Bladder cancer treatment and diagnosis methods

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 therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., atezolizumab). Also provided are compositions (e.g., PD-1 axis-binding antagonists (e.g., atezolizumab) and / or platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine), pharmaceutical compositions thereof, kits thereof, and articles of manufacture thereof) for use in treating bladder cancer (e.g., UC; including locally advanced or metastatic UC) in a patient. Additionally, 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 are provided.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, the entire contents of which are incorporated herein by reference. A copy of said XML, created on August 31, 2023, is named 50474-306WO2_Sequence_Listing_8_31_23.xml and is 12,341 bytes in size.

[0002] FIELD OF THE INVENTION The present invention relates to methods and compositions for use in treating bladder cancer (e.g., urothelial carcinoma (UC); including locally advanced or metastatic UC) in a subject, for example, by administering to the subject a therapeutic 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 technology]

[0003] Cancer remains one of the most deadly threats to human health. Cancer, or malignant tumors, rapidly metastasize and grow in an uncontrolled manner, making timely detection and treatment extremely difficult. Urothelial carcinoma (UC; also known as transitional cell carcinoma of the urinary tract (TCC), urothelial bladder cancer, or urothelial cell carcinoma (UCC)) is the most common urinary system cancer worldwide, with urothelial carcinoma of the bladder being the predominant histological type and location. Less commonly, urothelial carcinoma can also arise in the renal pelvis, ureter, or urethra. In the United States, bladder cancer was estimated to cause 74,000 cases and 16,000 deaths in 2015. Similar global data suggest that bladder cancer caused an estimated 123,000 deaths in men and 42,000 deaths in women in 2012. 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 stage at presentation, a Karnofsky Performance Status of less than 80%, and visceral metastases. Patients with these unfavorable features had a median survival of 4 months compared with 18 months for patients without these features.

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

[0005] Despite significant advances in the treatment of cancer (e.g., bladder cancer (e.g., UC; including locally advanced or metastatic UC)), improved therapies remain needed. Summary of the Invention

[0006] The present invention relates, inter alia, to methods of treating bladder cancer (e.g., UC; including locally advanced or metastatic UC) in a subject, and also to 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 of manufacture are also provided. Additionally, assays for determining the presence or expression level of PD-L1 in a tumor sample obtained from a subject afflicted with cancer, methods for labeling PD-L1 in a tumor sample, and methods for stratifying tumors are provided.

[0007] In certain aspects, provided herein is 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 the locally advanced or metastatic UC, and the method comprises administering to the patient a gene encoding one or more of 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). administering to the patient a therapeutic regimen comprising an anti-PD-L1 antibody, comprising:

[0008] In another aspect, 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) detecting a tumor sample obtained from the patient that has detectable expression levels of PD-L1 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 (b) detecting a tumor sample obtained from the patient that has detectable expression levels of PD-L1 in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody. (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 therapeutic regimen comprising an anti-PD-L1 antibody to the patient identified in step (a) as being likely to benefit from a therapeutic regimen comprising an anti-PD-L1 antibody.

[0009] (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 is immunohistochemically characterized. In another aspect, provided herein is an anti-PD-L1 antibody for use in treating locally advanced or metastatic UC in a patient in need of treatment, wherein the patient has not been previously treated for locally advanced or metastatic UC, and the treatment comprises administering to the patient a therapeutic regimen comprising 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), wherein a tumor sample obtained from the patient is immunohistochemically characterized. The patient has detectable expression levels of PD-L1 in tumor-infiltrating immune cells comprising 5% or more of the tumor sample, as determined using an IHC assay, and a CPS of 10 or greater, as determined using a PD-L1 IHC assay including the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as one who may benefit from a treatment regimen including an anti-PD-L1 antibody.

[0010] In another aspect, provided herein is an anti-PD-L1 antibody for use in a method 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) a tumor sample obtained from the patient has detectable expression levels of PD-L1 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 (b) a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody. (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 a therapeutic regimen comprising an anti-PD-L1 antibody to the patient identified in step (a) as being likely to benefit from a therapeutic regimen comprising an anti-PD-L1 antibody.

[0011] In another aspect, provided herein is a method for 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 the locally advanced or metastatic UC, the method comprising: (a) selecting a therapy for treating locally advanced or metastatic UC in a patient in need thereof, the patient having not been previously treated for the locally advanced or metastatic UC; and (b) selecting a therapy for treating locally advanced or metastatic UC in a patient in need thereof, the patient having not been previously treated for the locally advanced or metastatic UC; the method comprising: (a) selecting a therapy for treating locally advanced or metastatic UC in a patient having not been previously treated for the locally advanced or metastatic UC; and (b) selecting a therapy for treating locally advanced or metastatic UC in a patient having not been previously treated for the locally advanced or metastatic UC; (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) selecting a therapeutic regimen comprising an anti-PD-L1 antibody for the patient identified in step (a) as likely to benefit from a therapeutic regimen comprising an anti-PD-L1 antibody.

[0012] In another aspect, provided herein is a method of 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 the locally advanced or metastatic UC, and the method comprises: a tumor sample obtained from the patient having detectable expression levels of PD-L1 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 PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody. determining that the patient has a CPS of 10 or greater using an IHC assay, thereby identifying the patient as one who may benefit from a treatment regimen comprising an anti-PD-L1 antibody, where 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).

[0013] In some embodiments, the method further comprises administering to the subject a therapeutic regimen comprising an anti-PD-L1 antibody.

[0014] In some embodiments, the benefit from a treatment regimen comprising an anti-PD-L1 antibody is in terms of overall survival (OS).

[0015] In some embodiments, the treatment regimen extends the patient's OS by about 5.7 months to about 17 months compared to treatment with platinum-based chemotherapy that does not include an anti-PD-L1 antibody.

[0016] In some embodiments, the treatment regimen extends the subject's OS by about 11.3 months compared to treatment with platinum-based chemotherapy that does not include an anti-PD-L1 antibody.

[0017] In some aspects, the platinum-based chemotherapy comprises a platinum-based chemotherapeutic agent and a nucleoside analog.

[0018] In some embodiments, the platinum-based chemotherapeutic agent is cisplatin, carboplatin, or oxaliplatin.

[0019] In some embodiments, the platinum-based chemotherapeutic agent is cisplatin.

[0020] In some embodiments, the platinum-based chemotherapeutic agent is carboplatin.

[0021] In some embodiments, the nucleoside analog is gemcitabine.

[0022] In some embodiments, the platinum-based chemotherapy comprises cisplatin and gemcitabine, or carboplatin and gemcitabine.

[0023] In some embodiments, the platinum-based chemotherapy comprises cisplatin and gemcitabine.

[0024] In some embodiments, the platinum-based chemotherapy comprises carboplatin and gemcitabine.

[0025] In some embodiments, 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. Includes:

[0026] In some embodiments, the anti-PD-L1 antibody is atezolizumab.

[0027] In some embodiments, atezolizumab is administered intravenously to a patient at a dose of about 840 mg every two weeks, about 1200 mg every three weeks, or about 1680 mg every four weeks.

[0028] In some embodiments, atezolizumab is administered intravenously to a patient at a dose of about 1200 mg every three weeks.

[0029] In some embodiments, atezolizumab is administered to the patient in a 21-day dosing cycle, wherein atezolizumab is administered intravenously to the subject at a dose of about 1200 mg on days -2 to 4 of each 21-day dosing cycle.

[0030] In some embodiments, atezolizumab is administered intravenously to a patient at a dose of about 1200 mg on day 1 of each 21-day dosing cycle.

[0031] In some embodiments, the anti-PD-L1 antibody is administered to the subject as a monotherapy.

[0032] In other embodiments, the anti-PD-L1 antibody is administered to the subject in combination with one or more additional therapeutic agents.

[0033] In some embodiments, the one or more additional therapeutic agents comprises platinum-based chemotherapy.

[0034] In some aspects, the platinum-based chemotherapy comprises a platinum-based chemotherapeutic agent and a nucleoside analog.

[0035] In some embodiments, the platinum-based chemotherapeutic agent is cisplatin, carboplatin, or oxaliplatin.

[0036] In some embodiments, the platinum-based chemotherapeutic agent is cisplatin.

[0037] In some embodiments, the platinum-based chemotherapeutic agent is carboplatin.

[0038] In some embodiments, the nucleoside analog is gemcitabine.

[0039] In some embodiments, the platinum-based chemotherapy comprises cisplatin and gemcitabine, or carboplatin and gemcitabine.

[0040] In some embodiments, the platinum-based chemotherapy comprises cisplatin and gemcitabine.

[0041] In some embodiments, the platinum-based chemotherapy comprises carboplatin and gemcitabine.

[0042] In some embodiments, each dosing cycle of platinum-based chemotherapy is about 21 days.

[0043] In some embodiments, cisplatin is administered at about 70 mg / m on days -2 through 4 of each 21-day dosing cycle. 2 is administered intravenously to the subject at a dose of

[0044] In some embodiments, cisplatin is administered at about 70 mg / m on day 1 of each 21-day dosing cycle. 2 is administered intravenously to the subject at a dose of

[0045] In some embodiments, carboplatin is administered intravenously to a subject on days −2 to 4 of each 21-day dosing cycle at an area under the curve (AUC) of about 4.5.

[0046] In some embodiments, carboplatin is administered intravenously to a subject on day 1 of each 21-day dosing cycle at an AUC of about 4.5.

[0047] In some embodiments, gemcitabine is administered at about 1000 mg / m on days −2 to 4 and days 7 to 11 of each 21-day dosing cycle. 2 is administered intravenously to the subject at a dose of

[0048] In some embodiments, gemcitabine is administered at about 1000 mg / m on days 1 and 8 of each 21-day dosing cycle. 2 is administered intravenously to the subject at a dose of

[0049] In some embodiments, the patient has not previously received chemotherapy for locally advanced or metastatic UC.

[0050] In some embodiments, the patient has previously received adjuvant or neoadjuvant chemotherapy or chemoradiation therapy for urothelial carcinoma and has a treatment-free period of more than 12 months between the last dose of adjuvant or neoadjuvant chemotherapy or chemoradiation therapy and the date of recurrence.

[0051] In some embodiments, the locally advanced or metastatic UC is histologically confirmed locally advanced urothelial carcinoma (T4b, any N; or any T, N2-3) or metastatic urothelial carcinoma (mUC) (M1, stage IV).

[0052] In some embodiments, the UC is locally advanced UC.

[0053] In some embodiments, the locally advanced UC is inoperable.

[0054] In some embodiments, the UC is metastatic UC.

[0055] In some embodiments, the patient is eligible for treatment with platinum-based chemotherapy.

[0056] In some embodiments, the patient is eligible for treatment with cisplatin-based chemotherapy.

[0057] In some embodiments, the patient is a human.

[0058] In some embodiments, tumor samples obtained from patients have the presence of discernible PD-L1 staining of any intensity, as determined by a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody, in tumor-infiltrating immune cells covering 5% or more of the tumor area occupied by tumor cells, associated intratumoral stroma, and adjacent peritumoral stroma.

[0059] In some embodiments, the tumor sample obtained from the patient has a CPS of 10 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody.

[0060] In some embodiments, the tumor sample obtained from the patient has a CPS of 10 or greater using a PD-L1 IHC assay comprising the VENTANA SP263 anti-PD-L1 diagnostic antibody.

[0061] In some embodiments, the tumor sample obtained from the patient has a CPS of 10 or greater using a PD-L1 IHC assay comprising the 28-8 anti-PD-L1 diagnostic antibody.

[0062] In some embodiments, the tumor sample is a formalin-fixed, paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0063] In another aspect, provided herein is an assay for determining the presence or expression level of PD-L1 in a tumor sample obtained from a patient afflicted with cancer, the assay comprising: (a) 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 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, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody.

[0064] In some embodiments, using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody, a tumor sample obtained from a patient has detectable PD-L1 expression levels in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample.

[0065] In some embodiments, tumor samples obtained from patients have the presence of discernible PD-L1 staining of any intensity in tumor-infiltrating immune cells covering 5% or more of the tumor area occupied by tumor cells, associated intratumoral stroma, and adjacent peritumoral stroma, as determined by a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

[0066] In some embodiments, the tumor sample obtained from the patient has a CPS of 10 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody.

[0067] In some embodiments, a tumor sample obtained from a patient has detectable PD-L1 expression levels 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 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody.

[0068] In some embodiments, steps (a) and (b) are performed simultaneously.

[0069] In some embodiments, steps (a) and (b) are performed sequentially.

[0070] In some embodiments, steps (a) and (b) are performed on different sections of the tumor sample or on the same section of the tumor sample.

[0071] In some embodiments, the different sections of the tumor sample are serial sections.

[0072] In some aspects, the cancer is locally advanced or metastatic urothelial carcinoma.

[0073] In some aspects, the patient has not been previously treated for locally advanced or metastatic urothelial cancer.

[0074] In some embodiments, the assay is used to (i) select a therapy for treating locally advanced or metastatic UC in a patient in need thereof, or (ii) identify patients with locally advanced or metastatic UC who may benefit from a treatment regimen comprising an anti-PD-L1 antibody.

[0075] In some embodiments, 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).

[0076] In some embodiments, the tumor sample is an FFPE tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0077] In another aspect, provided herein is a method for labeling PD-L1 in a tumor sample, the method comprising the steps of: (a) contacting the tumor sample with VENTANA SP142 anti-PD-L1 diagnostic antibody; (b) contacting the tumor sample with Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody; and (c) visualizing the anti-PD-L1 diagnostic antibodies of steps (a) and (b) using one or more detectable reagents that produce a detectable signal for both of the anti-PD-L1 diagnostic antibodies.

[0078] In some embodiments, the detectable signal of the VENTANA SP142 anti-PD-L1 diagnostic antibody is an amplified signal.

[0079] In some embodiments, the amplified signal is generated by tyramide signal amplification.

[0080] In some embodiments, steps (a) and (b) are performed simultaneously.

[0081] In some embodiments, steps (a) and (b) are performed sequentially.

[0082] In some embodiments, steps (a) and (b) are performed on different sections of the tumor sample or on the same section of the tumor sample.

[0083] In some embodiments, the different sections of the tumor sample are serial sections.

[0084] In some embodiments, the visualizing step comprises IHC or immunofluorescence (IF).

[0085] In some embodiments, the visualizing step comprises IHC.

[0086] In some embodiments, the tumor sample is an FFPE tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0087] In some embodiments, the tumor sample is obtained from a patient with cancer.

[0088] In some aspects, the cancer is locally advanced or metastatic urothelial carcinoma.

[0089] In some aspects, the patient has not been previously treated for locally advanced or metastatic urothelial cancer.

[0090] In another aspect, provided herein is a kit comprising: (a) the VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) 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.

[0091] In some embodiments, the kit further comprises one or more reagents for visualizing the anti-PD-L1 diagnostic antibodies of (a) and (b).

[0092] In another aspect, provided herein is a method for identifying a tumor likely to respond to a PD-1 axis-binding antagonist, the method comprising the steps of: (a) staining a first portion of the tumor with an immune-directed 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 immune-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 cutoff and the second score to a second cutoff, wherein both when the first score meets or exceeds the first cutoff and when the second score meets or exceeds the second cutoff, the tumor is likely to respond to a PD-1 axis-binding antagonist.

[0093] In some embodiments, the immune-tropic PD-L1 assay has: (i) at least 80% overall agreement (OPA) with the SP142 assay using a first scoring algorithm at a first cutoff value; (ii) at least 80% positive agreement (PPA) with the SP142 assay using a first scoring algorithm at a first cutoff value; (iii) at least 80% negative agreement (NPA) with the SP142 assay using a first scoring algorithm at a first cutoff value; (iv) at least 80% PP agreement with the SP142 assay using a first scoring algorithm at a first cutoff value. (v) at least 80% PPA and at least 80% OPA with the SP142 assay using a first scoring algorithm at a first cutoff value; (vi) at least 80% NPA and at least 80% OPA with the SP142 assay using a first scoring algorithm at a first cutoff 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 cutoff value.

[0094] In some embodiments, the immune-independent PD-L1 assay has: (i) at least 80% overall agreement (OPA) with the 22C3 assay using a second scoring algorithm at a second cutoff value; (ii) at least 80% positive agreement (PPA) with the 22C3 assay using a second scoring algorithm at a second cutoff value; (iii) at least 80% negative agreement (NPA) with the 22C3 assay using a second scoring algorithm at a second cutoff value; (iv) at least 80% PP agreement with the 22C3 assay using a second scoring algorithm at a second cutoff value. (v) using a second scoring algorithm with a second cutoff value, at least 80% PPA and at least 80% OPA with the 22C3 assay; (vi) using a second scoring algorithm with a second cutoff value, at least 80% NPA and at least 80% OPA with the 22C3 assay; and / or (vii) using a second scoring algorithm with a second cutoff value, at least 80% OPA, at least 80% PPA, and at least 80% NPA with the 22C3 assay.

[0095] In another aspect, provided herein is a method for stratifying tumors having a score from an immune-independent PD-L1 assay that exceeds a predetermined cutoff, the method comprising: (a) staining a portion of the tumor with an immune-directed 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 cutoff, wherein the tumor is more likely to respond to a PD-1 axis-binding antagonist if the first score meets or exceeds the first cutoff.

[0096] In another aspect, provided herein is a method of stratifying tumors with a CPS of ≧10% as determined by the 22C3 assay, the method comprising: (a) staining a portion of the tumor with an immunotropic PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (e) comparing the score to a first cutoff and the second score to a second cutoff, wherein both the first score meeting or exceeding the first cutoff and the second score meeting or exceeding the second cutoff indicate that the tumor is likely to respond to a PD-1 axis-binding antagonist.

[0097] In another aspect, provided herein is a method of stratifying tumors with a CPS of ≧10% as determined by an SP263 assay, the method comprising: (a) staining a portion of the tumor with an immunotropic PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (e) comparing the score to a first cutoff and the second score to a second cutoff, wherein both the first score meeting or exceeding the first cutoff and the second score meeting or exceeding the second cutoff indicate that the tumor is likely to respond to a PD-1 axis-binding antagonist.

[0098] In another aspect, provided herein is 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 immunotropic PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (e) comparing the score to a first cutoff and comparing the second score to a second cutoff, wherein both the first score meeting or exceeding the first cutoff and the second score meeting or exceeding the second cutoff indicate that the tumor is likely to respond to a PD-1 axis-binding antagonist.

[0099] In some embodiments, the tumor is locally advanced or metastatic UC.

[0100] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows. [Brief explanation of the drawings]

[0101] [Figure 1] Schematic diagram of the phase III IMvigor130 trial (NCT02807636). mUC is metastatic urothelial carcinoma; R is randomized; ECOG PS is Eastern Cooperative Oncology Group performance status; 1L is first-line; platinum is investigator's choice of cisplatin or carboplatin. [Figure 2] This is a schematic diagram showing the change in the study design from two arms to three arms in the IMvigor130 study. The bottom row is a table summarizing the design changes. Atezo is atezolizumab; carbo is carboplatin; cis is cisplatin; mono is monotherapy; PFS is progression-free survival; OS is overall survival. [Figure 3] This is a schematic diagram showing the evolution of the study design for IMvigor130. FPI: first patient enrollment; LPI: last patient enrollment. [Figure 4] FIG. 1 is a schematic diagram showing the hierarchical relationships among test endpoints for the control of Type I error. [Figures 5A-5C] A series of images showing the results of immunohistochemistry (IHC) in urothelial carcinoma (UC) tumor tissue using anti-PD-L1 antibody SP142 (Figure 5A), anti-DC-LAMP antibody (Figure 5B), and a combination of anti-PD-L1 antibody SP142 and anti-DC-LAMP antibody (Figure 5C). Double labeling of SP142 (brown) and anti-DC-LAMP antibody (green) is shown (Figure 5C). [Figures 6A-6C]6A-6C are a series of graphs showing bulk RNA sequencing deconvolution data for the estimated population frequency percent of dendritic cells (FIG. 6A), CD8+ T cells (FIG. 6B), and CD4+ T cells (FIG. 6C) in urothelial carcinoma tissue samples identified as double negative (DN) for 22C3 and SP142, positive only for 22C3 (22C3), positive for SP142 (SP142), and double positive (DP) for 22C3 and SP142. [Figures 7A-7B] This is a series of graphs showing Kaplan-Meier plots of overall survival (OS) for 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 for patients with PD-L1-stained tumor-infiltrating immune cells (IC) scores of IC0 / 1 (red) and IC2 / 3 (blue) is shown for the SP142 IHC assay (Figure 7A). OS for patients with a combined positive score (CPS) <10 (red) and a CPS ≥10 (blue) is shown for the 22C3 assay (Figure 7B). CPS is combined positive score; CI is confidence interval; HR is hazard ratio; OS is overall survival; NE is not estimable. [Figure 8A-8B] Figures 8A-8D are a series of graphs showing Kaplan-Meier plots of OS for patients receiving atezolizumab monotherapy (Group B; Atez) and patients receiving placebo, platinum, and gemcitabine chemotherapy (Group C; Chemo) by PD-L1 IHC assay tumor tissue score using the SP142 assay (Figures 8A and 8C) and the PD-L1 IHC assay tumor tissue score using the 22C3 assay (Figures 8B and 8D). For patients with an IC score of IC0 / 1, OS for atezolizumab monotherapy (red) and chemotherapy (blue) is shown for the SP142 IHC assay (Figure 8A). For patients with a CPS <10, OS for atezolizumab monotherapy (red) and chemotherapy (blue) is shown for the 22C3 assay (Figure 8B). [Figure 8C-8D]The OS of patients with an IC score of IC2 / 3 receiving atezolizumab monotherapy (green) versus chemotherapy (purple) is shown for the SP142 IHC assay (Figure 8C), and the OS of patients with a CPS ≥ 10 receiving atezolizumab monotherapy (green) versus chemotherapy (purple) is shown for the 22C3 assay (Figure 8D). [Figure 9A-9B] Figures 9A–9D are a series of graphs showing Kaplan–Meier plots of OS for patients who received atezolizumab monotherapy (Group B; Atezo) and patients who were cisplatin-ineligible and received chemotherapy (i.e., placebo + carboplatin + gemcitabine) (Group C; Chemo). Study groups are shown by tumor tissue score for the SP142 assay (Figures 9A and 9C) and the 22C3 assay (Figures 9B and 9D). OS for patients with an IC score of IC0 / 1 who received atezolizumab monotherapy (red) and chemotherapy (blue) is shown for the SP142 IHC assay (Figure 9A), and OS for patients with a CPS <10 who received atezolizumab monotherapy (red) and chemotherapy (blue) is shown for the 22C3 assay (Figure 9B). [Figure 9C-9D] The OS of patients with an IC score of IC2 / 3 receiving atezolizumab monotherapy (green) versus chemotherapy (purple) is shown for the SP142 IHC assay (Figure 9C), and the OS of patients with a CPS ≥ 10 receiving atezolizumab monotherapy (green) versus chemotherapy (purple) is shown for the 22C3 assay (Figure 9D). [Figures 10A-10B]

[0033] Figure 10A is a series of graphs showing Kaplan-Meier plots of OS for patients receiving atezolizumab monotherapy (Group B; Atezo) (Figure 10A) and patients receiving placebo + platinum + gemcitabine (Group C; Chemo) (Figure 10B), further indicated by double-positive staining from the SP142 and 22C3 assays. Within each treatment group, OS for 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) is shown for patients receiving atezolizumab monotherapy (Figure 10A) and patients receiving placebo + carboplatin + gemcitabine (Figure 10B). [Figure 11] Schematic diagram showing an exemplary method using the OptiView DAB IHC Detection Kit. DAB is 3,3'-diaminobenzidine tetrahydrochloride, and HQ is a unique hapten conjugated to a goat antibody. DETAILED DESCRIPTION OF THE INVENTION

[0102] The present invention provides methods and compositions for treating cancer, such as bladder cancer (e.g., UC; including locally advanced or metastatic UC), including in patients who have not been previously treated for cancer. The invention is based, at least in part, on the discovery that PD-L1 expression on immune cells, as assessed by the SP142 antibody, colocalizes with dendritic cells and is associated with improved overall survival (OS) from treatment with the anti-PD-L1 antibody atezolizumab in patients with previously untreated locally advanced or metastatic UC. Unexpectedly, prolonged OS was associated with tumor status of SP142 IC2 / 3+22C3 CPS≧10, while shorter OS was observed in patients with SP142 IC0 / 1+22C3 CPS≧10 staining tumors. Therefore, analyzing tumor samples from cancer patients using assays and methods in which PD-L1 is detected by both immune-directed PD-L1 AHC assays (e.g., SP142 IHC assay) and immune-independent PD-L1 AHC assays (e.g., 22C3, SP263, or 28-8 IHC assays) may be useful in identifying patients who may benefit from immune checkpoint inhibitors, such as PD-1 axis-binding antagonists (e.g., atezolizumab).

[0103] B. Definition The following abbreviations are used herein: TIFF2025531738000001.tif83170

[0104] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, thereby relieving T cell dysfunction resulting from signaling along the PD-1 signaling axis, thereby restoring or enhancing 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, the PD-1 axis binding antagonist includes a PD-L1 binding antagonist or a PD-1 binding antagonist. In a preferred embodiment, the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

[0105] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L1 with one or more of its binding partners (e.g., 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 partners. In certain aspects, 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, abrogate, or prevent signaling resulting from the interaction of PD-L1 with one or more of its binding partners (e.g., PD-1 and / or B7-1). In one example, the PD-L1 binding antagonist reduces the negative costimulatory signal mediated by cell surface proteins expressed by T lymphocyte-mediated signaling through PD-L1, thereby alleviating dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some cases, the PD-L1 binding antagonist binds to PD-L1. In some cases, the 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, embafolimab, 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 examples, the anti-PD-L1 antibody is atezolizumab, MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab).In one particular embodiment, the PD-L1 binding antagonist is MDX-1105. In another particular embodiment, the PD-L1 binding antagonist is MEDI4736 (durvalumab). In another particular embodiment, the PD-L1 binding antagonist is MSB0010718C (avelumab). In other embodiments, the PD-L1 binding antagonist may be a small molecule, such as GS-4224, INCB086550, MAX-10181, INCB090244, CA-170, or ABSK041, which may 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 embodiment, the PD-L1 binding antagonist is atezolizumab.

[0106] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-1 with one or more of its binding partners (e.g., PD-L1 and / or PD-L2). PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2." An exemplary human PD-1 is set forth in UniProtKB / Swiss-Prot Accession No. 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 certain aspects, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signaling resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one example, the PD-1 binding antagonist reduces the negative costimulatory signal mediated by cell surface proteins expressed by T lymphocyte-mediated signaling via PD-1, thereby alleviating dysfunction of dysfunctional T cells (e.g., enhancing effector responses 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, prorugolimab, canrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, retifanlimab, sasanlimab, penprimab, CS1003, HLX10, SCT-I10A, zimberelimab, balstilimab, genolimuzumab, BI 754091, cetrelimab, YBL-006, BAT1306, HX008, budigalimab, AMG404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103, and hAb21. In a specific embodiment, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific embodiment, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In another specific embodiment, the PD-1 binding antagonist is a PD-L2 Fc fusion protein, e.g., AMP-224. In another specific embodiment, the PD-1 binding antagonist is MED1-0680. In another specific embodiment, the PD-1 binding antagonist is PDR001 (spartalizumab). In another specific embodiment, the PD-1 binding antagonist is REGN2810 (cemiplimab). In another specific embodiment, the PD-1 binding antagonist is BGB-108. In another specific embodiment, the PD-1 binding antagonist is prorugolimab. In another specific embodiment, the PD-1 binding antagonist is camrelizumab. In another specific embodiment, the PD-1 binding antagonist is sintilimab. In another specific embodiment, the PD-1 binding antagonist is tislelizumab. In another specific embodiment, the PD-1 binding antagonist is toripalimab. Other additional exemplary PD-1 binding antagonists include BION-004, CB201, AUNP-012, ADG104, and LBL-006.

[0107] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L2 with any one or more of its binding partners (e.g., PD-1). PD-L2 (programmed death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2," "PDCD1LG2," "CD273," "B7-DC," "Btdc," and "PDL2." An exemplary human PD-L2 is set forth in UniProtKB / Swiss-Prot Accession No. Q9BQ51. In some cases, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In certain aspects, a 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, abrogate, or interfere with signaling resulting from the interaction of PD-L2 with one or more of its binding partners (e.g., PD-1). In certain aspects, the PD-L2 binding antagonist reduces the negative costimulatory signal mediated by cell surface proteins expressed by T lymphocyte-mediated signaling through PD-L2, thereby alleviating dysfunction of dysfunctional T cells (e.g., enhancing effector responses 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.

[0108] The terms "programmed death-ligand 1" and "PD-L1," as used herein, refer to native sequence human PD-L1 polypeptide. Native sequence PD-L1 polypeptide is provided by Uniprot Accession No. Q9NZQ7. For example, native sequence PD-L1 may have the amino acid sequence set forth in Uniprot Accession No. Q9NZQ7-1 (isoform 1). In another example, native sequence PD-L1 may have the amino acid sequence set forth in Uniprot Accession No. Q9NZQ7-2 (isoform 2). In yet another example, native sequence PD-L1 may have the amino acid sequence set forth in Uniprot Accession No. Q9NZQ7-3 (isoform 3). PD-L1 is also referred to in the art as "programmed cell death 1 ligand 1," "PDCD1LG1," "CD274," "B7-H," and "PDL1."

[0109] The Kabat numbering system is commonly used when referring to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues within the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is typically used (e.g., the EU index as described in Kabat et al., supra). The "Kabat EU index" refers to the residue numbering of the human IgG1 EU antibody.

[0110] As used herein, "atezolizumab" refers to an Fc-engineered, humanized, aglycosylated IgG1 kappa immunoglobulin that binds 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 EU numbering of Fc region amino acid residues, resulting in an aglycosylated antibody with minimal binding to Fc receptors. Atezolizumab is also listed in WHO National Institute of Therapeutic Goods (Proposed International Nonproprietary Names (INN)) Vol. 28, No. 4, List 112, p. 488 (2014).

[0111] The term "cancer" refers to a disease caused by the uncontrolled division of abnormal cells in a part of the body. Forms of cancer include solid tumor cancer and non-solid tumor cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, 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); urinary tract cancer; breast cancer (e.g., HER2+ breast cancer and estrogen receptor negative (ER-), progesterone receptor negative (PR-) and HER2 negative (HER2-) triple-negative breast cancer (TNBC); prostate cancer, including castration-resistant prostate cancer (CRPC); peritoneal cancer; hepatocellular carcinoma; gastric or 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)); liver cancer; colon cancer; colorectal cancer; endometrial cancer or uterine cancer Uterine cancer; salivary gland cancer; prostate cancer; vulva cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma (including superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma, nodular and nodular melanoma); multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaving cell NHL; gigantoma Nodular lesions (NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal blood vessel growth associated with phacomatosis, edema (such as that associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and related metastases.In one example, the cancer is bladder cancer, such as urothelial carcinoma (UC) (e.g., locally advanced or metastatic UC). The cancer is locally advanced or metastatic. In some cases, the cancer is locally advanced. In other cases, the cancer is metastatic. In some cases, the 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, the locally advanced or metastatic UC is histologically confirmed locally advanced urothelial carcinoma (T4b, any N; or any T, N2-3) or metastatic urothelial carcinoma (mUC) (M1, stage IV).

[0112] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" do not exclude each other when referred to herein.

[0113] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In certain embodiments, the cell proliferative disorder is cancer. In certain embodiments, the cell proliferative disorder is a tumor.

[0114] The term "bladder cancer" includes, but is not limited to, urothelial carcinoma (UC), which may be, for example, locally advanced or metastatic. The methods described herein are suitable for treating various stages of cancer, including locally advanced and / or metastatic cancer. In cancer staging, locally advanced is generally defined as cancer that has spread from the local region to nearby tissues and / or lymph nodes. In the Roman numeral staging system, locally advanced cancer is usually classified as stage II or III. Metastatic cancer is a stage (stage IV) in which cancer has spread to distant tissues and organs throughout the body.

[0115] As used herein, "treating" 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 and either cisplatin or carboplatin)). Treatment herein includes, among others, adjuvant therapy, neoadjuvant therapy, non-metastatic cancer therapy (e.g., locally advanced cancer therapy), and metastatic cancer therapy. Treatment can be first-line therapy (e.g., the patient has not been previously treated or has not received prior systemic therapy) or second-line or subsequent therapy. In certain examples, treatment can be first-line therapy (e.g., the patient may not have been previously treated for locally advanced or metastatic urothelial carcinoma).

[0116] As used herein, an "effective amount" refers to the amount of a therapeutic agent (e.g., a PD-1 axis binding antagonist (e.g., atezolizumab) or combination of therapeutic agents, such as a PD-1 axis antagonist and one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., gemcitabine and either cisplatin or carboplatin)) that achieves a therapeutic result. In some examples, an effective amount of a therapeutic agent or combination of therapeutic agents may be an 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). The amount of therapeutic agent or combination of therapeutic agents that achieves the clinical endpoint of improved duration of response (OS), and / or improved duration of response (DOR). Improvement (e.g., in terms of response rate (e.g., ORR, CR, PR, and / or DCR), survival (e.g., PFS and / or OS) or DOR) can be relative to a suitable reference treatment, e.g., a treatment that does not include a PD-1 axis-binding antagonist. For example, treatment with an anti-cancer therapy that includes an anti-PD-L1 antibody (e.g., atezolizumab) can be compared to a reference treatment that is treatment with a platinum-based chemotherapy that does not include an anti-PD-L1 antibody.

[0117] As used herein, "complete response" and "CR" refer to the disappearance of all target lesions.

[0118] As used herein, "partial response" and "PR" refer to at least a 30% reduction in the sum of the longest diameters (SLD) of target lesions relative to the pre-treatment baseline SLD.

[0119] As used herein, "disease control rate" and "DCR" refer to the proportion of patients with a confirmed CR or PR as the best response, or stable disease (SD). For example, in some embodiments, DCR can be defined as the proportion of patients with a confirmed CR or PR as the best response, or patients who have maintained stable disease for 6 months or more, according to RECIST version 1.1.

[0120] As used herein, "stable state" or "SD" refers to neither sufficient shrinkage of target lesions to qualify as PR nor sufficient increase to qualify as PD, based on the smallest SLD since the start of treatment.

[0121] As used herein, "progressive disease" or "PD" refers to at least a 20% increase in the SLD of a target lesion, based on the smallest SLD recorded since the start of treatment or the presence of one or more new lesions.

[0122] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which the treated disease (e.g., cancer) does not worsen. Progression-free survival can include the amount of time a patient experiences a complete or partial response, as well as the amount of time a patient experiences stable disease. In some embodiments, PFS can be defined as the time from randomization or treatment initiation to first confirmed disease progression as assessed by RECIST version 1.1 or death from any cause, whichever occurs first.

[0123] As used herein, "objective response rate" or "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 with a confirmed objective response, either CR or PR, observed at two assessments separated by at least 28 days according to RECIST version 1.1, based on investigator assessment.

[0124] As used herein, "overall survival" and "OS" refer to the length of time a patient is still alive, from either the date of diagnosis or the date of initiation of treatment for a disease (e.g., cancer). In some embodiments, OS is defined as the time from randomization to death from any cause.

[0125] As used herein, the term "duration of response" (DOR) refers to the length of time from documentation of a tumor response to disease progression or death from any cause, whichever occurs first.

[0126] As used herein, the terms "inoperable" and "unresectable" are used interchangeably to 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, the bladder cancer (e.g., UC, including locally advanced or metastatic UC) is inoperable due to invasion of the pelvic sidewall or adjacent viscera (clinical stage T4b) or widespread lymph node metastasis (N2-N3).

[0127] The term "eligible for platinum-based chemotherapy treatment" means that a subject is eligible for platinum-based chemotherapy treatment according to the judgment of the attending clinician or according to the standardized criteria for platinum-based chemotherapy eligibility known in the art.For example, the criteria described in Galsky et al.Lancet Oncol.12(3):211-4,2011 can be used to determine whether a subject is eligible for cisplatin-based chemotherapy.Galsky et al. have described a consensus definition of metastatic UC (mUC) patients, in which patients who meet at least one of the following are considered to be unsuitable for cisplatin-based chemotherapy: (i) World Health Association (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 / sec; (iii) hearing loss by audiogram of National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v4.0 Grade 2 or greater; (iv) peripheral neuropathy of CTCAE v4.0 Grade 2 or greater; and / or New York Heart Association (NYHA) Class III heart failure. In one example, a patient is considered unsuitable for cisplatin-based chemotherapy if they have one or more of the following: Decreased renal function (e.g., glomerular filtration rate (GFR) ≥ 30 mL / min but < 60 mL / min); GFR can be assessed by direct measurement (i.e., creatinine clearance or ethyldediaminetetra-acetate, or, if unavailable, by calculation from serum / plasma creatinine (Cockcroft-Gault formula)); hearing loss (e.g., National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v4.0 Grade 2 or greater, hearing loss by audiogram of 25 decibels at two consecutive frequencies); peripheral neuropathy (e.g., NCI CTCAE v4.0 Grade 2 or greater 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, subjects may be eligible for carboplatin-based chemotherapy if they have any one of the following: reduced renal function (e.g., glomerular filtration rate (GFR) of 30 mL / min or greater but less than 60 mL / min); GFR can be assessed by direct measurement (i.e., creatinine clearance or ethyldiaminetetra-acetate, or, if unavailable, by calculation from serum / plasma creatinine (Cockcroft-Gault formula)); hearing loss (e.g., CTCAE v4.0 Grade 2 or greater, hearing loss by audiogram of 25 decibels at two consecutive frequencies); peripheral neuropathy (e.g., NCI CTCAE v4.0 Grade 2 or greater peripheral neuropathy (i.e., sensory changes or paresthesia, including tingling)); and / or ECOG performance status assessment (e.g., ECOG performance status 2).

[0128] As used herein, the term "chemotherapeutic agent" refers to a compound useful in the treatment of cancer, such as bladder cancer, for example, UC (e.g., locally advanced or metastatic UC). Examples of chemotherapeutic agents include EGFR inhibitors (small molecule inhibitors such as erlotinib (Tarceva®, Genentech / OSIPharm); PD183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer); ZD1839, gefitinib (Iressa®) 4-(3'-chloro-4'-fluoroanilino)-7-methoxybenzoate). -6-(3-morpholinopropoxy)quinazoline, AstraZeneca; ZM10518 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zena); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidine (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine; CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer) Inc.); AG1571 (SU5271; Pfizer Inc.); and dual EGFR / HER2 tyrosine kinase inhibitors such as lapatinib (Tykerb®, GSK572016, or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[(2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine); tyrosine kinase inhibitors (e.g., EGFR inhibitors; small molecule HER2 tyrosine kinase inhibitors such as TAK165 (Takeda Pharmaceuticals);Oral selective inhibitors of ErbB2 receptor tyrosine kinase, CP-724, 714 (Pfizer and OSI); dual HER inhibitors such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2-overexpressing and EGFR-overexpressing cells; PKI-166 (Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as ISIS-5132 (Isis Pharmaceuticals), an antisense agent that inhibits Raf-1 signaling; and imatinib mesylate. non-HER-targeted tyrosine kinase inhibitors such as thiazol-3-one (Gleevec®, GlaxoSmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (Sutent®, Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, Novartis / Schering); MAPK extracellular-regulated kinase I inhibitor CI-1040 (Pharmacia); quinazolines such as PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; CGP59326, Pyrrolopyrimidines such as CGP60261 and CGP62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to nucleic acids encoding HERs); quinoxalines (U.S. Patent No. 5,804,396); tryphostins (U.S. Patent No. 5,804,396); ZD6474 (AstraZeneca) ); PTK-787 (Novartis / Schering); pan-HER inhibitors such as CI-1033 (Pfizer); Afinitac (ISIS3521; Isis / Lilly); PKI166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering); INC-1C11 (ImClone); and rapamycin (sirolimus, Rapamune®);Proteasome inhibitors such as bortezomib (Velcade®, Millennium Pharma); disulfiram; epigallocatechin gallate; salinosporamide A; carfilzomib; 17-AAG (geldanamycin); radicicol; lactate dehydrogenase A (LDH-A); fulvestrant (FASLODEX®, AstraZeneca); letrozole (FEMARA®, Novartis), finasunate (vatalanib®, Novartis); oxazolidinone liplatin (Eloxatin®, Sanofi); 5-FU (5-fluorouracil); leucovorin; lonafamib (SCH66336); sorafenib (Nexavar®, Bayer Labs); alkylating agents such as AG1478, thiotepa, and Cytoxan® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; benzodopa, carboquone, meturedopa, and uredopa aziridines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethyleneethioniphosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins steroids (especially cryptophycin 1 and cryptophycin 8); corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductase enzymes (including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat, dolastatins; aldesleukin, talc, duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictiin; spongistatin;Nitrogen mustards such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, for example, enediyne antibiotics (e.g., calicheamicin, especially calicheamicin g1 and calicheamicin methylprednimustine); Dynemicin ω1; dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein (enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin Mitomycins such as bicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); denopterin, methotrexate, and pteropterin Folic acid analogues such as phosphate and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calucelone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergic drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid;Aceglatone; Aldophosphamide glycosides; Aminolevulinic acid; Eniluracil; Amsacrine; Bestravcil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elfomithine; Elliptinium acetate; Epothilones; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin; Maytansinoids such as maytansine and ansamitocin; Mitoguazone ;Mitoxantrone;Mopidamnol;Nitraerin;Pentostatin;Fenamet;Pirarubicin;Losoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK® Polysaccharide Complex (JHS Natural Products);Razoxane;Rhizoxin;Sizofuran;Spirogermanium;Tenuazonic acid;Triazicon;2,2',2''- Trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; chlorambucil; Gemzar® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; ethan toposide (VP-16); ifosfamide; mitoxantrone; novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (Xeloda®); ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, prodrugs, and derivatives of any of the above.

[0129] Chemotherapeutic agents also include (i) antihormonal agents that regulate or inhibit hormone action on tumors, such as antiestrogens, selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (Nolvadex®; tamoxifen citrate), raloxifene, droloxifene, iodoxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Fairston® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), Aromasin® (exemestane; estrogen, Pfizer), formestane, fadrozole, RIVISOR® estrogen (vorozole), Femara® (letrozole; Novartis), and arimide. (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retionic acid, fenretinide, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) (v) protein kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation (e.g., PKC-alpha, Ralf, and H-Ras); (vii) ribozymes such as VEGF expression inhibitors (e.g., Angiozyme®) and HER2 expression inhibitors; (viii) gene therapy vaccines, such as Allovectin®, Leuvectin®, and VAXID®;(ix) vincas (e.g., vincristine and vinblastine), Navelbine® (vinorelbine), taxanes (e.g., paclitaxel, nab-paclitaxel, and docetaxel), topoisomerase II inhibitors (e.g., doxorubicin, epirubicin, daunorubicin, 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 above; Includes:

[0130] As used herein, the term "cytotoxic agent" refers to any agent that is detrimental to cells (e.g., causing cell death, inhibiting growth, or otherwise interfering with cell function). Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212, and radioactive isotopes of Lu); chemotherapeutic agents; enzymes such as nucleases and fragments thereof; and toxins (including fragments and / or variants thereof) such as small molecule or enzymatically active toxins of bacterial, fungal, plant, or animal origin. Exemplary cytotoxic agents can be selected from anti-microtubule 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, proapoptotic agents, inhibitors of LDH-A, inhibitors of fatty acid biosynthesis, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism. In one example, the cytotoxic agent is a platinum-based chemotherapeutic agent (e.g., carboplatin or cisplatin). In one example, the cytotoxic agent is an EGFR antagonist, such as N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g., erlotinib). In one example, the cytotoxic agent is a RAF inhibitor, such as a BRAF and / or CRAF inhibitor. In one example, the RAF inhibitor is vemurafenib. In one example, the cytotoxic agent is a PI3K inhibitor.

[0131] Chemotherapeutic agents also include "platinum-based" chemotherapeutic agents, which include organic compounds containing platinum as an integral part of the molecule. Typically, platinum-based chemotherapeutic agents are coordination complexes of platinum. Platinum-based chemotherapeutic agents are sometimes referred to in the art as "platins." 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) may be administered in combination with one or more additional chemotherapeutic agents, such as nucleoside analogs (e.g., gemcitabine).

[0132] As used herein, "platinum-based chemotherapy" refers to a chemotherapy regimen that includes a platinum-based chemotherapy agent. For example, platinum-based chemotherapy can include a platinum-based chemotherapy agent (e.g., cisplatin or carboplatin) in combination with one or more additional chemotherapy agents, such as, for example, a nucleoside analog (e.g., gemcitabine).

[0133] As used herein, "nucleoside analog" refers to a nucleoside comprising a nucleic acid analog and a sugar. Nucleoside analogs can function as antimetabolites. Exemplary nucleoside analogs include, but are not limited to, gemcitabine, cytarabine, fludarabine, and cladribine.

[0134] The term "patient" refers to a human patient. For example, the patient may be an adult.

[0135] The term "antibody" as used herein specifically encompasses 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 example, the antibody is a full-length monoclonal antibody.

[0136] As used herein, the term "isotype" or "subclass" of IgG refers to any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions.

[0137] Antibodies (immunoglobulins) are classified into various classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IGA1, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are designated A, G, E, G, and M, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known and are generally described, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or noncovalent bonding of the antibody to one or more other proteins or peptides.

[0138] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment, as defined below. These terms refer to an antibody that includes an Fc region.

[0139] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term encompasses native-sequence Fc regions and variant Fc regions. In certain embodiments, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain a full-length heavy chain or a truncated variant of the full-length heavy chain. This is true even when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. The amino acid sequence of a heavy chain comprising an Fc region is shown herein without the C-terminal lysine (Lys447) unless otherwise indicated. In certain aspects, heavy chains comprising an Fc region as specified herein included in an antibody disclosed herein comprise an additional C-terminal glycine-lysine dipeptide (G446 and K447). In certain aspects, heavy chains comprising an Fc region as specified herein included in an antibody disclosed herein comprise an additional C-terminal glycine residue (G446). In certain aspects, heavy chains comprising an Fc region as specified herein included in an antibody disclosed herein comprise an additional C-terminal lysine residue (K447). In certain embodiments, the Fc region contains a single amino acid substitution, N297A, in the heavy chain. Unless otherwise specified herein, 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.

[0140] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0141] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding region thereof. In some cases, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, F(ab')2, and Fv fragments, diabodies, linear antibodies, single antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0142] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, except for variant antibodies that contain, for example, naturally occurring mutations or that may arise during the production of a monoclonal antibody preparation (such variants are generally present in minor amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies according to the invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0143] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and determines antigen-binding specificity, e.g., the "complementarity-determining region" (CDR).

[0144] Antibodies typically contain six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) hypervariable loops located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact sites present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0145] Unless otherwise specified, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that CDR nomenclature may also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature.

[0146] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining regions (CDRs). The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following VH (or VL) sequence: FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0147] The terms "Kabat variable domain residue numbering" or "Kabat amino acid position numbering" and variations thereof refer to the numbering system used for the light chain variable domains or heavy chain variable domains of the antibody compilation in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to truncations of, or insertions into, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the regions of homology of that antibody's sequence with the "standard" Kabat numbered sequences.

[0148] The term "package insert" is used to refer to instructions customarily included in commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or precautions regarding the use of such therapeutic product.

[0149] As used herein, "in combination with" refers to a treatment regimen that includes the administration of one therapeutic modality in addition to another, for example, the administration of 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" refers to the administration of one therapeutic modality before, during, or after the administration of the other therapeutic modality to a patient.

[0150] A drug that is administered "in parallel" with one or more other drugs is administered on the same treatment day during the same treatment cycle as the one or more other drugs, and optionally at the same time as the one or more other drugs. For example, in the case of a cancer therapy administered every three weeks, each of the drugs that are administered in parallel is administered on the first day of the three-week cycle.

[0151] The term "detection" includes all means of detection, including direct and indirect detection.

[0152] As used herein, the term "biomarker" refers to an indicator (e.g., a predictive, diagnostic, and / or prognostic indicator) that can be detected in a sample, such as PD-L1. Biomarkers can serve as indicators of particular subtypes of diseases or disorders (e.g., cancer) characterized by specific molecular, pathological, histological, and / or clinical features. In some embodiments, a biomarker is a gene. Biomarkers include, but are not limited to, molecular markers of polynucleotides (e.g., DNA and / or RNA), changes in polynucleotide copy number (e.g., DNA copy number), polypeptides, polypeptide modifications and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and / or glycolipids.

[0153] The "amount" or "level" of a biomarker associated with increased clinical benefit to an individual is the level detectable in a biological sample. These can be measured by methods known to those of skill in the art and disclosed herein. The expression level or amount of the biomarker assessed can be used to determine response to treatment.

[0154] The terms "level of expression" or "expression level" are commonly used interchangeably and generally refer to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., genetically encoded information and / or epigenetic information) is converted into structures present and functional in a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Also, fragments of a transcribed polynucleotide, a translated polypeptide, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide) shall be considered expressed, regardless of whether they are derived from a transcript generated by alternative splicing or a degraded transcript, or from post-translational processing of a polypeptide, for example, by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, as well as those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer RNA and ribosomal RNA).

[0155] "Increased expression," "increased expression level," "increased level," "elevated expression," "elevated expression level," or "elevated level" refers to increased expression or increased level of a biomarker in an individual compared to a control, such as an individual not afflicted with a disease or disorder (e.g., cancer) or an internal standard (e.g., a housekeeping biomarker). In some examples, the control is a baseline expression level.

[0156] "Decreased expression," "decreased expression level," "decreased level," "reduced expression," "decreased expression level," or "decreased level" refers to decreased expression or decreased level of a biomarker in an individual compared to a control, such as an individual not afflicted with a disease or disorder (e.g., cancer) or an internal standard (e.g., housekeeping biomarker). In some embodiments, decreased expression is little or no expression. In some examples, the control is a baseline expression level.

[0157] The term "housekeeping biomarker" refers to a biomarker or group of biomarkers (e.g., polynucleotides and / or polypeptides) that are typically present similarly in all cell types. In some embodiments, a housekeeping biomarker is a "housekeeping gene." As used herein, "housekeeping gene" refers to a gene or group of genes that encode a protein, whose activity is essential for maintaining cellular function, and that are typically present similarly in all cell types.

[0158] The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease, or condition (e.g., cancer (e.g., bladder cancer (e.g., UC, including locally advanced or metastatic UC))). For example, "diagnosis" may refer to the identification of a particular type of cancer. "Diagnosis" can also refer to the classification of a particular subtype of cancer, for example, by histopathological criteria or by molecular features (e.g., a subtype characterized by the expression of a biomarker (e.g., a particular gene or protein encoded by that gene) or a combination of biomarkers).

[0159] As used herein, the term "sample" refers to a composition obtained or derived from a subject and / or patient of interest that contains cells and / or other molecular entities to be characterized and / or identified, e.g., based on physical, biochemical, chemical, and / or physiological properties. For example, "disease sample" and variations thereof refer to any sample obtained from a subject of interest that is expected to contain or is known to contain the cells and / or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts, e.g., homogenized tissue, tumor tissue, cell extracts, and combinations thereof. In some examples, the sample is a tumor sample (e.g., a tumor tissue sample).

[0160] A "tissue sample" or "cell sample" refers to a collection of similar cells obtained from the tissue of a subject or individual. The source of a tissue or cell sample 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; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of a subject's pregnancy or development. A tissue sample can be primary or cultured cells or cell lines. Optionally, a tissue or cell sample is obtained from a diseased tissue / organ. For example, a "tumor sample" is a tissue sample obtained from a tumor (e.g., a bladder tumor, e.g., a UC tumor (e.g., a locally advanced or metastatic UC tumor)) or other cancerous tissue. A tissue sample can contain a mixed population of cell types (e.g., tumor and non-tumor cells, cancerous and non-cancerous cells). The tissue sample may contain compounds that are not naturally associated with tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. In some examples, the tissue sample is a tumor tissue sample.

[0161] 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, intratumoral immune cells, peritumoral immune cells, other tumor stromal cells (e.g., fibroblasts), or any combination thereof. Such tumor-infiltrating immune cells may be, for example, T lymphocytes (such as CD8+ T lymphocytes and / or CD4+ T lymphocytes), B lymphocytes, or other myeloid cells, including granulocytes (e.g., neutrophils, eosinophils, and basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer cells. In some examples, tumor-infiltrating immune cells may include dendritic cells (e.g., DC-LAMP-positive dendritic cells).

[0162] As used herein, "tumor cell" refers to any tumor cell present in a tumor or a sample thereof. Tumor cells can be distinguished from other cells, such as stromal cells and tumor-infiltrating immune cells, that may be present in a tumor sample using methods known in the art and / or described herein.

[0163] As used herein, a "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of the same subject or individual. For example, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue can be a healthy and / or non-diseased cell or tissue adjacent to a diseased cell or tissue (e.g., a cell or tissue adjacent to a tumor). In another embodiment, a reference sample is obtained from an untreated tissue and / or cell of the body of the same subject or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of an individual other than the test subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from intact tissues and / or cells from the body of an individual other than the test subject or individual.

[0164] As used herein, a "section" of a tissue sample refers to a single portion or piece of a tissue sample, e.g., a thin slice of tissue or cells cut from a tissue sample (e.g., a tumor sample). It is understood that multiple sections of a tissue sample may be taken and subjected to analysis, with the understanding that the same section of a tissue sample may be analyzed at both a morphological and molecular level, or may be analyzed for polypeptides (e.g., by immunohistochemistry) and / or for polynucleotides (e.g., by in situ hybridization). In some instances, the sections may be serial sections. In other instances, the sections may be non-serial sections.

[0165] "Correlating" or "correlating" means comparing, in any manner, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, the results of a first analysis or protocol may be used in performing a second protocol and / or may be used to determine whether a second analysis or protocol should be performed. With respect to embodiments of polypeptide analyses or protocols, the results of a polypeptide expression analysis or protocol may be used to determine whether a particular therapeutic regimen should be performed. With respect to embodiments of polynucleotide analyses or protocols, the results of a polynucleotide expression analysis or protocol may be used to determine whether a particular therapeutic regimen should be performed.

[0166] The phrase "based on," as used herein, means using information about one or more biomarkers to inform treatment decisions, information provided in package inserts, marketing / promotional policies, etc.

[0167] As used herein, the term "combined positive score" or "CPS" refers to the number of PD-L1-staining cells (e.g., tumor cells, lymphocytes, or macrophages) divided by the total number of viable tumor cells, multiplied by 100, in the context of an IHC assay (e.g., an IHC assay), such as an IHC assay using antibody SP142, SP263, 22C3, or 28-8 to stain PD-L1. In one example, the CPS can be calculated according to the formula above using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, such as the PD-L1 IHC 22C3 PHARMDX assay (Dako). In other examples, the CPS can be calculated using another anti-PD-L1 diagnostic antibody, such as SP263 or 28-8. In some examples, a sample (e.g., a tumor sample) is considered to have PD-L1 expression if the CPS is 1 or greater or 10 or greater. In certain instances, a sample (e.g., a tumor sample) is considered to have PD-L1 expression if the CPS is 10 or greater.

[0168] As used herein, "percentage of PD-L1-positive tumor cells," in the context of an IHC assay (e.g., an IHC assay), such as an IHC assay using antibodies SP142, SP263, 22C3, or 28-8 to stain PD-L1, refers to the percentage of viable tumor cells that exhibit partial or complete membrane staining (excluding cytoplasmic staining) of any intensity relative to all viable tumor cells present in the sample following staining of the sample. Thus, the percentage of PD-L1-positive tumor cells can be calculated using a PD-L1 IHC SP263 (Ventana) assay, for example, by the formula: percent PD-L1-positive tumor cells = (number of PD-L1-positive tumor cells) / (total number of PD-L1-positive and PD-L1-negative tumor cells), where PD-L1 cytoplasmic staining of tumor cells and all non-tumor cells (e.g., tumor-infiltrating immune cells, normal cells, necrotic cells, and debris) is excluded from evaluation and scoring. It will be understood that any given diagnostic PD-L1 antibody may correspond to a particular IHC assay protocol and / or scoring terminology that can be used to derive the percentage of PD-L1-positive tumor cells. For example, the percentage of PD-L1-positive tumor cells can be derived from tumor cell samples stained with SP263, 22C3, SP142, or 28-8 using OPTIVIEW® detection on Benchmark ULTRA®, EnVision Flex on AutostainerLink 48, OPTIVIEW® detection and amplification on Benchmark ULTRA®, or EnVision Flex on AutostainerLink 48, respectively. In another example, the PD-L1 IHC 22C3 PHARMDX assay (Dako) may be used to calculate the percentage of PD-L1-positive tumor cells according to the formula above. As used herein, the terms PD-L1-positive tumor cell percentage and "tumor percentage score" (TPS) are used interchangeably.

[0169] As used herein, the term "anti-PD-L1 diagnostic antibody" refers to an antibody that can bind to PD-L1 with sufficient affinity so as to be useful 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 some embodiments, the extent of binding of an anti-PD-L1 diagnostic antibody to an unrelated, non-PD-L1 protein is less than about 10% of the binding of the antibody to PD-L1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to PD-L1 has an 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, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 The anti-PD-L1 diagnostic antibody has a dissociation constant (Kd) of 0.05 M. 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), h5H1 (Advanced Cell Diagnostics), and 9A11. In some examples, the anti-PD-L1 diagnostic antibody is SP142. In other examples, the anti-PD-L1 diagnostic antibody is SP263, 22C3, or 28-8.

[0170] 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.

[0171] Heavy chain variable region: TIFF2025531738000002.tif25170Light chain variable region: TIFF2025531738000003.tif25170

[0172] 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 by reference in their entireties. 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, e.g., Example 1 and Table 1).

[0173] 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 incorporated by reference in their entireties. 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 is shown, for example, in U.S. Patent No. 9,709,568 (see, for example, Figures 2 and 3 and Table 2 in U.S. Patent No. 9,709,568).

[0174] 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 International Publication No. WO 2013 / 173223, which are incorporated herein by reference in their entireties. 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 set forth, for example, in U.S. Patent No. 9,212,224. For example, U.S. Patent No. 9,212,224 discloses that the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence of 28-8 are set forth in SEQ ID NO: 35 and SEQ ID NO: 36, respectively, of U.S. Patent No. 9,212,224.

[0175] As used herein, the term "immunotropic PD-L1 assay" refers to an affinity histochemical (AHC) assay (e.g., any IHC assay) specific for the human PD-L1 protein that is designed to highlight immune cell expression of PD-L1, for example, by preferentially staining PD-L1-expressing immune cells and PD-L1-expressing tumor cells. Immune cell highlighting can be the result of (a) inherent antibody specificity for immune-expressed PD-L1 compared with expression by other cell types; (b) careful selection of staining conditions, such as antigen retrieval method, choice of antibody diluent, choice of buffer, detection system, labeling time and temperature, etc.; or (c) a combination of (a) and (b). One example of a commercially available immunotropic PD-L1 assay is the VENTANA PD-L1 (SP142) assay (the "SP142 assay"). The SP142 assay is an affinity histochemical 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-amplified 3,3'-diaminobenzidine (DAB)-based detection system (OPTIVIEW DAB IHC Detection Kit and OPTIVIEW Amplification Kit (Roche)). As shown in Figures 5A-5C, the SP142 assay preferentially stains immune cells, particularly dendritic cells, in tumor sections. While the SP142 assay can also stain tumor cells, we optimized the antibody selection and assay conditions to emphasize immune cell staining.

[0176] As used herein, the term "immune-independent PD-L1 assay" refers to an affinity histochemical assay specific for human PD-L1 protein that is not an immunotropic PD-L1 assay. Exemplary commercially available immune-independent PD-L1 assays include the PD-L1 IHC 22C3 PHARMDX Assay (Agilent) (the "22C3 Assay"), the VENTANA PD-L1 (SP263) Assay (Roche) (the "SP263 Assay"), and the PD-L1 IHC 28-8 PHARMDX Assay (Agilent) (the "28-8 Assay").

[0177] II. Therapeutic and Diagnostic Methods and Compositions for Bladder Cancer 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, comprising administering to the subject a therapeutic 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), comprising administering to the subject a therapeutic 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 subject following treatment. For example, in some embodiments, the treatment increases the likelihood that the subject will have an objective response (e.g., a complete response (CR)), prolongs the subject's progression-free survival (PFS), and improves the subject's overall survival (OS), compared to, for example, a standard treatment (e.g., a treatment that does not include a PD-1 axis-binding antagonist) or treatment with a platinum-based chemotherapy that does not include a PD-1 axis-binding antagonist. Also provided herein is a method for enhancing immune function in a subject with bladder cancer, comprising administering to the subject a therapeutic 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 is a method for enhancing immune function in a subject with bladder cancer, comprising administering to the subject a therapeutic 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). and (b) administering to a subject a therapeutically effective amount of a PD-1 axis-binding antagonist to a subject having bladder cancer (e.g., UC, including locally advanced or metastatic UC). Also provided are methods for identifying tumors likely to respond to a PD-1 axis-binding antagonist. Further provided are methods for identifying and / or selecting patients, e.g., for treatment with a PD-1 axis-binding antagonist. Further provided are methods for stratifying tumors. Any of the PD-1 axis-binding antagonists and / or platinum-based chemotherapeutics known in the art or described herein can be used in the above methods.

[0178] In another aspect, provided herein is a method for identifying a tumor likely to respond to a PD-1 axis-binding antagonist, the method comprising: (a) staining a first portion of the tumor with an immune-directed PD-L1 assay to obtain a first-stained sample; (b) applying a first scoring algorithm to the first-stained sample to generate a first score; (c) staining a second portion of the tumor with an immune-independent PD-L1 assay to obtain a second-stained sample; (d) applying a second scoring algorithm to the second-stained sample to generate a second score; and (e) comparing the first score to a first cutoff and the second score to a second cutoff, wherein both when the first score meets or exceeds the first cutoff and when the second score meets or exceeds the second cutoff, the tumor is likely to respond to a PD-1 axis-binding antagonist.

[0179] Any suitable first and / or second scoring algorithms can be used. For example, the first scoring algorithm may be the immune cell scoring algorithm shown in Table 2 herein (e.g., as used in the SP142 assay). In some examples, the second scoring algorithm may be the combined positive score (CPS), such as that used in the 22C3 assay. It is understood that the CPS may be determined using other PD-L1 AHC assays (e.g., other PD-L1 IHC assays), including IHC assays using VENTANA SP263 or 28-8. Other scoring algorithms for PD-L1 assays, such as the TPS, tumor cell (TC) percentage, and the tumor cell scoring algorithm shown in Table 3 herein, are known in the art. A description of various exemplary scoring algorithms for PD-L1 assays that may be used is shown in Figure 1 of Zajac et al., Diagnostic Pathology.

[0180] Any suitable first and second cutoffs can be used. For example, in some instances, the first cutoff is IC≧5%, e.g., as described in Table 2 herein. In some instances, the second cutoff is CPS≧1 or CPS≧10. In some instances, the second cutoff is CPS≧10.

[0181] In some embodiments, the immune-tropic PD-L1 assay has: (i) at least 80% overall agreement (OPA) with the SP142 assay using a first scoring algorithm at a first cutoff value; (ii) at least 80% positive agreement (PPA) with the SP142 assay using a first scoring algorithm at a first cutoff value; (iii) at least 80% negative agreement (NPA) with the SP142 assay using a first scoring algorithm at a first cutoff value; (iv) at least 80% PP agreement with the SP142 assay using a first scoring algorithm at a first cutoff value. (v) at least 80% PPA and at least 80% OPA with the SP142 assay using a first scoring algorithm at a first cutoff value; (vi) at least 80% NPA and at least 80% OPA with the SP142 assay using a first scoring algorithm at a first cutoff 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 cutoff value.

[0182] In some embodiments, the immune-independent PD-L1 assay has: (i) an OPA of at least 80% with the 22C3 assay using a second scoring algorithm at a second cutoff value; (ii) a PPA of at least 80% with the 22C3 assay using a second scoring algorithm at a second cutoff value; (iii) an NPA of at least 80% with the 22C3 assay using a second scoring algorithm at a second cutoff value; (iv) a PPA of at least 80% and an NPA of at least 80% with the 22C3 assay using a second scoring algorithm at a second cutoff value. (v) at least 80% PPA and at least 80% OPA with the 22C3 assay using a second scoring algorithm at a second cutoff value; (vi) at least 80% NPA and at least 80% OPA with the 22C3 assay using a second scoring algorithm at a second cutoff 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 cutoff value.

[0183] In another aspect, provided herein is a method for stratifying tumors having a score from an immune-independent PD-L1 assay that exceeds a predetermined cutoff, the method comprising: (a) staining a portion of the tumor with an immune-directed 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 cutoff, wherein the tumor is more likely to respond to a PD-1 axis-binding antagonist if the first score meets or exceeds the first cutoff.

[0184] In another aspect, provided herein is a method of stratifying tumors with a CPS of ≧10% as determined by the 22C3 assay, the method comprising: (a) staining a portion of the tumor with an immunotropic PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (e) comparing the score to a first cutoff and the second score to a second cutoff, wherein both the first score meeting or exceeding the first cutoff and the second score meeting or exceeding the second cutoff indicate that the tumor is likely to respond to a PD-1 axis-binding antagonist.

[0185] In another aspect, provided herein is a method of stratifying tumors with a CPS of ≧10% as determined by an SP263 assay, the method comprising: (a) staining a portion of the tumor with an immunotropic PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (e) comparing the score to a first cutoff and the second score to a second cutoff, wherein both the first score meeting or exceeding the first cutoff and the second score meeting or exceeding the second cutoff indicate that the tumor is likely to respond to a PD-1 axis-binding antagonist.

[0186] In another aspect, provided herein is 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 immunotropic PD-L1 assay to obtain a stained sample; (b) generating a score by applying a scoring algorithm to the stained sample; and (e) comparing the score to a first cutoff and comparing the second score to a second cutoff, wherein both the first score meeting or exceeding the first cutoff and the second score meeting or exceeding the second cutoff indicate that the tumor is likely to respond to a PD-1 axis-binding antagonist.

[0187] Any suitable scoring algorithm may be used. For example, the scoring algorithm may be the immune cell scoring algorithm shown in Table 2 herein (e.g., as used in the SP142 assay). In some examples, the scoring algorithm may be the combined positive score (CPS), such as that used in the 22C3 assay. It is understood that the CPS may be determined using other PD-L1 AHC assays (e.g., other PD-L1 IHC assays), including IHC assays using VENTANA SP263 or 28-8. Other scoring algorithms for PD-L1 assays are known in the art, such as the TPS, tumor cell (TC) percentage, and the tumor cell scoring algorithm shown in Table 3 herein. A description of various exemplary scoring algorithms for PD-L1 assays that may be used is shown in Figure 1 of Zajac et al., Diagnostic Pathology.

[0188] Any suitable cutoff can be used. For example, in some instances, the cutoff is IC≧5% (e.g., IC2 / 3), e.g., as described in Table 2 herein. In some instances, the cutoff is CPS≧1 or CPS≧10. In some instances, the cutoff is CPS≧10.

[0189] The tumor may be of 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); urinary tract cancer; breast cancer (e.g., HER2+ breast cancer, as well as estrogen receptor-negative (ER-), progesterone receptor-negative (PR-), and HER2-negative (HER2-) breast cancers). triple-negative breast cancer (TNBC); prostate cancer, including castration-resistant prostate cancer (CRPC); peritoneal cancer; hepatocellular carcinoma; gastric or 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)); liver cancer; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; prostate cancer; vulvar cancer; thyroid cancer Cancer; Liver cancer; Anal cancer; Penile cancer; Melanoma (including superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma, nodular and nodular melanoma); Multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); Small lymphocytic (SL) NHL; Intermediate-grade / follicular NHL; Intermediate-grade diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-cleaved cell NHL; Giant mass disease NHL; Mantle cell lymphoma The tumor may be a bladder cancer, such as UC, UC, UC, UC (arterial ulcerative colitis), UC (cytoplasmic leukemia), UC (cytoplasmic lymphoma ...

[0190] In another aspect, provided herein is a method of treating a patient afflicted with cancer identified or stratified according to any of the foregoing methods, comprising administering to the patient a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)).

[0191] In one example, provided herein is a method of treating cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)) in a patient in need of treatment, comprising administering a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) to the patient in need of such treatment, wherein a tumor sample obtained from the patient has been determined to have detectable expression levels of PD-L1 on tumor-infiltrating immune cells using an immune-directed PD-L1 assay; and detectable expression levels of PD-L1 using an immune-independent PD-L1 assay, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist.

[0192] In another example, provided herein is a method of treating cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)) in a patient in need of such treatment, comprising the steps of: (a) determining that a tumor sample obtained from the patient has detectable expression levels of PD-L1 on tumor-infiltrating immune cells using an immune-directed PD-L1 assay; and detectable expression levels of PD-L1 using an immune-independent PD-L1 assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering a therapeutic regimen comprising a PD-1 axis-binding antagonist to the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. The method includes:

[0193] In one example, provided herein is a method of enhancing immune function in a patient with cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)), comprising administering to the patient a therapeutic 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 detectable expression levels of PD-L1 on tumor-infiltrating immune cells using an immune-directed PD-L1 assay; and detectable expression levels of PD-L1 using an immune-independent PD-L1 assay, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist.

[0194] In another example, provided herein is a method of enhancing immune function in a patient with cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)), comprising the steps of: (a) determining that a tumor sample obtained from the patient has detectable expression levels of PD-L1 on tumor-infiltrating immune cells using an immune-directed PD-L1 assay; and detectable expression levels of PD-L1 using an immune-independent PD-L1 assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering a therapeutic regimen comprising a PD-1 axis-binding antagonist to the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. The method includes:

[0195] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in treating cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)) in a patient in need of such treatment, wherein the treatment comprises administering to the patient a therapeutic regimen comprising the PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have detectable expression levels of PD-L1 on tumor-infiltrating immune cells using an immune-directed PD-L1 assay; and detectable expression levels of PD-L1 using an immune-independent PD-L1 assay, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising the PD-1 axis-binding antagonist.

[0196] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in a method for treating cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)) in a patient in need of such treatment, the method comprising: (a) determining that a tumor sample obtained from the patient has detectable expression levels of PD-L1 on tumor-infiltrating immune cells using an immune-directed PD-L1 assay; and detectable expression levels of PD-L1 using an immune-independent PD-L1 assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising the PD-1 axis-binding antagonist; and (b) administering a therapeutic regimen comprising the anti-PD-L1 antibody to the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising the PD-1 axis-binding antagonist. Includes:

[0197] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in enhancing immune function in a patient with cancer (e.g., bladder cancer (e.g., locally advanced or metastatic UC)), wherein the treatment involves administering to the patient a therapeutic regimen comprising the PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have detectable expression levels of PD-L1 on tumor-infiltrating immune cells using an immune-directed PD-L1 assay; and detectable expression levels of PD-L1 using an immune-independent PD-L1 assay, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising the PD-1 axis-binding antagonist.

[0198] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in a method of enhancing immune function in a patient with 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 detectable expression levels of PD-L1 on tumor-infiltrating immune cells using an immune-directed PD-L1 assay; and detectable expression levels of PD-L1 using an immune-independent PD-L1 assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising the PD-1 axis-binding antagonist; and (b) administering a therapeutic regimen comprising the anti-PD-L1 antibody to the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising the PD-1 axis-binding antagonist. Includes.

[0199] In another example, 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, comprising the steps of: (a) determining that a tumor sample obtained from the patient has a detectable expression level of PD-L1 on tumor-infiltrating immune cells using an immune-directed PD-L1 assay; and a detectable expression level of PD-L1 using an immune-independent PD-L1 assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) selecting a therapeutic regimen comprising a PD-1 axis-binding antagonist for the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. The method includes:

[0200] 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 therapeutic 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 detectable expression levels of PD-L1 on tumor-infiltrating immune cells using an immune-directed PD-L1 assay; and detectable expression levels of PD-L1 using an immune-independent PD-L1 assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist.

[0201] In some examples, the immune-directed PD-L1 assay is an AHC assay (e.g., an IHC assay).

[0202] In some examples, the immune-independent PD-L1 assay is an AHC assay (e.g., an IHC assay).

[0203] The cancer may 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); urinary tract cancer; breast cancer (e.g., HER2+ breast cancer, as well as ER-, PR-, and HER2-positive breast cancers). TNBC that is HER2-; prostate cancer, including CRPC; peritoneal cancer; hepatocellular carcinoma; gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., PDAC); glioblastoma; cervical cancer; ovarian cancer; liver cancer (e.g., HCC); hepatic carcinoma; 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 lentigo melanoma, nodular and nodular melanoma); multiple myeloma and B-cell lymphoma (including low-grade / follicular NHL; SL The cancer may be NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; CLL; ALL; AML; hairy cell leukemia; CML; PTLD; and MDS, as well as nevus syndrome, edema (such as that associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and abnormal blood vessel proliferation associated with related metastases. In some examples, the cancer is bladder cancer (e.g., locally advanced or metastatic UC).

[0204] In one example, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC), comprising administering a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) to a patient in need thereof, wherein a tumor sample obtained from the patient has been determined to have detectable expression levels of PD-L1 in tumor-infiltrating immune cells using a PD-L1 immunohistochemistry (IHC) assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody, and detectable expression levels of PD-L1 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist.

[0205] In another example, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment, comprising: (a) detecting a tumor sample obtained from the patient that has detectable expression levels of PD-L1 in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) detecting a tumor sample obtained from the patient that has detectable expression levels of PD-L1 in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody. determining that the patient has a detectable expression level of PD-L1 using an IHC assay, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering a therapeutic regimen comprising a PD-1 axis-binding antagonist to the patient identified in step (a) as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. The method includes:

[0206] In one example, provided herein is a method of enhancing immune function in a patient with bladder cancer (e.g., locally advanced or metastatic UC), comprising administering to the patient a therapeutic 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 detectable expression levels of PD-L1 in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody, and to have detectable expression levels of PD-L1 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist.

[0207] In another example, provided herein is a method of enhancing immune function in a patient with bladder cancer (e.g., locally advanced or metastatic UC), comprising: (a) administering to the patient a tumor sample that has detectable expression levels of PD-L1 in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) administering to the patient 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. determining that the patient has a detectable expression level of PD-L1 using an IHC assay, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering a therapeutic regimen comprising a PD-1 axis-binding antagonist to the patient identified in step (a) as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. The method includes:

[0208] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, wherein the treatment comprises administering to the patient a therapeutic regimen comprising the PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has detectable expression levels of PD-L1 in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody, and detectable expression levels of PD-L1 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody. It has been determined that the patient has a PD-1 axis-binding antagonist, which identifies the patient as one who may benefit from a treatment regimen that includes the PD-1 axis-binding antagonist.

[0209] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in a method for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of such treatment, the method comprising: (a) detecting a tumor sample obtained from the patient that has detectable expression levels of PD-L1 in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) detecting 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. (b) determining that the patient has a detectable expression level of PD-L1 using an IHC assay, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist; and (b) administering a therapeutic regimen comprising an anti-PD-L1 antibody to the patient identified in step (a) as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. Includes:

[0210] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in enhancing immune function in a patient with bladder cancer (e.g., locally advanced or metastatic UC), the treatment comprising administering to the patient a therapeutic regimen comprising the PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has been determined to have detectable expression levels of PD-L1 in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody, and detectable expression levels of PD-L1 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising the PD-1 axis-binding antagonist.

[0211] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in a method of enhancing immune function in a patient with bladder cancer (e.g., locally advanced or metastatic UC), the method comprising: (a) administering to the patient a tumor sample that has detectable expression levels of PD-L1 in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) administering to the patient 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. (b) determining that the patient has a detectable expression level of PD-L1 using an IHC assay, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist; and (b) administering a therapeutic regimen comprising an anti-PD-L1 antibody to the patient identified in step (a) as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. Includes.

[0212] In another example, provided herein is a method for selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment, comprising: (a) selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment, the method comprising: (a) selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment; and (b) selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment; (b) determining that the patient has a detectable expression level of PD-L1 using an IHC assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) selecting a therapeutic regimen comprising a PD-1 axis-binding antagonist for the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. The method includes:

[0213] 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 therapeutic 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 detectable expression levels of PD-L1 in tumor-infiltrating immune cells using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody, and detectable expression levels of PD-L1 using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist.

[0214] In some instances, the patient has not been previously treated for bladder cancer.

[0215] Any suitable reference expression level or cut-off for the presence or expression level of PD-L1 may be used, for example, any of the reference expression levels or cut-offs described below in Section IV.

[0216] In some embodiments, the patient has detectable expression levels of PD-L1 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.

[0217] In some instances, the patient has a CPS of 10 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody.

[0218] In some examples, the patient has detectable expression levels of PD-L1 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 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody.

[0219] In some instances, the presence and / or expression level of PD-L1 in a tumor sample identifies a patient as one who may benefit from a treatment regimen including a PD-1 axis-binding antagonist.

[0220] In one example, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC), comprising administering a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) to a patient in need thereof, wherein a tumor sample obtained from the patient has a detectable expression level of PD-L1 in tumor-infiltrating immune cells comprising 5% or more of the tumor sample, determined using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody, and a CPS of 10 or greater, determined using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist.

[0221] In another example, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment, comprising: (a) detecting a tumor sample obtained from the patient that has detectable expression levels of PD-L1 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 (b) detecting 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. (b) determining that the patient has a CPS of 10 or greater using an IHC assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering a therapeutic regimen comprising a PD-1 axis-binding antagonist to the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. The method includes:

[0222] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need thereof, the treatment comprising administering to the patient a therapeutic regimen comprising the PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has detectable expression levels of PD-L1 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 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. Using an IHC assay, a CPS of 10 or greater has been determined, which identifies the patient as one who may benefit from a treatment regimen that includes a PD-1 axis-binding antagonist.

[0223] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in a method for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of such treatment, the method comprising: (a) detecting a tumor sample obtained from the patient that has detectable expression levels of PD-L1 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 (b) detecting 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. (b) determining that the patient has a CPS of 10 or greater using an IHC assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist; and (b) administering a therapeutic regimen comprising an anti-PD-L1 antibody to the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. Includes:

[0224] In another example, provided herein is a method for selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment, comprising: (a) selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment, the method comprising: (a) selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment; and (b) selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment; (b) determining that the patient has a CPS of 10 or greater using the IHC assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) selecting a therapeutic regimen comprising a PD-1 axis-binding antagonist for the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. The method includes:

[0225] 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 therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), comprising determining that a tumor sample obtained from the patient has a detectable expression level of PD-L1 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 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist.

[0226] In some instances, the patient has not been previously treated for bladder cancer.

[0227] In one example, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC), comprising administering a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) to a patient who has not previously been treated for bladder cancer (e.g., locally advanced or metastatic UC) and is in need of treatment for bladder cancer, wherein a tumor sample obtained from the patient has a detectable expression level of PD-L1 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 has a CPS of 10 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist.

[0228] In another example, provided herein is a method of treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment and who has not been previously treated for bladder cancer, comprising: (a) a tumor sample obtained from the patient has detectable expression levels of PD-L1 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 (b) a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody. (b) determining that the patient has a CPS of 10 or greater using an IHC assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) administering a therapeutic regimen comprising a PD-1 axis-binding antagonist to the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. The method includes:

[0229] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment and who has not been previously treated for bladder cancer, wherein the treatment comprises administering to the patient a therapeutic regimen comprising the PD-1 axis-binding antagonist, wherein a tumor sample obtained from the patient has detectable expression levels of PD-L1 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 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. Using an IHC assay, a CPS of 10 or greater has been determined, which identifies the patient as one who may benefit from a treatment regimen that includes a PD-1 axis-binding antagonist.

[0230] In another example, provided herein is a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) for use in a method for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment and who has not been previously treated for bladder cancer, the method comprising: (a) detecting a tumor sample obtained from the patient that has detectable expression levels of PD-L1 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 (b) detecting 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. (b) determining that the patient has a CPS of 10 or greater using an IHC assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist; and (b) administering a therapeutic regimen comprising an anti-PD-L1 antibody to the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. Includes.

[0231] In another example, provided herein is a method for selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment and who has not been previously treated for bladder cancer, comprising: (a) selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment and who has not been previously treated for bladder cancer, the method comprising: (a) selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment and who has not been previously treated for bladder cancer, the method comprising: (i ...i) selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment and who has not been previously treated for bladder cancer, the method comprising: (a) selecting a therapy for treating bladder cancer (e.g., locally advanced or metastatic UC) in a patient in need of treatment and who has not (b) determining that the patient has a CPS of 10 or greater using the IHC assay, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)); and (b) selecting a therapeutic regimen comprising a PD-1 axis-binding antagonist for the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist. The method includes:

[0232] In another example, provided herein is a method for identifying a patient with bladder cancer (e.g., locally advanced or metastatic UC) who may benefit from a therapeutic 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 expression level of PD-L1 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 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist.

[0233] In some examples, the method further includes administering to the patient a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)).

[0234] Any suitable PD-1 axis binding antagonist may 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.

[0235] For example, in some instances, the PD-1 axis binding antagonist 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). and anti-PD-L1 antibodies, including

[0236] In certain examples, provided herein are methods of treating locally advanced or metastatic UC in a patient in need thereof, wherein the patient has not been previously treated for the locally advanced or metastatic UC, and the method comprises administering to the patient a gene encoding a nucleotide sequence ... administering to the patient a therapeutic regimen comprising an anti-PD-L1 antibody, comprising:

[0237] In another example, 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) detecting a tumor sample obtained from the patient that has detectable expression levels of PD-L1 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 (b) detecting a tumor sample obtained from the patient that has detectable expression levels of PD-L1 in tumor-infiltrating immune cells comprising 5% or more of the tumor sample 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. Using an IHC assay, it was determined to have 10 or more CPS, which identified the following HVRs: (i) HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO: 3); (ii) HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO: 4); (iii) HVR-H3 sequence of RHWPGGFDY (SEQ ID NO: 5); (iv) HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO: 6); (v) HVR-L2 sequence of SASFLYS (SEQ ID NO: 7); and (vi) HVR-L3 sequence of QQYLYHPAT (SEQ ID NO: 8). and (b) administering a therapeutic regimen comprising an anti-PD-L1 antibody to the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising an anti-PD-L1 antibody.

[0238] 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, wherein the patient has not been previously treated for the locally advanced or metastatic UC, the method comprising: (a) determining that a tumor sample obtained from the patient has a detectable expression level of PD-L1 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 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying an HVR with one of the following: (i) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO: 3); (ii) an 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). (b) selecting a therapeutic regimen comprising an anti-PD-L1 antibody for the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising an anti-PD-L1 antibody.

[0239] In another example, provided herein is 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 the locally advanced or metastatic UC, and the method comprises: a tumor sample obtained from the patient having detectable expression levels of PD-L1 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 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. determining that the patient has a CPS of 10 or greater using an IHC assay, thereby identifying the patient as one who may benefit from a treatment regimen comprising an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody has 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). Includes.

[0240] In some examples, the method further comprises administering to the patient a therapeutic regimen comprising an anti-PD-L1 antibody.

[0241] In some examples, the anti-PD-L1 antibody is atezolizumab.

[0242] In one example, provided herein is a method of treating locally advanced or metastatic UC, comprising administering a therapeutic regimen comprising atezolizumab to a patient who has not previously been treated for locally advanced or metastatic UC and who is in need of treatment for locally advanced or metastatic UC, wherein a tumor sample obtained from the patient has detectable expression levels of PD-L1 in tumor-infiltrating immune cells comprising 5% or more of the tumor sample using a PD-L1 immunohistochemistry (IHC) assay comprising VENTANA SP142 anti-PD-L1 diagnostic antibody, and has a CPS of 10 or greater using a PD-L1 IHC assay comprising Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising atezolizumab.

[0243] In another example, 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 the locally advanced or metastatic UC, the method comprising the steps of: (a) determining that a tumor sample obtained from the patient has a detectable expression level of PD-L1 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 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising atezolizumab; and (b) administering a therapeutic regimen comprising atezolizumab to the patient identified in step (a) as a patient who may benefit from a therapeutic regimen comprising atezolizumab.

[0244] In another example, provided herein is a method for 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 the locally advanced or metastatic UC, the method comprising: (a) selecting a therapy for treating locally advanced or metastatic UC in a patient in need thereof; (b) selecting a therapy for treating locally advanced or metastatic UC in a patient in need thereof; (c) selecting a therapy for treating locally advanced or metastatic UC in a patient in need thereof; (d) selecting a therapy for treating locally advanced or metastatic UC in a patient in need thereof; (b) determining that the patient has a CPS of 10 or greater using the IHC assay, thereby identifying the patient as one who may benefit from a therapeutic regimen comprising atezolizumab; and (b) selecting a therapeutic regimen comprising an anti-PD-L1 antibody for the patient identified in step (a) as one who may benefit from a therapeutic regimen comprising atezolizumab.

[0245] In another example, provided herein is a method for identifying a patient with locally advanced or metastatic UC who may benefit from a therapeutic regimen comprising atezolizumab, wherein the patient has not been previously treated for the locally advanced or metastatic UC, the method comprising determining that a tumor sample obtained from the patient has a detectable expression level of PD-L1 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 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, thereby identifying the patient as a patient who may benefit from a therapeutic regimen comprising atezolizumab.

[0246] In some instances, the method further comprises administering to the patient a therapeutic regimen comprising atezolizumab.

[0247] The benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) may be in terms of, for example, overall survival (OS), progression-free survival (PFS), objective response rate (ORR), complete response (CR), and / or duration of response (DOR). The benefit from a therapeutic regimen comprising a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) may be compared to an appropriate standard of care, for example, treatment with platinum-based chemotherapy without an anti-PD-L1 antibody. For example, in some embodiments, the treatment regimen increases the likelihood that the subject will have an objective response (e.g., CR), prolongs the subject's PFS, prolongs the subject's OS, and / or prolongs the subject's DOR compared to treatment with platinum-based chemotherapy that does not include a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). In some embodiments, the treatment regimen increases the likelihood that the subject will have an objective response compared to treatment with platinum-based chemotherapy that does not include a PD-1 axis binding antagonist. In some embodiments, the treatment regimen increases the likelihood that the subject will have a CR compared to treatment with platinum-based chemotherapy that does not include a PD-1 axis binding antagonist. In some embodiments, the treatment regimen prolongs the subject's PFS compared to treatment with platinum-based chemotherapy that does not include a PD-1 axis binding antagonist. In some embodiments, the treatment regimen prolongs the subject's OS compared to treatment with platinum-based chemotherapy that does not include a PD-1 axis binding antagonist. In some embodiments, the treatment regimen prolongs the subject's DOR compared to treatment with platinum-based chemotherapy that does not include a PD-1 axis-binding antagonist.

[0248] In some instances, benefit from a treatment regimen including a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is in terms of OS.

[0249] In some examples, the treatment regimen extends the patient's OS 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) compared to treatment with platinum-based chemotherapy that does not include a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). In some examples, the treatment regimen extends the patient's OS by about 5.7 months to about 17 months compared to treatment with platinum-based chemotherapy that does not include a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). In some examples, the treatment regimen extends the subject's OS by approximately 11.3 months compared to treatment with platinum-based chemotherapy that does not include a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)).

[0250] In some instances, the platinum-based chemotherapy comprises a platinum-based chemotherapeutic agent and a nucleoside analog.

[0251] In some instances, the platinum-based chemotherapeutic agent is cisplatin, carboplatin, or oxaliplatin.

[0252] In some instances, the platinum-based chemotherapeutic agent is cisplatin.

[0253] In some instances, the platinum-based chemotherapeutic agent is carboplatin.

[0254] In some examples, the nucleoside analog is gemcitabine.

[0255] In some instances, the platinum-based chemotherapy includes cisplatin and gemcitabine, or carboplatin and gemcitabine.

[0256] In some instances, the platinum-based chemotherapy includes cisplatin and gemcitabine.

[0257] In some instances, the platinum-based chemotherapy includes carboplatin and gemcitabine.

[0258] In some examples, 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. Includes:

[0259] In some examples, the anti-PD-L1 antibody is atezolizumab.

[0260] In some embodiments, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is administered as a monotherapy.

[0261] In other embodiments, the 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 an anti-tumor agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenic agent, radiation therapy, or a cytotoxic agent. In some embodiments, the one or more additional therapeutic agents is a platinum-based chemotherapy. In some embodiments, the treatment that does not include a PD-1 axis-binding antagonist includes treatment with platinum-based chemotherapy.

[0262] 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, the platinum-based chemotherapy comprises a platinum-based chemotherapeutic agent and a nucleoside analog. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin, carboplatin, or oxaliplatin.

[0263] For example, in some embodiments, the platinum-based chemotherapeutic agent is cisplatin. Any suitable dosing regimen for cisplatin known in the art can be used. In some embodiments, cisplatin is administered to the subject in a 21-day dosing cycle. In some embodiments, cisplatin is administered at a dose of about 35 mg / m 2 ~about 140mg / m 2 In some embodiments, cisplatin is administered intravenously to a subject at a dose of about 70 mg / m 2 In some embodiments, cisplatin is administered intravenously to a subject at a dose of about 70 mg / m on days -2 through 4 of each 21-day dosing cycle. 2 In some embodiments, cisplatin is administered intravenously to a subject at a dose of about 70 mg / m on day 1 of each 21-day dosing cycle. 2 is administered intravenously to the subject at a dose of

[0264] In another example, in other embodiments, the platinum-based chemotherapeutic agent is carboplatin. Any suitable dosing regimen for carboplatin known in the art may be used. In some embodiments, carboplatin is administered to a subject in a 21-day dosing cycle. In some embodiments, carboplatin is administered intravenously to a subject at an area under the curve (AUC) of about 2 to about 9. In some embodiments, carboplatin is administered intravenously to a subject at an area under the curve (AUC) of about 4.5. In some embodiments, carboplatin is administered intravenously to a subject on days -2 to 4 of each 21-day dosing cycle at an area under the curve (AUC) of about 4.5. In some embodiments, carboplatin is administered intravenously to a subject on day 1 of each 21-day dosing cycle at an AUC of about 4.5.

[0265] In any of the foregoing examples, the platinum-based chemotherapy may include a nucleoside analog. Any suitable nucleoside analog can be used, including any nucleoside analog known in the art or described herein (e.g., Section VII, below). Any suitable dosing regimen of gemcitabine known in the art may 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 at a dose of about 500 mg / m 2 ~about 2000mg / m 2 In some embodiments, gemcitabine is administered intravenously to a subject at a dose of about 1000 mg / m 2 In some embodiments, gemcitabine is administered intravenously to a subject at a dose of about 1000 mg / m on days -2 to 4 and days 7 to 11 of each 21-day dosing cycle. 2 In some embodiments, gemcitabine is administered intravenously to a subject at a dose of about 1000 mg / m on days 1 and 8 of each 21-day dosing cycle. 2 is administered intravenously to the subject at a dose of

[0266] In any of the foregoing examples, the platinum-based chemotherapy may include cisplatin and gemcitabine. In other examples, the platinum-based chemotherapy may include carboplatin and gemcitabine.

[0267] In some instances, the patient has not received prior chemotherapy for locally advanced or metastatic UC.

[0268] In some instances, the patient has previously received adjuvant or neoadjuvant chemotherapy or chemoradiation therapy for urothelial carcinoma and has a treatment-free interval of more than 12 months between the last dose of adjuvant or neoadjuvant chemotherapy or chemoradiation therapy and the date of recurrence.

[0269] In some instances, the locally advanced or metastatic UC is histologically confirmed locally advanced urothelial carcinoma (T4b, any N; or any T, N2-3) or metastatic urothelial carcinoma (mUC) (M1, stage IV).

[0270] In some instances, the UC is locally advanced UC.

[0271] In some instances, locally advanced UC is inoperable.

[0272] In some instances, the UC is metastatic UC.

[0273] In some examples, the patient may be eligible for any suitable platinum-based chemotherapy. Eligibility for platinum-based chemotherapy may be as described herein or according to criteria known in the art. For example, criteria for defining patients as cisplatin-eligible or cisplatin-ineligible are known in the art, e.g., as described in Galsky et al. Lancet. Oncol. 12:211-4, 2011, which is incorporated herein by reference in its entirety. In some embodiments, the subject is eligible for treatment with platinum-based chemotherapy including cisplatin. In some embodiments, the subject is eligible for treatment with platinum-based chemotherapy including carboplatin.

[0274] In other examples, the patient may be ineligible for platinum-based chemotherapy. In some embodiments, the subject is ineligible for treatment with platinum-based chemotherapy including cisplatin. In some embodiments, the subject is ineligible for treatment with platinum-based chemotherapy including carboplatin.

[0275] In some instances, the patient is a human.

[0276] In some instances, tumor samples obtained from patients have the presence of discernible PD-L1 staining of any intensity in tumor-infiltrating immune cells covering 5% or more of the tumor area occupied by tumor cells, associated intratumoral stroma, and adjacent peritumoral stroma, as determined by a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

[0277] In some instances, the tumor sample obtained from the patient has a CPS of 10 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody.

[0278] In some instances, the tumor sample obtained from the patient has a CPS of 10 or greater using a PD-L1 IHC assay comprising the VENTANA SP263 anti-PD-L1 diagnostic antibody.

[0279] In some instances, the tumor sample obtained from the patient has a CPS of 10 or greater using a PD-L1 IHC assay comprising the 28-8 anti-PD-L1 diagnostic antibody.

[0280] In any of the foregoing examples, the PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atezolizumab) may be administered in one or more dosing cycles.

[0281] In any of the foregoing examples, each dosing cycle can have any suitable length, for example, about 7 days, about 14 days, about 21 days, about 28 days, or longer, hi some embodiments, each dosing cycle is about 21 days.

[0282] Any suitable number of dosing cycles can be used, for example, 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 can be used. In some embodiments, 25 or fewer dosing cycles can be used.

[0283] In some embodiments, the tumor sample is a formalin-fixed, paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample. The presence and / or expression level of any biomarker described herein (e.g., PD-L1) can be determined using the methods described herein (e.g., in Section IV or Example 2, below) or using techniques known in the art.

[0284] 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 ranges from about 0.01 to about 50 mg / kg of patient body weight, whether administered in a single dose or multiple doses. 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))) may be administered in a range 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, or about 0. The antagonist is administered at a dose of 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, for example, daily, weekly, every two weeks, every three weeks, 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 15 mg / kg. However, other dosing regimens may be useful. In certain embodiments, the PD-1 axis-binding antagonist is administered at 15 mg / kg. The agonist (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)) is administered to a human at a dose of about 1000 mg to about 1400 mg every three weeks. The antagonist may be 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 (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.This dose can be administered as a single dose or multiple doses (e.g., two or three doses), for example, by infusion. The dose of the antibody administered in combination therapy may be reduced compared to monotherapy. In some embodiments, the treatment regimen comprises intravenously administering about 1200 mg of atezolizumab to a subject every three weeks. The progress of this therapy can be easily monitored by conventional techniques.

[0285] In some cases, the patient may receive a total of 1 to 50 doses of the PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), 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, Amount, 2~20 doses, 2~15 doses, 2~10 doses, 2~5 doses, 3~50 doses, 3~45 doses, 3~40 doses, 3~35 doses, 3~30 doses, 3~25 doses, 3~20 doses, 3~15 doses, 3~ 10 doses, 3~5 doses, 4~50 doses, 4~45 doses, 4~40 doses, 4~35 doses, 4~30 doses, 4~25 doses, 4~20 doses, 4~15 doses, 4~10 doses, 4~5 doses, 5~50 doses, 5 ~45 doses, 5~40 doses, 5~35 doses, 5~30 doses, 5~25 doses, 5~20 doses, 5~15 doses, 5~10 doses, 10~50 doses, 10~45 doses, 10~40 doses, 10~35 doses, 10~30 doses, 10~25 doses, 10~20 doses, 10~15 doses, 15~50 doses, 15~45 doses, 15~40 doses, 15~35 doses, 15~30 doses, 15~25 doses, 15~20 doses, 20-50 doses, 20-45 doses, 20-40 doses, 20-35 doses, 20-30 doses, 20-25 doses, 25-50 doses, 25-45 doses, 25-40 doses, 25-35 doses, 25-30 doses, 30-50 doses, 30-45 doses, 30-40 doses, 30-35 doses, 35-50 doses, 35-45 doses, 35-40 doses, 40-50 doses, 40-45 doses, or 45-50 doses. In certain cases, these doses may be administered intravenously.

[0286] Atezolizumab can be administered to a subject at any suitable dosage. In some embodiments, 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, atezolizumab is administered intravenously to a subject at a dose of about 1200 mg every three weeks. In some embodiments, atezolizumab is administered intravenously 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 on days -2 to 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.

[0287] In a preferred embodiment, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atezolizumab) is administered intravenously. In one example, atezolizumab may be administered intravenously over 60 minutes, and if the first infusion is tolerated, all subsequent infusions can be delivered over 30 minutes. In some examples, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atezolizumab) is not administered as an intravenous push or bolus.

[0288] In some embodiments, the 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. The agents may be administered concurrently or separately, depending on the dosing regimen.

[0289] The PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) and / or the one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) may be administered by any suitable method known in the art. For example, the PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) and the 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 concurrently (at the same time). In some embodiments, the PD-1 axis-binding antagonist is administered before the 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 some embodiments, the PD-1 axis-binding antagonist is administered after one or more of the additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)). In yet other embodiments, the PD-1 axis-binding antagonist is administered concurrently 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., an anti-PD-L1 antibody (e.g., atezolizumab)) is in a separate composition from the 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., an anti-PD-L1 antibody (e.g., atezolizumab)) is in the same composition as the one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)).

[0290] The PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and / or the one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) may be administered by the same or 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, implanted, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the PD-1 axis-binding antagonist (e.g., cisplatin or carboplatin and gemcitabine) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, 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)) may be administered to prevent or treat the disease. The choice of PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and / or one or more additional therapeutic agents (e.g., platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine)) can be based on the type of disease being treated, the type of PD-1 axis binding antagonist, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and 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) are administered intravenously by infusion.

[0291] For example, when administered with chemotherapy with or without bevacizumab, atezolizumab may be administered at a dose of 1200 mg every 3 weeks prior to chemotherapy and bevacizumab. In another example, after completion of 4 to 6 cycles of chemotherapy and when bevacizumab is discontinued, atezolizumab may be administered at a dose of 840 mg every 2 weeks, 1200 mg every 3 weeks, or 1680 mg every 4 weeks. In another example, atezolizumab may be administered at a dose of 840 mg, followed by 100 mg / m 2 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, atezolizumab, when administered with carboplatin and etoposide, can be administered at a dose of 1200 mg every three weeks prior to chemotherapy. In yet another example, atezolizumab can be administered at a dose of 840 mg every two weeks, 1200 mg every three weeks, or 1680 mg every four weeks after completion of four cycles of carboplatin and etoposide. In another example, after completion of a 28-day cycle of cobimetinib and vemurafenib, atezolizumab can be administered at a dose of 840 mg every 2 weeks, cobimetinib can be administered orally at a dose of 60 mg once daily (21 days on, 7 days off), and vemurafenib can be administered orally at a dose of 720 mg twice daily.

[0292] 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 radiation therapy, surgery (e.g., transurethral resection of bladder tumor (TURBT) or cystectomy (including partial cystectomy or radical cystectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, or a combination thereof. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is a side effect limiting agent (e.g., an anti-nausea agent) to reduce the occurrence of side effects of the treatment. and / or administration of a drug aimed at reducing the severity of the disease. In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy 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 chemopreventive agent. The additional therapy may be one or more chemotherapeutic agents described herein.

[0293] In some cases, the additional therapy is the administration of a side effect limiting agent (e.g., a drug intended to reduce the occurrence and / or severity of side effects of treatment, such as an anti-nausea agent, a corticosteroid (e.g., prednisone or equivalent, e.g., at a dose of 1-2 mg / kg / day), a hormone replacement agent, etc.).

[0294] III. Combination Therapy 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, comprising administering to the subject a therapeutic regimen comprising an effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) in combination with another anti-cancer agent or cancer therapy. For example, provided herein are therapeutic regimens 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). and methods for treating or delaying the progression of bladder cancer (e.g., UC, including locally advanced or metastatic UC) in a subject, comprising administering to the subject 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. Any of the combinations described below can be used in the methods, for example, as described in Section II above.

[0295] 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) may be administered in conjunction with additional chemotherapy or chemotherapeutic agents. 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) may be administered in conjunction with radiation therapy or a radiation therapeutic agent. In some embodiments, the PD1 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) may be administered in conjunction with a targeted therapy or targeted therapeutic agent. In some embodiments, a 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) may be administered in conjunction with immunotherapy or an immunotherapeutic agent (e.g., a monoclonal antibody).

[0296] Without wishing to be bound by theory, it is believed that enhancing T cell stimulation by promoting activation of costimulatory molecules or inhibiting negative costimulatory molecules may promote tumor cell death, thereby treating or delaying cancer progression. In some embodiments, a 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) may be administered in conjunction with an agonist to an activating costimulatory molecule. In some embodiments, the activating costimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist to an activating costimulatory molecule is an agonistic 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 platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) may be administered in conjunction with an antagonist to an inhibitory costimulatory molecule. In some embodiments, the inhibitory costimulatory molecule may 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 to an inhibitory costimulatory 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.

[0297] In some embodiments, a 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) may be administered in combination with an antagonist (e.g., a blocking antibody) to CTLA-4 (also known as CD152). In some embodiments, a 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) may be administered in combination with ipilimumab (also known as MDX-010, MDX-101, Yervoy®). In some embodiments, a 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) may 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 platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) may be administered in combination with an antagonist (e.g., a blocking antibody) against B7-H3 (also known as CD276). In some embodiments, a 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) may 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 platinum-based chemotherapy (e.g., cisplatin or carboplatin and gemcitabine) may be administered in combination with an antagonist to TGF-beta, such as meterimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.

[0298] 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) may be administered in conjunction with a treatment comprising the adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR). 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) may be administered in conjunction with a treatment comprising the adoptive transfer of T cells comprising a dominant-negative TGF beta receptor, e.g., a dominant-negative TGF beta type II receptor. In some embodiments, a 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) may be administered in conjunction with treatments comprising the HERCREEM protocol (see, e.g., ClinicalTrials.gov Identifier NCT00889954).

[0299] In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an agonist, e.g., an activating antibody, against CD137 (also known as TNFRSF9, 4-1BB, or ILA). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with urelumab (also known as BMS-663513). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an agonist, e.g., an activating antibody, against CD40. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with CP-870893. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an agonist, e.g., an activating antibody, against OX40 (also known as CD134). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an anti-OX40 antibody (e.g., AgonOX). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an agonist, such as an activating antibody, against CD27. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with CDX-1127. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an antagonist against indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).

[0300] In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises mertansine or monomethylauristatin E (MMAE). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or Kadceila®, Genentech). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with DMUC5754A. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in conjunction with an antibody-drug conjugate that targets endothelin B receptor (EDNBR), e.g., an antibody against EDNBR conjugated with MMAE.

[0301] In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an angiogenesis inhibitor. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an antibody against angiopoietin 2 (also known as Ang2). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with MEDI3617.

[0302] In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an anti-neoplastic agent. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an agent targeting CSF-1R (also known as M-CSFR or CD115). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with anti-CSF-1R (also known as IMC-CS4). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an interferon, such as interferon alpha or interferon gamma. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with Roferon A (also known as recombinant interferon alpha-2a). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with GM-CSF (also known as recombinant human granulocyte-macrophage colony-stimulating factor, rhu GM-CSF, sargramostim, or Leukine®). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with IL-2 (also known as aldesleukin or Proleukin®). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with IL-12. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an antibody targeting CD20. In some embodiments, the antibody targeting CD20 is obinutuzumab (also known as GA101 or Gazyva®) or rituximab. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in conjunction with an antibody that targets GITR. In some embodiments, the antibody that targets GITR is TRX518.

[0303] In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in conjunction with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, and in some embodiments, a personalized peptide vaccine. In some embodiments, the peptide cancer vaccine is a multivalent long peptide vaccine, a multi-peptide vaccine, a peptide cocktail vaccine, a hybrid peptide vaccine, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in conjunction with an adjuvant. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in conjunction with a treatment including a TLR agonist, e.g., poly-ICLC (also known as HILTONOL®), LPS, MPL, or CpG ODN. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with tumor necrosis factor (TNF) alpha. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with IL-1. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with HMGB1. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an IL-10 antagonist. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an IL-4 antagonist. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an IL-13 antagonist. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an HVEM antagonist. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy can be administered in conjunction with an ICOS agonist (e.g., administration of ICOS-L) or an agonistic antibody to ICOS.In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in conjunction with a therapy targeting CX3CL1. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in conjunction with a therapy targeting CXCL9. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in conjunction with a therapy targeting CXCL10. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in conjunction with a therapy targeting CCL5. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in conjunction with an LFA-1 or ICAM1 agonist. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in conjunction with a selectin agonist.

[0304] In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with a targeted therapy. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an inhibitor of B-Raf. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with vemurafenib (also known as Zelboraf®). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with dabrafenib (also known as Tafinlar®). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with erlotinib (also known as Tarceva®). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an inhibitor of MEK, such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with cobimetinib (also known as GDC-0973 or XL-518). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with trametinib (also known as Mekinist®). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an inhibitor of K-Ras. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an inhibitor of c-Met. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with onartuzumab (also known as MetMAb). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an inhibitor of Alk. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with AF802 (also known as CH5424802 or alectinib). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an inhibitor of phosphatidylinositol 3-kinase (PI3K).In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with BKM120. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with perifosine (also known as KRX-0401). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an inhibitor of Akt. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with MK2206. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with GSK690693. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with GDC-0941. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with an inhibitor of mTOR. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with sirolimus (also known as rapamycin). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with temsirolimus (also known as CCI-779 or Torisel®). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with everolimus (also known as RAD001). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with ridaforolimus (also known as AP-23573, MK-8669, or deforolimus). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with OSI-027. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with AZD8055. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with INK128.In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with a dual PI3K / mTOR inhibitor. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with XL765. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with GDC-0980. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with BEZ235 (also known as NVP-BEZ235). In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with BGT226. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with GSK2126458. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with PF-04691502. In some embodiments, the PD-1 axis binding antagonist and / or platinum-based chemotherapy may be administered in combination with PF-05212384 (also known as PKI-587).

[0305] In any of the foregoing embodiments, the PD-1 axis binding antagonist can be a human PD-1 axis binding antagonist.

[0306] In any of the foregoing embodiments, the PD-1 axis binding antagonist is an anti-PD-L1 antibody (e.g., atezolizumab).

[0307] In any of the foregoing embodiments, the platinum-based chemotherapy comprises a platinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin). In some embodiments, the platinum-based chemotherapy comprises cisplatin. In some embodiments, the platinum-based chemotherapy comprises carboplatin. In some embodiments, the platinum-based chemotherapy further comprises one or more additional chemotherapeutic agents, such as, for example, a nucleoside analog. In some embodiments, the nucleoside analog is gemcitabine. In some embodiments, the platinum-based chemotherapy comprises cisplatin and gemcitabine. In other embodiments, the platinum-based chemotherapy comprises carboplatin and gemcitabine.

[0308] IV. Evaluation of PD-L1 expression The presence and / or expression level of PD-L1 can be assessed in patients identified, selected, stratified, and / or treated according to any of the methods and compositions for use described herein. The methods and compositions for use may include determining the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the patient. In other examples, the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the patient has been determined before or after treatment has begun. PD-L1 expression may be determined using any suitable technique. For example, PD-L1 expression may be determined as described in U.S. Patent Application Publication Nos. 15 / 787988 and 15 / 790680. Any suitable tumor sample may be used, such as a formalin-fixed, paraffin-embedded (FFPE) tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0309] In some instances, assessment of the presence and / or expression levels of PD-L1 and / or patient selection may involve the use of two different affinity histochemical (AHC) assays (e.g., IHC assays) for PD-L1 protein: (a) an immune-directed PD-L1 assay; and (b) an immune-independent PD-L1 assay.

[0310] As described herein, an immune-directed PD-L1 assay is any AHC assay specific for the human PD-L1 protein that is designed to highlight immune cell expression of PD-L1, for example, by preferentially staining PD-L1-expressing immune cells and PD-L1-expressing tumor cells. Immune cell highlighting can be the result of (a) inherent antibody specificity for immune-expressed PD-L1 compared to expression by other cell types; (b) careful selection of staining conditions, such as antigen retrieval method, choice of antibody diluent, choice of buffer, detection system, labeling time and temperature, etc.; or (c) a combination of (a) and (b). One example of a commercially available immune-directed PD-L1 assay is the VENTANA PD-L1 (SP142) assay (the "SP142 assay"). The SP142 assay is an affinity histochemical assay that uses (a) a PD-L1 rabbit monoclonal antibody (clone SP142, see US10689445); (b) an automated IHC / ISH staining platform (BENCHMARK IHC / ISH staining platform (Roche)); and (c) a tyramide-amplified 3,3'-diaminobenzidine (DAB)-based detection system (OPTIVIEW DAB IHC Detection Kit and OPTIVIEW Amplification Kit (Roche)).

[0311] In certain embodiments, the immune-directed PD-L1 assay is an AHC assay (e.g., an IHC assay) that has, for a given scoring algorithm and cutoffs, at least 80% overall concordance (OPA), at least 80% positive concordance (PPA), and / or at least 80% negative concordance (NPA) with an SP142 assay using the same scoring algorithm and cutoffs for the same indication. In specific embodiments, the AHC has an OPA of at least 80%, at least 85%, at least 90%, or at least 95% with the SP142 assay. In another specific embodiment, the AHC has a PPA of at least 80%, at least 85%, at least 90%, or at least 95% with the SP142 assay. In another specific embodiment, the AHC has an NPA of at least 80%, at least 85%, at least 90%, or at least 95% with the SP142 assay. In another specific embodiment, the AHC has a PPA of at least 80%, at least 85%, at least 90%, or at least 95% with the SP142 assay and an NPA of at least 0%, at least 85%, at least 90%, or at least 95% with the SP142 assay. In certain embodiments, OPA, PPA, and / or NPA are measured using an immune fraction (IC) scoring method at a single cutoff shown to be predictive of response to PD-1 axis-binding antagonists in the indication tested. In another embodiment, OPA, PPA, and / or NPA are measured using an IC2 / 3 cutoff of ≥ 5% in a bladder cancer indication (e.g., locally advanced or metastatic UC).

[0312] Also, as used herein, an immune-independent PD-L1 assay is any AHC assay (e.g., an IHC assay) specific for the human PD-L1 protein that is not an immune-directed PD-L1 assay. Exemplary commercially available immune-independent 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 certain embodiments, the immune-independent 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, SP263 assay, and 28-8 assay, using the same scoring algorithm and cutoffs for the same indication. In certain embodiments, 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 a PPA of at least 80%, at least 85%, at least 90%, or at least 95% with the 22C3 assay and an NPA of at least 0%, at least 85%, at least 90%, or at least 95% with the 22C3 assay. In certain embodiments, OPA, PPA, and / or NPA are measured using a combined positive score (CPS) scoring method at a single cutoff shown to be predictive of response to PD-1 axis-binding antagonists in the indication tested. In another embodiment, OPA, PPA, and / or NPA are measured using a ≥ 10% CPS cutoff in a bladder cancer indication (e.g., locally advanced or metastatic UC).

[0313] In one aspect, provided herein is an assay for determining the presence or expression level of PD-L1 in a tumor sample obtained from a patient afflicted with cancer, the assay comprising: (a) determining the presence or expression level of PD-L1 in the tumor sample obtained from the patient using an immune-directed PD-L1 assay (e.g., the SP142 assay); and (b) determining the presence or expression level of PD-L1 in the tumor sample obtained from the patient using an immune-independent PD-L1 assay (e.g., the 22C3 assay, the SP263 assay, or the 28-8 assay).

[0314] In some examples, the assay is an AHC assay. In some examples, the AHC assay is an IHC assay.

[0315] 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 suffering from cancer, the assay comprising: (a) 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 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, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody.

[0316] Any suitable scoring algorithm may be used. For example, the scoring algorithm may be the immune cell scoring algorithm shown in Table 2 herein (e.g., as used in the SP142 assay). In some examples, the scoring algorithm may be the combined positive score (CPS), such as that used in the 22C3 assay. It is understood that the CPS may be determined using other PD-L1 AHC assays (e.g., other PD-L1 IHC assays), including the use of VENTANA SP263 or 28-8. Other scoring algorithms for PD-L1 assays are known in the art, such as the TPS, tumor cell (TC) percentage, and the tumor cell scoring algorithm shown in Table 3 herein. A description of various exemplary scoring algorithms for PD-L1 assays that may be used is shown in Figure 1 of Zajac et al., Diagnostic Pathology.

[0317] Any suitable cutoff can be used. For example, in some instances, the cutoff is IC≧5%, e.g., as described in Table 2 herein. In some instances, the cutoff is CPS≧1 or CPS≧10. In some instances, the cutoff is CPS≧10.

[0318] In some examples, using a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody, tumor samples obtained from patients have detectable expression levels of PD-L1 in tumor-infiltrating immune cells that comprise 5% or more of the tumor sample.

[0319] In some instances, tumor samples obtained from patients have the presence of discernible PD-L1 staining of any intensity in tumor-infiltrating immune cells covering 5% or more of the tumor area occupied by tumor cells, associated intratumoral stroma, and adjacent peritumoral stroma, as determined by a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody.

[0320] In some instances, using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody, a tumor sample obtained from a patient has a CPS of 10 or greater. In some instances, using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody, a tumor sample obtained from a patient has a CPS of 10 or greater.

[0321] In some instances, tumor samples obtained from patients have detectable expression levels of PD-L1 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 or greater using a PD-L1 IHC assay comprising the Dako 22C3 anti-PD-L1 diagnostic antibody.

[0322] In some instances, steps (a) and (b) are performed simultaneously.

[0323] In some instances, steps (a) and (b) are performed sequentially.

[0324] In some examples, steps (a) and (b) are performed on different sections of the tumor sample or on the same section of the tumor sample.

[0325] In some instances, the different sections of the tumor sample are serial sections.

[0326] In some instances, the cancer is locally advanced or metastatic urothelial carcinoma.

[0327] In some instances, the patient has not been previously treated for locally advanced or metastatic urothelial cancer.

[0328] In some examples, the assays are used to (i) select a therapy for treating locally advanced or metastatic UC in a patient in need thereof, or (ii) identify patients with locally advanced or metastatic UC who may benefit from a treatment regimen comprising an anti-PD-L1 antibody.

[0329] In some examples, the anti-PD-L1 antibody may have 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 examples, the anti-PD-L1 antibody is atezolizumab.

[0330] In some examples, the tumor sample is an FFPE tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0331] In another example, provided herein is a method for labeling PD-L1 in a tumor sample, the method comprising: (a) performing an immune-directed PD-L1 assay on the tumor sample; and (b) performing an immune-independent PD-L1 assay on the tumor sample.

[0332] Any suitable immune-directed PD-L1 assay may be used, for example the SP142 assay.

[0333] Any suitable immune-independent PD-L1 assay may be used, for example, the 22C3 assay, the SP263 assay, or the 28-8 assay.

[0334] In another example, provided herein is a method for labeling PD-L1 in a tumor sample, the method comprising the steps of: (a) contacting the tumor sample with VENTANA SP142 anti-PD-L1 diagnostic antibody; (b) contacting the tumor sample with Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody; and (c) visualizing the anti-PD-L1 diagnostic antibodies of steps (a) and (b) using one or more detectable reagents that produce a detectable signal for both of the anti-PD-L1 diagnostic antibodies.

[0335] In some instances, the detectable signal of the VENTANA SP142 anti-PD-L1 diagnostic antibody is an amplified signal.

[0336] In some examples, the amplified signal is generated by tyramide signal amplification.

[0337] In some instances, steps (a) and (b) are performed simultaneously.

[0338] In some instances, steps (a) and (b) are performed sequentially.

[0339] In some examples, steps (a) and (b) are performed on different sections of the tumor sample or on the same section of the tumor sample.

[0340] In other examples, the different sections of the tumor sample are serial sections.

[0341] In some examples, the visualizing step comprises AHC.

[0342] In some examples, the visualizing step comprises IHC or immunofluorescence (IF).

[0343] In some examples, the visualizing step comprises IHC.

[0344] In some examples, the tumor sample is an FFPE tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0345] In some examples, the tumor sample is obtained from a patient with cancer.

[0346] In some instances, the cancer is locally advanced or metastatic urothelial carcinoma.

[0347] In some instances, the patient has not been previously treated for locally advanced or metastatic urothelial cancer.

[0348] Any suitable scoring algorithm or method can be used to assess the presence and / or expression level of PD-L1 in a biological sample (e.g., a tumor sample).

[0349] For example, the scoring algorithm may be the immune cell scoring algorithm shown in Table 2 herein (e.g., as used in the SP142 assay). In some instances, the scoring algorithm may be the combined positive score (CPS), such as that used in the 22C3 assay. It is understood that the CPS may be determined using other PD-L1 AHC assays (e.g., other PD-L1 IHC assays), including the use of VENTANA SP263 or 28-8. Other scoring algorithms for PD-L1 assays, such as the TPS, tumor cell (TC) percentage, and the tumor cell scoring algorithm shown in Table 3 herein, are known in the art. A description of various exemplary scoring algorithms for PD-L1 assays that may be used is shown in Figure 1 of Zajac et al., Diagnostic Pathology.

[0350] In some instances, PD-L1 expression may be determined with respect to the proportion of a tumor sample comprised of tumor-infiltrating immune cells that express a detectable level of PD-L1, as the proportion of tumor-infiltrating immune cells in the tumor sample that express a detectable level of PD-L1, and / or as the proportion of tumor cells in the tumor sample that express a detectable level of PD-L1. In any of the foregoing examples, the proportion of a tumor sample comprised of tumor-infiltrating immune cells may be the proportion of tumor area covered by tumor-infiltrating immune cells in a section of a tumor sample obtained from a patient, as assessed by IHC using, for example, an anti-PD-L1 antibody (e.g., the SP142 antibody). Any suitable anti-PD-L1 antibody may be used, including, for example, SP142 (Ventana), SP263 (Ventana), 22C3 (Dako), 28-8 (Dako), E1L3N (Cell Signaling Technology), 4059 (Prosci), 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.

[0351] In some examples, a tumor sample obtained from a patient has detectable expression levels of PD-L1 in less than 1% of the tumor cells in the tumor sample, in more than 1% of the tumor cells in the tumor sample, in between 1% and less than 5% of the tumor cells in the tumor sample, in more than 5% of the tumor cells in the tumor sample, in between 5% and less than 50% of the tumor cells in the tumor sample, or in more than 50% of the tumor cells in the tumor sample.

[0352] In some examples, tumor samples obtained from a patient have detectable expression levels of PD-L1 in tumor-infiltrating immune cells that comprise less than 1% of the tumor sample, more than 1% of the tumor sample, between 1% and less than 5% of the tumor sample, more than 5% of the tumor sample, between 5% and less than 10% of the tumor sample, or more than 10% of the tumor sample.

[0353] In some examples, tumor samples may be scored for PD-L1 positivity in tumor-infiltrating immune cells and / or tumor cells according to the criteria for diagnostic evaluation set out in Table 2 and / or Table 3, respectively. In some examples, tumor samples may be scored for PD-L1 positivity in tumor-infiltrating immune cells and / or tumor cells for a PD-L1 IHC assay comprising the VENTANA SP142 anti-PD-L1 diagnostic antibody (e.g., the SP142 assay), according to the criteria for diagnostic evaluation set out in Table 2 and / or Table 3, respectively.

[0354] In some embodiments, the tumor sample obtained from the subject comprises 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) of the tumor sample. or greater, 34% or greater, 35% or greater, 36% or greater, 37% or greater, 38% or greater, 39% or greater, 40% or greater, 41% or greater, 42% or greater, 43% or greater, 44% or greater, 45% or greater, 46% or greater, 47% or greater, 48% or greater, 49% or greater, about 50% or greater, about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, about 99% or greater, or 100% or greater). For example, in some embodiments, a tumor sample obtained from a subject is determined to have detectable expression levels of PD-L1 in tumor-infiltrating immune cells that comprise between about 1% and less than about 5% of the tumor sample (e.g., between 1% and 4.9%, between 1% and 4.5%, between 1% and 4%, between 1% and 3.5%, between 1% and 3%, between 1% and 2.5%, or between 1% and 2%).

[0355] In some embodiments, a tumor sample obtained from a subject comprises 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) of tumor-infiltrating immune cells in the tumor sample. or greater, 32% or greater, 33% or greater, 34% or greater, 35% or greater, 36% or greater, 37% or greater, 38% or greater, 39% or greater, 40% or greater, 41% or greater, 42% or greater, 43% or greater, 44% or greater, 45% or greater, 46% or greater, 47% or greater, 48% or greater, 49% or greater, about 50% or greater, about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, about 99% or greater, or 100% or greater have been determined to have detectable expression levels of PD-L1. For example, in some embodiments, a tumor sample obtained from a subject is determined to have detectable expression levels of PD-L1 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 tumor-infiltrating immune cells in the tumor sample.

[0356] In other embodiments, a tumor sample obtained from a subject is determined to have detectable expression levels of PD-L1 in tumor-infiltrating immune cells that comprise about 5% or more of the tumor sample. For example, in some embodiments, a tumor sample obtained from a subject is determined to have detectable expression levels of PD-L1 in tumor-infiltrating immune cells that comprise about 5% to less than about 10% of the tumor sample (e.g., 5%-9.5%, 5%-9%, 5%-8.5%, 5%-8%, 5%-7.5%, 5%-7%, 5%-6.5%, 5%-6%, 5%-5.5%, 6%-9.5%, 6%-9%, 6%-8.5%, 6%-8%, 6%-7.5%, 6%-7%, 6%-6.5%, 7%-9.5%, 7%-9%, 7%-7.5%, 8%-9.5%, 8%-9%, or 8%-8.5%).

[0357] In still other embodiments, a tumor sample obtained from a subject is determined to have a detectable expression level of PD-L1 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 is determined to have a detectable expression level of PD-L1 in about 5% to less than about 10% of the tumor-infiltrating immune cells in the tumor sample (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%).

[0358] In further embodiments, the tumor sample obtained from the subject comprises 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, or greater than 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of tumor-infiltrating immune cells have been determined to have detectable levels of PD-L1 expression.

[0359] In further embodiments, the tumor sample obtained from the subject contains 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, 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 ≥ 4%, ≥ 35%, ≥ 36%, ≥ 37%, ≥ 38%, ≥ 39%, ≥ 40%, ≥ 41%, ≥ 42%, ≥ 43%, ≥ 44%, ≥ 45%, ≥ 46%, ≥ 47%, ≥ 48%, ≥ 49%, ≥ 50%, ≥ 60%, ≥ 70%, ≥ 80%, ≥ 90%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, ≥ 99%, or 100%) have been determined to have detectable expression levels of PD-L1.

[0360] In still other embodiments, the tumor sample obtained from the subject comprises 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%, ≥74%, ≥75%, ≥76%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% and / or 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) of the tumor sample or greater than 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of tumor-infiltrating immune cells.

[0361] In any of the foregoing examples, the proportion of a tumor sample comprised of tumor-infiltrating immune cells can be the proportion of tumor area covered by tumor-infiltrating immune cells in a section of a tumor sample obtained from a subject, as assessed, for example, by IHC using an anti-PD-L1 antibody (e.g., SP142 antibody). Any suitable anti-PD-L1 antibody may be used, including, for example, SP142 (Ventana), SP263 (Ventana), 22C3 (Dako), 28-8 (Dako), E1L3N (Cell Signaling Technology), 4059 (Prosci), 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.

[0362] In some embodiments, the tumor sample obtained from the subject comprises 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 Above, 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). For example, in some embodiments, a tumor sample obtained from a subject is determined to have detectable expression levels of PD-L1 in about 1% to less than about 5% (e.g., 1%-4.9%, 1%-4.5%, 1%-4%, 1%-3.5%, 1%-3%, 1%-2.5%, or 1%-2%) of the tumor cells in the tumor sample. In other embodiments, a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in less than about 1% of the tumor cells in the tumor sample.

[0363] In other embodiments, a tumor sample obtained from the subject is determined to have a detectable expression level of PD-L1 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 contains less than about 5% to 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%).

[0364] In yet other embodiments, the tumor sample obtained from the subject comprises 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) of the tumor cells in the tumor sample. , 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). In some embodiments, the tumor sample obtained from the subject contains between about 50% and 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%, between 60% and 65%, 65% and 99%, 65% and 95%, 65% and 90%, 65% and 85%, 65% and 80%, 65% and 75%, 65% and 70%, 70% and 99%, 70% and 95%, 70% and 90%, 70% and 85%, 70% and 80%, 70% and 75%, 75% and 99%, 75% and 95%, 75% and 90%, 75% and 85%, 75% and 80%, 80% and 99%, 80% and 95%, 80% and 90%, 80% and 85%, 85% and 99%, 85% and 95%, 85% and 90%, 90% and 99%, or 90% and 95%).

[0365] In some instances, the CPS is determined in a tumor sample obtained from the patient. In some embodiments, the CPS is determined by positive staining with an anti-PD-L1 diagnostic antibody (e.g., as part of an IHC assay), where the anti-PD-L1 diagnostic antibody is SP142, SP263, 22C3, or 28-8. In some embodiments, the CPS is greater than or equal to 1 when determined by positive staining with the anti-PD-L1 diagnostic antibody SP263 (e.g., as calculated using a Ventana SP263 IHC assay), 22C3 (e.g., as calculated using a PHARMDX 22C3 IHC assay), or 28-8 (e.g., as calculated using a PHARMDX 28-8 IHC assay). In some embodiments, the CPS is 10 or greater, as determined by positive staining with the anti-PD-L1 antibody SP263 (e.g., as calculated using a Ventana SP263 IHC assay), 22C3 (e.g., as calculated using a PHARMDX 22C3 IHC assay), or 28-8 (e.g., as calculated using a PHARMDX 28-8 IHC assay).

[0366] In some instances, the percentage of PD-L1-positive tumor cells in the subject is determined. In some embodiments, the percentage of PD-L1-positive tumor cells is determined by positive staining with an anti-PD-L1 diagnostic antibody (e.g., as part of an IHC assay), where the anti-PD-L1 antibody is SP142, SP263, 22C3, or 28-8. In some embodiments, the percentage of PD-L1-positive tumor cells is 1% or greater tumor cells (TC), as determined by positive staining with the anti-PD-L1 antibody SP263 (e.g., calculated using a Ventana SP263 IHC assay) or 22C3 (e.g., calculated using a PHARMDX 22C3 IHC assay). In some embodiments, the proportion of PD-L1 positive tumor cells is less than 1% TC (e.g., 0% to 1% TC, e.g., PD-L1 negative), as determined by positive staining with the anti-PD-L1 antibody SP263 (e.g., calculated using a Ventana SP263 IHC assay) or 22C3 (e.g., calculated using a PHARMDX 22C3 IHC assay).

[0367] In some cases, in any of the methods, uses, or compositions for use described herein, the tumor sample obtained from the 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.

[0368] In some cases, the sample is selected from the group consisting of a tissue sample, a whole blood sample, a serum sample, and a plasma sample.

[0369] 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.

[0370] In some embodiments, the tumor sample is a formalin-fixed, paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample. In some embodiments, the tumor sample is an FFPE tumor sample.

[0371] In other cases, the sample may be a cytology sample (eg, a fine needle aspiration).

[0372] The presence and / or expression level of any of the above biomarkers (e.g., PD-L1 (e.g., including PD-L1 expression on tumor-infiltrating immune cells (ICs) in a tumor sample obtained from a subject and / or PD-L1 expression on tumor cells (TCs) in a tumor sample obtained from a subject)) in a tumor sample obtained from a subject can be assessed qualitatively and / or quantitatively based on any suitable criteria known in the art, including, but not limited to, DNA, mRNA, cDNA, protein, protein fragments, and / or gene copy number. Methodologies for measuring such biomarkers are known in the art and understood by those of skill in the art. Such methodologies include, but are not limited to, IHC, Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, quantitative blood-based assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization (ISH), fluorescent in situ hybridization (FISH), Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (PCR) including quantitative real-time PCR (qRT-PCR) and other amplification-based detection methods (e.g., branched DNA, SISBA, and TMA, etc.), RNA-Seq, microarray analysis, gene expression profiling, whole genome sequencing (WGS), and / or serial analysis of gene expression ("SAGE"), as well as any one of a wide variety of assays that may be performed by protein, gene, and / or tissue array analysis. Exemplary protocols for assessing the status of genes and gene products are found, for example, in Parts 2 (Northern blotting), 4 (Southern blotting), 15 (immunoblotting), and 18 (PCR analysis) of Current Protocols in Molecular Biology, edited by Ausubel et al. (1995). Multiplex immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD") can also be used.

[0373] In some embodiments of any of the aforementioned methods, the expression level of a biomarker (e.g., PD-L1) may be a protein expression level.

[0374] Biomarker analysis can be performed, for example, by the AHC method or the ACC method. The AHC and ACC methods are typically achieved by contacting a sample from a tumor with a biomarker-specific reagent under conditions that facilitate 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 proximity to the biomarker, thereby generating a detectable signal localized to the biomarker. Typically, a washing step is performed between the application of different reagents to prevent undesired nonspecific labeling of tissues. The biomarker-labeled reagent can optionally be further labeled with an imaging agent to visualize the macromolecular structure.

[0375] The samples used in the AHC / ACC assay are typically tissue samples that have been processed in a manner compatible with affinity labeling and brightfield microscopic analysis of the sample. In certain embodiments, the samples are microtome sections of formalin-fixed, paraffin-embedded (FFPE) samples derived from tumors.

[0376] Exemplary biomarker-specific reagents useful in the AHC and ACC methods include antibodies and antigen-binding fragments thereof, adnectins (scaffolds based on the 10th unit of FN3 fibronectin; Bristol-Myers Squibb), affibodies (scaffolds based on the Z domain of protein A from S. aureus; Affibody, Solna, Sweden), AVIMER (scaffolds based on domain A / LD receptors; Amgen, Thousand Oaks, CA), dAbs (scaffolds based on VH or VL antibody domains; GlaxoSmithKline, Cambridge, UK), and dAbs (scaffolds based on VH or VL antibody domains; GlaxoSmithKline, Cambridge, UK). These include: DARPins (ankyrin repeat protein-based scaffolds; Molecular Partners, Zurich, Switzerland), anticalins (lipocalin-based scaffolds; Pieris, Freising, Germany), nanobodies (VHH (camelid Ig)-based scaffolds; Ablynx, Ghent, Belgium), transbodies (transferrin-based scaffolds; Pfizer, New York, NY), SMIPs (Emergent BioSolutions, Rockville, MD), and tetranectin (C-type lectin domain (CTLD)-based scaffolds; Borean Pharma, Aarhus, Denmark). These biomarker-specific reagents are reviewed in 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 contents of which are incorporated by reference.

[0377] Non-limiting examples of commercially available detection reagents or kits containing detection reagents suitable for use in the methods of the invention include: the VENTANA ULTRAVIEW detection system (secondary antibodies conjugated to enzymes including HRP and AP); the VENTANA IVIEW detection system (biotinylated anti-species secondary antibodies and streptavidin-conjugated enzymes); the VENTANA Amplification kit (unconjugated secondary antibodies that can be used with any of the aforementioned VENTANA detection systems to increase the number of enzymes deposited at the primary antibody binding sites); the OPTIVIEW detection system (anti-species secondary antibodies conjugated to haptens and anti-hapten tertiary antibodies conjugated to enzyme multimers); and the OPTIVIEW Amplification systems (hapten-conjugated anti-species secondary antibodies, enzyme multimer-conjugated anti-hapten tertiary antibodies, and tyramides conjugated to the same hapten, which can be used with OPTIVIEW kits to increase the number of enzymes deposited at primary antibody binding sites); POWERVISION and POWERVISION+ IHC detection systems (secondary antibodies directly polymerized with HRP or AP to form compact polymers with high enzyme-to-antibody ratios); DAKO ENVISION TM + System (enzyme-labeled polymer conjugated to secondary antibody); CELL IDx's ULTRAPLEX Multiplex Chromogenic IHC Technology (combining a hapten-labeled primary antibody with an enzyme- or fluorescent-labeled anti-hapten secondary antibody).

[0378] If desired, biomarker-labeled slides can be counterstained to aid in the identification of morphologically relevant regions for identifying ROIs, either manually or automatically. Examples of counterstains include brightfield nuclear counterstains such as hematoxylin (stains blue to purple), methylene blue (stains blue), toluidine blue (stains nuclei dark blue and polysaccharides pink to red), nuclear fast red (also known as Kern Echtrot dye, stains red), and methyl green (stains green); and non-nucleogenic stains such as eosin (stains pink).

[0379] An automated labeling system may be used to apply the AHC / ACC assay and counterstain to the sample. Prichard, Overview of Automated Immunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp. 1578-1582 (2014), incorporated herein by reference in its entirety, describes several specific examples of automated AHC labeling systems and their various features, including the intelliPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO AUTOSTAINER LINK 48 (Agilent Technologies), BENCHMARK (Ventana Medical Systems), Leica BOND and LAB VISION AUTOSTAINER (Thermo Scientific) automated AHC labeling systems. Commercially available labeling devices typically operate on one of the following principles: (1) open individual slide labeling, in which the slide is positioned horizontally and the reagent is dispensed as a puddle onto the surface of the slide containing the tissue sample (e.g., as implemented in the DAKO AUTOSTAINER Link 48 (Agilent Technologies) and INTELLIPATH (Biocare Medical) labeling machines); (2) liquid overlay technology, in which the reagent is dispensed over or through a layer of inert fluid deposited on the sample (e.g., as implemented in the BENCHMARK labeling machine); or (3) capillary gap staining, in which the slide surface is placed near another surface, creating a narrow gap through which capillary forces draw the liquid reagent into contact with the sample (e.g., as used in the DAKO TECHMATE, Leica BOND, and DAKO OMNIS labeling machines). Even when capillary gap labeling is repeated multiple times, the fluids in the gap do not mix (e.g., in the DAKO TECHMATE and Leica BOND).A variation on capillary gap labeling, called dynamic gap labeling, uses capillary forces to apply the sample to the slide, then translates the parallel surfaces relative to each other to agitate and mix the reagents during incubation (such as the labeling principle implemented in the DAKO OMNIS slide labeling machine (Agilent)). It has also been proposed to use inkjet technology to deposit reagents on the slide. See WO 2016 / 170008. This list of labeling techniques is not intended to be comprehensive, and any fully or semi-automated system or manual method for performing biomarker labeling may be incorporated into the methods of the present invention.

[0380] In certain embodiments, the methods of the invention comprise contacting a sample with an antibody that specifically binds to a biomarker described herein under conditions that allow binding of the biomarker, and detecting whether a complex forms between the antibody and the biomarker. Such methods can be in vitro or in vivo. In some embodiments, the antibody is used to select subjects eligible for treatment with an anti-cancer therapy that includes a PD-1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab), e.g., a biomarker for selecting subjects. In some embodiments, the antibody is used to select subjects eligible for treatment with an anti-cancer therapy that includes a 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), a biomarker for selecting subjects, etc.

[0381] Any method of measuring protein expression levels known in the art or described herein may be used. For example, in some embodiments, the protein expression levels of biomarkers may be measured using immunohistochemistry (IHC), flow cytometry (e.g., fluorescence activated cell sorting (FACS)), or other methods. TM)), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC.

[0382] In some embodiments, the protein expression level of a biomarker (e.g., PD-L1) is determined in tumor-infiltrating immune cells. In some embodiments, the protein expression level of a biomarker is determined in tumor cells. In some embodiments, the protein expression level of a biomarker is determined in tumor-infiltrating immune cells and / or tumor cells. In some embodiments, the protein expression level of a biomarker is determined in peripheral blood mononuclear cells (PBMCs).

[0383] In certain embodiments, the presence and / or expression level / amount of a biomarker protein (e.g., PD-L1) in a sample is examined using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method for determining or detecting the presence of a protein in a sample. In some embodiments of any of the methods, assays, and / or kits of the invention, the biomarker is one or more of the protein expression products of PD-L1. In certain embodiments, the expression level of the biomarker is determined using a method comprising: (a) performing an IHC analysis of a sample (e.g., 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 in comparison to a standard. In some embodiments, the standard is a reference value. In some embodiments, the standard is a reference sample (e.g., a control cell line staining sample, a tissue sample from a non-cancer subject, or a tumor sample determined to be negative for the biomarker of interest).

[0384] 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.

[0385] IHC can be performed in combination with additional techniques such as morphological staining and / or in situ hybridization (e.g., ISH). Two general methods of IHC are available: direct and indirect assays. According to the first assay, antibody binding to the target antigen is directly determined. This direct assay uses a labeled reagent, such as a fluorescent tag or enzyme-labeled primary antibody, which can be visualized without further antibody interaction. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. If the secondary antibody is conjugated to an enzyme label, a chromogenic or fluorogenic substrate is added to visualize the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody.

[0386] The primary and / or secondary antibodies used in IHC are typically labeled with a detectable moiety. Numerous labels are available, which generally fall into the following categories: (a) 35 S, 14 C. 125 1. 3 H, and 131(b) colloidal gold particles; (c) fluorescent labels, including, but not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycoglycerin, 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) a variety of enzyme-substrate labels are available, and U.S. Pat. No. 4,275,149 provides a review of some of these. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; see, e.g., U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.

[0387] Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate; alkaline phosphatase (AP) with para-nitrophenyl phosphate as a chromogenic substrate; and β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-BD-galactosidase) or a fluorogenic substrate (e.g., 4-methylumbelliferyl-BD-galactosidase). For reviews, see, e.g., U.S. Patent Nos. 4,275,149 and 4,318,980.

[0388] Specimens may be prepared, for example, manually or using an automated staining instrument (e.g., a Ventana BenchMark XT or Benchmark ULTRA instrument). Specimens prepared in this manner may be mounted and coverslipped. Slide evaluation may then be determined, for example, using a microscope, and staining intensity criteria routinely used in the art may be used. In certain embodiments, when IHC is used to examine cells and / or tissues derived from a tumor, it is understood that staining is generally determined or assessed in tumor cells and / or tissues (vs. stroma or surrounding tissue that may be present in the sample). In other embodiments, staining may be determined or assessed in stroma or surrounding tissue that may be present in the sample. In some embodiments, when IHC is used to examine cells and / or tissues derived from a tumor, it is understood that staining includes determination or assessment in tumor-infiltrating immune cells, including intratumoral or peritumoral immune cells. In some embodiments, the presence of the biomarker is detected by IHC in more than 0% of the samples, at least 1% of the samples, at least 5% of the samples, at least 10% of the samples, at least 15% of the samples, at least 15% of the samples, at least 20% of the samples, at least 25% of the samples, at least 30% of the samples, at least 35% of the samples, at least 40% of the samples, at least 45% of the samples, at least 50% of the samples, at least 55% of the samples, at least 60% of the samples, at least 65% of the samples, at least 70% of the samples, at least 75% of the samples, at least 80% of the samples, at least 85% of the samples, at least 90% of the samples, at least 95% of the samples, or more. Samples can be scored using any method known in the art, for example, by a pathologist or by automated image analysis.

[0389] In some embodiments of any of the methods of the invention, 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).

[0390] 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 of determining nucleic acid expression levels can be used. In some embodiments, the nucleic acid expression level is determined using RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, ISH, or a combination thereof.

[0391] 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, Northern blots, and related techniques using labeled riboprobes specific for one or more genes), 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-based detection methods such as branched DNA, SISBA, and TMA). Furthermore, these methods may include one or more steps that allow for the determination of the level of a target mRNA in a biological sample (e.g., by simultaneously examining the level of a control mRNA sequence of a "housekeeping" gene such as an actin family member). Optionally, the sequence of the amplified target cDNA can be determined. Optional methods include protocols for examining or detecting mRNA, such as a target mRNA, in a tissue or cell sample by microarray technology. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured so that the sequence and location of each array member is known. For example, a selection of genes whose expression correlates with increased or decreased clinical benefit of treatments, including immunotherapy and inhibitory stromal antagonists, can be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.

[0392] The sample can be obtained from the subject at any suitable time. For example, in some embodiments, the sample is obtained from the subject prior to administration of the therapeutic regimen (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years). In some embodiments of any of the foregoing methods, the sample from the subject is obtained about 2 to about 10 weeks (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks) after administration of the therapeutic regimen. In some embodiments, the sample from the subject is obtained about 4 to about 6 weeks after administration of the therapeutic regimen.

[0393] In some embodiments, the expression level or number 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 fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, and tissue culture medium, tissue extract (e.g., homogenized tissue), tumor tissue, cell extract, or a 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 comprises tumor cells, tumor-infiltrating immune cells, stromal cells, or a combination thereof. In some embodiments, the tumor tissue sample is a formalin-fixed, paraffin-embedded (FFPE) sample, an archived sample, a fresh sample, or a frozen sample.

[0394] In some instances, the sample may be a cytology sample (eg, a fine needle aspiration).

[0395] For example, in some embodiments, the expression level of a biomarker (e.g., PD-L1) is detected in tumor-infiltrating immune cells, tumor cells, PBMCs, or a combination thereof using known techniques (e.g., IHC, immunofluorescence microscopy, or flow cytometry). Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, or any combination thereof, and other tumor stromal cells (e.g., fibroblasts). Such tumor-infiltrating immune cells include T lymphocytes (e.g., CD8 + T lymphocytes (e.g., CD8 + T effector (Teff) cells) and / or CD4 + T lymphocytes (e.g., CD4 + The biomarkers may be Teff cells), B lymphocytes or other myeloid lineage cells (including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer (NK) cells). In some embodiments, staining of the biomarker is detected as membranous staining, cytoplasmic staining, or a combination thereof. In other embodiments, the absence of the biomarker is detected as a lack of staining or no staining in the sample compared to a reference sample.

[0396] In certain embodiments, the expression level of a biomarker is assessed in a sample containing or suspected to contain cancer cells. The sample can be, for example, a tissue biopsy or metastatic lesion obtained from a subject suffering from, suspected of suffering from, or diagnosed with cancer (e.g., bladder cancer (e.g., UC, including locally advanced or metastatic UC)). In some embodiments, the sample is a sample of tissue (e.g., renal pelvis, ureter, bladder, and / or urethral tissue), a biopsy of a tumor (e.g., locally advanced or metastatic UC tumor, including pelvic, ureter, bladder, and / or urethral tumor), a lesion or section of known or suspected metastatic bladder cancer (e.g., metastatic UC), or a blood sample, e.g., a peripheral blood sample, known or suspected to contain circulating cancer cells, e.g., bladder cancer cells (e.g., UC cells, including locally advanced or metastatic UC cells). ) samples may contain both cancer cells (i.e., tumor cells) and non-cancerous cells (e.g., lymphocytes such as T cells or NK cells), and in certain embodiments, contain both cancerous and non-cancerous cells. Methods for obtaining biological samples, including tissue resections, biopsies, and bodily fluids (e.g., blood samples containing cancer / tumor cells), are well known in the art.

[0397] The sample may be from 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); urinary tract cancer; breast cancer (e.g., HER2+ breast cancer and ER-, PR-, and HER2+ breast cancers); TNBC that is HER2-; prostate cancer, including CRPC; peritoneal cancer; hepatocellular carcinoma; gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., PDAC); glioblastoma; cervical cancer; ovarian cancer; liver cancer (e.g., HCC); hepatic cancer; 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 lentigo melanoma, nodular and nodular melanoma); multiple myeloma and B-cell lymphoma (low-grade / follicular NHL; SL The cancer may be NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; CLL; ALL; AML; hairy cell leukemia; CML; PTLD; and MDS, as well as nevus syndrome, edema (such as associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and abnormal blood vessel proliferation associated with related metastases. In some examples, the cancer is bladder cancer (e.g., UC, including locally advanced or metastatic UC).

[0398] In certain embodiments, the subject may have an advanced, refractory, recurrent, and / or chemotherapy-resistant form of cancer.

[0399] In certain embodiments, the presence and / or expression level / amount of the biomarker in the first sample is increased compared to its presence / absence and / or expression level / amount in the second sample. In certain embodiments, the presence / absence and / or expression level / amount of the biomarker in the first sample is decreased or reduced compared to its presence and / or expression level / amount in the second sample. In certain embodiments, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0400] In certain 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 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 an earlier time point than when the test sample is obtained. Such a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful when the reference sample is obtained during the initial diagnosis of cancer and the test sample is obtained when the cancer has become metastatic.

[0401] In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined plurality of samples from one or more healthy individuals who are not the subject. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined plurality of samples from one or more individuals who are not the subject and have a disease or disorder (e.g., cancer). In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from normal tissue, or a pooled plasma or serum sample from one or more individuals who are not the subject. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from tumor tissue, or a pooled plasma or serum sample from one or more individuals who are not the subject and have a disease or disorder (e.g., cancer).

[0402] In some embodiments, the methods of the invention further comprise administering to the subject an effective amount of a therapeutic regimen described herein (e.g., a therapeutic regimen comprising a 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)), e.g., based on the expression level of one or more biomarkers (e.g., PD-L1). The therapeutic regimen can be any therapeutic regimen described herein, e.g., in Section II above.

[0403] The presence and / or expression level of PD-L1 can be assessed in a subject treated according to any of the methods and compositions for use described herein. In some embodiments, the method comprises determining the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the subject. In other embodiments, the expression level of PD-L1 in the biological sample (e.g., a tumor sample) obtained from the subject has been determined before the initiation of treatment. In still other embodiments, the expression level of PD-L1 in the biological sample (e.g., a tumor sample) obtained from the subject can be determined after the initiation of treatment.

[0404] V. PD-1 Axis Binding Antagonists PD-1 axis binding antagonists can include PD-L1 binding antagonists, PD-1 binding antagonists, and PD-L2 binding antagonists. Any suitable PD-1 axis binding antagonist can be used.

[0405] 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, comprising administering to the subject a therapeutic 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, comprising administering to the subject a therapeutic 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 ... and a method for enhancing immune function in a subject with bladder cancer, the method comprising administering to the subject a therapeutic regimen comprising an anti-PD-L1 antibody (e.g., atezolizumab). Also provided herein is a method for enhancing immune function in a subject with bladder cancer (e.g., UC, including locally advanced or metastatic UC), the method comprising administering to the subject a therapeutic 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). Related compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture for use are also provided. Any of the methods, compositions for use, kits, or articles of manufacture described herein may comprise or be accompanied by any of the PD-1 axis-binding antagonists described below.

[0406] A. PD-L1 Binding Antagonists In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In still other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. The PD-L1 binding antagonist may be, but is not limited to, an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, oligopeptide, or 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 / or WO 2015 / 033299.

[0407] 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 of the cases herein, the isolated anti-PD-L1 antibody can bind to human PD-L1, for example, human PD-L1 as set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7-1, or a variant thereof. In some cases, the anti-PD-L1 antibody can inhibit the binding between PD-L1 and PD-1 and / or between PD-L1 and B7-1. In some cases, the anti-PD-L1 antibody is a monoclonal antibody. In some cases, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some cases, the anti-PD-L1 antibody is a humanized antibody. In some cases, the anti-PD-L1 antibody is a human antibody. Exemplary anti-PD-L1 antibodies include atezolizumab, MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), SHR-1316, CS1001, embafolimab, 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. Examples of anti-PD-L1 antibodies useful in the methods of the invention and methods for making them are described in WO 2010 / 077634 and U.S. Patent No. 8,217,149, each of which is incorporated by reference in its entirety.

[0408] In some instances, the anti-PD-L1 antibody: (a) the HVR-H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 3), AWISPYGGSTYYADSVKG (SEQ ID NO: 4), and RHWPGGFDY (SEQ ID NO: 5), respectively; and (b) comprising the HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 6), SASFLYS (SEQ ID NO: 7), and QQYLYHPAT (SEQ ID NO: 8), respectively.

[0409] In one embodiment, the anti-PD-L1 antibody is (a) a heavy chain variable region (VH) comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 9); (b) a light chain variable region (VL) comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 10).

[0410] In some examples, 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 a VH 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 a VL comprising the sequence of SEQ ID NO: 10; or (c) the VH of (a) and the VL of (b).

[0411] In one embodiment, the anti-PD-L1 antibody comprises atezolizumab, which comprises: (a) Heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1) and (b) Light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2)

[0412] In some instances, 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 Inc.).

[0413] In some instances, 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 (MedImmune, AstraZeneca) described in WO 2011 / 066389 and U.S. Patent Application Publication No. 2013 / 034559.

[0414] In some instances, the anti-PD-L1 antibody is MDX-1105 (Bristol-Myers Squibb). MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO 2007 / 005874.

[0415] In some instances, the anti-PD-L1 antibody is LY3300054 (Eli Lilly and Company).

[0416] In some instances, the anti-PD-L1 antibody is STI-A1014 (Sorrento). STI-A1014 is a human anti-PD-L1 antibody.

[0417] In some instances, the anti-PD-L1 antibody is KN035 (Suzhou Alphamab), which is a single domain antibody (dAB) generated from a camel phage display library.

[0418] In some cases, the anti-PD-L1 antibody comprises a cleavable moiety or linker that, when cleaved (e.g., by proteases in the tumor microenvironment), activates the antibody antigen-binding domain so that it is able to bind its antigen, e.g., by removing non-binding steric moieties. In some cases, the anti-PD-L1 antibody is CX-072 (CytomX Therapeutics).

[0419] In some instances, the anti-PD-L1 antibody comprises six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and / or a heavy chain variable domain and a light chain variable domain from the anti-PD-L1 antibodies described in US Patent Application Publication No. 20160108123, WO 2016 / 000619, WO 2012 / 145493, US Patent No. 9205148, WO 2013 / 181634, or WO 2016 / 061142.

[0420] In yet a further specific embodiment, the anti-PD-L1 antibody has reduced or minimal effector function. In a further specific embodiment, the minimal effector function is due to an "effector-less Fc mutation" or aglycosylation. In yet a further embodiment, the effector-less Fc mutation is an N297A or D265A / N297A substitution in the constant region. In a still further embodiment, the effector-less Fc mutation is an N297A substitution in the constant region. In some embodiments, the isolated anti-PD-L1 antibody is aglycosylated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the 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 the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of a glycosylation site from an antibody is conveniently accomplished by altering the amino acid sequence such that one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites) is deleted. This alteration 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).

[0421] B. PD-1 Binding Antagonists 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 may 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., an 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 small molecule. In some cases, the PD-1 binding antagonist is AUNP-12 (Pierre Fabre / Aurigene). See, e.g., WO 2012 / 168944, WO 2015 / 036927, WO 2015 / 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.

[0422] 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 of the cases herein, the PD-1 antibody can bind to human PD-1 or a variant thereof. In some embodiments, 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')2 fragments. 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, prorugolimab, canrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, retifanlimab, sasanlimab, penprimab, CS1003, HLX10, SCT-I10A, zimberelimab, balstilimab, genolimuzumab, BI 754091, cetrelimab, YBL-006, BAT1306, HX008, budigalimab, AMG404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103, and hAb21.

[0423] In some instances, 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 Opdivo®, is an anti-PD-1 antibody described in WO 2006 / 121168.

[0424] In some instances, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number 1374853-91-4). Pembrolizumab (Merck & Co.), also known as MK-3475, Merck 3475, lambrolizumab, SCH-900475, and Keytruda®, is an anti-PD-1 antibody described in WO 2009 / 114335.

[0425] In some instances, the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.

[0426] In some instances, the anti-PD-1 antibody is PDR001 (CAS Registry Number 1859072-53-9; Novartis). PDR001 is a humanized IgG4 anti-PD-1 antibody that blocks the binding of PD-L1 and PD-L2 to PD-1.

[0427] In some instances, the anti-PD-1 antibody is REGN2810 (Regeneron). REGN2810 is a human anti-PD-1 antibody.

[0428] In some instances, the anti-PD-1 antibody is BGB-108 (BeiGene).

[0429] In some instances, the anti-PD-1 antibody is BGB-A317 (BeiGene).

[0430] In some instances, the anti-PD-1 antibody is JS-001 (Shanghai Junxi Co., Ltd.), which is a humanized anti-PD-1 antibody.

[0431] In some instances, the anti-PD-1 antibody is STI-A1110 (Sorrento). STI-A1110 is a human anti-PD-1 antibody.

[0432] In some instances, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR-1210 is a human IgG4 anti-PD-1 antibody.

[0433] In some instances, the anti-PD-1 antibody is PF-06801591 (Pfizer).

[0434] In some instances, the anti-PD-1 antibody is TSR-042 (also known as ANB 011; Tesaro / AnaptysBio).

[0435] In some instances, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).

[0436] In some instances, the anti-PD-1 antibody is ENUM 244C8 (Enumeral Biomedical Holdings, Inc.), an anti-PD-1 antibody that inhibits PD-1 function without blocking the binding of PD-L1 to PD-1.

[0437] In some instances, the anti-PD-1 antibody is ENUM 388D4 (Enumeral Biomedical Holdings, Inc.). ENUM 388D4 is an anti-PD-1 antibody that competitively inhibits the binding of PD-L1 to PD-1.

[0438] In some instances, the anti-PD-1 antibody is a PD-1 antibody described in WO 2015 / 112800, WO 2015 / 112805, WO 2015 / 112900, U.S. Patent Application Publication Nos. 20150210769, WO 2016 / 089873, WO 2015 / 035606, WO 2015 / 085847, WO 2014 / 206107, WO 2012 / 145493, U.S. Patent and WO 2014 / 194302.

[0439] In still further particular embodiments, the anti-PD-1 antibody has reduced or minimal effector function. In further particular embodiments, the minimal effector function is due to an "effector-less Fc mutation" or aglycosylation. In still further instances, the effector-less Fc mutation is an N297A or D265A / N297A substitution in the constant region. In some instances, the isolated anti-PD-1 antibody is aglycosylated.

[0440] C. PD-L2 Binding Antagonists 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 certain embodiments, the PD-L2 binding ligand partner is PD-1. The PD-L2 binding antagonist may be, but is not limited to, an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, or a small molecule.

[0441] In some cases, the PD-L2 binding antagonist is an anti-PD-L2 antibody. In any of the cases 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')2 fragments. 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 still further particular embodiments, the anti-PD-L2 antibody has reduced or minimal effector function. In further particular embodiments, the minimal effector function results from an "effectorless Fc mutation" or aglycosylation. In still further instances, the effectorless Fc mutation is an N297A or D265A / N297A substitution in the constant region. In some cases, the isolated anti-PD-L2 antibody is aglycosylated.

[0442] 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 described herein for use in any of the cases listed above may have, alone or in combination, any of the characteristics described in Sections 1-7 below.

[0443] 1. Antibody affinity In some instances, an antibody described herein (e.g., an anti-PD-L1 antibody) may have a PD-L1 activity 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 It has a dissociation constant (Kd) of 1 M.

[0444] In some instances, Kd is measured by a radiolabeled antigen binding assay (RIA). In some instances, an RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the Kd is measured using a minimum concentration of ( 125 I) The solution binding affinity of Fab for antigen is measured by equilibrating the Fab with labeled antigen and then capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of 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 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 [I] The antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:45934599 (1997)). The Fab of interest is then incubated overnight, although incubation may be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with PBS containing 0.1% polysorbate 20 (TWEEN-20®). Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20 TM Packard) was added and the plate was then loaded onto a TOPCOUNT TM Count for 10 minutes in a gamma counter (Packard). Concentrations of each Fab that give 20% or less of maximal binding are selected for use in competitive binding assays.

[0445] In another example, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, assays using a BIACORE®-2000 or BIACORE®-3000 (Biacore, Piscataway, NJ) are performed at 25°C using approximately 10 response units (RU) of immobilized antigen CM5 chips. In one example, a carboxymethylated dextran biosensor chip (CM5, Biacore) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min, achieving approximately 10 response units (RU) of bound protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were incubated in 0.05% polysorbate 20 (TWEEN-20 TM ) in PBS containing surfactant (PBST) at 25°C at a flow rate of approximately 25 ul / min. on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple 1:1 Langmuir binding model (BIAcore Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is k off / k on See, for example, Chen et al., J. Mol. Biol. 293:865881 (1999). When the association rate by the surface plasmon resonance assay is 10 6 M -1 s -1If the association rate exceeds 100 kJ / s, the association rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen as measured with a spectrometer such as a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 Series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette.

[0446] 2. Antibody fragment In some instances, the antibodies (e.g., anti-PD-L1 antibodies) described herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0447] Diabodies are antibody fragments with two antigen-binding sites and may be bivalent or bispecific. See, e.g., EP 404097; WO 1993 / 01161; Hudson et al. Nat. Med. 9:129-134 (2003); and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003).

[0448] Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some cases, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, Massachusetts; see, e.g., U.S. Patent No. 6,248,516).

[0449] Antibody fragments can be produced 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.

[0450] 3. Chimeric and humanized antibodies In some instances, an antibody described herein (e.g., an anti-PD-L1 antibody) is a chimeric antibody. Particular chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al. Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0451] In some cases, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs (e.g., CDRs) (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody also optionally comprises at least a portion of a human constant region. In some cases, some FR residues of the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0452] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:2534 (2005) (describing specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0453] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:16191633 (2008)); and framework regions obtained from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:1067810684 (1997) and See Rosok et al., J. Biol. Chem. 271:2261122618 (1996)).

[0454] 4. Human antibodies In some instances, the antibodies described herein (e.g., anti-PD-L1 antibodies) are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0455] Human antibodies can be prepared by administering an immunogen to transgenic animals that have been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, XENOMOUSE. TM See also U.S. Patent Nos. 6,075,181 and 6,150,584, describing HuMab® technology; U.S. Patent No. 5,770,429, describing HuMab® technology; U.S. Patent No. 7,041,870, describing KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, describing VELOCIMOUSE® technology. The human variable regions of intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0456] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (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 by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0457] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0458] 5. Library-derived antibodies Antibodies (e.g., anti-PD-L1 antibodies) can be isolated by screening combinatorial libraries for antibodies with the desired activity. Various methods are known in the art, for example, for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, in 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. 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(12):119-132 (2004).

[0459] In some phage display methods, VH and VL gene repertoires can be separately cloned by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need for constructing hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J., 12:725-734 (1993). Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0460] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0461] 6. Multispecific antibodies In any one of the above embodiments, the antibodies described herein (e.g., anti-PD-L1 antibodies) may be multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In some cases, the antibodies described...

Claims

1. A kit for identifying tumors likely to respond to PD-1 axis-coupled antagonists, comprising the following means: (a) Stain the first portion of the tumor with an immunotropic PD-L1 assay to obtain the first stained sample; (b) To generate a first score by applying a first scoring algorithm to a first stained sample; (c) Stain a second portion of the tumor with an immunoindependent PD-L1 assay to obtain a second stained sample; (d) generating a second score by applying a second scoring algorithm to a second stained sample; and (e) Compare the first score to the first cutoff and the second score to the second cutoff, such that if the first score meets or exceeds the first cutoff and the second score meets or exceeds the second cutoff, the tumor is likely to respond to a PD-1 axially coupled antagonist. A kit that includes this.

2. A pharmaceutical product for treating cancer in a patient, comprising a PD-1 axially coupled antagonist, wherein a tumor sample obtained from the patient is (i) a first score obtained by applying a first scoring algorithm to a first stained portion of a tumor sample that satisfies or exceeds a first cutoff, wherein the first stained portion of the tumor sample is stained with an immunotropic PD-L1 assay; and (ii) A second score obtained by applying a second scoring algorithm to a second stained portion of a tumor sample that satisfies or exceeds a second cutoff, wherein the second stained portion of the tumor sample is stained with an immunoindependent PD-L1 assay. A pharmaceutical product that has been determined to possess [certain properties].

3. A kit according to claim 1 or a pharmaceutical product according to claim 2, wherein the immunotropic PD-L1 assay is (i) Using the first scoring algorithm with the first cutoff value, an overall agreement rate (OPA) of at least 80%, at least 85%, at least 90%, or at least 95% is obtained with the SP142 assay; (ii) Using the first scoring algorithm with the first cutoff value, a positive agreement rate (PPA) of at least 80%, at least 85%, at least 90%, or at least 95% with the SP142 assay is obtained; (iii) Using the first scoring algorithm with the first cutoff value, a negative agreement rate (NPA) of at least 80%, at least 85%, at least 90%, or at least 95% is obtained with the SP142 assay; (iv) Using the first scoring algorithm with the first cutoff value, the SP142 assay yields 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; (v) Using the first scoring algorithm with the first cutoff value, the SP142 assay yields at least 80%, at least 85%, at least 90%, or at least 95% of PPA and at least 80%, at least 85%, at least 90%, or at least 95% of OPA; (vi) Using the first scoring algorithm with the first cutoff value, the SP142 assay yields 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; and / or (vii) Using the first scoring algorithm with the first cutoff value, the SP142 assay yields 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. Having; and / or an immunoindependent PD-L1 assay, (i) Using the second scoring algorithm with the second cutoff value, the 22C3 assay yields at least 80%, at least 85%, at least 90%, or at least 95% of OPA; (ii) Using the second scoring algorithm with the second cutoff value, the 22C3 assay yields at least 80%, at least 85%, at least 90%, or at least 95% of PPA; (iii) Using the second scoring algorithm with the second cutoff value, the 22C3 assay yields at least 80%, at least 85%, at least 90%, or at least 95% NPA; (iv) Using the second scoring algorithm with the second cutoff value, the 22C3 assay yields 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; (v) Using the second scoring algorithm with the second cutoff value, the 22C3 assay yields at least 80%, at least 85%, at least 90%, or at least 95% of PPA and at least 80%, at least 85%, at least 90%, or at least 95% of OPA; (vi) Using the second scoring algorithm with the second cutoff value, the 22C3 assay yields 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; and / or (vii) Using the second scoring algorithm with the second cutoff value, the 22C3 assay yields 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. A kit or pharmaceutical product that possesses a medical device.

4. A method for stratifying tumors having scores from an immunoindependent PD-L1 assay that exceed a predetermined cutoff, (a) A step of obtaining a stained sample by staining a portion of the tumor with an immunotropic PD-L1 assay; (b) A step of generating an immunotropic PD-L1 assay score by applying a scoring algorithm to a stained sample; and (c) A step of comparing an immune-targeting PD-L1 assay score with a first cutoff, wherein if the immune-targeting PD-L1 assay score meets or exceeds the first cutoff, the tumor is likely to respond to a PD-1 axis-binding antagonist. Methods that include...

5. The method according to claim 4, wherein the scoring algorithm is a composite positive score (CPS), and the tumor is a tumor with a CPS ≥ 10% determined by the 22C3 assay, the SP263 assay, or the 28-8 assay.

6. The kit according to claim 1, the pharmaceutical product according to claim 2, or the method according to claim 4, wherein the immunotropic PD-L1 assay comprises a PD-L1 immunohistochemistry (IHC) assay containing the VENTANA SP142 anti-PD-L1 diagnostic antibody.

7. The kit, pharmaceutical, or method according to claim 6, wherein the first cutoff is a detectable expression level of PD-L1 in tumor-infiltrating immune cells constituting 5% or more of the tumor sample.

8. The kit according to claim 1, the pharmaceutical product according to claim 2, or the method according to claim 4, wherein the immunoindependent PD-L1 assay comprises a PD-L1 IHC assay containing 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.

9. The kit, pharmaceutical, or method according to claim 8, wherein the second cutoff is CPS ≥ 10%.

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

11. The method according to claim 10, wherein the detectable signal of the VENTANA SP142 anti-PD-L1 diagnostic antibody is an amplified signal.

12. The method according to claim 11, wherein the amplified signal is generated by tyramide signal amplification.

13. Processes (a) and (b) (i) At the same time; (ii) sequentially; or (iii) In different sections of the tumor sample or in the same section of the tumor sample The method according to claim 10, which is implemented.

14. The method according to claim 13, wherein the different sections of the tumor specimen are serial sections.

15. The method according to any one of claims 10 to 14, wherein the visualization step includes IHC or immunofluorescence (IF).

16. The method according to claim 15, wherein the visualization step includes IHC.

17. A kit according to claim 1, a pharmaceutical product according to claim 2, or a method according to claim 4, wherein the tumor or cancer is bladder cancer, kidney cancer, lung cancer, urinary tract cancer, breast cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, gastric or gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatic cancer, 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, multiple myeloma or B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), pilocytic cell leukemia, chronic myeloblastic leukemia (CML), post-transplant lymphoproliferative disorder (PTLD), myelodysplastic syndrome (MDS), Meigs syndrome, brain cancer or head and neck cancer.

18. The kit, pharmaceutical, or method according to claim 17, wherein the tumor or cancer is bladder cancer.

19. The kit, pharmaceutical product, or method according to claim 18, wherein the bladder cancer is urothelial carcinoma (UC).

20. The kit, pharmaceutical, or method according to claim 19, wherein the UC is locally progressive or metastatic UC.

21. The pharmacopoeia according to claim 2, or the method according to claim 4, wherein the cancer is locally advanced or metastatic UC, and the patient has not been previously treated for locally advanced or metastatic UC.

22. The pharmaceutical agent or method according to claim 21, wherein locally advanced or metastatic UC is histologically confirmed locally advanced urothelial carcinoma (T4b, any N; or any T, N2-3) or metastatic urothelial carcinoma (mUC) (M1, stage IV).

23. The pharmaceutical product according to claim 2, wherein the pharmaceutical product is to be administered to a patient.

24. The pharmaceutical product according to claim 23, wherein the PD-1 axis-binding antagonist is a PD-L1-binding antagonist, a PD-1-binding antagonist, or a PD-L2-binding antagonist.

25. The pharmaceutical product according to claim 24, wherein the PD-1 axis-binding antagonist is a PD-L1 binding antagonist.

26. The pharmaceutical product according to claim 25, wherein the PD-1 axis-binding antagonist is an anti-PD-L1 antibody.

27. Anti-PD-L1 antibodies are effective against the following HVRs: (i) HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO: 3); (ii) HVR-H2 sequence of AWISPYGGSTYYADSVKG (Sequence ID 4); (iii) HVR-H3 sequence of RHWPGGGFDY (SEQ ID NO: 5); (iv) HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO: 6); (v) HVR-L2 sequence of SASFLYS (SEQ ID NO: 7); and (vi) HVR-L3 sequence of QQYLYHPAT (SEQ ID NO: 8) The pharmaceutical product according to claim 26, including the above.

28. Anti-PD-L1 antibody, (a) VH domain containing the amino acid sequence of Sequence ID No. 9; and (b) VL domain containing the amino acid sequence of SEQ ID NO: 10 The pharmaceutical product according to claim 27, including the above.

29. The anti-PD-L1 antibody is atezolizumab. The pharmaceutical product according to claim 28.

30. The pharmaceutical product according to claim 29, wherein atezolizumab is administered intravenously to the patient in doses of approximately 840 mg every two weeks, approximately 1200 mg every three weeks, or approximately 1680 mg every four weeks.

31. The pharmaceutical product according to claim 30, wherein atezolizumab is administered intravenously to the patient at a dose of approximately 1200 mg every three weeks.

32. The pharmaceutical product according to claim 31, wherein atezolizumab is administered to the patient in a 21-day drug cycle, and atezolizumab is administered intravenously to the patient at a dose of approximately 1200 mg on day -2 to day -4 of each 21-day drug cycle.

33. The pharmaceutical product according to claim 32, wherein atezolizumab is to be administered intravenously to the patient at a dose of approximately 1200 mg on the first day of each 21-day drug cycle.

34. The pharmaceutical product according to claim 2, wherein one or more additional therapeutic agents are administered to the patient, and one or more additional therapeutic agents include platinum-based chemotherapy.

35. The pharmaceutical product according to claim 34, wherein the platinum-based chemotherapy comprises a platinum-based chemotherapeutic agent and a nucleoside analog.

36. The pharmaceutical product according to claim 35, wherein the platinum-based chemotherapeutic agent is cisplatin, carboplatin, or oxaliplatin; and / or the nucleoside analog is gemcitabine.

37. A kit for determining the presence or expression level of PD-L1 in tumor samples obtained from patients with cancer, (a) Determining the presence or expression level of PD-L1 in tumor samples obtained from patients using a PD-L1 IHC assay containing the VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) Determining the presence or expression level of PD-L1 in tumor samples obtained from patients using a PD-L1 IHC assay comprising Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody; A kit that includes the means to do so.

38. It's a kit, (a) VENTANA SP142 anti-PD-L1 diagnostic antibody; and (b) Dako 22C3 anti-PD-L1 diagnostic antibody, VENTANA SP263 anti-PD-L1 diagnostic antibody, or 28-8 anti-PD-L1 diagnostic antibody A kit that includes this.

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