Methods of treatment and diagnosis of bladder cancer

Abstract

The present invention provides methods and compositions for treating bladder cancer (e.g., urothelial carcinoma (UC), including locally advanced or metastatic UC) in a subject, for example, by administering to the patient a treatment regimen that includes a PD-1 axis binding antagonist (e.g., acterizumab). The present invention also provides compositions (e.g., PD-1 axis binding antagonists (e.g., atrizumab) and / or platinum based chemotherapies (e.g., cis-platinum or carboplatin and gemcitabine), pharmaceutical compositions thereof, kits thereof, and articles of manufacture thereof) for use in the treatment of bladder cancer (e.g., UC, including locally advanced or metastatic UC) in a patient. The invention also provides assays and methods for determining the presence and / or expression level of PD-L1 in a sample obtained from a patient and for labeling PD-L1 in a sample obtained from a patient.

Description

[0001] Sequence Listing

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on August 31, 2023, named 50474-306WO2_Sequence_Listing_8_31_23.xml, and is 12,341 bytes in size. Technical Field

[0003] The present invention relates to methods and compositions for treating a subject having bladder cancer (e.g., urothelial carcinoma (UC), including locally advanced or metastatic UC) in a subject, for example, by administering to the subject a treatment regimen comprising a PD-1 axis binding antagonist (e.g., atezolizumab). The present invention also relates to assays and methods for labeling PD-L1 in tumor samples obtained from a subject. Background Art

[0004] Cancer remains one of the most lethal threats to human health. Cancers or malignant tumors metastasize or grow rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult. Urothelial carcinoma (UC, also known as transitional cell carcinoma (TCC), urothelial carcinoma of the bladder, or urothelial cell carcinoma of the urethra (UCC)) is the most common cancer of the urinary system worldwide, with urothelial carcinoma of the bladder being the major histological type and site. Although less common, urothelial carcinoma can also originate in the renal pelvis, ureter, or urethra. It is estimated that in 2015, there will be 74,000 new cases of bladder cancer in the United States, and 16,000 deaths from bladder cancer. Similar global data estimate that in 2012, 123,000 men and 42,000 women died from bladder cancer. The overall 5-year survival rate for metastatic urothelial carcinoma is approximately 5.4%. Poor prognostic factors for survival in patients with metastatic urothelial carcinoma include advanced disease at initial diagnosis, Karnofsky performance status <80%, and visceral metastases. The median survival for patients with these unfavorable characteristics is 4 months, while the median survival for patients without these characteristics is 18 months.

[0005] Programmed death ligand 1 (PD-L1) is a protein that is involved in the inhibition of the immune system response during cancer, chronic infection, pregnancy, tissue allotransplantation, and autoimmune diseases. PD-L1 regulates the immune response by binding to an inhibitory receptor called programmed death 1 (PD-1), which is expressed on the surface of T cells, B cells, and monocytes. PD-L1 also negatively regulates T cell function by interacting 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, resulting in the downregulation of T cell activation and the inhibition of anti-tumor immune activity.

[0006] Despite significant progress in the treatment of cancer (e.g., bladder cancer (e.g., UC, including locally advanced or metastatic UC)), improved therapies are still being sought. Summary of the Invention

[0007] The present invention particularly relates to methods of treating bladder cancer (e.g., UC, including locally advanced or metastatic UC) in a subject and compositions (e.g., PD-1 axis-binding antagonists) or pharmaceutical compositions thereof for use in treating bladder cancer (e.g., UC, including locally advanced or metastatic UC) in a subject. Related kits and articles are also provided. Assays for determining the presence or expression level of PD-L1 in a tumor sample obtained from a subject with cancer, methods of labeling PD-L1 in a tumor sample, and methods of stratifying tumors are further provided.

[0008] In one aspect, the present disclosure provides a method of treating locally advanced or metastatic urothelial carcinoma (UC) in a patient in need thereof, wherein the patient has not been previously treated for locally advanced or metastatic UC, the method comprising administering to the patient a treatment regimen comprising an anti-PD-L1 antibody, the anti-PD-L1 antibody comprising the following hypervariable regions (HVRs): (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) an HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) an HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) an HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) an HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8), wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 immunohistochemistry (IHC) assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a combined positive score (CPS) of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody.

[0009] In another aspect, the present disclosure provides a method of treating locally advanced or metastatic UC in a patient in need thereof who has not been previously treated for locally advanced or metastatic UC, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample using a PD-L1 IHC assay that comprises the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay that comprises the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen that comprises an anti-PD-L1 antibody, the anti-PD-L1 antibody comprising the following HVRs: (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) an HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) an HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) an HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) an HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8); and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen that comprises an anti-PD-L1 antibody a treatment regimen that comprises an anti-PD-L1 antibody.

[0010] In another aspect, the present disclosure provides an anti-PD-L1 antibody for use in the treatment of locally advanced or metastatic UC in a patient in need thereof, wherein the patient has not been previously treated for locally advanced or metastatic UC, and the treatment comprises administering to the patient a treatment regimen comprising the anti-PD-L1 antibody, the anti-PD-L1 antibody comprising the following HVRs: (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) an HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) an HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) an HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) an HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8), wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising the anti-PD-L1 antibody.

[0011] In another aspect, the present disclosure provides an anti-PD-L1 antibody for use in a method of treating locally advanced or metastatic UC in a patient in need thereof, wherein the patient has not been previously treated for locally advanced or metastatic UC, and the method comprises: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising the anti-PD-L1 antibody, the anti-PD-L1 antibody comprising the following HVRs: (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4);

[0012] (iii) The HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) The HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) The HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) The HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8); and (b) Administering a treatment regimen comprising an anti-PD-L1 antibody to a patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody.

[0013] In another aspect, the present disclosure provides a method of selecting a therapy for treating locally advanced or metastatic UC in a patient in need thereof, wherein the patient has not been previously treated for locally advanced or metastatic UC, the method comprising: (a) Determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody, the anti-PD-L1 antibody comprising the following HVRs: (i) The HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) The HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4);

[0014] (iii) The HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) The HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) The HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) The HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8); and (b) Selecting a treatment regimen comprising an anti-PD-L1 antibody for a patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody.

[0015] In another aspect, the present disclosure provides a method for identifying a patient with locally advanced or metastatic UC who may benefit from a treatment regimen comprising an anti-PD-L1 antibody, wherein the patient has not been previously treated for locally advanced or metastatic UC, the method comprising: determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a treatment regimen comprising an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody comprises the following HVRs: (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) an HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) an HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) an HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) an HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8).

[0016] In some aspects, the method further comprises administering to the patient a treatment regimen comprising an anti-PD-L1 antibody.

[0017] In some aspects, benefiting from a treatment regimen comprising an anti-PD-L1 antibody is in terms of overall survival (OS).

[0018] In some aspects, compared to treatment with platinum-based chemotherapy without using an anti-PD-L1 antibody, the treatment regimen extends the patient's OS by about 5.7 months to about 17 months.

[0019] In some aspects, compared to treatment with platinum-based chemotherapy without using an anti-PD-L1 antibody, the treatment regimen extends the patient's OS by about 11.3 months.

[0020] In some aspects, the platinum-based chemotherapy comprises a platinum-based chemotherapeutic agent and a nucleoside analogue.

[0021] In some aspects, the platinum-based chemotherapeutic agent is cisplatin, carboplatin, or oxaliplatin.

[0022] In some aspects, the platinum-based chemotherapeutic agent is cisplatin.

[0023] In some aspects, the platinum-based chemotherapeutic agent is carboplatin.

[0024] In some aspects, the nucleoside analogue is gemcitabine.

[0025] In some aspects, the platinum-based chemotherapy includes cisplatin and gemcitabine or carboplatin and gemcitabine.

[0026] In some aspects, the platinum-based chemotherapy includes cisplatin and gemcitabine.

[0027] In some aspects, the platinum-based chemotherapy includes carboplatin and gemcitabine.

[0028] In some aspects, the anti-PD-L1 antibody comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO:9, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO:10.

[0029] In some aspects, the anti-PD-L1 antibody is atezolizumab.

[0030] In some aspects, atezolizumab is administered intravenously to a patient at a dose of about 840 mg every 2 weeks, about 1200 mg every 3 weeks, or about 1680 mg every 4 weeks.

[0031] In some aspects, atezolizumab is administered intravenously to a patient at a dose of about 1200 mg every 3 weeks.

[0032] In some aspects, atezolizumab is administered to a patient in a 21-day dosing cycle, and wherein atezolizumab is administered intravenously to the patient at a dose of about 1200 mg from day -2 to day 4 of each 21-day dosing cycle.

[0033] In some aspects, atezolizumab is administered intravenously to a patient at a dose of about 1200 mg on day 1 of each 21-day dosing cycle.

[0034] In some aspects, the anti-PD-L1 antibody is administered to a patient as a single therapy.

[0035] In other aspects, the anti-PD-L1 antibody is administered to a patient in combination with one or more additional therapeutic agents.

[0036] In some aspects, the one or more additional therapeutic agents include platinum-based chemotherapy.

[0037] In some aspects, the platinum-based chemotherapy includes a platinum-based chemotherapeutic agent and a nucleoside analogue.

[0038] In some aspects, the platinum-based chemotherapeutic agent is cisplatin, carboplatin, or oxaliplatin.

[0039] In some aspects, the platinum-based chemotherapeutic agent is cisplatin.

[0040] In some aspects, the platinum-based chemotherapeutic agent is carboplatin.

[0041] In some aspects, the nucleoside analogue is gemcitabine.

[0042] In some aspects, the platinum-based chemotherapy comprises cisplatin and gemcitabine or carboplatin and gemcitabine.

[0043] In some aspects, the platinum-based chemotherapy comprises cisplatin and gemcitabine.

[0044] In some aspects, the platinum-based chemotherapy comprises carboplatin and gemcitabine.

[0045] In some aspects, each dosing cycle of the platinum-based chemotherapy is about 21 days.

[0046] In some aspects, cisplatin is intravenously administered to the subject at a dose of about 70 mg / m 2 from day -2 to day 4 of each 21-day dosing cycle.

[0047] In some aspects, cisplatin is intravenously administered to the subject at a dose of about 70 mg / m 2 on day 1 of each 21-day dosing cycle.

[0048] In some aspects, carboplatin is intravenously administered to the subject at an area under the curve (AUC) of about 4.5 from day -2 to day 4 of each 21-day dosing cycle.

[0049] In some aspects, carboplatin is intravenously administered to the subject at an AUC of about 4.5 on day 1 of each 21-day dosing cycle.

[0050] In some aspects, gemcitabine is intravenously administered to the subject at a dose of about 1000 mg / m 2 from day -2 to day 4 and on days 7 to 11 of each 21-day dosing cycle.

[0051] In some aspects, gemcitabine is intravenously administered to the subject at a dose of about 1000 mg / m 2 on days 1 and 8 of each 21-day dosing cycle.

[0052] In some aspects, the patient has not received prior chemotherapy for locally advanced or metastatic UC.

[0053] In some aspects, the patient has previously received adjuvant or neoadjuvant chemotherapy or chemoradiotherapy for urothelial carcinoma and has had a treatment-free interval of more than 12 months between the last administration of adjuvant or neoadjuvant chemotherapy or chemoradiotherapy and the date of recurrence.

[0054] In some aspects, locally advanced or metastatic UC is histologically documented, locally advanced (T4b, any N; or any T, N2-3) or metastatic urothelial carcinoma (mUC) (M1, stage IV).

[0055] In some aspects, UC is locally advanced UC.

[0056] In some aspects, locally advanced UC is inoperable.

[0057] In some aspects, UC is metastatic UC.

[0058] In some aspects, the patient is eligible for treatment with platinum-based chemotherapy.

[0059] In some aspects, the patient is eligible for treatment with cisplatin-based chemotherapy.

[0060] In some aspects, the patient is a human.

[0061] In some aspects, the tumor sample obtained from the patient has discernible PD-L1 staining of any intensity in tumor-infiltrating immune cells covering ≥5% of the tumor area occupied by tumor cells, associated intratumoral stroma, and contiguous peritumoral stroma, as determined by a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

[0062] In some aspects, the tumor sample obtained from the patient has a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody.

[0063] In some aspects, the tumor sample obtained from the patient has a CPS of ≥10 using a PD-L1 IHC assay comprising the VENTANA SP263 anti-PD-L1 diagnostic antibody.

[0064] In some aspects, the tumor sample obtained from the patient has a CPS of ≥10 using a PD-L1 IHC assay comprising the 28-8 anti-PD-L1 diagnostic antibody.

[0065] In some aspects, the tumor sample is a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0066] In another aspect, the present disclosure provides an assay for determining the presence or expression level of PD-L1 in a tumor sample obtained from a patient with cancer, the assay comprising: (a) determining the presence or expression level of PD-L1 in a tumor sample obtained from the patient using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) determining the presence or expression level of PD-L1 in a tumor sample obtained from the patient using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody.

[0067] In some aspects, a tumor sample obtained from a patient has a detectable PD-L1 expression level in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

[0068] In some aspects, a tumor sample obtained from a patient has discernible PD-L1 staining of any intensity in tumor-infiltrating immune cells that cover ≥5% of the tumor area occupied by tumor cells, associated intratumoral stroma, and contiguous peritumoral stroma, as determined by a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

[0069] In some aspects, a tumor sample obtained from a patient has a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody.

[0070] In some aspects, a tumor sample obtained from a patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody.

[0071] In some aspects, steps (a) and (b) are performed simultaneously.

[0072] In some aspects, steps (a) and (b) are performed sequentially.

[0073] In some aspects, steps (a) and (b) are performed in different sections of the tumor sample or in the same section of the tumor sample.

[0074] In some aspects, different sections of the tumor sample are consecutive sections.

[0075] In some aspects, the cancer is locally advanced or metastatic urothelial carcinoma.

[0076] In some aspects, the patient has not been previously treated for locally advanced or metastatic urothelial carcinoma.

[0077] In some aspects, the assay is used for (i) selecting a therapy for locally advanced or metastatic UC in patients in need thereof, or (ii) identifying patients with locally advanced or metastatic UC who may benefit from a treatment regimen including an anti-PD-L1 antibody.

[0078] In some aspects, the anti-PD-L1 antibody comprises the following HVRs: (i) the HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) the HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) the HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) the HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) the HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) the HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8).

[0079] In some aspects, the tumor sample is an FFPE tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0080] In another aspect, provided herein is a method of labeling PD-L1 in a tumor sample, the method comprising the steps of: (a) contacting the tumor sample with a VENTANA SP142 anti-PD-L1 diagnostic antibody; (b) contacting the tumor sample with a Dako 22C3 anti-PD-L1 diagnostic antibody, a VENTANA SP263 anti-PD-L1 diagnostic antibody, or a 28-8 anti-PD-L1 diagnostic antibody; and (c) visualizing the anti-PD-L1 diagnostic antibodies of steps (a) and (b) with one or more detectable reagents that generate a detectable signal for the two anti-PD-L1 diagnostic antibodies.

[0081] In some aspects, the detectable signal for the VENTANA SP142 anti-PD-L1 diagnostic antibody is an amplified signal.

[0082] In some aspects, the amplified signal is generated by tyramide signal amplification.

[0083] In some aspects, steps (a) and (b) are performed simultaneously.

[0084] In some aspects, steps (a) and (b) are performed in sequence.

[0085] In some aspects, steps (a) and (b) are performed in different sections of the tumor sample or in the same section of the tumor sample.

[0086] In some aspects, the different sections of the tumor sample are consecutive sections.

[0087] In some aspects, the visualization includes IHC or immunofluorescence (IF).

[0088] In some aspects, the visualization includes IHC.

[0089] In some aspects, the tumor sample is an FFPE tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0090] In some aspects, the tumor sample is obtained from a patient with cancer.

[0091] In some aspects, the cancer is locally advanced or metastatic urothelial carcinoma.

[0092] In some aspects, the patient has not been previously treated for locally advanced or metastatic urothelial carcinoma.

[0093] In another aspect, the present disclosure provides a kit comprising: (a) a VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) a Dako 22C3 anti-PD-L1 diagnostic antibody, a VENTANA SP263 anti-PD-L1 diagnostic antibody, or a 28-8 anti-PD-L1 diagnostic antibody.

[0094] In some aspects, the kit further comprises one or more reagents for visualizing the anti-PD-L1 diagnostic antibodies of (a) and (b).

[0095] In another aspect, the present disclosure provides a method for identifying a tumor that may respond to a PD-1 axis-binding antagonist, the method comprising: (a) staining a first portion of the tumor with an immunoguided PD-L1 assay to obtain a first stained sample; (b) generating a first score by applying a first scoring algorithm to the first stained sample; (c) staining a second portion of the tumor with an immunagnostic PD-L1 assay to obtain a second stained sample; (d) generating a second score by applying a second scoring algorithm to the second stained sample; and (e) comparing the first score with a first cut-off value and the second score with a second cut-off value, wherein the tumor may respond to the PD-1 axis-binding antagonist when both the first score reaches or exceeds the first cut-off value and the second score reaches or exceeds the second cut-off value.

[0096] In some aspects, an immune-guided PD-L1 assay has: (i) at least 80% overall percent agreement (OPA) with the SP142 assay using a first scoring algorithm at a first cut-off value; (ii) at least 80% positive percent agreement (PPA) with the SP142 assay using a first scoring algorithm at a first cut-off value; (iii) at least 80% negative percent agreement (NPA) with the SP142 assay using a first scoring algorithm at a first cut-off value; (iv) at least 80% PPA and at least 80% NPA with the SP142 assay using a first scoring algorithm at a first cut-off value; (v) at least 80% PPA and at least 80% OPA with the SP142 assay using a first scoring algorithm at a first cut-off value; (vi) at least 80% NPA and at least 80% OPA with the SP142 assay using a first scoring algorithm at a first cut-off value; and / or (vii) at least 80% OPA, at least 80% PPA, and at least 80% NPA with the SP142 assay using a first scoring algorithm at a first cut-off value.

[0097] In some aspects, an immune-agnostic PD-L1 assay has: (i) at least 80% overall percent agreement (OPA) with the 22C3 assay using a second scoring algorithm at a second cut-off value; (ii) at least 80% positive percent agreement (PPA) with the 22C3 assay using a second scoring algorithm at a second cut-off value; (iii) at least 80% negative percent agreement (NPA) with the 22C3 assay using a second scoring algorithm at a second cut-off value; (iv) at least 80% PPA and at least 80% NPA with the 22C3 assay using a second scoring algorithm at a second cut-off value; (v) at least 80% PPA and at least 80% OPA with the 22C3 assay using a second scoring algorithm at a second cut-off value; (vi) at least 80% NPA and at least 80% OPA with the 22C3 assay using a second scoring algorithm at a second cut-off value; and / or (vii) at least 80% OPA, at least 80% PPA, and at least 80% NPA with the 22C3 assay using a second scoring algorithm at a second cut-off value.

[0098] In another aspect, provided herein is a method of stratifying tumors having a score above a predetermined cut-off value by an immune-agnostic PD-L1 assay, the method comprising: (a) staining a portion of the tumor with an immune-guided PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (c) comparing the score to a first cut-off value, wherein the tumor is likely to respond to a PD-1 axis binding antagonist when two first scores reach or exceed the first cut-off value.

[0099] In another aspect, the present disclosure provides a method for stratifying tumors with a CPS ≥ 10% as determined by the 22C3 assay, the method comprising: (a) staining a portion of the tumor with an immunoguided PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (c) comparing the score with a first cut-off value and a second score with a second cut-off value, wherein the tumor is likely to respond to a PD-1 axis-binding antagonist when both the first score reaches or exceeds the first cut-off value and the second score reaches or exceeds the second cut-off value.

[0100] In another aspect, the present disclosure provides a method for stratifying tumors with a CPS ≥ 10% as determined by the SP263 assay, the method comprising: (a) staining a portion of the tumor with an immunoguided PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (c) comparing the score with a first cut-off value and a second score with a second cut-off value, wherein the tumor is likely to respond to a PD-1 axis-binding antagonist when both the first score reaches or exceeds the first cut-off value and the second score reaches or exceeds the second cut-off value.

[0101] In another aspect, the present disclosure provides a method for stratifying tumors with a CPS ≥ 10% as determined by the 28-8 assay, the method comprising: (a) staining a portion of the tumor with an immunoguided PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (c) comparing the score with a first cut-off value and a second score with a second cut-off value, wherein the tumor is likely to respond to a PD-1 axis-binding antagonist when both the first score reaches or exceeds the first cut-off value and the second score reaches or exceeds the second cut-off value.

[0102] In some aspects, the tumor is locally advanced or metastatic UC.

[0103] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 is a schematic diagram of the Phase III IMvigor130 study (NCT02807636). mUC, metastatic urothelial carcinoma; R, random; ECOGPS, Eastern Cooperative Oncology Group Performance Status; 1L, first line; platinum, investigator's choice of cisplatin or carboplatin.

[0105] Figure 2 It is a schematic diagram showing that the study design in the IMvigor130 study changed from a two-group design to a three-group design. The bottom figure shows a table summarizing the design changes. Atezo, atezolizumab; carbo, carboplatin; cis, cisplatin; mono, monotherapy; PFS, progression-free survival; OS, overall survival.

[0106] Figure 3 It is a schematic diagram showing the evolution of the IMvigor130 study design. FPI: First patient admitted; LPI: Last patient admitted.

[0107] Figure 4 It is a schematic diagram depicting the hierarchical relationship between study endpoints for type I error control.

[0108] Figures 5A to 5C It shows the use of the anti-PD-L1 antibody SP142 ( Figure 5A ), the anti-DC-LAMP antibody ( Figure 5B ), and the combination of the anti-PD-L1 antibody SP142 and the anti-DC-LAMP antibody ( Figure 5C ) in immunohistochemistry (IHC) results in urothelial carcinoma (UC) tumor tissues. Double labeling from SP142 (brown) and the anti-DC-LAMP antibody (green) is shown ( Figure 5C ).

[0109] Figures 6A to 6C It is a series of graphs showing the inferred population frequency percentages of dendritic cells ( Figure 6A ), CD8+ T cells ( Figure 6B ), and CD4+ T cells ( Figure 6C ) in urothelial carcinoma tissue samples identified as 22C3 and SP142 double-negative (DN), 22C3 only positive (22C3), SP142 positive (SP142), and 22C3 and SP142 double-positive (DP) from bulk RNA sequencing deconvolution data.

[0110] Figure 7A and 7B are a series of graphs showing Kaplan-Meier plots of overall survival (OS) of patients receiving atezolizumab monotherapy comparing PD-L1 IHC assay tumor tissue scores by the SP142 assay ( Figure 7A ) and the 22C3 assay ( Figure 7B ). OS is shown for patients with PD-L1 staining tumor-infiltrating immune cell (IC) scores of IC0 / 1 (red) versus IC2 / 3 (blue) for the SP142 IHC assay ( Figure 7A)。Shows the OS of patients with combined positive score (CPS) < 10 (red) and CPS ≥ 10 (blue) for the 22C3 assay Figure 7B )。CPS, combined positive score; CI, confidence interval; HR, hazard ratio; OS, overall survival; NE, not estimable.

[0111] Figures 8A to 8D is a series of plots of the Kaplan-Meier curves showing the OS of patients receiving atezolizumab monotherapy (Group B; atezolizumab) and placebo plus platinum plus gemcitabine chemotherapy (Group C; chemotherapy) by the SP142 assay Figure 8A and 8C ) and the 22C3 assay Figure 8B and 8D ) PD-L1 IHC assay tumor tissue scores. Shows the OS of patients with an IC score of IC0 / 1 for the SP142 IHC assay receiving atezolizumab monotherapy (red) and chemotherapy (blue) Figure 8A ) and shows the OS of patients with a CPS < 10 for the 22C3 assay receiving atezolizumab monotherapy (red) and chemotherapy (blue) Figure 8B )。Shows the OS of patients with an IC score of IC2 / 3 for the SP142 IHC assay receiving atezolizumab monotherapy (green) and chemotherapy (purple) Figure 8C ) and shows the OS of patients with a CPS ≥ 10 for the 22C3 assay receiving atezolizumab monotherapy (green) and chemotherapy (purple) Figure 8D )。

[0112] Figures 9A to 9D is a series of plots of the Kaplan-Meier curves showing the OS of patients receiving atezolizumab monotherapy (Group B; atezolizumab) and patients receiving chemotherapy not eligible for cisplatin (i.e., placebo plus carboplatin plus gemcitabine) (Group C; chemotherapy). The study groups were scored by PD-L1 IHC assays of the SP142 assay Figure 9A and 9C ) and the 22C3 assay Figure 9B and 9D ) tumor tissue scores. Shows the OS of patients with an IC score of IC0 / 1 for the SP142 IHC assay receiving atezolizumab monotherapy (red) and chemotherapy (blue) Figure 9A ) and shows the OS of patients with a CPS < 10 for the 22C3 assay receiving atezolizumab monotherapy (red) and chemotherapy (blue) Figure 9B)。The OS of patients with an IC score of IC2 / 3 for the SP142 IHC assay receiving atezolizumab monotherapy (green) versus chemotherapy (purple) is shown ( Figure 9C ),and the OS of patients with a CPS ≥ 10 for the 22C3 assay receiving atezolizumab monotherapy (green) versus chemotherapy (purple) is shown ( Figure 9D )。

[0113] Figure 10A And 10B are a series of Kaplan-Meier plots showing the OS of patients receiving atezolizumab monotherapy (Group B; atezolizumab) ( Figure 10A ) and patients receiving placebo plus platinum plus gemcitabine (Group C; chemotherapy) ( Figure 10B ),further shown by double positive staining from the SP142 and 22C3 assays. Within each treatment group, the OS of patients with SP142 IC0 / 1 and 22C3 CPS < 10 (red), SP142 IC0 / 1 and 22C3 CPS ≥ 10 (green), SP142 IC2 / 3 and 22C3 CPS < 10 (blue), and SP142 IC2 / 3 and 22C3 CPS ≥ 10 (purple) receiving atezolizumab monotherapy ( Figure 10A ) and patients receiving placebo plus carboplatin plus gemcitabine ( Figure 10B ) is shown.

[0114] Figure 11 is a schematic diagram showing an exemplary method using the OptiView DAB IHC detection kit. DAB, 3,3'-diaminobenzidine tetrahydrochloride; HQ, a proprietary hapten attached to the goat antibody. Detailed Description

[0115] The present invention provides methods and compositions for the treatment of cancers such as bladder cancer (e.g., UC, including locally advanced or metastatic UC), including patients who have not previously been treated for their cancer. The present invention is at least in part based on the discovery that PD-L1 expression on immune cells, as assessed by the SP142 antibody, co-localizes with dendritic cells and is associated with improved overall survival (OS) in patients with untreated locally advanced or metastatic UC treated with the anti-PD-L1 antibody atezolizumab. Unexpectedly, longer OS was associated with the SP142 IC2 / 3+22C3 CPS≥10 tumor status, while shorter OS was observed in patients with tumor staining for SP142 IC0 / 1+22C3 CPS≥10. Thus, assays and methods for detecting PD-L1 using both an immune-guided PD-L1 AHC assay (e.g., the SP142 IHC assay) and an immune-agnostic PD-L1 AHC assay (e.g., the 22C3, SP263, or 28-8 IHC assays) to analyze tumor samples from cancer patients may be useful, e.g., for identifying patients who may benefit from immune checkpoint inhibitors such as PD-1 axis-binding antagonists (e.g., atezolizumab).

[0116] I. Definitions

[0117] The following abbreviations are used herein:

[0118] AHC Affinity histochemistry HVR Hypervariable region ACC Affinity cytochemistry IHC Immunohistochemistry or immunohistochemical CAS Chemical Abstracts Service ORR Overall response rate / Objective response rate CDR Complementary determining region OS Overall survival CR Complete response PD-1 Programmed death 1 DNA Deoxyribonucleic acid PD-L1 Programmed death ligand 1 DOR Duration of response PD-L2 Programmed death ligand 2 Fab Antigen-binding fragment PFS Progression-free survival Fc Crystallizable fragment PR Partial response FFPE Formalin-fixed paraffin-embedded RNA Ribonucleic acid FR Framework SLD Sum of longest diameters

[0119] The term "PD-1 axis-binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis-binding ligand with one or more of its binding partners to abrogate T cell dysfunction caused by signaling on the PD-1 signaling axis, resulting in the restoration or enhancement of T cell function (e.g., proliferation, cytokine production, and / or target cell killing). As used herein, PD-1 axis-binding antagonists include PD-L1-binding antagonists, PD-1-binding antagonists, and PD-L2-binding antagonists. In some cases, PD-1 axis-binding antagonists include PD-L1-binding antagonists or PD-1-binding antagonists. In a preferred aspect, the PD-1 axis-binding antagonist is a PD-L1-binding antagonist.

[0120] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction generated by the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In some cases, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some cases, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction generated by the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In one case, a PD-L1 binding antagonist reduces negative co-stimulatory signals mediated by signal transduction through PD-L1 mediated by cell surface proteins expressed on T lymphocytes, thereby making dysfunctional T cells less dysfunctional (e.g., enhancing the effector response to antigen recognition). In some cases, a PD-L1 binding antagonist binds to PD-L1. In some cases, a PD-L1 binding antagonist is an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody). Exemplary anti-PD-L1 antagonist antibodies include atezolizumab, MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), SHR-1316, CS1001, envafolimab, TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, cosibelimab, lodapolimab, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, RC98, PDL-GEX, KD036, KY1003, YBL-007, and HS-636. In some aspects, the anti-PD-L1 antibody is atezolizumab, MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab). In a specific aspect, the PD-L1 binding antagonist is MDX-1105. In another specific aspect, the PD-L1 binding antagonist is MEDI4736 (durvalumab). In another specific aspect, the PD-L1 binding antagonist is MSB0010718C (avelumab).In other aspects, the PD-L1 binding antagonist can be a small molecule, for example, GS-4224, INCB086550, MAX-10181, INCB090244, CA-170 or ABSK041, which can be administered orally in some cases. Other exemplary PD-L1 binding antagonists include AVA-004, MT-6035, VXM10, LYN192, GB7003 and JS-003. In a preferred aspect, the PD-L1 binding antagonist is atezolizumab.

[0121] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with the signal transduction generated by the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and / or PD-L2. PD-1 (programmed death 1) is also known in the art as "programmed cell death 1", "PDCD1", "CD279", and "SLEB2". An exemplary human PD-1 is shown in UniProtKB / Swiss-Prot accession number Q15116. In some cases, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies and their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with the signal transduction generated by the interaction of PD-1 with PD-L1 and / or PD-L2. In one case, the PD-1 binding antagonist reduces the negative co-stimulatory signal mediated by signal transduction through PD-1 mediated by a cell surface protein expressed on T lymphocytes, thereby making dysfunctional T cells less dysfunctional (e.g., enhancing the effector response to antigen recognition). In some cases, the PD-1 binding antagonist binds to PD-1. In some cases, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody). Exemplary anti-PD-1 antagonist antibodies include nivolumab, pembrolizumab, MEDI-0680, PDR001 (spartalizumab), REGN2810 (cemiplimab), BGB-108, palorolizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, rivulizumab, sasanlimab, penpulimab, CS1003, HLX10, SCT-I10A, zoliflodacin, btilimumab, genolimab, BI 754091, ciliflimab, YBL-006, BAT1306, HX008, buglizumab, AMG 404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103, and hAb21. In a specific 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 a PD-L2 fusion protein, such as AMP-224. In another specific aspect, the PD-1 binding antagonist is MED1-0680.In another specific aspect, the PD-1 binding antagonist is PDR001 (spartalizumab). In another specific aspect, the PD-1 binding antagonist is REGN2810 (cemiplimab). In another specific aspect, the PD-1 binding antagonist is BGB-108. In another specific aspect, the PD-1 binding antagonist is palorolizumab. In another specific aspect, the PD-1 binding antagonist is camrelizumab. In another specific aspect, the PD-1 binding antagonist is sintilimab. In another specific aspect, the PD-1 binding antagonist is tislelizumab. In another specific aspect, the PD-1 binding antagonist is toripalimab. Other additional exemplary PD-1 binding antagonists include BION-004, CB201, AUNP-012, ADG104, and LBL-006.

[0122] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with the signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). PD-L2 (programmed death ligand 2) is also known in the art as "programmed cell death 1 ligand 2", "PDCD1LG2", "CD273", "B7-DC", "Btdc", and "PDL2". Exemplary human PD-L2 is shown in UniProtKB / Swiss-Prot accession number Q9BQ51. In some cases, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. Exemplary PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with the signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In one aspect, the PD-L2 binding antagonist reduces the negative co-stimulatory signal mediated by or expressed through a cell surface protein expressed on T lymphocytes, and this surface protein makes dysfunctional T cells less dysfunctional through PD-L2-mediated signal transduction (e.g., enhances the effector response to antigen recognition). In some aspects, the PD-L2 binding antagonist binds to PD-L2. In some aspects, the PD-L2 binding antagonist is an immunoadhesin. In other aspects, the PD-L2 binding antagonist is an anti-PD-L2 antagonist antibody.

[0123] The terms "programmed death ligand 1" and "PD-L1" as used herein refer to the native sequence human PD-L1 polypeptide. The native sequence PD-L1 polypeptide is provided under Uniprot accession number Q9NZQ7. For example, the native sequence PD-L1 can have the amino acid sequence as described in Uniprot accession number Q9NZQ7-1 (isoform 1). In another example, the native sequence PD-L1 can have the amino acid sequence as described in Uniprot accession number Q9NZQ7-2 (isoform 2). In yet another example, the native sequence PD-L1 can have the amino acid sequence as described in Uniprot accession number Q9NZQ7-3 (isoform 3). PD-L1 is also known in the art as "programmed cell death 1 ligand 1", "PDCD1LG1", "CD274", "B7-H", and "PDL1".

[0124] When referring to residues in the variable domains (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th ed., U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, Maryland (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported by Kabat et al. supra). The "EU index as described by Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0125] For the purposes of this application, "atezolizumab" is an Fc-engineered, humanized, non-glycosylated IgG1κ immunoglobulin that binds to PD-L1 and comprises the heavy chain sequence of SEQ ID NO:1 and the light chain sequence of SEQ ID NO:2. Atezolizumab contains a single amino acid substitution (asparagine to alanine) (N297A) at position 297 on the heavy chain, using the EU numbering of the amino acid residues of the Fc region, which results in a minimally glycosylated antibody with minimal binding to Fc receptors. Atezolizumab is also described in the WHODrug Information (International Nonproprietary Name (Proposed INN)) list 112, volume 28, number 4, 2014, page 488.

[0126] The term "cancer" refers to a disease caused by the uncontrolled division of abnormal cells in part of the body. Aspects of cancer include solid tumor cancers and non-solid tumor cancers. Examples of cancers 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: bladder cancer (e.g., urothelial carcinoma (UC), including metastatic UC (mUC); muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC)); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; urothelial cancer; breast cancer (e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC), where TNBC refers to being negative for estrogen receptor (ER-), progesterone receptor (PR-), and HER2 (HER2-)); prostate cancer, such as castration-resistant prostate cancer (CRPC); peritoneal cancer; hepatocellular carcinoma; gastric / stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)); glioblastoma; cervical cancer; ovarian cancer; liver cancer (e.g., hepatocellular carcinoma (HCC)); hepatoma; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma, including superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous malignant melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate diffuse NHL; high-grade immunoblastic NHL; high-grade lymphocytic NHL; high-grade small non-cleaved cell NHL; large mass NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloid leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndrome (MDS), as well as abnormal blood vessel proliferation associated with phakomatosis, edema (such as diseases associated with brain tumors), Meigs' syndrome, brain cancer, head and neck cancer, and related metastases. In one case, the cancer is bladder cancer, e.g., urothelial carcinoma (UC) (e.g., locally advanced or metastatic UC). The cancer may be locally advanced or metastatic. In some cases, the cancer is locally advanced. In other cases, the cancer is metastatic.In some cases, cancer may be unresectable (e.g., unresectable locally advanced or metastatic cancer). In some embodiments, the UC is locally advanced UC. In some embodiments, locally advanced UC is inoperable. In some embodiments, the UC is metastatic UC (mUC). In some embodiments, locally advanced or metastatic UC is histologically documented, locally advanced (T4b, any N; or any T, N2-3) or metastatic urothelial carcinoma (mUC) (M1, stage IV).

[0127] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive herein.

[0128] The terms "cell proliferative disease" and "proliferative disease" refer to conditions associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In another embodiment, the cell proliferative disorder is a tumor.

[0129] The term "bladder cancer" includes, but is not limited to, urothelial carcinoma (UC), and it can be, for example, locally advanced or metastatic. The methods described herein are applicable to treating cancers at different stages, including locally advanced and / or metastatic cancers. In cancer staging, locally advanced is generally defined as cancer that has spread from a local area to nearby tissues and / or lymph nodes. In the Roman numeral staging system, locally advanced is generally classified as stage II or III. Metastatic cancer is the stage in which cancer has spread throughout the body to distant tissues and organs (stage IV).

[0130] As used herein, "treatment" includes effective cancer treatment with an effective amount of a therapeutic agent (e.g., a PD-1 axis binding antagonist (e.g., atezolizumab)) or a combination of therapeutic agents (e.g., a PD-1 axis antagonist and one or more additional therapeutic agents, such as platinum-based chemotherapy (e.g., gemcitabine in combination with cisplatin or carboplatin)). Treatment herein particularly includes adjuvant therapy, neoadjuvant therapy, non-metastatic cancer therapy (e.g., locally advanced cancer therapy) and metastatic cancer therapy. Treatment can be first-line treatment (e.g., the patient may not have been treated previously or have not received a prior systemic therapy), or second-line or subsequent treatment. In a particular instance, the treatment can be first-line treatment (e.g., the patient may not have been treated previously for locally advanced or metastatic urothelial carcinoma).

[0131] As used herein, "effective amount" refers to the amount of a therapeutic agent (e.g., a PD-1 axis binding antagonist (e.g., atezolizumab) or a combination of therapeutic agents (e.g., a PD-1 axis antagonist and one or more additional therapeutic agents, such as platinum-based chemotherapy (e.g., gemcitabine in combination with cisplatin or carboplatin))) that achieves a therapeutic effect. In some instances, the effective amount of a therapeutic agent or combination of therapeutic agents is the amount of the agent or combination of agents that achieves a clinical endpoint of improved overall response rate (ORR), complete response (CR), partial response (PR), disease control rate (DCR), improved survival (e.g., progression-free survival (PFS) and / or overall survival (OS)), and / or improved duration of response (DOR). An improvement (e.g., in response rate (e.g., ORR, CR, PR, and / or DCR), survival (e.g., PFS and / or OS), or DOR) may be relative to a suitable reference treatment, which may be, for example, a treatment that does not include a PD-1 axis binding antagonist. For example, the treatment with an anti-cancer therapy comprising an anti-PD-L1 antibody (e.g., atezolizumab) may be compared to a reference treatment, which may be a treatment with platinum-based chemotherapy without the anti-PD-L1 antibody.

[0132] As used herein, "complete response" and "CR" refer to the disappearance of all target lesions.

[0133] As used herein, "partial response" and "PR" refer to a reduction of at least 30% in the sum of the longest diameters (SLD) of target lesions, referenced to the sum of the longest diameters at baseline prior to treatment.

[0134] As used herein, "disease control rate" and "DCR" refer to the proportion of patients with confirmed CR or PR as best response or stable disease (SD). For example, in some embodiments, DCR may be defined as the proportion of patients with confirmed CR or PR as best response or disease stability maintained for ≥6 months according to RECIST v1.1.

[0135] As used herein, "stable disease" or "SD" refers to target lesions that have neither decreased sufficiently to qualify for PR nor increased sufficiently to qualify for PD, referenced to the smallest SLD since the start of treatment.

[0136] As used herein, "progressive disease" or "PD" refers to an increase of at least 20% in the SLD of target lesions, referenced to the smallest SLD recorded since the start of treatment or the appearance of one or more new lesions.

[0137] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment that the disease being treated (e.g., cancer) does not get worse. Progression-free survival can include the amount of time the patient experiences a complete response or a partial response, as well as the amount of time the patient experiences stable disease. In some embodiments, PFS can be defined as the time from randomization or start of treatment until the first documented disease progression as evaluated by RECIST v1.1 or death from any cause, whichever occurs first.

[0138] As used herein, "objective response rate" or "objective response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate. For example, in some embodiments, ORR refers to the proportion of patients who, based on investigator assessment, are observed to have a confirmed objective response (CR or PR) according to RECIST v1.1 in two assessments separated by greater than or equal to 28 days.

[0139] As used herein, "overall survival" and "OS" refer to the length of time the patient remains alive from the date of diagnosis or the start of treatment of the disease (e.g., cancer). In some embodiments, OS is defined as the time from randomization to death from any cause.

[0140] As used herein, the term "duration of response" (DOR) refers to the length of time from the documentation of tumor response until disease progression or death, whichever occurs first.

[0141] As used herein, the terms "inoperable" and "unresectable" are used interchangeably and refer to cancer (e.g., bladder cancer (e.g., UC, including locally advanced or metastatic UC)) for which surgical resection is not possible or cannot be performed safely. In some embodiments, bladder cancer (e.g., UC, including locally advanced or metastatic UC) is inoperable based on involvement of the pelvic sidewall or adjacent viscera (clinical T4b stage) or bulky nodal metastases (N2-N3).

[0142] The term "eligible for platinum-based chemotherapy" means that a subject is eligible for treatment with platinum-based chemotherapy, either according to the judgment of the treating clinician or according to the established criteria for platinum-based chemotherapy known in the art. For example, the criteria proposed by Galsky et al. in Lancet Oncol. 12(3):211-4, 2011 can be used to determine whether a subject is eligible for cisplatin-based chemotherapy. Galsky et al. described a consensus definition of patients with metastatic UC (mUC), in which patients who meet at least one of the following are considered ineligible for cisplatin-based chemotherapy: (i) World Health Organization (WHO) or Eastern Cooperative Oncology Group (ECOG) performance status of 2, or Karnofsky performance status of 60-70%; (ii) creatinine clearance (calculated or measured) less than 1 mL / s; (iii) National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v4.0 ≥ grade 2 hearing loss in audiometry; (iv) CTCAE v.4.0 ≥ grade 2 peripheral neuropathy; and / or New York Heart Association (NYHA) class III heart failure. In one example, patients are considered ineligible for cisplatin-based chemotherapy if they have one or more of the following: impaired renal function (e.g., glomerular filtration rate (GFR) > 30 but < 60 mL / min); GFR can be assessed by direct measurement (i.e., creatinine clearance or ethylenediaminetetraacetate), or, if not available, calculated from serum / plasma creatinine (Cockcroft-Gault formula); hearing loss (e.g., NCI CTCAE v4.0 ≥ grade 2 hearing loss in audiometry at 25 decibels at two consecutive frequencies); peripheral neuropathy (e.g., NCI CTCAE v4.0 ≥ grade 2 peripheral neuropathy (i.e., sensory changes or paresthesia, including tingling)); and / or ECOG performance status assessment (see Oken et al. Am. J. Clin. Oncol. 5:649-655, 1982) (e.g., ECOG performance status of 2).In some embodiments, a subject having one of the following may be eligible for carboplatin-based chemotherapy: impaired renal function (e.g., glomerular filtration rate (GFR) > 30 but < 60 mL / min); GFR can be assessed by direct measurement (i.e., creatinine clearance or ethylenediaminetetraacetate), or, if not available, calculated from serum / plasma creatinine (Cockcroft-Gault formula); hearing loss (e.g., audiometric hearing loss of CTCAE v4.0 ≥ grade 2 at 25 decibels at two consecutive frequencies); peripheral neuropathy (e.g., peripheral neuropathy of NCI CTCAE v4.0 ≥ grade 2 (i.e., sensory change or paresthesia, including tingling)); and / or ECOG performance status assessment (e.g., ECOG performance status is 2).

[0143] As used herein, the term "chemotherapeutic agent" refers to a compound that can be used to treat cancer, such as bladder cancer, e.g., UC (e.g., locally advanced or metastatic UC). Examples of chemotherapeutic agents include EGFR inhibitors (including small molecule inhibitors (e.g., erlotinib ( Genentech / OSI Pharm.); PD 183805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib ( 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluorophenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butanamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butanamide) (Wyeth); AG1478 (Pfizer Inc.); AG1571 (SU 5271; Pfizer Inc.); and dual EGFR / HER2 tyrosine kinase inhibitors, such as lapatinib ( GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[[[2-(methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine); tyrosine kinase inhibitors (e.g., EGFR inhibitors; small molecule HER2 tyrosine kinase inhibitors such as TAK165 (Takeda); CP-724,714, an orally selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors such as EKB-569 (purchased from Wyeth), which preferentially binds to EGFR but inhibits HER2 and EGFR overexpressing cells; PKI-166 (Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as the antisense agent ISIS-5132 (ISIS Pharmaceuticals), which inhibits Raf-1 signaling; non-HER targeted tyrosine kinase inhibitors such as imatinib mesylate( GlaxoSmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib( Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (Pharmacia); quinazolines such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(anilino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (dibenzoylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrosine containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., molecules that bind to nucleic acids encoding HER); quinoxaline (U.S. Patent No. 5,804,396); trypsin (U.S. Patent No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); afinitak (ISIS 3521; Isis / Eli Lilly); PKI 166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (ImClone); and rapamycin (sirolimus, )); proteasome inhibitors such as bortezomib( Millennium Pharmaceuticals); disulfiram; epigallocatechin gallate; salinosporamide A; carfilzomib; 17-AAG (geldanamycin); radicicol; lactate dehydrogenase A (LDH-A); fulvestrant( AstraZeneca); letrozole( Novartis), finasteride( Novartis AG); oxaliplatin( Sanofi); 5-FU (5-fluorouracil); leucovorin; lonafarnib (SCH 66336); sorafenib( Bayer Laboratories); AG1478, alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, amosulbazine, and peat; aziridines such as benzodopa, carboquone, metoprine, and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylamine; acetogenins (especially annonacin and annonacinone); camptothecin (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); microcystins (especially microcystin 1 and microcystin 8); adrenocortical steroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductases (including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mossytatin-dolastatin; aldesleukin, calicheamicin (including synthetic analogs KW-2189 and CB1-TM1); ellipticine; pancratistatin; botulinum; spongistatin; nitrogen mustard drugs such as chlorambucil, clomaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, neocarcinostatin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1 and calicheastatin ω1); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, anthramycin, azaserine, carzinophilin, carubicin, carminomycin, carzinophilin, chromomycin, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolidino-doxorubicin, and daunorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, methylmitomycin, puromycin, quelamycin, rhodomycin, streptothricin, streptozocin, tubercidin, ubenimex, zinostatin, daunorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as demethylfolate, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thioguanine, tioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine; androgens such as caproterone, desogestrel, cyproterone acetate, methenolone, testosterone enanthate; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; acetylacetone;Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; bestrabucil; Bisantrene; Edaravone; defofamine; Demecoxin; Diaziquone; Allyl imine; Lithium acetate; Epothilone; Ethoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Maytansine drugs, such as Maytansine and Ansamitocin; Mitoguazone; Mitoxantrone; Mopidamol; Nitramine; Tebuconazole; Benamecol; Pirarubicin; Loxapytone; Podophyllic acid; 2-Ethyl hydrazide; Procarbazine; Polysaccharide complex (JHS natural product); Razoxane; Rhizoxin; Sizofuran; Geomycin; Tenazolic acid; Triamcinolone acetonide; 2,2',2”-Trichloroethylamine; Cephalotaxenes (especially T-2 toxin, Verrucarin A, AAL toxin A and Anguidine); Urethane; Vinblastine; Dacarbazine; Mannomustine; Dibromomannitol; Dibromodulcitol; Pipobroman; gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Chlorpropamide; (Gemcitabine); 6-Thioguanine; Mercaptopurine; Methotrexate; Etoposide (Vp-16); Ifosfamide; Mitoxantrone; Mitoxantrone; Teniposide; Edatrexate; Daunorubicin; Aminopterin; Capecitabine Ibandronate; CPT-11; Topoisomerase inhibitor RFS2000; Difluoromethylornithine; Retinoids, such as retinoic acid; and pharmaceutically acceptable salts, acids, prodrugs and derivatives of any of the foregoing substances.

[0144] Chemotherapeutic agents also include: (i) antihormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including for example tamoxifen (including tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, raloxifene hydrochloride (keoxifene), LY117018, onapristone and (toremifine citrate); (ii) aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal gland, such as, for example, 4(5)-imidazole, aminoglutethimide, (megestrol acetate), (exemestane; Pfizer), formestanie, fadrozole, (vorozole), (Letrozole; Novartis) and (Anastrozole; AstraZeneca); (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, Premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and zalcitabine (a 1,3-dioxolane nucleoside cytosine analogue); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit gene expression in signal transduction pathways associated with abnormal cell proliferation, such as, for example, PKC-α, Ralf, and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ), and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, e.g., and (ix) growth inhibitors, including vinca alkaloids (e.g., vincristine and vinblastine), (vinorelbine), taxanes (e.g., paclitaxel, albumin-bound paclitaxel, and docetaxel), topoisomerase II inhibitors (e.g., doxorubicin, epirubicin, daunomycin, etoposide, and bleomycin), and DNA alkylating agents (e.g., tamoxigen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C); and (x) pharmaceutically acceptable salts, acids, prodrugs, and derivatives of any of the foregoing.

[0145] As used herein, the term "cytotoxic agent" refers to any agent that is harmful to cells (e.g., causes cell death, inhibits proliferation, or otherwise impedes cell function). Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212and radioactive isotopes of Lu); chemotherapeutic agents; enzymes and fragments thereof, such as nucleases; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. Exemplary cytotoxic agents can be selected from antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, LDH-A inhibitors, fatty acid biosynthesis inhibitors, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors and cancer metabolism inhibitors. In one case, the cytotoxic agent is a platinum-based chemotherapeutic agent (e.g., carboplatin or cisplatin). In one case, the cytotoxic agent is an antagonist of EGFR, e.g., N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g., erlotinib). In one case, the cytotoxic agent is a RAF inhibitor, e.g., a BRAF and / or CRAF inhibitor. In one case, the RAF inhibitor is vemurafenib. In one case, the cytotoxic agent is a PI3K inhibitor.

[0146] Chemotherapeutic agents also include "platinum-based" chemotherapeutic agents, which comprise organic compounds containing platinum as a molecular constituent. Generally, platinum-based chemotherapeutic agents are coordination complexes of platinum. Platinum-based chemotherapeutic agents are sometimes referred to in the art as "platinum drugs". Examples of platinum-based chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, lipoplatin and satraplatin. Platinum-based chemotherapeutic agents (e.g., cisplatin or carboplatin) can be administered in combination with one or more additional chemotherapeutic agents (e.g., nucleoside analogs (e.g., gemcitabine)).

[0147] As used herein, "platinum-based chemotherapy" refers to a chemotherapy regimen that includes a platinum-based chemotherapeutic agent. For example, platinum-based chemotherapy can include a platinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin) in combination with one or more additional chemotherapeutic agents (e.g., nucleoside analogs (e.g., gemcitabine)).

[0148] As used herein, "nucleoside analog" refers to a nucleoside that includes a nucleic acid analog and a sugar. Nucleoside analogs can act as antimetabolites. Exemplary nucleoside analogs include, but are not limited to, gemcitabine, cytarabine, fludarabine and cladribine.

[0149] The term "patient" refers to a human patient. For example, the patient can be an adult.

[0150] As used herein, the term "antibody" specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. In one case, the antibody is a full-length monoclonal antibody.

[0151] As used herein, the term IgG "isotype" or "subclass" refers to any subclass of immunoglobulins defined by the chemical and antigenic characteristics of the immunoglobulin constant region.

[0152] Antibodies (immunoglobulins) can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. Immunoglobulins are mainly divided into five classes: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al., Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody can be part of a larger fusion molecule formed by the covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0153] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein and refer to an antibody in its substantially intact form rather than an antibody fragment as defined below. The term refers to an antibody that includes an Fc region.

[0154] The term "Fc region" as used herein defines the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by a host cell can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain can include the full-length heavy chain, or the antibody can include a cleavage variant of the full-length heavy chain. This can be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447), may or may not be present. Unless otherwise indicated, the amino acid sequence of the heavy chain including the Fc region is represented herein as lacking the C-terminal lysine (Lys447). In one aspect, the heavy chain including the Fc region as specified herein is included in an antibody according to the disclosure herein, the heavy chain including an additional C-terminal glycine-lysine dipeptide (G446 and K447). In one aspect, the heavy chain including the Fc region as specified herein is included in an antibody according to the disclosure herein, the heavy chain including an additional C-terminal glycine residue (G446). In one aspect, the heavy chain including the Fc region as specified herein is included in an antibody according to the disclosure herein, the heavy chain including an additional C-terminal lysine residue (K447). In one embodiment, the Fc region contains a single amino acid substitution of N297A. 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 the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0155] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in a pharmaceutical composition.

[0156] An "antibody fragment" comprises a portion of a full-length antibody, preferably comprising its antigen-binding region. In some instances, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0157] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or arising during the production of a monoclonal antibody preparation, such variants generally being present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies according to the present invention can be prepared by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals that comprise all or part of the human immunoglobulin locus.

[0158] As used herein, the term "hypervariable region" or "HVR" refers to each region within the variable domain of an antibody that is highly variable in sequence and determines antigen-binding specificity, such as "complementary determining regions" ("CDRs").

[0159] Typically, an antibody comprises six CDRs; three in VH (CDR-H1, CDR-H2, CDR-H3), and three in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

[0160] (a) Hypervariable loops occurring at the following amino acid residues: 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0161] (b) CDRs occurring at the following 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)); and

[0162] (c) Antigen contact points that occur at the following amino acid residues: 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2), and 93 to 101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)).

[0163] Unless otherwise specified, CDRs are determined by the method described by Kabat et al., supra. Those skilled in the art will understand that CDR nomenclature can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted numbering system.

[0164] "Framework" or "FR" refers to the variable domain residues other than the complementarity determining regions (CDRs). The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, CDR and FR sequences typically occur in VH (or VL) in the following sequence: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0165] The term "Kabat numbering of variable domain residues" or "Kabat numbering of amino acid positions" and variations thereof refer to the numbering system for the compiled heavy chain variable domain or light chain variable domain of an antibody proposed in the above-mentioned Kabat et al. reference. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FRs or HVRs of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat numbering) and insertion residues after residue 82 of the heavy chain FR (e.g., residues 82a, 82b, and 82c, etc. according to Kabat numbering). The Kabat numbering of residues of a given antibody can be determined by aligning the antibody sequence with the homologous regions of the "standard" Kabat numbering sequence.

[0166] The term "package insert" is used to refer to the instructions for use that are typically included in the commercial packaging of a therapeutic product and that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings related to the use of such therapeutic product.

[0167] As used herein, "in combination with" means that, in addition to administering one mode of treatment, another mode of treatment is also administered. For example, a treatment regimen comprising administering a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atezolizumab) and platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine). Thus, "in combination with" means that another mode of treatment is administered before, during, or after administering one mode of treatment to a patient.

[0168] A drug administered "concurrently" with one or more other drugs is administered within the same treatment cycle, on the same day of treatment with one or more other drugs, and optionally concurrently with one or more other drugs. For example, for a cancer treatment administered every 3 weeks, the concurrently administered drugs are each administered on day 1 of the 3-week cycle.

[0169] The term "detect" includes any means of detection, including direct detection and indirect detection.

[0170] The term "biomarker" as used herein refers to an indicator that can be detected in a sample, such as predictive, diagnostic, and / or prognostic, such as PD-L1. Biomarkers can be used as indicators of specific subtypes of a disease or disorder (e.g., cancer) characterized by certain characteristics, molecular characteristics, pathological characteristics, histological characteristics, and / or clinical characteristics. 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, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and / or glycolipid-based molecular markers.

[0171] The "amount" or "level" of a biomarker associated with an increased clinical benefit to an individual is the level detectable in a biological sample. These can be measured by methods known to those of skill in the art and disclosed herein. The expression level or quantity of the biomarker being evaluated can be used to determine the response to treatment.

[0172] Generally, the terms "level of expression" or "expression level" are used interchangeably and typically refer to the amount of a biomarker in a biological sample. "Expression" generally refers to the process of converting information (e.g., gene-encoded and / or epigenetic information) into structures that are present and operative in a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of the transcribed polynucleotide, translated polypeptide, or 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 degradation of transcripts, or from post-translational processing of a polypeptide (e.g., by proteolysis). "Expressed genes" include those that are transcribed into a polynucleotide such as mRNA and then translated into a polypeptide, as well as those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer RNA and ribosomal RNA).

[0173] "Increased expression", "increased level of expression", "increased level", "elevated expression", "elevated level of expression", or "elevated level" refer to an increase in the expression or level of a biomarker in an individual relative to a control such as one or more individuals not suffering from a disease or disorder (e.g., cancer) or an internal control (e.g., a housekeeping biomarker). In some instances, the control is a reference expression level.

[0174] "Decreased expression", "decreased level of expression", "decreased level", "diminished expression", "diminished level of expression", or "diminished level" refer to a decrease in the expression or level of a biomarker in an individual relative to a control of one or more individuals not suffering from a disease or disorder (e.g., cancer) or an internal control (e.g., a housekeeping biomarker). In some embodiments, the decreased expression is little or no expression. In some instances, the control is a reference expression level.

[0175] The term "housekeeping biomarker" refers to a biomarker or a group of biomarkers (e.g., polynucleotides and / or polypeptides) that are generally similarly present in all cell types. In some embodiments, the housekeeping biomarker is a "housekeeping gene". A "housekeeping gene" as used herein refers to a gene or a group of genes encoding proteins whose activity is essential for maintaining cell function, and housekeeping genes are generally similarly present in all cell types.

[0176] As used herein, the term "diagnosis" is used to refer to the identification or classification of a molecular or pathological state, disease or disorder (e.g., cancer (e.g., bladder cancer (e.g., UC, including locally advanced or metastatic UC))). For example, "diagnosis" can refer to the identification of a specific type of cancer. "Diagnosis" can also refer to the classification of a specific subtype of cancer, e.g., by histopathological criteria or molecular signatures (e.g., a subtype characterized by the expression of a biomarker or combination of biomarkers (e.g., a specific gene or a protein encoded by said gene)).

[0177] As used herein, the term "sample" refers to a composition obtained from or derived from a subject of interest and / or patient, which comprises, for example, cells and / or other molecular entities to be characterized and / or identified based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase "disease sample" and variations thereof refer to any sample obtained from a subject of interest that is expected or known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous 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 media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof. In some instances, the sample is a tumor sample (e.g., a tumor tissue sample).

[0178] A "tissue sample" or "cell sample" refers to a collection of similar cells obtained from the tissue of a subject or individual. The source of the tissue or cell sample can be solid tissue from fresh, frozen, and / or preserved organs, tissue samples, biopsies, and / or aspirates; blood or any blood component, such as plasma; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells at any time during the pregnancy or development of the subject. The tissue sample can also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from diseased tissue / organs. For example, a "tumor sample" is a tissue sample obtained from a tumor (e.g., a bladder tumor, e.g., a UC tumor (e.g., a locally advanced or metastatic UC tumor)) or other cancerous tissue. The tissue sample can contain a mixed population of cell types (e.g., tumor cells and non-tumor cells, cancer cells and non-cancer cells). The tissue sample can contain compounds that are not naturally mixed with the tissue in its natural environment, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. In some instances, the tissue sample is a tumor tissue sample.

[0179] As used herein, "tumor-infiltrating immune cells" refers to any immune cells present in a tumor or a sample thereof. Tumor-infiltrating immune cells include, but are not limited to, intra-tumoral immune cells, peritumoral immune cells, other tumor stromal cells (e.g., fibroblasts), or any combination thereof. Such tumor-infiltrating immune cells can be, for example, T lymphocytes (such as CD8+ T lymphocytes and / or CD4+ T lymphocytes), B lymphocytes, or other myeloid lineage cells, including granulocytes (e.g., neutrophils, eosinophils, and basophils), monocytes, macrophages, dendritic cells (e.g., finger-like dendritic cells), histiocytes, and natural killer cells. In some instances, tumor-infiltrating immune cells can include dendritic cells (e.g., dendritic cells positive for DC-LAMP).

[0180] As used herein, "tumor cells" refers to any tumor cells present in a tumor or a sample thereof. Using methods known in the art and / or described herein, tumor cells can be distinguished from other cells that may be present in a tumor sample, such as stromal cells and tumor-infiltrating immune cells.

[0181] 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 a healthy and / or non-diseased part (e.g., tissue or cell) of the same subject's or individual's body. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue can be healthy and / or non-diseased cells or tissues adjacent to the diseased cells or tissues (e.g., cells or tissues adjacent to a tumor). In another embodiment, the reference sample is obtained from an untreated body tissue and / or cell of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased body part (e.g., tissue or cell) of an individual who is not the subject or the individual being studied. In another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated body tissue and / or cell of an individual who is not the subject or the individual being studied.

[0182] For the purposes of this article, a "section" of a tissue sample refers to an individual part or piece of the tissue sample, e.g., a thin slice of tissue or cells cut from a tissue sample (e.g., a tumor sample). It should be understood that multiple parts of the tissue sample can be obtained and analyzed, provided that it is understood that the same part of the tissue sample can be analyzed at the morphological and molecular levels, or can be analyzed for polypeptides (e.g., by immunohistochemistry) and / or polynucleotides (e.g., by in situ hybridization). In some instances, the section can be a serial section. In other instances, the section can be a non - serial section.

[0183] "Associating" or "correlating" means comparing the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol in any manner. For example, the results of a first analysis or protocol can be used when performing a second protocol, and / or the results of a first analysis or protocol can be used to determine whether a second analysis or protocol should be performed. In an embodiment regarding a polypeptide analysis or protocol, the results of a polypeptide expression analysis or protocol can be used to determine whether a specific treatment protocol should be performed. In an embodiment regarding a polynucleotide analysis or protocol, the results of a polynucleotide expression analysis or protocol can be used to determine whether a specific treatment protocol should be performed.

[0184] The phrase "based on" as used herein means that information regarding one or more biomarkers is used to inform the information provided on treatment decisions, package inserts, or marketing / promotion guides, etc.

[0185] As used herein, the term "Combined Positive Score" or "CPS" refers to, in the context of an AHC assay (e.g., an IHC assay) (e.g., an IHC assay for staining PD - L1 using antibodies SP142, SP263, 22C3, or 28 - 8), the number of PD - L1 stained cells (e.g., tumor cells, lymphocytes, or macrophages) divided by the total number of viable tumor cells, and then multiplied by 100. In one instance, a PD - L1 IHC assay containing the Dako 22C3 anti - PD - L1 diagnostic antibody, e.g., the PD - L1 IHC 22C3 PHARMDX assay (Dako), can be used to calculate the CPS according to the above formula. In other instances, another anti - PD - L1 diagnostic antibody such as SP263 or 28 - 8 can be used to calculate the CPS. In some instances, if the CPS is ≥1 or ≥10, the sample (e.g., a tumor sample) can be considered to have PD - L1 expression. In a specific instance, if the CPS is ≥10, the sample (e.g., a tumor sample) can be considered to have PD - L1 expression.

[0186] As used herein, "PD-L1 positive tumor cell fraction" is, in the context of an AHC assay (e.g., an IHC assay), such as an IHC assay that stains for PD-L1 using an antibody such as SP142, SP263, 22C3, or 28-8, the percentage of viable tumor cells that display partial or complete membranous staining (excluding cytoplasmic staining) at any intensity relative to all viable tumor cells present in a sample after staining the sample. Thus, the PD-L1 IHC SP263 (Ventana) assay can be used, for example, to calculate the PD-L1 positive tumor cell fraction by the formula PD-L1 positive tumor cell fraction = (number of PD-L1 positive tumor cells) / (total number of PD-L1 positive and PD-L1 negative tumor cells), where cytoplasmic staining of PD-L1 in tumor cells and all non-tumor cells (e.g., tumor-infiltrating immune cells, normal cells, necrotic cells, and debris) is excluded from the evaluation and scoring. It should be understood that any given diagnostic PD-L1 antibody can correspond to a specific IHC assay protocol and / or scoring terminology that can be used to derive the PD-L1 positive tumor cell fraction. For example, the PD-L1 positive tumor cell fraction can be derived using Benchmark on detection, EnVisionFlex on AutostainerLink 48, Benchmark on detection and amplification, or EnVision Flex on AutostainerLink 48 from a tumor cell sample stained with SP263, 22C3, SP142, or 28-8. In another example, the PD-L1 positive tumor cell fraction can be calculated using the PD-L1 IHC 22C3 PHARMDX assay (Dako) according to the above formula. As used herein, the terms PD-L1 positive tumor cell fraction and "tumor proportion score" (TPS) are used interchangeably.

[0187] For the purposes of this disclosure, an "anti-PD-L1 diagnostic antibody" refers to an antibody that binds to PD-L1 with sufficient affinity such that the antibody can be used as a diagnostic agent for detecting the presence and / or expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from a patient. In one embodiment, the degree of binding of the anti-PD-L1 diagnostic antibody to an unrelated non-PD-L1 protein is less than about 10% of the degree of binding of the antibody to PD-L1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to PD-L1 has a binding affinity of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, such as 10-8 M to 10 -13 M, such as 10 -9 M to 10 -13 dissociation constant (Kd) of the anti-PD-L1 diagnostic antibody. In certain embodiments, the anti-PD-L1 diagnostic antibody binds to an epitope of PD-L1 that is conserved among PD-L1 from different species. Exemplary anti-PD-L1 diagnostic antibodies include, but are not limited to, SP142 (Ventana), SP263 (Ventana), 22C3 (Dako), 28-8 (Dako), E1L3N (Cell Signaling Technology), 4059 (ProSci, Inc.), h5H1 (Advanced Cell Diagnostics), and 9A11. In some instances, the anti-PD-L1 diagnostic antibody is SP142. In other instances, the anti-PD-L1 diagnostic antibody is SP263, 22C3, or 28-8.

[0188] As used herein, "VENTANA SP142" or "SP142" refers to the anti-PD-L1 diagnostic antibody described in U.S. Patent No. 10,689,445, which is incorporated herein by reference in its entirety. The VENTANA PD-L1 (SP142) assay is commercially available. The heavy and light chain variable region sequences of the SP142 antibody are as follows (the hypervariable sequences HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are shown in underlined and italic text).

[0189] Heavy chain variable region:

[0190]

[0191] Light chain variable region:

[0192]

[0193] As used herein, "VENTANA SP263" or "SP263" refers to the anti-PD-L1 diagnostic antibody described in U.S. Patent No. 10,775,383 and WO 2015 / 181342, which are incorporated herein by reference in their entirety. The VENTANA PD-L1 (SP263) assay is commercially available. The amino acid sequence of the VENTANA SP263 anti-PD-L1 diagnostic antibody is shown, for example, in U.S. Patent No. 10,775,383 (see, for example, Example 1 and Table 1).

[0194] As used herein, "Dako 22C3" or "22C3" refers to a commercially available anti-PD-L1 diagnostic antibody. The Dako 22C3 anti-PD-L1 diagnostic antibody is described in U.S. Patent No. 9,709,568 and WO 2014 / 100079, which are hereby incorporated by reference in their entirety. The PD-L1 IHC 22C3 PHARMDX assay is commercially available (Agilent Dako). The amino acid sequence of the Dako 22C3 anti-PD-L1 diagnostic antibody sequence is shown, for example, in U.S. Patent No. 9,709,568 (see, for example, Figure 2 and 3 and Table 2).

[0195] As used herein, "28-8" refers to a commercially available anti-PD-L1 diagnostic antibody. The 28-8 anti-PD-L1 diagnostic antibody is described in U.S. Patent No. 9,212,224 and WO 2013 / 173223, which are hereby incorporated by reference in their entirety. The PD-L1 IHC 28-8 PHARMDX assay is commercially available (Agilent Dako). The amino acid sequence of the 28-8 anti-PD-L1 diagnostic antibody is shown, for example, in U.S. Patent No. 9,212,224. For example, U.S. Patent No. 9,212,224 describes that the amino acid sequences of the heavy and light chain variable regions of 28-8 are listed in SEQ ID NO:35 and SEQ ID NO:36 of U.S. Patent No. 9,212,224, respectively.

[0196] As used herein, the term "immunoguidance PD-L1 assay" refers to any affinity histochemistry (AHC) assay (e.g., any IHC assay) specific for human PD-L1 protein that has been designed to highlight the immune cell expression of PD-L1, e.g., by preferentially staining immune cells expressing PD-L1 relative to tumor cells expressing PD-L1. The highlighting of immune cells may be the result of: (a) the inherent antibody specificity of immunologically expressed PD-L1 compared to the expression of other cell types; (b) careful selection of staining conditions such as antigen retrieval process, antibody diluent selection, buffer selection, detection system, labeling time and temperature, etc.; or (c) a combination of (a) and (b). An example of a commercially available immunoguidance PD-L1 assay is the VENTANA PD-L1 (SP142) assay ("SP142 assay"). The SP142 assay is an affinity histochemistry assay that uses: (a) a PD-L1 rabbit monoclonal antibody (clone SP142, see U.S. Patent No. 10,689,445); (b) an automated IHC / ISH staining platform (BENCHMARK IHC / ISH staining platform (Roche)); and (c) a tyramide amplification-based 3,3'-diaminobenzidine (DAB) detection system (OPTIVIEW DAB IHC detection kit with OPTIVIEW amplification kit (Roche)). As Figures 5A to 5C shown, the SP142 assay preferentially stains immune cells, particularly dendritic cells, in tumor sections. The SP142 assay is also capable of staining tumor cells, but antibody selection and assay conditions are optimized to emphasize immune cell staining.

[0197] As used herein, the term "immunologically agnostic PD-L1 assay" refers to any affinity histochemistry assay specific for human PD-L1 protein that is not an immunoguidance PD-L1 assay. Exemplary commercially available immunologically agnostic PD-L1 assays include the PD-L1 IHC 22C3 PHARMDX assay (Agilent) ("22C3 assay"), the VENTANA PD-L1 (SP263) assay (Roche) ("SP263 assay"), and the PD-L1 IHC 28-8 PHARMDX assay (Agilent) ("28-8 assay").

[0198] II. Methods and Compositions for the Treatment and Diagnosis of Bladder Cancer

[0199] The present disclosure provides methods for treating bladder cancer (e.g., UC, including locally advanced or metastatic UC) in a subject or delaying its progression, the methods comprising administering to the subject a treatment regimen comprising an effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). The present disclosure also provides methods for treating bladder cancer (e.g., UC, including locally advanced or metastatic UC) in a subject or delaying its progression, the methods comprising administering to the subject a treatment regimen comprising an effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine). In some embodiments, the treatment results in a response in the treated subject. For example, in some embodiments, the treatment increases the likelihood that the subject has an objective response (e.g., a complete response (CR)), prolongs the progression-free survival (PFS) of the subject, prolongs the overall survival (OS) of the subject, and / or prolongs the duration of response (DOR) of the subject, e.g., as compared to a reference treatment (e.g., a treatment that does not use a PD-1 axis-binding antagonist or a treatment that uses platinum-based chemotherapy without using a PD-1 axis-binding antagonist). The present disclosure also provides methods for enhancing the immune function of a subject having bladder cancer (e.g., UC, including locally advanced or metastatic UC), the methods comprising administering to the subject a treatment regimen comprising an effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). The present disclosure further provides methods for enhancing the immune function of a subject having bladder cancer (e.g., UC, including locally advanced or metastatic UC), the methods comprising administering to the subject a treatment regimen comprising an effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine). Also provided are methods for identifying tumors that are likely to respond to a PD-1 axis-binding antagonist. Further provided are methods for patient identification and / or selection, e.g., for treatment with a PD-1 axis-binding antagonist. Further provided are methods for stratifying tumors. Any PD-1 axis-binding antagonist and / or platinum-based chemotherapy known in the art or described herein can be used in the methods.

[0200] In another aspect, provided herein is a method of identifying a tumor that may respond to a PD-1 axis-binding antagonist, the method comprising: (a) staining a first portion of the tumor with an immunoguided PD-L1 assay to obtain a first stained sample; (b) generating a first score by applying a first scoring algorithm to the first stained sample; (c) staining a second portion of the tumor with an immunoagnostic PD-L1 assay to obtain a second stained sample; (d) generating a second score by applying a second scoring algorithm to the second stained sample; and (e) comparing the first score with a first cut-off value and the second score with a second cut-off value, wherein the tumor may respond to the PD-1 axis-binding antagonist when both the first score meets or exceeds the first cut-off value and the second score meets or exceeds the second cut-off value.

[0201] Any suitable first scoring algorithm and / or second scoring algorithm can be used. For example, the first scoring algorithm can be the immune cell scoring algorithm listed in Table 2 herein, e.g., as used in the SP142 assay. In some instances, the second scoring algorithm can be the combined positive score (CPS), e.g., as used in the 22C3 assay. It should be understood that other PD-L1 AHC assays (e.g., other PD-L1 IHC assays) can be used, e.g., including IHC assays using VENTANA SP263 or 28-8 to determine CPS. Other scoring algorithms known in the art for PD-L1 assays, such as TPS, tumor cell percentage (TC), and the tumor cell scoring algorithm listed in Table 3 herein. Descriptions of different exemplary scoring algorithms of PD-L1 assays that can be used are shown in Zajac et al. Diagnostic Pathology. Figure 1 In.

[0202] Any suitable first cut-off value and second cut-off value can be used. For example, in some instances, the first cut-off value is IC ≥ 5%, e.g., as described in Table 2 herein. In some instances, the second cut-off value is CPS ≥ 1 or CPS ≥ 10. In some instances, the second cut-off value is CPS ≥ 10.

[0203] In some aspects, the immune-guided PD-L1 assay has: (i) at least 80% overall percent agreement (OPA) with the SP142 assay using a first scoring algorithm at a first cut-off value; (ii) at least 80% positive percent agreement (PPA) with the SP142 assay using a first scoring algorithm at a first cut-off value; (iii) at least 80% negative percent agreement (NPA) with the SP142 assay using a first scoring algorithm at a first cut-off value; (iv) at least 80% PPA and at least 80% NPA with the SP142 assay using a first scoring algorithm at a first cut-off value; (v) at least 80% PPA and at least 80% OPA with the SP142 assay using a first scoring algorithm at a first cut-off value; (vi) at least 80% NPA and at least 80% OPA with the SP142 assay using a first scoring algorithm at a first cut-off value; and / or (vii) at least 80% OPA, at least 80% PPA, and at least 80% NPA with the SP142 assay using a first scoring algorithm at a first cut-off value.

[0204] In some aspects, the immune-agnostic PD-L1 assay has: (i) at least 80% OPA with the 22C3 assay using a second scoring algorithm at a second cut-off value; (ii) at least 80% PPA with the 22C3 assay using a second scoring algorithm at a second cut-off value; (iii) at least 80% NPA with the 22C3 assay using a second scoring algorithm at a second cut-off value; (iv) at least 80% PPA and at least 80% NPA with the 22C3 assay using a second scoring algorithm at a second cut-off value; (v) at least 80% PPA and at least 80% OPA with the 22C3 assay using a second scoring algorithm at a second cut-off value; (vi) at least 80% NPA and at least 80% OPA with the 22C3 assay using a second scoring algorithm at a second cut-off value; and / or (vii) at least 80% OPA, at least 80% PPA, and at least 80% NPA with the 22C3 assay using a second scoring algorithm at a second cut-off value.

[0205] In another aspect, provided herein is a method of stratifying a tumor having a score above a predetermined cut-off value by an immune-agnostic PD-L1 assay, the method comprising: (a) staining a portion of the tumor with an immune-guided PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (c) comparing the score with a first cut-off value, wherein when two first scores reach or exceed the first cut-off value, the tumor is likely to respond to a PD-1 axis binding antagonist.

[0206] In another aspect, provided herein is a method of stratifying tumors with a CPS ≥ 10% as determined by, for example, the 22C3 assay, the method comprising: (a) staining a portion of the tumor with an immune-guided PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (c) comparing the score to a first cut-off value and a second score to a second cut-off value, wherein the tumor is likely to respond to a PD-1 axis-binding antagonist when both the first score meets or exceeds the first cut-off value and the second score meets or exceeds the second cut-off value.

[0207] In another aspect, provided herein is a method of stratifying tumors with a CPS ≥ 10% as determined by, for example, the SP263 assay, the method comprising: (a) staining a portion of the tumor with an immune-guided PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (c) comparing the score to a first cut-off value and a second score to a second cut-off value, wherein the tumor is likely to respond to a PD-1 axis-binding antagonist when both the first score meets or exceeds the first cut-off value and the second score meets or exceeds the second cut-off value.

[0208] In another aspect, provided herein is a method of stratifying tumors with a CPS ≥ 10% as determined by, for example, the 28-8 assay, the method comprising: (a) staining a portion of the tumor with an immune-guided PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (c) comparing the score to a first cut-off value and a second score to a second cut-off value, wherein the tumor is likely to respond to a PD-1 axis-binding antagonist when both the first score meets or exceeds the first cut-off value and the second score meets or exceeds the second cut-off value.

[0209] Any suitable scoring algorithm can be used. For example, the scoring algorithm can be the immune cell scoring algorithm listed in Table 2 herein, such as that used in the SP142 assay. In some instances, the scoring algorithm can be the combined positive score (CPS), such as that used in the 22C3 assay. It should be understood that other PD-L1 AHC assays (e.g., other PD-L1 IHC assays) can be used, such as IHC assays including those using VENTANA SP263 or 28-8 to determine CPS. Other scoring algorithms known in the art for PD-L1 assays, such as TPS, tumor cell percentage (TC), and the tumor cell scoring algorithm listed in Table 3 herein. Descriptions of different exemplary scoring algorithms for PD-L1 assays that can be used are shown in Zajac et al. Diagnostic Pathology. Figure 1In.

[0210] Any suitable cut-off value can be used. For example, in some instances, the cut-off value is IC ≥ 5%, such as, for example, as described in Table 2 herein (e.g., IC2 / 3). In some instances, the cut-off value is CPS ≥ 1 or CPS ≥ 10. In some instances, the cut-off value is CPS ≥ 10.

[0211] The tumor can be any suitable cancer type (e.g., bladder cancer (e.g., UC, including metastatic UC (mUC); muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC)); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; urothelial cancer; breast cancer (e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC), which is negative for estrogen receptor (ER-), progesterone receptor (PR-), and HER2 (HER2-)); prostate cancer, such as castration-resistant prostate cancer (CRPC); peritoneal cancer; hepatocellular carcinoma; gastric / stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)); glioblastoma; cervical cancer; ovarian cancer; liver cancer (e.g., hepatocellular carcinoma (HCC)); hepatoma; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma, including superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate diffuse NHL; high-grade immunoblastic NHL; high-grade lymphocytic NHL; high-grade small non-cleaved cell NHL; large mass NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloid leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndrome (MDS), as well as abnormal angiogenesis associated with phakomatosis, edema (such as diseases associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and related metastases). In some aspects, the tumor is bladder cancer, such as locally advanced or metastatic UC.

[0212] In another aspect, the present disclosure provides a method of treating a patient having cancer that has been identified or stratified according to any of the foregoing methods, the method comprising administering to the patient a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)).

[0213] In one example, the present disclosure provides a method of treating cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)) in a patient in need thereof, the method comprising administering to the patient a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), wherein a tumor sample obtained from the patient has been determined to have: a detectable level of PD-L1 expression in tumor-infiltrating immune cells using an immune-guided PD-L1 assay; and a detectable level of PD-L1 expression using an immune-agnostic PD-L1 assay, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0214] In another example, the present disclosure provides a method of treating cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)) in a patient in need thereof, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable level of PD-L1 expression in tumor-infiltrating immune cells using an immune-guided PD-L1 assay; and a detectable level of PD-L1 expression using an immune-agnostic PD-L1 assay, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist a treatment regimen comprising a PD-1 axis-binding antagonist.

[0215] In one example, the present disclosure provides a method of enhancing the immune function of a patient having cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)), the method comprising administering to the patient a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), wherein a tumor sample obtained from the patient has been determined to have: a detectable level of PD-L1 expression in tumor-infiltrating immune cells using an immune-guided PD-L1 assay; and a detectable level of PD-L1 expression using an immune-agnostic PD-L1 assay, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0216] In another instance, provided herein is a method of enhancing the immune function of a patient having cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)), the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells using an immune-guided PD-L1 assay; and a detectable PD-L1 expression level using an immune-agnostic PD-L1 assay, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist a treatment regimen comprising a PD-1 axis-binding antagonist.

[0217] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for treating cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)) in a patient in need thereof, the treatment comprising administering to the patient a treatment regimen comprising a PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells using an immune-guided PD-L1 assay; and a detectable PD-L1 expression level using an immune-agnostic PD-L1 assay, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0218] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) in a method for treating cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)) in a patient in need thereof, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells using an immune-guided PD-L1 assay; and a detectable PD-L1 expression level using an immune-agnostic PD-L1 assay, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist; and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist a treatment regimen comprising an anti-PD-L1 antibody.

[0219] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for enhancing the immune function of a patient having cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)), the treatment comprising administering to the patient a treatment regimen comprising the PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells using an immune-guided PD-L1 assay; and a detectable PD-L1 expression level using an immune-agnostic PD-L1 assay, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising the PD-1 axis-binding antagonist.

[0220] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) in a method for enhancing the immune function of a patient having cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)), the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells using an immune-guided PD-L1 assay; and a detectable PD-L1 expression level using an immune-agnostic PD-L1 assay, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising the PD-1 axis-binding antagonist; and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising the PD-1 axis-binding antagonist a treatment regimen comprising the anti-PD-L1 antibody.

[0221] In another instance, provided herein is a method of selecting a therapy for treating cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)) in a patient in need thereof, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells using an immune-guided PD-L1 assay; and a detectable PD-L1 expression level using an immune-agnostic PD-L1 assay, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) selecting for the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising the PD-1 axis-binding antagonist a treatment regimen comprising the PD-1 axis-binding antagonist.

[0222] In another example, provided herein is a method of identifying a patient having cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)) who may benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the method comprising: determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells using an immune-guided PD-L1 assay; and a detectable PD-L1 expression level using an immune-agnostic PD-L1 assay, thereby identifying the patient as a patient who may benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0223] In some examples, the immune-guided PD-L1 assay is an AHC assay (e.g., an IHC assay).

[0224] In some examples, the immune-agnostic PD-L1 assay is an AHC assay (e.g., an IHC assay).

[0225] The cancer can be any suitable type of cancer (e.g., bladder cancer (e.g., UC, including mUC; MIBC and NMIBC); kidney or renal cancer (e.g., RCC); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; urothelial cancer; breast cancer (e.g., HER2+ breast cancer and TNBC, which is ER-, PR-, and HER2 HER2-); prostate cancer, such as CRPC; peritoneal cancer; hepatocellular carcinoma; gastric / stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., PDAC); glioblastoma; cervical cancer; ovarian cancer; liver cancer (e.g., HCC); hepatoma; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma, including superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, and nodular melanoma; multiple myeloma and B cell lymphoma (including low-grade / follicular NHL; SL NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; large mass NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia); CLL; ALL; AML; hairy cell leukemia; CML; PTLD; and MDS, as well as abnormal angiogenesis associated with phakomatosis, edema (such as diseases associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and related metastases). In some examples, the cancer is bladder cancer (e.g., locally advanced or metastatic UC).

[0226] In one example, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, the method comprising administering to the patient a treatment regimen comprising a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells using a PD-L1 immunohistochemistry (IHC) assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a detectable PD-L1 expression level using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist.

[0227] In another example, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a detectable PD-L1 expression level using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist a treatment regimen comprising a PD-1 axis binding antagonist.

[0228] In one example, provided herein is a method of enhancing immune function in a patient having bladder cancer (e.g., locally advanced or metastatic UC), the method comprising administering to the patient a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a detectable PD-L1 expression level using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0229] In another example, provided herein is a method of enhancing immune function in a patient having bladder cancer (e.g., locally advanced or metastatic UC), the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a detectable PD-L1 expression level using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist a treatment regimen comprising a PD-1 axis-binding antagonist.

[0230] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, the treatment comprising administering to the patient a treatment regimen comprising the PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a detectable PD-L1 expression level using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0231] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) in a method for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a detectable PD-L1 expression level using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist; and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist a treatment regimen comprising an anti-PD-L1 antibody.

[0232] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for enhancing the immune function of a patient with bladder cancer (e.g., locally advanced or metastatic UC), the treatment comprising administering to the patient a treatment regimen comprising the PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have: a detectable level of PD-L1 expression in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a detectable level of PD-L1 expression using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0233] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) in a method for enhancing the immune function of a patient with bladder cancer (e.g., locally advanced or metastatic UC), the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable level of PD-L1 expression in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a detectable level of PD-L1 expression using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist; and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist a treatment regimen comprising an anti-PD-L1 antibody.

[0234] In another instance, provided herein is a method of selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a detectable PD-L1 expression level using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) selecting, for the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist, a treatment regimen comprising a PD-1 axis-binding antagonist.

[0235] In another instance, provided herein is a method of identifying a patient with bladder cancer (e.g., locally advanced or metastatic UC) who is likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the method comprising: determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a detectable PD-L1 expression level using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0236] In some instances, the patient has not been previously treated for bladder cancer.

[0237] Any suitable reference expression level or cut-off value for the presence or expression level of PD-L1 can be utilized, such as any of the reference expression levels or cut-off values described in Section IV below.

[0238] In some instances, the patient has a detectable PD-L1 expression level in 5% or more of tumor-infiltrating immune cells in the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

[0239] In some instances, the patient has a CPS of ≥10 using a PD-L1 IHC assay that includes the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody.

[0240] In some instances, the patient has a detectable PD-L1 expression level in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample using a PD-L1 IHC assay that includes the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay that includes the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody.

[0241] In some instances, the presence and / or expression level of PD-L1 in a tumor sample identifies the patient as a patient who may benefit from a treatment regimen that includes a PD-1 axis-binding antagonist.

[0242] In one instance, provided herein is a method of treating a patient in need thereof for bladder cancer (e.g., locally advanced or metastatic UC), the method comprising administering to the patient a treatment regimen that includes a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample using a PD-L1 IHC assay that includes the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay that includes the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a treatment regimen that includes a PD-1 axis-binding antagonist.

[0243] In another instance, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample using a PD-L1 IHC assay that comprises the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay that comprises the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen that comprises a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen that comprises a PD-1 axis-binding antagonist a treatment regimen that comprises a PD-1 axis-binding antagonist.

[0244] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, the treatment comprising administering to the patient a treatment regimen that comprises a PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample using a PD-L1 IHC assay that comprises the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay that comprises the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen that comprises a PD-1 axis-binding antagonist.

[0245] In another instance, provided herein is a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) in a method for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist; and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist a treatment regimen comprising an anti-PD-L1 antibody.

[0246] In another instance, provided herein is a method of selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) selecting for the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis binding antagonist a treatment regimen comprising a PD-1 axis binding antagonist.

[0247] In another example, provided herein is a method of identifying a patient with bladder cancer (e.g., locally advanced or metastatic UC) who may benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the method comprising: determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0248] In some examples, the patient has not been previously treated for bladder cancer.

[0249] In one example, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, wherein the patient has not been previously treated for bladder cancer, the method comprising administering to the patient a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0250] In another instance, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, wherein the patient has not been previously treated for bladder cancer, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist a treatment regimen comprising a PD-1 axis-binding antagonist.

[0251] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, wherein the patient has not been previously treated for bladder cancer, the treatment comprising administering to the patient a treatment regimen comprising a PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0252] In another instance, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) in a method for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, wherein the patient has not been previously treated for bladder cancer, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist; and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist a treatment regimen comprising an anti-PD-L1 antibody.

[0253] In another instance, provided herein is a method of selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, wherein the patient has not been previously treated for bladder cancer, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) selecting for the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist a treatment regimen comprising a PD-1 axis-binding antagonist.

[0254] In another example, provided herein is a method of identifying a patient with bladder cancer (e.g., locally advanced or metastatic UC) who may benefit from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), wherein the patient has not been previously treated for bladder cancer, the method comprising: determining that a tumor sample obtained from the patient has: a detectable level of PD-L1 expression in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

[0255] In some examples, the method further comprises administering to the patient a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)).

[0256] Any suitable PD-1 axis-binding antagonist can be used. In some embodiments, PD-1 axis-binding antagonists are described in Section VI below. Other PD-1 axis-binding antagonists are known in the art. In some embodiments, the PD-1 axis-binding antagonist is selected from the group consisting of a PD-L1-binding antagonist, a PD-1-binding antagonist, and a PD-L2-binding antagonist.

[0257] For example, in some examples, the PD-1 axis-binding antagonist is an anti-PD-L1 antibody comprising the following HVRs: (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) an HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) an HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) an HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) an HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8).

[0258] In one instance, provided herein is a method of treating locally advanced or metastatic UC in a patient in need thereof, where the patient has not been previously treated for locally advanced or metastatic UC, the method comprising administering to the patient a treatment regimen comprising an anti-PD-L1 antibody, the anti-PD-L1 antibody comprising the following hypervariable regions (HVRs): (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) an HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) an HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) an HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) an HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8), wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 immunohistochemistry (IHC) assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who would benefit from a treatment regimen comprising an anti-PD-L1 antibody.

[0259] In another instance, provided herein is a method of treating locally advanced or metastatic UC in a patient in need thereof, wherein the patient has not been previously treated for locally advanced or metastatic UC, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody, the anti-PD-L1 antibody comprising the following HVRs: (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) an HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) an HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) an HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) an HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8); and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody a treatment regimen comprising an anti-PD-L1 antibody.

[0260] In another instance, provided herein is a method of selecting a therapy for treating locally advanced or metastatic UC in a patient in need thereof, wherein the patient has not been previously treated for locally advanced or metastatic UC, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody, the anti-PD-L1 antibody comprising the following HVRs: (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4);

[0261] (iii) The HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) The HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) The HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) The HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8); and (b) Selecting a treatment regimen comprising an anti-PD-L1 antibody for a patient identified in step (a) as a patient who may benefit from a treatment regimen comprising an anti-PD-L1 antibody.

[0262] In another example, the present disclosure provides a method for identifying a patient with locally advanced or metastatic UC who may benefit from a treatment regimen comprising an anti-PD-L1 antibody, wherein the patient has not been previously treated for locally advanced or metastatic UC, the method comprising: determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a treatment regimen comprising an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody comprises the following HVRs: (i) The HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) The HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) The HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) The HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) The HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) The HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8).

[0263] In some examples, the method further comprises administering to the patient a treatment regimen comprising an anti-PD-L1 antibody.

[0264] In some examples, the anti-PD-L1 antibody is atezolizumab.

[0265] In one instance, provided herein is a method of treating locally advanced or metastatic UC in a patient in need thereof, wherein the patient has not been previously treated for locally advanced or metastatic UC, the method comprising administering to the patient a treatment regimen comprising atezolizumab, wherein a tumor sample obtained from the patient has been determined to have: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 immunohistochemistry (IHC) assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising atezolizumab.

[0266] In another instance, provided herein is a method of treating locally advanced or metastatic UC in a patient in need thereof, wherein the patient has not been previously treated for locally advanced or metastatic UC, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising atezolizumab; and (b) administering to the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising atezolizumab a treatment regimen comprising atezolizumab.

[0267] In another example, provided herein is a method of selecting a therapy for treating locally advanced or metastatic UC in a patient in need thereof, where the patient has not been previously treated for locally advanced or metastatic UC, the method comprising: (a) determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising atezolizumab; and (b) selecting, for the patient identified in step (a) as a patient likely to benefit from a treatment regimen comprising atezolizumab, a treatment regimen comprising an anti-PD-L1 antibody.

[0268] In another example, provided herein is a method of identifying a patient with locally advanced or metastatic UC who is likely to benefit from a treatment regimen comprising atezolizumab, where the patient has not been previously treated for locally advanced or metastatic UC, the method comprising: determining that a tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient likely to benefit from a treatment regimen comprising atezolizumab.

[0269] In some examples, the method further comprises administering to the patient a treatment regimen comprising atezolizumab.

[0270] Benefiting from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) can be, for example, in terms of overall survival (OS), progression-free survival (PFS), objective response rate (ORR), complete response (CR) rate, and / or duration of response (DOR). Benefiting from a treatment regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) can be compared to a suitable reference treatment (e.g., a treatment using platinum-based chemotherapy without an anti-PD-L1 antibody).

[0271] For example, in some embodiments, compared to treatment with platinum-based chemotherapy without a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the treatment regimen increases the likelihood that a subject has an objective response (e.g., CR), prolongs the PFS of the subject, prolongs the OS of the subject, and / or prolongs the DOR of the subject. In some embodiments, compared to treatment with platinum-based chemotherapy without a PD-1 axis-binding antagonist, the treatment regimen increases the likelihood that a subject has an objective response. In some embodiments, compared to treatment with platinum-based chemotherapy without a PD-1 axis-binding antagonist, the treatment regimen increases the likelihood that a subject has a CR. In some embodiments, compared to treatment with platinum-based chemotherapy without a PD-1 axis-binding antagonist, the treatment regimen prolongs the PFS of the subject. In some embodiments, compared to treatment with platinum-based chemotherapy without a PD-1 axis-binding antagonist, the treatment regimen prolongs the OS of the subject. In some embodiments, compared to treatment with platinum-based chemotherapy without a PD-1 axis-binding antagonist, the treatment regimen prolongs the DOR of the subject.

[0272] In some instances, a benefit of a treatment regimen that includes a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is in terms of OS.

[0273] In some instances, compared to treatment with platinum-based chemotherapy without a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the treatment regimen prolongs the OS of the patient by about 1 month to about 35 months (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 months). In some instances, compared to treatment with platinum-based chemotherapy without a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the treatment regimen prolongs the OS of the patient by about 5.7 months to about 17 months. In some specific instances, compared to treatment with platinum-based chemotherapy without a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the treatment regimen prolongs the OS of the patient by about 11.3 months.

[0274] In some instances, platinum-based chemotherapy includes a platinum-based chemotherapeutic agent and a nucleoside analogue.

[0275] In some instances, the platinum-based chemotherapeutic agent is cisplatin, carboplatin, or oxaliplatin.

[0276] In some instances, the platinum-based chemotherapeutic agent is cisplatin.

[0277] In some instances, the platinum-based chemotherapeutic agent is carboplatin.

[0278] In some instances, the nucleoside analogue is gemcitabine.

[0279] In some instances, platinum-based chemotherapy includes cisplatin and gemcitabine or carboplatin and gemcitabine.

[0280] In some instances, platinum-based chemotherapy includes cisplatin and gemcitabine.

[0281] In some instances, platinum-based chemotherapy includes carboplatin and gemcitabine.

[0282] In some instances, the anti-PD-L1 antibody comprises: (a) a VH domain that comprises the amino acid sequence of SEQ ID NO:9, and (b) a VL domain that comprises the amino acid sequence of SEQ ID NO:10.

[0283] In some instances, the anti-PD-L1 antibody is atezolizumab.

[0284] In some embodiments, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is administered as a single therapy.

[0285] In other embodiments, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is administered in combination with an effective amount of one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are selected from anti-tumor agents, chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, radiotherapy, or cytotoxic agents. In some embodiments, the one or more additional therapeutic agents are platinum-based chemotherapy. In some embodiments, treatment without using a PD-1 axis-binding antagonist includes treatment using platinum-based chemotherapy.

[0286] Any suitable platinum-based chemotherapy can be used, including any platinum-based chemotherapy known in the art or described herein (e.g., in Section VII below). In some embodiments, platinum-based chemotherapy comprises a platinum-based chemotherapeutic agent and a nucleoside analogue. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin, carboplatin, or oxaliplatin.

[0287] For example, in some embodiments, the platinum-based chemotherapeutic agent is cisplatin. Any suitable cisplatin dosing regimen known in the art can be used. In some embodiments, cisplatin is administered to a subject in a 21-day dosing cycle. In some embodiments, cisplatin is administered intravenously to a subject at a dose of about 35 mg / m 2 to about 140 mg / m 2 In some embodiments, cisplatin is administered intravenously to a subject at a dose of about 70 mg / m 2Cisplatin is administered intravenously to the subject at a dose of. In some embodiments, cisplatin is administered intravenously to the subject at a dose of about 70 mg / m² from day - 2 to day 4 of each 21 - day dosing cycle. 2 Cisplatin is administered intravenously to the subject at a dose of. In some embodiments, cisplatin is administered intravenously to the subject at a dose of about 70 mg / m² on day 1 of each 21 - day dosing cycle. 2 Cisplatin is administered intravenously to the subject at a dose of.

[0288] In another example, in other embodiments, the platinum - based chemotherapeutic agent is carboplatin. Any suitable carboplatin dosing regimen known in the art can be used. In some embodiments, carboplatin is administered to the subject in a 21 - day dosing cycle. In some embodiments, carboplatin is administered intravenously to the subject at an area under the curve (AUC) of about 2 to about 9. In some embodiments, carboplatin is administered intravenously to the subject at an AUC of about 4.5. In some embodiments, carboplatin is administered intravenously to the subject at an AUC of about 4.5 from day - 2 to day 4 of each 21 - day dosing cycle. In some embodiments, carboplatin is administered intravenously to the subject at an AUC of about 4.5 on day 1 of each 21 - day dosing cycle.

[0289] In any of the foregoing examples, platinum - based chemotherapy may include nucleoside analogs. Any suitable nucleoside analog can be used, including any nucleoside analog known in the art or described herein (e.g., in Section VII below). Any suitable gemcitabine dosing regimen known in the art can be used. In some embodiments, the nucleoside analog is gemcitabine. In some embodiments, gemcitabine is administered to the subject in a 21 - day dosing cycle. In some embodiments, gemcitabine is administered intravenously to the subject at a dose of about 500 mg / m² 2 to about 2000 mg / m² 2 In some embodiments, gemcitabine is administered intravenously to the subject at a dose of about 1000 mg / m² 2 In some embodiments, gemcitabine is administered intravenously to the subject at a dose of about 1000 mg / m² from day - 2 to day 4 and on days 7 to 11 of each 21 - day dosing cycle. 2 In some embodiments, gemcitabine is administered intravenously to the subject at a dose of about 1000 mg / m² on days 1 and 8 of each 21 - day dosing cycle. 2 In some embodiments, gemcitabine is administered intravenously to the subject at a dose of about 1000 mg / m².

[0290] In any of the foregoing examples, platinum - based chemotherapy may include cisplatin and gemcitabine. In other examples, platinum - based chemotherapy may include carboplatin and gemcitabine.

[0291] In some examples, the patient has not received prior chemotherapy for locally advanced or metastatic UC.

[0292] In some instances, the patient has previously received adjuvant or neoadjuvant chemotherapy or chemoradiation for urothelial carcinoma, and has had a treatment-free interval of greater than 12 months between the last administration of adjuvant or neoadjuvant chemotherapy or chemoradiation and the date of recurrence.

[0293] In some instances, locally advanced or metastatic UC is histologically documented, locally advanced (T4b, any N; or any T, N2-3) or metastatic urothelial carcinoma (mUC) (M1, stage IV).

[0294] In some instances, UC is locally advanced UC.

[0295] In some instances, locally advanced UC is inoperable.

[0296] In some instances, UC is metastatic UC.

[0297] In some instances, the patient may be eligible for any suitable platinum-based chemotherapy. Eligibility for platinum-based chemotherapy can be as described herein or according to criteria known in the art. For example, the criteria for defining patients eligible or ineligible for cisplatin are known in the art, e.g., as described in Galsky et al., Lancet Oncol. 12:211-4, 2011, the entire text of which is incorporated herein by reference. In some embodiments, the subject is eligible for treatment with a platinum-based chemotherapy comprising cisplatin. In some embodiments, the subject is eligible for treatment with a platinum-based chemotherapy comprising carboplatin.

[0298] In other instances, the patient may not be eligible for platinum-based chemotherapy. In some embodiments, the subject is ineligible for treatment with a platinum-based chemotherapy comprising cisplatin. In some embodiments, the subject is ineligible for treatment with a platinum-based chemotherapy comprising carboplatin.

[0299] In some instances, the patient is a human.

[0300] In some instances, any intensity of discernible PD-L1 staining is present in tumor-infiltrating immune cells in a tumor sample obtained from the patient that covers ≥5% of the tumor area occupied by tumor cells, associated intratumoral stroma, and contiguous peritumoral stroma, as determined by a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

[0301] In some instances, the tumor sample obtained from the patient has a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody.

[0302] In some instances, a tumor sample obtained from a patient has a CPS of ≥10 using a PD-L1 IHC assay that includes the VENTANA SP263 anti-PD-L1 diagnostic antibody.

[0303] In some instances, a tumor sample obtained from a patient has a CPS of ≥10 using a PD-L1 IHC assay that includes the 28-8 anti-PD-L1 diagnostic antibody.

[0304] In any of the foregoing instances, a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atezolizumab) can be administered in one or more dosing cycles.

[0305] In any of the foregoing examples, each dosing cycle can have any suitable length, e.g., about 7 days, about 14 days, about 21 days, about 28 days or longer. In some embodiments, each dosing cycle is about 21 days.

[0306] Any suitable number of dosing cycles can be used, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or more dosing cycles. In some embodiments, 10 or fewer dosing cycles can be used. In some embodiments, 20 or fewer dosing cycles are used. In some embodiments, 25 or fewer dosing cycles are used.

[0307] In some embodiments, the tumor sample is a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample. The presence and / or expression level of any of the biomarkers described herein (e.g., PD-L1) can be determined using any of the methods described herein (e.g., in Section IV or Example 2 below) or using methods known in the art.

[0308] As a general proposition, a therapeutically effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) administered to a human will be in the range of about 0.01 to about 50 mg / kg of patient body weight, whether administered in one or multiple administrations. In some embodiments, for example, the antagonist (e.g., a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab))) is administered at a dose of about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg, e.g., daily, weekly, bi-weekly, tri-weekly, or every four weeks. In some embodiments, the antagonist (e.g., a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab))) is administered at a dose of 15 mg / kg. However, other dosing regimens may be available. In one embodiment, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, or about 1800 mg. In some embodiments, the antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) may be administered at a dose of about 1000 mg to about 1400 mg every three weeks (e.g., about 1100 mg to about 1300 mg every three weeks, e.g., about 1150 mg to about 1250 mg every three weeks). In some embodiments, the antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is administered intravenously to a subject at a dose of about 840 mg every two weeks, about 1200 mg every three weeks, or about 1680 mg every four weeks. In some embodiments, the antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is administered at a dose of about 1200 mg of atezolizumab every three weeks. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as by infusion. The dose of the antibody in combination therapy can be reduced compared to single therapy. In some embodiments, the treatment regimen includes administering about 1200 mg of atezolizumab intravenously to a subject every three weeks. The progress of the therapy can be readily monitored by conventional techniques.

[0309] In some cases, a total of 1 to 50 doses of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) are administered to a patient, e.g., 1 to 50 doses, 1 to 45 doses, 1 to 40 doses, 1 to 35 doses, 1 to 30 doses, 1 to 25 doses, 1 to 20 doses, 1 to 15 doses, 1 to 10 doses, 1 to 5 doses, 2 to 50 doses, 2 to 45 doses, 2 to 40 doses, 2 to 35 doses, 2 to 30 doses, 2 to 25 doses, 2 to 20 doses, 2 to 15 doses, 2 to 10 doses, 2 to 5 doses, 3 to 50 doses, 3 to 45 doses, 3 to 40 doses, 3 to 35 doses, 3 to 30 doses, 3 to 25 doses, 3 to 20 doses, 3 to 15 doses, 3 to 10 doses, 3 to 5 doses, 4 to 50 doses, 4 to 45 doses, 4 to 40 doses, 4 to 35 doses, 4 to 30 doses, 4 to 25 doses, 4 to 20 doses, 4 to 15 doses, 4 to 10 doses, 4 to 5 doses, 5 to 50 doses, 5 to 45 doses, 5 to 40 doses, 5 to 35 doses, 5 to 30 doses, 5 to 25 doses, 5 to 20 doses, 5 to 15 doses, 5 to 10 doses, 10 to 50 doses, 10 to 45 doses, 10 to 40 doses, 10 to 35 doses, 10 to 30 doses, 10 to 25 doses, 10 to 20 doses, 10 to 15 doses, 15 to 50 doses, 15 to 45 doses, 15 to 40 doses, 15 to 35 doses, 15 to 30 doses, 15 to 25 doses, 15 to 20 doses, 20 to 50 doses, 20 to 45 doses, 20 to 40 doses, 20 to 35 doses, 20 to 30 doses, 20 to 25 doses, 25 to 50 doses, 25 to 45 doses, 25 to 40 doses, 25 to 35 doses, 25 to 30 doses, 30 to 50 doses, 30 to 45 doses, 30 to 40 doses, 30 to 35 doses, 35 to 50 doses, 35 to 45 doses, 35 to 40 doses, 40 to 50 doses, 40 to 45 doses or 45 to 50 doses. In certain cases, the doses may be administered intravenously.

[0310] Atezolizumab can be administered to a subject at any suitable dose. In some embodiments, atezolizumab is administered intravenously to a subject at a dose of about 840 mg every 2 weeks, about 1200 mg every 3 weeks, or about 1680 mg every 4 weeks. In some embodiments, atezolizumab is administered intravenously to a subject at a dose of about 1200 mg every 3 weeks. In some embodiments, atezolizumab is administered to a subject in a 21-day dosing cycle. In some embodiments, atezolizumab is administered intravenously to a subject at a dose of about 1200 mg from day -2 to day 4 of each 21-day dosing cycle. In some embodiments, atezolizumab is administered intravenously to a subject at a dose of about 1200 mg on day 1 of each 21-day dosing cycle.

[0311] In a preferred embodiment, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is administered intravenously. In one example, atezolizumab can be administered intravenously over 60 minutes; if the first infusion is tolerated, all subsequent infusions can be delivered over 30 minutes. In some instances, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is not administered as a bolus or rapid injection.

[0312] In some embodiments, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) are administered in a single dosing regimen. In the context of a dosing regimen, the administration of these agents can be simultaneous or separate.

[0313] PD-1 axis-binding antagonists (e.g., anti-PD-L1 antibodies (e.g., atezolizumab)) and / or one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) can be administered in any suitable manner known in the art. For example, PD-1 axis-binding antagonists (e.g., anti-PD-L1 antibodies (e.g., atezolizumab)) and one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the PD-1 axis-binding antagonist is administered before one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)). In other embodiments, the PD-1 axis-binding antagonist is administered after one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)). In still other embodiments, the PD-1 axis-binding antagonist is administered simultaneously with one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)). In some embodiments, the PD-1 axis-binding antagonist (e.g., anti-PD-L1 antibody (e.g., atezolizumab)) and one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) are in different compositions. In some embodiments, the PD-1 axis-binding antagonist (e.g., anti-PD-L1 antibody (e.g., atezolizumab)) and one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) are in the same composition.

[0314] A PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) can be administered by the same route of administration or by different routes of administration. In some embodiments, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, platinum-based chemotherapy (e.g., carboplatin or cisplatin and gemcitabine) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) can be administered for preventing or treating a disease. The appropriate dosage of the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) can be determined based on the type of disease to be treated, the type of PD-1 axis-binding antagonist, the severity and cause of the disease, the clinical condition of the individual, the clinical history of the individual, and the response to treatment, as well as the discretion of the attending physician. In some embodiments, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) is administered intravenously by infusion.

[0315] For example, when administered with chemotherapy with or without bevacizumab, atezolizumab can be administered at a dose of 1200 mg every 3 weeks before chemotherapy and bevacizumab. In another example, after completion of 4 to 6 cycles of chemotherapy and if bevacizumab is discontinued, atezolizumab can be administered at a dose of 840 mg every 2 weeks, 1200 mg every 3 weeks, or 1680 mg every four weeks. In another instance, atezolizumab can be administered at a dose of 840 mg, followed by 100 mg / m 2Protein-bound paclitaxel (e.g., albumin-bound paclitaxel); for each 28-day cycle, atezolizumab is administered on days 1 and 15, and protein-bound paclitaxel is administered on days 1, 8, and 15. In another example, when administered with carboplatin and etoposide, atezolizumab can be administered at a dose of 1200 mg every 3 weeks before chemotherapy. In yet another example, after completion of 4 cycles of carboplatin and etoposide, atezolizumab can be administered at a dose of 840 mg every 2 weeks, 1200 mg every 3 weeks, or 1680 mg every 4 weeks. In another instance, after completion of a 28-day cycle of cobimetinib and vemurafenib, atezolizumab can be administered at a dose of 840 mg every 2 weeks in combination with cobimetinib at a dose of 60 mg orally once daily (administered for 21 days, off for 7 days) and vemurafenib at a dose of 720 mg orally twice daily.

[0316] In some embodiments, the treatment may further comprise an additional therapy. Any suitable additional therapy known in the art or described herein may be used. The additional therapy may be radiotherapy, surgery (e.g., transurethral resection of bladder tumor (TURBT) or cystectomy (including partial or radical cystectomy)), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is the administration of a side effect limiting agent (e.g., an agent designed to reduce the incidence and / or severity of treatment side effects, such as an antiemetic, etc.). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PI3K / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemotherapeutic agent. The additional therapy may be one or more of the chemotherapeutic agents described herein.

[0317] In some cases, the additional therapy is the administration of a side effect limiting agent (e.g., an agent designed to reduce the occurrence and / or severity of treatment side effects, such as an anti-nausea agent, a corticosteroid (e.g., prednisone or an equivalent, e.g., at a dose of 1 to 2 mg / kg / day), a hormone replacement drug, etc.).

[0318] III. Combination Therapy

[0319] The present invention also provides methods for treating bladder cancer (e.g., UC, including locally advanced or metastatic UC) in a subject or delaying its progression, the methods comprising administering to the subject a treatment regimen in combination with another anti-cancer agent or cancer therapy, the treatment regimen comprising an effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). For example, the present invention provides methods for treating bladder cancer (e.g., UC, including locally advanced or metastatic UC) in a subject or delaying its progression, the methods comprising administering to the subject a treatment regimen in combination with another anti-cancer agent or cancer therapy, the treatment regimen comprising an effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine). In some embodiments, the methods comprise administering to an individual a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine), and an additional therapeutic agent. For example, any of the combinations described below can be used in the methods described in Section II above.

[0320] In some embodiments, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or the platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with an additional chemotherapy or chemotherapeutic agent. In some embodiments, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or the platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with radiation therapy or a radiation therapeutic agent. In some embodiments, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or the platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with a targeted therapy or a targeted therapeutic agent. In some embodiments, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or the platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with an immunotherapy or an immunotherapeutic agent (e.g., a monoclonal antibody).

[0321] Without wishing to be bound by theory, it is believed that enhancing T cell stimulation by promoting co-stimulatory molecules or by inhibiting negative co-stimulatory molecules can promote tumor cell death, thereby treating cancer or delaying its progression. In some embodiments, a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be co-administered with an agonist against an activating co-stimulatory molecule. In some embodiments, the activating co-stimulatory molecules can include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist against an activating co-stimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be co-administered with an antagonist against an inhibitory co-stimulatory molecule. In some embodiments, the inhibitory co-stimulatory molecules can include CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase. In some embodiments, the antagonist against an inhibitory co-stimulatory molecule is an antagonist antibody that binds to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase.

[0322] In some embodiments, a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be co-administered with an antagonist against CTLA-4 (also known as CD152) (e.g., a blocking antibody). In some embodiments, a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be co-administered with ipilimumab (also known as MDX-010, MDX-101, or )Combined administration. In some embodiments, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with tremelimumab (also known as ticilimumab or CP-675,206). In some embodiments, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with an antagonist against B7-H3 (also known as CD276) (e.g., a blocking antibody). In some embodiments, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with MGA271. In some embodiments, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with an antagonist against TGFβ (e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GC1008) or LY2157299).

[0323] In some embodiments, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with a treatment involving adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) (e.g., cytotoxic T cells or CTLs). In some embodiments, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with a treatment involving adoptive transfer of T cells comprising a dominant negative TGFβ receptor (e.g., a dominant negative TGFβ type II receptor). In some embodiments, a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) can be administered in combination with a treatment comprising the HERCREEM regimen (see, e.g., ClinicalTrials.gov Identifier NCT00889954).

[0324] In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy 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 embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with urelumab (also known as BMS-663513). In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with an agonist (e.g., an activating antibody) directed against CD40. In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with CP-870893. In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with an agonist (e.g., an activating antibody) directed against OX40 (also known as CD134). In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with an anti-OX40 antibody (e.g., AgonOX). In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with an agonist (e.g., an activating antibody) directed against CD27. In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with CDX-1127. In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with an antagonist directed against indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).

[0325] In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE). In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599). In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or Genentech). In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with DMUC5754A. In some embodiments, a PD-1 axis-binding antagonist and / or a platinum-based chemotherapy can be administered in combination with an antibody-drug conjugate that targets the endothelin B receptor (EDNBR) (e.g., a conjugate of an antibody directed against EDNBR and MMAE).

[0326] In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with an angiogenesis inhibitor. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with an antibody against angiopoietin 2 (also known as Ang2). In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with MEDI3617.

[0327] In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with an anti-tumor agent. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with an agent that targets CSF-1R (also known as M-CSFR or CD115). In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with anti-CSF-1R (also known as IMC-CS4). In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with an interferon (e.g., interferon α or interferon γ). In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with Roferon-A (also known as recombinant interferon α-2a). In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with GM-CSF (also known as recombinant human granulocyte macrophage colony-stimulating factor, rhuGM-CSF, sargramostim or )). In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with IL-2 (also known as aldesleukin or ). In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with IL-12. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with an antibody that targets CD20. In some embodiments, the antibody that targets CD20 is obinutuzumab (also known as GA101 or ) or rituximab. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in combination with an antibody that targets GITR. In some embodiments, the antibody that targets GITR is TRX518.

[0328] In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, and in some embodiments, it is an individualized peptide vaccine. In some embodiments, the peptide cancer vaccine is a multivalent long peptide, polypeptide, peptide mixture, hybrid peptide, or peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with an adjuvant. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with a treatment comprising a TLR agonist (e.g., Poly-ICLC (also known as ), LPS, MPL, or CpG ODN). In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with tumor necrosis factor (TNF) α. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with IL-1. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with HMGB1. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with an IL-10 antagonist. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with an IL-4 antagonist. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with an IL-13 antagonist. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with an HVEM antagonist. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with an ICOS agonist, e.g., by administering ICOS-L or an agonistic antibody against ICOS. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with a treatment targeting CX3CL1. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with a treatment targeting CXCL9. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with a treatment targeting CXCL10. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with a treatment targeting CCL5. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with an LFA-1 or ICAM1 agonist. In some embodiments, a PD-1 axis binding antagonist and / or platinum-based chemotherapy can be co-administered with a selectin agonist.

[0329] In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with a targeted therapy. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with an inhibitor of B-Raf. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with vemurafenib (also known as ). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with dabrafenib (also known as ). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with erlotinib (also known as ). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with an inhibitor of MEK (e.g., MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2)). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with cobimetinib (also known as GDC-0973 or XL-518). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with trametinib (also known as )Combined administration. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with an inhibitor of K-Ras. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with an inhibitor of c-Met. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with onartuzumab (also known as MetMAb). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with an inhibitor of Alk. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with AF802 (also known as CH5424802 or alectinib). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with an inhibitor of phosphatidylinositol 3-kinase (PI3K). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with BKM120. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with perifosine (also known as KRX-0401). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with an inhibitor of Akt. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with MK2206. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with GSK690693. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with GDC-0941. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with an inhibitor of mTOR. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with sirolimus (also known as rapamycin). In some embodiments, a PD-1 axis-binding antagonist can be administered in combination with temsirolimus (also known as CCI-779 or )Combined administration. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with everolimus (also known as RAD001). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with ridaforolimus (also known as AP-23573, MK-8669 or deforolimus). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with OSI-027. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with AZD8055. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with INK128. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with a dual PI3K / mTOR antagonist. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with XL765. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with GDC-0980. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with BEZ235 (also known as NVP-BEZ235). In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with BGT226. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with GSK2126458. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with PF-04691502. In some embodiments, a PD-1 axis-binding antagonist and / or platinum-based chemotherapy can be administered in combination with PF-05212384 (also known as PKI-587).

[0330] In any of the foregoing embodiments, the PD-1 axis-binding antagonist can be a human PD-1 axis-binding antagonist.

[0331] In any of the foregoing embodiments, the PD-1 axis-binding antagonist is an anti-PD-L1 antibody (e.g., atezolizumab).

[0332] In any of the foregoing embodiments, the platinum-based chemotherapy includes a platinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin). In some embodiments, the platinum-based chemotherapy includes cisplatin. In some embodiments, the platinum-based chemotherapy includes carboplatin. In some embodiments, the platinum-based chemotherapy further includes one or more additional therapeutic agents, e.g., a nucleoside analogue. In some embodiments, the nucleoside analogue is gemcitabine. In some embodiments, the platinum-based chemotherapy includes cisplatin and gemcitabine. In other embodiments, the platinum-based chemotherapy includes carboplatin and gemcitabine.

[0333] IV. Assessment of PD-L1 Expression

[0334] The presence and / or expression level of PD-L1 can be evaluated in patients identified, selected, stratified, and / or treated using any of the methods and compositions described herein. The methods and compositions for use can include determining the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from a patient. In other instances, the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from a patient has been determined before or after initiation of treatment. PD-L1 expression can be determined using any suitable method. For example, PD-L1 expression can be determined as described in U.S. Patent Application Nos. 15 / 787,988 and 15 / 790,680. Any suitable tumor sample can be used, e.g., a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0335] In some instances, the assessment of the presence and / or expression level of PD-L1 and / or patient selection may involve the use of the following two different affinity immunohistochemistry (AHC) assays (e.g., IHC assays) for PD-L1 protein: (a) an immune-guided PD-L1 assay; and (b) an immune-agnostic PD-L1 assay.

[0336] As described herein, an immune-guided PD-L1 assay is any AHC assay that is specific for human PD-L1 protein and has been designed to highlight the immune cell expression of PD-L1, e.g., by preferentially staining immune cells expressing PD-L1 relative to tumor cells expressing PD-L1. The highlighting of immune cells may be the result of: (a) the inherent antibody specificity of immune-expressed PD-L1 compared to the expression of other cell types; (b) careful selection of staining conditions such as antigen retrieval process, antibody diluent selection, buffer selection, detection system, labeling time, and temperature, etc.; or (c) a combination of (a) and (b). An example of a commercially available immune-guided PD-L1 assay is the VENTANA PD-L1 (SP142) assay ("SP142 assay"). The SP142 assay is an affinity immunohistochemistry assay that uses: (a) a PD-L1 rabbit monoclonal antibody (clone SP142, see US10,689,445); (b) an automated IHC / ISH staining platform (BENCHMARK IHC / ISH staining platform (Roche)); and (c) a tyramide amplification-based 3,3'-diaminobenzidine (DAB) detection system (OPTIVIEW DAB IHC detection kit with OPTIVIEW amplification kit (Roche)).

[0337] In an embodiment, the immune-guided PD-L1 assay is an AHC assay (e.g., an IHC assay) that has at least 80% overall percent agreement (OPA), at least 80% positive percent agreement (PPA), and / or at least 80% negative percent agreement (NPA) with an SP142 assay for the same indication and using the same scoring algorithm and cut-off value. In a specific embodiment, the AHC has at least 80%, at least 85%, at least 90%, or at least 95% OPA with the SP142 assay. In another specific embodiment, the AHC has at least 80%, at least 85%, at least 90%, or at least 95% PPA with the SP142 assay. In another specific embodiment, the AHC has at least 80%, at least 85%, at least 90%, or at least 95% NPA with the SP142 assay. In another specific embodiment, the AHC has at least 80%, at least 85%, at least 90%, or at least 95% PPA with the SP142 assay and at least 80%, at least 85%, at least 90%, or at least 95% NPA with the SP142 assay. In an embodiment, OPA, PPA, and / or NPA are measured using an immune proportion (IC) scoring method at a single cut-off point that has been shown to predict response to PD-1 axis binding antagonists in the test indication. In another embodiment, a ≥5% IC 2 / 3 cut-off value is used to measure OPA, PPA, and / or NPA in the bladder cancer indication (e.g., locally advanced or metastatic UC).

[0338] Also as described herein, an immune-agnostic PD-L1 assay is any AHC assay (e.g., an IHC assay) that is specific for human PD-L1 protein and that is not an immune-guided PD-L1 assay. Exemplary commercially available immune-agnostic PD-L1 assays include the PD-L1 IHC 22C3 PHARMDX assay (Agilent) (hereinafter referred to as the "22C3 assay"), the VENTANA PD-L1 (SP263) assay (Roche) (hereinafter referred to as the "SP263 assay"), and the PD-L1 IHC 28-8 PHARMDX assay (Agilent) (hereinafter referred to as the "28-8 assay"). In embodiments, the immune-agnostic PD-L1 assay is an AHC assay (e.g., an IHC assay) that has at least 80% OPA, at least 80% PPA, and / or at least 80% NPA with one or more of the 22C3 assay, the SP263 assay, and the 28-8 assay in the same indication and using the same scoring algorithm and cut-off value. In a specific embodiment, the AHC has at least 80%, at least 85%, at least 90%, or at least 95% OPA with the 22C3 assay. In another specific embodiment, the AHC has at least 80%, at least 85%, at least 90%, or at least 95% PPA with the 22C3 assay. In another specific embodiment, the AHC has at least 80%, at least 85%, at least 90%, or at least 95% NPA with the 22C3 assay. In another specific embodiment, the AHC has at least 80%, at least 85%, at least 90%, or at least 95% PPA with the 22C3 assay and at least 80%, at least 85%, at least 90%, or at least 95% NPA with the 22C3 assay. In embodiments, OPA, PPA, and / or NPA are measured using a combined positive score (CPS) scoring method at a single cut-off point, which has been shown to predict response to PD-1 axis-binding antagonists in the test indication. In another embodiment, a ≥10% CPS cut-off value is used to measure OPA, PPA, and / or NPA in the bladder cancer indication (e.g., locally advanced or metastatic UC).

[0339] In one example, provided herein is an assay for determining the presence or expression level of PD-L1 in a tumor sample obtained from a patient having cancer, the assay comprising: (a) determining the presence or expression level of PD-L1 in a tumor sample obtained from the patient using an immune-guided PD-L1 assay (e.g., the SP142 assay); and (b) determining the presence or expression level of PD-L1 in a tumor sample obtained from the patient using an immune-agnostic PD-L1 assay (e.g., the 22C3 assay, the SP263 assay, or the 28-8 assay).

[0340] In some instances, the assay is an AHC assay. In some instances, the AHC assay is an IHC assay.

[0341] For example, provided herein is an assay for determining the presence or expression level of PD-L1 in a tumor sample obtained from a patient with cancer, the assay comprising: (a) determining the presence or expression level of PD-L1 in a tumor sample obtained from the patient using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) determining the presence or expression level of PD-L1 in a tumor sample obtained from the patient using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody.

[0342] Any suitable scoring algorithm can be used. For example, the scoring algorithm can be the immune cell scoring algorithm listed in Table 2 herein, such as, as used in the SP142 assay. In some instances, the scoring algorithm can be the combined positive score (CPS), such as, as used in the 22C3 assay. It should be understood that other PD-L1 AHC assays (e.g., other PD-L1 IHC assays) can be used, such as IHC assays including the use of VENTANA SP263 or 28-8, to determine the CPS. Other scoring algorithms known in the art for PD-L1 assays, such as TPS, tumor cell percentage (TC), and the tumor cell scoring algorithm listed in Table 3 herein. A description of different exemplary scoring algorithms for PD-L1 assays that can be used is shown in Zajac et al. Diagnostic Pathology. Figure 1 in.

[0343] Any suitable cut-off value can be used. For example, in some instances, the cut-off value is IC ≥ 5%, such as, as described in Table 2 herein. In some instances, the cut-off value is CPS ≥ 1 or CPS ≥ 10. In some instances, the cut-off value is CPS ≥ 10.

[0344] In some instances, the tumor sample obtained from the patient has a detectable PD-L1 expression level in tumor-infiltrating immune cells that account for 5% or more of the tumor sample using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

[0345] In some instances, any discernible PD-L1 staining of any intensity is present in tumor-infiltrating immune cells in a tumor sample obtained from a patient that covers ≥5% of the tumor area occupied by tumor cells, associated intratumoral stroma, and contiguous peritumoral stroma, as determined by a PD-L1 IHC assay that includes the VENTANA SP142 anti-PD-L1 diagnostic antibody.

[0346] In some instances, a tumor sample obtained from a patient has a CPS of ≥10 using a PD-L1 IHC assay that includes the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody. In some instances, a tumor sample obtained from a patient has a CPS of ≥10 using a PD-L1 IHC assay that includes the Dako 22C3 anti-PD-L1 diagnostic antibody.

[0347] In some instances, a tumor sample obtained from a patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells that account for 5% or more of the tumor sample using a PD-L1 IHC assay that includes the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using a PD-L1 IHC assay that includes the Dako22C3 anti-PD-L1 diagnostic antibody.

[0348] In some instances, steps (a) and (b) are performed simultaneously.

[0349] In some instances, steps (a) and (b) are performed sequentially.

[0350] In some instances, steps (a) and (b) are performed in different sections of the tumor sample or in the same section of the tumor sample.

[0351] In some instances, the different sections of the tumor sample are consecutive sections.

[0352] In some instances, the cancer is locally advanced or metastatic urothelial carcinoma.

[0353] In some instances, the patient has not been previously treated for locally advanced or metastatic urothelial carcinoma.

[0354] In some instances, the assay is used for (i) selecting a therapy for locally advanced or metastatic UC in patients in need thereof, or (ii) identifying patients with locally advanced or metastatic UC who may benefit from a treatment regimen that includes an anti-PD-L1 antibody.

[0355] In some instances, the anti-PD-L1 antibody comprises the following HVRs: (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) an HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) an HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) an HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) an HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8). In some instances, the anti-PD-L1 antibody is atezolizumab.

[0356] In some instances, the tumor sample is an FFPE tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0357] In another instance, provided herein is a method of labeling PD-L1 in a tumor sample, the method comprising the steps of: (a) performing an immuno-guided PD-L1 assay on the tumor sample; and (b) performing an immuno-agnostic PD-L1 assay on the tumor sample.

[0358] Any suitable immuno-guided PD-L1 assay can be used, such as the SP142 assay.

[0359] Any suitable immuno-agnostic PD-L1 assay can be used, such as the 22C3 assay, the SP263 assay, or the 28-8 assay.

[0360] In another instance, provided herein is a method of labeling PD-L1 in a tumor sample, the method comprising the steps of: (a) contacting the tumor sample with a VENTANA SP142 anti-PD-L1 diagnostic antibody; (b) contacting the tumor sample with a Dako 22C3 anti-PD-L1 diagnostic antibody, a VENTANA SP263 anti-PD-L1 diagnostic antibody, or a 28-8 anti-PD-L1 diagnostic antibody; and (c) visualizing the anti-PD-L1 diagnostic antibodies of steps (a) and (b) with one or more detectable reagents that generate a detectable signal for both anti-PD-L1 diagnostic antibodies.

[0361] In some instances, the detectable signal for the VENTANA SP142 anti-PD-L1 diagnostic antibody is an amplified signal.

[0362] In some instances, the amplified signal is generated by tyramide signal amplification.

[0363] In some instances, steps (a) and (b) are performed simultaneously.

[0364] In some instances, steps (a) and (b) are carried out sequentially.

[0365] In some instances, steps (a) and (b) are carried out in different sections of the tumor sample or in the same section of the tumor sample.

[0366] In other instances, the different sections of the tumor sample are consecutive sections.

[0367] In some instances, visualization includes AHC.

[0368] In some instances, visualization includes IHC or immunofluorescence (IF).

[0369] In some instances, visualization includes IHC.

[0370] In some instances, the tumor sample is an FFPE tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0371] In some instances, the tumor sample is obtained from a patient with cancer.

[0372] In some instances, the cancer is locally advanced or metastatic urothelial carcinoma.

[0373] In some instances, the patient has not been previously treated for locally advanced or metastatic urothelial carcinoma.

[0374] Any suitable scoring algorithm or method can be used to evaluate the presence and / or expression level of PD-L1 in a biological sample (e.g., a tumor sample).

[0375] For example, the scoring algorithm can be the immune cell scoring algorithm listed in Table 2 herein, e.g., as used in the SP142 assay. In some instances, the scoring algorithm can be the combined positive score (CPS), e.g., as used in the 22C3 assay. It should be understood that other PD-L1 AHC assays (e.g., other PD-L1 IHC assays) can be used, e.g., IHC assays including those using VENTANA SP263 or 28-8 to determine CPS. Other scoring algorithms known in the art for PD-L1 assays, such as TPS, tumor cell percentage (TC), and the tumor cell scoring algorithm listed in Table 3 herein. Descriptions of different exemplary scoring algorithms for PD-L1 assays that can be used are shown in Zajac et al. Diagnostic Pathology. Figure 1 in.

[0376] In one example, PD-L1 expression can be determined based on the percentage of a tumor sample occupied by tumor-infiltrating immune cells that express a detectable level of PD-L1 expression, as the percentage of tumor-infiltrating immune cells in the tumor sample that express a detectable level of PD-L1 expression, and / or as the percentage of tumor cells in the tumor sample that express a detectable PD-L1 expression level. It should be understood that in any of the foregoing examples, the percentage of the tumor sample occupied by tumor-infiltrating immune cells can be the percentage of the tumor area covered by tumor-infiltrating immune cells in a section of the tumor sample obtained from a patient, e.g., as assessed by IHC using an anti-PD-L1 antibody (such as the SP142 antibody). Any suitable anti-PD-L1 antibody can be used, including, for example, SP142 (Ventana), SP263 (Ventana), 22C3 (Dako), 28-8 (Dako), E1L3N (Cell Signaling Technology), 4059 (ProSci, Inc.), h5H1 (Advanced Cell Diagnostics), and 9A11. In some examples, the anti-PD-L1 antibody is SP142. In other examples, the anti-PD-L1 antibody is SP263.

[0377] In some examples, a tumor sample obtained from a patient has a detectable level of PD-L1 expression in less than 1% of the tumor cells in the tumor sample, in 1% or more of the tumor cells in the tumor sample, in 1% to less than 5% of the tumor cells in the tumor sample, in 5% or more of the tumor cells in the tumor sample, in 5% to less than 50% of the tumor cells in the tumor sample, or in 50% or more of the tumor cells in the tumor sample.

[0378] In some examples, a tumor sample obtained from a patient has a detectable level of PD-L1 expression in tumor-infiltrating immune cells that occupy less than 1% of the tumor sample, more than 1% of the tumor sample, 1% to less than 5% of the tumor sample, more than 5% of the tumor sample, 5% to less than 10% of the tumor sample, or more than 10% of the tumor sample.

[0379] In some embodiments, the tumor sample can be scored for PD-L1 positivity in tumor-infiltrating immune cells and / or tumor cells according to the diagnostic assessment criteria shown in Table 2 and / or Table 3, respectively. In some examples, for a PD-L1 IHC assay that includes the VENTANA SP142 anti-PD-L1 diagnostic antibody (e.g., the SP142 assay), the tumor sample can be scored for PD-L1 positivity in tumor-infiltrating immune cells and / or tumor cells according to the diagnostic assessment criteria shown in Table 2 and / or Table 3, respectively.

[0380] In some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that account for about 1% or more (e.g., about 1% or more, 2% or more, 3% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or 100%) of the tumor sample. For example, in some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that account for from about 1% to less than about 5% (e.g., 1% to 4.9%, 1% to 4.5%, 1% to 4%, 1% to 3.5%, 1% to 3%, 1% to 2.5%, or 1% to 2%) of the tumor sample.

[0381] In some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 1% or more (e.g., about 1% or more, 2% or more, 3% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or 100%) of the tumor-infiltrating immune cells. For example, in some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 1% to less than about 5% (e.g., 1% to 4.9%, 1% to 4.5%, 1% to 4%, 1% to 3.5%, 1% to 3%, 1% to 2.5%, or 1% to 2%) of the tumor-infiltrating immune cells.

[0382] In other embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 5% or more of the tumor-infiltrating immune cells. For example, in some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 5% to less than about 10% (e.g., 5% to 9.5%, 5% to 9%, 5% to 8.5%, 5% to 8%, 5% to 7.5%, 5% to 7%, 5% to 6.5%, 5% to 6%, 5% to 5.5%, 6% to 9.5%, 6% to 9%, 6% to 8.5%, 6% to 8%, 6% to 7.5%, 6% to 7%, 6% to 6.5%, 7% to 9.5%, 7% to 9%, 7% to 7.5%, 8% to 9.5%, 8% to 9%, or 8% to 8.5%) of the tumor-infiltrating immune cells.

[0383] In yet other embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 5% or more of the tumor-infiltrating immune cells in the tumor sample. For example, in some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 5% to less than about 10% (e.g., 5% to 9.5%, 5% to 9%, 5% to 8.5%, 5% to 8%, 5% to 7.5%, 5% to 7%, 5% to 6.5%, 5% to 6%, 5% to 5.5%, 6% to 9.5%, 6% to 9%, 6% to 8.5%, 6% to 8%, 6% to 7.5%, 6% to 7%, 6% to 6.5%, 7% to 9.5%, 7% to 9%, 7% to 7.5%, 8% to 9.5%, 8% to 9%, or 8% to 8.5%) of the tumor-infiltrating immune cells in the tumor sample.

[0384] In still other embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 10% or more (e.g., 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) of the tumor-infiltrating immune cells in the tumor sample.

[0385] In still other embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 10% or more (e.g., 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 100%) of tumor-infiltrating immune cells in the tumor sample.

[0386] In yet other embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 50% or more (e.g., about 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more) of the tumor cells and / or in about 10% or more (e.g., 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 100%) of the tumor-infiltrating immune cells of the tumor sample.

[0387] It should be understood that in any of the foregoing examples, the percentage of the tumor sample occupied by tumor-infiltrating immune cells can be the percentage of the tumor area covered by tumor-infiltrating immune cells in a section of the tumor sample obtained from a subject, e.g., as assessed by IHC using an anti-PD-L1 antibody (e.g., the SP142 antibody). Any suitable anti-PD-L1 antibody can be used, including, for example, SP142 (Ventana), SP263 (Ventana), 22C3 (Dako), 28-8 (Dako), E1L3N (Cell Signaling Technology), 4059 (ProSci, Inc.), h5H1 (Advanced Cell Diagnostics), and 9A11. In some embodiments, the anti-PD-L1 antibody is SP142. In some embodiments, the anti-PD-L1 antibody is SP263.

[0388] In some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 1% or more (e.g., about 1% or more, 2% or more, 3% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more) of the tumor cells. For example, in some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 1% to less than about 5% (e.g., 1% to 4.9%, 1% to 4.5%, 1% to 4%, 1% to 3.5%, 1% to 3%, 1% to 2.5% or 1% to 2%) of the tumor cells in the tumor sample. In some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in less than about 1% of the tumor cells in the tumor sample.

[0389] In other embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 5% or more of the tumor cells in the tumor sample. For example, in some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 5% to less than about 50% (e.g., 5% to 49.5%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, 5% to 10%, 5% to 9%, 5% to 8%, 5% to 7%, 5% to 6%, 10% to 49.5%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, 10% to 15%, 15% to 49.5%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to 30%, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 49.5%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25%, 25% to 49.5%, 25% to 45%, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 49.5%, 30% to 45%, 30% to 40%, 30% to 35%, 35% to 49.5%, 35% to 45%, 35% to 40%, 40% to 49.5%, 40% to 45% or 45% to 49.5%) of the tumor cells in the tumor sample.

[0390] In still other embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 50% or more (e.g., about 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more) of the tumor cells in the tumor sample. In some embodiments, a tumor sample obtained from a subject has been determined to have a detectable level of PD-L1 expression in about 50% to about 99% (e.g., 50% to 99%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, 50% to 55%, 55% to 99%, 55% to 95%, 55% to 90%, 55% to 85%, 55% to 80%, 55% to 75%, 55% to 70%, 55% to 65%, 55% to 60%, 60% to 99%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 60% to 70%, 60% to 65%, 65% to 99%, 65% to 95%, 65% to 90%, 65% to 85%, 65% to 80%, 65% to 75%, 65% to 70%, 70% to 99%, 70% to 95%, 70% to 90%, 70% to 85%, 70% to 80%, 70% to 75%, 75% to 99%, 75% to 95%, 75% to 90%, 75% to 85%, 75% to 80%, 80% to 99%, 80% to 95%, 80% to 90%, 80% to 85%, 85% to 99%, 85% to 95%, 85% to 90%, 90% to 99% or 90% to 95%) of the tumor cells in the tumor sample.

[0391] In some cases, the CPS is determined in a tumor sample obtained from a patient. In some embodiments, the CPS is determined by positive staining with an anti-PD-L1 diagnostic antibody, where the anti-PD-L1 diagnostic antibody is SP142, SP263, 22C3, or 28-8 (e.g., as part of an IHC assay). In some embodiments, the CPS is greater than or equal to 1, as determined by positive staining with anti-PD-L1 diagnostic antibody SP263 (e.g., as calculated using the Ventana SP263 IHC assay), 22C3 (e.g., as calculated using the PHARMDX 22C3 IHC assay), or 28-8 (e.g., as calculated using the PHARMDX 28-8 IHC assay). In some embodiments, the CPS is greater than or equal to 10, as determined by positive staining with anti-PD-L1 antibody SP263 (e.g., as calculated using the Ventana SP263 IHC assay), 22C3 (e.g., as calculated using the PHARMDX 22C3 IHC assay), or 28-8 (e.g., as calculated using the PHARMDX 28-8 IHC assay).

[0392] In some cases, the PD-L1 positive tumor cell fraction of a subject is determined. In some embodiments, the PD-L1 positive tumor cell fraction is determined by positive staining with an anti-PD-L1 diagnostic antibody, where the anti-PD-L1 antibody is SP142, SP263, 22C3, or 28-8 (e.g., as part of an IHC assay). In some embodiments, the PD-L1 positive tumor cell fraction is greater than or equal to 1% tumor cells (TC), as determined by positive staining with anti-PD-L1 antibody SP263 (e.g., as calculated using the Ventana SP263 IHC assay) or 22C3 (e.g., as calculated using the PHARMDX 22C3 IHC assay). In some embodiments, the PD-L1 positive tumor cell fraction is less than 1% TC (e.g., 0% to 1% TC, e.g., PD-L1 negative), as determined by positive staining with anti-PD-L1 antibody SP263 (e.g., as calculated using the Ventana SP263 IHC assay) or 22C3 (e.g., as calculated using the PHARMDX 22C3 IHC assay).

[0393] In some cases, in any of the methods, uses, or compositions for use described herein, a tumor sample obtained from an individual has a detectable nucleic acid expression level of PD-L1. In some cases, the detectable nucleic acid expression level of PD-L1 has been determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

[0394] In some cases, the sample is selected from the group consisting of tissue samples, whole blood samples, serum samples, and plasma samples.

[0395] In some cases, the tissue sample is a tumor sample. Any suitable tumor sample can be used. In some cases, the tumor sample includes tumor-infiltrating immune cells, tumor cells, stromal cells, and any combination thereof.

[0396] In some embodiments, the tumor sample is a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample. In some embodiments, the tumor sample is an FFPE tumor sample.

[0397] In other cases, the sample can be a cytology sample (e.g., fine needle aspirate).

[0398] Any of the above biomarkers (including, for example, PD-L1 (e.g., PD-L1 expression on tumor-infiltrating immune cells (IC) in a tumor sample obtained from a subject and / or PD-L1 expression on tumor cells (TC) in a tumor sample obtained from a subject)) can be qualitatively and / or quantitatively assessed for the presence and / or expression level (e.g., in a tumor sample obtained from a subject) based on any suitable criteria known in the art, including but not limited to DNA, mRNA, cDNA, proteins, protein fragments, and / or gene copy number. Methods for measuring such biomarkers are known in the art and understood by those skilled in the art, including but not limited to IHC, Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence-activated cell sorting (“FACS”), MassARRAY, proteomics, blood-based quantitative assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), Southern analysis, Northern analysis, whole-genome sequencing, polymerase chain reaction (PCR) (including quantitative real-time PCR (qRT-PCR) and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA, etc.), RNASeq, microarray analysis, gene expression profiling, whole-genome sequencing (WGS), and / or serial analysis of gene expression (“SAGE”), and any one of a variety of assays that can be performed by protein, gene, and / or tissue array analysis. Typical protocols for assessing the status of genes and gene products can be found, for example, in Current Protocols In Molecular Biology, edited by Ausubel et al. (1995), Unit 2 (Northern blotting), Unit 4 (Southern blotting), Unit 15 (immunoblotting), and Unit 18 (PCR analysis). Multiplex immunoassays, such as those available from RulesBased Medicine or Meso Scale Discovery (“MSD”), can also be used.

[0399] In some embodiments of any of the foregoing methods, the expression level of the biomarker (e.g., PD-L1) can be the protein expression level.

[0400] Biomarker analysis can be performed, for example, by AHC or ACC methods. The AHC and ACC methods are typically accomplished by contacting a sample from a tumor with a biomarker-specific reagent under conditions that promote specific binding between the biomarker and the biomarker-specific reagent. The sample is then contacted with a set of detection reagents that interact with the biomarker-specific reagent to promote deposition of a detectable moiety in the vicinity of the biomarker, thereby generating a detectable signal localized to the biomarker. Typically, a washing step is performed between applications of different reagents to prevent unwanted non-specific labeling of the tissue. The biomarker-labeled sample can optionally be additionally labeled with a contrast agent to visualize macromolecular structures.

[0401] Samples for AHC / ACC assays are typically tissue samples that are processed in a manner compatible with affinity labeling and brightfield microscopy analysis of the sample. In a specific embodiment, the sample is a microtome section of a formalin-fixed paraffin-embedded (FFPE) sample derived from a tumor.

[0402] Exemplary biomarker-specific reagents for AHC and ACC methods include antibodies and their antigen-binding fragments, ADNECTIN (scaffold based on the 10th FN3 fibronectin domain; Bristol-Myers-Squibb Co.), AFFIBODY (scaffold based on the Z domain of protein A from Staphylococcus aureus; Affibody AB, Solna, Sweden), AVIMER (scaffold based on the domain A / LDL receptor; Amgen, Thousand Oaks, CA), dAb (scaffold based on VH or VL antibody domains; GlaxoSmithKline PLC, Cambridge, UK), DARPin (scaffold based on ankyrin repeat proteins; Molecular Partners AG, Zürich, CH), ANTICALIN (scaffold based on lipocalins; Pieris AG, Freising, DE), NANOBODY (scaffold based on VHH (camel Ig); Ablynx N / V, Ghent, BE), TRANS-BODY (scaffold based on transferrin; Pfizer Inc., New York, NY), SMIP (Emergent Biosolutions, Inc., Rockville, MD), and TETRANECTIN (scaffold based on the C-type lectin domain (CTLD), tetranectin; BoreanPharma A / S, Aarhus, DK). Such biomarker-specific reagents are reviewed by Wurch et al., Development of Novel Protein Scaffolds as Alternatives to Whole Antibodies for Imaging and Therapy: Status on Discovery Research and Clinical Validation, Current Pharmaceutical Biotechnology, Vol. 9, pp. 502-509 (2008), the content of which is incorporated herein by reference.

[0403] Non-limiting examples of commercially available detection reagents or kits containing detection reagents suitable for the methods of the present invention include: VENTANA ULTRAVIEW detection system (secondary antibodies conjugated with enzymes, including HRP and AP); VENTANA IVIEW detection system (biotinylated anti-species secondary antibodies and streptavidin-conjugated enzymes); VENTANA amplification kit (unconjugated secondary antibodies, which can be used with any of the foregoing VENTANA detection systems to increase the amount of enzyme deposited at the primary antibody binding site); OPTIVIEW detection system (anti-species secondary antibodies conjugated with haptens and anti-hapten tertiary antibodies conjugated with enzyme polymers); OPTIVIEW amplification system (anti-species secondary antibodies conjugated with haptens, anti-hapten tertiary antibodies conjugated with enzyme polymers, and tyramine conjugated with the same hapten, which can be used with the OPTIVIEW kit to increase the amount of enzyme deposited at the primary antibody binding site); POWERVISION and POWERVISION+ IHC detection systems (secondary antibodies directly polymerized with HRP or AP into compact polymers with a high enzyme-to-antibody ratio); DAKO ENVISION TM + system (enzyme-labeled polymers conjugated with secondary antibodies); ULTRAPLEX multiplex chromogenic IHC technology from CELL IDx (hapten-labeled primary antibodies combined with enzyme-labeled or fluorescent-labeled anti-hapten secondary antibodies).

[0404] If desired, biomarker-labeled slides can be counterstained to assist in identifying morphologically relevant regions for manual or automated identification of ROIs. Examples of counterstains include bright-field nuclear counterstains such as hematoxylin (staining from blue to purple), methylene blue (staining blue), toluidine blue (staining cell nuclei dark blue and polysaccharides pink to red), nuclear fast red (also known as Kernechtrot dye, staining red), and methyl green (staining green) and non-nuclear chromogenic stains such as eosin (staining pink).

[0405] An automated labeling system can be used to apply the AHC / ACC assay and counterstaining to a sample. Prichard, Overview of Automated Immunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp. 1578–1582 (2014), the entire content of which is incorporated herein by reference, describes several specific examples of automated AHC labeling systems and their various features, including intelliPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKO OMNIS, and DAKO Autostainer LINK 48 (Agilent Technologies), BENCHMARK (Ventana Medical Systems, Inc.), Leica BOND, and LAB VISION Autostainer (Thermo Scientific) automated AHC labeling systems. Commercially available labeling devices generally operate according to one of the following principles: (1) open single-slide labeling, in which the slide is placed horizontally and the reagent is dispensed as a pool on the surface of the slide containing the tissue sample (such as performed on the DAKO Autostainer Link 48 (Agilent Technologies) and INTELLIPATH (Biocare Medical) labelers); (2) liquid overlay technique, in which the reagent is covered by an inert fluid layer deposited on the sample or dispensed through an inert fluid layer deposited on the sample (such as performed on the BENCHMARK labeler); (3) capillary gap labeling, in which the slide surface is placed close to another surface to form a narrow gap, and capillary forces attract and bring the liquid reagent into contact with the sample through this gap (such as the labeling principle used by DAKO TECHMATE, Leica BOND, and DAKO OMNIS labelers). Some iterations of capillary gap labeling do not mix the fluid in the gap (such as on DAKO TECHMATE and Leica BOND). In a variant of capillary gap labeling called dynamic gap labeling, capillary forces are used to apply the sample to the slide, and then the parallel surfaces are translated relative to each other to agitate the reagent during incubation to achieve reagent mixing (such as the labeling principle performed on the DAKO OMNIS slide labeler (Agilent)). It has also been proposed to use inkjet technology to deposit reagents on the slide. See WO 2016 / 170008 A1. This list of labeling techniques is not intended to be comprehensive, and any fully automated or semi-automated system or manual method for performing biomarker labeling can be incorporated into this method.

[0406] In certain embodiments, the method includes contacting a sample with an antibody that binds to a biomarker of the invention under conditions that permit binding to the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such methods can be in vitro or in vivo methods. In some embodiments, an antibody is used to select a subject eligible for treatment with an anti-cancer therapy comprising a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the antibody being, for example, a biomarker for subject selection. In some embodiments, an antibody is used to select a subject eligible for treatment with an anti-cancer therapy comprising a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or a platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine), the antibody being, for example, a biomarker for subject selection.

[0407] Any method known in the art or provided herein for measuring protein expression levels can be used. For example, in some embodiments, a method selected from immunohistochemistry (IHC), flow cytometry (e.g., fluorescence-activated cell sorting (FACS TM ))), Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, and HPLC is used to determine the protein expression level of the biomarker.

[0408] In some embodiments, the protein expression level of a biomarker (e.g., PD-L1) in tumor-infiltrating immune cells is determined. In some embodiments, the protein expression level of the biomarker in tumor cells is determined. In some embodiments, the protein expression level of the biomarker in tumor-infiltrating immune cells and / or tumor cells is determined. In some embodiments, the protein expression level of the biomarker in peripheral blood mononuclear cells (PBMCs) is determined.

[0409] In certain embodiments, immunohistochemistry (IHC) and staining protocols are used to examine the presence and / or expression level / amount of a biomarker protein (e.g., PD-L1) in a sample. IHC staining of tissue sections has proven to be a reliable method for determining or detecting the presence of a protein in a sample. In some embodiments of any method, assay, and / or kit, one or more of the protein expression products of PD-L1 are the biomarker. In one embodiment, the expression level of the biomarker is determined using the following method: (a) performing IHC analysis of a sample (such as a tumor sample obtained from a subject) using an antibody; and (b) determining the expression level of the biomarker in the sample. In some embodiments, the IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control cell line stained sample, a tissue sample from a non-cancerous subject, or a tumor sample determined to be negative for the target biomarker).

[0410] For example, in some embodiments, the protein expression level of PD-L1 is determined using IHC. In some embodiments, the protein expression level of PD-L1 is detected using an anti-PD-L1 antibody. Any suitable anti-PD-L1 antibody can be used, including, for example, SP142, SP263, 22C3, 28-8, E1L3N, 4059, h5H1, and 9A11. In some embodiments, the anti-PD-L1 antibody is SP142. In some embodiments, the anti-PD-L1 antibody is SP263.

[0411] IHC can be performed in combination with other techniques such as morphological staining and / or in situ hybridization (e.g., ISH). There are two general IHC methods available: direct assays and indirect assays. According to the first assay, the binding of an antibody to a target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction. In a typical indirect assay, an un-conjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. When the secondary antibody is conjugated to an enzyme label, a chromogenic or fluorescent substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.

[0412] The primary antibody and / or secondary antibody used for IHC are typically labeled with a detectable moiety. Many labels are available, which are generally divided into the following categories: (a) radioisotopes, such as 35 S,[ 14 C,[ 125 1,[ 3 H and 131I; (b) colloidal gold particles; (c) fluorescent labels, including but not limited to rare earth chelates (e.g., europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, phycoerythrin, phycocyanin, or commercially available fluorophores (such as SPECTRUM ORANGE 7 and SPECTRUM GREEN 7) and / or derivatives of any one or more of the above; (d) various enzyme-substrate labels are available, and U.S. Patent No. 4,275,149 provides a review of some of them. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; see, e.g., U.S. Patent No. 4,737,456), fluorescein, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc.

[0413] Examples of enzyme-substrate combinations include: for example, horseradish peroxidase (HRPO) and catalase as a substrate; alkaline phosphatase (AP) and p-nitrophenyl phosphate as a chromogenic substrate; and β-D-galactosidase (β-D-Gal) and a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorescent substrate (e.g., 4-methylumbelliferyl-β-D-galactosidase). For a general review of these, see, e.g., U.S. Patent Nos. 4,275,149 and 4,318,980.

[0414] The specimens can be prepared, for example, manually or using an automated staining instrument (e.g., Ventana BenchMark XT or BenchMark ULTRA instrument). The specimens so prepared can be mounted and covered with a coverslip. Then, for example, the slide valuation can be determined using a microscope, and staining intensity criteria routinely used in the art can be employed. In one embodiment, it should be understood that when examining cells and / or tissues from a tumor using IHC, staining can be determined or evaluated in the tumor cells and / or tissues (as opposed to stroma or surrounding tissue that may be present in the sample). In other embodiments, staining can be determined or evaluated in the stroma or surrounding tissue that may be present in the sample. In some embodiments, it should be understood that when examining cells and / or tissues from a tumor using IHC, staining includes determining or evaluating tumor-infiltrating immune cells, including immune cells within or around the tumor. In some embodiments, the presence of the biomarker is detected by IHC in >0% of the samples, in at least 1% of the samples, in at least 5% of the samples, in at least 10% of the samples, in at least 15% of the samples, in at least 20% of the samples, in at least 25% of the samples, in at least 30% of the samples, in at least 35% of the samples, in at least 40% of the samples, in at least 45% of the samples, in at least 50% of the samples, in at least 55% of the samples, in at least 60% of the samples, in at least 65% of the samples, in at least 70% of the samples, in at least 75% of the samples, in at least 80% of the samples, in at least 85% of the samples, in at least 90% of the samples, in at least 95% or more of the samples. Any method known in the art can be used, e.g., scoring the samples by a pathologist or by automated image analysis.

[0415] In some embodiments of any method, the biomarker is detected by immunohistochemistry using a diagnostic antibody (i.e., a primary antibody). In some embodiments, the diagnostic antibody specifically binds to a human antigen. In some embodiments, the diagnostic antibody is a non-human antibody. In some embodiments, the diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the diagnostic antibody is a rabbit antibody. In some embodiments, the diagnostic antibody is a monoclonal antibody. In some embodiments, the diagnostic antibody is directly labeled. In other embodiments, the diagnostic antibody is indirectly labeled (e.g., via a secondary antibody).

[0416] In other embodiments of any of the foregoing methods, the expression level of the biomarker can be a nucleic acid expression level (e.g., a DNA expression level or an RNA expression level (e.g., an mRNA expression level)). Any suitable method for determining nucleic acid expression levels can be used. In some embodiments, nucleic acid expression levels are determined using RNAseq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or combinations thereof.

[0417] Methods for assessing mRNA in cells are well known and include, for example, serial analysis of gene expression (SAGE), whole genome sequencing (WGS), hybridization assays using complementary DNA probes (such as in situ hybridization using labeled ribonucleoproteins specific for one or more genes, Northern blotting, and related techniques), and various nucleic acid amplification assays (such as RT-PCR (e.g., qRT-PCR) using complementary primers specific for one or more genes, and other amplification type detection methods, e.g., branched DNA, SISBA, TMA, etc.). Additionally, such methods can include one or more steps that allow a person to determine the level of target mRNA in a biological sample (e.g., by simultaneously examining the levels of comparative control mRNA sequences of "housekeeping" genes such as actin family members). Optionally, the sequence of the amplified target cDNA can be determined. Optional methods include protocols for examining or detecting mRNA (e.g., target mRNA) in tissue or cell samples by microarray technology. Using a nucleic acid microarray, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to produce cDNA probes. The probes are then hybridized to a nucleic acid array immobilized on a solid support. The array is configured such that the order and position of each component of the array are known. For example, genes (whose expression is associated with an increase or decrease in the clinical benefit of treatments including immunotherapy and inhibitory matrix antagonists) can be selected and arranged on the solid support. Hybridization of the labeled probe to a specific array component indicates that the sample from which the probe was derived expresses that gene.

[0418] Samples can be obtained from a subject at any suitable time. For example, in some embodiments, samples can be obtained from a subject prior to administration of a treatment regimen (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years prior). In some embodiments of any of the foregoing methods, samples from a subject are obtained about 2 to about 10 weeks (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks) after administration of a treatment regimen. In some embodiments, samples from a subject are obtained about 4 to about 6 weeks after administration of a treatment regimen.

[0419] In some embodiments, the expression level or amount of a biomarker (e.g., PD-L1) is detected in a tissue sample, primary or cultured cells or cell lines, cell supernatant, cell lysate, 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 lysate, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, or any combination thereof. In some embodiments, the sample is a tissue sample (e.g., a tumor tissue sample), a cell sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the tumor tissue sample, wherein 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.

[0420] In some instances, the sample can be a cytology sample (e.g., a fine needle aspirate).

[0421] For example, in some embodiments, known techniques (e.g., IHC, immunofluorescence microscopy, or flow cytometry) are used to detect the expression level of a biomarker (e.g., PD-L1) in tumor-infiltrating immune cells, tumor cells, PBMCs, or a combination thereof. Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, or any combination thereof, or other tumor stromal cells (e.g., fibroblasts). Such tumor-infiltrating immune cells can be T lymphocytes (such as CD8 + T lymphocytes (e.g., CD8 + T effector (Teff) cells) and / or CD4 + T lymphocytes (e.g., CD4 + Teff cells)), B lymphocytes, or other myeloid lineage cells, including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (e.g., finger-like dendritic cells), histiocytes, and natural killer (NK) cells. In some embodiments, staining for the biomarker is detected due to membrane staining, cytoplasmic staining, or a combination thereof. In other embodiments, the absence of the biomarker is detected due to the absence or lack of staining in the sample.

[0422] In certain embodiments, the expression level of a biomarker is assayed in a sample that contains or is suspected of containing cancer cells. The sample can be, for example, a tissue biopsy or a metastatic lesion obtained from a subject having, suspected of having, or diagnosed with cancer (e.g., bladder cancer (e.g., UC, including locally advanced or metastatic UC)). In some embodiments, the sample is a tissue sample (e.g., renal pelvis, ureter, bladder, and / or urethra tissue), a biopsy of a tumor (e.g., a locally advanced or metastatic UC tumor, including pelvic, ureter, bladder, and / or urethra tumors), a known or suspected metastatic bladder cancer (e.g., metastatic UC) lesion or section, or a blood sample, e.g., a peripheral blood sample known or suspected of containing circulating cancer cells, such as bladder cancer cells (e.g., UC cells, including locally advanced or metastatic UC cells). The sample can contain both cancer cells (i.e., tumor cells) and non-cancer cells (e.g., lymphocytes, such as T cells or NK cells), and in certain embodiments, contains both cancer cells and non-cancer cells. Methods for obtaining biological samples (tissue resections, biopsy samples, and body fluids (e.g., blood samples containing cancer / tumor cells)) are well known in the art.

[0423] Samples can be obtained from patients with any suitable cancer (e.g., bladder cancer (e.g., UC, including mUC; MIBC and NMIBC); kidney or renal cancer (e.g., RCC); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; urothelial cancer; breast cancer (e.g., HER2+ breast cancer and TNBC, where TNBC is ER-, PR-, and HER2-); prostate cancer, such as CRPC; peritoneal cancer; hepatocellular carcinoma; gastric / stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., PDAC); glioblastoma; cervical cancer; ovarian cancer; liver cancer (e.g., HCC); hepatoma; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma, including superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade / follicular NHL; SLNHL; intermediate / follicular NHL; intermediate diffuse NHL; high-grade immunoblastic NHL; high-grade lymphocytic NHL; high-grade small non-cleaved cell NHL; large mass NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia); CLL; ALL; AML; hairy cell leukemia; CML; PTLD; and MDS, as well as abnormal angiogenesis associated with phakomatosis, edema (such as diseases associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and related metastases). In some instances, the cancer is bladder cancer (e.g., UC, including locally advanced or metastatic UC).

[0424] In certain embodiments, the subject may have advanced, refractory, recurrent, and / or chemotherapy-resistant forms of cancer.

[0425] In certain embodiments, the presence and / or expression level / amount of a biomarker in a first sample is increased or elevated compared to the presence / absence and / or expression level / amount in a second sample. In certain embodiments, the presence / absence and / or expression level / amount of a biomarker in a first sample is decreased or reduced compared to the presence and / or expression level / amount in a second sample. In certain embodiments, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0426] In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combination of multiple samples from the same subject, which are obtained at one or more time points different from when the test sample is obtained. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject at a time point earlier than when the test sample is obtained. If the reference sample is obtained during the initial diagnosis of cancer and the test sample is obtained later when cancer metastasizes, such reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful.

[0427] In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more healthy individuals who are not the subject. In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more individuals who have a disease or disorder (such as cancer) that is not the subject. In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from normal tissues or pooled plasma or serum samples from one or more individuals who are not the subject. In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from tumor tissues or a pooled plasma or serum sample from one or more individuals who have a disease or disorder (such as cancer) that is not the subject.

[0428] In some embodiments, the method further comprises, for example, administering to the subject an effective amount of a treatment regimen described herein (such as a treatment regimen comprising a PD-1 axis binding antagonist (such as an anti-PD-L1 antibody (such as atezolizumab)) and / or a platinum-based chemotherapy (such as cisplatin or carboplatin and gemcitabine)) based on the expression level of one or more biomarkers (such as PD-L1). The treatment regimen can be any treatment regimen described herein, for example, in Section II above.

[0429] The presence and / or expression level of PD-L1 in a subject being treated with any method and composition described herein can be evaluated. In one embodiment, the method comprises determining the expression level of PD-L1 in a biological sample (such as a tumor sample) obtained from the subject. In other embodiments, the expression level of PD-L1 in a biological sample (such as a tumor sample) obtained from the subject has been determined prior to the initiation of treatment. In still other embodiments, the expression level of PD-L1 in a biological sample (such as a tumor sample) obtained from the subject can be determined after the initiation of treatment.

[0430] V.PD-1 axis binding antagonist

[0431] The PD-1 axis binding antagonist may include a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist. Any suitable PD-1 axis binding antagonist can be used.

[0432] Provided herein are methods for treating or delaying the progression of bladder cancer (e.g., UC, including locally advanced or metastatic UC) in a subject, the method comprising administering to the subject a treatment regimen comprising an effective amount of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). Also provided herein are methods for treating or delaying the progression of bladder cancer (e.g., UC, including locally advanced or metastatic UC) in a subject, the method comprising administering to the subject a treatment regimen comprising an effective amount of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine). Also provided herein are methods for enhancing the immune function of a subject having bladder cancer (e.g., UC, including locally advanced or metastatic UC), the method comprising administering to the subject a treatment regimen comprising an effective amount of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). Further provided herein are methods for enhancing the immune function of a subject having bladder cancer (e.g., UC, including locally advanced or metastatic UC), the method comprising administering to the subject a treatment regimen comprising an effective amount of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine). Also provided are related compositions for use (e.g., pharmaceutical compositions), kits, and articles. Any method, composition for use, kit, or article described herein may include or relate to any of the following PD-1 axis binding antagonists.

[0433] A.PD-L1 binding antagonist

[0434] In some cases, a PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In other cases, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In still other cases, a PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some cases, a PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. The PD-L1 binding antagonist can be, but is not limited to, an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, or a small molecule. In some cases, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 (e.g., GS-4224, INCB086550, MAX-10181, INCB090244, CA-170, or ABSK041). In some cases, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 and VISTA. In some cases, the PD-L1 binding antagonist is CA-170 (also known as AUPM-170). In some cases, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 and TIM3. In some cases, the small molecule is a compound described in WO 2015 / 033301 and WO 2015 / 033299.

[0435] In some cases, the PD-L1 binding antagonist is an anti-PD-L1 antibody. A variety of anti-PD-L1 antibodies are contemplated and described herein. In any case herein, an isolated anti-PD-L1 antibody can bind to human PD-L1 (e.g., human PD-L1 as shown in UniProtKB / Swiss-Prot accession number Q9NZQ7-1, or a variant thereof). In some cases, the anti-PD-L1 antibody is capable of inhibiting the binding between PD-L1 and PD-1 and / or between PD-L1 and B7-1. In some 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 that are groups 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. Exemplary anti-PD-L1 antibodies include atezolizumab, MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), SHR-1316, CS1001, envafolimab, TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, cosibelimab, lodalisumab, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, RC98, PDL-GEX, KD036, KY1003, YBL-007, and HS-636. Examples of anti-PD-L1 antibodies useful in the methods of the invention and methods for their preparation are described in International Patent Application Publication No. WO2010 / 077634 and U.S. Patent No. 8,217,149, each of which is incorporated herein by reference in its entirety.

[0436] In some cases, the anti-PD-L1 antibody comprises:

[0437] (a) HVR-H1, HVR-H2, and HVR-H3 sequences that are GFTFSDSWIH (SEQ ID NO:3), AWISPYGGSTYYADSVKG (SEQ ID NO:4), and RHWPGGFDY (SEQ ID NO:5), respectively, and

[0438] (b) HVR-L1, HVR-L2, and HVR-L3 sequences that are RASQDVSTAVA (SEQ ID NO:6), SASFLYS (SEQ ID NO:7), and QQYLYHPAT (SEQID NO:8), respectively.

[0439] In one embodiment, the anti-PD-L1 antibody comprises:

[0440] (a) A heavy chain variable region (VH) comprising the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWV AWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:9), and

[0441] (b) A light chain variable region (VL) comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:10).

[0442] In some cases, the anti-PD-L1 antibody comprises (a) a VH 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:9, or comprising the sequence of SEQ ID NO:9; (b) a VL 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:10, or comprising the sequence of SEQ ID NO:10; or (c) a VH as described in (a) and a VL as described in (b).

[0443] In one embodiment, the anti-PD-L1 antibody comprises atezolizumab, which comprises:

[0444] (a) The following heavy chain amino acid sequence:

[0445] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:1) and

[0446] (b) The following light chain amino acid sequence:

[0447] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:2).

[0448] In some cases, the anti-PD-L1 antibody is avelumab (CAS Registry Number: 1537032-82-8). Avelumab, also known as MSB0010718C, is a human monoclonal IgG1 anti-PD-L1 antibody (Merck KGaA, Pfizer).

[0449] In some cases, the anti-PD-L1 antibody is durvalumab (CAS Registry Number: 1428935-60-7). Durvalumab, also known as MEDI4736, is an Fc-optimized human monoclonal IgG1 kappa anti-PD-L1 antibody as described in WO 2011 / 066389 and US2013 / 034559 (MedImmune, AstraZeneca).

[0450] In some cases, the anti-PD-L1 antibody is MDX-1105 (Bristol Myers Squibb). MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody as described in WO 2007 / 005874.

[0451] In some cases, the anti-PD-L1 antibody is LY3300054 (Eli Lilly).

[0452] In some cases, the anti-PD-L1 antibody is STI-A1014 (Sorrento). STI-A1014 is a human anti-PD-L1 antibody.

[0453] In some cases, the anti-PD-L1 antibody is KN035 (Suzhou Alphamab). KN035 is a single domain antibody (dAB) generated from a camelid phage display library.

[0454] In some cases, the anti-PD-L1 antibody comprises a cleavable moiety or linker that, when cleaved (e.g., by a protease in the tumor microenvironment), activates the antibody antigen-binding domain to bind its antigen, e.g., by removing a non-binding spacer portion. In some cases, the anti-PD-L1 antibody is CX-072 (CytomX Therapeutics).

[0455] In some cases, the anti-PD-L1 antibody comprises six HVR sequences (e.g., three heavy-chain HVRs and three light-chain HVRs) and / or heavy-chain variable domain and light-chain variable domain from anti-PD-L1 antibodies described in the following patents: US20160108123, WO 2016 / 000619, WO 2012 / 145493, U.S. Patent No. 9,205,148, WO 2013 / 181634, or WO 2016 / 061142.

[0456] In a further specific aspect, the anti-PD-L1 antibody has reduced or minimal effector function. In a further specific aspect, the minimal effector function results from an "effectorless Fc mutation" or a deglycosylation mutation. In a further case, the effectorless Fc mutation is an N297A or D265A / N297A substitution in the constant region. In a further case, the effectorless Fc mutation is an N297A substitution in the constant region. In some cases, the isolated anti-PD-L1 antibody is deglycosylated. Glycosylation of the antibody is generally N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid (most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used). Glycosylation sites can be conveniently removed from the antibody by altering the amino acid sequence to remove one of the above tripeptide sequences (for N-linked glycosylation sites). Mutations can be made by substituting the asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

[0457] B. PD-1 Binding Antagonist

[0458] In some cases, the PD-1 axis binding antagonist is a PD-1 binding antagonist. For example, in some cases, the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners. In some cases, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In other cases, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In still other cases, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. The PD-1 binding antagonist can be, but is not limited to, an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, or a small molecule. In some cases, 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)). For example, in some cases, the PD-1 binding antagonist is an Fc fusion protein. In some cases, the PD-1 binding antagonist is AMP-224. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342. In some cases, the PD-1 binding antagonist is a peptide or a small molecule compound. In some cases, the PD-1 binding antagonist is AUNP-12 (Pierre Fabre / Aurigene). See, for example, WO 2012 / 168944, WO 2015 / 036927, WO2015 / 044900, WO 2015 / 033303, WO 2013 / 144704, WO 2013 / 132317, and WO 2011 / 161699. In some cases, the PD-1 binding antagonist is a small molecule that inhibits PD-1.

[0459] In some cases, the PD-1 binding antagonist is an anti-PD-1 antibody. A variety of anti-PD-1 antibodies can be utilized in the methods and uses disclosed herein. In any case herein, the PD-1 antibody can bind to human PD-1 or a variant thereof. In some cases, the anti-PD-1 antibody is a monoclonal antibody. In some cases, the anti-PD-1 antibody is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, scFv, and (Fab') 2Fragment. In some cases, the anti-PD-1 antibody is a humanized antibody. In other cases, the anti-PD-1 antibody is a human antibody. Exemplary anti-PD-1 antagonist antibodies include nivolumab, pembrolizumab, MEDI-0680, PDR001 (spartalizumab), REGN2810 (cemiplimab), BGB-108, palorolizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, rivulizumab, sasanlimab, penpulimab, CS1003, HLX10, SCT-I10A, sepaclimab, batrilimab, genolimab, BI 754091, cilimab, YBL-006, BAT1306, HX008, bugulimab, AMG 404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103, and hAb21.

[0460] In some cases, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). Nivolumab (Bristol-Myers Squibb / Ono Pharmaceutical Co., Ltd.), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and is the anti-PD-1 antibody described in WO 2006 / 121168.

[0461] In some cases, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4). Pembrolizumab (Merck), also known as MK-3475, Merck 3475, pembrolizumab, SCH-900475, and is the anti-PD-1 antibody described in WO 2009 / 114335.

[0462] In some cases, the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.

[0463] In some cases, the anti-PD-1 antibody is PDR001 (CAS Registration Number 1859072-53-9; Novartis). PDR001 is a humanized IgG4 anti-PD-1 antibody that can block the binding of PD-L1 and PD-L2 to PD-1.

[0464] In some cases, the anti-PD-1 antibody is REGN2810 (Regeneron). REGN2810 is a human anti-PD-1 antibody.

[0465] In some cases, the anti-PD-1 antibody is BGB-108 (BeiGene).

[0466] In some cases, the anti-PD-1 antibody is BGB-A317 (BeiGene).

[0467] In some cases, the anti-PD-1 antibody is JS-001 (Shanghai Junshi Biosciences). JS-001 is a humanized anti-PD-1 antibody.

[0468] In some cases, the anti-PD-1 antibody is STI-A1110 (Sorrento Therapeutics). STI-A1110 is a human anti-PD-1 antibody.

[0469] In some cases, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR-1210 is a human IgG4 anti-PD-1 antibody.

[0470] In some cases, the anti-PD-1 antibody is PF-06801591 (Pfizer).

[0471] In some cases, the anti-PD-1 antibody is TSR-042 (also known as ANB011; Tesaro / AnaptysBio).

[0472] In some cases, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).

[0473] In some cases, the anti-PD-1 antibody is ENUM 244C8 (Enumeral Biomedical Holdings). ENUM 244C8 is an anti-PD-1 antibody that inhibits the function of PD-1 without preventing the binding of PD-L1 to PD-1.

[0474] In some cases, the anti-PD-1 antibody is ENUM 388D4 (Enumeral Biomedical Holdings). ENUM 388D4 is an anti-PD-1 antibody that competitively inhibits the binding of PD-L1 to PD-1.

[0475] In some cases, the anti-PD-1 antibody comprises six HVR sequences (e.g., three heavy-chain HVRs and three light-chain HVRs) and / or the heavy-chain variable domain and the light-chain variable domain from the anti-PD-1 antibodies described in the following patents: WO 2015 / 112800, WO 2015 / 112805, WO 2015 / 112900, US20150210769, WO2016 / 089873, WO 2015 / 035606, WO 2015 / 085847, WO 2014 / 206107, WO 2012 / 145493, US 9,205,148, WO 2015 / 119930, WO 2015 / 119923, WO 2016 / 032927, WO 2014 / 179664, WO 2016 / 106160, and WO 2014 / 194302.

[0476] In a further specific aspect, the anti-PD-1 antibody has reduced or minimal effector function. In a further specific aspect, the minimal effector function results from an "effectorless Fc mutation" or a deglycosylation mutation. In a further case, the effectorless Fc mutation is an N297A or D265A / N297A substitution in the constant region. In some cases, the isolated anti-PD-1 antibody is aglycosylated.

[0477] C. PD-L2 binding antagonist

[0478] In some cases, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some cases, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its ligand-binding partner. In a specific aspect, the PD-L2 binding ligand partner is PD-1. The PD-L2 binding antagonist can be, but is not limited to, an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, or a small molecule.

[0479] In some cases, the PD-L2 binding antagonist is an anti-PD-L2 antibody. In any case herein, the anti-PD-L2 antibody can bind to human PD-L2 or a variant thereof. In some cases, the anti-PD-L2 antibody is a monoclonal antibody. In some cases, the anti-PD-L2 antibody is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, scFv, and (Fab') 2Fragment. In some cases, the anti-PD-L2 antibody is a humanized antibody. In other cases, the anti-PD-L2 antibody is a human antibody. In a further specific aspect, the anti-PD-L2 antibody has reduced or minimal effector function. In a further specific aspect, the minimal effector function results from a "Fc mutation without effector" or a deglycosylation mutation. In a further case, the Fc mutation without effector is an N297A or D265A / N297A substitution in the constant region. In some cases, the isolated anti-PD-L2 antibody is deglycosylated.

[0480] It is expressly contemplated that such PD-L1 axis-binding antagonist antibodies (e.g., anti-PD-L1 antibodies, anti-PD-1 antibodies, and anti-PD-L2 antibodies) or other antibodies for any of the above-listed cases described herein may individually or in combination have any of the features described in Sections 1 to 7 below.

[0481] 1. Antibody Affinity

[0482] In certain cases, the antibodies described herein (e.g., anti-PD-L1 antibodies) have a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0483] In one case, the Kd is measured by a radiolabeled antigen-binding assay (RIA). In one example, the Fab form of the antibody of interest and its antigen are used for the RIA. For example, the solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration ( 125 I) of radiolabeled antigen in the presence of a series of unlabeled antigen titrations and then capturing the bound antigen with a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions for the assay, microtiter plates (Thermo Scientific) are coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM 125 ​The I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., following the assessment of the anti-VEGF antibody (Fab-12) in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., for one hour). Subsequently, the solution is removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN- ) in PBS. When the plate is dry, 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard) is added, and the plate is counted for ten minutes on a TOPCOUNT TM γ counter (Packard). The concentration of each Fab that gives less than or equal to 20% of the maximum binding is selected for use in the competitive binding assay.

[0484] In another case, the Kd is measured using surface plasmon resonance assay. For example, using -2000 or -3000 (BIAcore, Inc., Piscataway, NJ), the assay is performed at 25 °C with a fixed antigen CM5 chip at approximately 10 response units (RU). In one case, according to the supplier's instructions, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of the coupled protein. After injecting the antigen, 1 M ethanolamine is injected to block the unreacted groups. For kinetic measurements, at 25 °C, a two-fold serial dilution (0.78 nM to 500 nM) of the Fab in PBS containing 0.05% polysorbate 20 (TWEEN 20 TM ) surfactant (PBST) is injected at a flow rate of approximately 25 μL / min. Using a simple one-to-one Langmuir binding model ( Evaluation Software version 3.2), the association rate (k on ) and dissociation rate (k off ) are calculated by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k off / k onSee, e.g., Chen et al., J. Mol. Biol. 293:865 - 881 (1999). If the association rate obtained by the above surface plasmon resonance assay exceeds 10 6 M -1 s -1 , then the association rate can be determined by using fluorescence quenching techniques, i.e., as measured in a spectrometer such as a spectrophotometer equipped with a stopped - flow device (Aviv Instruments) or an 8000 series SLM - AMINCO TM spectrophotometer (ThermoSpectronic) with a stirred cuvette, by measuring the increase or decrease in the fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM antigen - specific antibody (Fab form) in PBS pH 7.2 at 25 °C in the presence of increasing concentrations of antigen.

[0485] 2. Antibody Fragments

[0486] In certain cases, the antibodies described herein (e.g., anti - PD - L1 antibodies) are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab' - SH, F(ab') 2 , Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129 - 134 (2003). For a review of scFv fragments, see, e.g., Pluckthün as described in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenberg and Moore, (Springer - Verlag, New York), pp. 269 - 315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') 2 fragments that contain salvage receptor - binding epitope residues and have an increased in vivo half - life, see U.S. Patent No. 5,869,046.

[0487] Bispecific antibodies are antibody fragments that have two antigen-binding sites and can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Trispecific and tetravalent antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0488] Single-domain antibodies are antibody fragments that comprise all or part of the heavy-chain variable domain or all or part of the light-chain variable domain of an antibody. In some cases, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0489] Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0490] 3. Chimeric and Humanized Antibodies

[0491] In some cases, the antibodies described herein (e.g., anti-PD-L1 antibodies) are chimeric antibodies. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class-switch" antibody in which the class or subclass has been changed from that of the parental antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0492] In some cases, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or parts thereof), are derived from a non-human antibody and the FRs (or parts thereof) are derived from human antibody sequences. A humanized antibody optionally also will comprise at least a portion of a human constant region. In some instances, some FR residues in a humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0493] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specific determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing "directed selection" methods for FR shuffling).

[0494] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions from consensus sequences of human antibodies from specific subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0495] 4. Human Antibodies

[0496] In some cases, the antibodies described herein (e.g., anti-PD-L1 antibodies) are human antibodies. A variety of techniques known in the art can be used to generate human antibodies. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0497] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a complete human antibody or a complete antibody with human variable regions in response to antigenic stimulation. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or is present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSE TM technology; U.S. Patent No. 5,770,429, which describes the technology; U.S. Patent No. 7,041,870, which describes the K-M technology, and U.S. Patent Application Publication No. US2007 / 0061900, which describes the technology. The human variable regions of the complete antibodies produced by such animals can be further modified, e.g., by combining with different human constant regions.

[0498] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines have been described for the production of human monoclonal antibodies. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0499] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0500] 5. Antibodies from Libraries

[0501] Antibodies (e.g., anti-PD-L1 antibodies) can be isolated by screening a combinatorial library of antibodies for one or more desired activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries to obtain antibodies with desired binding characteristics. Such methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001) and further described in the following references: for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34); 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0502] In some phage display methods, all components of the VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombi...

Claims

1. A method of identifying a tumor that may respond to a PD-1 axis-binding antagonist, the method comprising: (a) staining a first portion of the tumor with an immuno-guided PD-L1 assay to obtain a first stained sample; (b) generating a first score by applying a first scoring algorithm to the first stained sample; (c) staining a second portion of the tumor with an immuno-agnostic PD-L1 assay to obtain a second stained sample; (d) generating a second score by applying a second scoring algorithm to the second stained sample; and (e) comparing the first score to a first cut-off value and the second score to a second cut-off value, wherein the tumor may respond to the PD-1 axis-binding antagonist when both the first score meets or exceeds the first cut-off value and the second score meets or exceeds the second cut-off value.

2. The method according to claim 1, wherein the tumor is in a patient, and the method further comprises administering to the patient a treatment regimen comprising the PD-1 axis-binding antagonist.

3. A method of treating cancer in a patient, the method comprising administering to the patient a treatment regimen comprising a PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have: (i) a first score obtained by applying a first scoring algorithm to a first stained portion of the tumor sample that meets or exceeds a first cut-off value, wherein the first stained portion of the tumor sample is stained with an immuno-guided PD-L1 assay; and (ii) a second score obtained by applying a second scoring algorithm to a second stained portion of the tumor sample that meets or exceeds a second cut-off value, wherein the second stained portion of the tumor sample is stained with an immuno-agnostic PD-L1 assay.

4. The method according to any one of claims 1 to 3, wherein the immuno-guided PD-L1 assay has: (i) at least 80%, at least 85%, at least 90% or at least 95% overall percent agreement (OPA) with the SP142 assay using the first scoring algorithm at the first cut-off value; (ii) at least 80%, at least 85%, at least 90% or at least 95% positive percent agreement (PPA) with the SP142 assay using the first scoring algorithm at the first cut-off value; (iii) at least 80%, at least 85%, at least 90% or at least 95% negative percent agreement (NPA) with the SP142 assay using the first scoring algorithm at the first cut-off value; (iv) at least 80%, at least 85%, at least 90% or at least 95% PPA and at least 80%, at least 85%, at least 90% or at least 95% NPA with the SP142 assay using the first scoring algorithm at the first cut-off value; (v) at least 80%, at least 85%, at least 90% or at least 95% PPA and at least 80%, at least 85%, at least 90% or at least 95% OPA of the SP142 assay using the first scoring algorithm at the first cut-off value; (vi) at least 80%, at least 85%, at least 90% or at least 95% NPA and at least 80%, at least 85%, at least 90% or at least 95% OPA of the SP142 assay using the first scoring algorithm at the first cut-off value; and / or (vii) at least 80%, at least 85%, at least 90% or at least 95% OPA; at least 80%, at least 85%, at least 90% or at least 95% PPA; and at least 80%, at least 85%, at least 90% or at least 95% NPA of the SP142 assay using the first scoring algorithm at the first cut-off value.

5. The method according to any one of claims 1 to 4, wherein the immune-agnostic PD-L1 assay has: (i) at least 80%, at least 85%, at least 90% or at least 95% OPA of the 22C3 assay using the second scoring algorithm at the second cut-off value; (ii) at least 80%, at least 85%, at least 90% or at least 95% PPA of the 22C3 assay using the second scoring algorithm at the second cut-off value; (iii) at least 80%, at least 85%, at least 90% or at least 95% NPA of the 22C3 assay using the second scoring algorithm at the second cut-off value; (iv) at least 80%, at least 85%, at least 90% or at least 95% PPA and at least 80%, at least 85%, at least 90% or at least 95% NPA of the 22C3 assay using the second scoring algorithm at the second cut-off value; (v) at least 80%, at least 85%, at least 90% or at least 95% PPA and at least 80%, at least 85%, at least 90% or at least 95% OPA of the 22C3 assay using the second scoring algorithm at the second cut-off value; (vi) at least 80%, at least 85%, at least 90% or at least 95% NPA and at least 80%, at least 85%, at least 90% or at least 95% OPA of the 22C3 assay using the second scoring algorithm at the second cut-off value; and / or (vii) at least 80%, at least 85%, at least 90% or at least 95% OPA; at least 80%, at least 85%, at least 90% or at least 95% PPA; and at least 80%, at least 85%, at least 90% or at least 95% NPA of the 22C3 assay using the second scoring algorithm at the second cut-off value.

6. A method of stratifying tumors having a score above a predetermined cut-off value by an immune-agnostic PD-L1 assay, the method comprising: (a) staining a portion of the tumor with an immune-guided PD-L1 assay to obtain a stained sample; (b) Generating an immunoguided PD-L1 assay score by applying a scoring algorithm to the stained sample; and (c) Comparing the immunoguided PD-L1 assay score with a first cutoff value, wherein when the immunoguided PD-L1 assay score reaches or exceeds the first cutoff value, the tumor may respond to a PD-1 axis-binding antagonist.

7. A method of stratifying tumors with a combined positive score (CPS) ≥ 10% as determined by the 22C3 assay, the method comprising: (a) Staining a portion of the tumor with an immunoguided PD-L1 assay to obtain a stained sample; (b) Generating an immunoguided PD-L1 assay score by applying a scoring algorithm to the stained sample; and (c) Comparing the immunoguided PD-L1 assay score with a first cutoff value, wherein when the immunoguided PD-L1 assay score reaches or exceeds the first cutoff value, the tumor may respond to the PD-1 axis-binding antagonist.

8. A method of stratifying tumors with a CPS ≥ 10% as determined by the SP263 assay, the method comprising: (a) Staining a portion of the tumor with an immunoguided PD-L1 assay to obtain a stained sample; (b) Generating an immunoguided PD-L1 assay score by applying a scoring algorithm to the stained sample; and (c) Comparing the immunoguided PD-L1 assay score with a first cutoff value, wherein when the immunoguided PD-L1 assay score reaches or exceeds the first cutoff value, the tumor may respond to the PD-1 axis-binding antagonist.

9. A method of stratifying tumors with a CPS ≥ 10% as determined by the 28-8 assay, the method comprising: (a) Staining a portion of the tumor with an immunoguided PD-L1 assay to obtain a stained sample; (b) Generating an immunoguided PD-L1 assay score by applying a scoring algorithm to the stained sample; and (c) Comparing the immunoguided PD-L1 assay score with a first cutoff value, wherein when the immunoguided PD-L1 assay score reaches or exceeds the first cutoff value in two cases, the tumor may respond to the PD-1 axis-binding antagonist.

10. The method according to any one of claims 1 to 9, wherein the tumor is bladder cancer, kidney cancer, lung cancer, urothelial cancer, breast cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatic carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal cancer, penile cancer, melanoma, multiple myeloma or B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia, chronic myelogenous leukemia (CML), post-transplant lymphoproliferative disorder (PTLD), myelodysplastic syndrome (MDS), Meigs syndrome, brain cancer or head and neck cancer.

11. The method according to claim 10, wherein the tumor is bladder cancer.

12. The method according to claim 11, wherein the bladder cancer is urothelial carcinoma (UC).

13. The method according to claim 12, wherein the UC is locally advanced or metastatic UC.

14. The method according to any one of claims 1 to 13, wherein the immune-guided PD-L1 assay comprises a PD-L1 immunohistochemistry (IHC) assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

15. The method according to any one of claims 1 to 6 and 14, wherein the immune-agnostic PD-L1 assay comprises a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody or the 28-8 anti-PD-L1 diagnostic antibody.

16. The method according to any one of claims 1 to 15, wherein the first cut-off value is a detectable PD-L1 expression level in tumor-infiltrating immune cells accounting for 5% or more of the tumor sample.

17. The method according to any one of claims 1 to 5 and 10 to 16, wherein the second cut-off value is CPS ≥ 10%.

18. A method of labeling PD-L1 in a tumor sample, the method comprising the steps of: (a) contacting the tumor sample with the VENTANA SP142 anti-PD-L1 diagnostic antibody; (b) contacting the tumor sample with the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody or the 28-8 anti-PD-L1 diagnostic antibody; and (c) visualizing the anti-PD-L1 diagnostic antibodies of steps (a) and (b) with one or more detectable reagents that generate a detectable signal for both of the anti-PD-L1 diagnostic antibodies.

19. The method according to claim 18, wherein the detectable signal for the VENTANA SP142 anti-PD-L1 diagnostic antibody is an amplified signal.

20. The method according to claim 19, wherein the amplified signal is generated by tyramide signal amplification.

21. The method according to any one of claims 18 to 20, wherein steps (a) and (b) are carried out simultaneously.

22. The method according to any one of claims 18 to 20, wherein steps (a) and (b) are carried out sequentially.

23. The method according to any one of claims 18 to 22, wherein steps (a) and (b) are carried out in different sections or in the same section of the tumor sample.

24. The method according to claim 23, wherein the different sections of the tumor sample are consecutive sections.

25. The method according to any one of claims 18 to 24, wherein the visualization comprises IHC or immunofluorescence (IF).

26. The method according to claim 25, wherein the visualization comprises IHC.

27. The method according to any one of claims 18 to 26, wherein the tumor sample is obtained from a patient suffering from cancer.

28. The method according to any one of claims 1, 2, and 4 to 17, wherein the tumor is a tumor sample obtained from a patient suffering from cancer.

29. The method according to any one of claims 3 to 5 and 18 to 28, wherein the tumor sample is an FFPE tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

30. The method according to any one of claims 3 to 5 and 27 to 29, wherein the cancer is bladder cancer, kidney cancer, lung cancer, urothelial cancer, breast cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal cancer, penile cancer, melanoma, multiple myeloma or B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia, chronic myelogenous leukemia (CML), post-transplant lymphoproliferative disorder (PTLD), myelodysplastic syndrome (MDS), Meigs syndrome, brain cancer or head and neck cancer.

31. The method according to claim 30, wherein the cancer is bladder cancer.

32. The method according to claim 31, wherein the bladder cancer is urothelial carcinoma (UC).

33. The method according to claim 32, wherein the UC is locally advanced or metastatic UC.

34. The method according to claim 33, wherein the patient has not been previously treated for the locally advanced or metastatic UC.

35. The method according to claim 33 or 34, wherein the locally advanced or metastatic UC is histologically documented, locally advanced (T4b, any N; or any T, N2-3) or metastatic urothelial carcinoma (mUC) (M1, stage IV).

36. The method according to any one of claims 33 to 35, wherein the cancer is locally advanced UC.

37. The method according to claim 13 and any one of claims 33 to 36, wherein the locally advanced UC is inoperable.

38. The method according to any one of claims 33 to 35, wherein the cancer is metastatic UC.

39. The method according to any one of claims 33 to 38, wherein the patient is eligible for treatment with platinum-based chemotherapy.

40. The method according to claim 39, wherein the patient is eligible for treatment with cisplatin-based chemotherapy.

41. The method according to claims 33 to 40, wherein the patient has not previously received chemotherapy for the locally advanced or metastatic UC.

42. The method according to any one of claims 33 to 40, wherein the patient has previously received adjuvant or neoadjuvant chemotherapy or chemoradiotherapy for UC, and has had a treatment-free interval of more than 12 months between the last administration of the adjuvant or neoadjuvant chemotherapy or chemoradiotherapy and the date of recurrence.

43. The method according to any one of claims 1, 2, and 4 to 42, wherein the method identifies the patient as a patient likely to benefit from a treatment regimen comprising a PD-1 axis-binding antagonist.

44. The method according to any one of claims 4 to 43, further comprising administering to the patient a treatment regimen comprising a PD-1 axis-binding antagonist.

45. The method according to any one of claims 1 to 5, 43, and 44, wherein the PD-1 axis-binding antagonist is a PD-L1-binding antagonist, a PD-1-binding antagonist, or a PD-L2-binding antagonist.

46. The method according to claim 45, wherein the PD-1 axis-binding antagonist is a PD-L1-binding antagonist.

47. The method according to claim 46, wherein the PD-1 axis-binding antagonist is an anti-PD-L1 antibody.

48. The method according to claim 47, wherein the anti-PD-L1 antibody comprises the following HVRs: (i) The HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) The HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) The HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) The HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) The HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) The HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8).

49. The method according to claim 47 or 48, wherein the anti-PD-L1 antibody comprises: (a) A VH domain comprising the amino acid sequence of SEQ ID NO:9; and (b) A VL domain comprising the amino acid sequence of SEQ ID NO:

10.

50. The method according to any one of claims 47 to 49, wherein the anti-PD-L1 antibody is atezolizumab.

51. The method according to claim 50, wherein atezolizumab is administered intravenously to the patient at a dose of about 840 mg every 2 weeks, about 1200 mg every 3 weeks, or about 1680 mg every 4 weeks.

52. The method according to claim 51, wherein atezolizumab is administered intravenously to the patient at a dose of about 1200 mg every 3 weeks.

53. The method according to claim 52, wherein atezolizumab is administered to the patient in a 21-day dosing cycle, and wherein atezolizumab is administered intravenously to the patient at a dose of about 1200 mg from day -2 to day 4 of each 21-day dosing cycle.

54. The method according to claim 53, wherein atezolizumab is administered intravenously to the patient at a dose of about 1200 mg on day 1 of each 21-day dosing cycle.

55. The method according to any one of claims 1 to 5 and 43 to 54, wherein the PD-1 axis-binding antagonist is administered to the patient as a single therapy.

56. The method according to any one of claims 2 to 5 and 43 to 54, wherein the PD-1 axis-binding antagonist is administered to the patient in combination with one or more additional therapeutic agents.

57. The method according to claim 56, wherein the one or more additional therapeutic agents include platinum-based chemotherapy.

58. The method according to claim 57, wherein the platinum-based chemotherapy includes a platinum-based chemotherapeutic agent and a nucleoside analogue.

59. The method according to claim 58, wherein the platinum-based chemotherapeutic agent is cisplatin, carboplatin, or oxaliplatin.

60. The method according to claim 59, wherein the platinum-based chemotherapeutic agent is cisplatin.

61. The method according to claim 59, wherein the platinum-based chemotherapeutic agent is carboplatin.

62. The method according to any one of claims 58 to 61, wherein the nucleoside analogue is gemcitabine.

63. The method according to any one of claims 58 to 62, wherein the platinum-based chemotherapy includes cisplatin and gemcitabine or carboplatin and gemcitabine.

64. The method according to claim 63, wherein the platinum-based chemotherapy includes cisplatin and gemcitabine.

65. The method according to claim 63, wherein the platinum-based chemotherapy includes carboplatin and gemcitabine.

66. The method according to any one of claims 43 to 65, wherein the benefit from the treatment regimen comprising the PD-1 axis-binding antagonist is in terms of overall survival (OS).

67. The method according to claim 66, wherein the treatment regimen prolongs the OS of the patient by about 5.7 months to about 17 months compared to treatment with platinum-based chemotherapy without using the PD-1 axis-binding antagonist.

68. The method according to claim 67, wherein the treatment regimen prolongs the OS of the patient by about 11.3 months compared to treatment with platinum-based chemotherapy without using the PD-1 axis-binding antagonist.

69. The method according to any one of claims 2 to 6 and 27 to 68, wherein the patient is a human.

70. The method according to any one of claims 3 to 5 and 16 to 69, wherein any intensity of distinguishable PD-L1 staining is present in tumor-infiltrating immune cells of the tumor sample obtained from the patient, covering ≥5% of the tumor area occupied by tumor cells, associated intratumoral stroma, and continuous peritumoral stroma, as determined by the PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

71. The method according to any one of claims 3 to 5 and 16 to 69, wherein the tumor sample obtained from the patient has a CPS of ≥10 using the PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody.

72. The method according to any one of claims 3 to 5 and 16 to 69, wherein the tumor sample obtained from the patient has a CPS of ≥10 using the PD-L1 IHC assay comprising the VENTANA SP263 anti-PD-L1 diagnostic antibody.

73. The method according to any one of claims 3 to 5 and 16 to 69, wherein the tumor sample obtained from the patient has a CPS of ≥10 using the PD-L1 IHC assay comprising the 28-8 anti-PD-L1 diagnostic antibody.

74. An assay for determining the presence or expression level of PD-L1 in a tumor sample obtained from a patient with cancer, the assay comprising: (a) determining the presence or expression level of PD-L1 in a tumor sample obtained from the patient using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) determining the presence or expression level of PD-L1 in the tumor sample obtained from the patient using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody.

75. The assay according to claim 74, wherein the tumor sample obtained from the patient has a detectable PD-L1 expression level in tumor-infiltrating immune cells that account for 5% or more of the tumor sample using the PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

76. The assay according to claim 74 or 75, wherein any intensity of distinguishable PD-L1 staining is present in tumor-infiltrating immune cells of the tumor sample obtained from the patient, covering ≥5% of the tumor area occupied by tumor cells, associated intratumoral stroma, and continuous peritumoral stroma, as determined by the PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

77. The assay according to any one of claims 74 to 76, wherein the tumor sample obtained from the patient has a CPS of ≥10 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, the VENTANA SP263 anti-PD-L1 diagnostic antibody, or the 28-8 anti-PD-L1 diagnostic antibody.

78. The assay according to any one of claims 74 to 77, wherein the tumor sample obtained from the patient has: a detectable PD-L1 expression level in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using the PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and a CPS of ≥10 using the PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody.

79. The assay according to any one of claims 74 to 78, wherein steps (a) and (b) are performed simultaneously.

80. The assay according to any one of claims 74 to 79, wherein steps (a) and (b) are performed sequentially.

81. The assay according to any one of claims 74 to 80, wherein steps (a) and (b) are performed in different sections or in the same section of the tumor sample.

82. The assay according to claim 81, wherein the different sections of the tumor sample are consecutive sections.

83. The assay according to any one of claims 74 to 82, wherein the cancer is locally advanced or metastatic urothelial carcinoma.

84. The assay according to claim 83, wherein the patient has not previously been treated for the locally advanced or metastatic urothelial carcinoma.

85. The assay according to any one of claims 74 to 84, wherein the assay is for (i) selecting a therapy for locally advanced or metastatic UC in a patient in need thereof, or (ii) identifying a patient with locally advanced or metastatic UC who is likely to benefit from a treatment regimen comprising an anti-PD-L1 antibody.

86. The assay according to claim 85, wherein the anti-PD-L1 antibody comprises the following HVRs: (i) the HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:3); (ii) the HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:4); (iii) the HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:5); (iv) the HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:6); (v) the HVR-L2 sequence of SASFLYS (SEQ ID NO:7); and (vi) the HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:8).

87. The assay according to any one of claims 74 to 86, wherein the tumor sample is a FFPE tumor sample, an archived tumor sample, a fresh tumor sample or a frozen tumor sample.

88. A kit, which comprises: (a) a VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) a Dako 22C3 anti-PD-L1 diagnostic antibody, a VENTANA SP263 anti-PD-L1 diagnostic antibody or a 28-8 anti-PD-L1 diagnostic antibody.

89. The kit according to claim 88, further comprising one or more reagents for visualizing the anti-PD-L1 diagnostic antibodies of (a) and (b).

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