relative to an individual not expressing the biomarker at the specified level, and/or relative to an individual treated with an approved antineoplastic agent. An objective response refers to a measurable response, including a Complete Response (CR) or a Partial Response (PR).

By "reduce or inhibit" is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. For example, reduced or inhibited can refer to a symptom of the condition being treated (e.g., cancer, e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), the presence or size of a metastasis, or the size of a primary tumor.

As used herein, "reference sample", "reference cell", "reference tissue", "control sample", "control cell", or "control tissue" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual. In another embodiment, the reference sample is obtained from one or more individuals that are not the subject or individual. In any one of the preceding embodiments, one or more individuals from whom the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained have cancer. In certain embodiments, one or more individuals from whom the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained have cancer and have been previously treated with an anti-cancer therapy (e.g., one or more doses of a PD-L1 axis binding antagonist). In other embodiments, one or more individuals from whom the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained have cancer and have not been treated. In any of the preceding embodiments, the subject/individual and the one or more individuals that are not the subject or individual have the same cancer. 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 disease-free body part (e.g., tissue or cells) of the same subject or individual, such as healthy and/or disease-free cells or tissues adjacent to the diseased cells or tissues (e.g., cells or tissues adjacent to the tumor). In another embodiment, the reference sample is obtained from untreated tissues and/or cells of the same subject or individual's body. 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 disease-free body part (e.g., tissue or cell) of an individual that is not the subject or individual. In even another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and/or cell of the body of an individual that is not the subject or individual.

The term "sample" as used herein refers to a composition obtained or derived from a subject and/or individual of interest, which comprises cells and/or other molecular entities to be characterized and/or identified based on, for example, physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "disease sample" or variants thereof refers to any sample obtained from a subject of interest that is expected or known to contain the cellular and/or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph 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 fluids (tissue culture medium), tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

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

As used herein, "treatment" (and grammatical variations thereof, such as "treatment" or "treatment") refers to clinical intervention in an attempt to alter the natural course of the subject being treated, which may be for the purpose of prevention or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease (e.g., cancer, such as lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and sparing or improving prognosis. In some embodiments, the treatments described herein are used to delay the onset/progression of a disease (e.g., cancer, e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)), or to slow the progression of a disease. In some cases, treatment may extend Overall Survival (OS) (e.g., 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, about 96% or more, about 97% or more, about 98% or more, or about 99% or more). In some cases, the treatment may prolong OS, e.g., from about 5% to about 500%, e.g., from about 10% to about 450%, e.g., from about 20% to about 400%, e.g., from about 25% to about 350%, e.g., from about 30% to about 400%, e.g., from about 35% to about 350%, e.g., from about 40% to about 300%, e.g., from about 45% to about 250%, e.g., from about 50% to about 200%, e.g., from about 55% to about 150%, e.g., from about 60% to about 100%, e.g., from about 65% to about 100%, e.g., from about 70% to about 100%, e.g., from about 75% to about 100%, e.g., from about 80% to about 100%, e.g., from about 85% to about 100%, e.g., from about 90% to about 100. In some cases, treatment can extend Progression Free Survival (PFS) (e.g., 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, about 96% or more, about 97% or more, about 98% or more, or about 99% or more). In some cases, treatment may prolong PFS, e.g., from about 5% to about 500%, e.g., from about 10% to about 450%, e.g., from about 20% to about 400%, e.g., from about 25% to about 350%, e.g., from about 30% to about 400%, e.g., from about 35% to about 350%, e.g., from about 40% to about 300%, e.g., from about 45% to about 250%, e.g., from about 50% to about 200%, e.g., from about 55% to about 150%, e.g., from about 60% to about 100%, e.g., from about 65% to about 100%, e.g., from about 70% to about 100%, e.g., from about 75% to about 100%, e.g., from about 80% to about 100%, e.g., from about 85% to about 100%, e.g., from about 90% to about 100%.

By "tissue sample" or "cell sample" is meant a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue such as from a fresh, frozen and/or preserved organ, a tissue sample, a biopsy and/or an aspirate; blood or any blood component such as plasma; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid (interstitial fluid); cells from a subject at any time during pregnancy or development. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a disease (e.g., prostate cancer, e.g., CRPC, e.g., mCRPC, or locally restricted, non-surgical CRPC) tissue/organ. Tissue samples may contain compounds that are not naturally intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

For purposes herein, a "slice" of a tissue sample means a piece or sheet of the tissue sample, such as a thin slice of tissue or cells cut from the tissue sample. It is understood that multiple slices of the tissue sample may be made and analyzed, provided it is understood that the same slice of the tissue sample may be used for both morphological and molecular level analysis or for both polypeptide and polynucleotide analysis.

As used herein, "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancerous) and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain comprising4 conserved Framework Regions (FR) and 3 hypervariable regions (HVR). (see, e.g., Kindt et al, Kuby Immunology,6^thed., W.H.Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening libraries of complementary VL or VH domains using VH or VL domains, respectively, from antibodies that bind the antigen. See, e.g., Portolano et al, J.Immunol.150: 880-; clarkson et al, Nature352: 624-.

Diagnostic methods and assays

Provided herein are methods and assays for identifying individuals with cancer (e.g., lung cancer (e.g., non-small cell lung cancer (NSCLC)), bladder cancer (e.g., Urothelial Bladder Cancer (UBC)), kidney cancer (e.g., Renal Cell Carcinoma (RCC)), or breast cancer (e.g., Triple Negative Breast Cancer (TNBC)) that may benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). The methods and assays described herein are based on the discovery that samples from an individual can be used that contain PD-L1, CXCL9, IFNG, GZMB, CD8A, and at least one, at least two, at least three, at least four, at least five, or all six (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) to predict PD-L1 axis binding antagonist therapy, such as the discovery of therapeutic efficacy of a PD-L1 axis binding antagonist monotherapy or a combination therapy comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

Further provided herein are methods and assays for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)); a method for determining whether an individual having cancer is likely to respond to a treatment comprising a PD-L1 axis binding antagonist; a method for predicting responsiveness of an individual having cancer to a treatment comprising a PD-L1 axis binding antagonist; and methods for monitoring the response of an individual having cancer to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Any of the methods provided herein can further comprise administering to the individual a PD-L1 axis binding antagonist (e.g., as described in section III below).

A. Combination of one-gene immune score and two-gene immune score

In particular instances, the methods and assays provided herein can be used to determine the level of expression of an immune score for a single gene selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. For example, the determining step may comprise determining the expression level of any one gene selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

In some cases, the determining step comprises determining the expression level of any one gene selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and one or more additional genes associated with T effector cells, e.g., determining (i) one gene selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and (ii) one or more genes associated with T effector cells (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, 1, and/or at least one of TAP2, at least two, at least five, at least six, at least four, at least nine, at least ten, at least four, at least ten, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or nineteen), wherein one or more genes associated with T effector cells are different from one selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

In one aspect, provided herein are methods for identifying an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) that may benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining the expression level of any one gene selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a sample (e.g., a tumor tissue sample) from the individual, wherein an immune expression level of the gene selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in the sample that is higher than a reference immune expression level (e.g., an immune score of the same gene selected in the reference population is identified as identifying the individual as having a higher immune expression Individuals who can benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). Alternatively, an immune score expression level of any one of the genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of the same selected gene in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment with an antagonist comprising PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In another aspect, also provided herein are methods for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)), the method comprising determining the expression level of any one of the genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein the sample is selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and an immune score expression level of the gene for PD-1 that is higher than a reference immune score expression level (e.g., an immune score expression level of the same selected gene in a reference population) identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of any one of the genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of the same selected gene in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment with an antagonist comprising PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

The examples and embodiments described in sections II.B (i-vi), II.C (i-vi), II.D (i-vi), and II.E (i-vi) below are also specifically contemplated to apply to the one-gene immune score expression level of any one of the genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

In particular instances, the methods and assays provided herein can be used to determine the immune score expression level of two genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. For example, the determining step can include determining the expression level of any two gene combinations listed in table 1.

In some cases, the determining step comprises determining the expression level of a particular combination of two genes listed in table 1 and one or more additional T-effector cell-associated genes, e.g., determining the expression level of (i) two genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 (e.g., any one of the combinations of genes listed in table 1) and (ii) one or more T-effector cell-associated genes (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, TAP1, and/or TAP2 of at least one, at least two, at least three, at least four, at least five, at least six, at least eight, at least nine, at least ten, at least fifteen, at least sixteen, at least seventeen, or eighteen), wherein the one or more T-effector cell-associated genes are different from two genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

TABLE 1 exemplary two Gene immunization score combinations

In one aspect, provided herein are methods for identifying an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) comprising determining an expression level of a combination of two genes listed in table 1 in a sample (e.g., a tumor tissue sample) from the individual, wherein a combination of two genes listed in table 1 in the sample having an immune score expression level greater than a reference immune score expression level (e.g., an immune score expression level of a combination of two genes listed in table 1 in the reference population) identifies the individual as likely to benefit from comprising a PD-L1 axis binding antagonist (e E.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of a combination of two genes listed in table 1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of a combination of two genes listed in table 1 that is the same in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In another aspect, also provided herein is a method for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)), the method comprising determining the expression level of a combination of two genes listed in table 1 in a sample from the individual, wherein an immune score expression level of a combination of two genes listed in table 1 in the sample that is higher than a reference immune score expression level (e.g., an immune score expression level of a combination of two genes listed in table 1 that is the same in a reference population) identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of a combination of two genes listed in table 1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of a combination of two genes listed in table 1 that is the same in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

The examples and embodiments described in sections ii.b (i-vi), ii.c (i-vi), ii.d (i-vi), and ii.e (i-vi) below are also specifically contemplated to apply to the two-gene immune score expression level of any combination of two genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 as described in table 1 above.

B. Three-gene immune score combination

In particular instances, the methods and assays provided herein can be used to determine the immune score expression levels of three genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. For example, the determining step can include determining the expression level of any of the three gene combinations listed in table 2.

In some cases, the determining step comprises determining the expression level of a particular combination of three genes listed in table 2 and one or more additional T-effector cell-associated genes, e.g., determining the expression level of (i) three genes (e.g., any of the combinations of genes listed in table 2) selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and (ii) one or more T-effector cell-associated genes (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, TAP1, and/or TAP2 of at least one, at least two, at least three, at least four, at least five, at least six, at least eight, at least nine, at least ten, at least fifteen, at least sixteen, or seventeen), wherein one or more genes associated with T effector cells are different from three genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

TABLE 2 exemplary three-Gene immunization score combinations

In one aspect, provided herein are methods for identifying an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) comprising determining the expression level of a combination of three genes listed in table 2 in a sample from the individual (e.g., a tumor tissue sample), wherein a combination of three genes listed in table 2 in the sample having an immune score expression level greater than a reference immune score expression level (e.g., an immune score expression level of a combination of three genes listed in table 2 in the reference population) identifies the individual as likely to benefit from comprising a PD-L1 axis binding antagonist (e E.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of a combination of three genes listed in table 2 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of the same combination of three genes listed in table 2 in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In another aspect, also provided herein is a method for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)), the method comprising determining the expression level of a combination of the three genes listed in Table 2 in a sample from the individual, wherein an immune score expression level of the combination of the three genes listed in table 2 in the sample that is higher than a reference immune score expression level (e.g., an immune score expression level of the same combination of the three genes listed in table 2 in a reference population) identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of a combination of three genes listed in table 2 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of the same combination of three genes listed in table 2 in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

The examples and embodiments described below for the combination of genes PD-L1, CXCL9, and IFNG can also be applied to any of the three gene combinations listed in table 2.

(i) Expression of PD-L1, CXCL9, and IFNG

In particular instances, the methods and assays provided herein can be used to determine the immune score expression levels of PD-L1, CXCL9, and IFNG. Various diagnostic methods based on determining the expression levels of the immune scores for PD-L1, CXCL9, and IFNG are described further below.

In one aspect, provided herein are methods for identifying an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who may benefit from treatment with an antagonist of PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining a level of expression of PD-L1, CXCL9, and IFNG (e.g., a tumor tissue sample) in a sample from the individual, wherein a level of expression of immune scores for at least one, at least two, or all three of PD-L3526, CXCL9, and IFNG in the sample is greater than a level of expression of immune scores for a reference population (e.g., benefit of a level of expression of immune scores for PD-L1, CXCL9, IFNG in the reference population) identifying An antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In another aspect, provided herein are methods for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)), the method comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein at least one, at least two of PD-L1, CXCL9, and IFNG in the sample, or all three, above a reference immune score expression level (e.g., the immune score expression levels of PD-L1, CXCL9, and IFNG in a reference population) identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Further provided herein are methods for determining whether an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) is likely to respond to a treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining expression levels of PD-L1, CXCL9, and IFNG in a sample (e.g., a tumor tissue sample) from the individual, wherein at least one, at least two, or all three of the immune score expression levels in the sample are higher than a reference immune score expression level (e.g., the immune score expression levels of PD-L1, CXCL9, and IFNG in the reference population) indicates that the individual is likely to respond to a PD-L67 1 axis binding antagonist (e.g., a tumor cell-L Such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an expression level of the immune score for at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level for PD-L1, CXCL9, and IFNG in a reference population) indicates that the individual is unlikely to respond to a treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Further provided herein are methods for predicting responsiveness of an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) to a treatment comprising a PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining an expression level of PD-L1, CXCL9, and IFNG (e.g., tumor tissue) in a sample from the individual, wherein an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample is greater than an immune score expression level of a reference population (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in the reference population) indicates that the individual is more likely to respond to the treatment comprising a PD-L67 Treatment of PD-L1 binding antagonists (e.g., anti-PD-L1 antibodies, such as atlizumab (MPDL3280A)) or PD-1 binding antagonists (e.g., anti-PD-1 antibodies)). Alternatively, an expression level of the immune score for at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level for PD-L1, CXCL9, and IFNG in a reference population) indicates that the individual is unlikely to respond to a treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Further provided herein are methods for determining the likelihood that an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) will exhibit benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample (e.g., tumor tissue) from the individual, wherein at least one, at least two, or all three of the immune score expression levels in the sample are higher than the reference immune score expression level (e.g., the immune score expression levels of PD-L1, CXCL 26, and IFNG in the reference population) indicates that the individual will have an increased likelihood of benefit from PD- Treatment of an L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). Alternatively, an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population) indicates that the individual will have a reduced likelihood of benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In any of the foregoing methods, a recommendation may be provided to an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) based on determining the immune score expression level of PD-L1, CXCL9, and/or IFNG according to any of the above methods prior to administering a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the method further comprises providing a recommendation that the individual will likely respond to or benefit from treatment with a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). In some cases, the method comprises providing a recommendation that the therapy selected for the individual comprises treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In any of the foregoing methods, the method may further comprise administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the method further comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein the expression level of the immune score for at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in a sample from the individual is greater than the expression level of the reference immune score and (e.g., the expression level of the reference immune score is the expression level of the immune score for PD-L1, CXCL9, and IFNG in the reference population). The PD-L1 axis binding antagonist can be any PD-L1 axis binding antagonist known in the art or described herein, e.g., in section iii.f, below. For example, in some cases, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some cases, the PD-L1 binding antagonist is an antibody. In some cases, the antibody is selected from the group consisting of yw243.55.s70, MPDL3280A (atelizumab), MDX-1105, MEDI4736 (covaptumab), and MSB0010718C (avizumab). In some cases, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO. 9, the HVR-H2 sequence of SEQ ID NO. 10, and the HVR-H3 sequence of SEQ ID NO. 11; and a light chain comprising the HVR-L1 sequence of SEQ ID NO. 12, the HVR-L2 sequence of SEQ ID NO. 13, and the HVR-L3 sequence of SEQ ID NO. 14. In some cases, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 15 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 16.

In some cases, the method further comprises administering to the individual an effective amount of an additional therapeutic agent. In some cases, the additional therapeutic agent is selected from the group consisting of a cytotoxic agent, a growth inhibitory agent, radiation therapy, an anti-angiogenic agent, or a combination thereof, as described herein.

Alternatively, where the individual is determined to have an expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG that is reduced relative to a reference immune score expression level, the method can further comprise administering to the individual an effective amount of an anti-cancer therapy in place of or in addition to the PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). For example, an anti-cancer therapy that replaces or supplements a PD-L1 axis binding antagonist can include a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) alone or in addition to a PD-L1 axis binding antagonist and/or any additional therapeutic agent described herein such as a cytotoxic agent, growth inhibitory agent, radiation therapy, anti-angiogenic agent, or a combination thereof, described herein.

(ii) Increased expression levels of immune scores for PD-L1, CXCL9, and IFNG

An immune score expression level of PD-L1, CXCL9, and IFNG in a sample from an individual having cancer that is greater than or equal to a reference immune score expression level of PD-L1, CXCL9, and IFNG can indicate that the individual is more likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population.

For example, in some cases, about the top 99 th percentile (equal to or higher than about 1% prevalence level), about the top 95 th percentile (equal to or higher than about 5% prevalence level), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level) in a reference population in a sample, PD-L1 in about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level), the level of expression of the immunization score for CXCL9, and IFNG identifies the individual as an individual who is likely to benefit from treatment with an antagonist comprising PD-L1 axis binding, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, about the first 10 th to about the first 90 th percentile, about the first 20 th to about the first 80 th percentile, about the first 30 th to about the first 70 th percentile, about the first 40 th to about the first 60 th percentile, about the first 45 th to about the first 55 th percentile, about the first 48 th to about the first 52 th percentile, about the first 49.5 th to about the first 50.5 th percentile, about the first 49.9 th to about the first 50.1 th percentile of the immune score expression levels of PD-L1, CXCL9, and IFNG in the reference population in the sample, or the immune score expression levels of PD-L1, CXCL9, and IFNG in about the first 50 th percentile identify an individual as one who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). For example, in some cases, the sample has between about 10% to about 90% prevalence, about 15% to about 85% prevalence, about 20% to about 80% prevalence, about 25% to about 75% prevalence, about 30% to about 70% prevalence, about 35% to about 65% prevalence, about 40% to about 60% prevalence, about 45% to about 55% prevalence, about 48% to about 52% prevalence, about 49.5% to about 50.5% prevalence, about 49.9% to about 50.1% prevalence in a reference population, or an immune score expression level of PD-L1, CXCL9, and IFNG of about 50% prevalence identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, the expression levels of the immune scores for PD-L1, CXCL9, and IFNG in about the top 80 th percentile of the reference population (i.e., equal to or above the 20% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores for PD-L1, CXCL9, and IFNG in the sample in the first approximately 75 th percentile of the reference population (i.e., at or above the 25% prevalence level) identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores for PD-L1, CXCL9, and IFNG in about the top 50 th percentile of the reference population (i.e., equal to or above the 50% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores for PD-L1, CXCL9, and IFNG in the sample in the first 25 th percentile of the reference population (i.e., at or above the 75% prevalence level) identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores for PD-L1, CXCL9, and IFNG in about the top 20 th percentile of the reference population (i.e., equal to or above the 80% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, an immune score expression level that is higher than a reference immune score expression level refers to an overall increase in the immune score expression levels of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more of PD-L1, CXCL9, and IFNG as compared to the immune score expression levels of PD-L1, CXCL9, and IFNG in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard methods known in the art, such as those described herein. In certain instances, an expression level of an immune score that is higher than a reference immune score expression level refers to an increase in the expression level of an immune score of PD-L1, CXCL9, and IFNG in a sample, wherein the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold greater than the expression level of an immune score of PD-L1, CXCL9, and IFNG in a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue. In some cases, an expression level of an immune score that is higher than a reference immune score expression level refers to an overall increase in the expression level of the immune score of PD-L1, CXCL9, and IFNG of greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to the expression level of the immune score of PD-L1, CXCL9, and IFNG in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

In some cases, an immune score expression level of PD-L1, CXCL9, and IFNG that is higher than a reference immune score expression level refers to an overall increase in the immune score expression level of PD-L1, CXCL9, and IFNG of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more, as compared to a pre-assigned immune score expression level of PD-L1, CXCL9, and IFNG, as detected by standard methods known in the art, such as those described herein. In certain instances, an immune score expression level of PD-L1, CXCL9, and IFNG that is higher than a reference immune score expression level refers to an increase in the immune score expression level of PD-L1, CXCL9, and IFNG in the sample, wherein the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold of the pre-assigned immune score expression level of PD-L1, CXCL9, and IFNG. In some cases, an immune score expression level of PD-L1, CXCL9, and IFNG that is higher than a reference immune score expression level refers to an overall increase in the immune score expression level of PD-L1, CXCL9, and IFNG that is greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold as compared to a pre-assigned immune score expression level of PD-L1, CXCL9, and IFNG.

(iii) Reduced expression levels of immune scores for PD-L1, CXCL9, and IFNG

Immune score expression levels of PD-L1, CXCL9, and IFNG that are less than or lower than reference immune score expression levels of PD-L1, CXCL9, and IFNG in a sample from an individual having cancer can indicate that the individual is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein the reference immune score expression levels are immune score expression levels of PD-L1, CXCL9, and IFNG in a reference population.

In some cases, about the last 99 th percentile (equal to or less than about 99% prevalence level), about the last 95 th percentile (equal to or less than about 95% prevalence level), about the last 90 th percentile (equal to or less than about 90% prevalence level), about the last 85 th percentile (equal to or less than about 85% prevalence level), about the last 80 th percentile (equal to or less than about 80% prevalence level), about the last 75 th percentile (equal to or less than about 75% prevalence level), about the last 70 th percentile (equal to or less than about 70% prevalence level), about the last 65 th percentile (equal to or less than about 65% prevalence level), about the last 60 th percentile (equal to or less than about 60% prevalence level), about the last 55 th percentile (equal to or less than about 55% prevalence level) of the immune score expression level of PD-L1, CXCL9, and IFNG in the reference population in the sample, PD-L1 in about the last 50 th percentile (equal to or lower than about 50% prevalence level), about the last 45 th percentile (equal to or lower than about 45% prevalence level), about the last 40 th percentile (equal to or lower than about 40% prevalence level), about the last 35 th percentile (equal to or lower than about 35% prevalence level), about the last 30 th percentile (equal to or lower than about 30% prevalence level), about the last 25 th percentile (equal to or lower than about 25% prevalence level), about the last 20 th percentile (equal to or lower than about 20% prevalence level), about the last 15 th percentile (equal to or lower than about 15% prevalence level), about the last 10 th percentile (equal to or lower than about 10% prevalence level), about the last 5th percentile (equal to or lower than about 5% prevalence level), or about the last 1 st percentile (equal to or lower than about 1% prevalence level), the level of expression of the immunization score for CXCL9, and IFNG identifies the individual as an individual who is unlikely to benefit from treatment with an antagonist comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, about the last 10 th to about the last 90 th percentile, about the last 20 th to about the last 80 th percentile, about the last 30 th to about the last 70 th percentile, about the last 40 th to about the last 60 th percentile, about the last 45 th to about the last 55 th percentile, about the last 48 th to about the last 52 th percentile, about the last 49.5 th to about the last 50.5 th percentile, about the last 49.9 th to about the last 50.1 th percentile of the expression level of the immune scores for PD-L1, CXCL9, and IFNG in the reference population in the sample, or the immune score expression levels of PD-L1, CXCL9, and IFNG in the post-approximately 50 th percentile identify the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist, e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). For example, in some cases, the sample has between about 10% to about 90% prevalence, about 15 to about 85% prevalence, about 20% to about 80% prevalence, about 25% to about 75% prevalence, about 30% to about 70% prevalence, about 35% to about 65% prevalence, about 40% to about 60% prevalence, about 45% to about 55% prevalence, about 48% to about 52% prevalence, about 49.5% to about 50.5% prevalence, about 49.9% to about 50.1% prevalence in a reference population, or an immune score expression level of PD-L1, CXCL9, and IFNG of about 50% prevalence identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist, e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, an immune score expression level that is lower than a reference immune score expression level refers to a reduction in the immune score expression level of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more of PD-L1, CXCL9, and IFNG, as compared to the immune score expression level of PD-L1, CXCL9, and IFNG in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as measured by standard methods known in the art, such as those described herein. In certain instances, an expression level of an immune score that is lower than a reference immune score expression level refers to a reduction in the expression level of an immune score of PD-L1, CXCL9, and IFNG in a sample, wherein reducing the expression level of an immune score of PD-L1, CXCL9, and IFNG in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue 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. In some cases, an immune score expression level that is lower than a reference immune score expression level refers to a greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold reduction in the immune score expression level of PD-L1, CXCL9, and IFNG as compared to the immune score expression level of PD-L1, CXCL9, and IFNG in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

In some cases, an immune score expression level that is lower than a reference immune score expression level refers to an overall reduction in the immune score expression level of PD-L1, CXCL9, and IFNG of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more, as compared to a pre-assigned immune score expression level of PD-L1, CXCL9, and IFNG, as detected by standard methods known in the art, such as those described herein. In certain instances, an immune score expression level that is lower than a reference immune score expression level refers to a reduction in the immune score expression level of PD-L1, CXCL9, and IFNG in a sample, wherein the reduction 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 of the pre-assigned immune score expression level of PD-L1, CXCL9, and IFNG. In some cases, an immune score expression level that is lower than a reference immune score expression level refers to an overall reduction in immune score expression levels of PD-L1, CXCL9, and IFNG of greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold as compared to a pre-assigned immune score expression level of PD-L1, CXCL9, and IFNG.

(iv) Reference immune score expression levels of PD-L1, CXCL9, and IFNG

The reference immune score expression levels described herein can be based on the immune score expression levels of PD-L1, CXCL9, and IFNG in a reference population. In some cases, a reference immune score expression level described herein is an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population of individuals comprising two or more (e.g., two or more, three or more, four or more, or five or more) subsets.

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population, wherein the reference population comprises at least a subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in the reference population, wherein the reference population comprises at least one subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who have been administered one or more doses (e.g., at least one, two, three, four, five, six, seven, eight, nine, or ten or more doses) of a PD-L1 axis binding antagonist (e.g., as a monotherapy of a PD-L1 axis binding antagonist or as part of a combination therapy comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population, wherein the reference population comprises at least one subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who have been treated with a PD-L1 axis binding antagonist therapy, wherein the PD-L1 axis binding antagonist therapy is a monotherapy (e.g., a PD-L1 axis binding monotherapy comprising a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL 32A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population, wherein the reference population comprises at least one subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who have been treated with a PD-L1 axis binding antagonist therapy, wherein the PD-L1 axis binding antagonist therapy is a combination therapy (e.g., comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and an additional therapeutic agent (e.g., an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, a radiotherapy, an anti, or a combination thereof)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population, wherein the reference population comprises at least one subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who have been treated with a non-PD-L1 axis binding antagonist therapy, wherein the non-PD-L1 axis binding antagonist therapy does not include a PD-L1 axis binding antagonist and includes an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, radiation therapy, an anti-angiogenic agent, or a combination thereof))).

For example, in some cases, the reference population includes a first subset of individuals who have been treated with PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals who have been treated with non-PD-L1 axis binding antagonist therapy, wherein the non-PD-L1 axis binding antagonist therapy does not include a PD-L1 axis binding antagonist.

In some cases, the reference immune score expression levels of PD-L1, CXCL9, and IFNG are significantly separated from each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist therapy above the reference immune score expression level, wherein the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy. For example, in some cases, the reference immune score expression levels of PD-L1, CXCL9, and IFNG optimally separate each of the first and second subsets of individuals based on a maximum difference between the responsiveness of the individual to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist therapy above the reference immune score expression level, wherein the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression levels of PD-L1, CXCL9, and IFNG are significantly separated from each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist therapy below the reference immune score expression level, wherein the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy. For example, in some cases, the reference immune score expression levels of PD-L1, CXCL9, and IFNG optimally separate each of the first and second subsets of individuals based on the greatest difference between the responsiveness of the individual to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist therapy below the reference immune score expression level, wherein the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the optimal or significant separation may be based on a Hazard Ratio (HR) determined from an analysis of the expression levels of the immune scores of PD-L1, CXCL9, and IFNG in the first and second subsets of individuals, wherein HR is less than 1, e.g., an HR of about 0.95, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1 or less. For example, in a particular case, the optimal or significant separation may be based on a Hazard Ratio (HR) determined from an analysis of the expression levels of the immune scores of PD-L1, CXCL9, and IFNG in the first and second subsets of individuals, wherein the upper limit of the 95% confidence interval for HR is less than 1, e.g., the upper limit of the 95% confidence interval for HR is about 0.95, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1 or less.

Additionally or alternatively, the reference immune score expression level can be an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population, wherein the reference population comprises at least a subset of individuals that do not have cancer (e.g., individuals that do not have NSCLC, UBC, RCC, or TNBC) or that have cancer but have not been treated.

(v) Indications of

The methods described herein are useful for predicting the therapeutic response of an individual with cancer to treatment with a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, the cancer can be lung cancer, kidney cancer, bladder cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, stomach cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoides, merkel cell carcinoma, or hematological malignancy.

In some cases, the cancer may be lung cancer. For example, the lung cancer may be non-small cell lung cancer (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). For example, an individual having lung cancer (e.g., NSCLC) who may benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which include determining the immune score expression levels of PD-L1, CXCL9, and IFNG in a sample (e.g., a tumor tissue sample) from the individual, wherein the immune score expression levels of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample are greater than a reference immune score expression level (e.g., the immune score expression levels of PD-L1, CXCL9, and IFNG in the reference population) identifies the individual as likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the cancer may be bladder cancer. For example, the bladder cancer may be urothelial bladder cancer, including but not limited to non-muscle invasive urothelial bladder cancer, or metastatic urothelial bladder cancer. In some cases, the urothelial bladder cancer is metastatic urothelial bladder cancer. For example, an individual having bladder cancer (e.g., UBC) who may benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which comprise determining the immune score expression levels of PD-L1, CXCL9, and IFNG in a sample (e.g., a tumor tissue sample) from the individual, wherein the immune score expression levels of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample are higher than a reference immune score expression level (e.g., the immune score expression levels of PD-L1, CXCL9, and IFNG in the reference population) identifies the individual as likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the cancer may be renal cancer. In some cases, the renal cancer may be Renal Cell Carcinoma (RCC), including stage I RCC, stage II RCC, stage III RCC, stage IV RCC, or recurrent RCC. For example, an individual having renal cancer (e.g., RCC) who may benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which include determining the immune score expression levels of PD-L1, CXCL9, and IFNG in a sample (e.g., a tumor tissue sample) from the individual, wherein at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample have immune score expression levels that are higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population) identifies the individual as likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the cancer may be breast cancer. For example, the breast cancer can be TNBC, estrogen receptor positive breast cancer, estrogen receptor positive/HER 2 negative breast cancer, HER2 negative breast cancer, HER2 positive breast cancer, estrogen receptor negative breast cancer, progesterone receptor positive breast cancer, or progesterone receptor negative breast cancer. In some cases, the breast cancer may be TNBC. For example, an individual having breast cancer (e.g., TNBC) who may benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which include determining the immune score expression levels of PD-L1, CXCL9, and IFNG in a sample (e.g., a tumor tissue sample) from the individual, wherein at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample have immune score expression levels that are higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population) identifies the individual as likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, an individual having cancer, e.g., a cancer described herein, has not been previously treated for cancer (has not been treated). For example, in some cases, an individual with cancer has not previously received PD-L1 axis binding antagonist therapy (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). For example, in some cases, an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population) identifies an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) as an individual likely to benefit from first line therapy comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, an individual with cancer has previously received treatment for cancer. In some cases, an individual with cancer has previously received treatment comprising a non-PD-L1 axis binding antagonist therapy, such as an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, a radiation therapy, an anti-angiogenic agent, or a combination thereof). For example, in some cases, an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population) identifies an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) as an individual likely to benefit from second line therapy comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

(vi) Therapeutic benefits

An individual who benefits from being treated with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may experience, for example, delay or prevent the occurrence or recurrence of a cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), alleviate symptoms, attenuate any direct or indirect pathological consequences of the cancer, prevent metastasis, slow the rate of disease progression, improve or alleviate the disease state, or regress or improve prognosis. In some cases, the treatments described herein are used to delay the onset or slow the progression of cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)). In some cases, the benefit may be an increase in Overall Survival (OS), Progression Free Survival (PFS), Complete Response (CR), Partial Response (PR), or a combination thereof.

In some cases, an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population) identifies the individual as an individual who is likely to benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) or an individual who does not include a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein the benefit is, an increase in PFS, CR, PR, or a combination thereof.

In some cases, an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population) identifies the individual as an individual who is likely to benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) or an individual who does not include a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein the benefit is an increase in OS (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more).

In some cases, an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population) identifies the individual as an individual who is likely to benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) or an individual who does not include a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein the benefit is an increase in PFS (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more).

C. Four-gene immune score combination

In particular instances, the methods and assays provided herein can be used to determine the immune score expression levels of four genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. For example, the determining step may comprise determining the expression level of any one of the four gene combinations listed in table 3.

In some cases, the determining step comprises determining the expression level of a particular combination of four genes listed in table 3 and one or more additional T-effector cell-associated genes, e.g., determining the expression level of (i) four genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 (e.g., any one of the combinations of genes listed in table 3) and (ii) one or more T-effector cell-associated genes (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, TAP1, and/or TAP2 of at least one, at least two, at least three, at least four, at least five, at least six, at least eight, at least nine, at least ten, at least fifteen, or sixteen) wherein one or more of the T effector cell-associated genes is different from four genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

TABLE 3 exemplary four Gene immunization score combinations

Provided herein are methods for identifying an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining the expression level of any of a combination of four genes listed in table 3 in a sample from the individual (e.g., a tumor tissue sample), wherein the immune score expression level of the combination of four genes listed in table 3 in the sample is greater than a reference immune score expression level (e.g., the immune score expression level of the same combination of four genes listed in table 3 in the reference population) identifies the individual as likely to benefit from comprising a PD-L1 axis binding antagonist -treatment of an L1 binding antagonist (e.g. an anti-PD-L1 antibody, e.g. attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g. an anti-PD-1 antibody)). Alternatively, an immune score expression level of a combination of four genes listed in table 3 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of the same combination of four genes listed in table 3 in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Also provided herein are methods for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising determining the expression level of a combination of the four genes listed in table 3 in a sample from the individual, wherein an immune score expression level of the combination of four genes listed in table 3 in the sample that is higher than a reference immune score expression level (e.g., an immune score expression level of the same combination of four genes listed in table 3 in a reference population) identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of a combination of four genes listed in table 3 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of the same combination of four genes listed in table 3 in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

The examples and cases described below with respect to the combination of genes PD-L1, IFNG, GZMB, and CD8A may also be applied to any four-gene combination listed in table 3.

(i) Expression of PD-L1, IFNG, GZMB, and CD8A

The methods and assays provided herein can be used to determine the immune score expression levels of PD-L1, IFNG, GZMB, and CD 8A. Various diagnostic methods based on determining the expression levels of the immune scores for PD-L1, IFNG, GZMB, and CD8A are described further below.

Provided herein are methods for identifying an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) that may benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining expression levels of PD-L1, IFNG, GZMB, and CD8 ng A in a sample (e.g., a tumor tissue sample) from the individual, wherein expression levels of at least one, at least two, at least three, or all four of the immune scores in the sample are higher than the expression level of the reference immune score (e.g., PD-L1 in the reference population, the immune score expression levels of IFNG, GZMB, and CD8A) identifies the individual as an individual who is likely to benefit from treatment with an antagonist comprising PD-L1 axis binding, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). Alternatively, an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Also provided herein are methods for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), the methods comprising determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein at least one, at least two, at least three of PD-L1, IFNG, GZMB, and CD8A in the sample relative to a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population), or all four, identifies the individual as one who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). Alternatively, an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Further provided herein are methods for determining whether an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) is likely to respond to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the methods comprising determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample (e.g., a tumor tissue sample) from the individual, wherein the expression levels of PD-L1, IFNG, GZMB, and CD8A in the sample relative to a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the reference population), the expression levels of at least three, or all four, of the immune scores indicate that the individual is likely to respond to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population) indicates that the individual is unlikely to respond to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Further provided herein are methods for predicting responsiveness of an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the methods comprising determining expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample (e.g., tumor tissue) from the individual, wherein the expression levels of PD-L1, IFNG, GZMB, and CD8A relative to a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the reference population), or the expression levels of all four of the immune scores indicate that the individual is more likely to respond to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population) indicates that the individual is more likely to respond to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Further provided herein are methods for determining the likelihood that an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) will exhibit benefit from treatment with a PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist (e.g., anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or PD-1 binding antagonist (e.g., anti-PD-1 antibody)), comprising determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample (e.g., tumor tissue) from the individual, wherein the expression levels of at least one of PD-L1, IFNG, GZMB, and CD8A relative to a reference immune score expression level (e.g., immune score expression level of PD-L1, IFNG, zmgb, and CD A in the reference population), expression levels of at least three, or all four, of the immune scores indicate that the individual will have an increased likelihood of benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population) indicates that the individual will have a reduced likelihood of benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, a recommendation may be provided to an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) based on determining the immune score expression level of PD-L1, IFNG, GZMB, and CD8A according to any of the above methods prior to administration of a PD-L1 binding antagonist. In some cases, the method further comprises providing a recommendation that the individual will likely respond to or benefit from treatment with a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). In some cases, the method comprises providing a recommendation that the therapy selected for the individual comprises treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the method may further comprise administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) to the individual. In some cases, the method further comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein the expression level of the immune score of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A in the sample from the individual is higher than the expression level of a reference immune score (e.g., the expression level of the immune score of PD-L1, ifzmng, GZMB, and CD8A in the reference population). The PD-L1 axis binding antagonist can be any PD-L1 axis binding antagonist known in the art or described herein, e.g., in section iii.f, below. For example, in some cases, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some cases, the PD-L1 binding antagonist is an antibody. In some cases, the antibody is selected from the group consisting of yw243.55.s70, MPDL3280A (atelizumab), MDX-1105, MEDI4736 (covaptumab), and MSB0010718C (avizumab). In some cases, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO. 9, the HVR-H2 sequence of SEQ ID NO. 10, and the HVR-H3 sequence of SEQ ID NO. 11; and a light chain comprising the HVR-L1 sequence of SEQ ID NO. 12, the HVR-L2 sequence of SEQ ID NO. 13, and the HVR-L3 sequence of SEQ ID NO. 14. In some cases, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 15 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 16.

Alternatively, where the individual is determined to have an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A that is reduced relative to a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population), the method can further comprise administering to the individual an effective amount of an anti-cancer therapy that replaces or supplements the PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). For example, an anti-cancer therapy that replaces or supplements a PD-L1 axis binding antagonist can include a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) alone or in addition to a PD-L1 axis binding antagonist and/or any additional therapeutic agent described herein such as a cytotoxic agent, growth inhibitory agent, radiation therapy, anti-angiogenic agent, or a combination thereof, described herein.

(ii) Increased expression levels of immune scores for PD-L1, IFNG, GZMB, and CD8A

Immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from an individual with cancer that exceed or are above a reference immune score expression level of PD-L1, CXCL9, and/or IFNG (e.g., a reference population or pre-assigned score) may indicate that the individual is more likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

For example, in some cases, about the top 99 th percentile (equal to or higher than about 1% prevalence level), about the top 95 th percentile (equal to or higher than about 5% prevalence level), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level) of the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in a reference population in a sample, PD-L1 in about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level), the expression levels of the immune scores of IFNG, GZMB, and CD8A identify the individual as one who is likely to benefit from treatment with an antagonist comprising PD-L1 axis binding, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, the presence of PD-L1, IFNG, GZMB, and CD8A, from about the top 10 to about the top 90 percentile, from about the top 20 to about the top 80 percentile, from about the top 30 to about the top 70 percentile, from about the top 40 to about the top 60 percentile, from about the top 45 to about the top 55 percentile, from about the top 48 to about the top 52 percentile, from about the top 49.5 to about the top 50.5 percentile, from about the top 49.9 to about the top 50.1 percentile, or the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in about the first 50 th percentile identify an individual as one who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). For example, in some cases, the sample has between about 10% to about 90% prevalence, about 15% to about 85% prevalence, about 20% to about 80% prevalence, about 25% to about 75% prevalence, about 30% to about 70% prevalence, about 35% to about 65% prevalence, about 40% to about 60% prevalence, about 45% to about 55% prevalence, about 48% to about 52% prevalence, about 49.5% to about 50.5% prevalence, about 49.9% to about 50.1% prevalence in a reference population, or an immune score expression level of PD-L1, IFNG, GZMB, and CD8A of about 50% prevalence identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in about the first 80 th percentile of the reference population (i.e., at or above the 20% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in about the first 75 th percentile of the reference population (i.e., at or above the 25% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in about the first 50 th percentile of the reference population (i.e., at or above the 50% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in about the first 25 th percentile of the reference population (i.e., at or above the 75% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in about the first 20 th percentile of the reference population (i.e., at or above the 80% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, an expression level of an immune score that is higher than a reference immune score expression level refers to an overall increase in the expression levels of PD-L1, IFNG, GZMB, and CD8A of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more as compared to the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard methods known in the art, such as those described herein. In certain instances, an expression level of an immune score that is higher than an expression level of a reference immune score refers to an increase in the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample, wherein the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold greater than the expression levels of an immune score of PD-L1, IFNG, GZMB, and CD8A in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some cases, an expression level of an immune score that is higher than a reference immune score expression level refers to an overall increase in the expression levels of PD-L1, IFNG, GZMB, and CD8A in the immune score of greater than about 1.5 fold, about 1.75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 2.75 fold, about 3.0 fold, or about 3.25 fold compared to the expression levels of the immune score of PD-L1, IFNG, GZMB, and CD8A in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

In some cases, an immune score expression level of PD-L1, IFNG, GZMB, and CD8A that is higher than a reference immune score expression level refers to an overall increase in the expression level of PD-L1, IFNG, GZMB, and CD8A of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more as compared to a pre-assigned immune score expression level of PD-L1, IFNG, GZMB, and CD8A, as detected by standard methods known in the art, such as those described herein. In certain instances, an immune score expression level of PD-L1, IFNG, GZMB, and CD8A that is higher than a reference immune score expression level refers to an increase in the expression level of PD-L1, IFNG, GZMB, and CD8A in a sample, wherein the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold of the pre-assigned immune score expression level of PD-L1, IFNG, GZMB, and CD 8A. In some cases, an immune score expression level of PD-L1, IFNG, GZMB, and CD8A that is higher than a reference immune score expression level refers to an overall increase in the immune score expression level of PD-L1, IFNG, GZMB, and CD8A that is greater than about 1.5 fold, about 1.75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 2.75 fold, about 3.0 fold, or about 3.25 fold as compared to a pre-assigned immune score expression level of PD-L1, IFNG, GZMB, and CD 8A.

(iii) Reduced expression levels of immune scores for PD-L1, IFNG, GZMB, and CD8A

Immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from an individual with cancer that are less than or lower than the reference immune score expression levels of PD-L1, IFNG, GZMB, and CD8A (e.g., a score in a reference population or pre-assigned) may indicate that the individual is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL32 3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein the reference immune score expression levels are the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the reference population.

In some cases, about the last 99 th percentile (equal to or less than about 99% prevalence level), about the last 95 th percentile (equal to or less than about 95% prevalence level), about the last 90 th percentile (equal to or less than about 90% prevalence level), about the last 85 th percentile (equal to or less than about 85% prevalence level), about the last 80 th percentile (equal to or less than about 80% prevalence level), about the last 75 th percentile (equal to or less than about 75% prevalence level), about the last 70 th percentile (equal to or less than about 70% prevalence level), about the last 65 th percentile (equal to or less than about 65% prevalence level), about the last 60 th percentile (equal to or less than about 60% prevalence level), about the last 55 th percentile (equal to or less than about 55% prevalence level) of the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the reference population in the sample, PD-L1 in about the last 50 th percentile (equal to or lower than about 50% prevalence level), about the last 45 th percentile (equal to or lower than about 45% prevalence level), about the last 40 th percentile (equal to or lower than about 40% prevalence level), about the last 35 th percentile (equal to or lower than about 35% prevalence level), about the last 30 th percentile (equal to or lower than about 30% prevalence level), about the last 25 th percentile (equal to or lower than about 25% prevalence level), about the last 20 th percentile (equal to or lower than about 20% prevalence level), about the last 15 th percentile (equal to or lower than about 15% prevalence level), about the last 10 th percentile (equal to or lower than about 10% prevalence level), about the last 5 th percentile (equal to or lower than about 5% prevalence level), or about the last 1 st percentile (equal to or lower than about 1% prevalence level), the expression levels of the immune scores of IFNG, GZMB, and CD8A identify the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, the presence of PD-L1, IFNG, GZMB, and about the last 10 th to about the last 90 th percentile, about the last 20 th to about the last 80 th percentile, about the last 30 th to about the last 70 th percentile, about the last 40 th to about the last 60 th percentile, about the last 45 th to about the last 55 th percentile, about the last 48 th to about the last 52 th percentile, about the last 49.5 th to about the last 50.5 th percentile, about the last 49.9 th to about the last 50.1 th percentile of the expression level of the immune score for CD8A, or the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the later 50 th percentile, identify the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). For example, in some cases, the sample has between about 10% to about 90% prevalence, about 15 to about 85% prevalence, about 20% to about 80% prevalence, about 25% to about 75% prevalence, about 30% to about 70% prevalence, about 35% to about 65% prevalence, about 40% to about 60% prevalence, about 45% to about 55% prevalence, about 48% to about 52% prevalence, about 49.5% to about 50.5% prevalence, about 49.9% to about 50.1% prevalence in a reference population, or an immune score expression level of PD-L1, IFNG, GZMB, and CD8A of about 50% prevalence identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, an immune score expression level of PD-L1, IFNG, GZMB, and CD8A that is lower than a reference immune score expression level refers to a decrease in the expression level of PD-L1, IFNG, GZMB, and CD8A of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more as compared to the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard methods known in the art, such as those described herein. In certain instances, an immune score expression level of PD-L1, IFNG, GZMB, and CD8A that is lower than a reference immune score expression level refers to a decrease in the expression level of PD-L1, IFNG, GZMB, and CD8A in a sample, wherein decreasing the expression level of PD-L1, IFNG, GZMB, and CD8A in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue 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 greater than the immune score expression level in the reference sample, reference cell, control tissue, or control tissue. In some cases, an immune score expression level of PD-L1, IFNG, GZMB, and CD8A that is lower than a reference immune score expression level refers to a greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold decrease in the immune score expression level of PD-L1, IFNG, GZMB, and CD8A compared to the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

In some cases, an immune score expression level that is lower than a reference immune score expression level refers to an overall decrease in the expression level of PD-L1, IFNG, GZMB, and CD8A of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more as compared to the pre-assigned immune score expression level of PD-L1, IFNG, GZMB, and CD8A, as detected by standard methods known in the art, such as those described herein. In certain instances, an immune score expression level that is lower than a reference immune score expression level refers to a decrease in the expression level of PD-L1, IFNG, GZMB, and CD8A in a sample, wherein the decrease 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 of the pre-assigned immune score expression level of PD-L1, IFNG, GZMB, and CD 8A. In some cases, an immune score expression level that is lower than a reference immune score expression level refers to an overall decrease in immune score expression levels of PD-L1, IFNG, GZMB, and CD8A that is greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to the pre-assigned immune score expression levels of PD-L1, IFNG, GZMB, and CD 8A.

(iv) Reference immune score expression levels of PD-L1, IFNG, GZMB, and CD8A

The reference immune score expression levels described herein can be based on the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the reference population. In some cases, a reference immune score expression level described herein is an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population of individuals comprising two or more (e.g., two or more, three or more, four or more, or five or more) subsets.

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population, wherein the reference population comprises at least a subset of individuals with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the reference population, wherein the reference population comprises at least one subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who have been administered one or more doses (e.g., at least one, two, three, four, five, six, seven, eight, nine, or ten or more doses) of a PD-L1 axis binding antagonist (e.g., as a monotherapy of a PD-L1 axis binding antagonist or as part of a combination therapy comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population, wherein the reference population comprises at least a subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who have been treated with a PD-L1 axis binding antagonist therapy, wherein the PD-L1 axis binding antagonist therapy is a monotherapy (e.g., a PD-L1 axis binding antagonist monotherapy comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population, wherein the reference population comprises at least a subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who have been treated with a PD-L1 axis binding antagonist therapy, wherein the PD-L1 axis binding antagonist therapy is a combination therapy (e.g., comprises a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and an additional therapeutic agent (e.g., an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, radiation, anti-angiogenic agents, or combinations thereof)).

In some cases, the reference immune score expression levels of PD-L1, IFNG, GZMB, and CD8A significantly separate each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist therapy above the reference immune score expression level, wherein the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy. For example, in some cases, the reference immune score expression levels of PD-L1, IFNG, GZMB, and CD8A best separate each of the first and second subsets of individuals based on a maximum difference between the individual's responsiveness to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the individual's responsiveness to treatment with a non-PD-L1 axis binding antagonist therapy above the reference immune score expression level, wherein the individual's responsiveness to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to the individual's responsiveness to treatment with the non-PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression levels of PD-L1, IFNG, GZMB, and CD8A significantly separate each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist therapy below the reference immune score expression level, wherein the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy. For example, in some cases, the reference immune score expression levels of PD-L1, IFNG, GZMB, and CD8A best separate each of the first and second subsets of individuals based on the greatest difference between the individual's responsiveness to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the individual's responsiveness to treatment with a non-PD-L1 axis binding antagonist therapy below the reference immune score expression level, wherein the individual's responsiveness to treatment with the non-PD-L1 axis binding antagonist therapy is significantly improved relative to the individual's responsiveness to treatment with a PD-L1 axis binding antagonist therapy.

In some cases, the optimal or significant separation may be based on a Hazard Ratio (HR) determined from an analysis of the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the first and second subsets of individuals, wherein the HR is less than 1, e.g., an HR of about 0.95, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1 or less. For example, in particular instances, the optimal or significant separation may be based on a Hazard Ratio (HR) determined from an analysis of the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the first and second subsets of individuals, wherein the upper limit of the 95% confidence interval for HR is less than 1, e.g., the upper limit of the 95% confidence interval for HR is about 0.95, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1 or less.

Additionally or alternatively, the reference immune score expression level can be an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population, wherein the reference population includes at least a subset of individuals who do not have cancer (e.g., individuals who do not have NSCLC, UBC, RCC, or TNBC) or who have cancer but have not been treated.

(v) Indications of

In some cases, the cancer may be lung cancer. For example, the lung cancer may be non-small cell lung cancer (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). For example, an individual having lung cancer (e.g., NSCLC) who may benefit from treatment with a binding antagonist comprising PD-L1 axis (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which include determining the expression level of an immune score for PD-L1, IFNG, GZMB, and CD8A in a sample (e.g., a tumor tissue sample) from the individual, wherein at least one, at least two, at least three, or all four of the expression levels of an immune score for PD-L1, IFNG, GZMB, and CD8A in the sample are greater than a reference immune score expression level (e.g., an immune score for PD-L1, IFNG, GZMB, and CD8A in the reference population) identifies the individual as likely to comprise a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L A PD-L1 antibody, such as atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the cancer may be bladder cancer. For example, the bladder cancer may be urothelial bladder cancer, including but not limited to non-muscle invasive urothelial bladder cancer, or metastatic urothelial bladder cancer. In some cases, the urothelial bladder cancer is metastatic urothelial bladder cancer. For example, an individual having bladder cancer (e.g., UBC) who may benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which include determining the expression level of an immune score for PD-L1, IFNG, GZMB, and CD8A in a sample (e.g., a tumor tissue sample) from the individual, wherein at least one, at least two, at least three, or all four of the expression levels of an immune score for PD-L1, IFNG, GZMB, and CD8A in the sample are greater than a reference immune score expression level (e.g., the expression level of a PD-L1, IFNG, GZMB, and CD8A in the reference population) identifies the individual as comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 An anti-PD-L1 antibody, such as atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the cancer may be renal cancer. In some cases, the renal cancer may be Renal Cell Carcinoma (RCC), including stage I RCC, stage II RCC, stage III RCC, stage IV RCC, or recurrent RCC. For example, a method described herein can be used to identify an individual having a renal cancer (e.g., RCC) who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) comprising determining the expression level of an immune score for PD-L1, IFNG, GZMB, and CD8A in a sample (e.g., a tumor tissue sample) from the individual, wherein at least one, at least two, at least three, or all four of the expression levels of the immune score in the sample are greater than a reference immune score expression level (e.g., a reference population in which the expression level of the immune score for PD-L1, IFNG, GZMB, and CD8A is greater than a reference immune score expression level) identifies the individual as likely to comprise a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L58 A PD-L1 antibody, such as atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the cancer may be breast cancer. For example, the breast cancer can be TNBC, estrogen receptor positive breast cancer, estrogen receptor positive/HER 2 negative breast cancer, HER2 negative breast cancer, HER2 positive breast cancer, estrogen receptor negative breast cancer, progesterone receptor positive breast cancer, or progesterone receptor negative breast cancer. In some cases, the breast cancer may be TNBC. For example, an individual having breast cancer (e.g., TNBC) who may benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which include determining the expression level of an immune score for PD-L1, IFNG, GZMB, and CD8A in a sample (e.g., a tumor tissue sample) from the individual, wherein at least one, at least two, at least three, or all four of the expression levels of an immune score for PD-L1, IFNG, GZMB, and CD8A in the sample are greater than a reference immune score expression level (e.g., an immune score for PD-L1, IFNG, GZMB, and CD8A in the reference population) that identifies the individual as likely to comprise a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD- An anti-PD-L1 antibody, such as atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, an individual having cancer, e.g., a cancer described herein, has not been previously treated for cancer (has not been treated). For example, in some cases, an individual with cancer has not previously received PD-L1 axis binding antagonist therapy (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). For example, in some cases, an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population) identifies an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) as an individual likely to benefit from first-line treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, an individual with cancer has previously received treatment for cancer. In some cases, an individual with cancer has previously received treatment comprising a non-PD-L1 axis binding antagonist therapy, such as an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, a radiation therapy, an anti-angiogenic agent, or a combination thereof). For example, in some cases, an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population) identifies an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) as an individual likely to benefit from second line therapy comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

(vi) Therapeutic benefits

In some cases, an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population) identifies the individual as an individual who is likely to benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., an attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) relative to treatment without the PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., an attrituximab (MPDL3280A)) or a PD-1 binding antagonist, wherein the benefit is an increase in OS, PFS, CR, PR, or a combination thereof.

In some cases, an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population) identifies the individual as an individual who is likely to benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., an attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) relative to treatment without the PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., an attrituximab (MPDL3280A)) or a PD-1 binding antagonist, wherein the benefit is an increase in OS (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more).

In some cases, an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population) identifies the individual as an individual who is likely to benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., an attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) relative to treatment without the PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., an attrituximab (MPDL3280A)) or a PD-1 binding antagonist, wherein the benefit is an increase in PFS (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more).

D. Five-gene immune score combination

In particular instances, the methods and assays provided herein can be used to determine the immune score expression levels of five genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. For example, the determining step can include determining the expression level of any one of the five combinations of genes listed in table 4.

In some cases, the determining step comprises determining the expression levels of a particular combination of five genes listed in table 4 and one or more additional T effector-associated genes, e.g., determining the expression levels of (i) five genes (e.g., any one of the combinations of genes listed in table 4) selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and (ii) one or more T effector-associated genes (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, TAP1, and/or TAP2 of at least one, at least two, at least three, at least four, at least five, at least six, at least eight, at least ten, or fifteen) wherein one or more genes associated with the T effector cell are different from five genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

TABLE 4 exemplary five Gene immunization score combinations

PD-L1; CXCL 9; IFNG; GZMB; and CD8A
	PD-L1; CXCL 9; IFNG; GZMB; and PD-1
PD-L1; CXCL 9; IFNG; CD 8A; and PD-1
	PD-L1; CXCL 9; GZMB; CD 8A; and PD-1
PD-L1; IFNG; GZMB; CD 8A; and PD-1
	CXCL 9; IFNG; GZMB; CD 8A; and PD-1

Provided herein are methods for identifying an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who may benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) comprising determining expression levels of combinations of five genes listed in table 4 in a sample (e.g., a tumor tissue sample) from the individual, wherein a combination of five genes listed in table 4 in the sample having an immune score expression level greater than a reference immune score expression level (e.g., an immune score expression level of a combination of five genes listed in table 4 that is the same in the reference population) will identify the individual as likely to benefit from treatment with a PD-L1 axis binding antagonist (e.g., PD- An L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of a combination of five genes listed in table 4 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of a combination of five genes listed in table 4 that is the same in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Also provided herein are methods for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising determining the expression level of a combination of five genes listed in table 4 in a sample from the individual, wherein an immune score expression level of the combination of five genes listed in table 4 in the sample that is higher than a reference immune score expression level (e.g., an immune score expression level of the same combination of five genes listed in table 4 in a reference population) identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of a combination of five genes listed in table 4 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of a combination of five genes listed in table 4 that is the same in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

The examples and cases described below with respect to the combination of genes PD-L1, IFNG, GZMB, CD8A, and PD-1 may also be applied to any of the five combinations of genes listed in table 4.

(i) Expression of PD-L1, IFNG, GZMB, CD8A, and PD-1

The methods and assays provided herein can be used to determine the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1. Various diagnostic methods based on determining the expression levels of the immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1 are described further below.

Provided herein are methods for identifying an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) that may benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining the expression levels of PD-L1, IFNG, GZMB, CD8 ng A, and PD-1 in a sample (e.g., a tumor tissue sample) from the individual, wherein the immune expression levels of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8 ng 25, and PD-1 in the sample are higher than the reference immune score expression level (e.g., PD-L1 in the reference population, IFNG, GZMB, CD8A, and PD-1 immune score expression levels) identifies the individual as an individual who is likely to benefit from treatment with an antagonist comprising PD-L1 axis binding, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL32 3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). Alternatively, an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Also provided herein are methods for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising determining expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein expression levels of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample relative to a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies the individual as likely to comprise a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment with an antagonist comprising a PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Further provided herein are methods for determining whether an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) is likely to respond to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the methods comprising determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample (e.g., a tumor tissue sample) from the individual, wherein the IFNG, GZMB, CD8 ng A, and the expression level of the immune score of at least one, at least two, at least three, at least four, or all five of PD-1 indicates that the individual is likely to respond to a treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) indicates that the individual is unlikely to respond to a treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Further provided herein are methods for predicting responsiveness of an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample (e.g., tumor tissue) from the individual, wherein the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 relative to a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population), expression levels of the immune scores of at least two, at least three, at least four, or all five indicate that the individual is more likely to respond to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) indicates that the individual is more likely to respond to treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Further provided herein are methods for determining the likelihood that an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) will exhibit benefit from treatment with a PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist (e.g., anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or PD-1 binding antagonist (e.g., anti-PD-1 antibody)), comprising determining the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample (e.g., tumor tissue) from the individual, wherein the IFNG, GZMB, and PD-1 are expressed relative to a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population), the expression levels of at least one, at least two, at least three, at least four, or all five of CD8A, and PD-1, indicate that the individual will have an increased likelihood of benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). Alternatively, an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) indicates that the individual will have a reduced likelihood of benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, a recommendation may be provided to an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)) based on determining the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-L1 according to any of the above methods prior to administration of a PD-L1 binding antagonist. In some cases, the method further comprises providing a recommendation that the individual will likely respond to or benefit from treatment with a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). In some cases, the method comprises providing a recommendation that the therapy selected for the individual comprises treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the method may further comprise administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) to the individual. In some cases, the method further comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein the expression level of the immune score in a sample from the individual of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 is greater than the expression level of a reference immune score (e.g., the expression level of the immune score in the reference population of PD-L1, IFNG, GZMB, CD8A, and PD-1). The PD-L1 axis binding antagonist can be any PD-L1 axis binding antagonist known in the art or described herein, e.g., in section iii.f, below. For example, in some cases, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some cases, the PD-L1 binding antagonist is an antibody. In some cases, the antibody is selected from the group consisting of yw243.55.s70, MPDL3280A (atelizumab), MDX-1105, MEDI4736 (covaptumab), and MSB0010718C (avizumab). In some cases, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO. 9, the HVR-H2 sequence of SEQ ID NO. 10, and the HVR-H3 sequence of SEQ ID NO. 11; and a light chain comprising the HVR-L1 sequence of SEQ ID NO. 12, the HVR-L2 sequence of SEQ ID NO. 13, and the HVR-L3 sequence of SEQ ID NO. 14. In some cases, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 15 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 16.

Alternatively, where the individual is determined to have an immune score expression level that is reduced relative to a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population), the method can further comprise administering to the individual an effective amount of an anti-cancer therapy that replaces or supplements at least one, at least two, at least three, at least four, or all five of PD-L1 axis 387 binding antagonists (e.g., PD-L2 binding antagonists (e.g., anti-PD-L1 antibodies, such as attritumab (MPDL3280A)) or PD-1 binding antagonists (e.g., anti-PD-1 antibodies)). For example, an anti-cancer therapy that replaces or supplements a PD-L1 axis binding antagonist can include a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) alone or in addition to a PD-L1 axis binding antagonist and/or any additional therapeutic agent described herein such as a cytotoxic agent, growth inhibitory agent, radiation therapy, anti-angiogenic agent, or a combination thereof, described herein.

(ii) Increased expression levels of immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1

An immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from an individual with cancer that exceeds or is higher than a reference immune score expression level of PD-L1, CXCL9, and/or IFNG (e.g., a reference population or pre-assigned score) may indicate that the individual is more likely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

For example, in some cases, about the top 99 th percentile (equal to or higher than about 1% prevalence level), about the top 95 th percentile (equal to or higher than about 5% prevalence level), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level) of the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population in a sample, PD-L1 in about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level), the expression levels of IFNG, GZMB, CD8A, and PD-1 immune scores identify an individual as one who is likely to benefit from treatment with an antagonist comprising PD-L1 axis binding, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, the presence of PD-L1, IFNG, GZMB, CD8A, and about the first 10 to about the first 90 percentile, about the first 20 to about the first 80 percentile, about the first 30 to about the first 70 percentile, about the first 40 to about the first 60 percentile, about the first 45 to about the first 55 percentile, about the first 48 to about the first 52 percentile, about the first 49.5 to about the first 50.5 percentile, about the first 49.9 to about the first 50.1 percentile of the expression level of the immune score for PD-1, or about PD-L1, IFNG, GZMB, CD8A, and the immune score expression level of PD-1 identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). For example, in some cases, the sample has between about 10% to about 90% prevalence, about 15% to about 85% prevalence, about 20% to about 80% prevalence, about 25% to about 75% prevalence, about 30% to about 70% prevalence, about 35% to about 65% prevalence, about 40% to about 60% prevalence, about 45% to about 55% prevalence, about 48% to about 52% prevalence, about 49.5% to about 50.5% prevalence, about 49.9% to about 50.1% prevalence in a reference population, or about 50% prevalence of PD-L1, IFNG, GZMB, CD8A, and the immune score expression level of PD-1 identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, the expression levels of the immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1 in about the first 80 th percentile of the reference population (i.e., at or above the 20% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1 in about the first 75 th percentile of the reference population (i.e., at or above the 25% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1 in about the first 50 th percentile of the reference population (i.e., equal to or above the 50% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1 in about the first 25 th percentile of the reference population (i.e., at or above the 75% prevalence level) in the sample identify the individual as an individual who is likely to benefit from treatment with an antagonist comprising PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the expression levels of the immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1 in the first 20 th percentile of the reference population (i.e., at or above the 80% prevalence level) identify the individual as an individual who is likely to benefit from treatment with an antagonist of PD-L1 axis binding (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, an expression level of an immune score that is higher than a reference immune score expression level refers to an overall increase in the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 immune scores of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more as compared to the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 immune scores in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard methods known in the art, such as those described herein. In certain instances, an expression level of an immune score that is higher than an expression level of a reference immune score refers to an increase in the expression level of an immune score of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample, wherein the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold of the expression level of an immune score of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some cases, an expression level of an immune score that is higher than a reference immune score expression level refers to an overall increase in the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 immune scores that is greater than about 1.5 fold, about 1.75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 2.75 fold, about 3.0 fold, or about 3.25 fold greater than the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 immune scores in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

In some cases, an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is higher than a reference immune score expression level refers to an overall increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more in the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 as compared to a pre-assigned immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1, as detected by standard methods known in the art, such as those described herein. In certain instances, an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is higher than a reference immune score expression level refers to an increase in an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample, wherein the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold of a pre-assigned immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1. In some cases, an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is higher than a reference immune score expression level refers to an overall increase in the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is greater than about 1.5 fold, about 1.75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 2.75 fold, about 3.0 fold, or about 3.25 fold as compared to a pre-assigned immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1.

(iii) Reduced expression levels of immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1

Immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 that are less than or lower than reference immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 (e.g., a pre-assigned score in a reference population) in a sample from an individual with cancer may indicate that the individual is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein the reference immune score expression levels are immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

For example, in some cases, about the last 99 th percentile (equal to or less than about 99% prevalence level), about the last 95 th percentile (equal to or less than about 95% prevalence level), about the last 90 th percentile (equal to or less than about 90% prevalence level), about the last 85 th percentile (equal to or less than about 85% prevalence level), about the last 80 th percentile (equal to or less than about 80% prevalence level), about the last 75 th percentile (equal to or less than about 75% prevalence level), about the last 70 th percentile (equal to or less than about 70% prevalence level), about the last 65 th percentile (equal to or less than about 65% prevalence level), about the last 60 th percentile (equal to or less than about 60% prevalence level), about the last 55 th percentile (equal to or less than about 55% prevalence level) in a reference population in a sample, PD-L1 in about the last 50 th percentile (equal to or lower than about 50% prevalence level), about the last 45 th percentile (equal to or lower than about 45% prevalence level), about the last 40 th percentile (equal to or lower than about 40% prevalence level), about the last 35 th percentile (equal to or lower than about 35% prevalence level), about the last 30 th percentile (equal to or lower than about 30% prevalence level), about the last 25 th percentile (equal to or lower than about 25% prevalence level), about the last 20 th percentile (equal to or lower than about 20% prevalence level), about the last 15 th percentile (equal to or lower than about 15% prevalence level), about the last 10 th percentile (equal to or lower than about 10% prevalence level), about the last 5 th percentile (equal to or lower than about 5% prevalence level), or about the last 1 st percentile (equal to or lower than about 1% prevalence level), the expression levels of the IFNG, GZMB, CD8A, and PD-1 immune scores identify the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, the presence of PD-L1, IFNG, GZMB, CD8A, and about the last 10 th to about the last 90 th percentile, about the last 20 th to about the last 80 th percentile, about the last 30 th to about the last 70 th percentile, about the last 40 th to about the last 60 th percentile, about the last 45 th to about the last 55 th percentile, about the last 48 th to about the last 52 th percentile, about the last 49.5 th to about the last 50.5 th percentile, about the last 49.9 th to about the last 50.1 th percentile of the expression level of the immune score for PD-1, or about the last 50 th percentile, PD-L1, IFNG, GZMB, CD8A, and the immune score expression level of PD-1 identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). For example, in some cases, the sample has between about 10% to about 90% prevalence, about 15 to about 85% prevalence, about 20% to about 80% prevalence, about 25% to about 75% prevalence, about 30% to about 70% prevalence, about 35% to about 65% prevalence, about 40% to about 60% prevalence, about 45% to about 55% prevalence, about 48% to about 52% prevalence, about 49.5% to about 50.5% prevalence, about 49.9% to about 50.1% prevalence in a reference population, or about 50% prevalence of PD-L1, IFNG, GZMB, CD8A, and the immune score expression level of PD-1 identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is lower than a reference immune score expression level refers to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more in the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 as compared to the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard methods known in the art, such as those described herein. In some cases, an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is lower than a reference immune score expression level refers to a decrease in an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample, wherein decreasing an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue 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 greater than the immune score expression level of PD-L1, IFNG, GZMB, CD 8-A, and PD-1. In some cases, an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is lower than a reference immune score expression level refers to a greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold decrease in an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 compared to an immune score expression level of PD-1 in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

In some cases, an immune score expression level that is lower than a reference immune score expression level refers to an overall decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more in the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 as compared to the pre-assigned immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1, as detected by standard methods known in the art, such as those described herein. In certain instances, an immune score expression level that is lower than a reference immune score expression level refers to a decrease in the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample, wherein the decrease 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 of the pre-assigned immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1. In some cases, an immune score expression level that is lower than a reference immune score expression level refers to an overall decrease in the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is greater than about 1.5 fold, about 1.75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 2.75 fold, about 3.0 fold, or about 3.25 fold compared to the pre-assigned immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1.

(iv) Reference immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and/or PD-1

The reference immune score expression levels described herein can be based on the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population. In some cases, a reference immune score expression level described herein is an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population of individuals comprising two or more (e.g., two or more, three or more, four or more, or five or more) subsets.

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population, wherein the reference population comprises at least a subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population, wherein the reference population comprises at least one subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who have been administered one or more doses (e.g., at least one, two, three, four, five, six, seven, eight, nine, or ten or more doses) of a PD-L1 axis binding antagonist (e.g., as a monotherapy of a PD-L1 axis binding antagonist or as part of a combination therapy comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population, wherein the reference population comprises at least one subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who have been treated with a PD-L1 axis binding antagonist therapy, wherein the PD-L1 axis binding antagonist therapy is a monotherapy (e.g., a PD-L1 axis binding antagonist monotherapy including a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population, wherein the reference population comprises at least a subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who have been treated with a PD-L1 axis binding antagonist therapy, wherein the PD-L1 axis binding antagonist therapy is a combination therapy (e.g., comprises a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and an additional therapeutic agent (e.g., an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory, radiation therapy, anti-angiogenic agents, or combinations thereof)).

In some cases, the reference immune score expression level is an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population, wherein the reference population comprises at least a subset of individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)) that have been treated with a non-PD-L1 axis binding antagonist therapy, wherein the non-PD-L1 axis binding antagonist therapy does not include a PD-L1 axis binding antagonist and includes an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, a radiation therapy, an anti-angiogenic agent, or a combination thereof).

In some cases, the reference immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 significantly separate each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist therapy above the reference immune score expression level, wherein the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy. For example, in some cases, the reference immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 optimally separate each of the first and second subsets of individuals based on a maximum difference between the responsiveness of the individual to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist therapy above the reference immune score expression level, wherein the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 significantly separate each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist therapy below the reference immune score expression levels, wherein the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy. For example, in some cases, the reference immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 optimally separate each of the first and second subsets of individuals based on the greatest difference between the responsiveness of the individual to treatment with a PD-L1 axis binding antagonist therapy (e.g., ORR, PFS, or OS) and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist therapy below the reference immune score expression level, wherein the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the optimal or significant separation may be based on a Hazard Ratio (HR) determined from analysis of the expression levels of the immune scores of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the first and second subsets of individuals, wherein HR is less than 1, e.g., an HR of about 0.95, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1 or less. For example, in a particular case, the optimal or significant split may be based on a Hazard Ratio (HR) determined from analysis of the expression levels of the immune scores of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the first and second subsets of individuals, wherein the upper limit of the 95% confidence interval for HR is less than 1, e.g., the upper limit of the 95% confidence interval for HR is about 0.95, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1 or less.

Additionally or alternatively, the reference immune score expression level can be an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population, wherein the reference population includes at least a subset of individuals who do not have cancer (e.g., individuals who do not have NSCLC, UBC, RCC, or TNBC) or who have cancer but have not been treated.

(v) Indications of

In some cases, the cancer may be lung cancer. For example, the lung cancer may be non-small cell lung cancer (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). For example, an individual having lung cancer (e.g., NSCLC) who may benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which include determining the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample (e.g., a tumor tissue sample) from the individual, wherein at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, gb, CD8A, and PD-1 in the sample are higher than the reference immune score expression level (e.g., the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population) can identify the individual as likely to include PD-L1 A treated individual with an axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, the cancer may be bladder cancer. For example, the bladder cancer may be urothelial bladder cancer, including but not limited to non-muscle invasive urothelial bladder cancer, or metastatic urothelial bladder cancer. In some cases, the urothelial bladder cancer is metastatic urothelial bladder cancer. For example, an individual having bladder cancer (e.g., UBC) who is likely to benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which include determining the level of immune score expression of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample (e.g., a tumor tissue sample) from the individual, wherein at least one, at least two, at least three, at least four, or all five of the levels of immune expression in the sample are higher than the level of immune score expression of reference immune score expression (e.g., the levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population) identifies the individual as likely to comprise An L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the cancer may be renal cancer. In some cases, the renal cancer may be Renal Cell Carcinoma (RCC), including stage I RCC, stage II RCC, stage III RCC, stage IV RCC, or recurrent RCC. For example, an individual having a renal cancer (e.g., RCC) who is likely to benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which include determining the expression level of an immune score for PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample (e.g., a tumor tissue sample) from the individual, wherein the expression level of an immune score for at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, gb, CD8A, and PD-1 in the sample is higher than the expression level of a reference immune score (e.g., the expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population) identifies the individual as likely to comprise the immune score for PD-L1 A treated individual with an axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody).

In some cases, the cancer may be breast cancer. For example, the breast cancer can be TNBC, estrogen receptor positive breast cancer, estrogen receptor positive/HER 2 negative breast cancer, HER2 negative breast cancer, HER2 positive breast cancer, estrogen receptor negative breast cancer, progesterone receptor positive breast cancer, or progesterone receptor negative breast cancer. In some cases, the breast cancer may be TNBC. For example, an individual having breast cancer (e.g., TNBC) who may benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be identified using the methods described herein, which include determining the level of expression of an immune score for PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample (e.g., a tumor tissue sample) from the individual, wherein the level of expression of an immune score for at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, gb, CD8A, and PD-1 in the sample is higher than the level of expression of an immune score for a reference (e.g., the level of expression of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population) identifies the individual as likely to benefit from the level of expression of an immune score for the An L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, an individual having cancer, e.g., a cancer described herein, has not been previously treated for cancer (has not been treated). For example, in some cases, an individual with cancer has not previously received PD-L1 axis binding antagonist therapy (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). For example, in some cases, an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) as an individual likely to benefit from first line treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, an individual with cancer has previously received treatment for cancer. In some cases, an individual with cancer has previously received treatment comprising a non-PD-L1 axis binding antagonist therapy, such as an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, a radiation therapy, an anti-angiogenic agent, or a combination thereof). For example, in some cases, an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) as an individual likely to benefit from second line treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

(vi) Therapeutic benefits

In some cases, an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies the individual as having a potential benefit from treatment with a 387 antagonist comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) 387-L5 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL 32A)) or a PD-1 binding antagonist (e.g., anti-PD-1 antibody)), wherein the benefit is an increase in OS, PFS, CR, PR, or a combination thereof.

In some cases, an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies the individual as having a potential benefit from treatment with a 387 antagonist comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) 387-L5 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL 32A)) or a PD-1 binding antagonist (e.g., anti-PD-1 antibody)), wherein the benefit is an increase in OS (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more).

In some cases, an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 that is higher than a reference immune score expression level (e.g., an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies the individual as having a potential benefit from treatment with a 387 antagonist comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) 387-L5 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL 32A)) or a PD-1 binding antagonist (e.g., anti-PD-1 antibody)), wherein the benefit is an increase in PFS (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more).

E. Six-gene immune score combination

In particular instances, the methods and assays provided herein can be used to determine the immune score expression levels of all six of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

In some cases, the determining step comprises determining the expression levels of all six genes and one or more additional T-effector cell-associated genes, e.g., determining the expression levels of all six of (i) PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and (ii) one or more T-effector cell-associated genes (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least eleven, at least twelve, at least ten, or more) of CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, TAP1, and/or 2, wherein the T-effector cell-associated genes are different from the expression levels of four, at least ten T-effector cell-1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

Provided herein are methods for identifying an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) that may benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), the method comprising determining the expression levels of all six of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a sample (e.g., a tumor tissue sample) from the individual, wherein the expression levels of at least one, at least two, at least three, at least four, at least five, or all six of the PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in the sample are higher than the PD-expression levels of the PD-L1 in a reference immune score, CXCL9, IFNG, GZMB, CD8A, and PD-1 expression levels of the immune score) identifies an individual as one who is likely to benefit from treatment comprising a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). Alternatively, an immune score expression level of at least one, at least two, at least three, at least four, at least five, or all six of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

Also provided herein are methods for selecting a therapy for an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising determining the expression level of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein at least one, at least two, at least three, at least four, at least five, or all six of the immune score expression levels in the sample are higher than a reference immune score expression level (e.g., the immune score expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies the individual as likely to comprise a PD-L35 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Alternatively, an immune score expression level of at least one, at least two, at least three, at least four, at least five, or all six of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level (e.g., an immune score expression level of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a reference population) identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

The examples and embodiments described in sections II.B (i-vi), II.C (i-vi), II.D (i-vi), and II.E (i-vi) below are also specifically contemplated to apply to the immune score expression levels of all six of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

F. Determination of expression levels

(i) Detection method

The immune score expression level of a gene described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the combinations of genes listed in tables 1-4)) can be based on a nucleic acid expression level, preferably an mRNA expression level. The presence and/or expression level/amount of a gene described herein can be determined qualitatively and/or quantitatively based on any suitable standard known in the art, including but not limited to DNA, mRNA, cDNA, protein fragment, and/or gene copy number.

In some cases, the genes described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof) can be measured by Polymerase Chain Reaction (PCR) -based assays(e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the combinations of genes listed in tables 1-4)) nucleic acid expression levels, such as quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (RT-PCR), and reverse transcriptase quantitative PCR (RT-qPCR). Platforms for performing quantitative PCR assays include Fluidigm (e.g., BIOMARK)^TMHD System). Other amplification-based methods include, for example, transcript-mediated amplification (TMA), Strand Displacement Amplification (SDA), nucleic acid sequence-based amplification (NASBA), and signal amplification methods such as bDNA.

In some cases, the nucleic acid expression level of a gene described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)) can also be measured by sequencing-based techniques, such as, for example, RNA-seq, Sequential Analysis of Gene Expression (SAGE), high throughput sequencing techniques (e.g., massively parallel sequencing), and SequenomProvided is a technique. Nucleic acid expression levels (e.g., expression levels of at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or combinations thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, gb, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)) can also be measured, for example, by NanoString nCounter, and high coverage expression profiling (HiCEP)). Additional Protocols for assessing the status of genes and gene products can be found, for example, In Ausubel et al, eds.,1995, Current Protocols In Molecular Biology, Unit 2(Northern blot), 4(Southern blot), 15 (immunoblot) and 18(PCR analysis).

Other methods for detecting the nucleic acid level of a gene described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or combinations thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the combinations of genes listed in tables 1-4)) include protocols for examining or detecting mRNA, such as a target mRNA, in a tissue or cell sample by microarray technology. Using a nucleic acid microarray, test and control mRNA samples from the test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and location of each member of the array is known. Hybridization of a labeled probe to a particular array member indicates that the sample from which the probe was derived expresses the gene.

The primers and probes may be labeled with detectable markers, such as, for example, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes. Such probes and primers can be used to detect the presence of expressed genes in a sample, such as at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or combinations thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the gene combinations listed in tables 1-4. as will be appreciated by the skilled artisan, many different primers and probes can be prepared based on the sequences provided herein (or, in the case of genomic DNA, their adjacent sequences) and are effective for amplification, cloning, and/or determining the presence and/or expression levels of the genes described herein.

Other methods of detecting the level of nucleic acid expression of a gene described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the combinations of genes listed in tables 1-4)) include electrophoresis, Northern and Southern blot analysis, in situ hybridization (e.g., single or multiplex nucleic acid in situ hybridization), RNase protection assays, and microarrays (e.g., Illumina BEADARRAY)^TMA technique; for detecting genesExpressed Bead Array (BADGE)).

In some cases, the immune score expression levels of the genes described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or combinations thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, ifzmb, CD8A, and PD-1; or the immune score expression levels of any combination of the genes listed in tables 1-4)) can be analyzed by a variety of methodologies, including but not limited to RNA-seq, PCR, RT-qPCR, multiplex RT-qPCR,gene expression assays, microarray analysis, gene expression profiling (SAGE), Northern blot analysis, MassARRAY, ISH, and whole genome sequencing, or combinations thereof.

In still other instances, the level of expression of an immune score of a gene described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFPD-L1, IFNG, GZMN B, and CD8 85NG; PD-8A; IFL 1, IFL 3583, GZMB, CD8A, and PD-1; or the immune score expression level of any one of the combinations of genes listed in tables 1-4)).

In some cases, RT-qPCR is used to detect the immune score expression level of a gene described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or combinations thereof (e.g., the immune score expression level of PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)). For example, in some cases, RT-qPCR is used to detect the level of expression of an immune score of at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof, based on the mRNA expression level. In some cases, RT-qPCR was used to detect the expression level of the immune score based on the mRNA expression level of either of the combinations of two genes listed in table 1. In some cases, RT-qPCR was used to detect the level of immune score expression based on the mRNA expression level of any of the three combinations of genes listed in table 2 (e.g., PD-L1, IFNG, and CXCL 9). In some cases, RT-qPCR was used to detect the expression level of the immune score based on the mRNA expression level of any of the four combinations of genes listed in table 3 (e.g., PD-L1, IFNG, GZMB, and CD 8A). In some cases, RT-qPCR was used to detect immune score expression levels based on the expression levels of mRNA of any of the five genes listed in table 3 (e.g., PD-L1, IFNG, GZMB, CD8A, and PD-1). In some cases, RT-qPCR was used to detect immune score expression levels based on mRNA expression levels of all six of PD-L1, CXCL9, IFNG, GZMB, and CD 8A.

In some cases, RNA-seq is used to detect the expression level of an immune score of at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the combinations of genes listed in tables 1-4)). For example, in some cases, RNA-seq is used to detect the expression level of an immune score based on the mRNA expression level of any of the combinations of PD-L1, CXCL9, IFNG, GZMB, or one of CD 8A. In some cases, RNA-seq is used to detect the expression level of an immune score based on the mRNA expression level of either of the combinations of two genes listed in table 1. In some cases, RNA-seq is used to detect the level of expression of an immune score based on the mRNA expression level of any of the three combinations of genes listed in table 2 (e.g., PD-L1, IFNG, and CXCL 9). In some cases, RNA-seq was used to detect the expression level of an immune score based on the mRNA expression level of any of the four combinations of genes listed in table 3 (e.g., PD-L1, IFNG, GZMB, and CD 8A). In some cases, RNA-seq was used to detect the expression level of an immune score based on the mRNA expression level of any of the five genes listed in table 4 (e.g., PD-L1, IFNG, GZMB, CD8A, and PD-1). In some cases, RNA-seq was used to detect immune score expression levels based on mRNA expression levels of all six of PD-L1, CXCL9, IFNG, GZMB, and CD 8A.

(ii)RT-qPCR

In some cases, reverse transcription quantitative polymerase chain reaction (RT-qPCR) can be used to detect the level of nucleic acid expression of a gene described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)). The RT-qPCR technique is a form of PCR in which the nucleic acid to be amplified is RNA, which is first reverse transcribed into cDNA and the amount of PCR product is measured at each step of the PCR reaction. Since RNA cannot serve as a template for PCR, the first step in gene expression profiling by PCR is reverse transcription of the RNA template into cDNA, followed by its amplification in a PCR reaction. For example, the reverse transcriptase may comprise avian myeloblastosis Virus reverse transcriptase (AMV-RT) or Moloney murine leukemia Virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamer, or oligo dT primers, depending on the circumstances and goals of the expression profiling. For example, GENEAMP can be used following the manufacturer's instructions^TMThe extracted RNA was reverse transcribed by RNA PCR kit (Perkin Elmer, Calif, USA). The derived cDNA can then be used as a template in subsequent PCR reactions.

One variation of the PCR technique is quantitative real-time PCR (qRT-PCR), which generates probes (i.e., dual-labeled fluorescence)Probe) to measure PCR product accumulation. Quantitative real-time polymerase chain reaction technology 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 a number of publications including Cronin et al, am.j.pathol.164(1)35-42 (2004); and Ma et al, Cancer Cell 5: 607-. Real-time PCR is compatible with both quantitative competitive PCR (in which normalization is performed using internal competitors for each target sequence) and/or quantitative comparative PCR (PCR is performed using normalization genes or housekeeping genes contained within the sample). For more details see, for example, Held et al, Genome Research 6: 986-.

Various published journal papers show steps of representative protocols for gene expression profiling using fixed, paraffin-embedded tissues as a source of RNA, including mRNA isolation, purification, primer extension and amplification (e.g., godfreyeet al, molecular diagnostics 2:84-91 (2000); Specht et al, am.j. pathol.158:419-29 (2001)). Briefly, one representative method begins by cutting out a section of a paraffin-embedded tumor tissue sample (approximately 10 micron section). Then, RNA is extracted, and proteins and DNA are removed. After analysis of the RNA concentration, RNA repair and/or amplification steps may be included, if necessary, and the RNA is reverse transcribed using gene specific promoters, followed by PCR.

The level of nucleic acid expression determined by an amplification-based method (e.g., RT-qPCR) can be expressed as a cycle threshold (Ct). From this value, the normalized expression level of each gene can be determined, for example, using the Δ Ct (dCt) method, Ct (control/reference gene) -Ct (gene/target gene of interest) ═ dCt (gene/target gene of interest). Those skilled in the art will appreciate that the resulting dCt value may be a negative dCt value or a positive dCt value. As defined herein, a higher dCt value indicates a higher expression level of the gene of interest relative to a control gene. Conversely, a lower dCt value indicates a lower expression level of the gene of interest relative to the control gene. In cases where the expression levels of multiple genes have been determined, the expression level of each gene (e.g., expressed as a dCt value) can then be used to determine a single value (e.g., an immune score expression level) that represents the aggregate or composite expression level of the multiple genes. The immune score expression level can be the mean or median of the dCt values determined for each target/gene of interest. Thus, in some instances, the immune score expression level described herein can be the mean or median of dCt values determined for at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the combinations of genes listed in tables 1-4). As defined herein, a higher mean dCt or median dCt value indicates a higher aggregate expression level of the plurality of target genes relative to the control gene (or genes). A lower mean dCt or median dCt value indicates a lower level of expression of the plurality of target genes in total relative to the control gene (or genes). As described herein, the immune score expression level may then be compared to a reference immune score expression level as further defined herein.

In one particular case, the expression level of a nucleic acid described herein can be determined using a method comprising the steps of:

(a) obtaining or providing a sample from an individual, wherein the sample comprises a tumor tissue sample (e.g., a paraffin-embedded, formalin-fixed NSCLC, UBC, RCC, or TNBC tumor tissue sample);

(b) isolating mRNA from the sample;

(c) performing reverse transcription of mRNA into cDNA (e.g., for at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the combinations of genes listed in tables 1-4));

(d) amplifying cDNA (e.g., for at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4)) using PCR; and

(e) quantifying the level of nucleic acid expression (e.g., for at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4)).

One or more genes (e.g., one, two, three, four, five, or six genes selected from PD-L1, IFNG, GZMB, CD8A, CXCL9, or PD-1) can be detected in a single assay, depending on the primers or probes used. Also, assays may be performed between one or more tubes (e.g., one, two, three, four, five, or six or more tubes).

In some cases, the method further comprises (f) normalizing the nucleic acid expression level of a gene (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)) in the sample to the expression level of one or more reference genes (e.g., one, two, three, four, five, six, seven, eight, nine, or more reference genes, e.g., a housekeeping gene (e.g., TMEM 55B)). For example, the immune score expression levels of the genes described herein ((e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the gene combinations listed in tables 1-4)) can be analyzed using RT-qPCR to generate immune score expression levels that reflect the normalized average dCT value of the genes analyzed.

(iii)RNA-seq

In some cases, RNA-seq can be used to detect the level of nucleic acid expression of a gene described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)). RNA-seq, also known as Whole Transcriptome Shotgun Sequencing (WTSS), refers to sequencing and/or quantifying cDNA using high-throughput sequencing techniques to obtain information about the RNA content of a sample. Publications describing RNA-Seq include Wang et al, "RNA-Seq: arevolutionway tools for transcriptitomics", Nature Reviews Genetics 10(1):57-63(January 2009); ryan et al, BioTechniques 45(1):81-94 (2008); and Maher et al, "transaction sequencing to detection gene fusions in cancer", Nature 458(7234):97-101(January 2009).

(iv) Sample (I)

Samples may be taken from individuals suspected of having or diagnosed with cancer and thus likely in need of treatment, or from healthy individuals who are not suspected of having cancer or not but who have a family history of cancer. For assessing gene expression, samples (such as those containing cells, or proteins or nucleic acids produced by such cells) can be used in the methods of the invention. The expression level of a gene can be determined by assessing the amount (e.g., absolute amount or concentration) of a marker in a sample (e.g., a tissue sample, e.g., a tumor tissue sample, such as a biopsy). In addition, the level of the gene can be assessed in a bodily fluid or excretion containing a detectable level of the gene. Body fluids or excretions useful as samples in the present invention include, for example, blood, urine, saliva, stool, pleural fluid, lymph, sputum, ascites, prostatic fluid, cerebrospinal fluid (CSF), or any other bodily secretion or derivative thereof. The word blood/blood is intended to include whole blood, plasma, serum, or any derivative of blood. Assessing genes in such bodily fluids or excreta may sometimes be preferred in situations where invasive sampling methods are inappropriate or inconvenient. In other embodiments, tumor tissue samples are preferred.

The sample may be frozen, fresh, fixed (e.g., formalin fixed), centrifuged, and/or embedded (e.g., paraffin embedded), etc. The cell sample may be subjected to a variety of well-known post-collection preparation and storage techniques (e.g., nucleic acid and/or protein extraction, immobilization, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of marker in the sample. Likewise, biopsies may also be submitted for collection for preparation and storage techniques, e.g., fixation, such as formalin fixation.

In one particular case, the sample is a clinical sample. In another aspect, the sample is used in a diagnostic assay, such as a diagnostic assay or diagnostic method of the invention. In some cases, the sample is obtained from a primary or metastatic tumor. Tissue biopsies are often used to obtain representative blocks of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of a tissue or fluid known or believed to contain tumor cells of interest. For example, samples of lung cancer lesions may be obtained by resection, bronchoscopy, fine needle aspiration, bronchial lavage, or from sputum, pleural fluid, or blood. The gene or gene product can be detected from cancer or tumor tissue or from other body samples, such as urine, sputum, serum or plasma. The same techniques discussed above with respect to detecting target genes or gene products in cancerous samples can be applied to other body samples. Cancer cells may shed from cancer lesions and appear in such body samples. By screening such body samples, a simple early diagnosis of these cancers can be achieved. In addition, the progress of therapy can be more easily monitored by testing such body samples for target genes or gene products.

In some cases, the sample from the individual is a tissue sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some cases, the sample is a tissue sample. In some cases, the sample is a tumor tissue sample. In some cases, the sample is obtained prior to treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL 3280A)). In some cases, the tissue sample is a formalin-fixed and paraffin-embedded (FFPE) sample, an archived sample, a fresh sample, or a frozen sample.

In some cases, the sample from the individual is a tissue sample. In some cases, the tissue sample is a tumor tissue sample (e.g., biopsy). In some cases, the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, or a combination thereof. In some cases, the tissue sample is lung tissue. In some cases, the tissue sample is bladder tissue. In some cases, the tissue sample is kidney tissue. In some cases, the tissue sample is breast tissue. In some cases, the tissue sample is skin tissue. In some cases, the tissue sample is pancreatic tissue. In some cases, the tissue sample is stomach tissue. In some cases, the tissue sample is esophageal tissue. In some cases, the tissue sample is mesothelial tissue. In some cases, the tissue sample is thyroid tissue. In some cases, the tissue sample is colorectal tissue. In some cases, the tissue sample is head or neck tissue. In some cases, the tissue sample is osteosarcoma tissue. In some cases, the tissue sample is prostate tissue. In some cases, the tissue sample is ovarian tissue, HCC (liver), blood cells, lymph nodes, or bone/bone marrow.

In some cases, the tumor tissue sample is extracted from a malignant cancerous tumor (i.e., cancer). In some cases, the cancer is a solid tumor, or a non-solid or soft tissue tumor. Examples of soft tissue tumors include leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, adult acute lymphoblastic leukemia, acute myelogenous leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, polymorphocellular leukemia (polymorphocellular leukemia), or hairy cell leukemia) or lymphoma (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). Solid tumors include any cancer of body tissues other than blood, bone marrow, or lymphatic system. Solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin. Examples of epithelial solid tumors include tumors of the gastrointestinal tract, colon, colorectal (e.g., basal-like colorectal cancer), breast, prostate, lung, kidney, liver, pancreas, ovary (e.g., endometrioid ovarian cancer), head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, labia, nasopharynx, skin, uterus, genitourinary organs, urinary organs (e.g., urothelial cancer, dysplastic urothelial cancer, transitional cell cancer), bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors. In some cases, the cancer is non-small cell lung cancer (NSCLC). In some cases, the cancer is second or third line locally advanced or metastatic non-small cell lung cancer. In some cases, the cancer is adenocarcinoma. In some cases, the cancer is squamous cell carcinoma.

(v) RNA extraction

mRNA can be isolated from a target sample prior to detecting the level of nucleic acid. In some cases, the mRNA is total RNA isolated from a tumor or tumor cell line, or, alternatively, a normal tissue or cell line. RNA can be isolated from a variety of tumor tissues, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, stomach, gall bladder, spleen, thymus, testis, ovary, uterus, etc., corresponding normal tissues, or tumor cell lines. If the source of the mRNA is a primary tumor, the mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g., formalin-fixed) tissue samples. General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular Biology, including Ausubel et al, Current Protocols of molecular Biology, John Wiley and Sons (1997). Methods for extracting RNA from paraffin-embedded tissue are disclosed, for example, in Rupp and Locker, Lab invest.56: A67(1987), and De Andres et al, BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kits, buffer kits, and proteases from manufacturers, such as Qiagen, following the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits includeComplete DNA and RNA purification kit (Madison, Wis.), and paraffin block RNA isolation kit (Ambion, Inc.). Total RNA from tissue samples can be isolated, for example, by using RNA Stat-60 (TelTest). RNA prepared from tumor tissue samples can also be isolated, for example, by cesium chloride density gradient centrifugation.

(vi) Immune score expression level

The immune score expression level can reflect the expression level of one or more genes described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)). In some cases, to determine the immune score expression level, the detected expression level of each gene is normalized using any standard normalization method known in the art. One skilled in the art will appreciate that the normalization method used may depend on the gene expression methodology used (e.g., one or more housekeeping genes may be used for normalization in the context of RT-qPCR methodology, but a whole genome or substantially whole genome may be used as a normalization baseline in the context of RNA-seq methodology). For example, the detected expression level of each gene assayed can be normalized to both the difference in the amount of gene assayed, the variability in the mass of the sample used, and/or the variability between assay runs.

In some cases, normalization can be achieved by detecting the expression of certain one or more normalization genes, including a reference gene (e.g., a housekeeping gene (e.g., TMEM 55B)). For example, in some cases, the expression levels of a nucleic acid detected using the methods described herein (e.g., for at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)) can be normalized to the expression level of one or more reference genes (e.g., one, two, three, four, five, six, seven, eight, nine, or more reference genes, e.g., a housekeeping gene (e.g., TMEM 55B)). Alternatively, normalization can be based on the mean or median signal of all genes assayed. The measured normalized amount of test tumor mRNA can be compared to the amount found in a reference immune score expression level on a gene-by-gene basis. The presence and/or expression level/amount measured in the particular test sample to be analyzed will fall within some percentile of this range, which may be determined by methods well known in the art.

In other cases, the detected expression level of each gene assayed is not normalized in order to determine the immune score expression level.

The immune score expression level can reflect the aggregate or comprehensive expression level of a single gene or multiple genes described herein (e.g., for at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)). Any statistical approach known in the art can be used to determine the level of expression of an immune score.

For example, the immune score expression level can reflect a median expression level, a mean expression level, or a numerical value reflecting an aggregate Z-score expression level of the determined gene combinations (e.g., for at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the gene combinations listed in tables 1-4)).

In some cases, the immune score expression level reflects a median normalized expression level, a mean normalized expression level, or a numerical value that reflects an aggregate Z-score normalized expression level of the measured gene combinations (e.g., for at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the gene combinations listed in tables 1-4)).

For example, the immune score expression level may reflect the average (mean) of the expression levels of each of the genes in the combination of two genes listed in table 1. In some cases, the immune score expression level reflects the average (mean) of the normalized expression levels of each of the two genes listed in table 1 in the combination (e.g., normalized against a reference gene, e.g., a housekeeping gene, e.g., TMEM 55B). In some cases, the immune score expression level reflects the median value of the expression level of each gene in the combination of two genes listed in table 1. In some cases, the immune score expression level reflects the median of the normalized expression levels for each of the two genes listed in table 1 in the combination (e.g., normalized against a reference gene, e.g., a housekeeping gene, e.g., TMEM 55B). In some cases, the immune score expression level reflects the Z-score of each gene in the combination of two genes listed in table 1. In some cases, the immune score expression level is a numerical value reflecting the aggregate Z-score expression level of the combination of two genes listed in table 1.

For example, the immune score expression level can reflect the average (mean) of the expression levels of each of the three genes listed in table 2 in the combination (e.g., each of PD-L1, CXCL9, and IFNG). In some cases, the immune score expression level reflects the average (mean) of the normalized expression levels (e.g., normalized against a reference gene, e.g., a housekeeping gene, e.g., TMEM55B) for each of the three gene combinations listed in table 2 (e.g., each of PD-L1, CXCL9, and IFNG). In some cases, the immune score expression level reflects the median of the expression levels of each of the three gene combinations listed in table 2 (e.g., each of PD-L1, CXCL9, and IFNG). In some cases, the immune score expression level reflects the median of the normalized expression levels (e.g., normalized against a reference gene, e.g., a housekeeping gene, e.g., TMEM55B) for each of the three gene combinations listed in table 2 (e.g., each of PD-L1, CXCL9, and IFNG). In some cases, the immune score expression level reflects the Z-score of each of the three gene combinations listed in table 2 (e.g., each of PD-L1, CXCL9, and IFNG). In some cases, the immune score expression level is a numerical value reflecting the aggregate Z-score expression level of the combination of three genes listed in table 2 (e.g., each of PD-L1, CXCL9, and IFNG).

In another specific case, the immune score expression level may reflect the average (mean) of the expression levels of each of the four gene combinations listed in table 3 (e.g., each of PD-L1, IFNG, GZMB, and CD 8A). In some cases, the immune score expression level reflects the average (mean) of the normalized expression levels (e.g., normalized against a reference gene, e.g., a housekeeping gene, e.g., TMEM55B) for each of the four gene combinations listed in table 3 (e.g., each of PD-L1, IFNG, GZMB, and CD 8A). In some cases, the immune score expression level reflects the median of the expression levels of each of the four gene combinations listed in table 3 (e.g., each of PD-L1, IFNG, GZMB, and CD 8A). In some cases, the immune score expression level reflects the median of the normalized expression levels (e.g., normalized against a reference gene, e.g., a housekeeping gene, e.g., TMEM55B) for each of the four gene combinations listed in table 3 (e.g., each of PD-L1, IFNG, GZMB, and CD 8A). In some cases, the immune score expression level reflects the Z-score for each gene in the four gene combinations listed in table 3 (e.g., each of PD-L1, IFNG, GZMB, and CD 8A). In some cases, the immune score expression level is a numerical value reflecting the aggregate Z-score expression level of the four gene combinations listed in table 3 (e.g., each of PD-L1, IFNG, GZMB, and CD 8A).

In yet another instance, the immune score expression level reflects the average (mean) of the expression levels of each of the genes (e.g., each of PD-L1, IFNG, GZMB, CD8A, and PD-1) in the combination of five genes listed in table 4. In some cases, the immune score expression level reflects the average (mean) of the normalized expression levels (e.g., normalized against a reference gene, e.g., a housekeeping gene, e.g., TMEM55B) for each of the five gene combinations listed in table 4 (e.g., each of PD-L1, IFNG, GZMB, CD8A, and PD-1). In some cases, the immune score expression level reflects the median of the expression levels of each of the genes (e.g., each of PD-L1, IFNG, GZMB, CD8A, and PD-1) in the combination of five genes listed in table 4. In some cases, the immune score expression level reflects the median of the normalized expression levels (e.g., normalized against a reference gene, e.g., a housekeeping gene, e.g., TMEM55B) for each of the five gene combinations listed in table 4 (e.g., each of PD-L1, IFNG, GZMB, CD8A, and PD-1). In some cases, the immune score expression level reflects the Z-score of each gene (e.g., each of PD-L1, IFNG, GZMB, CD8A, and PD-1) in the combination of five genes listed in table 4. In some cases, the immune score expression level is a numerical value reflecting the aggregate Z-score expression level of the combination of five genes listed in table 4 (e.g., each of PD-L1, IFNG, GZMB, CD8A, and PD-1).

In yet another instance, the immune score expression level reflects the mean (mean) of the expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1. In some cases, the immune score expression level reflects the mean (average) of the normalized expression levels for each of PD-L1, IFNG, GZMB, CD8A, and PD-1 (e.g., normalized against a reference gene, e.g., a housekeeping gene, e.g., TMEM 55B). In some cases, the immune score expression level reflects the median of the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1. In some cases, the immune score expression level reflects the median of the normalized expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 (e.g., normalized against a reference gene, e.g., a housekeeping gene, e.g., TMEM 55B). In some cases, the immune score expression level reflects the Z score of PD-L1, IFNG, GZMB, CD8A, and PD-1. In some cases, the immune score expression level is a numerical value reflecting the aggregate Z-score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1.

(vii) Reference immune score expression level

The reference immune score expression level can be a value derived from analysis of any reference population described herein. In some cases, the reference immune score expression level may be a "cut-off" value selected based on dividing the reference population into subsets, e.g., subsets that exhibit a significant difference (e.g., a statistically significant difference) in therapeutic response to PD-L1 axis binding antagonist therapy and non-PD-L1 axis binding antagonist therapy. In such cases, relative treatment response, expressed as, for example, Hazard Ratio (HR), may be assessed based on Progression Free Survival (PFS) or Overall Survival (OS) (e.g., progression free survival HR (PFS HR) or overall survival HR (OS HR)).

In certain instances, the reference immune score expression level is that of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in a reference population that has been separated from a population of reference populations in which the PD-L1 binding antagonist has been used (e.g., a statistically significant PD-1 binding antagonist has been used) (e.g., a statistically significant PD-1 binding antagonist has been used) (e.g., a PD-L1 binding antagonist is used) based on the immune score expression level that is higher than the reference immune score expression level (i.g., higher than the cutoff) of IFNG, GZMB, CD8, CD A, and PD-1, or any combination of genes listed in tables 1-4) Such as an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population that have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is that of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that best separates the reference population already with PD-L23 (e.g., PD-L1 binding antagonist (e.g., anti-PD-L1 binding antagonist) (e.g., PD-L1 binding antagonist) based on a substantial difference between the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist and the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist) above the reference immune score expression level (i.g., above cutoff) A first subset of individuals treated with a PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy.

In certain particular instances, the reference immune score expression level is the maximum difference between at least one, at least two, at least three, at least four, at least five, or all six genes, or combinations thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the combinations of genes listed in tables 1-4) in the reference population that optimally separates the PD-1 binding antagonist (e.g., anti-PD-L1 binding) in the reference population with the PD-L1 axis based on the responsiveness of the individual above the reference immune score expression level (i.e., above the cutoff) to treatment with the PD-L1 axis binding antagonist and the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist (e.g., PD-L1 binding anti-PD-L1 binding antagonist) A first subset of individuals treated with an L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy.

In certain instances, the reference immune score expression level is that of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in a reference population that has been separated by a significant (e.g., a statistically significant) difference between the responsiveness of the individual below the reference immune score expression level (i.e., below cutoff) to treatment with a PD-L1 axis binding antagonist and the responsiveness of the individual to treatment with a non-PD-L1 axis binding antagonist (e.g., PD-1 axis binding antagonist) in the reference population that has been treated with a PD-L1 (e.g., PD-1 axis binding antagonist) Such as an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is the maximum difference between at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the combinations of genes listed in tables 1-4)) in the reference population that most optimally separates the PD-L1 (e.g., anti-PD-L1) axis binding antagonist from the reference population based on the responsiveness of a substantial individual below the reference immune score expression level (i.e., below cutoff) to treatment with the PD-L1 axis binding antagonist and the responsiveness of an individual to treatment with the non-PD-L1 axis binding antagonist (e.g., anti-PD-L1 axis binding antagonist) A first subset of individuals treated with a PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy.

In certain particular instances, the reference immune score expression level is that of at least one, at least two, at least three, at least four, at least five, or all six genes, or combinations thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that best separates the PD-L1 binding (e.g., anti-PD-L1 binding) axis of the PD-L1 population from the PD-L1 binding axis of the reference antagonist based on the maximum difference between the responsiveness of the individual below the reference immune score expression level (i.e., below cutoff) to treatment with the PD-L1 axis binding antagonist and the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist (e.g., anti-PD-L1 binding) A first subset of individuals treated with an L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is defined by an immune score expression level having a certain prevalence in the reference population. For example, in certain instances, the reference immune score expression level is that of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that is significantly (e.g., statistically significantly) separate a PD-L637 axis binding antagonist (e.g., PD-L1 binding antibody (e.g., anti-PD-L1 binding antibody), e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, at least one, at least two, at least three, at least four, at least five, or all six genes in the reference population selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4)), about the top 99 th percentile (equal to or higher than about 1% prevalence level), about the top 95 th percentile (equal to or higher than about 5% prevalence level), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 45% prevalence level), about the first 50 percentile (equal to or greater than about 50% prevalence level), about the first 45 percentile (equal to or greater than about 55% prevalence level), about the first 40 percentile (equal to or greater than about 60% prevalence level), about the first 35 percentile (equal to or greater than about 65% prevalence level), about the first 30 percentile (equal to or greater than about 70% prevalence level), about the first 25 percentile (equal to or greater than about 75% prevalence level), about the first 20 percentile (equal to or greater than about 80% prevalence level), about the first 15 percentile (equal to or greater than about 85% prevalence level), about the first 10 percentile (equal to or greater than about 90% prevalence level), about the first 5 percentile (equal to or greater than about 95% prevalence level), or about the first 1 percentile (equal to or greater than about 99% prevalence level), wherein the responsiveness of an individual to a PD-L1 axis antagonist binding therapy is relative to the responsiveness to non-individual to non-response to non-therapeutic treatment with a PD-L1 axis antagonist binding therapy The responsiveness of treatment with PD-L1 axis binding antagonist therapy was significantly improved.

In some cases, the reference immune score expression level is the level of immune score expression of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that most closely separates the level of immune score expression in the reference population that has been treated with a PD-L637 axis binding antagonist (e.g., PD-L1-1 binding antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, at least one, at least two, at least three, at least four, at least five, or all six genes in the reference population selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4)), about the top 99 th percentile (equal to or higher than about 1% prevalence level), about the top 95 th percentile (equal to or higher than about 5% prevalence level), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 45% prevalence level), about the first 50 percentile (equal to or greater than about 50% prevalence level), about the first 45 percentile (equal to or greater than about 55% prevalence level), about the first 40 percentile (equal to or greater than about 60% prevalence level), about the first 35 percentile (equal to or greater than about 65% prevalence level), about the first 30 percentile (equal to or greater than about 70% prevalence level), about the first 25 percentile (equal to or greater than about 75% prevalence level), about the first 20 percentile (equal to or greater than about 80% prevalence level), about the first 15 percentile (equal to or greater than about 85% prevalence level), about the first 10 percentile (equal to or greater than about 90% prevalence level), about the first 5 percentile (equal to or greater than about 95% prevalence level), or about the first 1 percentile (equal to or greater than about 99% prevalence level), wherein the responsiveness of an individual to a PD-L1 axis antagonist binding therapy is relative to the responsiveness to non-individual to non-response to non-therapeutic treatment with a PD-L1 axis antagonist binding therapy The responsiveness of treatment with PD-L1 axis binding antagonist therapy was significantly improved.

In certain particular instances, the reference immune score expression level is the level of immune score expression of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that best separates the immune score expression levels in the reference population that have been treated with a PD-L637 axis binding antagonist (e.g., PD-L1 binding antagonist (e.g., anti-PD-L1 antibody), e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, at least one, at least two, at least three, at least four, at least five, or all six genes in the reference population selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4)), about the top 99 th percentile (equal to or higher than about 1% prevalence level), about the top 95 th percentile (equal to or higher than about 5% prevalence level), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 45% prevalence level), about the first 50 percentile (equal to or greater than about 50% prevalence level), about the first 45 percentile (equal to or greater than about 55% prevalence level), about the first 40 percentile (equal to or greater than about 60% prevalence level), about the first 35 percentile (equal to or greater than about 65% prevalence level), about the first 30 percentile (equal to or greater than about 70% prevalence level), about the first 25 percentile (equal to or greater than about 75% prevalence level), about the first 20 percentile (equal to or greater than about 80% prevalence level), about the first 15 percentile (equal to or greater than about 85% prevalence level), about the first 10 percentile (equal to or greater than about 90% prevalence level), about the first 5 percentile (equal to or greater than about 95% prevalence level), or about the first 1 percentile (equal to or greater than about 99% prevalence level), wherein the responsiveness of an individual to a PD-L1 axis antagonist binding therapy is relative to the responsiveness to non-individual to non-response to non-therapeutic treatment with a PD-L1 axis antagonist binding therapy A significant (e.g., statistically significant) improvement in responsiveness of treatment with the PD-L1 axis binding antagonist therapy.

In certain instances, the reference immune score expression level is that of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in a reference population that separates (e.g., statistically significantly) the level of immune score expression in the reference population that has been bound with a PD-L antagonist (e.g., PD-L1-5392 binding antibody (e.g., anti-PD-L1 antibody), e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, at least one, at least two, at least three, at least four, at least five, or all six genes in the reference population selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4)) about the last 99 th percentile (equal to or less than about 99% prevalence level), about the last 95 th percentile (equal to or less than about 95% prevalence level), about the last 90 th percentile (equal to or less than about 90% prevalence level), about the last 85 th percentile (equal to or less than about 85% prevalence level), about the last 80 th percentile (equal to or less than about 80% prevalence level), about the last 75 th percentile (equal to or less than about 75% prevalence level), about the last 70 th percentile (equal to or less than about 70% prevalence level), about the last 65 th percentile (equal to or less than about 65% prevalence level), about the last 60 th percentile (equal to or less than about 60% prevalence level), about the last 55 th percentile (equal to or less than about 55% prevalence level), about the posterior 50 th percentile (equal to or less than about 50% prevalence level), about the posterior 45 th percentile (equal to or less than about 45% prevalence level), about the posterior 40 th percentile (equal to or less than about 40% prevalence level), about the posterior 35 th percentile (equal to or less than about 35% prevalence level), about the posterior 30 th percentile (equal to or less than about 30% prevalence level), about the posterior 25 th percentile (equal to or less than about 25% prevalence level), about the posterior 20 th percentile (equal to or less than about 20% prevalence level), about the posterior 15 th percentile (equal to or less than about 15% prevalence level), about the posterior 10 th percentile (equal to or less than about 10% prevalence level), about the posterior 5 th percentile (equal to or less than about 5% prevalence level), or about the posterior 1 st percentile (equal to or less than about 1% prevalence level), wherein the responsiveness of an individual to treatment with a non-PD-L78 axis binding antagonist therapy is relative to the responsiveness to the individual to treatment with the non-PD-L1 axis binding antagonist therapy A significant (e.g., statistically significant) improvement in responsiveness of treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is the level of immune score expression of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that most closely separates the level of immune score expression in the reference population that has been treated with a PD-L637 axis binding antagonist (e.g., PD-L1-1 binding antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, at least one, at least two, at least three, at least four, at least five, or all six genes in the reference population selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4)) about the last 99 th percentile (equal to or less than about 99% prevalence level), about the last 95 th percentile (equal to or less than about 95% prevalence level), about the last 90 th percentile (equal to or less than about 90% prevalence level), about the last 85 th percentile (equal to or less than about 85% prevalence level), about the last 80 th percentile (equal to or less than about 80% prevalence level), about the last 75 th percentile (equal to or less than about 75% prevalence level), about the last 70 th percentile (equal to or less than about 70% prevalence level), about the last 65 th percentile (equal to or less than about 65% prevalence level), about the last 60 th percentile (equal to or less than about 60% prevalence level), about the last 55 th percentile (equal to or less than about 55% prevalence level), about the posterior 50 th percentile (equal to or less than about 50% prevalence level), about the posterior 45 th percentile (equal to or less than about 45% prevalence level), about the posterior 40 th percentile (equal to or less than about 40% prevalence level), about the posterior 35 th percentile (equal to or less than about 35% prevalence level), about the posterior 30 th percentile (equal to or less than about 30% prevalence level), about the posterior 25 th percentile (equal to or less than about 25% prevalence level), about the posterior 20 th percentile (equal to or less than about 20% prevalence level), about the posterior 15 th percentile (equal to or less than about 15% prevalence level), about the posterior 10 th percentile (equal to or less than about 10% prevalence level), about the posterior 5 th percentile (equal to or less than about 5% prevalence level), or about the posterior 1 st percentile (equal to or less than about 1% prevalence level), wherein the responsiveness of an individual to treatment with a non-PD-L78 axis binding antagonist therapy is relative to the responsiveness to the individual to treatment with the non-PD-L1 axis binding antagonist therapy A significant (e.g., statistically significant) improvement in responsiveness of treatment with the PD-L1 axis binding antagonist therapy.

In certain particular instances, the reference immune score expression level is the level of immune score expression of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that best separates the immune score expression levels in the reference population that have been treated with a PD-L637 axis binding antagonist (e.g., PD-L1 binding antagonist (e.g., anti-PD-L1 antibody), e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, at least one, at least two, at least three, at least four, at least five, or all six genes in the reference population selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4)) about the last 99 th percentile (equal to or less than about 99% prevalence level), about the last 95 th percentile (equal to or less than about 95% prevalence level), about the last 90 th percentile (equal to or less than about 90% prevalence level), about the last 85 th percentile (equal to or less than about 85% prevalence level), about the last 80 th percentile (equal to or less than about 80% prevalence level), about the last 75 th percentile (equal to or less than about 75% prevalence level), about the last 70 th percentile (equal to or less than about 70% prevalence level), about the last 65 th percentile (equal to or less than about 65% prevalence level), about the last 60 th percentile (equal to or less than about 60% prevalence level), about the last 55 th percentile (equal to or less than about 55% prevalence level), about the posterior 50 th percentile (equal to or less than about 50% prevalence level), about the posterior 45 th percentile (equal to or less than about 45% prevalence level), about the posterior 40 th percentile (equal to or less than about 40% prevalence level), about the posterior 35 th percentile (equal to or less than about 35% prevalence level), about the posterior 30 th percentile (equal to or less than about 30% prevalence level), about the posterior 25 th percentile (equal to or less than about 25% prevalence level), about the posterior 20 th percentile (equal to or less than about 20% prevalence level), about the posterior 15 th percentile (equal to or less than about 15% prevalence level), about the posterior 10 th percentile (equal to or less than about 10% prevalence level), about the posterior 5 th percentile (equal to or less than about 5% prevalence level), or about the posterior 1 st percentile (equal to or less than about 1% prevalence level), wherein the responsiveness of an individual to treatment with a non-PD-L78 axis binding antagonist therapy is relative to the responsiveness to the individual to treatment with the non-PD-L1 axis binding antagonist therapy A significant (e.g., statistically significant) improvement in responsiveness of treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is a median immune score expression level (e.g., normalized immune score expression level) of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in the reference population based on a significant difference (e.g., has been statistically significantly separated in the reference population) between the responsiveness of an individual to treatment with a PD-L4 axis binding antagonist and the responsiveness of an individual to treatment with a non-PD-L4 axis binding antagonist (e.g., has been statistically significantly separated in the reference population) A first subset of individuals treated with an L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population that have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individuals to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to responsiveness of the individuals to treatment with the non-PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is a median immune score expression level (e.g., median normalized immune score expression level) of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD 85, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that has been optimally separated by the reference axis 1 based on a maximum difference between the individual's responsiveness to treatment with the PD-L1 axis binding antagonist and the individual's responsiveness to treatment with the non-PD-L1 axis binding antagonist based on the median immune score expression level being higher than the reference immune score expression level (i.e., higher than the median score expression level) A first subset of individuals treated with a therapy of a binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individuals to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to responsiveness of the individuals to treatment with the non-PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is the median immune score expression level (e.g., the median normalized immune score expression level) for at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD 85, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that has optimally divided the reference axis 1 binding with the reference axis based on the maximum difference between the responsiveness of an individual to treatment with the PD-L1 axis binding antagonist and the responsiveness of an individual to treatment with the non-PD-L1 axis binding antagonist (i.e., above the median immune score expression level) For example, a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population that have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is a median immune score expression level (e.g., normalized immune score expression level) of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4) in a reference population based on a significant difference between the responsiveness of an individual to treatment with a PD-L1 axis binding antagonist and the responsiveness of an individual to treatment with a non-PD-L1 axis binding antagonist (e.g., a statistically significant difference between the responses of the population has been separated by the reference population) A first subset of individuals treated with an L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population that have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is a median immune score expression level (e.g., median normalized immune score expression level) for at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD 85, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that has been optimally separated by the reference axis 1 from the reference population based on a maximum difference between the individual's responsiveness to treatment with the PD-L1 axis binding antagonist and the individual's responsiveness to treatment with the non-PD-L1 axis binding antagonist A first subset of individuals treated with a therapy of a binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individuals to treatment with the non-PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to responsiveness of the individuals to treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is a median immune score expression level (e.g., median normalized immune score expression level) of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD 85, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that best separates the reference population having bound with the PD-L1 axis based on the maximum difference between the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist and the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist (i.e., below the median cutoff) For example, a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population that have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is an average (e.g., average of normalized immune score expression levels) (mean)) expression level of at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4) in the reference population based on a significant difference (e.g., a statistically significant split in the population) between the responsiveness of an individual to treatment with a PD-L1 axis binding antagonist therapy above the reference immune score expression level (i.g., above the mean) cut-off the responsiveness of an individual to treatment with a non-PD-L1 axis binding antagonist A first subset of individuals treated with an L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population that have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individuals to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to responsiveness of the individuals to treatment with the non-PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is an average (e.g., average of normalized immune score expression levels (mean)) expression level of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD 85, and PD-1; or any combination of genes listed in tables 1-4) in a reference population that has been optimally separated by a substantial reference L1 from a substantial reference population based on the responsiveness of an individual to treatment with a PD-L1 axis binding antagonist that is higher than the reference immune score expression level (i.e., higher than the mean) and the responsiveness of an individual to treatment with a non-PD-L1 axis binding antagonist A first subset of individuals treated with an axis-binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individuals to treatment with the PD-L1 axis binding antagonist therapy is significantly improved relative to responsiveness of the individuals to treatment with the non-PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is an average (e.g., average of normalized immune score expression levels (mean)) expression level of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD 85, and PD-1; or any combination of the genes listed in tables 1-4) in a reference population that has been optimally separated by the reference axis 1 from the reference population based on the maximum difference between the response of an individual to treatment with a PD-L1 axis binding antagonist therapy above the reference immune score expression level (i.e., above the mean) and the response of an individual to treatment with a PD-L1 axis binding antagonist A first subset of individuals treated with a (e.g., PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is an average (e.g., average of normalized immune score expression levels)) expression level of at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4) in a reference population based on a significant difference between the responsiveness of an individual to treatment with a PD-L1 axis binding antagonist below the reference immune score expression level (i.e., below the mean cutoff) and the responsiveness of an individual to treatment with a non-PD-L1 axis binding antagonist (e.g., the reference population has been statistically divided) A first subset of individuals treated with an L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population that have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is the average (e.g., mean) of normalized immune score expression levels) of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD 85, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that has been optimally separated by 1 from the reference population based on the maximum difference between the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist below the reference immune score expression level (i.e., below the mean cutoff) and the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist A first subset of individuals treated with an axis-binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individuals to treatment with the non-PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to responsiveness of the individuals to treatment with the PD-L1 axis binding antagonist therapy.

In some cases, the reference immune score expression level is the average (e.g., mean value) of normalized immune score expression levels of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD 85, and PD-1; or any combination of genes listed in tables 1-4) in the reference population that has optimally separated the reference axis 1 from the reference population based on the maximum difference between the responsiveness of an individual to treatment with a PD-L1 axis binding antagonist and the responsiveness of an individual to treatment with a non-PD-L1 axis binding antagonist in the reference population A first subset of individuals treated with a (e.g., PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise a PD-L1 axis binding antagonist, wherein responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist therapy is significantly (e.g., statistically significantly) improved relative to responsiveness of the individual to treatment with the PD-L1 axis binding antagonist therapy.

In some cases, a reference immune score expression level is defined by an immune score expression level with a certain prevalence in a reference population as discussed further herein. In some cases, the reference immune score expression level is a pre-assigned value (e.g., previously determined to be a significant (e.g., statistically significant) difference between the responsiveness of an individual to treatment with a PD-L1 axis binding antagonist and the responsiveness of an individual to treatment with a non-PD-L1 axis binding antagonist based on a cut-off value above and/or below the cut-off value that separates a first subset of individuals in a reference population that have been treated with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) therapy and a second subset of individuals in the same reference population that have been treated with a non-PD-L1 axis binding therapy that does not include a PD-L1 axis binding antagonist), wherein the responsiveness of the individual above the cut-off to treatment with the PD-L1 axis binding antagonist is significantly (e.g., statistically) improved relative to the responsiveness of the individual to treatment with the non-PD-L1 axis binding antagonist and/or the responsiveness of the individual below the cut-off to treatment with the non-PD-L1 axis binding antagonist is significantly (e.g., statistically) improved relative to the responsiveness of the individual to treatment with the PD-L1 axis binding antagonist.

In some cases, a reference immune score expression level may also be determined at one or more time points from one or more samples obtained from an individual undergoing testing and/or treatment using the methods and/or assays described herein. In some cases, the reference immune score expression level is an expression level of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and ng; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of genes listed in tables 1-4)) in a sample previously obtained from the individual at a time point prior to administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL 3280A)).

In some cases, the reference immune score expression level is an expression level of at least one, at least two, at least three, at least four, at least five, or all six genes, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, ifzmb, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1) in a sample obtained from the individual at a time point after administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., altlizumab (MPDL3280A)), or a combination of any of the genes listed in tables 1-4). Such reference immune score expression levels obtained from an individual may be useful for monitoring the response of the individual over time to treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL 3280A)).

The reference immune score expression level can be determined from any number of individuals in a reference population and/or any number of reference samples (e.g., reference cells, reference tissue, control sample, control cells, or control tissue). The reference sample may be a single sample or a combination of multiple samples. The reference immune score expression level based on a reference sample can be based on any number of reference samples (e.g., 2 or more, 5 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1000 or more reference samples). In some cases, the reference sample comprises pooled mRNA samples derived from samples obtained from multiple individuals. Moreover, the reference immune score expression level based on the reference population or a sample therefrom can be based on any number of individuals in the reference population (e.g., 2 or more, 5 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1000 or more individuals in the reference population). The reference immune score expression level can be determined from measurements based on multiple individuals or multiple samples in a reference population using any statistical method known in the art. See, e.g., Sokal R.R.and Rholf, F.J (1995) "Biometry: the principles and practice of statistics informatics research," W.H.Freeman and Co.New York, N.Y..

(viii) Reference population

The reference immune score expression level can reflect the expression level of one or more genes described herein (e.g., at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD8 zm 8A; PD-L1, IFNG, gb, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)) in one or more reference populations (or reference samples), or as a pre-assigned reference value.

In some cases, the reference immune score expression level is an immune score expression level of at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4)) in the reference population.

In some cases, the reference population is a population of individuals with cancer. In some cases, the reference population is a population of individuals having lung cancer (e.g., NSCLC). In some cases, the reference population is a population of individuals with renal cancer (e.g., RCC). In some cases, the reference population is a population of individuals having bladder cancer (e.g., UBC). In some cases, the reference population is a population of individuals having breast cancer (e.g., TNBC). In some cases, the reference population is a population of individuals without cancer.

Further, the reference population can include one or more subsets of individuals (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more subsets).

In some cases, the reference population is a population of individuals having cancer, wherein the population of individuals comprises a subset of individuals that have been treated with therapy comprising at least one dose (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten doses) of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, a therapy comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is a monotherapy. In other instances, a therapy comprising a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is a combination therapy comprising at least one additional therapeutic agent (e.g., an anti-cancer therapy (e.g., an anti-tumor agent, a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, radiation therapy, or a combination thereof)) in addition to the PD-L1 axis binding antagonist.

In some cases, the reference population is a population of individuals having cancer, wherein the population of individuals includes a subset of individuals that have been treated with a non-PD-L1 axis binding antagonist therapy (e.g., an anti-cancer therapy (e.g., an anti-tumor agent, a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, radiation therapy, or a combination thereof)) that does not include a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, the reference population includes a combination of individuals from different subsets. For example, in some cases, the reference population can be a population of individuals having cancer consisting of (i) a first subset of individuals who have been treated with a PD-L1 axis binding antagonist therapy (e.g., PD-L1 binding antagonist therapy) that includes a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) and (ii) a second subset of individuals who have been treated with a non-PD-L1 axis binding antagonist therapy (e.g., a non-PD-L1 binding antagonist therapy) that does not include a PD-L1 axis binding antagonist (e.g., an anti-cancer therapy (e.g., an anti-tumor agent, a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, radiotherapy, or a combination thereof) Antagonist therapy) may have been administered as monotherapy or as combination therapy.

Methods of treatment

Provided herein are methods, medicaments, and uses thereof, for treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., an antagonist of PD-L1 axis binding (e.g., an antagonist of PD-L1 binding (e.g., an anti-PD-L48325-L antibody), such as astuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In one aspect, provided herein are methods for treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising (a) determining the expression level of at least one, at least two, at least three, at least four, at least five, or all six genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein the expression level of the immune score in the sample of at least one, at least two, at least three, at least four, at least five, or all six of PD-L1, CXCL9, IFNG, GZMB, CD8A, or PD-1 has been determined to be greater than the expression level of the reference immune score (e.g., at least one selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in the reference population, at least two, at least three, at least four, at least five, or all six genes, and (b) administering an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) to the individual based on the immune score expression levels of at least one, at least two, at least three, at least four, at least five, or all six genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 determined in step (a).

In another aspect, provided herein are methods for treating an individual having cancer, the methods comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, the expression level of at least one, at least two, at least three, at least four, at least five, or all six genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual have been determined and have been determined to be above a reference immune score expression level (e.g., at least one, at least two, at least three, CD8A, and PD-1 in a reference population selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, immune score expression levels of at least four, at least five, or all six genes) in a sample selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be administered as a first line therapy. Alternatively, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be administered as a second line therapy.

A. Single and two-gene immune scores

In particular instances, individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) can be treated using the methods and medicaments provided herein based on determining the expression level of an immune score for any one of the genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. In some cases, the determining step comprises determining the expression level of a particular combination of any one gene selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and one or more additional T-effector cell-associated genes, e.g., determining the expression level of (i) any one gene selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and (ii) one or more T-effector cell-associated genes (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L TAP1, PD-1, CXCL9, CD27, FOXP3, CTLA, TIGIT, IDO1, CXCL10, CXCL11, PSMB9, 1, and/or 2, at least one, at least two, at least five, at least six, at least four, at least nine, at least ten, at least four, at least ten, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or nineteen), wherein the one or more genes associated with T effector cells are different from one gene selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

The examples and embodiments of the methods of treatment, medicaments, and uses thereof described in sections iii.b (i-iii), iii.c (i-iii), iii.d (i-iii), and iii.e (i-iii) may also be applied to the immune score expression level of any one gene selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

In particular instances, individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)) can be treated using the methods and medicaments provided herein based on determining the expression level of an immune score for two genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. For example, the determining step can include determining the expression level of any two gene combinations listed in table 1. In some cases, the determining step comprises determining the expression level of a particular combination of two genes listed in table 1 and one or more additional T-effector cell-associated genes, e.g., determining the expression level of (i) two genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 (e.g., any one of the combinations of genes listed in table 1) and (ii) one or more T-effector cell-associated genes (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, TAP1, and/or TAP2 of at least one, at least two, at least three, at least four, at least five, at least six, at least eight, at least nine, at least ten, at least fifteen, at least sixteen, at least seventeen, or eighteen), wherein the one or more T-effector cell-associated genes are different from two genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

The examples and embodiments of the methods of treatment, medicaments, and uses thereof described in sections iii.b (i-iii), iii.c (i-iii), iii.d (i-iii), and iii.e (i-iii) may also be applied to the expression level of the immune score of any two gene combinations listed in table 1.

B. Three-gene immune score combination

In particular instances, individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)) can be treated using the methods and medicaments provided herein based on determining the expression levels of immune scores for three genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. For example, the determining step may comprise determining the expression level of any three gene combination listed in table 2. In some cases, the determining step comprises determining the expression level of a particular combination of three genes listed in table 2 and one or more additional T-effector cell-associated genes, e.g., determining the expression level of (i) three genes (e.g., any of the combinations of genes listed in table 2) selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and (ii) one or more T-effector cell-associated genes (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, TAP1, and/or TAP2 of at least one, at least two, at least three, at least four, at least five, at least six, at least eight, at least nine, at least ten, at least fifteen, at least sixteen, or seventeen), wherein one or more genes associated with T effector cells are different from three genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

The examples and cases described below for the PD-L1, CXCL9, and IFNG gene sets can also be applied to any of the three gene combinations listed in table 2.

(i) Expression of PD-L1, CXCL9, and IFNG

In some cases, an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) can be treated using a method comprising (a) determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein the expression levels of the immune scores for at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample have been determined to be higher than a reference immune score expression level (e.g., the expression levels of the immune scores for PD-L1, CXCL9, and IFNG in a reference population), and (b) determining the expression levels of the immune scores for at least one, at least two, or all three of the immune score expression levels based on at least one, at least two, or all three of the PD-L1, CXCL9, and IFNG determined in step (a) based on the cancer having one or more of the cancer treated with the PD-L1 axis binding antagonist therapy or a non-PD-L1 binding antagonist About the top 99 th percentile (equal to or higher than about 1% prevalence level), about the top 95 th percentile (equal to or higher than about 5% prevalence level), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level) of the expression level of the immune score for PD-L1, CXCL9, and IFNG in an individual (e.g., a population of patients with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the immune score expression level of PD-L1, CXCL9, and IFNG in the sample in the first 1 th percentile (equal to or above about 99% prevalence level), administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL 3280A)).

In some cases, an individual having cancer can be treated using the methods provided herein, the methods comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, expression levels of PD-L1, CXCL9, and IFNG have been determined in a sample from the individual and expression levels of PD-L1, CXCL9, and IFNG have been determined in samples that are higher than a reference immune score expression level (e.g., an individual having cancer (e.g., lung cancer (e.g., NSCLC), a population of patients with bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), about the top 99 th percentile (equal to or higher than about 1% prevalence level), about the top 95 th percentile (equal to or higher than about 5% prevalence level), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level) of samples from 1L in PD-L, CXCL9, and IFNG immune score expression level).

(ii) Medicament and use thereof

In yet another aspect, the invention provides the use of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) in the manufacture or preparation of a medicament for treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)).

In some cases, the medicament for use in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising (a) determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein the expression levels of the immune scores for at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample have been determined to be greater than a reference immune score expression level (e.g., the expression levels of the immune scores for at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in a reference population), and (b) determining the immune score expression levels for at least one, at least two, or all three of PD-L1, CXCL9, and IFNG based on step (a) (e.g., in a reference population (e.g., an individual having cancer has been subjected to one or more of PD-L1 binding to one or more of the axes of PD-L1) A population of patients with PD-L1, CXCL9, and IFNG immune score expression levels in a cancer (e.g., a lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) treated with an anti-agent therapy or a non-PD-L1 axis binding antagonist therapy) about the first 99 th percentile (equal to or greater than about 1% prevalence level), about the first 95 th percentile (equal to or greater than about 5% prevalence level), about the first 90 th percentile (equal to or greater than about 10% prevalence level), about the first 85 th percentile (equal to or greater than about 15% prevalence level), about the first 80 th percentile (equal to or greater than about 20% prevalence level), about the first 75 th percentile (equal to or greater than about 25% prevalence level), about the first 70 th percentile (equal to or greater than about 30% prevalence level), about the first 65 th percentile (equal to or greater than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the immune score expression level of PD-L1, CXCL9, and IFNG in the sample in the first 1 th percentile (equal to or above about 99% prevalence level), administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL 3280A)).

In some cases, the medicament is for use in a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual have been determined and the expression levels of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in a sample that is higher than a reference immune score expression level have been determined (e.g., in a reference population (e.g., an individual having cancer who has undergone one or more treatments with a PD-L1 axis binding antagonist therapy or a non-PD-L1 axis binding antagonist therapy having cancer (e.g., lung cancer (e.g., NSCLC, a population of patients with bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), about the top 99 th percentile (equal to or higher than about 1% prevalence level), about the top 95 th percentile (equal to or higher than about 5% prevalence level), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level) of samples from 1L in PD-L, an immune score expression level of at least one, at least two, or all three of CXCL9, and IFNG).

(iii) Use of PD-L1 axis binding antagonists

In yet another aspect, the invention provides the use of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) in the treatment of an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)).

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is used in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising (a) determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein the expression levels of the immune scores of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the sample have been determined to be greater than a reference immune score expression level (e.g., the immune score expression levels of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in the reference population), and (b) based on the immune score expression levels of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG determined in step (a) (e.g., PD-L1, CXCL9, and about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level) of the immune score expression levels of IFNG in a reference population (e.g., a population of individuals with cancer (e.g., patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy) with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., RCC), kidney cancer (e.g., TNBC)), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), an effective amount of an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in a sample in about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level) is administered to the individual a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL 3280A)).

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is for use in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, expression levels of PD-L1, CXCL9, and IFNG have been determined in a sample from the individual and expression levels of PD-L1 have been determined to, CXCL9, and IFNG (e.g., in a reference population (e.g., a population of individuals with cancer (e.g., patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy) who have cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)), about the first 99 th percentile of the immune score expression levels of PD-L1, CXCL9, and IFNG (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), about the first 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), an immune score expression level of at least one, at least two, or all three of PD-L1, CXCL9, and IFNG in a sample in about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level).

C. Four-gene immune score combination

In particular instances, individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)) can be treated using the methods and medicaments provided herein based on determining the expression levels of immune scores for four genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. For example, the determining step may comprise determining the expression level of any four gene combination listed in table 3. In some cases, the determining step comprises determining the expression level of a particular combination of four genes listed in table 3 and one or more additional T-effector cell-associated genes, e.g., determining (i) four genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 (e.g., any four combinations of genes listed in table 3) and (ii) one or more T-effector cell-associated genes (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, TAP1, and/or TAP2 at least one, at least two, at least three, at least four, at least five, at least six, at least eight, at least nine, at least ten, at least fifteen, or sixteen) wherein one or more of the T effector cell-associated genes is different from four genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

The examples and conditions described below with respect to the PD-L1, IFNG, GZMB, and CD8A gene sets may also be applied to any four-gene combination listed in table 3.

(i) Expression of PD-L1, IFNG, GZMB, and CD8A

In some cases, an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) can be treated using a method comprising (a) determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein the expression levels of PD-L1, IFNG, GZMB, and CD8A in the sample have been determined to be greater than a reference immune score expression level (e.g., the immune score expression levels of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A in a reference population), and (b) determining the immune score expression level based on step (a) of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A (e.g., the immune score in the reference population is not present (e.g., the reference population has no cancer) About the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), about the first 80 th percentile (equal to or higher than about 20% prevalence level), about the first 75 th percentile (equal to or higher than about 25% prevalence level) of the immune score expression level of PD-L1, GZMB, and CD8A in a population having cancer (e.g., a population of patients having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) that have undergone one or more treatments with a PD-L1 axis binding antagonist therapy or a non-PD-L1 axis binding antagonist therapy, about the first 70 th percentile (equal to or higher than about 30% prevalence level), about the first 65 th percentile (equal to or higher than about 35% prevalence level), about the first 60 th percentile (equal to or higher than about 40% prevalence level), about the first 55 th percentile (equal to or higher than about 10% prevalence level), about the first 50 th percentile (equal to or higher than about 50% prevalence level), about the first 45 th percentile (equal to or higher than about 55% prevalence level), about the first 40 th percentile (equal to or higher than about 60% prevalence level), about the first 35 th percentile (equal to or higher than about 65% prevalence level), about the first 30 th percentile (equal to or higher than about 70% prevalence level), about the first 25 th percentile (equal to or higher than about 75% prevalence level), about the first 20 th percentile (equal to or higher than about 80% prevalence level), about the first 15 th percentile (equal to or higher than about 85% prevalence level), an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)), is administered to the individual at about the top 10 th percentile (equal to or greater than about 90% prevalence level), about the top 5 th percentile (equal to or greater than about 95% prevalence level), or about the top 1 st percentile (equal to or greater than about 99% prevalence level) in the sample.

In some cases, an individual having cancer can be treated using the methods provided herein, the methods comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual have been determined and the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample that has been determined to be higher than a reference immune score expression level (e.g., at least one, two, at least three, or all four immune score expression levels in a reference population (e.g., a population of individuals that do not have cancer or individuals that have cancer (e.g., have undergone treatment with one or more PD-L1 axis binding or non-PD-L1 axis binding antagonist therapies) A population having an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a population of patients having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC))), about the first 99 th percentile (equal to or above about 1% prevalence level), about the first 95 th percentile (equal to or above about 5% prevalence level), about the first 90 th percentile (equal to or above about 10% prevalence level), about the first 85 th percentile (equal to or above about 15% prevalence level), about the first 80 th percentile (equal to or above about 20% prevalence level), about the first 75 th percentile (equal to or above about 25% prevalence level), about the first 70 th percentile (equal to or above about 30% prevalence level), about the first 65 th percentile (equal to or above about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or an immune score expression level of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A in a sample in about the top 1 th percentile (equal to or above about 99% prevalence level).

(ii) Medicament and use thereof

In some cases, the medicament for use in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising (a) determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein the expression levels of the immune scores of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A in the sample have been determined to be greater than a reference immune score expression level (e.g., the expression levels of the immune scores of at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD 3878 2 in a reference population), and (b) determining the expression levels of the immune scores based on step (a), or all four immune score expression levels (e.g., about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), about the first 80 th percentile (equal to or higher than about 20% prevalence level) in a reference population (e.g., a population of individuals without cancer or a population of individuals with cancer (e.g., patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy) who have cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), an effective amount of a PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritor (MPDL3280A)) is administered to the individual at about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level) in the sample.

In some cases, the medicament is for use in a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual have been determined and the expression levels of PD-L1, IFNG, gb, and CD8A in a sample that is higher than a reference immune score expression level have been determined (e.g., a cancer-bearing expression level in a reference population (e.g., a population of individuals that do not have cancer or individuals that have cancer (e.g., have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding therapy) (e.g., patients of lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC))) in a population of PD-L1, IFNG, GZMB, and CD8A, about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), about the first 80 th percentile (equal to or higher than about 20% prevalence level), about the first 75 th percentile (equal to or higher than about 25% prevalence level), about the first 70 th percentile (equal to or higher than about 30% prevalence level), about the first 65 th percentile (equal to or higher than about 35% prevalence level), about the first 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level) of samples from 1L in PD-L, immune score expression levels of at least one, at least two, at least three, or all four of IFNG, GZMB, and CD 8A).

(iii) Use of PD-L1 axis binding antagonists

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is used in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising (a) determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein the expression levels of PD-L1, IFNG, GZMB, and CD8A in the sample have been determined to be higher than the expression level of a reference immune score (e.g., at least one of PD-L1, ifzmb, GZMB, and CD8 ng 8A in the reference population, at least two, at least three, or all four immune score expression levels), and (b) based on at least one, at least two, at least three, or all four immune score expression levels determined in step (a) (e.g., about the first 99 th percentile (equal to or higher than about the 1% prevalence level), about the first 95 th percentile (equal to or higher than about the 5% prevalence level) of the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in a reference population (e.g., a population of individuals without cancer or a population of individuals with cancer (e.g., a population of patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy), for example, lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the first 30 th percentile (equal to or higher than about 70% prevalence level), about the first 25 th percentile (equal to or higher than about 75% prevalence level), about the first 20 th percentile (equal to or higher than about 80% prevalence level), about the first 15 th percentile (equal to or higher than about 85% prevalence level), about the first 10 th percentile (equal to or higher than about 90% prevalence level), about the first 5 th percentile (equal to or higher than about 95% prevalence level), or about the first 1 th percentile (equal to or above about 99% prevalence level), at least one, at least two, at least three, or expression levels of all four immune scores) an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) is administered to the individual.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is used in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein, prior to treatment, expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual have been determined and a higher level of PD-L1 than a reference immune, immune score expression levels of at least one, at least two, at least three, or all four of IFNG, GZMB, and CD8A (e.g., about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), or all four immune score expression levels (e.g., PD-L1, IFNG, GZMB, and CD 8A) in a reference population (e.g., a population of individuals not having cancer or a population of individuals having cancer (e.g., a population of patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), at least one, at least two, at least three, or all four immune score expression levels in the sample in about the top 20 th percentile (equal to or greater than about 80% prevalence level), about the top 15 th percentile (equal to or greater than about 85% prevalence level), about the top 10 th percentile (equal to or greater than about 90% prevalence level), about the top 5 th percentile (equal to or greater than about 95% prevalence level), or about the top 1 st percentile (equal to or greater than about 99% prevalence level).

D. Five-gene immune score combination

In particular instances, individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)) can be treated using the therapeutic methods and medicaments provided herein based on determining the expression levels of immune scores of five genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. For example, the determining step may comprise determining the expression level of any one of the five combinations of genes listed in table 4. In some cases, the determining step comprises determining the expression levels of a particular combination of five genes listed in table 4 and one or more additional T effector-associated genes, e.g., determining the expression levels of (i) five genes (e.g., any one of the combinations of genes listed in table 4) selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and (ii) one or more T effector-associated genes (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB8, PSMB9, TAP1, and/or TAP2 of at least one, at least two, at least three, at least four, at least five, at least six, at least eight, at least ten, or fifteen) wherein one or more genes associated with the T effector cell are different from five genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

The examples and embodiments described below with respect to the PD-L1, IFNG, GZMB, CD8A, and PD-1 gene sets may also be applied to any five-gene combination listed in table 4.

(i) Expression of PD-L1, IFNG, GZMB, CD8A, and PD-1

In some cases, a method can be used to treat an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising (a) determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein the expression levels of the immune scores in the sample for at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 have been determined to be higher than a reference immune score expression level (e.g., the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population), and (b) determining the expression levels of the immune scores based on step (a) for at least one, at least two, at least three, at least four, or all five immune score expression levels (e.g., PD-L1, IFNG, GZMB, CD8A, and about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level) of immune score expression levels of PD-1 in a reference population (e.g., a population of individuals without cancer or a population of individuals with cancer (e.g., patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy) of a population with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., TNBC)), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or above about 80% prevalence level), about the top 15 th percentile (equal to or above about 85% prevalence level), about the top 10 th percentile (equal to or above about 90% prevalence level), about the top 5 th percentile (equal to or above about 95% prevalence level), or about the top 1 st percentile (equal to or above about 99% prevalence level) in a sample at least one, at least two, at least three, at least four, or all five immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1) will be administered to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritol mab (MPDL 3280A)).

In some cases, an individual having cancer can be treated using the methods provided herein, the methods comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample from the individual and expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample above a reference immune score expression level (e.g., in a reference population (e.g., a population of individuals that do not have cancer or individuals that have cancer (e.g., have undergone one or more axis binding therapies with a PD-L1 antagonist or not have cancer) A population of PD-L1 axis-binding antagonist therapy treated patients with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) patients) having PD-L1, IFNG, GZMB, CD8A, and PD-1 in the first 99 th percentile of the immune score expression level (equal to or above about 1% prevalence level), the first 95 th percentile (equal to or above about 5% prevalence level), the first 90 th percentile (equal to or above about 10% prevalence level), the first 85 th percentile (equal to or above about 15% prevalence level), the first 80 th percentile (equal to or above about 20% prevalence level), the first 75 th percentile (equal to or above about 25% prevalence level), the first 70 th percentile (equal to or above about 30% prevalence level), about the top 65 percentile (equal to or higher than about 35% prevalence level), about the top 60 percentile (equal to or higher than about 40% prevalence level), about the top 55 percentile (equal to or higher than about 10% prevalence level), about the top 50 percentile (equal to or higher than about 50% prevalence level), about the top 45 percentile (equal to or higher than about 55% prevalence level), about the top 40 percentile (equal to or higher than about 60% prevalence level), about the top 35 percentile (equal to or higher than about 65% prevalence level), about the top 30 percentile (equal to or higher than about 70% prevalence level), about the top 25 percentile (equal to or higher than about 75% prevalence level), about the top 20 percentile (equal to or higher than about 80% prevalence level), about the top 15 percentile (equal to or higher than about 85% prevalence level), about the top 10 percentile (equal to or higher than about 90% prevalence level), an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample in about the top 5 th percentile (equal to or greater than about 95% prevalence level), or about the top 1 th percentile (equal to or greater than about 99% prevalence level).

(ii) Medicament and use thereof

In some cases, the medicament for use in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising (a) determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein the expression levels of the immune scores for at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample have been determined to be greater than a reference immune score expression level (e.g., the immune expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population), and (b) determining the expression levels of the immune scores for at least one, at least two, PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population, at least three, at least four, or all five immune score expression levels (e.g., PD-L1, IFNG, GZMB, CD8A, and about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level) of immune score expression levels of PD-1 in a reference population (e.g., a population of individuals without cancer or a population of individuals with cancer (e.g., patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy) of a population with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., TNBC)), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or above about 80% prevalence level), about the top 15 th percentile (equal to or above about 85% prevalence level), about the top 10 th percentile (equal to or above about 90% prevalence level), about the top 5 th percentile (equal to or above about 95% prevalence level), or about the top 1 st percentile (equal to or above about 99% prevalence level) in a sample at least one, at least two, at least three, at least four, or all five immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1) will be administered to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritol mab (MPDL 3280A)).

In some cases, the medicament is for use in a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample from the individual and expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample above the reference immune score expression level (e.g., in a reference population (e.g., a population of individuals without cancer or individuals with cancer (e.g., have undergone one or more axial binding antagonist therapies with PD-L1 or non-active agents) A population of patients with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) treated with an L1 axis binding antagonist therapy) having an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 of about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), about the first 80 th percentile (equal to or higher than about 20% prevalence level), about the first 75 th percentile (equal to or higher than about 25% prevalence level), about the first 70 th percentile (equal to or higher than about 30% prevalence level), about the first 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or an immune score expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample in about the top 1 th percentile (equal to or above about 99% prevalence level).

(iii) Use of PD-L1 axis binding antagonists

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is for use in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising (a) determining expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein expression levels of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample have been determined to be higher than the reference immune expression level (e.g., PD-L1 in the reference population, immune score expression levels of IFNG, GZMB, CD8A, and PD-1), and (b) immune score expression levels based on at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 determined in step (a) (e.g., the first 99 th percentile (equal to or higher than about 1% prevalence level) of immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population (e.g., a population of individuals without cancer or a population of individuals with cancer (e.g., patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy) with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC))))), about the top 95 th percentile (equal to or higher than about 5% prevalence level), about the top 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the first 35 th percentile (equal to or greater than about 65% prevalence level), about the first 30 th percentile (equal to or greater than about 70% prevalence level), about the first 25 th percentile (equal to or greater than about 75% prevalence level), about the first 20 th percentile (equal to or greater than about 80% prevalence level), about the first 15 th percentile (equal to or greater than about 85% prevalence level), about the first 10 th percentile (equal to or greater than about 90% prevalence level), about the first 5 th percentile (equal to or greater than about 95% prevalence level), or about the first 1 st percentile (equal to or greater than about 99% prevalence level) in the sample will bind an effective amount of a PD-L1 axis binding antagonist (e.g., an antagonist such as PD-L6335, e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) is administered to the individual.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) for use in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprises administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein, prior to treatment, expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample from the individual and expression levels of PD-L1 have been determined that are, immune score expression levels of at least one, at least two, at least three, at least four, or all five of IFNG, GZMB, CD8A, and PD-1 (e.g., PD-L1, IFNG, gb, CD8A, and the pre-99 th percentile (equal to or above about 1% prevalence level), the pre-95 th percentile (equal to or above about 5% prevalence level), the pre-90 th percentile (equal to or above about 10% prevalence level) of immune score expression levels of PD-1 in a reference population (e.g., a population of individuals without cancer or a population of individuals with cancer (e.g., a population of patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy) with cancer (e.g., patients with lung cancer (e.g., NSCLC), bladder cancer (e.g., RCC), or breast cancer (e.g., TNBC)), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), immune score expression levels of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample of about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level).

E. Six-gene immune score combination

In particular instances, individuals having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)) can be treated using the treatment methods and medicaments provided herein based on determining the expression levels of immune scores for all six genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1. In some cases, the determining step comprises determining the expression levels of all six genes selected from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and one or more additional T-effector cell-associated genes, e.g., by determining (i) all six genes (e.g., any combination of genes listed in table 4) selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 and (ii) one or more T-effector cell-associated genes (e.g., CD8A, GZMA, GZMB, IFNG, EOMES, PRF1, PD-L1, PD-1, CXCL9, CD27, FOXP3, CTLA4, TIGIT, IDO1, CXCL10, CXCL11, PSMB 6, PSMB9, TAP 27, and/or TAP 3673729, at least one, at least six, at least two, at least five, at least nine, at least ten, at least seven, at least twelve, at least thirteen, or fourteen), wherein one or more of the T effector cell-associated genes is different from PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1.

(i) Expression of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1

In some cases, an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) can be treated using a method comprising (a) determining the expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein the immune score expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in the sample have been determined to be higher than a reference immune score expression level (e.g., the immune score expression levels of PD-L1, CXCL9, ifzmb, GZMB, CD8A, and PD-1 in a reference population), and (b) determining the immune score expression levels of PD-L1, CXCL9, ifzmb, CD8A, and PD-1 based on the immune score expression levels of PD-L1 in the population determined in step (e.g., the population having one or more cancer (e.g., the population having no cancer) in the reference population has undergone one or more cancer (e.g., the population has undergone the presence of the same cancer in the subject A population of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a population of patients having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)) treated with antagonist therapy or non-PD-L1 axis binding antagonist therapy) about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), about the first 80 th percentile (equal to or higher than about 20% prevalence level), about the first 75 th percentile (equal to or higher than about 25% prevalence level), about the first 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 percentile (equal to or higher than about 35% prevalence level), about the top 60 percentile (equal to or higher than about 40% prevalence level), about the top 55 percentile (equal to or higher than about 10% prevalence level), about the top 50 percentile (equal to or higher than about 50% prevalence level), about the top 45 percentile (equal to or higher than about 55% prevalence level), about the top 40 percentile (equal to or higher than about 60% prevalence level), about the top 35 percentile (equal to or higher than about 65% prevalence level), about the top 30 percentile (equal to or higher than about 70% prevalence level), about the top 25 percentile (equal to or higher than about 75% prevalence level), about the top 20 percentile (equal to or higher than about 80% prevalence level), about the top 15 percentile (equal to or higher than about 85% prevalence level), about the top 10 percentile (equal to or higher than about 90% prevalence level), an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered to the individual at about the top 5 th percentile (equal to or greater than about 95% prevalence level), or at about the top 1 st percentile (equal to or greater than about 99% prevalence level) in the sample to the PD-L1, CXCL9, IFNG, GZMB, CD8A, and the expression level of the immune score of PD-1.

In some cases, an individual having cancer can be treated using the methods provided herein, the methods comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, the expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample from the individual and the expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample above a reference immune score expression level (e.g., in a population of individuals that do not have cancer or individuals that have cancer (e.g., have undergone one or more treatment with PD-L1 axis binding antagonist therapy or non-L1 axis binding therapy with PD-L binding antagonist: (e.g., (ii) E.g., a population of patients with lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), about the first 80 th percentile (equal to or higher than about 20% prevalence level), about the first 75 th percentile (equal to or higher than about 25% prevalence level), about the first 70 th percentile (equal to or higher than about 30% prevalence level), about the first 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or the immune score expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in samples in about the first 1 th percentile (equal to or above about 99% prevalence level).

(ii) Medicament and use thereof

In some cases, the medicament for use in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising (a) determining the expression level of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein the expression level of the immune score of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in the sample has been determined to be higher than the expression level of a reference immune score (e.g., the expression level of the immune score of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in the reference population), and (b) determining the immune score expression level of PD-L1, CXCL9, ifcl 9, ifzmb, CD8A, and PD-1 based on step (e.g., in the population having one or more cancer (e.g., the population having no cancer) in the reference population (e.g., the individual has undergone one or more cancer(s) (e.g., the population has undergone the An axis-binding antagonist therapy or a non-PD-L1 axis-binding antagonist therapy treated population with PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a population of patients with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC))), about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), about the first 80 th percentile (equal to or higher than about 20% prevalence level), about the first 75 th percentile (equal to or higher than about 25% prevalence level), about the first 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 percentile (equal to or higher than about 35% prevalence level), about the top 60 percentile (equal to or higher than about 40% prevalence level), about the top 55 percentile (equal to or higher than about 10% prevalence level), about the top 50 percentile (equal to or higher than about 50% prevalence level), about the top 45 percentile (equal to or higher than about 55% prevalence level), about the top 40 percentile (equal to or higher than about 60% prevalence level), about the top 35 percentile (equal to or higher than about 65% prevalence level), about the top 30 percentile (equal to or higher than about 70% prevalence level), about the top 25 percentile (equal to or higher than about 75% prevalence level), about the top 20 percentile (equal to or higher than about 80% prevalence level), about the top 15 percentile (equal to or higher than about 85% prevalence level), about the top 10 percentile (equal to or higher than about 90% prevalence level), an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered to the individual at about the top 5 th percentile (equal to or greater than about 95% prevalence level), or at about the top 1 st percentile (equal to or greater than about 99% prevalence level) in the sample to the PD-L1, CXCL9, IFNG, GZMB, CD8A, and the expression level of the immune score of PD-1.

In some cases, the medicament is for use in a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, the expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample from the individual and the expression levels of PD-L1, CXCL9, ifcl 9, GZMB, CD8A, and PD-1 have been determined in a sample above a reference immune score expression level (e.g., a reference population (e.g., a population of individuals not having cancer or individuals having cancer (e.g., having undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy (e.g., lung cancer) (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) in a population of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1), about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), about the first 80 th percentile (equal to or higher than about 20% prevalence level), about the first 75 th percentile (equal to or higher than about 25% prevalence level), about the first 70 th percentile (equal to or higher than about 30% prevalence level), about the first 65 th percentile (equal to or higher than about 35% prevalence level), about the first 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level) of samples from 1L in PD-L, the level of expression of CXCL9, IFNG, GZMB, CD8A, and PD-1 immune scores).

(iii) Use of PD-L1 axis binding antagonists

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is for use in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising (a) determining the expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein the immune score expression levels of PD-L1, CXCL9, ifzmng, GZMB, CD8A, and PD-1 in the sample have been determined to be higher than the reference immune score expression level (e.g., the immune expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, PD-1 in the reference population), and (b) based on the immune score expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 determined in step (a) (e.g., PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in a reference population (e.g., a population of individuals without cancer or a population of individuals with cancer (e.g., a population of patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy) with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the top 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level) of the sample in PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 immune score expression levels will be administered to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL 3280A)).

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is used in a method of treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) comprising administering to the individual an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), wherein prior to treatment, expression levels of PD-L1, CXCL9, IFNG, GZMB, CD A, and PD-1 have been determined in a sample from the individual and a reference immune expression level has been determined that is higher than the PD-, CXCL9, IFNG, GZMB, CD8A, and the immune score expression level of PD-1 (e.g., PD-L1, CXCL9, IFNG, GZMB, CD8A, and the immune score expression level of PD-1 in a reference population (e.g., a population of individuals without cancer or a population of individuals with cancer (e.g., patients who have undergone one or more treatments with PD-L1 axis binding antagonist therapy or non-PD-L1 axis binding antagonist therapy) with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC))), about the first 99 th percentile (equal to or higher than about 1% prevalence level), about the first 95 th percentile (equal to or higher than about 5% prevalence level), about the first 90 th percentile (equal to or higher than about 10% prevalence level), about the first 85 th percentile (equal to or higher than about 15% prevalence level), about the top 80 th percentile (equal to or higher than about 20% prevalence level), about the top 75 th percentile (equal to or higher than about 25% prevalence level), about the top 70 th percentile (equal to or higher than about 30% prevalence level), about the top 65 th percentile (equal to or higher than about 35% prevalence level), about the top 60 th percentile (equal to or higher than about 40% prevalence level), about the top 55 th percentile (equal to or higher than about 10% prevalence level), about the top 50 th percentile (equal to or higher than about 50% prevalence level), about the top 45 th percentile (equal to or higher than about 55% prevalence level), about the top 40 th percentile (equal to or higher than about 60% prevalence level), about the top 35 th percentile (equal to or higher than about 65% prevalence level), about the top 30 th percentile (equal to or higher than about 70% prevalence level), about the top 25 th percentile (equal to or higher than about 75% prevalence level), immune score expression levels of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1 in samples in about the top 20 th percentile (equal to or higher than about 80% prevalence level), about the top 15 th percentile (equal to or higher than about 85% prevalence level), about the top 10 th percentile (equal to or higher than about 90% prevalence level), about the top 5 th percentile (equal to or higher than about 95% prevalence level), or about the top 1 st percentile (equal to or higher than about 99% prevalence level).

PD-L1 axis binding antagonists

PD-L1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. PD-1 (apoptosis 1) is also known in the art as "apoptosis 1", "PDCD 1", "CD 279", and "SLEB 2". An exemplary human PD-1 is shown in UniProtKB/Swiss-Prot accession number Q15116. PD-L1 (apoptosis ligand 1) is also known in the art as "apoptosis 1 ligand 1", "PDCD 1LG 1", "CD 274", "B7-H", and "PDL 1". An exemplary human PD-L1 is shown in UniProtKB/Swiss-Prot accession No. Q9NZQ7.1. PD-L2 (apoptosis ligand 2) is also known in the art as "apoptosis 1 ligand 2", "PDCD 1LG 2", "CD 273", "B7-DC", "Btdc", and "PDL 2". An exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot accession number Q9BQ 51. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2. In some cases, the PD-1 axis binding antagonist is a PD-1 binding antagonist, a PD-L1 binding antagonist, or a PD-L2 binding antagonist.

(i) PD-L1 binding antagonists

In some cases, the PD-L1 binding antagonist inhibits PD-L1 from binding to one or more of its ligand binding partners. In other instances, the PD-L1 binding antagonist inhibits PD-L1 from binding to PD-1. In still other instances, a PD-L1 binding antagonist inhibits binding of PD-L1 to B7-1. In some cases, the PD-L1 binding antagonist inhibits PD-L1 from binding to both PD-1 and B7-1. In some cases, the PD-L1 binding antagonist is an antibody. In some cases, the antibody is selected from the group consisting of yw243.55.s70, MPDL3280A (atelizumab), MDX-1105, MEDI4736 (covaptumab), and MSB0010718C (avizumab).

In some cases, the anti-PD-L1 antibody is a monoclonal antibody. In some cases, the anti-PD-L1 antibody is selected from the group consisting of Fab, Fab '-SH, Fv, scFv, and (Fab')₂Antibody fragments of the group consisting of fragments. In some cases, the anti-PD-L1 antibody is a humanized antibody. In some cases, the anti-PD-L1 antibody is a human antibody. In some cases, an anti-PD-L1 antibody described herein binds to human PD-L1. In some particular instances, the anti-PD-L1 antibody is atezumab (CAS registry number: 1422185-06-5). Astuzumab (Genentech) is also known as MPDL 3280A.

In some cases, the anti-PD-L1 antibody comprises a heavy chain variable region (HVR-H) comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

(a) the HVR-H1 sequence is GFTFSDSWIH (SEQ ID NO: 9);

(b) the HVR-H2 sequence is AWISPYGGSTYYADSVKG (SEQ ID NO: 10); and is

(c) The HVR-H3 sequence was RHWPGGFDY (SEQ ID NO: 11).

In some cases, the anti-PD-L1 antibody further comprises a light chain variable region (HVR-L) comprising HVR-L1, HVR-L2, and HVR-L3 sequences, wherein:

(a) the HVR-L1 sequence is RASQDVSTAVA (SEQ ID NO: 12);

(b) the HVR-L2 sequence is SASFLYS (SEQ ID NO: 13); and is

(c) The HVR-L3 sequence was QQYLYHPAT (SEQ ID NO: 14).

In some cases, the anti-PD-L1 antibody comprises heavy and light chain sequences, wherein:

(a) the heavy chain Variable (VH) region sequence comprises the amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 15); and is

(b) The light chain Variable (VL) region sequence comprises the amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:16)。

(a) the heavy chain comprises the amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 17); and is

(b) The light chain comprises the amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:18)。

in some cases, an anti-PD-L1 antibody comprises (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID No. 15 or the sequence of SEQ ID No. 15; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID No. 16 or the sequence of SEQ ID No. 16; or (c) a VH domain as in (a) and a VL domain as in (b). In other instances, the anti-PD-L1 antibody is selected from the group consisting of yw243.55.s70, MDX-1105, MEDI4736 (dulvacizumab), and MSB0010718C (avizumab). Antibody yw243.55.s70 is an anti-PD-L1 antibody described in PCT publication No. wo 2010/077634. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in PCT publication No. WO 2007/005874. MEDI4736 (DOVACULAM) is an anti-PD-L1 monoclonal antibody described in PCT publication No. WO 2011/066389 and U.S. publication No. 2013/034559. Examples of anti-PD-L1 antibodies useful for the methods of the invention, and methods for their production, are described in PCT publications nos. WO 2010/077634, WO 2007/005874, and WO 2011/066389, as well as U.S. patent No.8,217,149 and U.S. publication No.2013/034559, which are incorporated herein by reference.

(ii) PD-1 binding antagonists

In some cases, the PD-L1 axis binding antagonist is a PD-1 binding antagonist. For example, in some cases, a PD-1 binding antagonist inhibits PD-1 from binding to one or more of its ligand binding partners. In some cases, the PD-1 binding antagonist inhibits PD-1 from binding to PD-L1. In other instances, the PD-1 binding antagonist inhibits PD-1 binding to PD-L2. In still other instances, the PD-1 binding antagonist inhibits PD-1 from binding to both PD-L1 and PD-L2. In some cases, the PD-1 binding antagonist is an antibody. In some cases, the antibody is selected from the group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (piditumumab), 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 a further aspect, the invention provides the use of a PD-L1 axis binding antagonist in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating cancer. In yet another embodiment, the medicament is for use in a method of treating cancer, comprising administering an effective amount of the medicament to a patient suffering from kidney cancer (e.g., Renal Cell Carcinoma (RCC), e.g., advanced RCC or metastatic RCC (mRCC), e.g., previously untreated advanced RCC or mRCC). In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

In some embodiments, the PD-1 binding antagonist is a molecule that inhibits binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PD-L1 and/or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits binding of PD-L1 to its binding ligand. In a particular aspect, the PD-L1 binding partner is PD-1 and/or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits binding of PD-L2 to its ligand binding partner. In a specific aspect, the PD-L2 binding ligand pair is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), e.g., as described below. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (piditumumab), MEDI-0680(AMP-514), PDR001, REGN2810, and BGB-108. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in WO 2006/121168. MK-3475, also known as pembrolizumab or lambrolizumab, is an anti-PD-1 antibody described in WO 2009/114335. CT-011, also known as hBAT, hBAT-1 or pidumab, is an anti-PD-1 antibody described in WO 2009/101611. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO 2011/066342.

In some embodiments, the anti-PD-1 antibody is MDX-1106. Alias names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558, and nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registry number 946414-94-4). In yet another embodiment, provided is an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO:19 and/or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO: 20.

In yet another embodiment, provided is an isolated anti-PD-1 antibody comprising heavy and/or light chain sequences, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:19), and

(b) the light chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a light chain sequence that is:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:20)。

(iii) substitution, insertion, and deletion variants

In certain instances, anti-PD-L1 antibody (e.g., atlizumab (MPDL3280A)) variants having one or more amino acid substitutions are provided for use in the methods, compositions, and/or kits of the present invention. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table 5 under the heading of "preferred substitutions". More substantial changes are provided in table 5 under the heading of "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

TABLE 5 exemplary and preferred amino acid substitutions

According to common side chain properties, amino acids can be grouped as follows:

(1) hydrophobic norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic Cys, Ser, Thr, Asn, Gln;

(3) acidic Asp, Glu;

(4) basic His, Lys, Arg;

(5) residues affecting chain orientation Gly, Pro;

(6) aromatic, Trp, Tyr, Phe.

Non-conservative substitutions may entail replacing one of these classes with a member of the other class.

One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have an alteration (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. An exemplary surrogate variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Changes (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such changes can be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods mol. biol.207: 179. 196(2008)), and/or antigen-contacting residues, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and re-selection of secondary libraries has been described, for example, in Hoogenboom et al, in Methods in Molecular Biology 178:1-37 (O' Brien et al, ed., Human Press, Totowa, NJ (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-guided approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. For example, such changes may be outside of antigen-contacting residues in HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered, or contains no more than 1,2, or 3 amino acid substitutions.

One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-. In this method, a residue or set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that indicate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. As alternative candidates, such contact and adjacent residues may be targeted or eliminated. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

(iv) Glycosylation variants

In some cases, anti-PD-L1 antibody (e.g., atlizumab (MPDL3280A)) variants are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites of an anti-PD-L1 antibody, such as atlizumab (MPDL3280A), can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

In the case of antibodies comprising an Fc region, the carbohydrate to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched, bi-antennary oligosaccharides, which are generally N-linked to Asn297 of the CH2 domain attached to the Fc region. See, e.g., Wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the bi-antennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.

In some cases, an anti-PD-L1 antibody (e.g., atlizumab (MPDL3280A)) variant has a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all sugar structures (e.g. complexed, heterozygous and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g. as described in WO 2008/077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. patent publication No. us2003/0157108(Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); Yamane-Ohnuki et al, Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13 CHO cells (Ripka et al, Arch. biochem. Biophys.249:533-545 (1986); U.S. patent application No. US 2003/0157108A1, Presta, L; and WO 2004/056312A 1, Adams et al, inter alia, example 11), and knock-out cell lines, such as alpha-1, 6-fucosyltransferase gene FUT8 knock-out CHO cells (see, e.g., Yamane-Ohnuki et al, Biotech. Bioeng.87:614 (2004); Kanda, Y.et al, Biotechnol. Bioeng.94(4):680-688 (2006); and WO 2003/085107).

In view of the above, in some cases, the methods of the invention involve administering to a subject an anti-PD-L1 antibody (e.g., atlizumab (MPDL3280A)) variant comprising an aglycosylation site mutation in the context of a fractionated, dose escalation dosing regimen. In some cases, the aglycosylation site mutation reduces effector function of the antibody. In some cases, the aglycosylation site mutation is a substitution mutation. In some cases, the antibody comprises a substitution mutation in the Fc region that reduces effector function. In some cases, the substitution mutation is at amino acid residue N297, L234, L235, and/or D265(EU numbering). In some cases, the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, D265A, and P329G. In some cases, the substitution mutation is at amino acid residue N297. In a preferred embodiment, the substitution mutation is N297A.

In other cases, antibody variants having bisected oligosaccharides are used in accordance with the methods of the present invention, e.g., where the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878(Jean-Mairet et al); U.S. Pat. No.6,602,684(Umana et al); and US 2005/0123546(Umana et al). Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087(Patel et al); WO 1998/58964(Raju, S.); and WO1999/22764(Raju, S.).

(v) Fc region variants

In some cases, an anti-PD-L1 antibody, e.g., an attritumab (MPDL3280A) variant (i.e., an Fc region variant (see e.g., US 2012/0251531)) having one or more amino acid modifications introduced into the Fc region of the antibody, may be administered to a subject having cancer (e.g., prostate cancer, e.g., CRPC, e.g., mCRPC, or locally restricted, non-surgical CRPC) according to the methods of the invention. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In some cases, Fc region antibody variants possess some, but not all, effector functions, making them desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that antibodies lack fcyr binding (thus potentially allowing forLack ADCC activity) but retain FcRn binding ability. The major cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of ravech and Kinet, Annu. Rev. immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No.5,500,362 (see, e.g., Hellstrom, I.et al, Proc. nat' l Acad. Sci. USA 83: 7059-; 5,821,337 (see Bruggemann, M.et., J.Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry)^TMNon-radioactive cytotoxicity assays (CellTechnology, inc., Mountain View, CA); and CYTOTOXNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models such as that disclosed in Clynes et al, proc.nat' l acad.sci.usa 95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q, and thus lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al, J.Immunol.methods 202:163 (1996); Cragg, M.S.et al, Blood 101: 1045-. FcRn binding and in vivo clearance/half-life assays may also be performed using methods known in the art (see, e.g., Petkova, s.b.et al, Int' l.immunol.18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those having substitutions of one or more of residues 238,265,269,270,297,327 and 329 of the Fc region (U.S. Pat. nos. 6,737,056 and 8,219,149). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265,269,270,297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. nos. 7,332,581 and 8,219,149).

In certain instances, the proline at position 329 of the wild-type human Fc region in the antibody is replaced with glycine or arginine or an amino acid residue that is large enough to disrupt the proline sandwich formed between the proline 329 of Fc and the tryptophan residues Trp87 and Trp110 of Fc γ RIII within the Fc/Fc γ receptor interface (Sondermann et al, Nature 406:267-273 (2000)). In certain embodiments, the antibody comprises at least one additional amino acid substitution. In one embodiment, the additional amino acid substitutions are S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S, and in yet another embodiment, at least one additional amino acid substitution is L234A and L235A of the Fc region of human IgG1 or S228P and L235E of the Fc region of human IgG4 (see, e.g., US 2012/0251531), and in yet another embodiment, at least one additional amino acid substitution is L234A and L235A and P329G of the Fc region of human IgG 1.

Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., U.S. Pat. No.6,737,056; WO 2004/056312; and Shields et al, J.biol. chem.9(2):6591-6604 (2001)).

In certain instances, an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A), comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., a substitution at position 298,333, and/or 334 (EU numbering of residues) of the Fc region.

In some cases, alterations are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO 99/51642, and Idusogene et al, J.Immunol.164: 4178-.

Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn) responsible for transfer of maternal IgG to the fetus (Guyer et al, j.immunol.117:587(1976) and Kim et al, j.immunol.24:249(1994)) are described in US 2005/0014934a1(Hinton et al). Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of residues 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434 of the Fc region, for example, at residue 434 of the Fc region (U.S. patent No.7,371,826).

Also seen are Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260; U.S. Pat. Nos. 5,624,821; and WO 94/29351, which concerns other examples of Fc region variants.

(vi) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create a cysteine engineered anti-PD-L1 antibody, e.g., a "thioMAb," in which one or more residues of the antibody are replaced with a cysteine residue. In particular embodiments, the substituted residues are present at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol groups are thus localized at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, one may substitute cysteine for any one or more of V205(Kabat numbering) of the light chain; a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No.7,521,541.

(vii) Other antibody derivatives

In some cases, an anti-PD-L1 antibody (e.g., atlizumab (MPDL3280A)) can be modified to contain additional non-protein property modules known and readily available in the art and administered to a subject according to the methods described herein. Suitable moieties for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, and the like.

G. Administration of

The methods, uses, assays, and kits described herein utilize PD-L1 axis binding antagonists (e.g., PD-L1 binding antagonists (e.g., anti-PD-L1 antibodies, such as attrituximab (MPDL3280A)) or PD-1 binding antagonists (e.g., anti-PD-1 antibodies)), or compositions thereof, which can be formulated for administration or administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subconjunctivally, intravesicularly, intraperically, 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 the target cells, by catheter, by lavage, in an emulsion, or in a lipid composition. The compositions utilized in the methods described herein may also be administered systemically or topically. The method of administration can vary depending on a variety of factors, such as the compound or composition being administered and the severity of the condition, disease, or disorder being treated. In some embodiments, the PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. Dosing may 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 long-term. Various dosing schedules are contemplated herein, including but not limited to a single administration or multiple administrations over multiple time points, bolus administration, and pulse infusion.

The PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist, e.g., anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) and any additional therapeutic agents may be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. PD-L1 axis binding antagonists (e.g., PD-L1 binding antagonists, e.g., anti-PD-L1 antibodies, e.g., atuzumab (MPDL3280A)) are not necessarily, but optionally, formulated and/or administered concurrently with one or more agents currently used for preventing or treating the disorder in question. The effective amount of such other agents depends on the amount of PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist, e.g., anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and administration routes as described herein, or about 1 to 99% of the dosages described herein, or at any dosage and any route empirically/clinically determined to be appropriate.

For the prevention or treatment of cancer (e.g., lung cancer (NSCLC), bladder cancer (UBC), kidney cancer (RCC), or breast cancer (TNBC)), the appropriate dosage of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) (when used alone or in combination with one or more other additional therapeutic agents) described herein will depend on the type of disease being treated, the severity and course of the disease, whether a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) is administered for prophylactic or therapeutic purposes, prior therapy, the clinical history of the patient, and a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)), and the discretion of the attending physician. A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) is suitably administered to the patient at one time or in a series of treatments. A typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until a desired suppression of disease symptoms occurs. Such doses may be administered intermittently, e.g., weekly or every three weeks (e.g., such that the patient receives, e.g., about two to about 20 doses, or, e.g., about six doses of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody))). A higher initial loading agent may be administered followed by a lower dose or doses. However, other dosage regimens may be useful. The progress of this therapy is readily monitored by conventional techniques and assays.

In some cases, an effective amount of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be between about 60mg to about 5000mg (e.g., between about 60mg to about 4500mg, between about 60mg to about 4000mg, between about 60mg to about 3500mg, between about 60mg to about 3000mg, between about 60mg to about 2500mg, between about 650mg to about 2000mg, between about 60mg to about 1500mg, between about 100mg to about 1500mg, between about 300mg to about 1500mg, between about 500mg to about 1500mg, between about 700mg to about 1500mg, between about 1000mg to about 1400mg, between about 1100mg to about 1300mg, between about 1150mg to about 1250mg, between about 1175mg to about 1225mg, or between about 1190mg to about 1210mg, such as about 1200mg ± 5mg, about 1200 ± 2.5mg, about 1200 ± 1.0mg, about 1200 ± 0.5mg, about 1200 ± 0.2mg, or about 1200 ± 0.1 mg). In some cases, the methods comprise administering to the individual a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) at about 1200mg (e.g., a fixed dose of about 1200mg or about 15 mg/kg).

In some cases, the amount of PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL3280A)) or PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) administered to an individual (e.g., a human) can be between about 0.01 to about 50mg/kg of the individual's body weight (e.g., between about 0.01 to about 45mg/kg, between about 0.01mg/kg to about 40mg/kg, between about 0.01mg/kg to about 35mg/kg, between about 0.01mg/kg to about 30mg/kg, between about 0.1mg/kg to about 30mg/kg, between about 1mg/kg to about 30mg/kg, between about 2mg/kg to about 30mg/kg, between about 5mg/kg to about 30mg/kg, between about 5mg/kg and about 25mg/kg, between about 5mg/kg and about 20mg/kg, between about 10mg/kg and about 20mg/kg, or between about 12mg/kg and about 18mg/kg, such as about 15 + -2 mg/kg, about 15 + -1 mg/kg, about 15 + -0.5 mg/kg, about 15 + -0.2 mg/kg, or about 15 + -0.1 mg/kg). In some cases, the method comprises administering to the individual a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) at about 15 mg/kg.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is administered to an individual (e.g., a human) at 1200mg intravenously every three weeks (q3 w). The dose may be administered as a single dose or as multiple doses (e.g., 2,3,4,5,6,7, or more than 7 doses), such as infusion. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) alone or in combination with additional therapeutic agents described herein (e.g., a VEGF antagonist (e.g., bevacizumab) and/or a chemotherapeutic agent (e.g., carboplatin and paclitaxel)) can be administered to an individual (e.g., a human) in four to six doses (e.g., every three weeks). The dose of antibody administered in the combination therapy can be reduced compared to monotherapy. The progress of this therapy is readily monitored by conventional techniques. In one instance, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atlizumab (e.g., MPDL3280A)) is administered to an individual as a monotherapy to treat cancer. In other instances, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (e.g., MPDL3280A)) is administered to an individual as a combination therapy as described herein to treat cancer.

H. Indications of

The methods and medicaments described herein are useful for treating a patient having cancer, such as lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC), by administering to the individual an effective amount of a PD-L1 axis binding antagonist, such as a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). For example, the cancer may be lung cancer, kidney cancer, bladder cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, stomach cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoides, merkel cell carcinoma, or hematological malignancy.

In some cases, the cancer is lung cancer. For example, the lung cancer may be non-small cell lung cancer (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 some cases, the lung cancer is chemotherapy-naive lung cancer (e.g., chemotherapy-naive metastatic nsclc (mcnsclc)). In some cases, the lung cancer is non-squamous lung cancer (e.g., non-squamous msnsclc). In some cases, the lung cancer is stage IV lung cancer (e.g., stage IV nsclc). In some cases, the lung cancer is recurrent lung cancer (e.g., recurrent mcnscc). In some cases, patients with lung cancer (e.g., NSCLC) have EGFR or ALK genomic alterations. In some cases, patients with lung cancer with EGFR or ALK genomic alterations have disease progression/treatment intolerance to one or more approved Tyrosine Kinase Inhibitors (TKIs).

In some cases, the cancer may be bladder cancer. For example, the bladder cancer may be Urothelial Bladder Cancer (UBC), including but not limited to non-muscle invasive urothelial bladder cancer, or metastatic urothelial bladder cancer. In some cases, the urothelial bladder cancer is metastatic urothelial bladder cancer.

In some cases, the cancer may be renal cancer. For example, the renal cancer may be Renal Cell Carcinoma (RCC), including stage I RCC, stage II RCC, stage III RCC, stage IV RCC, or recurrent RCC.

In some cases, the cancer may be breast cancer. In some cases, the breast cancer may be triple negative breast cancer. For example, the breast cancer can be triple negative breast cancer, estrogen receptor positive/HER 2 negative breast cancer, HER2 negative breast cancer, HER2 positive breast cancer, estrogen receptor negative breast cancer, progesterone receptor positive breast cancer, or progesterone receptor negative breast cancer.

In some cases, an individual having cancer, e.g., a cancer described herein, has not been previously treated for cancer. For example, an individual with cancer has not previously received PD-L1 axis binding antagonist therapy (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, an individual with cancer has previously received treatment for cancer. In some cases, an individual with cancer has previously received treatment comprising a non-PD-L1 axis binding antagonist therapy, such as an anti-cancer therapy (e.g., a cytotoxic agent, a growth inhibitory agent, a radiation therapy, an anti-angiogenic agent, or a combination thereof).

I. Combination therapy

In any of the methods herein, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be administered in combination with an effective amount of one or more additional therapeutic agents. Suitable additional therapeutic agents include, for example, antineoplastic agents, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, radiation therapy, or combinations thereof.

In some cases, the method further involves administering to the patient an effective amount of one or more additional therapeutic agents. In some cases, the additional therapeutic agent is selected from the group consisting of cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, radiotherapy agents, anti-angiogenic agents, and combinations thereof. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with a chemotherapeutic or chemotherapeutic agent. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with a radiotherapy agent. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with a targeted therapy or targeted therapeutic. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an immunotherapy or immunotherapeutic agent, e.g., a monoclonal antibody. In some cases, the additional therapeutic agent is an agonist to an activating costimulatory molecule. In some cases, the additional therapeutic agent is an antagonist against an inhibitory co-stimulatory molecule.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same formulation or in separate formulations), and separate administration, in which case administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may occur prior to, concurrently with, and/or after administration of one or more additional therapeutic agents. In one instance, administration of the PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) 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.

Without wishing to be bound by theory, it is believed that enhancing T cell stimulation by promoting activating costimulatory molecules or by inhibiting negative costimulatory molecules can promote tumor cell death, thereby treating or delaying cancer progression. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an agonist against an activating costimulatory molecule. In some cases, the activating costimulatory molecule can include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD 127. In some cases, the agonist to an activating costimulatory molecule is an agonistic antibody that binds CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD 127. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an antagonist against an inhibitory co-stimulatory molecule. In some cases, the inhibitory co-stimulatory molecule may comprise CTLA-4 (also known as CD152), TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some cases, the antagonist against the inhibitory co-stimulatory molecule is an antagonistic antibody that binds CTLA-4, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an antagonist, e.g., a blocking antibody, against CTLA-4 (also known as CD 152). In some cases, it may be combined with ipilimumab (also known as MDX-010, MDX-101, or) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered in combination. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) can be administered in combination with tremelimumab (also known as ticilimumab or CP-675,206). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an antagonist, e.g., a blocking antibody, against B7-H3 (also referred to as CD 276). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with MGA 271. In some cases, it may be combined with an antagonist against TGF- β, such as metelilimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with a therapy comprising adoptive transfer of T cells expressing a Chimeric Antigen Receptor (CAR), e.g., cytotoxic T cells or CTLs. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with a therapy comprising adoptive transfer of T cells comprising a dominant negative TGF β receptor, e.g., a dominant negative TGF β type II receptor. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) (see, e.g., clinical trials. gov identifier NCT00889954) can be administered in combination with a treatment comprising a HERCREEM regimen.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) can be administered in combination with an agonist, e.g., an activating antibody, directed against CD137 (also known as TNFRSF9,4-1BB, or ILA). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) can be administered in combination with urelumab (also referred to as BMS-663513). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) may be administered in combination with an agonist, e.g., an activating antibody, directed against CD 40. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with CP-870893. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an agonist, e.g., an activating antibody, directed against OX40 (also referred to as CD 134). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) can be administered in combination with an anti-OX 40 antibody (e.g., AgonOX). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) may be administered in combination with an agonist, e.g., an activating antibody, directed against CD 27. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with CDX-1127. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an antagonist against indoleamine-2, 3-dioxygenase (IDO). In some cases, the IDO antagonist is 1-methyl-D-tryptophan (also referred to as 1-D-MT).

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with the antibody-drug conjugate. In some cases, the antibody-drug conjugate comprises mertansine or monomethyl auristatin e (mmae). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an anti-NaPi 2b antibody-MMAE conjugate (also referred to as DNIB0600A or RG 7599). In some cases, it may be combined with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, orGenentech) in combination with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL 3280A)). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with DMUC 5754A. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an antibody-drug conjugate targeting endothelin B receptors (EDNBRs), e.g., an antibody to EDNBRs conjugated with MMAE.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., a/v) can be administered in combination with an anti-angiogenic agentTezumab (MPDL 3280A)). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an antibody directed against VEGF, e.g., VEGF-a. In some cases, bevacizumab (also known as bevacizumab)Genentech) in combination with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL 3280A)). For example, attritzumab (MPDL3280A) may be administered in combination with bevacizumab. In still other cases, atuzumab (MPDL3280A) may be administered in combination with bevacizumab and one or more chemotherapeutic agents (e.g., carboplatin and/or paclitaxel). In certain instances, attrituximab (MPDL3280A) may be administered in combination with bevacizumab, carboplatin, and paclitaxel. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) can be administered in combination with an antibody to angiopoietin 2 (also known as Ang 2). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with MEDI 3617.

A VEGF antagonist (e.g., bevacizumab) administered to an individual (e.g., a human) in combination with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., an atuzumab (MPDL3280A)) can be between about 0.01 to about 50mg/kg of the individual's body weight (e.g., between about 0.01 to about 45mg/kg, between about 0.01mg/kg to about 40mg/kg, between about 0.01mg/kg to about 35mg/kg, between about 0.01mg/kg to about 30mg/kg, between about 0.1mg/kg to about 30mg/kg, between about 1mg/kg to about 30mg/kg, between about 2mg/kg to about 30mg/kg, between about 5mg/kg to about 25mg/kg, between about 5mg/kg to about 20mg/kg, between about 10mg/kg and about 20mg/kg, or between about 12mg/kg and about 18mg/kg, such as about 15 + -2 mg/kg, about 15 + -1 mg/kg, about 15 + -0.5 mg/kg, about 15 + -0.2 mg/kg, or about 15 + -0.1 mg/kg). For example, in some cases, the methods comprise administering to the individual about 1200mg of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) in combination with about 15mg/kg of the individual's weight of a VEGF antagonist (e.g., bevacizumab). The method may further comprise administering one or more chemotherapeutic agents, such as carboplatin and/or paclitaxel.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an anti-tumor agent. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, such as atuzumab (MPDL3280A)) may be administered in combination with an agent targeting CSF-1R (also referred to as M-CSFR or CD 115). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) can be administered in combination with anti-CSF-1R (also referred to as IMC-CS 4). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an interferon, e.g., interferon alpha or interferon gamma. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) may be administered in combination with Roferon-a (also known as recombinant interferon alpha-2 a). In some cases, it may be combined with GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargrastim, or) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered in combination. In some cases, it may be combined with IL-2 (also known as aldesleukin or) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered in combination. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with IL-12. In some cases, it may be associated with targeting CD20The antibody is administered in combination with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL 3280A)). In some cases, the antibody targeting CD20 is obinutuzumab (obinutuzumab) (also known as GA101 or) Or rituximab (rituximab). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an antibody targeting GITR. In some cases, the antibody targeting GITR is TRX 518.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with a cancer vaccine. In some cases, the cancer vaccine is a peptide cancer vaccine, which in some cases is a personalized peptide vaccine. In some cases, the peptide Cancer vaccine is a multivalent long peptide, a multiple peptide, a mixture of peptides, a hybrid peptide, or a peptide pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci.104:14-21,2013). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) may be administered in combination with an adjuvant. In some cases, a TLR agonist, such as Poly-ICLC (also known as) Treatment with LPS, MPL, or CpG ODN is administered in combination with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL 3280A)). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with Tumor Necrosis Factor (TNF) alpha (TNF-a). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with IL-1. In some cases, a PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MP) may be administered in combination with HMGB1DL 3280A)). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an IL-10 antagonist. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an IL-4 antagonist. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an IL-13 antagonist. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an HVEM antagonist. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an ICOS agonist, e.g., by administering ICOS-L, or an agonistic antibody to ICOS. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with a treatment targeting CX3CL 1. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with a therapy targeting CXCL 9. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with a therapy targeting CXCL 10. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with a therapy targeting CCL 5. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) can be administered in combination with an LFA-1 or ICAM1 agonist. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with a selectin agonist.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody,such as astuzumab (MPDL 3280A)). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with an inhibitor of B-Raf. In some cases, it may be combined with vemurafenib (also known as vemurafenib)) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered in combination. In some cases, it may be combined with dabrafenib (also known as dabrafenib)) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered in combination. In some cases, it may be combined with erlotinib (also known as) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered in combination. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) can be administered in combination with an inhibitor of a MEK, such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K 2). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with cobitinib (also referred to as GDC-0973 or XL-518). In some cases, it may be combined with trametinib (also known as trametinib)) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered in combination. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an inhibitor of K-Ras. In some cases, PD-L1 axis binding may be administered in combination with an inhibitor of c-MetAntagonists (e.g., PD-L1 binding antagonists, e.g., anti-PD-L1 antibodies, e.g., atlizumab (MPDL 3280A)). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) can be administered in combination with onartuzumab (aka MetMAb). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an inhibitor of Alk. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) may be administered in combination with AF802 (also known as CH5424802 or altanib). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) can be administered in combination with an inhibitor of phosphatidylinositol 3-kinase (PI 3K). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) may be administered in combination with BKM 120. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) may be administered in combination with piperacillin (also referred to as KRX-0401). In some embodiments, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with an inhibitor of Akt. In some embodiments, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) may be administered in combination with MK 2206. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with GSK 690693. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with GDC-0941. In some cases, administration may be in combination with an inhibitor of mTORPD-L1 axis binding antagonists (e.g., PD-L1 binding antagonists, e.g., anti-PD-L1 antibodies, e.g., attritumab (MPDL3280A)) are used. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with sirolimus (also referred to as rapamycin). In some cases, with temsirolimus (also known as CCI-779 or) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, such as an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) is administered in combination. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with everolimus (also referred to as RAD 001). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with ridaforolimus (also known as AP-23573, MK-8669, or ridaforolimus). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with OSI-027. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with AZD 8055. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with INK 128. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with a dual PI3K/mTOR inhibitor. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with XL 765. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with GDC-0980. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist) can be administered in combination with BEZ235 (also known as NVP-BEZ235)E.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL 3280A)). In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with BGT 226. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) may be administered in combination with GSK 2126458. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) can be administered in combination with PF-04691502. In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) can be administered in combination with PF-05212384 (also known as PKI-587).

(i) Combination therapy in clinical trials

A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be administered to an individual in combination with one or more additional therapeutic agents, wherein the individual, prior to or after treatment, has undergone diagnostic testing according to any of the diagnostic methods described herein and has been identified as an individual who is likely to benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). As described further below, the additional therapeutic agent may be a therapeutic agent that has been tested or is undergoing testing in a clinical trial for cancer therapy including attentizumab.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with the obinutuzumab and the polatuzumab vedotin (e.g., in the treatment of lymphoma (e.g., relapsed or refractory follicular lymphoma or diffuse large B-cell lymphoma), as in clinical trial NCT 02729896).

In some cases, it may be combined with paclitaxel (e.g., albumin-bound paclitaxel (nab-paclitaxel)) Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of breast cancer (e.g., TNBC)), as in clinical trial NCT 02530489.

In some cases, bevacizumab (also known as bevacizumab)) Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of locally advanced or metastatic tumors (e.g., in breast, cervical, renal, gastric, ovarian, or bladder cancer)), as in clinical trial NCT 01633970.

In some cases, bevacizumab (also known as bevacizumab)) The PD-L1 axis binding antagonist (e.g., PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is administered in combination with folinic acid/oxaliplatin/5-fluorouracil (FOLFOX) (e.g., in the treatment of locally advanced or metastatic tumors (e.g., in breast, cervical, renal, gastric, ovarian, or bladder cancer)) as in clinical trial NCT 01633970.

In some cases, it may be combined with paclitaxel (e.g., albumin-bound paclitaxel (nab-paclitaxel)) And carboplatin (e.g. of) Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist: (E.g., an anti-PD-1 antibody)) (e.g., in the treatment of locally advanced or metastatic tumors, e.g., in the treatment of lung cancer (NSCLC), breast cancer, cervical cancer, renal cancer, gastric cancer, ovarian cancer, or bladder cancer), as in clinical trial NCT 01633970.

In some cases, it may be combined with paclitaxel (e.g., albumin-bound paclitaxel (nab-paclitaxel)) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of locally advanced or metastatic tumors, e.g., in the treatment of lung cancer (NSCLC), breast cancer, cervical cancer, renal cancer, gastric cancer, ovarian cancer, or bladder cancer), as in clinical trial NCT 01633970.

In some cases, it may be combined with pemetrexed (e.g.) And carboplatin (e.g. of) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is administered in combination (e.g., in the treatment of locally advanced or metastatic tumors, e.g., in the treatment of breast, cervical, renal, gastric, ovarian, or bladder cancer), as in clinical trial NCT 01633970.

In some cases, with etoposide (e.g.) And carboplatin (e.g. of) Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in lung cancer (e.g., small-sized lung cancer)Cell Lung Cancer (SCLC)) as in clinical trial NCT 02748889.

In some cases, it may be combined with paclitaxel (e.g., albumin-bound paclitaxel (nab-paclitaxel)) And carboplatin (e.g. of) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is administered in combination (e.g., in the treatment of locally advanced or metastatic tumors, e.g., in the treatment of lung cancer (NSCLC)), as in clinical trial NCT 02716038.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with an epacadostat (e.g., INCB024360), e.g., in the treatment of lung cancer (e.g., NSCLC) or bladder cancer (e.g., urothelial cancer), as in clinical trial NCT 02298153.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with radiation therapy and chemotherapy (e.g., carboplatin and/or paclitaxel) (e.g., in the treatment of lung cancer (e.g., NSCLC), as in clinical trial NCT 02525757.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with veliparib (e.g., in the treatment of a breast cancer, e.g., TNBC, a BRCA1 gene mutation, a BRCA2 gene mutation, an estrogen receptor negative breast cancer, Her2/Neu negative breast cancer, stage III breast cancer, stage IIIB breast cancer, stage IIIC breast cancer, or stage IV breast cancer), as in clinical trial NCT 02849496.

In some cases, it may be combined with Alanib (also known as Acranium)) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is administered in combination (e.g., in the treatment of lung cancer (e.g., NSCLC)), as in clinical trial NCT 02013219.

In some cases, it may be combined with erlotinib (also known as) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is administered in combination (e.g., in the treatment of lung cancer (e.g., NSCLC)), as in clinical trial NCT 02013219.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with MTIG7192A (e.g., in the treatment of advanced metastatic tumors), as in clinical trial NCT 02794571.

In some cases, it may be combined with vemurafenib (also known as vemurafenib)) A PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) is administered in combination (e.g., in the treatment of skin cancer (e.g., malignant melanoma)) as in clinical trial NCT 01656642.

In some cases, it may be combined with vemurafenib (also known as vemurafenib)) And cobicistinib (also known as cobicistinib)Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., a C. sub.D)An anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of skin cancer (e.g., malignant melanoma)), as in clinical trial NCT 01656642.

In some cases, bevacizumab (also known as bevacizumab)Genentech) in combination with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of ovarian, fallopian, or peritoneal cancer), as in clinical trial NCT 0283907.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with the obinutuzumab, e.g., in the treatment of lymphoma (e.g., lymphocytic lymphoma or relapsed refractory or Chronic Lymphocytic Leukemia (CLL)), as in clinical trial NCT 46028623.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with carboplatin and pemetrexed (e.g., in the treatment of lung cancer (e.g., NSCLC), as in clinical trial NCT 57434.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with cisplatin and pemetrexed (e.g., in the treatment of lung cancer (e.g., NSCLC)), as in clinical trial NCT 02657434.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with tazemetostat (e.g., in the treatment of lymphoma (e.g., follicular lymphoma or diffuse large B-cell lymphoma), as in clinical trial NCT 20802242.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with the obinutuzumab, e.g., in the treatment of lymphoma (e.g., follicular lymphoma or diffuse large B-cell lymphoma), as in clinical trial NCT 20802242.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., altlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with lenalidomide (e.g., in the treatment of multiple myeloma), as in clinical trial NCT 02431208.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with daratumumab, e.g., in the treatment of multiple myeloma, as in clinical trial NCT 02431208.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with daratumumab and lenalidomide (e.g., in the treatment of multiple myeloma), as in clinical trial NCT 02431208.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with daratumumab and pomidomide (e.g., in the treatment of multiple myeloma), as in clinical trial NCT 02431208.

In some cases, bevacizumab (also known as bevacizumab)Genentech) in combination with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., a monoclonal antibody, e.g., a monoclonalSuch as an anti-PD-1 antibody)) (e.g., in the treatment of kidney cancer (e.g., renal cell carcinoma)), such as in clinical trial NCT 02420821.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with stereotactic radiation (e.g., in the treatment of lung cancer (e.g., NSCLC), as in clinical trial NCT 02400814.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with rociletinib (e.g., in the treatment of lung cancer (e.g., NSCLC)), as in clinical trial NCT 02630186.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with GDC-0919 (e.g., in the treatment of a solid tumor, e.g., Renal Cell Carcinoma (RCC), Urothelial Bladder Cancer (UBC), Triple Negative Breast Cancer (TNBC), non-small cell lung cancer (NSCLC), melanoma, Head and Neck Squamous Cell Carcinoma (HNSCC), gastric cancer, ovarian cancer, cervical cancer, endometrial cancer, or merkel cell carcinoma), as in clinical trial NCT 02471846.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with radium dichloride-223 (e.g., in the treatment of lung prostate cancer (e.g., castration resistant prostate cancer)), as in clinical trial NCT 02814669.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with MOXR0916 (e.g., in the treatment of a solid tumor, e.g., a locally advanced or metastatic solid tumor), as in clinical trial NCT 02410512.

In some cases, bevacizumab (also known as bevacizumab)Genentech) and MOXR0916 in combination with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of a solid tumor (e.g., a locally advanced or metastatic solid tumor)), as in clinical trial NCT 02410512.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with azacitidine (e.g., in the treatment of a solid tumor, e.g., myelodysplastic syndrome), as in clinical trial NCT 02508870.

In some cases, it may be combined with paclitaxel (e.g., albumin-bound paclitaxel (nab-paclitaxel)) Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of breast cancer (e.g., TNBC)), as in clinical trial NCT 02425891.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with lenalidomide and obinutuzumab (e.g., in the treatment of lymphoma), as in clinical trial NCT 02631577.

In some cases, with etoposide (e.g.) And carboplatin (e.g. of) Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., Attributab (MPD))L3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of lung cancer (e.g., Small Cell Lung Cancer (SCLC)), as in clinical trial NCT 02763579.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with ipilimumab (e.g., in the treatment of locally advanced or metastatic solid tumors), as in clinical trial NCT 02174172.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with interferon alpha-2 b (e.g., in the treatment of locally advanced or metastatic solid tumors (e.g., NSCLC, melanoma, or RCC), as in clinical trial NCT 02174172.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with low-segmentation image-guided radiotherapy (e.g., in the treatment of lung cancer (e.g., NSCLC), as in clinical trial NCT 02463994.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with CDX-1401 (e.g., in the treatment of lung cancer (e.g., NSCLC), as in clinical trial NCT 02495636.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with trastuzumab and pertuzumab (e.g., in the treatment of breast cancer (e.g., Her2 positive breast cancer)), as in clinical trial NCT 02605915.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with trastuzumab emtansine (e.g., in the treatment of breast cancer, e.g., Her2 positive breast cancer), as in clinical trial NCT 02605915.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with doxorubicin and cyclophosphamide (e.g., in the treatment of breast cancer (e.g., Her2 positive breast cancer)), as in clinical trial NCT 02605915.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with trastuzumab, pertuzumab, and docetaxel, e.g., in the treatment of breast cancer (e.g., Her2 positive breast cancer), as in clinical trial NCT 02605915.

In some cases, bevacizumab (also known as bevacizumab)) Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of renal cancer (e.g., advanced non-clear cell renal cancer)), as in clinical trial NCT 02724878.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atelizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) can be administered in combination with CMB305 (e.g., in the treatment of a sarcoma (e.g., myxoid/round cell liposarcoma, synovial sarcoma, metastatic sarcoma, recurrent adult soft tissue sarcoma, local advanced sarcoma, or liposarcoma), as in clinical trial NCT 02609984.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with RO7009789 (e.g., in the treatment of solid cancers (e.g., locally advanced and metastatic solid tumors), as in clinical trial NCT 02304393).

In some cases, it may be combined with BCG (also known as BCG)) Co-administration of a PD-L1 axis binding antagonist (e.g. a PD-L1 binding antagonist (e.g. an anti-PD-L1 antibody, e.g. attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g. an anti-PD-1 antibody)) (e.g. in the treatment of bladder cancer (e.g. non-muscle invasive bladder cancer)), as in clinical trial NCT 02792192.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with stereotactic radiotherapy (e.g., in the treatment of lung cancer (e.g., NSCLC), as in clinical trial NCT 02599454.

In some cases, it may be combined with carboplatin, and nab-paclitaxel (also known as) Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of breast cancer (e.g., invasive ductal breast cancer)), as in clinical trial NCT 02620280.

In some cases, it may be combined with carboplatin, nab-paclitaxel (also known as carboplatin)) And co-chemotherapy comprising AC or EC (doxorubicin or epirubicin and cyclophosphamide) or FEC (fluorouracil, epirubicin, and cyclophosphamide) with a PD-L1 axis binding antagonist (e.g.PD-L1 binding antagonists (e.g., anti-PD-L1 antibodies, e.g., atlizumab (MPDL3280A)) or PD-1 binding antagonists (e.g., anti-PD-1 antibodies)) (e.g., in the treatment of breast cancer (e.g., invasive ductal breast cancer)), as in clinical trial NCT 02620280.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with gemcitabine and carboplatin or cisplatin (e.g., in the treatment of urothelial cancer), as in clinical trial NCT 02807636.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with paclitaxel and carboplatin (e.g., in the treatment of lung cancer (e.g., NSCLC, e.g., non-squamous NSCLC), as in clinical trial NCT 02366143.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with bevacizumab, paclitaxel, and carboplatin (e.g., in the treatment of lung cancer (e.g., NSCLC, e.g., non-squamous NSCLC), as in clinical trial NCT 02366143).

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with cerglutuzumab (also referred to as RO6895882) (e.g., in the treatment of locally advanced and/or metastatic solid tumors), as in clinical trial NCT 02350673.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with bendamustine and obinutuzumab (e.g., in the treatment of lymphoma (e.g., diffuse large B-cell lymphoma or follicular lymphoma), as in clinical trial NCT 02596971.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., astuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with bendamustine, cyclophosphamide, orbitumumab, prednisone, and vincristine (e.g., in the treatment of lymphoma (e.g., diffuse large B-cell lymphoma or follicular lymphoma), as in clinical trial NCT 02596971.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with cyclophosphamide, doxorubicin, orbin, ortuzumab, prednisone, and vincristine (e.g., in the treatment of lymphoma (e.g., diffuse large B-cell lymphoma or follicular lymphoma), as in clinical trial NCT 02596971).

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with cyclophosphamide, doxorubicin, prednisone, vincristine, and rituximab (e.g., in the treatment of lymphoma (e.g., diffuse large B-cell lymphoma or follicular lymphoma), as in clinical trial NCT 02596971.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with RO6958688 (e.g., in the treatment of locally advanced and/or metastatic solid tumors (e.g., carcinoembryonic antigen (CEA) -positive solid tumors), as in clinical trial NCT 02650713).

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with acetylsalicylic acid (e.g., in the treatment of ovarian cancer (e.g., ovarian neoplasm)), as in clinical trial NCT 02659384.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atuzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with bevacizumab (e.g., in the treatment of ovarian cancer (e.g., ovarian neoplasm)), as in clinical trial NCT 02659384.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with vanucizumab (also known as RO5520985) (e), e.g., in the treatment of locally advanced and/or metastatic solid tumors, as in clinical trial NCT 01688206.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with carboplatin and nab-paclitaxel (e.g., in the treatment of lung cancer (e.g., non-squamous NSCLC), as in clinical trial NCT 02367781.

In some cases, bevacizumab (also known as bevacizumab)) Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of renal cancer (e.g., renal cell carcinoma)), as in clinical trial NCT 01984242.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with cobitinib (also referred to as GDC-0973), e.g., in the treatment of locally advanced or metastatic solid tumors, as in clinical trial NCT 01988896.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with RO5509554 (e.g., in the treatment of locally advanced solid tumors (e.g., locally advanced and/or metastatic triple negative breast cancer, ovarian cancer, bladder cancer, gastric cancer, or soft tissue sarcoma), as in clinical trial NCT 02323191.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with varliumab (e.g., in the treatment of advanced cancer (e.g., melanoma, RCC, triple negative breast cancer, bladder cancer, head and neck cancer, or non-small cell lung cancer)) as in clinical trial NCT 02543645.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with cobitinib (e.g., in the treatment of colorectal cancer), as in clinical trial NCT 02788279.

In some cases, bevacizumab (also known as bevacizumab)) Co-administration of a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of a solid tumor), as in clinical trial NCT 02715531.

In some cases, bevacizumab (also known as bevacizumab)) Leucovorin, oxaliplatin, and optionally capecitabine, in combination with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) (e.g., in the treatment of a solid tumor), as in clinical trial NCT 02715531.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with nab-paclitaxel and gemcitabine (e.g., in the treatment of a solid tumor), as in clinical trial NCT 02715531.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with oxaliplatin, leucovorin, 5-fluorouracil (5-FU), oxaliplatin, and cisplatin (e.g., in a clinical trial NCT 02715531).

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with nab-paclitaxel and carboplatin (e.g., in the treatment of lung cancer, e.g., squamous NSCLC), as in clinical trial NCT 02367794.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with paclitaxel and carboplatin (e.g., in the treatment of lung cancer (e.g., squamous NSCLC), as in clinical trial NCT 02367794.

In some cases, a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)) may be administered in combination with CPI-444 (e.g., in the treatment of advanced cancers (e.g., non-small cell lung cancer, malignant melanoma, renal cell carcinoma, triple negative breast cancer, colorectal cancer with microsatellite instability (MSI), and bladder cancer), as in clinical trial NCT 02655822).

Pharmaceutical compositions and formulations

The Pharmaceutical compositions can be prepared by mixing an active ingredient having the desired purity, such as an anti-PD-L1 antibody (MPDL3280A), with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16 th edition,osol, a. eds (1980)) pharmaceutical compositions and formulations as described herein are prepared in the form of a lyophilized formulation or an aqueous solution. Generally, pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; hydrocarbyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (r: (r) ())Baxter International, Inc.). Certain exemplary shasegps and methods of use (including rHuPH20) are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase. It is understood that any of the above pharmaceutical compositions or formulations may include an immunoconjugate described herein, in place of or in addition to a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

An exemplary lyophilized antibody formulation is described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO 2006/044908, the latter formulation including histidine-acetate buffer.

The compositions and formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitory agents, and/or anti-hormonal agents, such as those described herein above). Suitably, such active ingredients are present in combination in an amount effective for the intended purpose.

The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. eds (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

V. articles and kits

In another aspect of the invention, an article of manufacture or kit containing materials useful for treating, preventing, and/or diagnosing an individual is provided.

In some cases, such articles of manufacture or kits can be used to identify individuals with cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) who may benefit from a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). Such articles or kits can include (a) an agent for determining the expression level of an immune score in a sample from an individual selected from at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any combination of the genes listed in tables 1-4), and (b) an antibody (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL 3280) or a PD-1 binding antagonist (e.g., PD-1)) for identifying an antibody (e.g., an anti-NSCLC antibody) having the benefit of treating cancer (e.g., lung cancer), instructions for an individual with bladder cancer (e.g., UBC), renal cancer (e.g., RCC), or breast cancer (e.g., TNBC)).

For example, in some cases, an article of manufacture or kit includes (a) an agent for determining the expression level of an immune score for PD-L1, CXCL9, and IFNG in a sample from an individual, and (b) instructions for using the agent to identify an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) that can benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the article of manufacture or kit includes (a) an agent for determining the expression level of an immune score of PD-L1, IFNG, GZMB, and CD8A in a sample from an individual, and (b) instructions for using the agent to identify an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) that can benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atlizumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)). In some cases, the article of manufacture or kit includes (a) an agent for determining the expression level of an immune score for PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from an individual, and (b) instructions for using the agent to identify an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) that may benefit from treatment with a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)).

In some cases, such articles of manufacture or kits include a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituximab (MPDL3280A)), for treating an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)). In some cases, the article of manufacture or kit comprises (a) a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) and (b) a package insert, including instructions for administering a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) to an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), wherein, prior to treatment, at least one, at least two, at least three, at least four, at least five, or all six genes selected from the group consisting of PD-L1, CXCL9, IFNG, GZMB, CD8A, and PD-1) in a sample from the individual have been determined, or a combination thereof (e.g., PD-L1, CXCL9, and IFNG; PD-L1, IFNG, GZMB, and CD 8A; PD-L1, IFNG, GZMB, CD8A, and PD-1; or any of the combinations of genes listed in tables 1-4)) and at least one, at least two, at least three, at least four, at least five, or all six of PD-L1, CXCL9, IFNG, GZMB, CD8A, or PD-1 in the sample is higher than the reference immune score expression level.

For example, in some cases, the article of manufacture or kit comprises (a) a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) and (b) a package insert comprising instructions for administering a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) to an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)), wherein, prior to treatment, a sample from the subject has been determined to have a high degree of specificity for PD-L1, CXCL9, and IFNG and the level of expression of the immune score in the sample PD-L1, CXCL9, and IFNG, wherein at least one, at least two, or all three of the IFNG are expressed at a level greater than the reference immune score. In some cases, the article of manufacture or kit comprises (a) a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)); and (b) a package insert comprising instructions for administering a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) to an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) wherein, prior to treatment, the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual and at least one, at least two, at least three, or all four of PD-L1, IFNG, GZMB, and CD8A in the sample are determined to be greater than a reference immune score expression level. In some cases, the article of manufacture or kit comprises (a) a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, e.g., attritumab (MPDL3280A)) or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody)); and (b) a package insert comprising instructions for administering a PD-L1 axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., an anti-PD-L1 antibody, e.g., attrituzumab (MPDL3280A)) to an individual having cancer (e.g., lung cancer (e.g., NSCLC), bladder cancer (e.g., UBC), kidney cancer (e.g., RCC), or breast cancer (e.g., TNBC)) wherein, prior to treatment, the expression level of an immune score for PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual and the expression level of at least one, at least two, at least three, at least four, or all five of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample have been determined to be higher than the reference immune score expression level.

Any of the articles or kits described may include a loading device that is compartmentalized to receive in close confinement one or more container devices, such as vials, tubes, and the like, each container device containing one of the different elements to be used in the method. In the case of articles or kits that utilize nucleic acid hybridization to detect a target nucleic acid, the kit can also have a container containing nucleotides for amplifying the target nucleic acid sequence and/or a container containing a reporter means such as an enzymatic, fluorescent, or radioisotope label.

In some cases, the article of manufacture or kit comprises the container described above and one or more other containers containing materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with printed instructions for use. A label may be present on the container indicating that the composition is for a particular application, but may also indicate instructions for in vivo or in vitro use, such as those described above. For example, the article of manufacture or kit may further comprise a container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution.

The articles of manufacture or kits described herein can have many embodiments. In one aspect, an article of manufacture or a kit comprises a container, a label on the container, and a composition contained within the container, wherein the composition comprises one or more polynucleotides that hybridize under stringent conditions to the complement of a gene listed herein (e.g., PD-L1, CXCL9, IFNG, GZMB, CD8A, or PD-1), and the label on the container indicates that the composition can be used to assess a sample for the presence of a gene listed herein (e.g., PD-L1, CXCL9, IFNG, GZMB, CD8A, or PD-1), and wherein the kit comprises instructions for using the polynucleotides to assess the presence of a gene's RNA or DNA in a particular sample type.

For oligonucleotide-based articles or kits, the articles or kits can include, for example, (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, that hybridizes to a nucleic acid sequence encoding a protein, or (2) a pair of primers that are useful for amplifying a nucleic acid molecule. The article of manufacture or kit can also include, for example, a buffer, a preservative, or a protein stabilizer. The article of manufacture or kit may further comprise components (e.g., an enzyme or substrate) necessary for detection of the detectable label. The article of manufacture or kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the article of manufacture or kit may be enclosed in a separate container, and each container may be in a single package with instructions (for interpreting the results of the assays performed using the kit).

VI. examples

The following are examples of the method of the present invention. It is understood that various other embodiments may be implemented in view of the general description provided above.

Example 1 correlation between immune score expression levels of (i) PD-L1, CXCL9, and IFNG or (ii) PD-L1, IFNG, GZMB, and CD8A and clinical response to treatment with Attributab (MPDL3280A) in patients with non-small cell lung cancer (NSCLC)

An RNA-based molecular assay was used to assess the association between clinical response to treatment with the anti-PD-L1 antibody attritumab (MPDL3280A) and the expression levels of immune scores of (i) PD-L1, CXCL9, and IFNG or (ii) PD-L1, IFNG, GZMB, and CD8A in patients with non-small cell lung cancer (NSCLC) enrolled in a phase III clinical trial in which attritumab was administered as a monotherapy.

Design of research

The OAK (clinical trial ID No.: NCT02008227) patient population that evaluated the expression levels of (i) PD-L1, CXCL9, and IFNG and (ii) PD-L1, IFNG, GZMB, and CD8A consisted of 753 patients. If the patient has locally advanced or metastatic (e.g., stage IIIB, stage IV, or recurrent) NSCLC; disease progression during or after treatment with a platinum-containing prior regimen for locally advanced, unresectable/inoperable, or metastatic NSCLC, or disease recurrence within 6 months of treatment with a platinum-based adjuvant/neoadjuvant regimen; measurable disease as defined by RECIST v 1.1; and Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, they were eligible for registration in the OAK trial. Participants were randomized to receive either a 1200mg dose of attrituximab intravenously every three weeks or 75mg per square meter (mg/m) intravenously every three weeks²) Docetaxel. Treatment with atelizumab may continue as long as the participant experiences clinical benefit, i.e., no unacceptable toxicity or symptomatic worsening due to disease progression.

Analysis of PD-L1, CXCL9, and IFNG expression and MPDL3280A efficacy

To assess whether PD-L1, CXCL9, and IFNG gene expression status correlated with patient response to treatment with attrituximab (MPDL3280A), the immune score expression levels of PD-L1, CXCL9, and IFNG were assessed in pre-treatment formalin-fixed and paraffin-embedded (FFPE) tumor samples obtained from each patient. RNA was isolated from FFPE tumor sections and PD-L1, CXCL9, and IFNG gene expression were measured using PCR-based methodology. The expression level (expressed as the cycle threshold (Ct)) of each of PD-L1, CXCL9, and IFNG was normalized to the expression level of a housekeeping gene (e.g., TMEM 55B). Normalized expression values dCt (where dCt (target gene) ═ Ct (control gene) -Ct (target gene)) for each of PD-L1, CXCL9, and IFNG were then averaged to obtain single-value mean dCt values for the expression levels of the immune scores for PD-L1, CXCL9, and IFNG.

The cut-off values relative to a given percentile of the population (e.g., the 25.5 th percentile, the 50.2 th percentile, the 70.3 th percentile, and the 75.3 th percentile) classify tumor specimens obtained from patients into different high or low expression level subsets based on the immune score expression levels of PD-L1, CXCL9, and IFNG. The 25.5 th percentile cut-off value is defined by the expression level of the immune score of PD-L1, CXCL9, and IFNG being greater than or equal to 25.5% of the expression level of all immune scores of PD-L1, CXCL9, and IFNG in the population analyzed. The 50.2 th percentile cut-off is defined by the expression level of the immune scores of PD-L1, CXCL9, and IFNG being greater than or equal to 50.2% of the expression level of all immune scores of PD-L1, CXCL9, and IFNG in the population analyzed. The 70.3 th percentile cutoff is defined by the expression level of the immune scores of PD-L1, CXCL9, and IFNG being greater than or equal to 70.3% of the expression level of all immune scores of PD-L1, CXCL9, and IFNG in the population analyzed. The 75.3 th percentile cutoff is defined by the expression level of the immune scores of PD-L1, CXCL9, and IFNG being greater than or equal to 75.3% of the expression level of all immune scores of PD-L1, CXCL9, and IFNG in the population analyzed.

Efficacy results for attrituzumab and docetaxel arms on OAK trials were compared for high expression levels and low expression level subsets of each percentile cutoff. High expression levels were defined as PD-L1, CXCL9, and IFNG immune score expression levels at or above each percentile cut-off. Low expression levels were defined as PD-L1, CXCL9, and IFNG immune score expression levels below each percentile cut-off. The immune score expression levels of PD-L1, CXCL9, and IFNG between percentile cutoffs are presented in table 6.

TABLE 6 PD-L1, CXCL9, and IFNG immune score expression levels between percentile cutoffs in OAK assays

Overall Survival (OS) and Progression Free Survival (PFS) endpoints from the OAK assay were evaluated against PD-L1, CXCL9, and IFNG expression level cutoffs (e.g., 25.5 th, 50.2 th, 70.3 th, or 75.3 th expression level percentile cutoffs) as defined herein. The analysis showed a trend towards an improved correlation of the expression levels of the immune scores of PD-L1, CXCL9, and IFNG in patients with NSCLC in randomized OAK trials compared to the efficacy of treatment with astuzumab compared to treatment with docetaxel (fig. 1-4). A gradient of increasing PFS and OS benefit was observed with increasing PD-L1, CXCL9, and IFNG expression levels (fig. 1-4). A summary of the associations of expression levels of PD-L1, CXCL9, and IFNG with efficacy endpoints in the OAK patient population is presented in table 7.

TABLE 7 summary of the association of PD-L1, CXCL9, and IFNG expression levels with efficacy endpoints in OAK assays

Taken together, these data show that the immune score expression levels of PD-L1, CXCL9, and IFNG can serve as predictive biomarkers for predicting therapeutic efficacy of treatments comprising PD-L1 binding antagonists, such as anti-PD-L1 antibodies, e.g., attritumab (MPDL 3280A). Thus, assessment of the expression levels of PD-L1, CXCL9, and IFNG (e.g., the immune score expression levels of PD-L1, CXCL9, and IFNG) can be used, for example, to identify patients with cancer (e.g., NSCLC) who derive PFS benefits and OS benefits from treatment that includes a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL 3280A)).

Analysis of PD-L1, IFNG, GZMB, and CD8A expression and MPDL3280A efficacy

To assess whether PD-L1, IFNG, GZMB, and CD8A gene expression status correlated with patient response to atuzumab (MPDL3280A) treatment, PD-L1, IFNG, GZMB, and CD8A gene expression levels were assessed in pretreatment FFPE tumor sections and PD-L1, IFNG, GZMB, and CD8A gene expression was measured using PCR-based methodology. The expression level (expressed as the cycle threshold (Ct)) of each of PD-L1, IFNG, GZMB, and CD8A was normalized to the expression level of a housekeeping gene (e.g., TMEM 55B). Normalized expression values dCt (where dCt (target gene) ═ Ct (control gene) -Ct (target gene)) for each of PD-L1, IFNG, GZMB, and CD8A were then averaged to obtain a single numerical average dCt value for the aggregate expression level of PD-L1, IFNG, GZMB, and CD 8A.

The cutoff values relative to a given percentile of the population (e.g., the 25.4 percentile, the 50.2 percentile, the 70.1 percentile, or the 75 percentile) classify tumor specimens obtained from patients into different high or low expression level subsets based on the immune score expression levels of PD-L1, IFNG, GZMB, and CD 8A. The 25.4 percentile is defined by the expression level of the immune scores for PD-L1, IFNG, GZMB, and CD8A that is greater than or equal to 25.4% of the expression level of all immune scores for PD-L1, IFNG, GZMB, and CD8A in the population analyzed. The 50.2 percentile cutoff is defined by the expression level of the immune scores for PD-L1, IFNG, GZMB, and CD8A being greater than or equal to 50.2% of the expression level of all immune scores for PD-L1, IFNG, GZMB, and CD8A in the population analyzed. The 70.1 th percentile cutoff is defined by the expression level of the immune scores for PD-L1, IFNG, GZMB, and CD8A being greater than or equal to 70.1% of the expression level of all immune scores for PD-L1, IFNG, GZMB, and CD8A in the population analyzed. The 75 th percentile cutoff is defined by the expression level of the immune scores of PD-L1, IFNG, GZMB, and CD8A being greater than or equal to 75% of the expression level of all immune scores of PD-L1, IFNG, GZMB, and CD8A in the population analyzed.

Efficacy results for attrituzumab and docetaxel arms on OAK trials were compared for high expression levels and low expression level subsets of each percentile cutoff. High expression levels were defined as PD-L1, IFNG, GZMB, and CD8A immune score expression levels at or above each percentile cutoff. Low expression levels were defined as expression levels below the PD-L1, IFNG, GZMB, and CD8A immune scores of each percentile cutoff. The immune score expression levels of PD-L1, IFNG, GZMB, and CD8A between percentile cutoffs are presented in table 8.

TABLE 8 expression levels of PD-L1, IFNG, GZMB, and CD8A immune scores between percentile cutoffs in OAK assays

OS and PFS endpoints from OAK assays were evaluated for PD-L1, IFNG, GZMB, and CD8A expression level cutoffs (e.g., 25.4 th, 50.2 th, 70.1 th, and 75 th expression level percentile cutoffs) as defined herein. The analysis showed a trend towards an improved correlation of the expression levels of the immune scores for PD-L1, IFNG, GZMB, and CD8A in patients with NSCLC in randomized OAK trials compared to the efficacy of treatment with attrituzumab and treatment with docetaxel (figures 5 and 6). A gradient of increasing PFS and OS benefit was observed with increasing PD-L1, IFNG, GZMB, and CD8A expression levels (fig. 5 and 6). A summary of the associations of expression levels of PD-L1, IFNG, GZMB, and CD8A with efficacy endpoints in the OAK patient population is presented in table 9.

TABLE 9 summary of the correlation of PD-L1, IFNG, GZMB, and CD8A expression levels with efficacy endpoints in OAK assays

Taken together, these data show that the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A can serve as predictive biomarkers for predicting the efficacy treatment of treatments comprising PD-L1 binding antagonists, such as anti-PD-L1 antibodies, e.g., attrituximab (MPDL 3280A). Thus, assessment of expression levels of PD-L1, IFNG, GZMB, and CD8A (e.g., the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A) may be used, for example, to identify patients with cancer (e.g., NSCLC) who derive PFS benefit and OS benefit from treatment comprising a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL 3280A)).

Analysis of expression levels of five-and six-Gene immune scores

The expression levels of five genes (e.g., CD8A, GZMB, PD-L1, IFNG, and CXCL9) or six genes (e.g., CD8A, GZMB, PD-L1, IFNG, CXCL9, and PD-1) in patients in OAK trials were also assessed using the methodology described above. Consistent with the analysis of immune score expression levels based on three genes (e.g., PD-L1, CXCL9, and IFNG) and four genes (e.g., PD-L1, IFNG, GZMB, and CD8A), the five-and six-gene analysis showed a correlation between (i) CD8A, GZMB, PD-L1, IFNG, and CXCL9 or (ii) CD8A, GZMB, PD-L1, IFNG, CXCL9, and the immune score expression levels of PD-1 in patients with NSCLC in the OAK trial and the improved efficacy of treatment with attritumab compared to treatment with docetaxel (fig. 7). Gradients of improved PFS and OS benefits were observed with increasing expression levels of (i) CD8A, GZMB, PD-L1, IFNG, and CXCL9 or (ii) CD8A, GZMB, PD-L1, IFNG, CXCL9, and PD-1 immune scores (i.e., scores with increasing prevalence) (fig. 7). Taken together, these data show that the expression level of the immune score for a biomarker combination comprising five genes or all six genes can serve as a predictive biomarker for predicting the therapeutic efficacy of a treatment comprising a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL 3280A)).

Example 2 correlation between expression levels of PD-L1, CXCL9, and IFNG in patients with NSCLC and clinical response to treatment with Attributab (MPDL3280A)

RNA-based molecular assays were used to assess the association between clinical response of individuals with NSCLC enrolled in a phase II clinical trial in which atuzumab was administered as a monotherapy and the expression levels of PD-L1, CXCL9, and IFNG to treatment with the anti-PD-L1 antibody atuzumab (MPDL 3280A).

Design of research

The POPLAR (clinical trial ID No.: NCT01903993) patient population that evaluated the expression levels of PD-L1, CXCL9, and IFNG consisted of 215 patients. If the patient has locally advanced or metastatic (e.g., stage IIIB, stage IV, or recurrent) NSCLC; disease progression during or after treatment with a platinum-containing prior regimen for locally advanced, unresectable/inoperable, or metastatic NSCLC, or disease recurrence within 6 months of treatment with a platinum-based adjuvant/neoadjuvant regimen; measurable disease as defined by RECIST v 1.1; and ECOG performance status of 0 or 1, they are eligible for enrollment in the POPLAR study. Participants were randomized to receive either a 1200mg dose of attrituximab intravenously every three weeks or 75mg per square meter (mg/m) intravenously every three weeks²) Docetaxel. Can be continuously treated by the atelizumabAs long as the participants experience clinical benefit, i.e., no unacceptable toxicity or symptomatic exacerbation due to disease progression.

Analysis of PD-L1, CXCL9, and IFNG expression and MPDL3280A efficacy

To assess whether PD-L1, CXCL9, and IFNG gene expression status correlated with patient response to treatment with attrituximab (MPDL3280A), the gene expression levels of PD-L1, CXCL9, and IFNG were assessed in pretreatment FFPE tumor samples obtained from each patient. RNA was isolated from FFPE tumor sections and PD-L1, CXCL9, and IFNG gene expression were measured using PCR-based methodology (Fluidigm). The expression level (expressed as the cycle threshold (Ct)) of each of PD-L1, CXCL9, and IFNG was normalized to the expression level of a housekeeping gene (e.g., TMEM 55B). Normalized expression values dCt (where dCt (target gene) ═ Ct (control gene) -Ct (target gene)) for each of PD-L1, CXCL9, and IFNG were then averaged to obtain single-value mean dCt values for the aggregate expression levels of PD-L1, CXCL9, and IFNG.

The cut-off values relative to a given percentile of the population (25 th, 50 th, or 75 th percentile) classify tumor specimens obtained from patients into different high or low expression level subsets based on the immune score expression levels of PD-L1, CXCL9, and IFNG. The 25 th percentile is defined by the expression level of the immune scores of PD-L1, CXCL9, and IFNG being greater than or equal to 25% of the expression level of all the immune scores of PD-L1, CXCL9, and IFNG in the population analyzed. The 50 th percentile is defined by the expression level of the immune scores of PD-L1, CXCL9, and IFNG being greater than or equal to 50% of the expression level of all the immune scores of PD-L1, CXCL9, and IFNG in the population analyzed. The 75 th percentile is defined by the expression level of the immune scores of PD-L1, CXCL9, and IFNG being greater than or equal to 75% of the expression level of all the immune scores of PD-L1, CXCL9, and IFNG in the population analyzed.

Efficacy results on the attrituzumab and docetaxel arms were compared for high expression levels and low expression level subsets of each percentile cutoff. High expression levels were defined as PD-L1, CXCL9, and IFNG immune score expression levels at or above each percentile cut-off. Low expression levels were defined as PD-L1, CXCL9, and IFNG immune score expression levels below each percentile cut-off. The immune score expression levels of PD-L1, CXCL9, and IFNG between percentile cutoffs are presented in table 10.

TABLE 10 expression levels of PD-L1, CXCL9, and IFNG immune score between percentile cutoffs in the POPLAR assay

OS, PFS, and ORR endpoints from POPLAR clinical trials were evaluated against PD-L1, CXCL9, and IFNG expression level cutoffs (e.g., 25%, 50%, and 75% expression level quartiles) as defined herein. The analysis showed a trend towards an improved correlation of the immune score expression levels of PD-L1, CXCL9, and IFNG in patients with NSCLC in a randomized POPLAR study compared to the efficacy of treatment including astuzumab to treatment including docetaxel (fig. 7A-7B,8A-8B, and 9). At each percentile cut-off, a higher Objective Response Rate (ORR) was observed in the high expression level subgroup compared to the low expression level subgroup (table 11). A gradient of increasing PFS and OS benefit was observed with increasing PD-L1, CXCL9, and IFNG expression levels (fig. 7A-7B,8A-8B, and 9). A summary of the associations of expression levels of PD-L1, CXCL9, and IFNG with efficacy endpoints in the POPLAR patient population is presented in table 11. Higher ORR in patients treated with atuzumab was associated with increased expression levels of the immune scores of PD-L1, CXCL9, and IFNG, whereas docetaxel treated patients did not experience an improvement in ORR with increasing expression levels of the immune scores of PD-L1, CXCL9, and IFNG.

TABLE 11 summary of the association of PD-L1, CXCL9, and IFNG expression levels with efficacy endpoints in the POPLAR assay

Taken together, these data show that the immune score expression levels of PD-L1, CXCL9, and IFNG can serve as predictive biomarkers for predicting therapeutic efficacy of treatments comprising PD-L1 binding antagonists, such as anti-PD-L1 antibodies, e.g., attritumab (MPDL 3280A). Thus, assessment of expression levels of PD-L1, CXCL9, and IFNG (e.g., immune score expression levels of PD-L1, CXCL9, and IFNG) can be used, for example, to identify patients with cancer (e.g., NSCLC) who have treatment-derived PFS and OS benefits including a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL 3280A)).

Example 3 correlation between expression levels of PD-L1, CXCL9, and IFNG in patients with UBC and clinical response to treatment with Atlizumab (MPDL3280A)

An RNA-based molecular assay was used to assess the association between clinical response to treatment with astuzumab (MPDL3280A), anti-PD-L1 antibody and expression levels of PD-L1, CXCL9, and IFNG in individuals with advanced Urothelial Bladder Cancer (UBC) enrolled in a phase II clinical trial (IMvigor210 trial) in which the atuzumab was administered as a monotherapy.

Design of research

Expression levels of PD-L1, CXCL9, and IFNG were evaluated on pre-treatment tumor specimens from patients with advanced UBC in cohort 2 of the phase II IMvigor210 trial (clinical trial ID No.: NCT 02108652). If the patient has histologically or cytologically proven locally advanced or metastatic transitional cell carcinoma or urothelium (e.g., renal pelvis, ureter, bladder, or urethra); disease progression during or after a platinum-based prior chemotherapy regimen; an ECOG performance state of 0 or 1; a life expectancy of greater than or equal to 12 weeks; measurable disease as defined by RECIST v 1.1; and adequate hematology and end organ function, they were eligible for registration in cohort 2 of the IMvigor210 trial. In this one-arm study, all participants received a 1200mg dose of atlizumab intravenously every three weeks on day 1 of the 21-day cycle. Treatment of participants in cohort 2 of the trial can be continued as long as the participants experience clinical benefit, i.e., no unmanageable toxicity.

Analysis of PD-L1, CXCL9, and IFNG expression and MPDL3280A efficacy

To assess whether PD-L1, CXCL9, and IFNG gene expression status correlated with patient response to treatment with astuzumab (MPDL3280A), the gene expression levels of PD-L1, CXCL9, and IFNG were assessed in pretreatment FFPE tumor samples obtained from each patient. RNA was isolated from FFPE tumor sections and PD-L1, CXCL9, and IFNG gene expression were measured and normalized using RNA sequencing (RNA-seq).

The cut-off values relative to a given percentile of the population (percentile 66) were assigned to different high or low expression level subgroups based on the immune score expression levels of PD-L1, CXCL9, and IFNG. The 66 th percentile cut-off is defined by the expression level of the immune scores of PD-L1, CXCL9, and IFNG being greater than or equal to 66% of the expression level of all immune scores of PD-L1, CXCL9, and IFNG in the population analyzed.

Efficacy results of the individual attritumab arms of the IMvigor210 assay were compared between high and low expression level subgroups. High expression levels were defined as the immune score expression levels of PD-L1, CXCL9, and IFNG at or above the 66 th percentile cut-off. Low expression levels were defined as the immune score expression levels below PD-L1, CXCL9, and IFNG at the 66 th percentile cut-off.

OS from patients in the IMvigor210 trial was evaluated for PD-L1, CXCL9, and IFNG expression level cutoffs (e.g., 66 th percentile cutoff) as defined herein. As shown in the Kaplan-Meier curve for Overall Survival (OS) shown in figure 10, the analysis showed an improved correlation of the expression levels of the immune scores for PD-L1, CXCL9, and IFNG in patients with UBC in cohort 2 of the IMvigor210 trial with the therapeutic benefit of atlizumab. Increased OS benefit (OS HR (95% CI) ═ 0.66(0.46-0.93)) was observed in patients with high immune score expression levels of PD-L1, CXCL9, and IFNG (i.e., at or above 66% percentile cut-off) compared to patients with low normalized expression levels of PD-L1, CXCL9, and IFNG (i.e., below 66% percentile cut-off) (fig. 10).

Taken together, these data show that the immune score expression levels of PD-L1, CXCL9, and IFNG can serve as predictive biomarkers for predicting therapeutic efficacy of treatments comprising PD-L1 binding antagonists, such as anti-PD-L1 antibodies, e.g., attritumab (MPDL 3280A). Thus, assessment of expression levels of PD-L1, CXCL9, and IFNG (e.g., the immune score expression levels of PD-L1, CXCL9, and IFNG) can be used, for example, to identify patients with cancer (e.g., UBC) who have treatment-derived OS benefits that include a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL 3280A)).

Example 4 correlation between expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 and clinical response to treatment with MPDL3280A and bevacizumab in patients with Renal Cell Carcinoma (RCC)

Evaluation of bevacizumab with RNA-based molecular assaysCorrelation between clinical response to treatment with the anti-PD-L1 antibody attritumab (MPDL3280A) in combination with bevacizumab and expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in individuals with advanced Renal Cell Carcinoma (RCC) enrolled in a phase II clinical trial (IMmotion150 trial) administered attritumab in combination.

Design of research

(ii) if the patient has unresectable advanced or metastatic RCC and components of clear cell histology and/or components of sarcoma-like histology that have not been previously treated with any systemic agent (including treatment in an adjuvant setting); measurable disease as defined by RECIST v 1.1; a Karnofsky performance score greater than or equal to 70; and adequate hematology and end organ function, they were eligible for enrollment in the IMmotion150 trial (clinical trial ID No.: NCT 01984242). Participants were randomized to receive (i) a 15mg/kg intravenous dose of atuzumab and bevacizumab every three weeks on days 1 and 22 of each 6-week cycle; (ii) an intravenous 1200mg dose of atezumab every three weeks on days 1 and 22 of each 6-week cycle; or (iii) sunitinib in a dose of 50mg orally once daily on days 1 to 28 per 6 week cycle. Treatment in each arm of the study can be continued as long as the participants experience clinical benefit, i.e., no unacceptable toxicity or symptomatic worsening due to disease progression.

Analysis of PD-L1, IFNG, GZMB, CD8A, and PD-1 expression and MPDL3280A efficacy

To assess whether PD-L1, IFNG, GZMB, CD8A, and PD-1 gene expression status correlate with patient response to treatment with pertuzumab (MPDL3280A) in combination with bevacizumab, the gene expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 were assessed in pretreatment FFPE tumor samples obtained from each patient. RNA was isolated from FFPE tumor sections and PD-L1, IFNG, GZMB, CD8A, and PD-1 gene expression were measured and normalized using RNA sequencing (RNA-seq).

The cut-off values relative to a given percentile of the population (50 th percentile) classify tumor specimens obtained from patients into different high or low expression level subgroups based on the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1. The 50 th percentile cut-off is defined by the expression level of the immune score for PD-L1, IFNG, GZMB, CD8A, and PD-1 being greater than or equal to 50% of the expression level of all immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1 in the population analyzed.

Efficacy results for the attritumab and bevacizumab combinatorial arms and sunitinib arms tested on IMmotion150 were higher in expression levels and lower in expression levels subgroups. High expression levels were defined as PD-L1, IFNG, GZMB, CD8A, and PD-1 immune score expression levels at or above the 50 th percentile cut-off. Low expression levels were defined as PD-L1, IFNG, GZMB, CD8A, and PD-1 immune score expression levels below the 50 th percentile cutoff.

Patients were evaluated for the PD-L1, IFNG, GZMB, CD8A, and PD-1 expression level cutoff (i.e., 50 th percentile cutoff) as defined herein for PFS from the IMMotion150 trial. The analysis showed a trend towards an improved correlation of the immune score expression levels towards PD-L1, IFNG, GZMB, CD8A, and PD-1 in patients with RCC in the randomized IMmotion150 trial compared to the efficacy of treatment with atelizumab and bevacizumab to treatment with sunitinib (figure 11). Elevated PFS benefit (PFS HR (95% CI) ═ 0.54(0.33-0.9)) was observed in patients with high immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 relative to the 50% percentile retention (fig. 11).

These data show that the expression levels of the immune scores for PD-L1, IFNG, GZMB, CD8A, and PD-1 can serve as predictive biomarkers for predicting the therapeutic efficacy of treatments that include PD-L1 binding antagonists, such as anti-PD-L1 antibodies, e.g., attritumab (MPDL 3280A). Thus, assessment of expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 (e.g., the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1) can be used, for example, to identify patients with cancer (e.g., RCC) who have treatment-derived PFS benefits that include a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attritumab (MPDL 3280A)).

Example 5 correlation between expression levels of PD-L1, CXCL9, and IFNG and clinical response to treatment with Atlizumab (MPDL3280A) in patients with Triple Negative Breast Cancer (TNBC)

RNA sequencing (RNA-seq) was used to assess the expression levels of PD-L1, CXCL9, and IFNG in patients with Triple Negative Breast Cancer (TNBC) enrolled in a phase I clinical trial in which the anti-PD-L1 antibody attritumab (MPDL3280A) was administered as a monotherapy.

Expression levels of PD-L1, CXCL9, and IFNG were evaluated on pre-treatment FFPE tumor specimens from patients with TNBC in phase I PCD4989g trial (clinical trial ID No.: NCT 01375842). RNA was isolated from tumor specimens and RNA-seq was used to measure PD-L1, CXCL9, and IFNG gene expression.

Tumor specimens obtained from patients were classified into high or low expression level subgroups based on their immune score expression levels of PD-L1, CXCL9, and IFNG relative to cut-off values. The cut-off value is defined by the immune score expression level of PD-L1, CXCL9, and IFNG being greater than or equal to 50% of all immune score expression levels of PD-L1, CXCL9, and IFNG in the population analyzed (i.e., the 50 th percentile cut-off). High expression level subgroups were defined by the immune score expression levels of PD-L1, CXCL9, and IFNG at or above the 50 th percentile cut-off. The low expression level subgroup was defined by the immune score expression levels of PD-L1, CXCL9, and IFNG below the 50 th percentile cut-off.

As shown in the Kaplan-Meier curve for Overall Survival (OS) in figure 12, the analysis showed an improved correlation of the expression levels of the immune scores for PD-L1, CXCL9, and IFNG in patients with TNBC in the PCD4989g trial with the efficacy of treatment with atlizumab. Increased OS benefit (OS HR (95% CI) ═ 0.55(0.33-0.93)) was observed in patients with high immune score expression levels of PD-L1, CXCL9, and IFNG (i.e., at or above the 50 th percentile cut-off) compared to patients with low normalized expression levels of PD-L1, CXCL9, and IFNG (i.e., below the 50 th percentile cut-off) (fig. 12). Further, as shown in the block diagram in fig. 13, the analysis showed that the association of the immune score expression levels of PD-L1, CXCL9, and IFNG with increased ORR benefit (e.g., complete or partial response (CR/PR), Stable Disease (SD), or Progressive Disease (PD)) correlated with higher immune score expression levels of PD-L1, CXCL9, and IFNG.

Taken together, these data show that the immune score expression levels of PD-L1, CXCL9, and IFNG can serve as predictive biomarkers for predicting therapeutic efficacy of treatments comprising PD-L1 binding antagonists, such as anti-PD-L1 antibodies, e.g., attritumab (MPDL 3280A). Thus, assessment of expression levels of PD-L1, CXCL9, and IFNG (e.g., the immune score expression levels of PD-L1, CXCL9, and IFNG) can be used, for example, to identify patients with cancer (e.g., TNBC) who have treatment-derived OS and/or ORR benefits that include a PD-L1 binding antagonist (e.g., an anti-PD-L1 antibody, such as attrituximab (MPDL 3280A)).

Example 6 correlation between expression levels of PD-L1, CXCL9, and IFNG in patients with NSCLC and clinical response to treatment with combination therapy including Attributab (MPDL3280A)

A phase III IMpower150 trial (clinical trial ID No. nct02366143) was designed to address whether the addition of atuzumab to bevacizumab and chemotherapy regimens would provide clinical benefit, as well as whether atuzumab could replace bevacizumab in bevacizumab and chemotherapy regimens.

Method of producing a composite material

Patient's health

Patients had non-chemotherapeutic, non-squamous, stage IV or recurrent mcnscc. The patient also had RECIST v1.1 measurable disease, baseline ECOG performance status 0/1, tumor tissue was available for biomarker testing, and was bevacizumab qualified. Patients with EGFR/ALK genomic alterations have disease progression/treatment intolerance to ≧ 1 approved Tyrosine Kinase Inhibitor (TKI). Patients were excluded if they had untreated central nervous system metastases, autoimmune diseases, or received prior immunotherapy/anti-CTLA-4 therapy <6 weeks prior to randomization or systemic immunosuppressive medication <2 weeks prior to randomization. Patients receiving prior (new) adjuvant therapy are eligible if their last treatment is ≧ 6 months prior to randomization.

Study design and treatment

IMpower150 is a global, open label, phase III trial. Patients were randomized 1:1:1 to receive altlizumab, carboplatin, and paclitaxel (ACP); alemtuzumab, bevacizumab, carboplatin, and paclitaxel (ABCP); or bevacizumab, carboplatin, and paclitaxel (BCP). Randomization was stratified by gender, presence of liver metastases at baseline, and PD-L1 expression.

Induction therapy was administered for 4 or 6 (at the discretion of the investigator prior to randomization) 21-day periods. The dosage is 1200mg of attrituzumab, 15mg/kg of bevacizumab and 200mg/m²Parietai (175 mg/m)²For asian race), and the area under the concentration-time curve (AUC)6mg/mL/min carboplatin, were both administered on day 1 of each cycle. After induction, patients continued with atuzumab/bevacizumab until unmanageable toxicity/RECIST v1.1 disease progression. Allowing continued atlizumab after progression if evidence of clinical benefit exists. Crossover to attrituzumab was not allowed.

Endpoint and evaluation

The common primary endpoints were in ITT-WT (patients without EGFR/ALK genomic alterations) and ITT-WT patients with high PD-L1, CXCL9, and IFNG immune score expression levels (high Immune Score Expression Levels (ISEL)^{Height of}) -RECIST v1.1) in WT), and OS in ITT-WT. The nucleic acid expression levels of PD-L1, CXCL9, and IFNG were defined by PDL1, CXCL9, and IFNG mRNA expression, measured using RNA isolated from baseline tumor tissue and using quantitative real-time polymerase chain reaction (Roche Molecular Systems). The immune score expression level reflecting the normalized mean dCT value for the gene analyzed was derived from the mean expression of each target gene relative to the control gene. In this study, high Immune Score Expression Levels (ISEL)^{Height of}) Defined as an immune score expression level greater than or equal to a predefined cut-off value (i.e., mean normalized dCt) -1.91 and a low Immune Score Expression Level (ISEL)^{Is low in}) Defined as less than-1.91, based on previous data (Kowanetz et al, j.thorac. oncol.12: S1817-8,2017).

Key secondary objectives include PFS and OS in ITT, PFS as assessed by the independent review agency (IRF) in ITT-WT, Objective Response Rate (ORR) and duration of response (DOR; RECIST v1.1), and security.

Patients underwent tumor assessment during screening from the first 48 weeks of cycle 1 day 1, every 6 weeks, and thereafter every 9 weeks until RECIST v1.1 disease progression or (for patients who continued atzumab after initial disease progression) loss of clinical benefit. Adverse Events (AE) were assessed using NCI-CTCAE v 4.0.

Statistical analysis

In short, a common primary endpoint was first tested between ABCP and BCP, since bevacizumab could not have significant PFS or OS benefit instead of atuzumab (ACP versus BCP), if the addition of atuzumab to the BCP regimen did not exhibit benefit. To tightly control the overall type I error rate to a one-sided significance level of 0.025, one-sided α of 0.006 was assigned to PFS (further split into 0.003 of 2 main analysis populations) and 0.019 was assigned to OS (ITT-WT) (fig. 14) (dmitteringko et al, stat.med.32: 1079-. If any PFS comparison is statistically significant, the OS is compared for recycled α (Dmitrienko et al, Stat. Med.32:1079-111,2013 and Dmitrienko et al, Stat. Med.32:5172-218, 2013). The residual alpha is passed to test PFS and OS between ACP and BCP if ABCP is statistically significant to OS of BCP, followed by PFS and OS in ITT, including EGFR/ALK mutant population (fig. 14), if significant (dmitrinko et al, stat.med.32: 1079-.

When ABCP and BCP merge occurs ≈ 516 PFS and 507 OS events in ITT-WT, final PFS and OS analysis is planned. mid-OS analysis was planned at the time of final PFS analysis, and ABCP and BCP mergers were expected to occur with ≈ 370 OS events in the ITT-WT population. If the OS events are significant <370 cases at the final PFS analysis, then a nominal two-sided alpha of 0.0001 would be spent for the first mid-OS analysis. In this case, formal statistical testing of ACP for PFS and OS in BCP will be performed later when ABCP matures for BCP OS data.

Treatment comparisons of PFS and OS were based on a tiered log rank test; HR was estimated using a hierarchical Cox regression model and 95% CI was calculated using the Brookmeyer-Crowley methodology. Median values were estimated using the Kaplan-Meier methodology.

A pre-defined subgroup analysis was performed to assess the consistency of treatment effect using Kaplan-Meier estimates of unfractionated HR and median values estimated from the Cox proportional hazards model.

Results

Patient's health

1202 patients were enrolled at 240 sites (26 countries) and 402,400, and 400 patients were randomized to ACP, ABCP, and BCP, respectively (fig. 15). ITT-WT contained 1040 patients (86.5% of ITT; ACP, 348; ABCP, 356; BCP, 336). The immune score expression level can be assessed in 95.6% of ITT-WT patients. ISEL^{Height of}WT contained 445 patients (42.8% of ITT-WT; ACP, 161; ABCP, 155; BCP, 129).

The baseline characteristics were balanced globally between ABCP and BCP (tables 12 and 13). 3 patients in ABCP (12.0%) and 4 patients in BCP (12.5%) with EGFR/ALK genomic alterations do not have reported prior TKI therapy, primarily due to the lack of availability of approved TKI therapies in their respective countries.

TABLE 12 Baseline characteristics of the ITT population

ISEL cut-off of-1.91 was used.

Others include adenocarcinomas with neuroendocrine features, adenosquamous, bronchioloalveolar carcinoma, large cell, sarcoid, and undifferentiated.

ABCP represents alemtuzumab + bevacizumab + carboplatin + paclitaxel; BCP, bevacizumab + carboplatin + paclitaxel; ECOG, eastern oncology cooperative group; IC, tumor infiltrating immune cells; ITT, intent to treat; PD-L1, apoptosis ligand 1; TC, tumor cells; WT, wild type.

TABLE 13 Baseline characteristics of the major analysis population

ISEL cut-off of-1.91 was used.

Major PFS analysis-ABCP vs BCP arms

At the data cutoff, the minimum survival follow-up was 9.5 months (median ITT-WT follow-up for ABCP and BCP was 15.4 and 15.5 months, respectively).

In ITT-WT (ABCP and BCP combined), 517/692 patients (74.7%) had PFS events. Significant PFS benefit of ABCP on BCP was observed; stratified (following randomization factor) HR was 0.617 (95% CI, 0.517-0.737; P < 0.0001; ABCP:241/356 (67.7%) vs BCP:276/336 (82.1%) events) and median PFS was 8.3 vs 6.8 months, respectively (fig. 16). At 6 months, the PFS rate of ABCP to BCP was 66.9% versus 56.1%; at 12 months, 36.5% vs 18.0%. These results were confirmed by central IRF evaluation (fig. 17A and 17B).

In ISEL^{Height of}Of WT, 200/284 patients (70.4%) had PFS events. Stratified (by sex and liver transfer) HR is 0.505 (95% CI, 0.377-0.675; P<0.0001; ABCP:97/155 (62.6%) versus BCP:103/129 (79.8%) cases), median PFS of ABCP versus BCP was 11.3 months versus 6.8 months (fig. 18A and 18B). At 6 months, the PFS rate of ABCP to BCP was 71.7% versus 57.0%; at 12 months, the PFS rate of ABCP to BCP was 46.0% versus 18.0%. A cut-off value of-0.24 was used, corresponding to a prevalence of 15.7%, at ISEL^{Height of}Median PFS was observed in patients up to about 21.8 months versus 5.5 months for ABCP versus BCP (fig. 19).

Patients with EGFR mutations or ALK translocations (EGFR/ALK +) also exhibited PFS benefits of ABCP on BCP (fig. 20). All enrolled patients (ITTs), including patients with EGFR/ALK genetic alterations, also benefit from ABCP therapy compared to BCP therapy (figure 21). HR was 0.610 (95% CI, 0.517-0.720; P <0.0001), and the median PFS of ABCP to BCP was 8.3 months to 6.8 months. At 6 months, the PFS rate of ABCP to BCP was 66.7% versus 55.6%; at 12 months, the PFS rate of ABCP to BCP was 36.5% versus 18.6%.

In addition, PFS benefits of ABCP on BCP were observed in a key clinical and biomarker subgroup, including patients with liver metastases and KRAS mutations (fig. 22). The benefits observed in patients with genetic alterations to EGFR/ALK are notable given that clinical trials investigating the use of PD-L1/PD-1 inhibitors as monotherapy after TKI failure did not show improved efficacy in these patients compared to standard chemotherapy (Rittmeyer et al, Lancet.389: 255-. Moreover, such patients have limited proven treatment options and the effectiveness of platinum-based regimens ± PD-L1/PD-1 inhibitors has not been previously investigated in phase 3 trials (Peters et al, j.clin.oncol.35: 2781-.

Preliminary OS-ABCP to BCP arm

At the data cutoff, 310/692 patients (44.8%) in the ITT-WT ABCP and BCP arms died. The stratified (following randomization factor) HR for OS was 0.775 (95% CI, 0.619-0.970; P ═ 0.0262; ABCP:144/356 (40.4%) versus BCP:166/336 (49.4%) events) and median OS for ABCP versus BCP was 19.2 versus 14.4 months (fig. 23). Thus, a numerical improvement of the ABCP arm compared to the BCP arm was observed for OS in ITT-WT patients.

ORR and DOR-ABCP on BCP arms

In ITT-WT, the unacknowledged ORR of ABCP and BCP were 63.5% and 48.0%; more complete responses were observed to ABCP. ISEL^{Height of}The results in WT are similar (table 14). In ITT-WT, median DOR of ABCP and BCP was 9.0 months and 5.7 months. In ISEL^{Height of}Median DOR in WT was 11.2 months and 5.7 months, respectively (table 14).

TABLE 14 Objective Response Rate (ORR) and duration of response (DOR)

Response duration was assessed in patients who achieved an objective response determined by investigators according to RECIST v 1.1.

The value examined.

The results of this phase III randomization trial revealed clinically and statistically significant PFS improvement with the addition of atuzumab to BCP as a first line treatment of non-squamous mslc. ABCP significantly prolonged PFS, resulting in a 38.3% reduction in risk of disease progression or death compared to BCP, a doubling of PFS rate from 18.0% to 36.5% at 12 months, and an increase in ORR (48.0% versus 63.5%, respectively). Although not yet mature (44.8% event to patient ratio), preliminary OS data appears promising.

PFS assay-ACP vs BCP arms

When PFS enrichment was assessed in the ACP arm compared to the BCP arm, the high immune expression score level was not enriched for PFS at the initial cut-off of-1.91 (fig. 24 and 25A). However, at higher immune score expression levels, corresponding to a prevalence of about 25% or less (dCt ═ 0.91), PFS enrichment was observed (fig. 24 and 25B).

Safety feature

787 patients received ABCP (393) or BCP (394). For ABCP, the median treatment duration was 8.2 months (range, 0-26) for Attuzumab (median dose, 12[ range, 1-38]) and 6.7 months (0-26) for Bevacizumab (median dose, 10[1-38 ]). For BCP, the median treatment duration was 5.1 months (0-22) of bevacizumab (median dose, 8[1-33 ]). Median duration of chemotherapy treatment between arms was 2.2 months (0-5). 31.7% of patients receiving BCP received subsequent immunotherapy.

Treatment-related AEs occurred in 94.4% and 95.4% of patients receiving ABCP and BCP, respectively (table 15). The incidence of grade 1-2 treatment-related AEs in ABCP and BCP was 35.9% and 45.4% and transient; the most common grade 3-4 treatment-related AEs were neutropenia, reduced neutrophil counts, febrile neutropenia, and hypertension. The incidence of rashes, stomatitis, febrile neutropenia and hemoptysis was < 10% elevated in patients receiving ABCP versus BCP. 11 (2.8%) and 9 (2.3%) patients of ABCP and BCP experienced treatment-related deaths (Table 15). 5 deaths of ABCP were due to pulmonary hemorrhage, 4 of which occurred in patients with high risk features such as tumor infiltration or cavity formation of large blood vessels. These events occurred early in the trial and resulted in a change in eligibility criteria to prevent the enrollment of patients with high risk characteristics. The incidence of treatment-related severe AEs for ABCP and BCP was 25.4% and 19.3%, respectively (tables 15 and 16).

Most immune-related aes (irAE) were grade 1-2, and ABCP reported no grade 5 irAE. The most common iraes observed are rash, hepatitis, hypothyroidism, hyperthyroidism, pneumonia, and colitis.

TABLE 15 treatment-related adverse events

TABLE 16 safety summary

Treatment-related Adverse Events (AEs), severe treatment-related AEs and the incidence of AEs leading to withdrawal from any treatment were for any treatment.

Example 7 correlation between expression levels of PD-L1, CXCL9, and IFNG in patients with mUC and clinical response to treatment with Attributab (MPDL3280A)

IMvigor211 (clinical trial ID No. nct02302807) is a large, randomized, phase III clinical trial comparing attritumab therapy with chemotherapy (taxane or vinflunine) in platinum-treated metastatic urothelial cancer (mUC). The purpose of this analysis is to assess clinical outcome in the subgroups defined by the expression level of the immune score.

The IMvigor211 trial enrolled patients with ≦ 2 prior line number therapies for mUC whose disease progressed during or after treatment with platinum-based chemotherapy (Powles et al, lancet: S0140-6736(17)33297-X, 2017). Patients were randomized 1:1 to receive either alemtuzumab 1200mg or investigator-selected chemotherapy (vinflunine, paclitaxel, or docetaxel) administered intravenously every 3 weeks. Atuzumab is administered until clinical benefit is lost, and chemotherapy is administered until RECIST v1.1 progressive disease.

Randomization was stratified by the number of risk factors (< 3 months from time of prior chemotherapy; eastern cooperative oncology group performance status > 0; hemoglobin <10g/dL):0 vs 1/2/3; presence of liver metastasis, yes to no; the investigator pre-defined chemotherapy choice was vinflunine versus taxane.

The primary endpoint was Overall Survival (OS). Key secondary endpoints include objective response rate, duration of response and progression-free survival. Exploratory endpoints include correlations between tumor immunospecific or disease-associated biomarkers and efficacy. RNA sequencing was used to assess the expression levels of immune scores based on IFNG, CXCL9, and PD-L1.

First, OS was analyzed in ITT population (N931). Results in the ITT population demonstrated improved OS in the attlizumab treated arm compared to the chemotherapy treated arm (figure 26).

OS was also assessed as a function of the expression level of the immune score (fig. 27A and 27B). The biomarker evaluable population included 793 patients. To assess whether PD-L1, CXCL9, and IFNG gene expression status correlated with patient response to treatment with attrituximab (MPDL3280A), the immune score expression levels of PD-L1, CXCL9, and IFNG were assessed in tumor samples obtained from each patient. Relative toThe cut-off value defined by the median immune score expression level of the population analyzed classified tumor specimens obtained from patients into high or low expression level subgroups based on the immune score expression levels of PD-L1, CXCL9, and IFNG. High Immune Score Expression Level (ISEL)^{Height of}) PD-L1, CXCL9, and IFNG immune score expression levels defined as at or above median cut-off. Low Immune Score Expression Level (ISEL)^{Is low in}) PD-L1, CXCL9, and IFNG immune score expression levels defined as below median cut-off. ISEL in both arms, as shown in FIGS. 27A and 27B^{Height of}Tumor status is associated with better prognosis, but in ISEL^{Height of}A separation of the Kaplan-Meier curves was observed in the population.

Other embodiments

Although the foregoing invention has been described in some detail by way of illustration for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Sequence listing

<110> Gene Tak corporation (Genentech, Inc.)

Haofume Roche GmbH (F. Hoffmann-La Roche AG)

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<130> 50474-158WO3

<150> US 62/628,227

<151> 2018-02-08

<150> US 62/485,874

<151> 2017-04-14

<160> 42

<170> PatentIn version 3.5

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gcacaucgcg guagggcgug gaaaggggca ggccagagcu acccgcagag uucucagaau 1080

caugcugaga gagcuggagg cacccaugcc aucucaaccu cuuccccgcc cguuuuacaa 1140

agggggaggc uaaagcccag agacagcuug aucaaaggca cacagcaagu caggguugga 1200

gcaguagcug gagggaccuu gucucccagc ucagggcucu uuccuccaca ccauucaggu 1260

cuuucuuucc gaggccccug ucucagggug aggugcuuga gucuccaacg gcaagggaac 1320

aaguacuucu ugauaccugg gauacugugc ccagagccuc gaggagguaa ugaauuaaag 1380

aagagaacug ccuuuggcag aguucuauaa uguaaacaau aucagacuuu uuuuuuuuau 1440

aaucaagccu aaaauuguau agaccuaaaa uaaaaugaag uggugagcuu aacccuggaa 1500

aaugaauccc ucuaucucua aagaaaaucu cugugaaacc ccuacgugga ggcggaauug 1560

cucucccagc ccuugcauug cagaggggcc caugaaagag gacaggcuac cccuuuacaa 1620

auagaauuug agcaucagug agguuaaacu aaggcccucu ugaaucucug aauuugagau 1680

acaaacaugu uccugggauc acugaugacu uuuuauacuu uguaaagaca auuguuggag 1740

agccccucac acagcccugg ccuccgcuca acuagcagau acagggauga ggcagaccug 1800

acucucuuaa ggaggcugag agcccaaacu gcugucccaa acaugcacuu ccuugcuuaa 1860

gguaugguac aagcaaugcc ugcccauugg agagaaaaaa cuuaaguaga uaaggaaaua 1920

agaaccacuc auaauucuuc accuuaggaa uaaucuccug uuaauauggu guaca 1975

<210> 2

<211> 235

<212> PRT

<213> human (Homo sapiens)

<400> 2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr

20 25 30

Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser

35 40 45

Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala

50 55 60

Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala

65 70 75 80

Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp

85 90 95

Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr

100 105 110

Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe

115 120 125

Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg

130 135 140

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

145 150 155 160

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

165 170 175

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

180 185 190

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His

195 200 205

Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser

210 215 220

Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val

225 230 235

<210> 3

<211> 934

<212> RNA

<213> human (Homo sapiens)

<400> 3

ugagaagaug caaccaaucc ugcuucugcu ggccuuccuc cugcugccca gggcagaugc 60

aggggagauc aucgggggac augaggccaa gccccacucc cgccccuaca uggcuuaucu 120

uaugaucugg gaucagaagu cucugaagag gugcgguggc uuccugauac aagacgacuu 180

cgugcugaca gcugcucacu guuggggaag cuccauaaau gucaccuugg gggcccacaa 240

uaucaaagaa caggagccga cccagcaguu uaucccugug aaaagaccca ucccccaucc 300

agccuauaau ccuaagaacu ucuccaacga caucaugcua cugcagcugg agagaaaggc 360

caagcggacc agagcugugc agccccucag gcuaccuagc aacaaggccc aggugaagcc 420

agggcagaca ugcagugugg ccggcugggg gcagacggcc ccccugggaa aacacucaca 480

cacacuacaa gaggugaaga ugacagugca ggaagaucga aagugcgaau cugacuuacg 540

ccauuauuac gacaguacca uugaguugug cgugggggac ccagagauua aaaagacuuc 600

cuuuaagggg gacucuggag gcccucuugu guguaacaag guggcccagg gcauugucuc 660

cuauggacga aacaauggca ugccuccacg agccugcacc aaagucucaa gcuuuguaca 720

cuggauaaag aaaaccauga aacgcuacua acuacaggaa gcaaacuaag cccccgcugu 780

aaugaaacac cuucucugga gccaagucca gauuuacacu gggagaggug ccagcaacug 840

aauaaauacc ucucccagug uaaaucugga gccaagucca gauuuacacu gggagaggug 900

ccagcaacug aauaaauacc ucuuagcuga gugg 934

<210> 4

<211> 247

<212> PRT

<213> human (Homo sapiens)

<400> 4

Met Gln Pro Ile Leu Leu Leu Leu Ala Phe Leu Leu Leu Pro Arg Ala

1 5 10 15

Asp Ala Gly Glu Ile Ile Gly Gly His Glu Ala Lys Pro His Ser Arg

20 25 30

Pro Tyr Met Ala Tyr Leu Met Ile Trp Asp Gln Lys Ser Leu Lys Arg

35 40 45

Cys Gly Gly Phe Leu Ile Arg Asp Asp Phe Val Leu Thr Ala Ala His

50 55 60

Cys Trp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His Asn Ile Lys

65 70 75 80

Glu Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys Arg Pro Ile Pro

85 90 95

His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp Ile Met Leu Leu

100 105 110

Gln Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gln Pro Leu Arg

115 120 125

Leu Pro Ser Asn Lys Ala Gln Val Lys Pro Gly Gln Thr Cys Ser Val

130 135 140

Ala Gly Trp Gly Gln Thr Ala Pro Leu Gly Lys His Ser His Thr Leu

145 150 155 160

Gln Glu Val Lys Met Thr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp

165 170 175

Leu Arg His Tyr Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro

180 185 190

Glu Ile Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val

195 200 205

Cys Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly

210 215 220

Met Pro Pro Arg Ala Cys Thr Lys Val Ser Ser Phe Val His Trp Ile

225 230 235 240

Lys Lys Thr Met Lys Arg Tyr

245

<210> 5

<211> 1193

<212> RNA

<213> human (Homo sapiens)

<400> 5

ugaagaucag cuauuagaag agaaagauca guuaaguccu uuggaccuga ucagcuugau 60

acaagaacua cugauuucaa cuucuuuggc uuaauucucu cggaaacgau gaaauauaca 120

aguuauaucu uggcuuuuca gcucugcauc guuuuggguu cucuuggcug uuacugccag 180

gacccauaug uaaaagaagc agaaaaccuu aagaaauauu uuaaugcagg ucauucagau 240

guagcggaua auggaacucu uuucuuaggc auuuugaaga auuggaaaga ggagagugac 300

agaaaaauaa ugcagagcca aauugucucc uuuuacuuca aacuuuuuaa aaacuuuaaa 360

gaugaccaga gcauccaaaa gaguguggag accaucaagg aagacaugaa ugucaaguuu 420

uucaauagca acaaaaagaa acgagaugac uucgaaaagc ugacuaauua uucgguaacu 480

gacuugaaug uccaacgcaa agcaauacau gaacucaucc aagugauggc ugaacugucg 540

ccagcagcua aaacagggaa gcgaaaaagg agucagaugc uguuucaagg ucgaagagca 600

ucccaguaau gguuguccug ccugcaauau uugaauuuua aaucuaaauc uauuuauuaa 660

uauuuaacau uauuuauaug gggaauauau uuuuagacuc aucaaucaaa uaaguauuua 720

uaauagcaac uuuuguguaa ugaaaaugaa uaucuauuaa uauauguauu auuuauaauu 780

ccuauauccu gugacugucu cacuuaaucc uuuguuuucu gacuaauuag gcaaggcuau 840

gugauuacaa ggcuuuaucu caggggccaa cuaggcagcc aaccuaagca agaucccaug 900

gguugugugu uuauuucacu ugaugauaca augaacacuu auaagugaag ugauacuauc 960

caguuacugc cgguuugaaa auaugccugc aaucugagcc agugcuuuaa uggcauguca 1020

gacagaacuu gaauguguca ggugacccug augaaaacau agcaucucag gagauuucau 1080

gccuggugcu uccaaauauu guugacaacu gugacuguac ccaaauggaa aguaacucau 1140

uuguuaaaau uaucaauauc uaauauauau gaauaaagug uaaguucaca acu 1193

<210> 6

<211> 166

<212> PRT

<213> human (Homo sapiens)

<400> 6

Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe Gln Leu Cys Ile Val Leu

1 5 10 15

Gly Ser Leu Gly Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu

20 25 30

Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn

35 40 45

Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp

50 55 60

Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe

65 70 75 80

Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile

85 90 95

Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg

100 105 110

Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val

115 120 125

Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser

130 135 140

Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg

145 150 155 160

Gly Arg Arg Ala Ser Gln

165

<210> 7

<211> 2545

<212> RNA

<213> human (Homo sapiens)

<400> 7

auccaauaca ggagugacuu ggaacuccau ucuaucacua ugaagaaaag ugguguucuu 60

uuccucuugg gcaucaucuu gcugguucug auuggagugc aaggaacccc aguagugaga 120

aagggucgcu guuccugcau cagcaccaac caagggacua uccaccuaca auccuugaaa 180

gaccuuaaac aauuugcccc aagcccuucc ugcgagaaaa uugaaaucau ugcuacacug 240

aagaauggag uucaaacaug ucuaaaccca gauucagcag augugaagga acugauuaaa 300

aagugggaga aacaggucag ccaaaagaaa aagcaaaaga augggaaaaa acaucaaaaa 360

aagaaaguuc ugaaaguucg aaaaucucaa cguucucguc aaaagaagac uacauaagag 420

accacuucac caauaaguau ucuguguuaa aaauguucua uuuuaauuau accgcuauca 480

uuccaaagga ggauggcaua uaauacaaag gcuuauuaau uugacuagaa aauuuaaaac 540

auuacucuga aauuguaacu aaaguuagaa aguugauuuu aagaauccaa acguuaagaa 600

uuguuaaagg cuaugauugu cuuuguucuu cuaccaccca ccaguugaau uucaucaugc 660

uuaaggccau gauuuuagca auacccaugu cuacacagau guucacccaa ccacauccca 720

cucacaacag cugccuggaa gagcagcccu aggcuuccac guacugcagc cuccagagag 780

uaucugaggc acaugucagc aaguccuaag ccuguuagca ugcuggugag ccaagcaguu 840

ugaaauugag cuggaccuca ccaagcugcu guggccauca accucuguau uugaaucagc 900

cuacaggccu cacacacaau gugucugaga gauucaugcu gauuguuauu ggguaucacc 960

acuggagauc accagugugu ggcuuucaga gccuccuuuc uggcuuugga agccauguga 1020

uuccaucuug cccgcucagg cugaccacuu uauuucuuuu uguuccccuu ugcuucauuc 1080

aagucagcuc uucuccaucc uaccacaaug cagugccuuu cuucucucca gugcaccugu 1140

cauaugcucu gauuuaucug agucaacucc uuucucaucu uguccccaac accccacaga 1200

agugcuuucu ucucccaauu cauccucacu caguccagcu uaguucaagu ccugccucuu 1260

aaauaaaccu uuuuggacac acaaauuauc uuaaaacucc uguuucacuu gguucaguac 1320

cacaugggug aacacucaau gguuaacuaa uucuugggug uuuauccuau cucuccaacc 1380

agauugucag cuccuugagg gcaagagcca caguauauuu cccuguuucu uccacagugc 1440

cuaauaauac uguggaacua gguuuuaaua auuuuuuaau ugauguuguu augggcagga 1500

uggcaaccag accauugucu cagagcaggu gcuggcucuu uccuggcuac uccauguugg 1560

cuagccucug guaaccucuu acuuauuauc uucaggacac ucacuacagg gaccagggau 1620

gaugcaacau ccuugucuuu uuaugacagg auguuugcuc agcuucucca acaauaagaa 1680

gcacguggua aaacacuugc ggauauucug gacuguuuuu aaaaaauaua caguuuaccg 1740

aaaaucauau aaucuuacaa ugaaaaggac uuuauagauc agccagugac caaccuuuuc 1800

ccaaccauac aaaaauuccu uuucccgaag gaaaagggcu uucucaauaa gccucagcuu 1860

ucuaagaucu aacaagauag ccaccgagau ccuuaucgaa acucauuuua ggcaaauaug 1920

aguuuuauug uccguuuacu uguuucagag uuuguauugu gauuaucaau uaccacacca 1980

ucucccauga agaaagggaa cggugaagua cuaagcgcua gaggaagcag ccaagucggu 2040

uaguggaagc augauuggug cccaguuagc cucugcagga uguggaaacc uccuuccagg 2100

ggagguucag ugaauugugu aggagagguu gucuguggcc agaauuuaaa ccuauacuca 2160

cuuucccaaa uugaaucacu gcucacacug cugaugauuu agagugcugu ccgguggaga 2220

ucccacccga acgucuuauc uaaucaugaa acucccuagu uccuucaugu aacuucccug 2280

aaaaaucuaa guguuucaua aauuugagag ucugugaccc acuuaccuug caucucacag 2340

guagacagua uauaacuaac aaccaaagac uacauauugu cacugacaca cacguuauaa 2400

ucauuuauca uauauauaca uacaugcaua cacucucaaa gcaaauaauu uuucacuuca 2460

aaacaguauu gacuuguaua ccuuguaauu ugaaauauuu ucuuuguuaa aauagaaugg 2520

uaucaauaaa uagaccauua aucag 2545

<210> 8

<211> 125

<212> PRT

<213> human (Homo sapiens)

<400> 8

Met Lys Lys Ser Gly Val Leu Phe Leu Leu Gly Ile Ile Leu Leu Val

1 5 10 15

Leu Ile Gly Val Gln Gly Thr Pro Val Val Arg Lys Gly Arg Cys Ser

20 25 30

Cys Ile Ser Thr Asn Gln Gly Thr Ile His Leu Gln Ser Leu Lys Asp

35 40 45

Leu Lys Gln Phe Ala Pro Ser Pro Ser Cys Glu Lys Ile Glu Ile Ile

50 55 60

Ala Thr Leu Lys Asn Gly Val Gln Thr Cys Leu Asn Pro Asp Ser Ala

65 70 75 80

Asp Val Lys Glu Leu Ile Lys Lys Trp Glu Lys Gln Val Ser Gln Lys

85 90 95

Lys Lys Gln Lys Asn Gly Lys Lys His Gln Lys Lys Lys Val Leu Lys

100 105 110

Val Arg Lys Ser Gln Arg Ser Arg Gln Lys Lys Thr Thr

115 120 125

<210> 9

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 9

Gly Phe Thr Phe Ser Asp Ser Trp Ile His

1 5 10

<210> 10

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 10

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly

<210> 11

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 11

Arg His Trp Pro Gly Gly Phe Asp Tyr

1 5

<210> 12

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 12

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 13

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 13

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> 14

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 14

Gln Gln Tyr Leu Tyr His Pro Ala Thr

1 5

<210> 15

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 16

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 17

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 18

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 19

<211> 440

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 19

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 20

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400> 20

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 21

<211> 1204

<212> RNA

<213> human (Homo sapiens)

<400> 21

ggggugcaaa gaagagacag cagcgcccag cuuggaggug cuaacuccag aggccagcau 60

cagcaacugg gcacagaaag gagccgccug ggcagggacc auggcacggc cacaucccug 120

guggcugugc guucugggga cccugguggg gcucucagcu acuccagccc ccaagagcug 180

cccagagagg cacuacuggg cucagggaaa gcugugcugc cagaugugug agccaggaac 240

auuccucgug aaggacugug accagcauag aaaggcugcu cagugugauc cuugcauacc 300

gggggucucc uucucuccug accaccacac ccggccccac ugugagagcu gucggcacug 360

uaacucuggu cuucucguuc gcaacugcac caucacugcc aaugcugagu gugccugucg 420

caauggcugg cagugcaggg acaaggagug caccgagugu gauccucuuc caaacccuuc 480

gcugaccgcu cggucgucuc aggcccugag cccacacccu cagcccaccc acuuaccuua 540

ugucagugag augcuggagg ccaggacagc ugggcacaug cagacucugg cugacuucag 600

gcagcugccu gcccggacuc ucucuaccca cuggccaccc caaagauccc ugugcagcuc 660

cgauuuuauu cgcauccuug ugaucuucuc uggaauguuc cuuguuuuca cccuggccgg 720

ggcccuguuc cuccaucaac gaaggaaaua uagaucaaac aaaggagaaa guccugugga 780

gccugcagag ccuugucguu acagcugccc cagggaggag gagggcagca ccauccccau 840

ccaggaggau uaccgaaaac cggagccugc cugcuccccc ugagccagca ccugcgguag 900

cugcacuaca gcccuggccu ccacccccac cccgccgacc auccaaggga gagugagacc 960

uggcagccac aacugcaguc ccauccucuu gucagggccc uuuccugugu acacgugaca 1020

gagugccuuu ucgagacugg cagggacgag gacaaauaug gaugaggugg agagugggaa 1080

gcaggagccc agccagcugc gccugcgcug caggagggcg ggggcucugg uuguaaaaca 1140

cacuuccugc ugcgaaagac ccacaugcua caagacgggc aaaauaaagu gacagaugac 1200

cacc 1204

<210> 22

<211> 260

<212> PRT

<213> human (Homo sapiens)

<400> 22

Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val

1 5 10 15

Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr

20 25 30

Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe

35 40 45

Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro

50 55 60

Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His

65 70 75 80

Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys

85 90 95

Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys

100 105 110

Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu

115 120 125

Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His

130 135 140

Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met

145 150 155 160

Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr

165 170 175

His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile

180 185 190

Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala

195 200 205

Leu Phe Leu His Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser

210 215 220

Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu

225 230 235 240

Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro

245 250 255

Ala Cys Ser Pro

260

<210> 23

<211> 2397

<212> RNA

<213> human (Homo sapiens)

<400> 23

gcacacacuc aucgaaaaaa auuuggauua uuagaagaga gaggucugcg gcuuccacac 60

cguacagcgu gguuuuucuu cucgguauaa aagcaaaguu guuuuugaua cgugacaguu 120

ucccacaagc caggcugauc cuuuucuguc aguccacuuc accaagccug cccuuggaca 180

aggacccgau gcccaacccc aggccuggca agcccucggc cccuuccuug gcccuuggcc 240

cauccccagg agccucgccc agcuggaggg cugcacccaa agccucagac cugcuggggg 300

cccggggccc agggggaacc uuccagggcc gagaucuucg aggcggggcc caugccuccu 360

cuucuuccuu gaaccccaug ccaccaucgc agcugcagcu gcccacacug ccccuaguca 420

ugguggcacc cuccggggca cggcugggcc ccuugcccca cuuacaggca cuccuccagg 480

acaggccaca uuucaugcac cagcucucaa cgguggaugc ccacgcccgg accccugugc 540

ugcaggugca cccccuggag agcccagcca ugaucagccu cacaccaccc accaccgcca 600

cuggggucuu cucccucaag gcccggccug gccucccacc ugggaucaac guggccagcc 660

uggaaugggu guccagggag ccggcacugc ucugcaccuu cccaaauccc agugcaccca 720

ggaaggacag cacccuuucg gcugugcccc agagcuccua cccacugcug gcaaauggug 780

ucugcaagug gcccggaugu gagaaggucu ucgaagagcc agaggacuuc cucaagcacu 840

gccaggcgga ccaucuucug gaugagaagg gcagggcaca augucuccuc cagagagaga 900

ugguacaguc ucuggagcag cagcuggugc uggagaagga gaagcugagu gccaugcagg 960

cccaccuggc ugggaaaaug gcacugacca aggcuucauc uguggcauca uccgacaagg 1020

gcuccugcug caucguagcu gcuggcagcc aaggcccugu cgucccagcc uggucuggcc 1080

cccgggaggc cccugacagc cuguuugcug uccggaggca ccuguggggu agccauggaa 1140

acagcacauu cccagaguuc cuccacaaca uggacuacuu caaguuccac aacaugcgac 1200

ccccuuucac cuacgccacg cucauccgcu gggccauccu ggaggcucca gagaagcagc 1260

ggacacucaa ugagaucuac cacugguuca cacgcauguu ugccuucuuc agaaaccauc 1320

cugccaccug gaagaacgcc auccgccaca accugagucu gcacaagugc uuugugcggg 1380

uggagagcga gaagggggcu guguggaccg uggaugagcu ggaguuccgc aagaaacgga 1440

gccagaggcc cagcaggugu uccaacccua caccuggccc cugaccucaa gaucaaggaa 1500

aggaggaugg acgaacaggg gccaaacugg ugggaggcag aggugguggg ggcagggaug 1560

auaggcccug gaugugccca cagggaccaa gaagugaggu uuccacuguc uugccugcca 1620

gggccccugu ucccccgcug gcagccaccc ccucccccau cauauccuuu gccccaaggc 1680

ugcucagagg ggccccgguc cuggccccag cccccaccuc cgccccagac acacccccca 1740

gucgagcccu gcagccaaac agagccuuca caaccagcca cacagagccu gccucagcug 1800

cucgcacaga uuacuucagg gcuggaaaag ucacacagac acacaaaaug ucacaauccu 1860

gucccucacu caacacaaac cccaaaacac agagagccug ccucaguaca cucaaacaac 1920

cucaaagcug caucaucaca caaucacaca caagcacagc ccugacaacc cacacacccc 1980

aaggcacgca cccacagcca gccucagggc ccacaggggc acugucaaca caggggugug 2040

cccagaggcc uacacagaag cagcgucagu acccucagga ucugaggucc caacacgugc 2100

ucgcucacac acacggccug uuagaauuca ccuguguauc ucacgcauau gcacacgcac 2160

agccccccag ugggucucuu gagucccgug cagacacaca cagccacaca cacugccuug 2220

ccaaaaauac cccgugucuc cccugccacu caccucacuc ccauucccug agcccugauc 2280

caugccucag cuuagacugc agaggaacua cucauuuauu ugggauccaa ggcccccaac 2340

ccacaguacc guccccaaua aacugcagcc gagcucccca caaaaaaaaa aaaaaaa 2397

<210> 24

<211> 431

<212> PRT

<213> human (Homo sapiens)

<400> 24

Met Pro Asn Pro Arg Pro Gly Lys Pro Ser Ala Pro Ser Leu Ala Leu

1 5 10 15

Gly Pro Ser Pro Gly Ala Ser Pro Ser Trp Arg Ala Ala Pro Lys Ala

20 25 30

Ser Asp Leu Leu Gly Ala Arg Gly Pro Gly Gly Thr Phe Gln Gly Arg

35 40 45

Asp Leu Arg Gly Gly Ala His Ala Ser Ser Ser Ser Leu Asn Pro Met

50 55 60

Pro Pro Ser Gln Leu Gln Leu Pro Thr Leu Pro Leu Val Met Val Ala

65 70 75 80

Pro Ser Gly Ala Arg Leu Gly Pro Leu Pro His Leu Gln Ala Leu Leu

85 90 95

Gln Asp Arg Pro His Phe Met His Gln Leu Ser Thr Val Asp Ala His

100 105 110

Ala Arg Thr Pro Val Leu Gln Val His Pro Leu Glu Ser Pro Ala Met

115 120 125

Ile Ser Leu Thr Pro Pro Thr Thr Ala Thr Gly Val Phe Ser Leu Lys

130 135 140

Ala Arg Pro Gly Leu Pro Pro Gly Ile Asn Val Ala Ser Leu Glu Trp

145 150 155 160

Val Ser Arg Glu Pro Ala Leu Leu Cys Thr Phe Pro Asn Pro Ser Ala

165 170 175

Pro Arg Lys Asp Ser Thr Leu Ser Ala Val Pro Gln Ser Ser Tyr Pro

180 185 190

Leu Leu Ala Asn Gly Val Cys Lys Trp Pro Gly Cys Glu Lys Val Phe

195 200 205

Glu Glu Pro Glu Asp Phe Leu Lys His Cys Gln Ala Asp His Leu Leu

210 215 220

Asp Glu Lys Gly Arg Ala Gln Cys Leu Leu Gln Arg Glu Met Val Gln

225 230 235 240

Ser Leu Glu Gln Gln Leu Val Leu Glu Lys Glu Lys Leu Ser Ala Met

245 250 255

Gln Ala His Leu Ala Gly Lys Met Ala Leu Thr Lys Ala Ser Ser Val

260 265 270

Ala Ser Ser Asp Lys Gly Ser Cys Cys Ile Val Ala Ala Gly Ser Gln

275 280 285

Gly Pro Val Val Pro Ala Trp Ser Gly Pro Arg Glu Ala Pro Asp Ser

290 295 300

Leu Phe Ala Val Arg Arg His Leu Trp Gly Ser His Gly Asn Ser Thr

305 310 315 320

Phe Pro Glu Phe Leu His Asn Met Asp Tyr Phe Lys Phe His Asn Met

325 330 335

Arg Pro Pro Phe Thr Tyr Ala Thr Leu Ile Arg Trp Ala Ile Leu Glu

340 345 350

Ala Pro Glu Lys Gln Arg Thr Leu Asn Glu Ile Tyr His Trp Phe Thr

355 360 365

Arg Met Phe Ala Phe Phe Arg Asn His Pro Ala Thr Trp Lys Asn Ala

370 375 380

Ile Arg His Asn Leu Ser Leu His Lys Cys Phe Val Arg Val Glu Ser

385 390 395 400

Glu Lys Gly Ala Val Trp Thr Val Asp Glu Leu Glu Phe Arg Lys Lys

405 410 415

Arg Ser Gln Arg Pro Ser Arg Cys Ser Asn Pro Thr Pro Gly Pro

420 425 430

<210> 25

<211> 2033

<212> RNA

<213> human (Homo sapiens)

<400> 25

cuucugugug ugcacaugug uaauacauau cugggaucaa agcuaucuau auaaaguccu 60

ugauucugug uggguucaaa cacauuucaa agcuucagga uccugaaagg uuuugcucua 120

cuuccugaag accugaacac cgcucccaua aagccauggc uugccuugga uuucagcggc 180

acaaggcuca gcugaaccug gcuaccagga ccuggcccug cacucuccug uuuuuucuuc 240

ucuucauccc ugucuucugc aaagcaaugc acguggccca gccugcugug guacuggcca 300

gcagccgagg caucgccagc uuugugugug aguaugcauc uccaggcaaa gccacugagg 360

uccgggugac agugcuucgg caggcugaca gccaggugac ugaagucugu gcggcaaccu 420

acaugauggg gaaugaguug accuuccuag augauuccau cugcacgggc accuccagug 480

gaaaucaagu gaaccucacu auccaaggac ugagggccau ggacacggga cucuacaucu 540

gcaaggugga gcucauguac ccaccgccau acuaccuggg cauaggcaac ggaacccaga 600

uuuauguaau ugauccagaa ccgugcccag auucugacuu ccuccucugg auccuugcag 660

caguuaguuc gggguuguuu uuuuauagcu uucuccucac agcuguuucu uugagcaaaa 720

ugcuaaagaa aagaagcccu cuuacaacag gggucuaugu gaaaaugccc ccaacagagc 780

cagaauguga aaagcaauuu cagccuuauu uuauucccau caauugagaa accauuauga 840

agaagagagu ccauauuuca auuuccaaga gcugaggcaa uucuaacuuu uuugcuaucc 900

agcuauuuuu auuuguuugu gcauuugggg ggaauucauc ucucuuuaau auaaaguugg 960

augcggaacc caaauuacgu guacuacaau uuaaagcaaa ggaguagaaa gacagagcug 1020

ggauguuucu gucacaucag cuccacuuuc agugaaagca ucacuuggga uuaauauggg 1080

gaugcagcau uaugaugugg gucaaggaau uaaguuaggg aauggcacag cccaaagaag 1140

gaaaaggcag ggagcgaggg agaagacuau auuguacaca ccuuauauuu acguaugaga 1200

cguuuauagc cgaaaugauc uuuucaaguu aaauuuuaug ccuuuuauuu cuuaaacaaa 1260

uguaugauua caucaaggcu ucaaaaauac ucacauggcu auguuuuagc cagugaugcu 1320

aaagguugua uugcauauau acauauauau auauauauau auauauauau auauauauau 1380

auauauauau auauauauuu uaauuugaua guauugugca uagagccacg uauguuuuug 1440

uguauuuguu aaugguuuga auauaaacac uauauggcag ugucuuucca ccuugggucc 1500

cagggaaguu uuguggagga gcucaggaca cuaauacacc agguagaaca caaggucauu 1560

ugcuaacuag cuuggaaacu ggaugagguc auagcagugc uugauugcgu ggaauugugc 1620

ugaguuggug uugacaugug cuuuggggcu uuuacaccag uuccuuucaa ugguuugcaa 1680

ggaagccaca gcugguggua ucugaguuga cuugacagaa cacugucuug aagacaaugg 1740

cuuacuccag gagacccaca gguaugaccu ucuaggaagc uccaguucga ugggcccaau 1800

ucuuacaaac augugguuaa ugccauggac agaagaaggc agcagguggc agaauggggu 1860

gcaugaaggu uucugaaaau uaacacugcu uguguuuuua acucaauauu uuccaugaaa 1920

augcaacaac auguauaaua uuuuuaauua aauaaaaauc uguggugguc guuuuaaaaa 1980

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 2033

<210> 26

<211> 223

<212> PRT

<213> human (Homo sapiens)

<400> 26

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro

20 25 30

Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala

35 40 45

Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly

50 55 60

Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln

65 70 75 80

Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr

85 90 95

Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe

165 170 175

Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys

180 185 190

Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu

195 200 205

Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn

210 215 220

<210> 27

<211> 2978

<212> RNA

<213> human (Homo sapiens)

<400> 27

cguccuaucu gcagucggcu acuuucagug gcagaagagg ccacaucugc uuccuguagg 60

cccucugggc agaagcaugc gcuggugucu ccuccugauc ugggcccagg ggcugaggca 120

ggcuccccuc gccucaggaa ugaugacagg cacaauagaa acaacgggga acauuucugc 180

agagaaaggu ggcucuauca ucuuacaaug ucaccucucc uccaccacgg cacaagugac 240

ccaggucaac ugggagcagc aggaccagcu ucuggccauu uguaaugcug acuuggggug 300

gcacaucucc ccauccuuca aggaucgagu ggccccaggu cccggccugg gccucacccu 360

ccagucgcug accgugaacg auacagggga guacuucugc aucuaucaca ccuacccuga 420

ugggacguac acugggagaa ucuuccugga gguccuagaa agcucagugg cugagcacgg 480

ugccagguuc cagauuccau ugcuuggagc cauggccgcg acgcuggugg ucaucugcac 540

agcagucauc gugguggucg cguugacuag aaagaagaaa gcccucagaa uccauucugu 600

ggaaggugac cucaggagaa aaucagcugg acaggaggaa uggagcccca gugcucccuc 660

acccccagga agcugugucc aggcagaagc ugcaccugcu gggcucugug gagagcagcg 720

gggagaggac ugugccgagc ugcaugacua cuucaauguc cugaguuaca gaagccuggg 780

uaacugcagc uucuucacag agacugguua gcaaccagag gcaucuucug gaagauacac 840

uuuugucuuu gcuauuauag augaauauau aagcagcugu acucuccauc agugcugcgu 900

gugugugugu guguguaugu gugugugugu ucaguugagu gaauaaaugu cauccucuuc 960

uccaucuuca uuuccuuggc cuuuucguuc uauuccauuu ugcauuaugg caggccuagg 1020

gugaguaacg uggaucuuga ucauaaaugc aaaauuaaaa aauaucuuga ccugguuuua 1080

aaucuggcag uuugagcaga uccuaugucu cugagagaca cauuccucau aauggccagc 1140

auuuugggcu acaagguuuu gugguugaug augaggaugg caugacugca gagccauccu 1200

caucucauuu uuucacguca uuuucaguaa cuuucacuca uucaaaggca gguuauaagu 1260

aaguccuggu agcagccucu auggggagau uugagaguga cuaaaucuug guaucugccc 1320

ucaagaacuu acaguuaaau ggggagacaa uguugucaug aaaagguauu auaguaagga 1380

gagaaggaga cauacacagg ccuucaggaa gagacgacag uuugggguga gguaguuggc 1440

auaggcuuau cugugaugaa guggccuggg agcaccaagg ggauguugag gcuagucugg 1500

gaggagcagg aguuuugucu agggaacuug uaggaaauuc uuggagcuga aagucccaca 1560

aagaaggccc uggcaccaag ggagucagca aacuucagau uuuauucucu gggcaggcau 1620

uucaaguuuc cuuuugcugu gacauacuca uccauuagac agccugauac aggccuguag 1680

ccucuuccgg ccgugugugc uggggaagcc ccaggaaacg cacaugccca cacagggagc 1740

caagucguag cauuugggcc uugaucuacc uuuucugcau caauacacuc uugagccuuu 1800

gaaaaaagaa cguuucccac uaaaaagaaa auguggauuu uuaaaauagg gacucuuccu 1860

aggggaaaaa ggggggcugg gagugauaga ggguuuaaaa aauaaacacc uucaaacuaa 1920

cuucuucgaa cccuuuuauu cacucccuga cgacuuugug cugggguugg gguaacugaa 1980

ccgcuuauuu cuguuuaauu gcauucaggc uggaucuuag aagacuuuua uccuuccacc 2040

aucucucuca gaggaaugag cggggagguu ggauuuacug gugacugauu uucuuucaug 2100

ggccaaggaa cugaaagaga augugaagca agguuguguc uugcgcaugg uuaaaaauaa 2160

agcauugucc ugcuuccuaa gacuuagacu gggguugaca auuguuuuag caacaagaca 2220

auucaacuau uucuccuagg auuuuuauua uuauuauuuu uucacuuuuc uaccaaaugg 2280

guuacauagg aagaaugaac ugaaaucugu ccagagcucc aaguccuuug gaagaaagau 2340

uagaugaacg uaaaaauguu guuguuugcu guggcaguuu acagcauuuu ucuugcaaaa 2400

uuagugcaaa ucuguuggaa auagaacaca auucacaaau uggaagugaa cuaaaaugua 2460

augacgaaaa gggaguagug uuuugauuug gaggaggugu auauucggca gagguuggac 2520

ugagaguugg guguuauuua acauaauuau gguaauuggg aaacauuuau aaacacuauu 2580

gggaugguga uaaaauacaa aagggccuau agauguuaga aaugggucag guuacugaaa 2640

ugggauucaa uuugaaaaaa auuuuuuuaa auagaacuca cugaacuaga uucuccucug 2700

agaaccagag aagaccauuu cauaguugga uuccuggaga caugcgcuau ccaccacgua 2760

gccacuuucc acauguggcc aucaaccacu uaagaugggg uuaguuuaaa ucaagaugug 2820

cuguuauaau ugguauaagc auaaaaucac acuagauucu ggagauuuaa uaugaauaau 2880

aagaauacua uuucaguagu uuugguauau ugugugucaa aaaugauaau auuuuggaug 2940

uauuggguga aauaaaauau uaacauuaaa aaaaaaaa 2978

<210> 28

<211> 244

<212> PRT

<213> human (Homo sapiens)

<400> 28

Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala

1 5 10 15

Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn

20 25 30

Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser

35 40 45

Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln

50 55 60

Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser

65 70 75 80

Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln

85 90 95

Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr

100 105 110

Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu

115 120 125

Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Leu Leu Gly

130 135 140

Ala Met Ala Ala Thr Leu Val Val Ile Cys Thr Ala Val Ile Val Val

145 150 155 160

Val Ala Leu Thr Arg Lys Lys Lys Ala Leu Arg Ile His Ser Val Glu

165 170 175

Gly Asp Leu Arg Arg Lys Ser Ala Gly Gln Glu Glu Trp Ser Pro Ser

180 185 190

Ala Pro Ser Pro Pro Gly Ser Cys Val Gln Ala Glu Ala Ala Pro Ala

195 200 205

Gly Leu Cys Gly Glu Gln Arg Gly Glu Asp Cys Ala Glu Leu His Asp

210 215 220

Tyr Phe Asn Val Leu Ser Tyr Arg Ser Leu Gly Asn Cys Ser Phe Phe

225 230 235 240

Thr Glu Thr Gly

<210> 29

<211> 1944

<212> RNA

<213> human (Homo sapiens)

<400> 29

aauuucucac ugccccugug auaaacugug gucacuggcu guggcagcaa cuauuauaag 60

augcucugaa aacucuucag acacugaggg gcaccagagg agcagacuac aagaauggca 120

cacgcuaugg aaaacuccug gacaaucagu aaagaguacc auauugauga agaagugggc 180

uuugcucugc caaauccaca ggaaaaucua ccugauuuuu auaaugacug gauguucauu 240

gcuaaacauc ugccugaucu cauagagucu ggccagcuuc gagaaagagu ugagaaguua 300

aacaugcuca gcauugauca ucucacagac cacaagucac agcgccuugc acgucuaguu 360

cugggaugca ucaccauggc auaugugugg ggcaaagguc auggagaugu ccguaagguc 420

uugccaagaa auauugcugu uccuuacugc caacucucca agaaacugga acugccuccu 480

auuuugguuu augcagacug ugucuuggca aacuggaaga aaaaggaucc uaauaagccc 540

cugacuuaug agaacaugga cguuuuguuc ucauuucgug auggagacug caguaaagga 600

uucuuccugg ucucucuauu gguggaaaua gcagcugcuu cugcaaucaa aguaauuccu 660

acuguauuca aggcaaugca aaugcaagaa cgggacacuu ugcuaaaggc gcuguuggaa 720

auagcuucuu gcuuggagaa agcccuucaa guguuucacc aaauccacga ucaugugaac 780

ccaaaagcau uuuucagugu ucuucgcaua uauuugucug gcuggaaagg caacccccag 840

cuaucagacg gucuggugua ugaaggguuc ugggaagacc caaaggaguu ugcagggggc 900

agugcaggcc aaagcagcgu cuuucagugc uuugacgucc ugcugggcau ccagcagacu 960

gcugguggag gacaugcugc ucaguuccuc caggacauga gaagauauau gccaccagcu 1020

cacaggaacu uccugugcuc auuagaguca aaucccucag uccgugaguu uguccuuuca 1080

aaaggugaug cuggccugcg ggaagcuuau gacgccugug ugaaagcucu ggucucccug 1140

aggagcuacc aucugcaaau cgugacuaag uacauccuga uuccugcaag ccagcagcca 1200

aaggagaaua agaccucuga agacccuuca aaacuggaag ccaaaggaac uggaggcacu 1260

gauuuaauga auuuccugaa gacuguaaga aguacaacug agaaaucccu uuugaaggaa 1320

gguuaaugua acccaacaag agcacauuuu aucauagcag agacaucugu augcauuccu 1380

gucauuaccc auuguaacag agccacaaac uaauacuaug caauguuuua ccaauaaugc 1440

aauacaaaag accucaaaau accugugcau uucuuguagg aaaacaacaa aagguaauua 1500

uguguaauua uacuagaagu uuuguaaucu guaucuuauc auuggaauaa aaugacauuc 1560

aauaaauaaa aaugcauaag auauauucug ucggcugggc gcgguggcuc acgccuguaa 1620

ucccagcacu uugggaggcc gaggcgggcg gaucacaagg ucaggagauc gagaccaucu 1680

uggcuaacac ggugaaaccc cgucucuacu aaaaauacaa aaaauuagcc gggcgcggug 1740

gcgggcaccu guagucccag cuacucggga ggcugaggca ggagaauggc gugaaccugg 1800

gaggcggagc uugcagugag ccaagauugu gccacugcaa uccggccugg gcuaaagagc 1860

gggacuccgu cucaaaaaaa aaaaaaaaaa gauauauucu gucauaauaa auaaaaaugc 1920

auaagauaua aaaaaaaaaa aaaa 1944

<210> 30

<211> 403

<212> PRT

<213> human (Homo sapiens)

<400> 30

Met Ala His Ala Met Glu Asn Ser Trp Thr Ile Ser Lys Glu Tyr His

1 5 10 15

Ile Asp Glu Glu Val Gly Phe Ala Leu Pro Asn Pro Gln Glu Asn Leu

20 25 30

Pro Asp Phe Tyr Asn Asp Trp Met Phe Ile Ala Lys His Leu Pro Asp

35 40 45

Leu Ile Glu Ser Gly Gln Leu Arg Glu Arg Val Glu Lys Leu Asn Met

50 55 60

Leu Ser Ile Asp His Leu Thr Asp His Lys Ser Gln Arg Leu Ala Arg

65 70 75 80

Leu Val Leu Gly Cys Ile Thr Met Ala Tyr Val Trp Gly Lys Gly His

85 90 95

Gly Asp Val Arg Lys Val Leu Pro Arg Asn Ile Ala Val Pro Tyr Cys

100 105 110

Gln Leu Ser Lys Lys Leu Glu Leu Pro Pro Ile Leu Val Tyr Ala Asp

115 120 125

Cys Val Leu Ala Asn Trp Lys Lys Lys Asp Pro Asn Lys Pro Leu Thr

130 135 140

Tyr Glu Asn Met Asp Val Leu Phe Ser Phe Arg Asp Gly Asp Cys Ser

145 150 155 160

Lys Gly Phe Phe Leu Val Ser Leu Leu Val Glu Ile Ala Ala Ala Ser

165 170 175

Ala Ile Lys Val Ile Pro Thr Val Phe Lys Ala Met Gln Met Gln Glu

180 185 190

Arg Asp Thr Leu Leu Lys Ala Leu Leu Glu Ile Ala Ser Cys Leu Glu

195 200 205

Lys Ala Leu Gln Val Phe His Gln Ile His Asp His Val Asn Pro Lys

210 215 220

Ala Phe Phe Ser Val Leu Arg Ile Tyr Leu Ser Gly Trp Lys Gly Asn

225 230 235 240

Pro Gln Leu Ser Asp Gly Leu Val Tyr Glu Gly Phe Trp Glu Asp Pro

245 250 255

Lys Glu Phe Ala Gly Gly Ser Ala Gly Gln Ser Ser Val Phe Gln Cys

260 265 270

Phe Asp Val Leu Leu Gly Ile Gln Gln Thr Ala Gly Gly Gly His Ala

275 280 285

Ala Gln Phe Leu Gln Asp Met Arg Arg Tyr Met Pro Pro Ala His Arg

290 295 300

Asn Phe Leu Cys Ser Leu Glu Ser Asn Pro Ser Val Arg Glu Phe Val

305 310 315 320

Leu Ser Lys Gly Asp Ala Gly Leu Arg Glu Ala Tyr Asp Ala Cys Val

325 330 335

Lys Ala Leu Val Ser Leu Arg Ser Tyr His Leu Gln Ile Val Thr Lys

340 345 350

Tyr Ile Leu Ile Pro Ala Ser Gln Gln Pro Lys Glu Asn Lys Thr Ser

355 360 365

Glu Asp Pro Ser Lys Leu Glu Ala Lys Gly Thr Gly Gly Thr Asp Leu

370 375 380

Met Asn Phe Leu Lys Thr Val Arg Ser Thr Thr Glu Lys Ser Leu Leu

385 390 395 400

Lys Glu Gly

<210> 31

<211> 1227

<212> RNA

<213> human (Homo sapiens)

<400> 31

cuuugcagau aaauauggca cacuagcccc acguuuucug agacauuccu caauugcuua 60

gacauauucu gagccuacag cagaggaacc uccagucuca gcaccaugaa ucaaacugcc 120

auucugauuu gcugccuuau cuuucugacu cuaaguggca uucaaggagu accucucucu 180

agaacuguac gcuguaccug caucagcauu aguaaucaac cuguuaaucc aaggucuuua 240

gaaaaacuug aaauuauucc ugcaagccaa uuuuguccac guguugagau cauugcuaca 300

augaaaaaga agggugagaa gagaugucug aauccagaau cgaaggccau caagaauuua 360

cugaaagcag uuagcaagga aaggucuaaa agaucuccuu aaaaccagag gggagcaaaa 420

ucgaugcagu gcuuccaagg auggaccaca cagaggcugc cucucccauc acuucccuac 480

auggaguaua ugucaagcca uaauuguucu uaguuugcag uuacacuaaa aggugaccaa 540

ugauggucac caaaucagcu gcuacuacuc cuguaggaag guuaauguuc aucauccuaa 600

gcuauucagu aauaacucua cccuggcacu auaauguaag cucuacugag gugcuauguu 660

cuuaguggau guucugaccc ugcuucaaau auuucccuca ccuuucccau cuuccaaggg 720

uacuaaggaa ucuuucugcu uugggguuua ucagaauucu cagaaucuca aauaacuaaa 780

agguaugcaa ucaaaucugc uuuuuaaaga augcucuuua cuucauggac uuccacugcc 840

auccucccaa ggggcccaaa uucuuucagu ggcuaccuac auacaauucc aaacacauac 900

aggaagguag aaauaucuga aaauguaugu guaaguauuc uuauuuaaug aaagacugua 960

caaaguagaa gucuuagaug uauauauuuc cuauauuguu uucaguguac auggaauaac 1020

auguaauuaa guacuaugua ucaaugagua acaggaaaau uuuaaaaaua cagauagaua 1080

uaugcucugc auguuacaua agauaaaugu gcugaauggu uuucaaaaua aaaaugaggu 1140

acucuccugg aaauauuaag aaagacuauc uaaauguuga aagaucaaaa gguuaauaaa 1200

guaauuauaa cuaagaaaaa aaaaaaa 1227

<210> 32

<211> 98

<212> PRT

<213> human (Homo sapiens)

<400> 32

Met Asn Gln Thr Ala Ile Leu Ile Cys Cys Leu Ile Phe Leu Thr Leu

1 5 10 15

Ser Gly Ile Gln Gly Val Pro Leu Ser Arg Thr Val Arg Cys Thr Cys

20 25 30

Ile Ser Ile Ser Asn Gln Pro Val Asn Pro Arg Ser Leu Glu Lys Leu

35 40 45

Glu Ile Ile Pro Ala Ser Gln Phe Cys Pro Arg Val Glu Ile Ile Ala

50 55 60

Thr Met Lys Lys Lys Gly Glu Lys Arg Cys Leu Asn Pro Glu Ser Lys

65 70 75 80

Ala Ile Lys Asn Leu Leu Lys Ala Val Ser Lys Glu Arg Ser Lys Arg

85 90 95

Ser Pro

<210> 33

<211> 1610

<212> RNA

<213> human (Homo sapiens)

<400> 33

agagaacaaa acagaaacuc uuggaagcag gaaaggugca ugacucaaag agggaaauuc 60

cugugccaua aaaggauugc ugguguauaa aaugcucuau auaugccaau uaucaauuuc 120

cuuucauguu cagcauuucu acuccuucca agaagagcag caaagcugaa guagcagcag 180

cagcaccagc agcaacagca aaaaacaaac augaguguga agggcauggc uauagccuug 240

gcugugauau ugugugcuac aguuguucaa ggcuucccca uguucaaaag aggacgcugu 300

cuuugcauag gcccuggggu aaaagcagug aaaguggcag auauugagaa agccuccaua 360

auguacccaa guaacaacug ugacaaaaua gaagugauua uuacccugaa agaaaauaaa 420

ggacaacgau gccuaaaucc caaaucgaag caagcaaggc uuauaaucaa aaaaguugaa 480

agaaagaauu uuuaaaaaua ucaaaacaua ugaaguccug gaaaagagca ucugaaaaac 540

cuagaacaag uuuaacugug acuacugaaa ugacaagaau ucuacaguag gaaacugaga 600

cuuuucuaug guuuugugac uuucaacuuu uguacaguua ugugaaggau gaaagguggg 660

ugaaaggacc aaaaacagaa auacagucuu ccugaaugaa ugacaaucag aauuccacug 720

cccaaaggag uccaacaauu aaauggauuu cuaggaaaag cuaccuuaag aaaggcuggu 780

uaccaucgga guuuacaaag ugcuuucacg uucuuacuug uugcauuaua cauucaugca 840

uuucuaggcu agagaaccuu cuagauuuga ugcuuacaac uauucuguug ugacuaugag 900

aacauuucug ucucuagaag ucaucugucu guauugaucu uuaugcuaua uuacuaucug 960

ugguuacggu ggagacauug acauuauuac uggagucaag cccuuauaag ucaaaagcau 1020

cuaugugucg uaaaacauuc cucaaacauu uuuucaugca aauacacacu ucuuucccca 1080

aacaucaugu agcacaucaa uauguaggga gacauucuua ugcaucauuu gguuuguuuu 1140

auaaccaauu cauuaaaugu aauucauaaa auguacuaug aaaaaaauua uacgcuaugg 1200

gauacuggca aaagugcaca uauuucauaa ccaaauuagu agcaccaguc uuaauuugau 1260

guuuuucaac uuuuauucau ugagauguuu ugaagcaauu aggauaugug uguuuacugu 1320

acuuuuuguu uugauccguu uguauaaaug auagcaauau cuuggacaca ucugaaauac 1380

aaaauguuuu ugucuaccaa agaaaaaugu ugaaaaauaa gcaaauguau accuagcaau 1440

cacuuuuacu uuuuguaauu cugucucuua gaaaaauaca uaaucuaauc aauuucuuug 1500

uucaugccua uauacuguaa aauuuaggua uacucaagac uaguuuaaag aaucaaaguc 1560

auuuuuuucu cuaauaaacu accacaaccu uucuuuuuua aaaaaaaaaa 1610

<210> 34

<211> 94

<212> PRT

<213> human (Homo sapiens)

<400> 34

Met Ser Val Lys Gly Met Ala Ile Ala Leu Ala Val Ile Leu Cys Ala

1 5 10 15

Thr Val Val Gln Gly Phe Pro Met Phe Lys Arg Gly Arg Cys Leu Cys

20 25 30

Ile Gly Pro Gly Val Lys Ala Val Lys Val Ala Asp Ile Glu Lys Ala

35 40 45

Ser Ile Met Tyr Pro Ser Asn Asn Cys Asp Lys Ile Glu Val Ile Ile

50 55 60

Thr Leu Lys Glu Asn Lys Gly Gln Arg Cys Leu Asn Pro Lys Ser Lys

65 70 75 80

Gln Ala Arg Leu Ile Ile Lys Lys Val Glu Arg Lys Asn Phe

85 90

<210> 35

<211> 1135

<212> RNA

<213> human (Homo sapiens)

<400> 35

uuccuccucc gagagcggac agaucucugg gugcugggcg gucauggcgc uacuagaugu 60

augcggagcc ccccgagggc agcggccgga aucggcucuc ccgguugcgg gaagcgggcg 120

ucgcucggac ccaggacacu acaguuucuc uaugcgaucu ccagagcucg cuuuaccccg 180

gggaaugcag cccacagaau ucuuccaguc ccuggguggg gacggagaaa ggaacguuca 240

gauugagaug gcccauggca ccaccacgcu cgccuucaag uuccagcaug gagugauugc 300

agcaguggau ucucgggccu cagcuggguc cuacauuagu gccuuacggg ugaacaaggu 360

gauugagauu aacccuuacc ugcuuggcac caugucuggc ugugcagcag acugucagua 420

cugggagcgc cugcuggcca aggaaugcag gcuguacuau cugcgaaaug gagaacguau 480

uucagugucg gcagccucca agcugcuguc caacaugaug ugccaguacc ggggcauggg 540

ccucucuaug ggcaguauga ucuguggcug ggauaagaag gguccuggac ucuacuacgu 600

ggaugaacau gggacucggc ucucaggaaa uauguucucc acggguagug ggaacacuua 660

ugccuacggg gucauggaca guggcuaucg gccuaaucuu agcccugaag aggccuauga 720

ccuuggccgc agggcuauug cuuaugccac ucacagagac agcuauucug gaggcguugu 780

caauauguac cacaugaagg aagaugguug ggugaaagua gaaaguacag augucaguga 840

ccugcugcac caguaccggg aagccaauca auaauggugg ugguggcagc ugggcagguc 900

uccucuggga ggucuuggcc gacucaggga ccuaagccac guuaagucca aggagaagaa 960

gaggccuagc cugagccaaa gagagaguac gggcucagca gccagaggag gccggugaag 1020

ugcaucuucu gcguguucuc uauuugaaca agcauuuccc ccagggaagu uucugggugc 1080

cccacuaagu agaauaaaga aaaacgguua uaaauaaaaa aaaaaaaaaa aaaaa 1135

<210> 36

<211> 276

<212> PRT

<213> human (Homo sapiens)

<400> 36

Met Ala Leu Leu Asp Val Cys Gly Ala Pro Arg Gly Gln Arg Pro Glu

1 5 10 15

Ser Ala Leu Pro Val Ala Gly Ser Gly Arg Arg Ser Asp Pro Gly His

20 25 30

Tyr Ser Phe Ser Met Arg Ser Pro Glu Leu Ala Leu Pro Arg Gly Met

35 40 45

Gln Pro Thr Glu Phe Phe Gln Ser Leu Gly Gly Asp Gly Glu Arg Asn

50 55 60

Val Gln Ile Glu Met Ala His Gly Thr Thr Thr Leu Ala Phe Lys Phe

65 70 75 80

Gln His Gly Val Ile Ala Ala Val Asp Ser Arg Ala Ser Ala Gly Ser

85 90 95

Tyr Ile Ser Ala Leu Arg Val Asn Lys Val Ile Glu Ile Asn Pro Tyr

100 105 110

Leu Leu Gly Thr Met Ser Gly Cys Ala Ala Asp Cys Gln Tyr Trp Glu

115 120 125

Arg Leu Leu Ala Lys Glu Cys Arg Leu Tyr Tyr Leu Arg Asn Gly Glu

130 135 140

Arg Ile Ser Val Ser Ala Ala Ser Lys Leu Leu Ser Asn Met Met Cys

145 150 155 160

Gln Tyr Arg Gly Met Gly Leu Ser Met Gly Ser Met Ile Cys Gly Trp

165 170 175

Asp Lys Lys Gly Pro Gly Leu Tyr Tyr Val Asp Glu His Gly Thr Arg

180 185 190

Leu Ser Gly Asn Met Phe Ser Thr Gly Ser Gly Asn Thr Tyr Ala Tyr

195 200 205

Gly Val Met Asp Ser Gly Tyr Arg Pro Asn Leu Ser Pro Glu Glu Ala

210 215 220

Tyr Asp Leu Gly Arg Arg Ala Ile Ala Tyr Ala Thr His Arg Asp Ser

225 230 235 240

Tyr Ser Gly Gly Val Val Asn Met Tyr His Met Lys Glu Asp Gly Trp

245 250 255

Val Lys Val Glu Ser Thr Asp Val Ser Asp Leu Leu His Gln Tyr Arg

260 265 270

Glu Ala Asn Gln

275

<210> 37

<211> 1048

<212> RNA

<213> human (Homo sapiens)

<400> 37

gcgcguugug cgcuguccca gguuggaaac cagugcccca ggcggcgagg agagcggugc 60

cuugcaggga ugcugcgggc gggagcacca accggggacu uaccccgggc gggagaaguc 120

cacaccggga ccaccaucau ggcaguggag uuugacgggg gcguugugau ggguucugau 180

ucccgagugu cugcaggcga ggcgguggug aaccgagugu uugacaagcu guccccgcug 240

cacgagcgca ucuacugugc acucucuggu ucagcugcug augcccaagc cguggccgac 300

auggccgccu accagcugga gcuccauggg auagaacugg aggaaccucc acuuguuuug 360

gcugcugcaa auguggugag aaauaucagc uauaaauauc gagaggacuu gucugcacau 420

cucaugguag cuggcuggga ccaacgugaa ggaggucagg uauauggaac ccugggagga 480

augcugacuc gacagccuuu ugccauuggu ggcuccggca gcaccuuuau cuaugguuau 540

guggaugcag cauauaagcc aggcaugucu cccgaggagu gcaggcgcuu caccacagac 600

gcuauugcuc uggccaugag ccgggauggc ucaagcgggg gugucaucua ccuggucacu 660

auuacagcug ccggugugga ccaucgaguc aucuugggca augaacugcc aaaauucuau 720

gaugagugaa ccuuccccag acuucucuuu cuuauuuugu aauaaacucu cuagggccaa 780

aaccugguau ggucauuggg aaaugagugc ucagggagau ggagcuuagg ggaggugggu 840

gcuucccucc uagaugucag cauacacucu uucuucuuuu gucccagguc uaaaacaucu 900

uuccuagaga aaacaaaagg gacuaaacua gaaauauaaa gagcccuaua caugacaggu 960

gaucacguac ugaaugauuu ugaaguagua caaacaauaa aaauucucau uccgcaucau 1020

caugcggucc augaugauga ggccgcaa 1048

<210> 38

<211> 219

<212> PRT

<213> human (Homo sapiens)

<400> 38

Met Leu Arg Ala Gly Ala Pro Thr Gly Asp Leu Pro Arg Ala Gly Glu

1 5 10 15

Val His Thr Gly Thr Thr Ile Met Ala Val Glu Phe Asp Gly Gly Val

20 25 30

Val Met Gly Ser Asp Ser Arg Val Ser Ala Gly Glu Ala Val Val Asn

35 40 45

Arg Val Phe Asp Lys Leu Ser Pro Leu His Glu Arg Ile Tyr Cys Ala

50 55 60

Leu Ser Gly Ser Ala Ala Asp Ala Gln Ala Val Ala Asp Met Ala Ala

65 70 75 80

Tyr Gln Leu Glu Leu His Gly Ile Glu Leu Glu Glu Pro Pro Leu Val

85 90 95

Leu Ala Ala Ala Asn Val Val Arg Asn Ile Ser Tyr Lys Tyr Arg Glu

100 105 110

Asp Leu Ser Ala His Leu Met Val Ala Gly Trp Asp Gln Arg Glu Gly

115 120 125

Gly Gln Val Tyr Gly Thr Leu Gly Gly Met Leu Thr Arg Gln Pro Phe

130 135 140

Ala Ile Gly Gly Ser Gly Ser Thr Phe Ile Tyr Gly Tyr Val Asp Ala

145 150 155 160

Ala Tyr Lys Pro Gly Met Ser Pro Glu Glu Cys Arg Arg Phe Thr Thr

165 170 175

Asp Ala Ile Ala Leu Ala Met Ser Arg Asp Gly Ser Ser Gly Gly Val

180 185 190

Ile Tyr Leu Val Thr Ile Thr Ala Ala Gly Val Asp His Arg Val Ile

195 200 205

Leu Gly Asn Glu Leu Pro Lys Phe Tyr Asp Glu

210 215

<210> 39

<211> 2974

<212> RNA

<213> human (Homo sapiens)

<400> 39

gugugcguga uggagaaaau ugggcaccag ggcugcuccc gagauucuca gaucugauuu 60

ccacgcuugc uaccaaaaua gucugggcag gccacuuuug gaaguaggcg uuaucuagug 120

agcaggcggc cgcuuucgau uucgcuuucc ccuaaauggc ugagcuucuc gccagcgcag 180

gaucagccug uuccugggac uuuccgagag ccccgcccuc guucccuccc ccagccgcca 240

guaggggagg acucggcggu acccggagcu ucaggcccca ccggggcgcg gagaguccca 300

ggcccggccg ggaccgggac ggcguccgag ugccaauggc uagcucuagg ugucccgcuc 360

cccgcgggug ccgcugccuc cccggagcuu cucucgcaug gcuggggaca guacugcuac 420

uucucgccga cugggugcug cuccggaccg cgcugccccg cauauucucc cugcuggugc 480

ccaccgcgcu gccacugcuc cgggucuggg cggugggccu gagccgcugg gccgugcucu 540

ggcugggggc cugcgggguc cucagggcaa cgguuggcuc caagagcgaa aacgcaggug 600

cccagggcug gcuggcugcu uugaagccau uagcugcggc acugggcuug gcccugccgg 660

gacuugccuu guuccgagag cugaucucau ggggagcccc cggguccgcg gauagcacca 720

ggcuacugca cuggggaagu cacccuaccg ccuucguugu caguuaugca gcggcacugc 780

ccgcagcagc ccuguggcac aaacucggga gccucugggu gcccggcggu cagggcggcu 840

cuggaaaccc ugugcgucgg cuucuaggcu gccugggcuc ggagacgcgc cgccucucgc 900

uguuccuggu ccuggugguc cucuccucuc uuggggagau ggccauucca uucuuuacgg 960

gccgccucac ugacuggauu cuacaagaug gcucagccga uaccuucacu cgaaacuuaa 1020

cucucauguc cauucucacc auagccagug cagugcugga guucgugggu gacgggaucu 1080

auaacaacac caugggccac gugcacagcc acuugcaggg agagguguuu ggggcugucc 1140

ugcgccagga gacggaguuu uuccaacaga accagacagg uaacaucaug ucucggguaa 1200

cagaggacac guccacccug agugauucuc ugagugagaa ucugagcuua uuucuguggu 1260

accuggugcg aggccuaugu cucuugggga ucaugcucug gggaucagug ucccucacca 1320

uggucacccu gaucacccug ccucugcuuu uccuucugcc caagaaggug ggaaaauggu 1380

accaguugcu ggaagugcag gugcgggaau cucuggcaaa guccagccag guggccauug 1440

aggcucuguc ggccaugccu acaguucgaa gcuuugccaa cgaggagggc gaagcccaga 1500

aguuuaggga aaagcugcaa gaaauaaaga cacucaacca gaaggaggcu guggccuaug 1560

cagucaacuc cuggaccacu aguauuucag guaugcugcu gaaaguggga auccucuaca 1620

uuggugggca gcuggugacc aguggggcug uaagcagugg gaaccuuguc acauuuguuc 1680

ucuaccagau gcaguucacc caggcugugg agguacugcu cuccaucuac cccagaguac 1740

agaaggcugu gggcuccuca gagaaaauau uugaguaccu ggaccgcacc ccucgcugcc 1800

cacccagugg ucuguugacu cccuuacacu uggagggccu uguccaguuc caagaugucu 1860

ccuuugccua cccaaaccgc ccagaugucu uagugcuaca ggggcugaca uucacccuac 1920

gcccuggcga ggugacggcg cuggugggac ccaauggguc ugggaagagc acaguggcug 1980

cccugcugca gaaucuguac cagcccaccg ggggacagcu gcuguuggau gggaagcccc 2040

uuccccaaua ugagcaccgc uaccugcaca ggcagguggc ugcaguggga caagagccac 2100

agguauuugg aagaagucuu caagaaaaua uugccuaugg ccugacccag aagccaacua 2160

uggaggaaau cacagcugcu gcaguaaagu cuggggccca uaguuucauc ucuggacucc 2220

cucagggcua ugacacagag guagacgagg cugggagcca gcugucaggg ggucagcgac 2280

aggcaguggc guuggcccga gcauugaucc ggaaaccgug uguacuuauc cuggaugaug 2340

ccaccagugc ccuggaugca aacagccagu uacaggugga gcagcuccug uacgaaagcc 2400

cugagcggua cucccgcuca gugcuucuca ucacccagca ccucagccug guggagcagg 2460

cugaccacau ccucuuucug gaaggaggcg cuauccggga ggggggaacc caccagcagc 2520

ucauggagaa aaaggggugc uacugggcca uggugcaggc uccugcagau gcuccagaau 2580

gaaagccuuc ucagaccugc gcacuccauc ucccucccuu uucuucucuc ugugguggag 2640

aaccacagcu gcagaguagg cagcugccuc caggaugagu uacuugaaau uugccuugag 2700

uguguuaccu ccuuuccaag cuccucguga uaaugcagac uuccuggagu acaaacacag 2760

gauuuguaau uccuuacugu aacggaguuu agagccaggg cugaugcuuu gguguggcca 2820

gcacucugaa acugagaaau guucagaaug uacggaaaga ugaucagcua uuuucaacau 2880

aacugaaggc auaugcuggc ccauaaacac ccuguagguu cuugauauuu auaauaaaau 2940

ugguguuuug uaaaaaaaaa aaaaaaaaaa aaaa 2974

<210> 40

<211> 808

<212> PRT

<213> human (Homo sapiens)

<400> 40

Met Ala Glu Leu Leu Ala Ser Ala Gly Ser Ala Cys Ser Trp Asp Phe

1 5 10 15

Pro Arg Ala Pro Pro Ser Phe Pro Pro Pro Ala Ala Ser Arg Gly Gly

20 25 30

Leu Gly Gly Thr Arg Ser Phe Arg Pro His Arg Gly Ala Glu Ser Pro

35 40 45

Arg Pro Gly Arg Asp Arg Asp Gly Val Arg Val Pro Met Ala Ser Ser

50 55 60

Arg Cys Pro Ala Pro Arg Gly Cys Arg Cys Leu Pro Gly Ala Ser Leu

65 70 75 80

Ala Trp Leu Gly Thr Val Leu Leu Leu Leu Ala Asp Trp Val Leu Leu

85 90 95

Arg Thr Ala Leu Pro Arg Ile Phe Ser Leu Leu Val Pro Thr Ala Leu

100 105 110

Pro Leu Leu Arg Val Trp Ala Val Gly Leu Ser Arg Trp Ala Val Leu

115 120 125

Trp Leu Gly Ala Cys Gly Val Leu Arg Ala Thr Val Gly Ser Lys Ser

130 135 140

Glu Asn Ala Gly Ala Gln Gly Trp Leu Ala Ala Leu Lys Pro Leu Ala

145 150 155 160

Ala Ala Leu Gly Leu Ala Leu Pro Gly Leu Ala Leu Phe Arg Glu Leu

165 170 175

Ile Ser Trp Gly Ala Pro Gly Ser Ala Asp Ser Thr Arg Leu Leu His

180 185 190

Trp Gly Ser His Pro Thr Ala Phe Val Val Ser Tyr Ala Ala Ala Leu

195 200 205

Pro Ala Ala Ala Leu Trp His Lys Leu Gly Ser Leu Trp Val Pro Gly

210 215 220

Gly Gln Gly Gly Ser Gly Asn Pro Val Arg Arg Leu Leu Gly Cys Leu

225 230 235 240

Gly Ser Glu Thr Arg Arg Leu Ser Leu Phe Leu Val Leu Val Val Leu

245 250 255

Ser Ser Leu Gly Glu Met Ala Ile Pro Phe Phe Thr Gly Arg Leu Thr

260 265 270

Asp Trp Ile Leu Gln Asp Gly Ser Ala Asp Thr Phe Thr Arg Asn Leu

275 280 285

Thr Leu Met Ser Ile Leu Thr Ile Ala Ser Ala Val Leu Glu Phe Val

290 295 300

Gly Asp Gly Ile Tyr Asn Asn Thr Met Gly His Val His Ser His Leu

305 310 315 320

Gln Gly Glu Val Phe Gly Ala Val Leu Arg Gln Glu Thr Glu Phe Phe

325 330 335

Gln Gln Asn Gln Thr Gly Asn Ile Met Ser Arg Val Thr Glu Asp Thr

340 345 350

Ser Thr Leu Ser Asp Ser Leu Ser Glu Asn Leu Ser Leu Phe Leu Trp

355 360 365

Tyr Leu Val Arg Gly Leu Cys Leu Leu Gly Ile Met Leu Trp Gly Ser

370 375 380

Val Ser Leu Thr Met Val Thr Leu Ile Thr Leu Pro Leu Leu Phe Leu

385 390 395 400

Leu Pro Lys Lys Val Gly Lys Trp Tyr Gln Leu Leu Glu Val Gln Val

405 410 415

Arg Glu Ser Leu Ala Lys Ser Ser Gln Val Ala Ile Glu Ala Leu Ser

420 425 430

Ala Met Pro Thr Val Arg Ser Phe Ala Asn Glu Glu Gly Glu Ala Gln

435 440 445

Lys Phe Arg Glu Lys Leu Gln Glu Ile Lys Thr Leu Asn Gln Lys Glu

450 455 460

Ala Val Ala Tyr Ala Val Asn Ser Trp Thr Thr Ser Ile Ser Gly Met

465 470 475 480

Leu Leu Lys Val Gly Ile Leu Tyr Ile Gly Gly Gln Leu Val Thr Ser

485 490 495

Gly Ala Val Ser Ser Gly Asn Leu Val Thr Phe Val Leu Tyr Gln Met

500 505 510

Gln Phe Thr Gln Ala Val Glu Val Leu Leu Ser Ile Tyr Pro Arg Val

515 520 525

Gln Lys Ala Val Gly Ser Ser Glu Lys Ile Phe Glu Tyr Leu Asp Arg

530 535 540

Thr Pro Arg Cys Pro Pro Ser Gly Leu Leu Thr Pro Leu His Leu Glu

545 550 555 560

Gly Leu Val Gln Phe Gln Asp Val Ser Phe Ala Tyr Pro Asn Arg Pro

565 570 575

Asp Val Leu Val Leu Gln Gly Leu Thr Phe Thr Leu Arg Pro Gly Glu

580 585 590

Val Thr Ala Leu Val Gly Pro Asn Gly Ser Gly Lys Ser Thr Val Ala

595 600 605

Ala Leu Leu Gln Asn Leu Tyr Gln Pro Thr Gly Gly Gln Leu Leu Leu

610 615 620

Asp Gly Lys Pro Leu Pro Gln Tyr Glu His Arg Tyr Leu His Arg Gln

625 630 635 640

Val Ala Ala Val Gly Gln Glu Pro Gln Val Phe Gly Arg Ser Leu Gln

645 650 655

Glu Asn Ile Ala Tyr Gly Leu Thr Gln Lys Pro Thr Met Glu Glu Ile

660 665 670

Thr Ala Ala Ala Val Lys Ser Gly Ala His Ser Phe Ile Ser Gly Leu

675 680 685

Pro Gln Gly Tyr Asp Thr Glu Val Asp Glu Ala Gly Ser Gln Leu Ser

690 695 700

Gly Gly Gln Arg Gln Ala Val Ala Leu Ala Arg Ala Leu Ile Arg Lys

705 710 715 720

Pro Cys Val Leu Ile Leu Asp Asp Ala Thr Ser Ala Leu Asp Ala Asn

725 730 735

Ser Gln Leu Gln Val Glu Gln Leu Leu Tyr Glu Ser Pro Glu Arg Tyr

740 745 750

Ser Arg Ser Val Leu Leu Ile Thr Gln His Leu Ser Leu Val Glu Gln

755 760 765

Ala Asp His Ile Leu Phe Leu Glu Gly Gly Ala Ile Arg Glu Gly Gly

770 775 780

Thr His Gln Gln Leu Met Glu Lys Lys Gly Cys Tyr Trp Ala Met Val

785 790 795 800

Gln Ala Pro Ala Asp Ala Pro Glu

805

<210> 41

<211> 5732

<212> RNA

<213> human (Homo sapiens)

<400> 41

agaaugaagg ccuuggcugg ggaagcgaaa gcgaaagcug cccgagcccu gacgcccgcc 60

cuggccgagc guagcuggcg gaccagagcc gguagcgagg uugggagaga cggagcggac 120

cucagcgcug aagcagaagu ccccggagcu gcggucuccc cgccgcggcu gagccaugcg 180

gcucccugac cugagacccu ggaccucccu gcugcuggug gacgcggcuu uacuguggcu 240

gcuucagggc ccucugggga cuuugcuucc ucaagggcug ccaggacuau ggcuggaggg 300

gacccugcgg cugggagggc ugugggggcu gcuaaagcua agagggcugc ugggauuugu 360

ggggacacug cugcucccgc ucugucuggc caccccccug acugucuccc ugagagcccu 420

ggucgcgggg gccucacgug cucccccagc cagagucgcu ucagccccuu ggagcuggcu 480

gcuggugggg uacggggcug cggggcucag cuggucacug ugggcuguuc ugagcccucc 540

uggagcccag gagaaggagc aggaccaggu gaacaacaaa gucuugaugu ggaggcugcu 600

gaagcucucc aggccggacc ugccucuccu cguugccgcc uucuucuucc uuguccuugc 660

uguuuugggu gagacauuaa ucccucacua uucuggucgu gugauugaca uccugggagg 720

ugauuuugac ccccaugccu uugccagugc caucuucuuc augugccucu ucuccuuugg 780

cagcucacug ucugcaggcu gccgaggagg cugcuucacc uacaccaugu cucgaaucaa 840

cuugcggauc cgggagcagc uuuucuccuc ccugcugcgc caggaccucg guuucuucca 900

ggagacuaag acaggggagc ugaacucacg gcugagcucg gauaccaccc ugaugaguaa 960

cuggcuuccu uuaaaugcca augugcucuu gcgaagccug gugaaagugg uggggcugua 1020

uggcuucaug cucagcauau cgccucgacu cacccuccuu ucucugcugc acaugcccuu 1080

cacaauagca gcggagaagg uguacaacac ccgccaucag gaagugcuuc gggagaucca 1140

ggaugcagug gccagggcgg ggcagguggu gcgggaagcc guuggagggc ugcagaccgu 1200

ucgcaguuuu ggggccgagg agcaugaagu cugucgcuau aaagaggccc uugaacaaug 1260

ucggcagcug uauuggcgga gagaccugga acgcgccuug uaccugcucg uaaggagggu 1320

gcugcacuug ggggugcaga ugcugaugcu gagcuguggg cugcagcaga ugcaggaugg 1380

ggagcucacc cagggcagcc ugcuuuccuu uaugaucuac caggagagcg uggggagcua 1440

ugugcagacc cugguauaca uauaugggga uaugcucagc aacgugggag cugcagagaa 1500

gguuuucucc uacauggacc gacagccaaa ucugccuuca ccuggcacgc uugcccccac 1560

cacucugcag gggguuguga aauuccaaga cgucuccuuu gcauauccca aucgcccuga 1620

caggccugug cucaaggggc ugacguuuac ccuacguccu ggugagguga cggcgcuggu 1680

gggacccaau gggucuggga agagcacagu ggcugcccug cugcagaauc uguaccagcc 1740

cacaggggga caggugcugc uggaugaaaa gcccaucuca caguaugaac acugcuaccu 1800

gcacagccag gugguuucag uugggcagga gccugugcug uucuccgguu cugugaggaa 1860

caacauugcu uaugggcugc agagcugcga agaugauaag gugauggcgg cugcccaggc 1920

ugcccacgca gaugacuuca uccaggaaau ggagcaugga auauacacag auguagggga 1980

gaagggaagc cagcuggcug cgggacagaa acaacgucug gccauugccc gggcccuugu 2040

acgagacccg cggguccuca uccuggauga ggcuacuagu gcccuagaug ugcagugcga 2100

gcaggcccug caggacugga auucccgugg ggaucgcaca gugcugguga uugcucacag 2160

gcugcagaca guucagcgcg cccaccagau ccuggugcuc caggagggca agcugcagaa 2220

gcuugcccag cucuaggagg gacaggaccu cuauucccgc cuggugcagc agcggcugau 2280

ggacugaggc cccagggaua cugggcccuc uucucagggg cgucuccagg acccagagcu 2340

guuccugcuu ugaguuuccc uagagcugug cggccagaua gcuguuccug aguugcaggc 2400

acgauggaga uuuggacacu gugugcuuuu ggugggguag agaggugggg uggggugggg 2460

ugggggcugu cuguguccag gaaacuuaau ucccugguga cuagagcuuu gccuggugau 2520

gaggaguauu uuguggcaua auacauauau uuuaaaauau uuuccuucuu acaugaacug 2580

uauacauuca uauagaaaau uuagacaaua uaaaaaagua caaagaagaa aaguaaaagu 2640

acccauuguu ucacuuccug gagauaacca uaguugcuau uuugcugccu gucccaucag 2700

ucguuuaucu guuguuugag auagaaauua accaaaaaug acauaaauau ucaugagauu 2760

gccuuccuau auccuuccuu guuccuacca gugucugcua uuuugaagaa gcuagggucu 2820

ggagggacag agaacaguuc ccugauuaac aguauuaaua gcgacauugg uaacagcuac 2880

cauuuauaga guuuuaaugg gaguaggagc uaugcuaagu guuuuucaug uauuaucguu 2940

uuuaaucauu auccccaacc cuaugagguu gguuauuauc cccauuuuac agaugaggaa 3000

acugaagcuc aaagaggcuc aaugacuuuc ccaagguggu cguaguggug gaguuggagu 3060

uugaacacag gccugacccu agaguccaca cccugaccca aucaauuaua uugcaucuug 3120

gguccauaaa cccuaaucca uaaucccauc aagaaaagcu cugcugcucu uagcucuaaa 3180

uaauucagaa ucuauucucu ucucuccagu cccguuguua uagucuucac ucauagacuu 3240

aagaugaucc caucaccaga gagguuucuc uaccauuagc uucccucuuc cggccauucu 3300

ucacaaaguc auuuuucuaa auucuguguc acauacgaug auggcauuuc uggaaauucc 3360

uucaggugcu cucaagcccu gcugcagaga uccuuuucag agcacacacu guuccagccc 3420

aucugucuca cccucuccug uuguauccag cuccacgaca aacuucugcc uuccccaaca 3480

ccuuugugcc uuugcauaug guguuuucuu gcccauuuuc ugcucgacuc gccccugauu 3540

uucaaguuca agacuuaacu caggguucag gucuuccagg aggccuuacu uaugucguca 3600

gucuggggaa cucuccaugu gcuucuauca cugugcgguu accucuuuca cagcccuuuu 3660

aaaguucuau cuucccuuuc ccaccuuuuu ugaccuucca cuagaccaug agcaccuggg 3720

cggaaagcca uauaucuuau uaagcuuuau aucugcuacc uggccgaggg ccuaauucau 3780

aguggagaau aaauagucaa uugaauaaau gaauaaauau cuccaccauc guacuaaucu 3840

uaauccuccc ugcccacucc caccacugaa aaugcaacau uguacacauc acugguuguu 3900

gggagggacu uaccuuggaa aguugcuauu cuaggaaaga gaaaccuuca uauuccugga 3960

aacagcaggu aguuuccagu gcuggcaaug aauuccccag aacugcuguu uuggauuuuu 4020

ucuugccugg cagcuguugg gagcagggug cagugaggau ggggugagag ugggcaguuu 4080

cuugugcaga uuugccuuuc uuucauccug gggcugacuu gcagcuccac acccauccau 4140

cucucaaauu ucacagaggg uaaaauaggc auuuggagag aaagaacucu ggccugauuc 4200

cuuucucucc cacaaauguc cuuuauucau aaaacaggaa uaauaauucc uguaucuccc 4260

aacuacaugg aagcugcagc ccucacagaa gaagaugauc ugagaaauuc uuugauuucc 4320

ucaguacagu uauacccaug caucauaaua cuuuaagccu ggaaggcauc uuaaaaauaa 4380

ugcaacaguc aaaccuaauu uuacagagaa acugacauga aaucacgcag cuaaucauga 4440

uaaagcuggg uggaaaacuu aucuugaugg gcaguacagg aagaugcagu agaccuuaag 4500

auguccugaa aguuucuuau cucaggggaa acucccaggu aggcuuuaug ucagggacac 4560

agaaaaaugc ucccugaaag ucaaaauauu cgggcuagac agacaaauuc cuguaagugu 4620

gguuugucug ggaaccacag augucacuaa uccugguuug cuccagaguu cuuuuuguuc 4680

acuccuaccc cccaucacca uuugauugau cuccuuaccc uguaauuucc ccuucuuguc 4740

gcuuaccugc aguaucuuuc ccacccaggc augccuuauu cuuucuaaag gaaaguauga 4800

auggagaggg gaaagcuugg gaaacugaua gauuuccuug gaugccaaaa caccuccaua 4860

gccugucugc ccggcccuau guggaaacag cauugaguuu caaguccuuu augccuccac 4920

ccagggauag ccacuuguaa uccacauggc aauugugaaa caagcaggaa augcguaauu 4980

gucagaauuu uguggggaaa ggacuaggga auaaggaaaa caaagaucuu ccuuguguuu 5040

uagagcuguc agcuagagga gcaccugcuu gagucugaug ccaucuaaug gucccagaag 5100

aaacuggguu uugaaccuag aguuccaugg acucuuagga auuagacuac uacuacuacu 5160

aagcauucac uggugcuuac uaugugcuau ugcugugcca aguaucugaa accugucuuc 5220

uuaccuuauu uuucaagaua auucuaugug gcagguauua cuaucucaau ucuaagagug 5280

agaaaaugga guuuuagaaa cauuuacuaa cuugccuggg ucacauagcu aaggaagagg 5340

uggacuugcc cagcuuugca uaaaacuccu caaaagaguu gccuauacuc ccugacucca 5400

cuuaucuucc uacuauccuc uuuuuaaaau auauuauuua uuuauuuaaa uaagcaauau 5460

augaaugugg uuugaaauuc aaaagacaca aagaaguaua cagaggaaag ccucacucuc 5520

aauccuucuc aagguuugcu aauuccucuu gcauaggcaa uccguucuuc cagcuuugug 5580

uuuaucuuuc cagagaaguu uacuguguau uaagcaaaua uguauaucuu uauucuugcu 5640

caguauuuuc gcaaacagca gcugucuaag uucacuguuc ugaacuuuau uuuuuaaauu 5700

aaaaauauau ggcuauguag uauucuauuu ua 5732

<210> 42

<211> 686

<212> PRT

<213> human (Homo sapiens)

<400> 42

Met Arg Leu Pro Asp Leu Arg Pro Trp Thr Ser Leu Leu Leu Val Asp

1 5 10 15

Ala Ala Leu Leu Trp Leu Leu Gln Gly Pro Leu Gly Thr Leu Leu Pro

20 25 30

Gln Gly Leu Pro Gly Leu Trp Leu Glu Gly Thr Leu Arg Leu Gly Gly

35 40 45

Leu Trp Gly Leu Leu Lys Leu Arg Gly Leu Leu Gly Phe Val Gly Thr

50 55 60

Leu Leu Leu Pro Leu Cys Leu Ala Thr Pro Leu Thr Val Ser Leu Arg

65 70 75 80

Ala Leu Val Ala Gly Ala Ser Arg Ala Pro Pro Ala Arg Val Ala Ser

85 90 95

Ala Pro Trp Ser Trp Leu Leu Val Gly Tyr Gly Ala Ala Gly Leu Ser

100 105 110

Trp Ser Leu Trp Ala Val Leu Ser Pro Pro Gly Ala Gln Glu Lys Glu

115 120 125

Gln Asp Gln Val Asn Asn Lys Val Leu Met Trp Arg Leu Leu Lys Leu

130 135 140

Ser Arg Pro Asp Leu Pro Leu Leu Val Ala Ala Phe Phe Phe Leu Val

145 150 155 160

Leu Ala Val Leu Gly Glu Thr Leu Ile Pro His Tyr Ser Gly Arg Val

165 170 175

Ile Asp Ile Leu Gly Gly Asp Phe Asp Pro His Ala Phe Ala Ser Ala

180 185 190

Ile Phe Phe Met Cys Leu Phe Ser Phe Gly Ser Ser Leu Ser Ala Gly

195 200 205

Cys Arg Gly Gly Cys Phe Thr Tyr Thr Met Ser Arg Ile Asn Leu Arg

210 215 220

Ile Arg Glu Gln Leu Phe Ser Ser Leu Leu Arg Gln Asp Leu Gly Phe

225 230 235 240

Phe Gln Glu Thr Lys Thr Gly Glu Leu Asn Ser Arg Leu Ser Ser Asp

245 250 255

Thr Thr Leu Met Ser Asn Trp Leu Pro Leu Asn Ala Asn Val Leu Leu

260 265 270

Arg Ser Leu Val Lys Val Val Gly Leu Tyr Gly Phe Met Leu Ser Ile

275 280 285

Ser Pro Arg Leu Thr Leu Leu Ser Leu Leu His Met Pro Phe Thr Ile

290 295 300

Ala Ala Glu Lys Val Tyr Asn Thr Arg His Gln Glu Val Leu Arg Glu

305 310 315 320

Ile Gln Asp Ala Val Ala Arg Ala Gly Gln Val Val Arg Glu Ala Val

325 330 335

Gly Gly Leu Gln Thr Val Arg Ser Phe Gly Ala Glu Glu His Glu Val

340 345 350

Cys Arg Tyr Lys Glu Ala Leu Glu Gln Cys Arg Gln Leu Tyr Trp Arg

355 360 365

Arg Asp Leu Glu Arg Ala Leu Tyr Leu Leu Val Arg Arg Val Leu His

370 375 380

Leu Gly Val Gln Met Leu Met Leu Ser Cys Gly Leu Gln Gln Met Gln

385 390 395 400

Asp Gly Glu Leu Thr Gln Gly Ser Leu Leu Ser Phe Met Ile Tyr Gln

405 410 415

Glu Ser Val Gly Ser Tyr Val Gln Thr Leu Val Tyr Ile Tyr Gly Asp

420 425 430

Met Leu Ser Asn Val Gly Ala Ala Glu Lys Val Phe Ser Tyr Met Asp

435 440 445

Arg Gln Pro Asn Leu Pro Ser Pro Gly Thr Leu Ala Pro Thr Thr Leu

450 455 460

Gln Gly Val Val Lys Phe Gln Asp Val Ser Phe Ala Tyr Pro Asn Arg

465 470 475 480

Pro Asp Arg Pro Val Leu Lys Gly Leu Thr Phe Thr Leu Arg Pro Gly

485 490 495

Glu Val Thr Ala Leu Val Gly Pro Asn Gly Ser Gly Lys Ser Thr Val

500 505 510

Ala Ala Leu Leu Gln Asn Leu Tyr Gln Pro Thr Gly Gly Gln Val Leu

515 520 525

Leu Asp Glu Lys Pro Ile Ser Gln Tyr Glu His Cys Tyr Leu His Ser

530 535 540

Gln Val Val Ser Val Gly Gln Glu Pro Val Leu Phe Ser Gly Ser Val

545 550 555 560

Arg Asn Asn Ile Ala Tyr Gly Leu Gln Ser Cys Glu Asp Asp Lys Val

565 570 575

Met Ala Ala Ala Gln Ala Ala His Ala Asp Asp Phe Ile Gln Glu Met

580 585 590

Glu His Gly Ile Tyr Thr Asp Val Gly Glu Lys Gly Ser Gln Leu Ala

595 600 605

Ala Gly Gln Lys Gln Arg Leu Ala Ile Ala Arg Ala Leu Val Arg Asp

610 615 620

Pro Arg Val Leu Ile Leu Asp Glu Ala Thr Ser Ala Leu Asp Val Gln

625 630 635 640

Cys Glu Gln Ala Leu Gln Asp Trp Asn Ser Arg Gly Asp Arg Thr Val

645 650 655

Leu Val Ile Ala His Arg Leu Gln Thr Val Gln Arg Ala His Gln Ile

660 665 670

Leu Val Leu Gln Glu Gly Lys Leu Gln Lys Leu Ala Gln Leu

675 680 685

Claims

1. A method of identifying an individual having cancer who is likely to benefit from treatment with a PD-L1 binding antagonist, the method comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment with a PD-L1 binding antagonist, wherein the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in the reference population.

2. A method for selecting a therapy for an individual having cancer, the method comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 binding antagonist, wherein the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population.

3. The method of claim 1 or2, wherein the immune score expression level of PD-L1, CXCL9, and IFNG in the sample is greater than the reference immune score expression level and the method further comprises administering to the individual an effective amount of a PD-L1 binding antagonist.

4. The method of claim 1 or2, wherein an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is lower than the reference immune score expression level identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 binding antagonist.

5. The method of any one of claims 1-4, wherein the immune score expression level of PD-L1, CXCL9, and IFNG in the sample is below a reference immune score expression level and the method further comprises administering to the individual an effective amount of an anti-cancer therapy in place of or in addition to the PD-L1 binding antagonist.

6. A method of treating an individual having cancer, the method comprising:

(a) determining expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein the immune score expression levels of PD-L1, CXCL9, and IFNG in the sample have been determined to be higher than a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, CXCL9, and IFNG in a reference population, and

(b) administering to the individual an effective amount of a PD-L1 binding antagonist based on the immune score expression levels of PD-L1, CXCL9, and IFNG determined in step (a).

7. A method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 binding antagonist, wherein the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual have been determined prior to treatment and the immune score expression levels of PD-L1, CXCL9, and IFNG in the sample have been determined to be greater than a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, CXCL9, and IFNG in a reference population.

8. The method of any one of claims 1-3,6, and 7, wherein the immune score expression levels of PD-L1, CXCL9, and IFNG in the sample are in the top 80 th percentile of the immune score expression levels of PD-L1, CXCL9, and IFNG in the reference population.

9. The method of claim 8, wherein the immune score expression levels of PD-L1, CXCL9, and IFNG in the sample are in the top 50 th percentile of the immune score expression levels of PD-L1, CXCL9, and IFNG in the reference population.

10. The method of claim 9, wherein the immune score expression levels of PD-L1, CXCL9, and IFNG in the sample are in the top 20 th percentile of the immune score expression levels of PD-L1, CXCL9, and IFNG in the reference population.

11. The method of any one of claims 1-10, wherein the reference population is a population of individuals with cancer consisting of a first subset of individuals who have been treated with PD-L1 binding antagonist therapy and a second subset of individuals who have been treated with non-PD-L1 binding antagonist therapy, wherein the non-PD-L1 binding antagonist therapy does not comprise a PD-L1 binding antagonist.

12. The method of claim 11, wherein the reference immune score expression level significantly separates each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy and the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy above the reference immune score expression level, wherein the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy.

13. The method of claim 11 or 12, wherein the reference immune score expression level significantly separates each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy and the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy below the reference immune score expression level, wherein the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy.

14. The method of claim 12 or 13, wherein responsiveness to treatment is an extension of Progression Free Survival (PFS).

15. The method of claim 12 or 13, wherein responsiveness to treatment is an extension of Overall Survival (OS).

16. The method of any one of claims 1-15, wherein the immune score expression levels of PD-L1, CXCL9, and IFNG are averages of the expression levels of each of PD-L1, CXCL9, and IFNG.

17. The method of claim 16, wherein the average of the expression levels for each of PD-L1, CXCL9, and IFNG is the average of the normalized expression levels for each of PD-L1, CXCL9, and IFNG.

18. The method of any one of claims 1-15, wherein the immune score expression level of PD-L1, CXCL9, and IFNG is the median of the expression level of each of PD-L1, CXCL9, and IFNG.

19. The method of claim 18, wherein the immune score expression level of PD-L1, CXCL9, and IFNG is the median of the normalized expression levels of each of PD-L1, CXCL9, and IFNG.

20. The method of claim 17 or 19, wherein the normalized expression level for each of PD-L1, CXCL9, and IFNG is the expression level for each of PD-L1, CXCL9, and IFNG normalized to a reference gene.

21. The method of any one of claims 1-20, wherein the reference immune score expression level is a pre-assigned expression level of PD-L1, CXCL9, and IFNG.

22. A method of identifying an individual having cancer who is likely to benefit from treatment with a binding antagonist comprising PD-L1, the method comprising determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment with the binding antagonist comprising PD-L1, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the reference population.

23. A method for selecting a therapy for an individual having cancer, the method comprising determining expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein an expression level of an immune score for PD-L1, IFNG, GZMB, and CD8A in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 binding antagonist, wherein the reference immune score expression level is an immune score expression level for PD-L1, IFNG, GZMB, and CD8A in a reference population.

24. The method of claim 22 or 23, wherein the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample is greater than the reference immune score expression level and the method further comprises administering to the individual an effective amount of a PD-L1 binding antagonist.

25. The method of claim 22 or 23, wherein an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample that is lower than a reference immune score expression level identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 binding antagonist.

26. The method of any one of claims 22-25, wherein the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample is below the reference immune score expression level and the method further comprises administering to the individual an effective amount of an anti-cancer therapy that replaces or supplements the PD-L1 binding antagonist.

27. A method of treating an individual having cancer, the method comprising:

(a) determining expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the sample have been determined to be higher than a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in a reference population, and

(b) administering to the individual an effective amount of a PD-L1 binding antagonist based on the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A determined in step (a).

28. A method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 binding antagonist, wherein the expression levels of PD-L1, IFNG, GZMB, and CD8A have been determined in a sample from the individual prior to treatment and the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the sample have been determined to be higher than the expression level of the reference immune score, wherein the expression level of the reference immune score is the expression level of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the reference population.

29. The method of any one of claims 22-24,27, and 28, wherein the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the sample are in the first 80 th percentile of the expression levels of the immune scores of PD-L1, IFNG, GZMB, and CD8A in the reference population.

30. The method of claim 29, wherein the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the sample are in the first 50 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the reference population.

31. The method of claim 30, wherein the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the sample are in the top 20 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A in the reference population.

32. The method of any one of claims 22-31, wherein the reference population is a population of individuals with cancer consisting of a first subset of individuals who have been treated with PD-L1 binding antagonist therapy and a second subset of individuals who have been treated with non-PD-L1 binding antagonist therapy, wherein the non-PD-L1 binding antagonist therapy does not comprise a PD-L1 binding antagonist.

33. The method of claim 32, wherein the reference immune score expression level significantly separates each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy and the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy above the reference immune score expression level, wherein the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy.

34. The method of claim 32 or 33, wherein the reference immune score expression level significantly separates each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy and the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy below the reference immune score expression level, wherein the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy.

35. The method of claim 33 or 34, wherein responsiveness to treatment is prolongation of PFS.

36. The method of claim 33 or 34, wherein responsiveness to treatment is prolongation of OS.

37. The method of any one of claims 22-36, wherein the immune score expression levels of PD-L1, IFNG, GZMB, and CD8A are averages of the expression levels of each of PD-L1, IFNG, GZMB, and CD 8A.

38. The method of claim 37, wherein the average of the expression levels of each of PD-L1, IFNG, GZMB, and CD8A is the average of the normalized expression levels of each of PD-L1, IFNG, GZMB, and CD 8A.

39. The method of any one of claims 22-36, wherein the immune score expression level of PD-L1, IFNG, GZMB, and CD8A is the median of the expression levels of each of PD-L1, IFNG, GZMB, and CD 8A.

40. The method of claim 39, wherein the immune score expression level of PD-L1, IFNG, GZMB, and CD8A is the median of the normalized expression levels of each of PD-L1, IFNG, GZMB, and CD 8A.

41. The method of claim 38 or 40, wherein the normalized expression level of each of PD-L1, IFNG, GZMB, and CD8A is the expression level of each of PD-L1, IFNG, GZMB, and CD8A normalized to a reference gene.

42. The method of any one of claims 22-41, wherein the reference immune score expression level is a pre-assigned expression level of PD-L1, IFNG, GZMB, and CD 8A.

43. A method of identifying an individual having cancer who is likely to benefit from treatment with a binding antagonist comprising PD-L1, the method comprising determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment with the binding antagonist comprising PD-L1, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population.

44. A method for selecting a therapy for an individual having cancer, the method comprising determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment with a PD-L1 binding antagonist, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

45. The method of claim 43 or 44, wherein the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample is greater than the reference immune score expression level and the method further comprises administering to the individual an effective amount of a PD-L1 binding antagonist.

46. The method of claim 43 or 44, wherein an immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is lower than a reference immune score expression level identifies the individual as an individual who is unlikely to benefit from treatment comprising a PD-L1 binding antagonist.

47. The method of any one of claims 43-46, wherein the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample is less than the reference immune score expression level and the method further comprises administering to the individual an effective amount of an anti-cancer therapy that replaces or supplements the PD-L1 binding antagonist.

48. A method of treating an individual having cancer, the method comprising:

(a) determining expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample have been determined relative to a reference immune score expression level, wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population, and

(b) administering to the individual an effective amount of a PD-L1 binding antagonist based on the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 determined in step (a).

49. A method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a PD-L1 binding antagonist, wherein the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 have been determined in a sample from the individual prior to treatment and the expression levels of the immune scores of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample have been determined to be higher than the expression level of a reference immune score, wherein the expression level of the reference immune score is the expression level of the immune scores of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

50. The method of any one of claims 43-45,48, and 49, wherein the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample are in the first 80 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

51. The method of claim 50, wherein the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample are in the first 50 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

52. The method of claim 51, wherein the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample are in the first 20 th percentile of the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in the reference population.

53. The method of any one of claims 43-52, wherein the reference population is a population of individuals with cancer consisting of a first subset of individuals who have been treated with PD-L1 binding antagonist therapy and a second subset of individuals who have been treated with non-PD-L1 binding antagonist therapy, wherein the non-PD-L1 binding antagonist therapy does not comprise a PD-L1 binding antagonist.

54. The method of claim 53, wherein the reference immune score expression level significantly separates each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy and the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy above the reference immune score expression level, wherein the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy.

55. The method of claim 53 or 54, wherein the reference immune score expression level significantly separates each of the first and second subsets of individuals based on a significant difference between the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy and the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy below the reference immune score expression level, wherein the responsiveness of the individual to treatment with the non-PD-L1 binding antagonist therapy is significantly improved relative to the responsiveness of the individual to treatment with the PD-L1 binding antagonist therapy.

56. The method of claim 54 or 55, wherein responsiveness to treatment is prolongation of PFS.

57. The method of claim 54 or 55, wherein responsiveness to treatment is prolongation of OS.

58. The method of any one of claims 43-57, wherein the immune score expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 are averages of the expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1.

59. The method of claim 58, wherein the average of the expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1 is the average of the normalized expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1.

60. The method of any one of claims 43-57, wherein the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 is the median of the expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1.

61. The method of claim 60, wherein the immune score expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1 is the median of the normalized expression levels of each of PD-L1, IFNG, GZMB, CD8A, and PD-1.

62. The method of claim 59 or 61, wherein the normalized expression level for each of PD-L1, IFNG, GZMB, CD8A, and PD-1 is the expression level for each of PD-L1, IFNG, GZMB, CD8A, and PD-1 normalized to a reference gene.

63. The method of any one of claims 43-62, wherein the reference immune score expression level is a pre-assigned expression level of PD-L1, IFNG, GZMB, CD8A, and PD-1.

64. The method of any one of claims 20,41, and 62, wherein the reference gene is a housekeeping gene.

65. The method of claim 64, wherein the housekeeping gene is TMEM 55B.

66. The method of any one of claims 1-65, wherein the benefit from treatment comprising a PD-L1 binding antagonist is prolongation of OS.

67. The method of any one of claims 1-65, wherein the benefit from treatment comprising a PD-L1 binding antagonist is prolongation of PFS.

68. The method of claim 66 or 67, wherein the benefit from treatment comprising a PD-L1 binding antagonist is prolongation of OS and PFS.

69. The method of any one of claims 1-68, wherein the expression level is a nucleic acid expression level.

70. The method of claim 69, wherein the nucleic acid expression level is an mRNA expression level.

71. The method of claim 70, wherein mRNA expression levels are determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

72. The method of claim 71, wherein mRNA expression levels are detected using RNA-seq.

73. The method of claim 71, wherein mRNA expression levels are detected using RT-qPCR.

74. The method of any one of claims 69-73, wherein the expression level is detected in a tumor cell, a tumor-infiltrating immune cell, a stromal cell, or a combination thereof.

75. The method of any one of claims 1-74, wherein the sample is a tissue sample, a cell sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof.

76. The method of claim 75, wherein the tissue sample is a tumor tissue sample.

77. The method of claim 76, wherein the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, or a combination thereof.

78. The method of claim 76 or 77, wherein the tumor tissue sample is a Formalin Fixed and Paraffin Embedded (FFPE) sample, an archived sample, a fresh sample, or a frozen sample.

79. The method of claim 78, wherein the tumor tissue sample is an FFPE sample.

80. The method of any one of claims 1-79, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, bladder cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, stomach cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, or hematological malignancy.

81. The method of claim 80, wherein the cancer is lung cancer, renal cancer, bladder cancer, or breast cancer.

82. The method of claim 81, wherein the lung cancer is non-small cell lung cancer (NSCLC).

83. The method of claim 81, wherein the renal cancer is Renal Cell Carcinoma (RCC).

84. The method of claim 81, wherein the bladder cancer is Urothelial Bladder Cancer (UBC).

85. The method of claim 81, wherein the breast cancer is Triple Negative Breast Cancer (TNBC).

86. The method of any one of claims 1-85, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1, the binding of PD-L1 to B7-1, or the binding of PD-L1 to both PD-1 and B7-1.

87. The method of any one of claims 1-86, wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.

88. The method of claim 87, wherein the anti-PD-L1 antibody is selected from the group consisting of atelizumab (atezolizumab) (MPDL3280A), YW243.55.S70, MSB0010718C, MDX-1105, and MEDI 4736.

89. The method of claim 87 or 88, wherein the anti-PD-L1 antibody comprises the following hypervariable regions:

(a) GFTFSDSWIH (SEQ ID NO:9) of HVR-H1 sequence;

(b) AWISPYGGSTYYADSVKG (SEQ ID NO:10) of HVR-H2 sequence;

(c) RHWPGGFDY (SEQ ID NO:11) of HVR-H3 sequence;

(d) RASQDVSTAVA (SEQ ID NO:12) of HVR-L1 sequence;

(e) the HVR-L2 sequence of SASFLYS (SEQ ID NO: 13); and

(f) QQYLYHPAT (SEQ ID NO:14) HVR-L3 sequence.

90. The method of any one of claims 87-89, wherein the anti-PD-L1 antibody comprises:

(a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO 16;

(b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO. 17; or

(c) A VH domain as in (a) and a VL domain as in (b).

91. The method of claim 90, wherein the anti-PD-L1 antibody comprises:

(a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 16;

(b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 17; or

(c) A VH domain as in (a) and a VL domain as in (b).

92. The method of claim 91, wherein the anti-PD-L1 antibody comprises:

(a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO 16;

(b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO. 17; or

(c) A VH domain as in (a) and a VL domain as in (b).

93. The method of claim 92, wherein the anti-PD-L1 antibody comprises:

(a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO 16;

(b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO 17; or

(c) A VH domain as in (a) and a VL domain as in (b).

94. The method of claim 93, wherein the anti-PD-L1 antibody comprises:

(a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO 16;

(b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO 17; or

(c) A VH domain as in (a) and a VL domain as in (b).

95. The method of claim 94, wherein the anti-PD-L1 antibody comprises:

(a) a heavy chain Variable (VH) domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO 16;

(b) a light chain Variable (VL) domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 17; or

(c) A VH domain as in (a) and a VL domain as in (b).

96. The method of claim 95, wherein the anti-PD-L1 antibody comprises:

(a) a VH domain comprising the amino acid sequence of SEQ ID NO 16;

(b) a VL domain comprising the amino acid sequence of SEQ ID NO 17; or

(c) A VH domain as in (a) and a VL domain as in (b).

97. The method of claim 96, wherein the anti-PD-L1 antibody comprises:

(a) a VH domain comprising the amino acid sequence of SEQ ID NO 16; and

(b) a VL domain comprising the amino acid sequence of SEQ ID NO 17.

98. The method of claim 97, wherein the anti-PD-L1 antibody is atezumab.

99. The method of any one of claims 11-21,32-42, and 53-98, wherein the non-PD-L1 binding antagonist is an anti-neoplastic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, or a cytotoxic agent.

100. The method of any one of claims 5,26, and 47, wherein the anti-cancer therapy is an anti-tumor agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, or a cytotoxic agent.

101. The method of any one of claims 1-100, wherein the subject has not previously been treated for cancer.

102. The method of claim 101, wherein the individual has not previously been administered a PD-L1 binding antagonist.

103. The method of any one of claims 1-102, wherein the treatment comprising a PD-L1 binding antagonist is monotherapy.

104. The method of any one of claims 1-102, wherein the treatment comprising a PD-L1 binding antagonist is a combination therapy.

105. The method of any one of claims 1-3,6-24,27-45,48-102, and 104, further comprising administering to the individual an effective amount of an additional therapeutic agent.

106. The method of claim 105, wherein the additional therapeutic agent is an anti-neoplastic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, a cytotoxic agent, or a combination thereof.

107. The method of claim 106, wherein the additional therapeutic agent is a chemotherapeutic agent.

108. The method of claim 107 wherein the chemotherapeutic agent is carboplatin; -Parietai; or carboplatin and paclitaxel.

109. The method of claim 108 wherein the chemotherapeutic agents are carboplatin and paclitaxel.

110. The method of claim 106, wherein the additional therapeutic agent is an anti-angiogenic agent.

111. The method of claim 106, wherein the additional therapeutic agent is a combination of an anti-angiogenic agent and a chemotherapeutic agent.

112. The method of claim 111 wherein the chemotherapeutic agent is carboplatin; -Parietai; or carboplatin and paclitaxel.

113. The method of claim 112 wherein the chemotherapeutic agents are carboplatin and paclitaxel.

114. The method of any one of claims 110-113, wherein the anti-angiogenic agent is an anti-VEGF antibody.

115. The method of claim 114, wherein the anti-VEGF antibody is bevacizumab (bevacizumab).

116. The method of any one of claims 1-115, wherein the subject is a human.

117. A kit for identifying an individual having cancer who is likely to benefit from treatment with a binding antagonist comprising PD-L1, the kit comprising:

(a) reagents for determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from a subject; and, optionally,

(b) instructions for using the agent to identify an individual having cancer who may benefit from treatment comprising a PD-L1 binding antagonist.

118. A kit for identifying an individual having cancer who is likely to benefit from treatment with a binding antagonist comprising PD-L1, the kit comprising:

(a) reagents for determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from a subject; and, optionally,

119. A kit for identifying an individual having cancer who is likely to benefit from treatment with a binding antagonist comprising PD-L1, the kit comprising:

(a) reagents for determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from a subject; and, optionally,

120. An assay for identifying an individual having cancer as a candidate for treatment comprising a PD-L1 binding antagonist, the assay comprising determining the expression levels of PD-L1, CXCL9, and IFNG in a sample from the individual, wherein an immune score expression level of PD-L1, CXCL9, and IFNG in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 binding antagonist, and wherein the reference immune score expression level is an immune score expression level of PD-L1, CXCL9, and IFNG in a reference population.

121. An assay for identifying an individual having cancer as a candidate for treatment comprising a PD-L1 binding antagonist, the assay comprising determining the expression levels of PD-L1, IFNG, GZMB, and CD8A in a sample from the individual, wherein an immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 binding antagonist, and wherein the reference immune score expression level is the immune score expression level of PD-L1, IFNG, GZMB, and CD8A in the reference population.

122. An assay for identifying an individual having cancer as a candidate for treatment comprising a PD-L1 binding antagonist, the assay comprising determining the expression levels of PD-L1, IFNG, GZMB, CD8A, and PD-1 in a sample from the individual, wherein an expression level of an immune score for PD-L1, IFNG, GZMB, CD8A, and PD-1 in the sample that is higher than a reference immune score expression level identifies the individual as an individual who is likely to benefit from treatment comprising a PD-L1 binding antagonist, and wherein the reference immune score expression level is an expression level of an immune score for PD-L1, IFNG, GZMB, CD8A, and PD-1 in a reference population.