HK1236961B - Anti-pd-l1 antibodies and diagnostic uses thereof - Google Patents

Description

Anti-PD-L1 antibodies and their diagnostic uses

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Application No. 62/023,741, filed on July 11, 2014, the disclosure of which is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to anti-programmed death ligand 1 (PD-L1) antibodies and methods of using the same.

background

Programmed death ligand 1 (PD-L1) is a protein that has been implicated in suppressing immune system responses during chronic infection, pregnancy, tissue allografts, autoimmune diseases, and cancer. PD-L1 regulates immune responses by binding to an inhibitory receptor called programmed death 1 (PD-1), which is expressed on the surface of T cells, B cells, and monocytes. PD-L1 also negatively regulates T cell function through interaction with another receptor, B7.1 (also known as B7-1 or CD80). The formation of PD-L1/PD-1 and PD-L1/B7.1 complexes negatively regulates T cell receptor signaling, leading to subsequent downregulation of T cell activation and inhibition of anti-tumor immune activity. PD-L1 is overexpressed in many cancers, including a wide variety of solid tumors such as bladder, breast, colon, lung, melanoma, ovarian, salivary, gastric, and thyroid tumors. Overexpression of PD-L1 in tumor cells can promote tumor invasion and is often associated with a poor prognosis.

Given the role of PD-L1 in cancer development and immune system regulation, additional tools to detect the presence of PD-L1, for example, for diagnosis and/or patient selection, are desirable.

Overview

The present invention relates to anti-programmed death ligand 1 (PD-L1) antibodies and methods of using the same.

In one aspect, the invention features an isolated antibody that specifically binds to PD-L1, wherein the antibody binds to an epitope comprising amino acid residues 279-290 of the human PD-L1 polypeptide (SEQ ID NO: 1). In some embodiments, the antibody comprises the following hypervariable regions (HVRs): (a) HVR-H1 comprising the amino acid sequence of SNGLT (SEQ ID NO: 2); (b) HVR-H2 comprising the amino acid sequence of TINKDASAYYASWAKG (SEQ ID NO: 3); and (c) HVR-H3 comprising the amino acid sequence of IAFKTGTSI (SEQ ID NO: 4). In some embodiments, the antibody further comprises the following heavy chain variable domain framework region (FR): (a) FR-H1 comprising the amino acid sequence of QSLEESGGRLVKPDETLTITCTVSGIDLS (SEQ ID NO: 5); (b) FR-H2 comprising the amino acid sequence of WVRQAPGEGLEWIG (SEQ ID NO: 6); (c) FR-H3 comprising the amino acid sequence of RLTISKPSSTKVDLKITSPTTEDTATYFCGR (SEQ ID NO: 7); and (d) FR-H4 comprising the amino acid sequence of WGPGTLVTVSS (SEQ ID NO: 8). In some embodiments, the antibody further comprises the following HVRs: (a) HVR-L1 comprising the amino acid sequence of QASESVYSNNYLS (SEQ ID NO: 9); (b) HVR-L2 comprising the amino acid sequence of LASTLAS (SEQ ID NO: 10); and (c) HVR-L3 comprising the amino acid sequence of IGGKSSSTDGNA (SEQ ID NO: 11). In some embodiments, the antibody further comprises the following light chain variable domain FRs: (a) FR-L1 comprising the amino acid sequence of AIVMTQTPSPVSAAVGGTVTINC (SEQ ID NO: 12); (b) FR-L2 comprising the amino acid sequence of WFQQKPGQPPKLLIY (SEQ ID NO: 13); (c) FR-L3 comprising the amino acid sequence of GVPSRFKGSGSGTQFTLTISGVQCDDAATYYC (SEQ ID NO: 14); and (d) FR-L4 comprising the amino acid sequence of FGGGTEVVVR (SEQ ID NO: 15). In some embodiments, the antibody comprises: (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17; or (c) a VH sequence as in (a), and a VL sequence as in (b). In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises the VL sequence of SEQ ID NO: 17.

In other embodiments, the antibody comprises the following HVRs: (a) HVR-L1 comprising the amino acid sequence of QASESVYSNNYLS (SEQ ID NO: 9); (b) HVR-L2 comprising the amino acid sequence of LASTLAS (SEQ ID NO: 10); and (c) HVR-L3 comprising the amino acid sequence of IGGKSSSTDGNA (SEQ ID NO: 11). In some embodiments, the antibody further comprises the following light chain variable domain FR: (a) FR-L1 comprising the amino acid sequence of AIVMTQTPSPVSAAVGGTVTINC (SEQ ID NO: 12); (b) FR-L2 comprising the amino acid sequence of WFQQKPGQPPKLLIY (SEQ ID NO: 13); (c) FR-L3 comprising the amino acid sequence of GVPSRFKGSGSGTQFTLTISGVQCDDAATYYC (SEQ ID NO: 14); and (d) FR-L4 comprising the amino acid sequence of FGGGTEVVVR (SEQ ID NO: 15).

In another aspect, the invention features an isolated antibody that specifically binds PD-L1, wherein the antibody comprises the following HVRs: (a) an HVR-H1 comprising the amino acid sequence of SNGLT (SEQ ID NO: 2); (b) an HVR-H2 comprising the amino acid sequence of TINKDASAYYASWAKG (SEQ ID NO: 3); (c) an HVR-H3 comprising the amino acid sequence of IAFKTGTSI (SEQ ID NO: 4); (d) an HVR-L1 comprising the amino acid sequence of QASESVYSNNYLS (SEQ ID NO: 9); (e) an HVR-L2 comprising the amino acid sequence of LASTLAS (SEQ ID NO: 10); and (f) an HVR-L3 comprising the amino acid sequence of IGGKSSSTDGNA (SEQ ID NO: 11). In some embodiments, the antibody further comprises the following heavy chain variable domain and light chain variable domain FR: (a) FR-H1 comprising the amino acid sequence of QSLEESGGRLVKPDETLTITCTVSGIDLS (SEQ ID NO: 5); (b) FR-H2 comprising the amino acid sequence of WVRQAPGEGLEWIG (SEQ ID NO: 6); (c) FR-H3 comprising the amino acid sequence of RLTISKPSSTKVDLKITSPTTEDTATYFCGR (SEQ ID NO: 7); (d) FR-H4 comprising the amino acid sequence of WGPGTLVTVSS (SEQ ID NO: 8); (e) FR-L1 comprising the amino acid sequence of AIVMTQTPSPVSAAVGGTVTINC (SEQ ID NO: 12); (f) FR-L2 comprising the amino acid sequence of WFQQKPGQPPKLLIY (SEQ ID NO: 13); In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 17.

In another aspect, the invention features an isolated antibody that competes with any of the aforementioned antibodies for binding to PD-L1.

In another aspect, the invention features an isolated antibody that binds to the same epitope as any of the aforementioned antibodies.

In some embodiments, any of the aforementioned antibodies can be a monoclonal antibody. In some embodiments, the monoclonal antibody can be a rabbit monoclonal antibody.

In some embodiments, any of the aforementioned antibodies can be an IgG antibody (eg, an IgG1 antibody).

In some embodiments, any of the aforementioned antibodies may be an antibody fragment that specifically binds to PD-L1. In some embodiments, the antibody fragment is selected from the group consisting of: Fab, single-chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab') ₂ , and diabodies.

In another aspect, the invention features an immunoconjugate comprising any of the foregoing antibodies.

In another aspect, the present invention features an isolated nucleic acid encoding any one of the antibodies described herein. In another aspect, the present invention features a vector (e.g., an expression vector) comprising a nucleic acid for expressing the antibody. In another aspect, the present invention features a host cell comprising the aforementioned nucleic acid and/or vector.

In some aspects, any of the aforementioned antibodies can be used to detect the presence or expression level of PD-L1 in a biological sample. In some embodiments, detection is performed by immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, ELISA, or immunoblotting. In some embodiments, detection is performed by IHC. In some embodiments, the sample comprises fixed tissue. In some embodiments, the fixed tissue is formalin-fixed paraffin-embedded (FFPE) tissue. In some embodiments, the sample is from a subject suffering from cancer or immune dysfunction or at risk thereof. In some embodiments, the immune dysfunction is a T cell dysfunction disorder. In some embodiments, the T cell dysfunction disorder is an unresolved acute infection, chronic infection, or tumor immunity.

Yet another aspect of the present invention is a method for detecting the presence or expression level of PD-L1 in a biological sample, the method comprising contacting the biological sample with any one of the aforementioned antibodies and detecting the presence of bound antibody. In some embodiments, detection is performed by IHC, IF, flow cytometry, ELISA, or immunoblotting. In some embodiments, detection is performed by IHC. In some embodiments, the sample comprises fixed tissue. In some embodiments, the fixed tissue is FFPE tissue. In some embodiments, the sample is from a subject having or at risk of cancer or an immune disorder. In some embodiments, the immune disorder is a T cell dysfunction disorder. In some embodiments, the T cell dysfunction disorder is an unresolved acute infection, a chronic infection, or tumor immunity. In some embodiments, the sample is from a subject having cancer. In some embodiments, the presence or expression level of PD-L1 in the sample indicates that the subject is likely to respond to treatment with an anti-cancer therapy. In some embodiments, the presence or expression level of PD-L1 in the sample indicates that the subject is more likely to respond to treatment with an anti-cancer therapy. In some embodiments, the presence or expression level of PD-L1 in the sample indicates that the subject will show the possibility of benefit from treatment with anticancer therapy. In some embodiments, the method further comprises selecting an anticancer therapy for the subject based on the presence or expression level of PD-L1 in the sample. In some embodiments, the method further comprises administering a therapeutically effective amount of anticancer therapy to the subject. In some embodiments, the cancer is selected from the group consisting of: non-small cell lung cancer, squamous cell carcinoma, small cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia and head and neck cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is lung adenocarcinoma or lung squamous cell carcinoma. In some embodiments, the sample is a tumor sample. In some embodiments, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, or any combination thereof. In some embodiments, the tumor sample has a detectable expression level of PD-L1 in tumor-infiltrating immune cells, and the area of the tumor-infiltrating immune cells accounts for about 1% or more of the tumor sample. In some embodiments, the tumor sample has a detectable expression level of PD-L1 in tumor-infiltrating immune cells, and the area of the tumor-infiltrating immune cells accounts for about 5% or more of the tumor sample. In some embodiments, the tumor sample has a detectable expression level of PD-L1 in tumor-infiltrating immune cells, and the area of the tumor-infiltrating immune cells accounts for about 10% or more of the tumor sample. In some embodiments, the tumor sample has a detectable expression level of PD-L1 in about 1% or more of the tumor cells in the tumor sample. In some embodiments, the tumor sample has a detectable expression level of PD-L1 in about 5% or more of the tumor cells in the tumor sample. In some embodiments, the tumor sample has a detectable expression level of PD-L1 in about 10% or more of the tumor cells in the tumor sample. In some embodiments, the anti-cancer therapy comprises a PD-1 axis binding antagonist. 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. In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is selected from the group consisting of: YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the antibody is selected from the group consisting of: MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. In some embodiments, the PD-1 binding antagonist is an Fc fusion protein. In some embodiments, the Fc fusion protein is AMP-224. In some embodiments, the method further comprises administering to the patient an effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of: a cytotoxic agent, a growth inhibitory agent, a radiation therapy agent, an anti-angiogenic agent, and a combination thereof. In some embodiments, the subject is human.

BRIEF DESCRIPTION OF THE DRAWINGS

This application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG1 is a schematic diagram showing a general antibody production method for the SP142 anti-PD-L1 antibody.

FIG2A is an image showing the results of immunohistochemistry (IHC) performed using the anti-PD-L1 antibody SP142 on formalin-fixed paraffin-embedded (FFPE) HEK-293 cells transfected with an empty vector (negative control).

FIG2B is an image showing the results of IHC performed on FFPE DOR-13 cells (low to moderate expression) using the anti-PD-L1 antibody SP142.

FIG2C is an image showing the results of IHC performed on FFPE colon cancer RKO cells (moderate expression) using the anti-PD-L1 antibody SP142.

FIG2D is an image showing the results of IHC performed on FFPE HEK-293 cells transfected with full-length human PD-L1 (high expression) using the anti-PD-L1 antibody SP142.

FIG3A is an image showing the results of IHC performed on FFPE placenta tissue sections using the anti-PD-L1 antibody SP142.

FIG3B is an image showing the results of IHC performed on FFPE tonsil tissue sections using the anti-PD-L1 antibody SP142.

FIG3C is an image showing the results of IHC performed on FFPE Hodgkin's (HK) lymphoma patient tissue sections using the anti-PD-L1 antibody SP142.

4 is a Western blot showing PD-L1 expression in cell lysates from FFPE NIH H820 lung adenocarcinoma cell line (high expression), Karpas 299 T cell lymphoma cell line (intermediate expression), and Calu-3 lung adenocarcinoma cell line (negative control) using the anti-PD-L1 antibody SP142.

FIG5A is an image showing the results of IHC performed on FFPE placenta tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 0.11 μg/ml.

FIG5B is an image showing the results of IHC performed on FFPE placenta tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 0.44 μg/ml.

FIG5C is an image showing the results of IHC performed on FFPE placenta tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 1.75 μg/ml.

FIG5D is an image showing the results of IHC performed on FFPE placenta tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 7.0 μg/ml.

FIG5E is an image showing the results of IHC performed on FFPE placenta tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 28.0 μg/ml.

FIG5F is an image showing the results of IHC performed on FFPE placental tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 0.11 μg/ml.

FIG5G is an image showing the results of IHC performed on FFPE placental tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 0.44 μg/ml.

FIG5H is an image showing the results of IHC performed on FFPE placental tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 1.75 μg/ml.

FIG5I is an image showing the results of IHC performed on FFPE placental tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 7.0 μg/ml.

FIG5J is an image showing the results of IHC performed on FFPE placental tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 28.0 μg/ml.

FIG6A is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 0.11 μg/ml.

FIG6B is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 0.44 μg/ml.

FIG6C is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 1.75 μg/ml.

FIG6D is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 7.0 μg/ml.

FIG6E is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 28.0 μg/ml.

FIG6F is an image showing the results of IHC performed on FFPE neural tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 0.11 μg/ml.

FIG6G is an image showing the results of IHC performed on FFPE neural tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 0.44 μg/ml.

FIG6H is an image showing the results of IHC performed on FFPE neural tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 1.75 μg/ml.

FIG6I is an image showing the results of IHC performed on FFPE neural tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 7.0 μg/ml.

FIG6J is an image showing the results of IHC performed on FFPE neural tissue sections using the anti-PD-L1 antibody E1L3N at a concentration of 28.0 μg/ml.

FIG6K is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 0.11 μg/ml.

FIG6L is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 0.44 μg/ml.

FIG6M is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 1.75 μg/ml.

FIG6N is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 7.0 μg/ml.

FIG6O is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 28.0 μg/ml.

FIG6P is an image showing the results of IHC performed on FFPE neural tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 0.11 μg/ml.

FIG6Q is an image showing the results of IHC performed on FFPE neural tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 0.44 μg/ml.

FIG6R is an image showing the results of IHC performed on FFPE neural tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 1.75 μg/ml.

FIG6S is an image showing the results of IHC performed on FFPE neural tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 7.0 μg/ml.

FIG6T is an image showing the results of IHC performed on FFPE neural tissue sections using the anti-PD-L1 antibody SP142 at a concentration of 28.0 μg/ml.

FIG7A is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody E1L3N.

FIG7B is an image showing the results of IHC performed on FFPE kidney tissue sections using the anti-PD-L1 antibody E1L3N.

FIG7C is an image showing the results of IHC performed on FFPE bladder transitional cell carcinoma (TCC) tissue sections using the anti-PD-L1 antibody E1L3N.

FIG7D is an image showing the results of IHC performed on FFPE breast ductal carcinoma (Ca) tissue sections using the anti-PD-L1 antibody E1L3N.

FIG7E is an image showing the results of IHC performed on FFPE lung squamous cell carcinoma tissue sections using the anti-PD-L1 antibody E1L3N.

FIG7F is an image showing the results of IHC performed on FFPE gastric epithelial tissue sections using the anti-PD-L1 antibody SP142.

FIG7G is an image showing the results of IHC performed on FFPE kidney tissue sections using the anti-PD-L1 antibody SP142.

FIG7H is an image showing the results of IHC performed on FFPE bladder transitional cell carcinoma (TCC) tissue sections using the anti-PD-L1 antibody SP142.

FIG7I is an image showing the results of IHC performed on FFPE breast ductal carcinoma (Ca) tissue sections using the anti-PD-L1 antibody SP142.

FIG7J is an image showing the results of IHC performed on FFPE lung squamous cell carcinoma tissue sections using the anti-PD-L1 antibody SP142.

FIG8A is an image showing the results of IHC performed on FFPE tonsil tissue sections using the anti-PD-L1 antibody E1L3N.

FIG8B is an image showing the results of IHC performed on FFPE cervical squamous cell carcinoma (SCC) tissue sections using the anti-PD-L1 antibody E1L3N.

FIG8C is an image showing the results of IHC performed on FFPE Hodgkin lymphoma (HK lymphoma) tissue sections using the anti-PD-L1 antibody E1L3N.

FIG8D is an image showing the results of IHC performed on FFPE pancreatic adenocarcinoma tissue sections using the anti-PD-L1 antibody E1L3N.

FIG8E is an image showing the results of IHC performed on FFPE prostate adenocarcinoma tissue sections using the anti-PD-L1 antibody E1L3N.

FIG8F is an image showing the results of IHC performed on FFPE skin SCC tissue sections using the anti-PD-L1 antibody E1L3N.

FIG8G is an image showing the results of IHC performed on FFPE tonsil tissue sections using the anti-PD-L1 antibody SP142.

FIG8H is an image showing the results of IHC performed on FFPE cervical squamous cell carcinoma (SCC) tissue sections using the anti-PD-L1 antibody SP142.

FIG8I is an image showing the results of IHC performed on FFPE Hodgkin lymphoma (HK lymphoma) tissue sections using the anti-PD-L1 antibody SP142.

FIG8J is an image showing the results of IHC performed on FFPE pancreatic adenocarcinoma tissue sections using the anti-PD-L1 antibody SP142.

FIG8K is an image showing the results of IHC performed on FFPE prostate adenocarcinoma tissue sections using the anti-PD-L1 antibody SP142.

FIG8L is an image showing the results of IHC performed on FFPE skin SCC tissue sections using the anti-PD-L1 antibody SP142.

FIG9A is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody E1L3N.

FIG9B is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody E1L3N.

FIG9C is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody E1L3N.

FIG9D is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody E1L3N.

FIG9E is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody E1L3N.

FIG9F is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody SP142.

FIG9G is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody SP142.

FIG9H is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody SP142.

FIG9I is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody SP142.

FIG9J is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody SP142.

Figure 10 is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody SP142. The image shows PD-L1-positive tumor-infiltrating immune cells (IC, dark brown staining) presenting as aggregates in the tumor stroma. The tissue sections were counterstained with hematoxylin (blue).

Figure 11 is an image showing the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody SP142. The figure shows PD-L1 positive tumor cell (TC) staining. The PD-L1 signal is shown in dark brown. The tissue sections were counterstained with hematoxylin (blue).

FIG12A is an image showing the stromal and tumor regions in an H&E-stained NSCLC tumor resection specimen. Arrows indicate the peritumoral stroma, tumor cell clusters, and intratumoral stroma. Black lines outline the edges of the tumor region. Images were taken at a higher magnification.

Figures 12B-12C are images showing tumor areas of NSCLC resection specimens. Serial sections of the tumor specimens were stained with H&E (Figure 12B) or by PD-L1 IHC using the SP142 antibody (Figure 12C). The PD-L1 signal in Figure 12C is shown as dark brown. The blue line outlines the tumor area (see Example 5). These images correspond to the same specimens shown in Figure 12A, but were taken at a lower magnification.

Figure 13 is a schematic diagram showing an exemplary workflow for determining the percentage of tumor area covered by PD-L1 positive tumor infiltrating immune cells (IC%). In this example, the IC% was visually estimated to be 10%.

Figure 14A-14B shows an exemplary scoring method for PD-L1 IHC using a PD-L1 positive IC aggregate staining pattern. Figure 14A shows that the PD-L1 positive IC aggregate (dark brown signal) is visually observed as closely as possible, and the outline of each PD-L1 positive IC aggregate is generated (blue outline). These areas are combined, and the combined area is estimated as a percentage of the tumor area (Figure 14B). In Figure 14B, the tumor area is divided into 1/10 (frame), and the outlines of the PD-L1 positive IC aggregates from Figure 14A are combined. The outline fills one of the frames, and therefore in this example, the percentage of the tumor area covered by the PD-L1 positive IC is estimated to be 10%. The image shows the results of IHC performed on FFPE tissue sections from NSCLC patients using anti-PD-L1 antibody SP142.

Figures 15A-15B show images with single cell spread PD-L1 positive IC staining patterns. In this example, the images were scored based on the density of single cell spreads by comparison with a reference image (see, e.g., Figure 16). Figure 15A shows an image with a cell density of 1% for single cell spread PD-L1 positive ICs. Figure 15B shows an image with a cell density of 5% for single cell spread PD-L1 positive ICs. The images show the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody SP142.

Figures 16A-16H show exemplary reference images of single-cell spread PD-L1 positive IC staining patterns at the indicated PD-L1 IC expression cutoffs. The images show the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody SP142. Figures 16A-16B show images with a PD-L1 positive IC% of less than 1%. Figures 16C-16D show images with a PD-L1 positive IC% of greater than or equal to 1% to less than 5%. Figures 16E-16F show images with a PD-L1 positive IC% of greater than or equal to 5% to less than 10%. Figures 16G-16H show images with a PD-L1 positive IC% of greater than or equal to 10%.

Figure 17A shows images of serial sections of FFPE tissue from a NSCLC patient that were stained with H&E (top) or processed for IHC (bottom) using the anti-PD-L1 antibody SP142. In this example, no intratumoral ICs were detected in the H&E-stained sections, and strong TC PD-L1 staining was detected by IHC. ICs in the stroma were scored (arrows).

Figure 17B shows images of serial sections of FFPE tissue from NSCLC patients that were stained with H&E (top) or processed for IHC (bottom) using the anti-PD-L1 antibody SP142. In this example, intratumoral ICs were detected in H&E-stained sections, and weak to moderate TC PD-L1 staining was detected by IHC. ICs were scored in both stromal and tumor cell groups.

Figure 17C shows images of serial sections of FFPE tissue from NSCLC patients stained with H&E (top) or processed for IHC (bottom) using the anti-PD-L1 antibody SP142. In this example, no intratumoral ICs were detected in the H&E-stained sections, and granular PD-L1 TC staining was detected by IHC. Granular staining was scored as PD-L1-positive TC as long as the staining was arranged in a linear manner (i.e., along the outline of the cell membrane).

Figures 18A-18F show exemplary images of PD-L1 positive TC staining patterns at the indicated PD-L1 TC% cutoff values. The images show the results of IHC performed on FFPE tissue sections from NSCLC patients using the anti-PD-L1 antibody SP142. Figures 18A-18B show images with TC% less than 5%. Figures 18C-18D show images with TC% greater than or equal to 5% to less than 50%. Figures 18E-18F show images with TC% greater than or equal to 50%.

Detailed description of embodiments of the present invention

I. Definition

As used herein, the term "about" refers to the usual error range of the corresponding value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments for the value or parameter itself. For example, description of "about X" includes description of "X."

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

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

The term "anergy" refers to the state of anergy to antigenic stimulation caused by incomplete or insufficient signals (e.g., in the absence of ras activation) delivered by the T cell receptor. T cell anergy can also be produced when stimulated by ^antigens in the absence of costimulation, causing cells to become refractory to the subsequent activation carried out by the antigen, even in the case of costimulation. Anergy can often be overwritten by the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and/or obtain effector functions.

The term "anti-cancer therapy" refers to a therapy for treating cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitors, agents for radiotherapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as PD-1 axis binding antagonists, anti-CD20 antibodies, platelet-derived growth factor inhibitors (e.g., GLEEVEC ^™ (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: PDGFR-β, BlyS, APRIL, BCMA receptors, TRAIL/Apo2, other biologically active and organic chemical agents, etc. Compositions thereof are also included in the present invention.

The terms "anti-PD-L1 antibody,""anti-PD-L1antibody,""antibody that specifically binds to PD-L1," and "antibody that binds to PD-L1" refer to antibodies that can bind to PD-L1 with an affinity sufficient to allow the antibody to be used as a diagnostic and/or therapeutic agent in targeting PD-L1. In one embodiment, the extent of binding of the anti-PD-L1 antibody to unrelated, non-PD-L1 proteins is less than about 10% of the binding of the antibody to PD-L1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the antibody that binds to PD-L1 is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹³ M, e.g., 10 ⁻⁹ M to 10 ⁻¹³ M). In certain embodiments, the anti-PD-L1 antibody binds to an epitope of PD-L1 that is conserved among PD-L1 from different species.

The terms "anti-PD-1 antibody" and "antibody that binds to PD-1" refer to antibodies that can bind to PD-1 with an affinity sufficient to allow the antibody to be used as a diagnostic and/or therapeutic agent in targeting PD-1. In one embodiment, the extent of binding of the anti-PD-1 antibody to unrelated non-PD-1 proteins is less than about 10% of the binding of the antibody to PD-1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the antibody that binds to PD-1 is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ^-8 M or less, e.g., 10 ^-8 M to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M). In certain embodiments, the anti-PD-1 antibody binds to an epitope of PD-1 that is conserved among PD-1 from different species. Exemplary anti-PD-1 antibodies include, but are not limited to, MDX 1106 (nivolumab), MK-3475 (pembrolizumab), and CT-011 (pidilizumab).

The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

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

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

"Fv" is the smallest antibody fragment containing a complete antigen binding site. In one embodiment, the double-chain Fv material is composed of a dimer of a heavy chain variable domain and a light chain variable domain in a tight, non-covalent association. In single-chain Fv (scFv) material, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker so that the light chain and the heavy chain can associate with a "dimerization" structure similar to the "dimerization" structure in the double-chain Fv material. In this configuration, the three HVRs of each variable domain interact to limit the antigen binding site on the surface of the VH-VL dimer. In short, six HVRs confer antibody antigen-binding specificity. However, even a single variable domain (or only half an Fv comprising three HVRs specific for an antigen) has the ability to recognize and bind antigens, although at a lower affinity than the entire binding site.

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

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

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

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

An "autoimmune disease" is a disease or condition that arises from or is directed against an individual's own tissues or organs, or a co-isolation or manifestation thereof, or a condition resulting therefrom. An autoimmune disease can be an organ-specific disease (i.e., the immune response is specifically directed against an organ system, such as the endocrine system, hematopoietic system, skin, cardiopulmonary system, gastrointestinal and hepatic system, renal system, thyroid, ear, neuromuscular system, central nervous system, etc.) or a systemic disease that can affect multiple organ systems (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis, etc.). Non-limiting exemplary autoimmune diseases include autoimmune rheumatic disorders (such as RA, Sjögren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis-dermatomyositis, cryoglobulinemia, antiphospholipid antibody syndrome, and psoriatic arthritis), autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-negative vasculitis and ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and vasculitis). granulomatosis and microscopic polyangiitis), autoimmune neurologic disorders (e.g., multiple sclerosis, opsoclonus-myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathy), renal disorders (e.g., glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (e.g., psoriasis, urticaria, urticaria, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (e.g., thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing disorders (e.g., inner ear disease and hearing loss), Behçet's disease, Raynaud's syndrome, and other autoimmune hearing disorders. syndrome), organ transplantation, and autoimmune endocrine disorders (e.g., diabetes-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid diseases (e.g., Graves' disease and thyroiditis). More preferred such diseases include, for example, RA, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

For example, as used herein, the phrase "based on" means that information about one or more biomarkers (e.g., PD-L1) is used to inform treatment decisions, provide information on a package insert, or marketing/promotional guidance.

"Biological sample" means a collection of similar cells obtained from a subject or patient. A biological sample can be a tissue or cell sample. The source of the tissue or cell sample can be solid tissue such as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from any time during the subject's pregnancy or development. Biological samples can also be obtained from in vitro tissue or cell cultures. Tissue samples can contain compounds that are not naturally mixed with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of biological samples herein include, but are not limited to, tumor biopsies, circulating tumor cells, serum or plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed paraffin-embedded tumor samples or frozen tumor samples.

As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, such as PD-L1, for example, that is predictive, diagnostic, and/or prognostic. A biomarker can be used as an indicator of a specific subtype of a disease or condition (e.g., cancer) characterized by certain molecular, pathological, histological, and/or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polynucleotide copy number changes (e.g., DNA copy number), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based molecular markers.

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

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphomas), blastomas, sarcomas, and leukemias. More specific examples of such cancers include non-small cell lung cancer (NSCLC) (including lung adenocarcinoma and lung squamous cell carcinoma), squamous cell carcinoma, small cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, gliomas, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer. In specific embodiments, the cancer is NSCLC. In some embodiments, the NSCLC is lung adenocarcinoma or lung squamous cell carcinoma. NSCLC can be squamous NSCLC or non-squamous NSCLC.

"Chemotherapeutic agents" are chemical compounds used to treat cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (particularly bullatacin and bullatacinone); delta-9- Tetrahydrocannabinol (dronabinol); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including the synthetic analogs topotecan CPT-11 (irinotecan), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid acid; teniposide; cryptophycin (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil. cil), chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Nicolaou et al., Angew. Chem. Am. Am., 1996; 1996). Intl. Ed. Engl., 33:183-186 (1994); dynemicins, including dynemicin A; esperamicin; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid (such as mycophenolic acid), acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate, purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgen analogs such as calusterone, dromostanolone propionate propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folicacid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansines, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, OR; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, A) and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");thiotepa; taxoids, such as taxanes, including paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE ^™ Cremophor-free albumin-engineered paclitaxel nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, Illinois, and docetaxel (Rorer, Antony, France); chlorambucil; gemcitabine (6-thioguanine); mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine (platinum); etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (oxaliplatin); leucovovin; vinorelbine (novantrone); edatrexate; daunorubicin; aminopterin; ibandronate; and the topoisomerase inhibitor RFS. 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine, a pharmaceutically acceptable salt, acid or derivative of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, an abbreviation for the treatment regimen of oxaliplatin (ELOXATIN ^™ ) in combination with 5-FU and folinic acid. Additional chemotherapeutic agents include cytotoxic agents used as antibody drug conjugates, such as, for example, maytansinoids (e.g., DM1) and the auristatins MMAE and MMAF.

"Chemotherapeutic agents" also include "antihormonal agents" or "endocrine therapeutic agents" that are used to regulate, reduce, block or inhibit the effects of hormones that promote cancer growth and are often in the form of systemic or systemic treatment. They may be hormones themselves. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including raloxifene, droloxifene, 4-hydroxytamoxifen, traloxifene, keoxifene, LY117018, onapristone and toremifene; antiprogestins; estrogen receptor downregulators (ERDs); agents used to suppress or stop the ovaries, for example, luteinizing hormone-releasing hormone (LH-RH); hormone-releasing hormone (LHRH) agonists such as leuprorelin acetate, goserelin acetate, buserelin acetate, and tripterelin; other antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole. In addition, such definition of chemotherapeutic agents includes bisphosphonates, such as clodronate (e.g., or etidronate, NE-58095, zoledronic acid/zoledronate, alendronate, pamidronate, tiludronate, or risedronate; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes involved in signaling pathways involving abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGFR); vaccines, such as vaccines and gene therapy vaccines, for example, vaccines, vaccines, and vaccines; topoisomerase 1 inhibitors; rmRH; lapatinib ditosylate (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases, also known as GW572016); and pharmaceutically acceptable salts, acids, or derivatives of any of the above chemotherapeutic agents.

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

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these antibodies are further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . The heavy chain constant domains corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively.

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

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, dapoxetine ... C), chlorambucil, daunombicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed below.

The term "detecting" includes detection by any means, including direct and indirect detection.

The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease, or condition (e.g., cancer). For example, "diagnosis" can refer to the identification of a particular type of cancer. "Diagnosis" can also refer to the classification of a particular subtype of cancer, for example, by histopathological criteria or by molecular signatures (e.g., a subtype characterized by the expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).

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

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

The term "Fc region" herein is used to define the C-terminal region of at least a portion of an immunoglobulin heavy chain containing a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al. Sequences of Proteins of Immunological Interest. 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

"Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of a variable domain is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

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

The terms "level of expression" or "expression level" are often used interchangeably and generally refer to the amount of polynucleotides, mRNA or amino acid products or proteins in a biological sample. "Expression" generally refers to the process by which the information encoded by a gene is converted into a structure that exists in and operates in a cell. Thus, according to the present invention, "expression" of a gene (e.g., a PD-L1 gene) can refer to transcription into polynucleotides, translation into protein, or even post-translational modification of a protein. Fragments of transcribed polynucleotides, translated proteins, or post-translationally modified proteins should also be considered to be expressed, regardless of whether they originate from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of proteins, such as by proteolysis. In some embodiments, "expression level" refers to the amount of a protein (e.g., PD-L1) in a biological sample as determined using methods known in the art or described herein, including but not limited to immunohistochemistry (IHC), immunoblotting (e.g., Western blot), immunofluorescence (IF), flow cytometry, such as fluorescence activated cell sorting (FACS ^™ ), or enzyme-linked immunosorbent assay (ELISA).

"Increased expression," "increased expression level," "increased level," "elevated expression," "elevated expression level," or "elevated level" refers to increased expression or increased levels of a biomarker in an individual relative to a control, such as one or more individuals not suffering from a disease or disorder (e.g., cancer) or an internal control (e.g., a housekeeping biomarker).

"Decreased expression," "decreased expression level," "decreased level," "lower expression," "lower expression level," or "reduced level" refers to less expression or reduced levels of a biomarker in an individual relative to a control, such as one or more individuals without a disease or condition (e.g., cancer) or an internal control (e.g., a housekeeping biomarker). In some embodiments, decreased expression is almost no expression.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the original transformed cell are included herein.

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

A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, a human immunoglobulin VL or VH sequence is selected from a subgroup of variable domain sequences. Generally, the sequence subgroups are as described in Kabat et al., Sequences of Proteins of Immunological Interest. Fifth Edition, NIH Publication 91-3242, Bethesda MD, Volumes 1-3, 1991. In one embodiment, for VL, the subgroup is subgroup κI as described in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al. (supra).

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

As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or form structure-determining loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen contacts"). Generally, antibodies comprise six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

(a) Hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia et al. 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) (Kaba et al., Sequences of Proteins of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991);

(c) antigenic contacts present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745, 1996); and

(d) A combination of (a), (b), and/or (c) comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. (supra).

"Immune dysfunction" is a disorder or condition that affects the immune system and includes, for example, autoimmune diseases and T-cell dysfunctional disorders.

As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous protein ("adhesin") with the effector functions of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence with the desired binding specificity that is different from the antigen recognition and binding site of an antibody (i.e., is "heterologous") and an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a continuous amino acid sequence that comprises at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin can be obtained from any immunoglobulin such as IgG1, IgG2 (including IgG2A and IgG2B), IgG3 or IgG4 subtypes, IgA (including IgA1 and IgA2), IgE, IgD or IgM. Ig fusions preferably include replacing the domains of a polypeptide or antibody as described herein in the position of at least one variable region within an Ig molecule. In particularly preferred embodiments, immunoglobulin fusions include the hinge, CH2 and CH3 or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions, see also U.S. Patent No. 5,428,130. For example, useful immunoadhesins for use as drugs in the present therapy include polypeptides comprising an extracellular domain (ECD) or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant domain of an immunoglobulin sequence (such as PD-L1 ECD-Fc, PD-L2 ECD-Fc, and PD-1 ECD-Fc), or an extracellular or PD-L1 binding portion or PD-L2 binding portion of PD-1. The immunoadhesin combination of Ig Fc and ECD of a cell surface receptor is sometimes referred to as a soluble receptor.

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

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

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

"Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-PD-L1 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of the antibody (or fragments thereof), including said nucleic acid molecules in a single vector or separate vectors and said nucleic acid molecules present at one or more locations in a host cell.

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

As used herein, the term "monoclonal antibody" refers to the antibody obtained from a substantially homogeneous antibody colony, i.e., except for possible variant antibodies (such as those containing naturally occurring mutations or those produced during the manufacture of monoclonal antibody preparations, these variants are generally present in a small amount), the individual antibodies constituting the colony are identical and/or in conjunction with identical epitopes. Unlike the polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is for a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates the characteristic of the antibody obtained from a substantially homogeneous antibody colony, and should not be construed as needing to manufacture antibodies by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by various techniques, including but not limited to hybridoma method, recombinant DNA method, phage display method, and a method or a combination thereof utilizing a transgenic animal containing all or a portion of a human immunoglobulin locus.

" Amino acid sequence identity percentage (%) " with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and, if necessary, introducing gaps to achieve maximum sequence identity percentage, and not taking into account any conservative substitutions as part of sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in a variety of ways that are within the skill of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. However, for purposes herein, amino acid sequence identity % values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, and the source code has been filed with the U.S. Copyright Office (U.S. Copyright Office, Washington D.C., 20559) along with user files, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and do not change.

Where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with or relative to a given amino acid sequence B (which can alternatively be phrased as the given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with or relative to the given amino acid sequence B) is calculated as follows:

100 × fraction X/Y

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

The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

"Pharmaceutically acceptable carriers" refer to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

Unless otherwise indicated, the terms "programmed cell death 1 ligand 1," "PD-L1," "programmed death ligand 1," "cluster of differentiation 274," "CD274," or "B7 homolog 1" as used herein refer to any native PD-L1 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The terms encompass "full-length," unprocessed PD-L1 as well as any form of PD-L1 produced by processing in cells. PD-L1 can exist as a transmembrane protein or as a soluble protein. The terms also encompass naturally occurring variants of PD-L1, such as splice variants or allelic variants. The basic structure of PD-L1 includes four domains: an extracellular Ig-like V-type domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain. Additional information about the human PD-L1 gene, including the genomic DNA sequence, can be found under NCBI Gene ID No. 29126. Additional information about the mouse PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 60533. The amino acid sequence of an exemplary full-length human PD-L1 protein is shown in SEQ ID NO: 18. The amino acid sequence of an exemplary full-length human PD-L1 protein can be found, for example, under NCBI Accession No. NP_001254653 or UniProt Accession No. Q9NZQ7, while an exemplary full-length mouse PD-L1 protein sequence can be found, for example, under NCBI Accession No. NP_068693 or UniProt Accession No. Q9EP73.

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, so as to remove T cell dysfunction resulting from signaling on the PD-1 signaling axis - resulting in restoration or enhancement of T cell function (e.g., proliferation, cytokine production and/or target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

As used herein, a "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates, or interferes with the signal transduction generated by the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and/or B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In specific aspects, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules that reduce, block, inhibit, eliminate, or interfere with the signal transduction generated by the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and/or B7-1. In one embodiment, the PD-L1 binding antagonist reduces negative signals mediated by or through cell surface proteins expressed on T lymphocytes and other cells, and the signaling mediated by PD-L1 or PD-1 makes dysfunctional T cells less dysfunctional. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific aspect, the anti-PD-L1 antibody is YW243.55.S70. In another specific aspect, the anti-PD-L1 antibody is MDX-1105. In another specific aspect, the anti-PD-L1 antibody is MPDL3280A (atezolizumab). In another specific aspect, the anti-PD-L1 antibody is MEDI4736 (durvalumab). In another specific aspect, the anti-PD-L1 antibody is MSB0010718C (avelumab). MDX-1105 (also known as BMS-936559) is an anti-PD-L1 antibody described in WO 2007/005874. Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO 2010/077634 and US 8,217,149, the entire contents of each of which are incorporated herein by reference.

As used herein, a "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with the signal transduction generated by the interaction of PD-1 with one or more of its binding partners (such as PD-L1 and/or PD-L2). In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In specific aspects, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules that reduce, block, inhibit, eliminate or interfere with the signal transduction generated by the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative signals mediated by or through cell surface proteins expressed on T lymphocytes and other cells, and the signal transduction mediated by PD-1 or PD-L1 makes dysfunctional T cells less dysfunctional. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In another specific aspect, the PD-1 binding antagonist is CT-011 (pidilizumab). In another specific aspect, the PD-1 binding antagonist is MEDI-0680 (AMP-514). In another specific aspect, the PD-1 binding antagonist is PDR001. In another specific aspect, the PD-1 binding antagonist is REGN2810. In another specific aspect, the PD-1 binding antagonist is BGB-108. In another specific aspect, the PD-1 binding antagonist is AMP-224. MDX-1106 (also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab) is an anti-PD-1 antibody described in WO2006/121168. AMP-224 (also known as B7-DCIg) is a soluble PD-L2-Fc fusion receptor described in WO2010/027827 and WO2011/066342.

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

"Individual response" or "response" can be assessed using any endpoint indicative of a benefit to the individual, including, but not limited to, (1) inhibition of disease progression (e.g., cancer progression) to some extent, including slowing and complete arrest; (2) reduction in tumor size; (3) inhibition (i.e., reduction, slowing, or complete cessation) of cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition (i.e., reduction, slowing, or complete cessation) of metastasis; (5) alleviation to some extent of one or more symptoms associated with a disease or condition (e.g., cancer); (6) increase or prolongation of the length of survival (including overall survival and progression-free survival); and/or (9) reduction in mortality at a given time point after treatment.

A patient's "effective response" or a patient's "responsiveness" to treatment with a drug and similar expressions refer to a clinical or therapeutic benefit conferred to a patient at risk for or suffering from a disease or condition, such as cancer. In one embodiment, such benefits include any one or more of: prolonging survival (including overall survival and progression-free survival); causing an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer. In one embodiment, a biomarker (e.g., PD-L1 expression as determined using IHC) is used to identify patients who are predicted to have an increased likelihood of responding to treatment with a drug (e.g., treatment including anti-cancer therapy) relative to patients who do not express the biomarker. In one embodiment, a biomarker (e.g., PD-L1 expression as determined using IHC) is used to identify patients who are predicted to have an increased likelihood of responding to treatment with a drug (e.g., anti-cancer therapy) relative to patients who do not express the same level of the biomarker. In one embodiment, the presence of a biomarker is used to identify patients who are more likely to respond to treatment with a drug relative to patients who do not have the presence of a biomarker. In another embodiment, the presence of a biomarker is used to determine an increased likelihood that a patient will have a benefit from treatment with a drug relative to a patient who does not have the presence of the biomarker.

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

"Complete response" or "CR" means the disappearance of all signs of cancer in response to treatment (eg, disappearance of all target lesions). This does not always mean that the cancer has been cured.

"Sustained response" refers to a persistent effect on reducing tumor growth after treatment has stopped. For example, the tumor size can remain the same or smaller than the size at the beginning of the administration period. In some embodiments, the sustained response has a duration that is at least the same as the duration of treatment, at least 1.5X, 2.0X, 2.5X, or 3.0X the length of treatment, or longer.

As used herein, "reducing or inhibiting cancer recurrence" means reducing or inhibiting tumor or cancer recurrence, or tumor or cancer progression. As disclosed herein, cancer recurrence and/or cancer progression include, but are not limited to, cancer metastasis.

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

As used herein, "stable disease" or "SD" refers to insufficient shrinkage of target lesions to qualify as a PR and insufficient increase to qualify as a PD, taking the minimum SLD since the start of treatment as reference.

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

The term "survival" refers to whether the patient remains alive and includes overall survival as well as progression-free survival

As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment that the disease being treated (e.g., cancer) does not get worse. Progression-free survival can include the amount of time a patient has experienced a complete response or partial response, as well as the amount of time a patient has experienced stable disease.

As used herein, "overall survival" (OS) refers to the percentage of individuals in a group who are likely to be alive after a specific duration of time.

By "prolonging survival" is meant increasing overall or progression-free survival in treated patients relative to untreated patients (i.e., relative to patients not treated with the drug), or relative to patients who do not express a specified level of a biomarker, and/or relative to patients treated with an approved anti-tumor agent.

For the purposes herein, a "section" of a tissue sample means a single portion or piece of a tissue sample, such as a thin slice of tissue or cells cut from a tissue sample (e.g., a tumor sample). It will be understood that multiple tissue sample sections can be collected and analyzed, provided that it will be understood that the same tissue sample section can be analyzed at the morphological and molecular levels, or for peptides (e.g., by immunohistochemistry) and/or polynucleotides (e.g., by in situ hybridization).

As used herein, the term "specifically binds to" or "specific for..." refers to a measurable and reproducible interaction, such as the binding of a target to an antibody, that determines the presence of a target in the presence of a heterogeneous population of molecules including biomolecules. For example, an antibody that specifically binds to a target (which can be an epitope, such as amino acid residues 279-290 of human PD-L1 (SEQ ID NO: 1)) is an antibody that binds to this target with greater affinity, avidity more readily, and/or for a longer duration than it binds to other targets. In one embodiment, the degree of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the antibody that specifically binds to the target is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding may include but does not require exclusive binding.

A "subject" or "individual" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

As used herein, the term "substantially the same" refers to a sufficiently high degree of similarity between two values that one skilled in the art would consider the difference between the two values to be of little or no biological and/or statistical significance in the context of the biological property measured by the value (e.g., Kd value or expression level). The difference between the two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as the reference/comparison value changes.

As used herein, the phrase "substantially different" refers to a sufficiently high difference between two values that one skilled in the art would consider the difference between the two values to be statistically significant in the context of the biological property measured by the value (e.g., Kd value or expression level). The difference between the two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% over the value of the reference/comparator molecule.

"T cell dysfunction disorder" is a disorder or condition characterized by increased or decreased responsiveness to antigenic stimulation of T cells. In a specific embodiment, a T cell dysfunction disorder is a disorder specifically associated with inappropriately increased signaling through PD-1/PD-L1 and/or PD-L1/B7.1. In another embodiment, a T cell dysfunction disorder is a disorder in which T cells are anergic or have a decreased ability to secrete cytokines, proliferate, or perform cytolytic activity. In another embodiment, T cell dysfunction is T cell exhaustion caused by persistent TCR signaling that occurs during many chronic infections and cancers. It is distinguished from anergy because it is not caused by incomplete or absent signaling but by persistent signaling. It is defined as poor effector function, persistent expression of inhibitory receptors, and a transcriptional state that is different from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Exhaustion can be caused by extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) as well as cell-intrinsic negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.). In particular aspects, reduced responsiveness renders control of pathogens or tumors expressing immunogens ineffective. Examples of T cell dysfunctional disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity.

As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to an attempt to alter the natural course of the disease in the individual being treated, and can be for prevention or prophylaxis or for clinical intervention during the course of a clinical disorder. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or relieving the disease state, and alleviating or improving prognosis. In some embodiments, antibodies (e.g., anti-PD-1 antibodies and/or anti-PD-L1 antibodies) are used to delay disease development or slow disease progression.

As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues. As mentioned herein, the terms "cancer," "cancerous," and "tumor" are not mutually exclusive.

As used herein, "tumor infiltrating immune cells" refers to any immune cell 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 bone marrow lineage cells, including granulocytes (e.g., neutrophils, eosinophils, and basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), tissue cells, and natural killer cells.

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 that may be present in a tumor sample (e.g., stromal cells and tumor-infiltrating immune cells) using methods known in the art and/or described herein.

"Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Therefore, as a therapeutic concept, tumor immunity is "cured" when this evasion is weakened, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

"Therapeutically effective amount" refers to the amount of a therapeutic agent for treating or preventing a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a therapeutic agent can reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow down and preferably prevent to some extent) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow down and preferably prevent to some extent) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with the disorder to some extent. To the extent that a drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing the duration of survival, time to disease progression (TTP), response rate (e.g., CR and PR), duration of response, and/or quality of life.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al. Kuby Immunology. 6th ed., p. 91, W.H. Freeman and Co., 2007. A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using VH or VL domains from antibodies that bind to that antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al. J. Immunol. 150: 880-887, 1993, and Clarkson et al. Nature. 352: 624-628, 1991.

The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variants thereof refer to the numbering system used for the heavy chain variable domain or light chain variable domain compiled in Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues for a given antibody can be determined by aligning the antibody sequence with the "standard" Kabat numbering sequence at homologous regions.

When referring to residues in the variable domain, the Kabat numbering system is generally used (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)). The "EU numbering system" or "EU index" is generally used when referring to residues in the constant region of an immunoglobulin heavy chain (e.g., the EU index as reported in Kabat et al. (supra)). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

As used herein, "growth inhibitory agent" refers to a compound or composition that inhibits the growth and/or proliferation of cells (e.g., cells whose growth depends on PD-L1 expression) in vitro or in vivo. Thus, a growth inhibitory agent may be a growth inhibitor that significantly reduces the percentage of cells in the S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a location other than the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vinca alkaloids (vincas) (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as anthracycline antibiotics doxorubicin ((8S-cis) -10- [(3-amino-2,3,6-trideoxy-α-L-lyso-hexopyranosyl) oxygen] -7,8,9,10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5,12-tetraphenyldione), epirubicin, daunomycin, etoposide, and bleomycin. Those agents that arrest G1 also spill over to S phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and cytarabine. Additional information can be found in "The Molecular Basis of Cancer," edited by Mendelsohn and Israel, Chapter 1, "Cell cycle regulation, oncogenes, and antineoplastic drugs," by Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel (Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analog of paclitaxel (Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which leads to the inhibition of mitosis in cells.

"Radiotherapy" refers to the use of directed gamma or beta rays to induce sufficient damage to cells to limit their ability to function normally or to destroy the cells completely. It will be understood that there are many methods known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one-time administration, and typical dosages range from 10 to 200 units (Gy) per day.

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

As used herein, "administering" means a method of administering a dose of a compound (e.g., an anticancer therapeutic) or a pharmaceutical composition (e.g., a pharmaceutical composition comprising an anticancer therapeutic) to a subject (e.g., a patient). Administration can be performed in any suitable manner, including parenteral, intrapulmonary, and intranasal, and (if desired for local treatment) intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be performed by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at various time points, bolus administration, and pulse infusions.

The term "simultaneously" herein refers to the administration of two or more therapeutic agents wherein at least some of the administration overlaps in time. Thus, simultaneous administration includes dosing regimens when administration of one or more agents is continued after administration of one or more other agents has ceased.

II. Compositions and Methods

The present invention provides novel antibodies that bind to PD-L1. The antibodies of the present invention are used, for example, to detect the presence of PD-L1 or the expression level of PD-L1 (e.g., in biological samples including tumor samples).

A. Exemplary Anti-PD-L1 Antibodies

The present invention provides anti-PD-L1 antibodies for use in, for example, diagnostic applications (e.g., immunohistochemistry (IHC), immunofluorescence (IF), and immunoblotting (e.g., Western blot)). In one example, the present invention provides anti-PD-L1 antibodies that bind to an epitope comprising amino acid residues 279-290 of PD-L1 (e.g., amino acid residues 279-290 of human PD-L1 SKKQSDTHLEET (SEQ ID NO: 1)), which is part of the N-terminal cytoplasmic region of human PD-L1. The epitope on PD-L1 can be recognized in a conformation-dependent or conformation-independent manner.

In some instances, an anti-PD-L1 antibody that binds to amino acid residues 279-290 of PD-L1 comprises at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. For example, in some instances, an anti-PD-L1 antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4. In some instances, the anti-PD-L1 antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In some cases, in which the anti-PD-L1 antibody binds to amino acid residues 279-290 of PD-L1 and comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4, the anti-PD-L1 antibody further comprises the following heavy chain variable domain framework region (FR): (a) FR-H1 comprising the amino acid sequence of QSLEESGGRLVKPDETLTITCTVSGIDLS (SEQ ID NO: 5); (b) FR-H2 comprising the amino acid sequence of WVRQAPGEGLEWIG (SEQ ID NO: 6); (c) FR-H3 comprising the amino acid sequence of RLTISKPSSTKVDLKITSPTTEDTATYFCGR (SEQ ID NO: 7); or (d) FR-H4 comprising the amino acid sequence of WGPGTLVTVSS (SEQ ID NO: 8). In some cases, in which the anti-PD-L1 antibody binds to amino acid residues 279-290 of PD-L1 and comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4, the anti-PD-L1 antibody further comprises the following heavy chain variable domain framework regions (FRs): (a) FR-H1 comprising the amino acid sequence of QSLEESGGRLVKPDETLTITCTVSGIDLS (SEQ ID NO: 5); (b) FR-H2 comprising the amino acid sequence of WVRQAPGEGLEWIG (SEQ ID NO: 6); (c) FR-H3 comprising the amino acid sequence of RLTISKPSSTKVDLKITSPTTEDTATYFCGR (SEQ ID NO: 7); and (d) FR-H4 comprising the amino acid sequence of WGPGTLVTVSS (SEQ ID NO: 8).

In some cases, the anti-PD-L1 antibody binds to amino acid residues 279-290 of PD-L1, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4; (d) an HVR-L1 comprising the amino acid sequence of QASESVYSNNYLS (SEQ ID NO: 9); (e) an HVR-L2 comprising the amino acid sequence of LASTLAS (SEQ ID NO: 10); and (f) an HVR-L3 comprising the amino acid sequence of IGGKSSSTDGNA (SEQ ID NO: 11). In some instances, these anti-PD-L1 antibodies comprise the following FRs: (a) FR-H1 comprising the amino acid sequence of QSLEESGGRLVKPDETLTITCTVSGIDLS (SEQ ID NO: 5); (b) FR-H2 comprising the amino acid sequence of WVRQAPGEGLEWIG (SEQ ID NO: 6); (c) FR-H3 comprising the amino acid sequence of RLTISKPSSTKVDLKITSPTTEDTATYFCGR (SEQ ID NO: 7); and (d) FR-H4 comprising the amino acid sequence of WGPGTLVTVSS (SEQ ID NO: 8), and may additionally or alternatively comprise: (e) FR-L1 comprising the amino acid sequence of AIVMTQTPSPVSAAVGGTVTINC (SEQ ID NO: 12); (f) FR-L2 comprising WFQQKPGQPPKLLIY (SEQ ID NO: 13). (g) FR-L3 comprising the amino acid sequence of GVPSRFKGSGSGTQFTLTISGVQCDDAATYYC (SEQ ID NO: 14); and (h) FR-L4 comprising the amino acid sequence of FGGGTEVVVR (SEQ ID NO: 15).

In some instances, an anti-PD-L1 antibody that binds to amino acid residues 279-290 of PD-L1 can further comprise a heavy chain variable domain (VH) sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequence identity to, or having the sequence of, the amino acid sequence of SEQ ID NO: 16. In certain embodiments, a VH sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence (SEQ ID NO: 16), but an anti-PD-L1 antibody comprising said sequence retains the ability to bind to PD-L1. In certain embodiments, a total of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) have been substituted, inserted, and/or deleted in SEQ ID NO: 16. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-PD-L1 antibody comprises the VH sequence in SEQ ID NO: 16, including post-translational modifications of said sequence. In a specific embodiment, VH comprises one, two or three HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4.

In some instances, an anti-PD-L1 antibody that binds to amino acid residues 279-290 of PD-L1 can further comprise a light chain variable domain (VL) having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequence identity to, or having the sequence of, the amino acid sequence of SEQ ID NO: 17. In certain embodiments, a VL sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence (SEQ ID NO: 17) contains substitutions (e.g., conservative substitutions), insertions, or deletions, but an anti-PD-L1 antibody comprising said sequence retains the ability to bind to PD-L1. In certain embodiments, a total of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) have been substituted, inserted, and/or deleted in SEQ ID NO: 17. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-PD-L1 antibody comprises the VL sequence in SEQ ID NO: 17, including post-translational modifications of said sequence. In a specific embodiment, VL comprises one, two or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In some instances, an anti-PD-L1 antibody that binds to amino acid residues 279-290 of PD-L1 comprises VH and VL sequences having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequences of SEQ ID NOs: 16 and 17, respectively, or sequences having the amino acid sequences of SEQ ID NOs: 16 and 17, respectively, and may or may not contain post-translational modifications of these sequences.

In other instances, the present invention provides an antibody that specifically binds to PD-L1, wherein the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4; (d) HVR-L1 comprising the amino acid sequence of QASESVYSNNYLS (SEQ ID NO: 9); (e) HVR-L2 comprising the amino acid sequence of LASTLAS (SEQ ID NO: 10); and (f) HVR-L3 comprising the amino acid sequence of IGGKSSSTDGNA (SEQ ID NO: 11). In some instances, these anti-PD-L1 antibodies comprise the following FRs: (a) FR-H1 comprising the amino acid sequence of QSLEESGGRLVKPDETLTITCTVSGIDLS (SEQ ID NO: 5); (b) FR-H2 comprising the amino acid sequence of WVRQAPGEGLEWIG (SEQ ID NO: 6); (c) FR-H3 comprising the amino acid sequence of RLTISKPSSTKVDLKITSPTTEDTATYFCGR (SEQ ID NO: 7); and (d) FR-H4 comprising the amino acid sequence of WGPGTLVTVSS (SEQ ID NO: 8), and may additionally or alternatively comprise: (e) FR-L1 comprising the amino acid sequence of AIVMTQTPSPVSAAVGGTVTINC (SEQ ID NO: 12); (f) FR-L2 comprising WFQQKPGQPPKLLIY (SEQ ID NO: 13). (g) FR-L3 comprising the amino acid sequence of GVPSRFKGSGSGTQFTLTISGVQCDDAATYYC (SEQ ID NO: 14); and (h) FR-L4 comprising the amino acid sequence of FGGGTEVVVR (SEQ ID NO: 15). For example, in some embodiments, the anti-PD-L1 antibody comprises VH and VL sequences comprising the amino acid sequences of SEQ ID NOs: 16 and 17, respectively, and may or may not comprise post-translational modifications.

For example, the invention features an anti-PD-L1 antibody, such as anti-PD-L1 antibody SP142, having the following heavy chain variable region sequence and light chain variable region sequence.

The amino acid sequence of the heavy chain variable region of SP142 is as follows:

The amino acid sequence of the light chain variable region of SP142 is as follows:

In some cases, the anti-PD-L1 antibodies of the present invention are antibodies that compete with any one or more of the above-mentioned anti-PD-L1 antibodies for binding to PD-L1. In some cases, the anti-PD-L1 antibodies of the present invention are antibodies that bind to the same epitope or substantially the same epitope as any one or more of the above-mentioned anti-PD-L1 antibodies.

In some cases, the anti-PD-L1 antibody according to any of the above embodiments may be a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-PD-L1 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, a diabody, or a F(ab') ₂ fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG antibody (e.g., a complete IgG1 antibody) or other antibody classes or isotypes as defined herein.

It should be understood that the anti-PD-L1 antibodies of the present invention, while used to detect the presence or expression level of PD-L1 in biological samples as exemplified by the following examples, can also be used or adapted for therapeutic use.

In other aspects, the anti-PD-L1 antibodies according to any of the above embodiments may incorporate any of the features described in Sections 1-5 below, either singly or in combination.

1. Antibody affinity

In certain embodiments, the dissociation constant (Kd) of an antibody provided herein is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹³ M, e.g., 10 ⁻⁹ M to 10 ⁻¹³ M).

In one embodiment, Kd is measured by performing a radiolabeled antigen binding assay (RIA) using a Fab version of the antibody of interest and its antigen as described in the following assay. The solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen, followed by 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 assay conditions, multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked for 2 to 5 hours at room temperature (approximately 23° C.) with 2% (w/v) bovine serum albumin in PBS. In a non-adsorbent plate (Nunc No. 269620), 100 pM or 26 pM [ ¹²⁵ I]-antigen is mixed with a serial dilution of the target Fab (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al. Cancer Res. 57:4593-4599, 1997). The target Fab is then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation 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 ^™ ). When the plate has dried, 150 μl of scintillant (MICROSCINT-20 ^™ ; Packard) is added to each well and the plate is counted for 10 minutes on a TOPCOUNT ^™ gamma counter (Packard). Concentrations of each Fab that yielded less than or equal to 20% of maximal binding were chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a surface plasmon resonance assay at about 10 response units (RU) at 25° C. using a BIAcore ® -2000 or -3000 (BIAcore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg/ml (-0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl/min to achieve about 10 response units (RU) of coupled protein. Following the injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS containing 0.05% polysorbate 20 (TWEEN-20 ^™ ) surfactant (PBST) were injected at 25° C. at a flow rate of approximately 25 μl/min. The association rate (k on ) and dissociation rate (k _off ) were calculated using a simple one-to-one Langmuir binding model (Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation _constant (Kd) was calculated as the ratio k _off / k _on . See, e.g., Chen et al. J. Mol. Biol. 293: 865-881, 1999. If the on-rate as determined above by surface plasmon resonance exceeds 10 ⁶ M ⁻¹ s ⁻¹ , the on-rate can then be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen ^antibody (Fab form) in PBS (pH 7.2) at 25° C. in the presence of increasing concentrations of antigen as measured in a spectrometer such as a stopped-flow equipped spectrophotometer (Aviv Instruments) or a 8000 series SLM-AMINCO™ spectrophotometer with a stirred cuvette (ThermoSpectronic).

2. Antibody fragments

In certain embodiments, the antibodies provided 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 certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134, 2003. For a review of scFv fragments, see, for example, Pluckthun. The Pharmacology of Monoclonal Antibodies. Vol. 113, pp. 269-315, Rosenburg and Moore, eds. Springer-Verlag, New York, 1994; see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments that contain salvage receptors that bind to epitope residues and have increased in vivo half-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments that can be bivalent or bispecific and have two antigen-binding sites. See, for example, EP 404,097; WO 1993/01161; Hudson et al. Nat. Med. 9:129-134, 2003; and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993. Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134, 2003.

Single-domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

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

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA. 81: 6851-6855, 1984. In one embodiment, 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 yet another embodiment, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed compared to that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally speaking, humanized antibodies include one or more variable domains, wherein the HVR (or part thereof) of, for example, CDR is derived from non-human antibodies, and FR (or part thereof) is derived from human antibody sequences. Humanized antibodies are optionally also comprised of at least a portion of human constant regions. In some embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for making them are reviewed in, for example, Almagro et al. Front. Biosci. 13: 1619-1633, 2008, and are also described in, for example, Riechmann et al. Nature. 332: 323-329, 1988; Queen et al. Proc. Natl. Acad. Sci. USA. 86: 10029-10033, 1989; U.S. Pat. Nos. 5,821,337, 7,527,79 1, 6,982,321 and 7,087,409; Kashmiri et al. Methods. 36:25-34, 2005 (describing SDR (a-CDR) grafting); Padlan. Mol. Immunol. 28:489-498, 1991 (describing "surfacing"); DaU'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 the "directed selection" method of FR shuffling).

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 the consensus sequence of human antibodies having a particular subgroup of light chain variable regions 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: 2 623, 1993); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro et al. Front. Biosci. 13: 1619-1633, 2008); and framework regions derived from screening FR libraries (see, e.g., Baca et al. J. Biol. Chem. 272: 10678-10684, 1997, and Rosok et al. J. Biol. Chem. 271: 22611-22618, 1996).

4. Multispecific Antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different sites. In certain embodiments, one binding specificity is for PD-L1 and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of PD-L1. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing PD-L1. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, Milstein et al. Nature. 305:537, 1983, WO 93/08829, and Traunecker et al. EMBO J. 10:3655, 1991) and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects to prepare antibody Fc heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al. Science. 229:81, 1985); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al. J. Immunol. 148(5):1547-1553, 1992); using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Hollinger et al. Proc. Natl. Acad. Sci. USA., 90:6444-6448, 1993); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al. J. Immunol. 152:5368, 1994); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147:60, 1991.

Also included herein are engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies" (see, eg, US 2006/0025576A1).

The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs," which comprise an antigen binding site that binds to PD-L1 as well as another, different antigen (see, e.g., US 2008/0069820).

5. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be necessary to improve the binding affinity and/or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Deletion, insertion, and substitution can be carried out in any combination to obtain the final construct, with the limiting condition that the final construct has the desired characteristics, such as antigen binding.

a) Substitution, insertion and deletion variants

In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 1. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 1, and are further described below with respect to amino acid side chain categories. Amino acid substitutions can be introduced into target antibodies and products screened for desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

Table 1. Exemplary and preferred amino acid substitutions

Amino acids can be grouped according to common side chain properties:

(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues that affect chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally speaking, the resulting variant selected for further study will have modifications (e.g., improvements) in certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and/or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently produced, for example, using affinity maturation techniques based on phage display (such as those described herein). In short, one or more HVR residues are mutated, and the variant antibody is displayed on phage and screened for specific biological activity (e.g., binding affinity).

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

In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as these changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in HVRs. Such changes may be outside HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham et al., Science. 244: 1081-1085, 1989. In this method, a residue or group of residues of interest (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitutions. Alternatively or in addition, a crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

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

b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Adding glycosylation sites to antibodies or deleting glycosylation sites from antibodies can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

When an antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically contain a branched biantennary oligosaccharide attached, generally via an N-link, to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH. 15: 26-32, 1997. Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to produce antibody variants with certain improved properties.

In one embodiment, antibody variants are provided that lack a carbohydrate structure that is (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain on Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as described, for example, in WO 2008/077546. Asn297 refers to an asparagine residue located at approximately position 297 (Eu numbering of Fc region residues) in the Fc region; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Nos. US 2003/0157108 and US 2004/0093621. Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249, 2004; and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614, 2004. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545, 1986; US 2003/0157108; and WO 2004/056312, particularly at Example 11) and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614, 2004; Kanda et al. Biotechnol. Bioeng. 94(4): 680-688, 2006; and WO 2003/085107).

Further provided are antibody variants having bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; U.S. Patent No. 6,602,684; and US 2005/0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

c) Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an anti-PD-L1 antibody of the invention (e.g., SP142) provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human _IgG1 , _IgG2 , _IgG3 , or _IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the present invention encompasses antibody variants having some, but not all, effector functions that make them candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/reduction of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells used to mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch et al. Annu. Rev. Immunol. 9: 457-492, 1991. Non-limiting examples of in vitro assays to assess ADCC activity of target molecules are described in U.S. Pat. Nos. 5,500,362 and 5,821,337; Hellstrom et al. Proc. Natl. Acad. Sci. USA. 83:7059-7063, 1986; Hellstrom et al. Proc. Natl Acad. Sci. USA. 82:1499-1502, 1985; and Bruggemann et al. J. Exp. Med. 166:1351-1361, 1987. Alternatively, non-radioactive assays can be employed (see, e.g., the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc., Mountain View, CA); and the CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the target molecule can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA. 95:652-656, 1998. C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO C1q and C3c binding ELISA in 2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods. 202: 163, 1996; Cragg et al. Blood. 101: 1045-1052, 2003; and Cragg et al. Blood 103: 2738-2743, 2004. FcRn binding and in vivo clearance/half-life can also be determined using methods known in the art (see, e.g., Petkova et al. Intl. Immunol. 18(12): 1759-1769, 2006).

Antibodies with reduced effector function include those with substitutions in one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Pat. No. 6,737,056 ). Such Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine ( U.S. Pat. No. 7,332,581 ).

Certain antibody variants with improved or impaired FcR binding have been described. See, for example, U.S. Patent No. 6,737,056; WO 2004/056312; and Shields et al. J. Biol. Chem. 9(2): 6591-6604, 2001.

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 (EU residue numbering) of the Fc region.

In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or impaired) CIq binding and/or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184, 2000.

U.S. Patent Application No. 2005/0014934 describes antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587, 1976, and Kim et al., J. Immunol. 24:249, 1994). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, such as substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ). See also Duncan et al. Nature. 322: 738-740, 1988; U.S. Pat. Nos. 5,648,260 and 5,624,821; and WO 94/29351 for other examples of Fc region variants.

d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to produce cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are replaced by cysteine residues. In a specific embodiment, the replaced residues are present at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to produce immunoconjugates as further described herein. In certain embodiments, any one or more of the following residues may be replaced by cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies can be generated as described, for example, in U.S. Patent No. 7,521,541.

e) Antibody derivatives

In certain embodiments, the anti-PD-L1 antibodies of the present invention provided herein (e.g., SP142) may be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally speaking, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under certain conditions, etc.

In another embodiment, a conjugate of an antibody with a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al. Proc. Natl. Acad. Sci. USA. 102: 11600-11605, 2005). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but will heat the non-protein moiety to a temperature that kills cells adjacent to the antibody-non-protein moiety.

B. Recombinant Methods and Compositions

Antibodies can be produced using, for example, recombinant methods and compositions as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-PD-L1 antibody described herein (e.g., SP142) is provided. Such nucleic acid may encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (e.g., a light chain and/or a heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NSO, Sp20 cell). In one embodiment, a method of preparing an anti-PD-L1 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-PD-L1 antibodies (e.g., SP142), nucleic acids encoding antibodies such as those described above are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expressing antibody encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. Also see Charlton. Methods in Molecular Biology. Vol. 248, pp. 245-254, B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003, describing expression of antibody fragments in E. coli. After expression, the antibodies can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross. Nat. Biotech. 22: 1409-1414, 2004, and Li et al. Nat. Biotech. 24: 210-215, 2006.

Suitable host cells for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES ^™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney line (such as 293 or 293 cells as described, for example, in Graham et al. J. Gen Virol. 36:59, 1977); baby hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells as described, for example, in Mather. Biol. Reprod. 23:243-251, 1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells as described, for example, in Mather et al. Annals N.Y. Acad. Sci. 383:44-68, 1982; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR" CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA. 77:4216, 1980); and myeloma cell lines such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki et al., Methods in Molecular Biology. Vol. 248, pp. 255-268, B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003.

C. Determination

The physical/chemical properties and/or biological activities of the anti-PD-L1 antibodies provided herein can be identified, screened, or characterized by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, the antigen binding activity of the antibodies of the invention is tested, for example, by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (eg, Western blot), flow cytometry (eg, FAGS ^™ ), immunohistochemistry, immunofluorescence, and the like.

In another aspect, a competition assay can be used to identify antibodies that compete with any of the antibodies of the present invention (e.g., anti-PD-L1 antibody SP142) for binding to PD-L1. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) bound by any of the antibodies of the present invention (e.g., anti-PD-L1 antibody SP142). Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris' "Epitope Mapping Protocol" in Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ, 1996).

In an exemplary competition assay, immobilized PD-L1 is incubated in a solution comprising a first labeled antibody that binds to PD-L1 (e.g., anti-PD-L1 antibody SP142) and a second unlabeled antibody (which will be tested for its ability to compete with the first antibody for binding to PD-L1). The second antibody may be present in the hybridoma supernatant. As a control, immobilized PD-L1 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to PD-L1, excess unbound antibody is removed and the amount of label associated with the immobilized PD-L1 is measured. If the amount of label associated with the immobilized PD-L1 is substantially reduced in the test sample relative to the control sample, then it is indicated that the second antibody competes with the first antibody for binding to PD-L1. See, e.g., Harlow et al. Antibodies: A Laboratory Manual. Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

2. Detection and Assay

In one aspect, assays are provided for identifying anti-PD-L1 antibodies for use in detecting the presence of PD-L1 in, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS ^™ ), or enzyme-linked immunosorbent assay (ELISA) assays. In certain embodiments, the antibodies of the invention are tested for such activity.

D. Immunoconjugates

The present invention also provides immunoconjugates comprising an anti-PD-L1 antibody herein conjugated to one or more labels and/or agents, such as radioisotopes.

In one embodiment, the immunoconjugate comprises an antibody conjugated to a radioactive atom as described herein to form a radioconjugate. Various radioisotopes can be used to produce radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu. When the radioconjugate is used for detection, it can include radioactive atoms for scintigraphy research, such as tc 99m or I ¹²³ , or spin labels for nuclear magnetic resonance (NMR) imaging (also referred to as magnetic resonance imaging, MRI), such as iodine-123 (again), iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of anti-PD-L1 antibodies and labels or agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3-(2-pyridyldithiol) propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl diimidoadipate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94/11026. The linker can be a "cleavable linker" that facilitates release of the label or agent. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020).

The immunoconjugates herein specifically encompass, but are not limited to, such conjugates prepared with cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio-MBS, thio-SIAB, thio-SMCC, and thio-SMPB, as well as SVSB (succinimidyl (4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Rockford, IL., U.S.A.).

E. Methods and compositions for diagnosis and detection

In certain embodiments, the anti-PD-L1 antibodies provided herein (e.g., SP142 or any other anti-PD-L1 antibodies described in Section A, "Exemplary Anti-PD-L1 Antibodies," above) are used to detect the presence of PD-L1 in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection.

In one embodiment, an anti-PD-L1 antibody (e.g., SP142) for use in a diagnostic or detection method is provided. In another embodiment, the present invention provides a use of an anti-PD-L1 antibody (e.g., SP142) in the preparation of a reagent for a diagnostic or detection method.

For example, in one instance, a method for detecting the presence of PD-L1 in a biological sample described below is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-PD-L1 antibody as described herein under conditions that allow the anti-PD-L1 antibody to bind to PD-L1, and detecting whether a complex is formed between the anti-PD-L1 antibody and PD-L1. Such methods may be in vitro or in vivo methods. The anti-PD-L1 antibodies of the present invention (e.g., SP142) can be used, for example, in immunoassays, including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (e.g., Western blot), flow cytometry (e.g., FAGS ^™ ), and enzyme-linked immunosorbent assay (ELISA). In one embodiment, for example, when PD-L1 is a biomarker for selecting a patient, the anti-PD-L1 antibody is used to select a subject suitable for therapy with the anti-PD-L1 antibody. The present invention also provides use of an anti-PD-L1 antibody in a method for diagnosing a subject having a disorder (e.g., cancer or immune dysfunction), the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibody.

For example, the method provides the use of an anti-PD-L1 antibody in a method for diagnosing a subject with cancer, the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibody. 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. In some cases, the tissue sample is a tumor sample. In some cases, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, and any combination thereof.

The present invention further provides use of an anti-PD-L1 antibody in preparing a reagent for use in a method for diagnosing a subject suffering from a disorder (e.g., cancer or immune dysfunction), the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with the anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibody.

For example, the method provides the use of an anti-PD-L1 antibody in the preparation of a reagent for use in a method for diagnosing a subject with cancer, the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibody. 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. In some cases, the tissue sample is a tumor sample. In some cases, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, and any combination thereof.

In another embodiment, the present invention provides a method for identifying a subject having a condition (e.g., cancer or immune dysfunction) who may respond to treatment, the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibody, wherein the presence or expression level of PD-L1 in the sample indicates that the subject may respond to treatment.

For example, the present invention provides a method for identifying a subject with cancer who may respond to treatment with an anti-cancer therapy, the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibodies, wherein the presence or expression level of PD-L1 in the sample indicates that the subject may respond to treatment with an anti-cancer therapy. 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. In some cases, the tissue sample is a tumor sample. In some cases, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, and any combination thereof.

In yet another aspect, the present invention provides a method for predicting responsiveness to treatment in an individual having a disorder (e.g., cancer or an immune disorder), the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibody, wherein the presence or expression level of PD-L1 in the sample indicates that the subject is more likely to respond to treatment.

For example, the present invention provides a method for predicting the responsiveness of an individual with cancer to treatment with an anti-cancer therapy, the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from a subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibodies, wherein the presence or expression level of PD-L1 in the sample indicates that the subject is more likely to respond to treatment with an anti-cancer therapy. 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. In some cases, the tissue sample is a tumor sample. In some cases, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, and any combination thereof.

In yet another aspect, the invention provides a method for determining the likelihood that a subject having a disorder (e.g., cancer or an immune disorder) will exhibit a benefit from treatment, the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the invention (e.g., SP142) and detecting the presence of bound antibody, wherein the presence or expression level of PD-L1 in the sample indicates the likelihood that the subject will exhibit a benefit from treatment with the treatment.

For example, the present invention provides a method for determining the likelihood that a subject with cancer will exhibit a benefit from treatment with an anti-cancer therapy, the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibody, wherein the presence or expression level of PD-L1 in the sample indicates the likelihood that the subject will exhibit a benefit from treatment with an anti-cancer therapy. 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. In some cases, the tissue sample is a tumor sample. In some cases, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, and any combination thereof.

In another embodiment, the present invention provides a method for selecting a therapy for a subject having a disorder (e.g., cancer or immune dysfunction), the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibody; and selecting an anti-cancer therapy for the subject based on the presence or expression level of PD-L1 in the sample.

For example, the present invention provides a method for selecting a therapy for a subject with cancer, the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibodies; and selecting an anti-cancer therapy for the subject based on the presence or expression level of PD-L1 in the sample. 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. In some cases, the tissue sample is a tumor sample. In some cases, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, and any combination thereof.

In any of the foregoing methods, the tumor sample can have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise about 1% or more (e.g., by area) of the tumor sample (e.g., about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more). For example, in some cases, the tumor sample can have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise (e.g., by area) from about 1% to less than about 99% (e.g., from about 1% to less than about 95%, from about 1% to less than about 90%, from about 1% to less than about 85%, from about 1% to less than about 80%, from about 1% to less than about 75%, from about 1% to less than about 70%, from about 1% to less than about 65%, from about 1% to less than about 80%) of the tumor sample. about 60%, about 1% to less than about 55%, about 1% to less than about 50%, about 1% to less than about 40%, about 1% to less than about 35%, about 1% to less than about 30%, about 1% to less than about 25%, about 1% to less than about 20%, about 1% to less than about 15%, about 1% to less than about 10%, about 1% to less than about 5%, about 5% to less than about 95%, about 5% to less than about 90%, about 5% to less than about 85%, about 5% to less than about 80%, about 5% to less than about 75%, about 5% to less than about 70%, about 5% to less than about 65%, about 5% to less than about 60%, about 5% to less than about 55%, about 5% to less than about 50%, about 5% to less than about 40%, about 5% to less than about 35%, about 5% to less than about 30%, about 5% to less than about 25%, about 5% to less than about 20%, about 5% to less than about 15%, about 5% to less than about 10%, about 10% to less than about 95%, about 10% to less than about 90%, about 10% To the extent that the tumor is expressed in an amount greater than about 10%, the tumor may be expressed in an amount less than about 10%. To the extent that the tumor is expressed in an amount less than about 10%, the tumor may be expressed in an amount less than about 10%. To the extent that the tumor is expressed in an amount less than about 10%, the tumor may be expressed in an amount less than about 10%. To the extent that the tumor is expressed in an amount less than about 10%, the tumor may be expressed in an amount less than about 10%. To the extent that the tumor is expressed in an amount less than about 10%, the tumor may be expressed in an amount less than about 10%. In other cases, the tumor may have a detectable expression level of PD-L1 in tumor-infiltrating immune cells, and the tumor-infiltrating immune cells (e.g., area) account for about 5% or more to less than about 10% of the tumor sample. In other cases, the tumor may have a detectable expression level of PD-L1 in tumor-infiltrating immune cells, and the tumor-infiltrating immune cells (e.g., area) account for about 10% or more of the tumor sample.

In any of the foregoing methods, the tumor sample can have a detectable level of PD-L1 expression in about 1% or more (e.g., about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more) of the tumor cells in the tumor sample. For example, in some cases, the tumor sample comprises from about 1% to less than about 99% (e.g., from about 1% to less than about 95%, from about 1% to less than about 90%, from about 1% to less than about 85%, from about 1% to less than about 80%, from about 1% to less than about 75%, from about 1% to less than about 70%, from about 1% to less than about 65%, from about 1% to less than about 60%, from about 1% to less than about 55%, from about 1% to less than about 50%, from about 1% to less than about 40%, from about 1% to less than about 55%) of the tumor sample. about 1% to less than about 35%, about 1% to less than about 30%, about 1% to less than about 25%, about 1% to less than about 20%, about 1% to less than about 15%, about 1% to less than about 10%, about 1% to less than about 5%, about 5% to less than about 95%, about 5% to less than about 90%, about 5% to less than about 85%, about 5% to less than about 80%, about 5% to less than about 75%, about 5% to less than about 70%, about 5% to less than about 65%, about 5% to less than about 60%, about 5% to less than about 55%, about 5% to less than about 50%, about 5% to less than about 40%, about 5% to less than about 35%, about 5% to less than about 30%, about 5% to less than about 25%, about 5% to less than about 20%, about 5% to less than about 15%, about 5% to less than about 10%, about 10% to less than about 95%, about 10% to less than about 90%, about 10% to less than about 85%, about 10% to less than about 80%, about 10% to less than About 75%, about 10% to less than about 70%, about 10% to less than about 65%, about 10% to less than about 60%, about 10% to less than about 55%, about 10% to less than about 50%, about 10% to less than about 40%, about 10% to less than about 35%, about 10% to less than about 30%, about 10% to less than about 25%, about 10% to less than about 20%, about 10% to less than about 15%) of the tumor cells may have a detectable expression level of PD-L1. For example, in some cases, the tumor sample may have a detectable expression level of PD-L1 in about 1% or more to less than 5% of the tumor cells in the tumor sample. In other cases, the tumor may have a detectable expression level of PD-L1 in about 5% or more to less than 10% of the tumor cells in the tumor sample. In other cases, the tumor may have a detectable expression level of PD-L1 in about 10% or more of the tumor cells in the tumor sample.

In any of the foregoing methods, the cancer can be non-small cell lung cancer (NSCLC), squamous cell carcinoma (including lung adenocarcinoma and lung squamous carcinoma), small cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia, and head and neck cancer. In some cases, the cancer is NSCLC. In some embodiments, the NSCLC is lung adenocarcinoma or lung squamous carcinoma. In some embodiments, the NSCLC is squamous NSCLC or non-squamous NSCLC.

The benefit and/or response in any of the foregoing methods can be any benefit and/or response known in the art and/or described herein, such as (1) inhibiting disease progression (e.g., cancer progression) to some extent, including slowing and complete arrest; (2) reduction in tumor size; (3) inhibiting (i.e., reducing, slowing, or completely stopping) cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibiting (i.e., reducing, slowing, or completely stopping) metastasis; (5) alleviating to some extent one or more symptoms associated with a disease or condition (e.g., cancer); (6) increasing or extending the length of survival (including overall survival and progression-free survival); and/or (9) reducing mortality at a given time point after treatment. In some cases, such benefits include any one or more of the following: prolonging survival (including overall survival and progression-free survival); causing an objective response (including a complete response or a partial response); or ameliorating signs or symptoms of cancer.

Any of the foregoing methods further comprises administering to the subject a therapeutically effective amount of an anti-cancer therapy based on the expression level of PD-L1 in the sample. In some cases, the anti-cancer therapy comprises a PD-1 axis binding antagonist. The PD-1 axis binding antagonist can be any PD-1 axis binding antagonist known in the art or described herein.

For example, in some cases, 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. In some cases, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In other cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some cases, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some cases, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is selected from the group consisting of: YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab).

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

In some cases, the method further comprises administering to the patient an effective amount of a second therapeutic agent. In some cases, the second therapeutic agent is selected from the group consisting of a cytotoxic agent, a growth inhibitory agent, a radiation therapy agent, an anti-angiogenic agent, and combinations thereof.

The sample used in any of the foregoing embodiments can be as described below in Section F "Biological Samples". In some embodiments, the tumor sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, or any combination thereof. In any of the foregoing embodiments, the tumor sample can be a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

In certain embodiments, the presence and/or expression level/amount of PD-L1 in a sample can be determined 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 proteins in a sample. In one embodiment, the expression level of PD-L1 is determined using a method comprising the following: (a) performing IHC analysis of a sample (such as a tumor sample obtained from a subject) using an anti-PD-L1 antibody of the present invention (e.g., SP142); and (b) determining the presence and/or expression level of PD-L1 in the sample. In some embodiments, the IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control cell line staining sample, a tissue sample from a non-cancerous patient, a reference sample known to have a predetermined PD-L1 expression level (e.g., a reference sample with a determined IC% or TC%), or a PD-L1 negative tumor sample).

IHC can be performed in combination with other techniques such as morphological staining and/or in situ hybridization (e.g., FISH). Two general methods of IHC are available: direct assay and indirect assay. According to the first assay, the binding of the antibody to the target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent tag or enzyme-labeled primary antibody, which can be visualized without the need for 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. When the secondary antibody is conjugated to an enzymatic label, a chromogenic substrate or a fluorescent substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody. The primary and/or secondary antibodies used for IHC will typically be labeled with a detectable moiety. Many labels are available, which generally fall into the following categories: (a) radioisotopes, such as ³⁵ S, ¹⁴ C, ¹²⁵ 1, ³ H, and ¹³¹ I; (b) colloidal gold particles; (c) fluorescent labels, including but not limited to rare earth metal chelates (europium chelates), Texas Red, rhodamine, fluorescein, monoacyl, lissamine, umbelliferone, phycoerythrin, phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more of the above; (d) various enzyme substrate labels are available, and U.S. Patent No. 4,275,149 provides a review of some of these labels. Examples of enzymatic 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.

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

The specimen can be prepared, for example, manually, or using an automatic staining instrument (e.g., Ventana BenchMark XT or Benchmark ULTRA instrument). The specimen thus prepared can be mounted and covered with a coverslip. The slide evaluation is then determined, for example, using a microscope, and the staining intensity standards conventionally used in the art can be used. In one embodiment, it will be understood that when using IHC to examine cells and/or tissues from a tumor, staining is typically measured or assessed in tumor cells and/or tissues (as opposed to the matrix or surrounding tissue that may be present in the sample). In some embodiments, it will be understood that when using IHC to examine cells and/or tissues from a tumor, staining is included in measuring or assessing in tumor-infiltrating immune cells (including immune cells within or around the tumor). In some embodiments, the presence of PD-L1 is detected by IHC in >0% of the samples, in at least 1% of the samples, in at least 5% of the samples, in at least 10% of the samples, in at least 15% of the samples, in at least 15% of the samples, in at least 20% of the samples, in at least 25% of the samples, in at least 30% of the samples, in at least 35% of the samples, in at least 40% of the samples, in at least 45% of the samples, in at least 50% of the samples, in at least 55% of the samples, in at least 60% of the samples, in at least 65% of the samples, in at least 70% of the samples, in at least 75% of the samples, in at least 80% of the samples, in at least 85% of the samples, in at least 90% of the samples, in at least 95% of the samples, or more. The samples can be scored using any of the criteria described herein (e.g., by a pathologist or using automated image analysis).

In some embodiments of any of the aforementioned methods, the expression level of PD-L1 is detected in tumor cells, tumor-infiltrating immune cells, or a combination thereof, for example using IHC. Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, or any combination thereof, and other tumor stromal cells (e.g., fibroblasts). Such tumor-infiltrating immune cells may be T lymphocytes (such as CD8+ T lymphocytes and/or CD4+ T lymphocytes), B lymphocytes, or other myeloid lineage cells, including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), tissue cells, and natural killer cells. In some embodiments, staining of PD-L1 is detected as membrane staining, cytoplasmic staining, and combinations thereof. In other embodiments, the absence of PD-L1 is detected in the sample as a lack of staining or absence of staining. In some embodiments, e.g., as described herein, the method comprises determining the percentage of tumor area covered by tumor-infiltrating immune cells that express a detectable amount of PD-L1 (see, e.g., the IC % as described in Example 5). In some embodiments, e.g., as described herein, the method comprises determining the percentage of tumor cells in a tumor sample that express a detectable amount of PD-L1 (see, e.g., Example 5).

In some cases, labeled anti-PD-L1 antibodies are provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent, chromophore, electron-dense, chemiluminescent, and radioactive labels) and moieties that are indirectly detected, for example, via an enzymatic reaction or molecular interaction, such as an enzyme or ligand. Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores (such as rare earth chelates or fluorescein and its derivatives), rhodamine and its derivatives, dansyl, umbelliferone, luciferases (e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456)), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase) coupled to enzymes that utilize hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase, or microperoxidase), biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

It will also be understood that any of the above methods for diagnosis and/or detection may be performed using the immunoconjugates of the invention as described above instead of or in addition to unconjugated anti-PD-L1 antibodies.

F. Biological samples

In certain embodiments, an anti-PD-L1 antibody of the invention (e.g., SP142 or any other anti-PD-L1 antibody described above in Section A, "Exemplary Anti-PD-L1 Antibodies") can be used to detect the presence of PD-L1 in a biological sample using methods known in the art or described herein.

In some cases, a biological sample comprises a tissue or cell sample. For example, a biological sample can include cells or tissues from a normal or cancer patient, such as normal and cancerous tissues of the breast, colon, lung, kidney, bone, brain, muscle, stomach, pancreas, bladder, ovary, uterus, and heart, embryonic and placental tissue.

In some cases, the source of the tissue or cell sample can be solid tissue such as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from any time of pregnancy or development of the subject. In some embodiments, the biological sample is obtained from an in vitro tissue or cell culture. Examples of biological samples herein include, but are not limited to, tumor biopsies, circulating tumor cells, serum or plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples such as formalin-fixed paraffin-embedded (FFPE) tumor samples or frozen tumor samples.

In some embodiments, the biological sample may contain compounds that are not naturally intermixed with tissue, such as preservatives, anticoagulants, buffers, nutrients, antibiotics, etc. In certain embodiments, the biological sample has been exposed to and/or contains one or more fixatives. Fixatives that can be used with the methods and compositions of the present invention include formalin, glutaraldehyde, osmium tetroxide, acetic acid, ethanol, acetone, picric acid, chloroform, potassium dichromate, and mercuric chloride, and/or are stabilized by microwave heating or freezing.

In some embodiments, the biological sample is from a subject suffering from, prone to, or being tested for an immune disorder. In certain embodiments, the immune disorder is a T cell dysfunction disorder. In some embodiments, the T cell dysfunction disorder is an unresolved acute infection, chronic infection, or tumor immunity. In certain embodiments, the immune disorder is an autoimmune disease. In some embodiments, the autoimmune disease is an autoimmune rheumatic disorder (including rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis-dermatomyositis, cryoglobulinemia, antiphospholipid antibody syndrome, and psoriatic arthritis), autoimmune gastrointestinal and liver disorders (including inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (including ANCA-negative vasculitis and ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and vasculitis). granulomatosis and microscopic polyangiitis), autoimmune neurologic disorders (including multiple sclerosis, opsoclonus-myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathy), renal disorders (including glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (including psoriasis, urticaria, urticaria, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (including thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing disorders (including inner ear disease and hearing loss), Behçet's disease, Raynaud's syndrome, and other autoimmune hearing disorders. syndrome), organ transplantation, or autoimmune endocrine disorders (including diabetes-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid diseases (including Graves' disease and thyroiditis)).

In other embodiments, the biological sample is from a subject suffering from, prone to, or being tested for cancer. In certain embodiments, the cancer is non-small cell lung cancer (NSCLC) (including lung adenocarcinoma and lung squamous cell carcinoma), carcinoma, lymphoma (including Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders or various types of head and neck cancer. In specific embodiments, the cancer is NSCLC. In some embodiments, the NSCLC is lung adenocarcinoma or lung squamous cell carcinoma. In some embodiments, the NSCLC is non-squamous NSCLC or squamous NSCLC.

G. Treatment approaches based on the presence and/or expression level of PD-L1

The present invention provides methods for treating a subject having a condition (e.g., cancer or immune dysfunction) based on the presence and/or expression level of PD-L1 in a sample obtained from the subject as determined using an anti-PD-L1 antibody of the present invention. In some cases, the method involves administering a treatment (e.g., an anti-cancer therapy) based on the presence and/or expression level of PD-L1 in a sample obtained from the subject as determined using an anti-PD-L1 antibody of the present invention. In some cases, the treatment method involves correlating the presence and/or expression level of PD-L1 as determined using an anti-PD-L1 antibody of the present invention with the likelihood that the subject will benefit from or respond to an anti-cancer therapy, e.g., as described herein.

The method provides a method of treating a subject having a disorder (e.g., cancer or immune dysfunction), comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibody; and administering a therapeutically effective amount of therapy to the subject.

For example, the method provides a method for treating a subject with cancer, the method comprising: determining the presence or expression level of PD-L1 in a sample obtained from the subject by contacting the sample with an anti-PD-L1 antibody of the present invention (e.g., SP142) and detecting the presence of bound antibodies; and administering a therapeutically effective amount of anti-cancer therapy to the subject. 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. In some cases, the tissue sample is a tumor sample. In some cases, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, and any combination thereof. In any of the foregoing methods, the tumor sample can have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise about 1% or more (e.g., by area) of the tumor sample (e.g., about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more). For example, in some cases, the tumor sample can have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise (e.g., by area) from about 1% to less than about 99% (e.g., from about 1% to less than about 95%, from about 1% to less than about 90%, from about 1% to less than about 85%, from about 1% to less than about 80%, from about 1% to less than about 75%, from about 1% to less than about 70%, from about 1% to less than about 65%, from about 1% to less than about 80%) of the tumor sample. Less than about 60%, about 1% to less than about 55%, about 1% to less than about 50%, about 1% to less than about 40%, about 1% to less than about 35%, about 1% to less than about 30%, about 1% to less than about 25%, about 1% to less than about 20%, about 1% to less than about 15%, about 1% to less than about 10%, about 1% to less than about 5%, about 5% to less than about 95%, about 5% to less than about 90%, about 5% to less than about 85%, about 5% to less than about 80%, about 5% to less than about 75%, about 5% to less than about 70%, about 5% to less than about 65%, about 5% to less than about 60%, about 5% to less than about 55%, about 5% to less than about 50%, about 5% to less than about 40%, about 5% to less than about 35%, about 5% to less than about 30%, about 5% to less than about 25%, about 5% to less than about 20%, about 5% to less than about 15%, about 5% to less than about 10%, about 10% to less than about 95%, about 10% to less than about 90%, about 10 For example, in some cases, the tumor can have a detectable level of PD-L1 expression in tumor-infiltrating immune cells that comprise (e.g., by area) from about 1% or more to less than about 5% of the tumor sample. In other cases, the tumor may have a detectable expression level of PD-L1 in tumor-infiltrating immune cells, and the tumor-infiltrating immune cells (e.g., area) account for about 5% or more to less than about 10% of the tumor sample. In other cases, the tumor may have a detectable expression level of PD-L1 in tumor-infiltrating immune cells, and the tumor-infiltrating immune cells (e.g., area) account for about 10% or more of the tumor sample.

In any of the foregoing methods, the tumor sample can have a detectable level of PD-L1 expression in about 1% or more (e.g., about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more) of the tumor cells in the tumor sample. For example, in some cases, the tumor sample comprises from about 1% to less than about 99% (e.g., from about 1% to less than about 95%, from about 1% to less than about 90%, from about 1% to less than about 85%, from about 1% to less than about 80%, from about 1% to less than about 75%, from about 1% to less than about 70%, from about 1% to less than about 65%, from about 1% to less than about 60%, from about 1% to less than about 55%, from about 1% to less than about 50%, from about 1% to less than about 40%, from about 1% to less than about 55%) of the tumor sample. about 1% to less than about 35%, about 1% to less than about 30%, about 1% to less than about 25%, about 1% to less than about 20%, about 1% to less than about 15%, about 1% to less than about 10%, about 1% to less than about 5%, about 5% to less than about 95%, about 5% to less than about 90%, about 5% to less than about 85%, about 5% to less than about 80%, about 5% to less than about 75%, about 5% to less than about 70%, about 5% to less than about 65%, about 5% to less than about 60%, about 5% to less than about 55%, about 5% to less than about 50%, about 5% to less than about 40%, about 5% to less than about 35%, about 5% to less than about 30%, about 5% to less than about 25%, about 5% to less than about 20%, about 5% to less than about 15%, about 5% to less than about 10%, about 10% to less than about 95%, about 10% to less than about 90%, about 10% to less than about 85%, about 10% to less than about 80%, about 10% to less than About 75%, about 10% to less than about 70%, about 10% to less than about 65%, about 10% to less than about 60%, about 10% to less than about 55%, about 10% to less than about 50%, about 10% to less than about 40%, about 10% to less than about 35%, about 10% to less than about 30%, about 10% to less than about 25%, about 10% to less than about 20%, about 10% to less than about 15%) of the tumor cells may have a detectable expression level of PD-L1. For example, in some cases, the tumor sample may have a detectable expression level of PD-L1 in about 1% or more to less than 5% of the tumor cells in the tumor sample. In other cases, the tumor may have a detectable expression level of PD-L1 in about 5% or more to less than 10% of the tumor cells in the tumor sample. In other cases, the tumor may have a detectable expression level of PD-L1 in about 10% or more of the tumor cells in the tumor sample.

In any of the foregoing methods, the cancer can be non-small cell lung cancer (NSCLC) (including lung adenocarcinoma and lung squamous carcinoma), squamous cell carcinoma, small cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia, and head and neck cancer. In some cases, the cancer is NSCLC. In some embodiments, the NSCLC is lung adenocarcinoma or lung squamous carcinoma. In some embodiments, the NSCLC is non-squamous NSCLC or squamous NSCLC.

In any of the foregoing methods, the anti-cancer therapy may include a PD-1 axis binding antagonist. In some cases, the method may include administering a therapeutically effective amount of a PD-1 axis binding antagonist to the patient based on the expression level of PD-L1 in tumor cells and/or tumor-infiltrating immune cells in a tumor sample. The PD-1 axis binding antagonist may be any PD-1 axis binding antagonist known in the art or described herein.

For example, in some cases, the PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. PD-1 (programmed death 1) is also referred to as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2" in the art. Exemplary human PD-1 is shown in UniProtKB/Swiss-Prot accession number Q15116. PD-L1 (programmed death ligand 1) is also referred to as "programmed cell death 1 ligand 1," "PDCD1LG1," "CD274," "B7-H," and "PDL1" in the art. Exemplary human PD-L1 is shown in UniProtKB/Swiss-Prot accession number Q9NZQ7.1. PD-L2 (programmed death ligand 2) is also referred to as "programmed cell death 1 ligand 2," "PDCD1LG2," "CD273," "B7-DC," "Btdc," and "PDL2" in the art. An exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot Accession No. Q9BQ51. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

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

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), such as described below. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of: MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. MDX-1106 (also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab) is an anti-PD-1 antibody described in WO2006/121168. MK-3475 (also known as pembrolizumab or lambrolizumab) is an anti-PD-1 antibody described in WO 2009/114335. CT-011 (also known as hBAT, hBAT-1, or pidilizumab) is an anti-PD-1 antibody described in WO 2009/101611. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. AMP-224 (also known as B7-DCIg) is a PD-L2-Fc fusion soluble receptor described in WO 2010/027827 and WO 2011/066342.

In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternative names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558, and nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4).

In some embodiments, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2 binding antagonist is an anti-PD-L2 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is capable of inhibiting the binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, 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 embodiments, the anti-PD-L1 antibody is a humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody. In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of: YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO 2010/077634 and U.S. Patent No. 8,217,149, the entire contents of each of which are incorporated herein by reference. MDX-1105 (also known as BMS-936559) is an anti-PD-L1 antibody described in WO 2007/005874. MEDI4736 is an anti-PD-L1 monoclonal antibody described in WO 2011/066389 and US 2013/034559. Examples of anti-PD-L1 antibodies for use in the methods of the invention and methods for making the same are described in PCT patent applications WO 2010/077634, WO 2007/005874, WO 2011/066389, U.S. Pat. No. 8,217,149, and US 2013/034559, each of which is incorporated herein by reference in its entirety.

In any of the foregoing embodiments, the isolated anti-PD-L1 antibody can bind to human PD-L1, e.g., human PD-L1 as set forth in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof.

In yet another embodiment, an isolated nucleic acid encoding any of the antibodies described herein is provided. In some embodiments, the nucleic acid further comprises a vector suitable for expressing the nucleic acid encoding any of the aforementioned anti-PD-L1 antibodies. In yet another specific aspect, the vector is in a host cell suitable for nucleic acid expression. In yet another specific aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In yet another specific aspect, the eukaryotic cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

The antibodies or antigen-binding fragments thereof can be prepared using methods known in the art, for example, by a method comprising culturing a host cell containing a nucleic acid encoding any of the aforementioned anti-PD-L1 antibodies or antigen-binding fragments in a form suitable for expression under conditions suitable for production of such antibodies or fragments, and recovering the antibody or fragment. It is expressly contemplated that the PD-1 axis binding antagonist antibodies (e.g., anti-PD-L1 antibodies, anti-PD-1 antibodies, and anti-PD-L2 antibodies) used in any of the above-enumerated embodiments or other antibodies described herein (e.g., anti-PD-L1 antibodies for detecting PD-L1 expression levels) may have any of the features described in subsections 1-5 of Section A, "Exemplary Anti-PD-L1 Antibodies," either singly or in combination.

In some cases, any of the foregoing methods further comprise administering to the patient an effective amount of a second therapeutic agent. In some cases, the second therapeutic agent is selected from the group consisting of a cytotoxic agent, a growth inhibitory agent, a radiotherapy agent, an anti-angiogenic agent, and combinations thereof.

The compositions used in the methods described herein can be administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subconjunctivally, intracapsularly, mucosally, intrapericardially, intraumbilicalally, intraocularly, intraorbitally, orally, topically, transdermally, intravitreally (e.g., by intravitreal injection), by eye drops, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by local perfusion that directly bathes target cells, by catheter, by lavage, in the form of a cream, or in the form of a lipid composition. The compositions used in the methods described herein can also be administered systemically or topically. The method of administration can vary according to various factors (e.g., the compound or composition administered and the severity of the condition, disease or illness being treated). In some embodiments, anticancer therapy (e.g., PD-1 axis binding antagonist) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly or intranasally. Depending in part on whether the administration is short-lived or long-term, administration can be performed by any suitable route, for example, by injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including but not limited to multiple administrations, push administrations, and pulse infusions in a single administration or various time points.

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

For the prevention or treatment of cancer (e.g., non-small cell lung cancer), the appropriate dosage of the therapeutic agents described herein (e.g., PD-1 axis binding antagonists) (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the severity and course of the disease, whether the therapeutic agent (e.g., PD-1 axis binding antagonist) is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the therapeutic agent (e.g., PD-1 axis binding antagonist), and the judgment of the attending physician. The therapeutic agent (e.g., PD-1 axis binding antagonist) is suitably administered to the patient once or over a series of treatments. Depending on the factors mentioned above, a typical daily dosage may range from about 1 μg/kg to 100 mg/kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. Such doses can be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives, for example, from about two to about twenty doses, or, for example, about six doses of the therapeutic agent (e.g., a PD-1 axis binding antagonist)). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing schedules can be used. The progress of this therapy is readily monitored by conventional techniques and assays.

For example, as a general proposition, whether or not performed by one or more administrations, the therapeutically effective amount of the antagonist antibody (e.g., PD-1 axis binding antagonist antibody) administered to a human will be in the range of about 0.01 to about 50 mg/kg of patient body weight. In some embodiments, the antibody used is, for example, about 0.01 mg/kg to about 45 mg/kg, about 0.01 mg/kg to about 40 mg/kg, about 0.01 mg/kg to about 35 mg/kg, about 0.01 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 25 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about 0.01 mg/kg to about 15 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 5 mg/kg, or about 0.01 mg/kg to about 1 mg/kg, for daily, weekly, biweekly, three-weekly, or monthly administration. In some embodiments, the antibody is administered at 15 mg/kg. However, other dosing regimens can be used. In one embodiment, on the first day of a 21-day cycle (every three weeks, q3w), an antagonist antibody described herein (e.g., PD-1 axis binding antagonist antibody) is administered to a human at a dosage 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. The dosage can be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as an infusion. Compared to a single treatment, the dosage of the antibody administered in the combination therapy can be reduced. The progress of this therapy is easily monitored by conventional techniques.

In some embodiments, the method further involves administering an effective amount of a second therapeutic agent to the patient. In some embodiments, the second therapeutic agent is selected from the group consisting of: a cytotoxic agent, a chemotherapeutic agent, a growth inhibitor, a radiotherapy agent, an anti-angiogenic agent, and a combination thereof. In some embodiments, the therapeutic agent (e.g., a PD-1 axis binding antagonist) can be administered in combination with chemotherapy or a chemotherapeutic agent. In some embodiments, the therapeutic agent (e.g., a PD-1 axis binding antagonist) can be administered in combination with a radiotherapy agent. In some embodiments, the therapeutic agent (e.g., a PD-1 axis binding antagonist) can be administered in combination with a targeted therapy or a targeted therapeutic agent. In some embodiments, the therapeutic agent (e.g., a PD-1 axis binding antagonist) can be administered in combination with an immunotherapy or an immunotherapeutic agent (e.g., a monoclonal antibody).

Such combination therapies indicated above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), as well as separate administration, in which case administration of the therapeutic agent of the invention (e.g., a PD-1 axis binding antagonist) can be performed prior to, concurrently with, and/or after administration of the additional therapeutic agent or agent. In one embodiment, administration of the therapeutic agent (e.g., a PD-1 axis binding antagonist) and administration of the additional therapeutic agent are performed within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

Therapeutic formulations of therapeutic agents (eg, PD-1 axis binding antagonists) used in accordance with the present invention are prepared by mixing the antagonist having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers in the form of a lyophilized formulation or aqueous solution for storage. For general information on formulations, see, e.g., Gilman et al. (eds.) The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Pennsylvania, 1990; Avis et al. (eds.) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York, 1993; Lieberman et al. (eds.) Pharmaceutical Dosage Forms: Tablets Dekker, New York, 1990; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York, 1990; and Walters (ed.) Dermatological and Transdermal Formulations (Drugs and the Pharmaceutical Sciences), Vol. 119, Marcel Dekker, 2002.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, and methylbenzyl ammonium chloride. chloride), phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium ions; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN ^™ , PLURONICS ^™ or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compound, preferably those with complementary activities that do not adversely affect each other. The type and effective amount of such drugs depends, for example, on the amount and type of antagonist present in the formulation, and the clinical parameters of the subject.

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

Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing antagonists, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Patent number 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ^™ (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

It should be understood that any of the above articles of manufacture may include an immunoconjugate described herein instead of or in addition to an antibody (e.g., an anti-PD-L1 antibody or an anti-PD-1 antibody).

III. Examples

The following are examples of methods and compositions of the present invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Example 1. Generation of anti-PD-L1 antibodies

Anti-PD-L1 rabbit monoclonal antibodies were generated as schematically depicted in FIG1 . Briefly, a peptide fragment of the N-terminal cytoplasmic region (amino acid residues 279-290) of human PD-L1 (SKKQSDTHLEET; SEQ ID NO: 1) was synthesized. The 12-amino acid fragment intended for immunization was conjugated to keyhole limpet hemocyanin (KLH), a widely used carrier protein for stimulating substantial immune responses through antibody production. Two amino acids (Cys-Gly) were appended to the naturally occurring N-terminus of the sequence that allows conjugation to the carrier protein KLH. New Zealand white rabbits were immunized with KLH-conjugated PD-L1 antigen emulsified in complete Freund's adjuvant, followed by a series of PD-L1 antigen boosters emulsified in incomplete Freund's adjuvant. Antibody-expressing cells were screened using PD-L1 antigen by enzyme-linked immunosorbent assay (ELISA). All ELISA-positive clones were further screened by immunohistochemistry (IHC), and clones producing antibodies with the highest specificity were selected. For recombinant production of anti-PD-L1 antibodies, cDNAs encoding the heavy and light chain sequences of the antibody were cloned, expressed by co-transfection, and screened for binding to PD-L1 (SEQ ID NO: 1) by IHC. An anti-PD-L1 monoclonal antibody (SP142) was produced using these methods and subsequently purified by protein A affinity chromatography. The heavy and light chain variable region sequences of the SP142 antibody are shown below.

Heavy chain variable region:

Light chain variable region:

Example 2. Diagnostic Use of Anti-PD-L1 Antibodies

SP142 anti-PD-L1 antibody was used for further IHC and western blot analysis. For IHC analysis, tissue sections were incubated with SP142 for 16 minutes, followed by standard washing and secondary detection with goat anti-rabbit biotinylated antibody. The specificity of SP142 on formalin-fixed paraffin-embedded (FFPE) cells with different PD-L1 expression levels (human embryonic kidney HEK-293 cells transfected with empty vector as control (negative), as well as DOR-13 (low to moderate expression), colon cancer RKO cells (moderate expression) and PD-L1 transfected 293 cells (high expression); see Figures 2A-2D) or formalin-fixed paraffin-embedded (FFPE) cells from different tissue types (placenta tissue, tonsil tissue and Hodgkin (HK) lymphoma; see Figures 3A-3C) was evaluated. To further determine the specificity of SP142, Western blot analysis was performed on cell lysates from cell lines with different PD-L1 expression levels (NIH H820 lung adenocarcinoma cell line (high expression), Karpas 299 T cell lymphoma cell line (intermediate expression), and Calu-3 lung adenocarcinoma cell line (negative control); see Figure 4).

Example 3. Comparison of SP142 anti-PD-L1 antibody and E1L3N anti-PD-L1 antibody

SP142 anti-PD-L1 antibody was compared with anti-PD-L1 antibody E1L3N (Cell Signaling Technology) in IHC analysis of normal tissue sections and tumor tissue sections, including tissue from patients with non-small cell lung cancer (NSCLC). Briefly, for IHC experiments, SP142 and E1L3N anti-PD-L1 antibodies were serially diluted (0.11-28 μg/ml). Slides of tissue sections were dewaxed using xylene substitute and graded alcohols. Antigen retrieval was achieved by boiling tissue sections in EDTA buffer (pH 8.0) for 10 minutes, followed by cooling at room temperature for 20 minutes. Tissue sections were incubated with SP142 or E1L3N for 10 minutes, followed by standard washes and secondary detection with goat anti-rabbit antibody for 15 minutes. The final incubation was performed for 10 minutes with the reporter molecule 3,3′-diaminobenzidine (DAB). To assess sensitivity, different concentrations of E1L3N and SP142 were compared on FFPE tissue sections of placental tissue (see Figures 5A-5J). The specificity of E1L3N and SP142 was determined using normal tissue types and tumor tissue types (gastric epithelial tissue, neural tissue, kidney tissue, bladder transitional cell carcinoma (TCC), breast ductal carcinoma (Ca), and lung squamous cell carcinoma (lung SCCa); see Figures 6A-6T and Figures 7A-7J). IHC analysis was also performed on sections of different FFPE tissue types stained with E1L3N or SP142 (tonsil tissue, cervical squamous cell carcinoma (SCC), Hodgkin lymphoma (HK lymphoma), pancreatic adenocarcinoma, prostate adenocarcinoma, and skin SCC; see Figures 8A-8L).

The detection of PD-L1 expression using E1L3N and SP142 was then compared in FFPE tissue sections from NSCLC patients. E1L3N detected 50 PD-L1-positive cases from 119 NSCLC samples whose PD-L1 status had not been previously determined, and SP142 detected 58 PD-L1-positive cases, where 5% or more of tumor cells that were positively stained were considered to indicate PD-L1-positive cases. To further evaluate PD-L1 detection using these antibodies, FFPE tissue sections from NSCLC patients were sequentially stained with E1L3N and SP142 (Figures 9A-9J). The degree of membrane immunoreactivity (i.e., H-score) was then calculated for 119 NSCLC patient tissue sections stained with E1L3N or SP142. The H-score for PD-L1 was calculated using the following formula: H-score = 3 (percentage of strongly stained membranes) + 2 (percentage of moderately stained membranes) + 1 (percentage of weakly stained membranes), resulting in an H-score range of 0 to 300. The mean H-score of E1L3N from these 119 NSCLC cases (56 ± 8) was significantly lower than that of SP142 (100 ± 11). These collective data confirm that SP142 is more sensitive and specific than E1L3N.

Example 4. Use of anti-PD-L1 antibody SP142 in IHC analysis to confirm PD-L1 staining in immune cells (IC) and tumor cells (TC)

In representative cases from all cancer types tested (approximately 28-30 types), the anti-PD-L1 antibody SP142 detected PD-L1 expression in tumor-infiltrating immune cells (ICs) and tumor cells, including but not limited to non-small cell lung cancer (NSCLC), cervical squamous cell carcinoma, HK lymphoma, pancreatic adenocarcinoma, prostate adenocarcinoma, and skin squamous cell carcinoma (see, e.g., Figures 8A-8L, 9A-9J, 10, 11, 15A-15B, 16A-16H, and 18A-18F).

Immune cells (IC)

Immune cells (IC) are tumor-infiltrating immune cells present in the tumor and adjacent peritumoral stroma. The immune cells include a morphologically heterogeneous population of cell types, including lymphocytes, macrophages, dendritic cells, and cells with a reticular morphology. IHC analysis of tumor tissue sections was performed using the SP142 antibody. In tumor tissue sections, PD-L1-positive ICs often show dark brown punctate or discontinuous membrane staining associated with lymphocytes (Figure 10). PD-L1-positive ICs are typically observed as aggregates within the tumor and in the peritumoral stroma and/or as diffuse spread in single cells or tumor cell groups (tumor cell groupings based on location in the tumor section). PD-L1-positive IC staining was also observed at the tumor-stroma interface and as reticular staining in tertiary lymphoid structures.

Tumor cells (TC)

In addition to the PD-L1-positive ICs described above, IHC analysis using the SP142 antibody also confirmed the presence of PD-L1-positive tumor cells (TCs) in tumor samples. PD-L1-positive TCs were typically characterized by membrane staining (Figure 11), which was occasionally associated with cytoplasmic staining.

The results demonstrated that the SP-142 antibody could be used to obtain sensitive and specific PD-L1 staining of tumor tissue sections when used for IHC analysis of PD-L1 expression, and also revealed the presence of PD-L1-positive ICs as well as PD-L1-positive TCs in tumor tissue sections.

Materials and methods

FFPE tissue sections were dewaxed and heated in EDTA antigen retrieval buffer, after which rabbit anti-human PD-L1 (SP142) monoclonal antibody was added to the tissue sections. IHC was processed using an automated staining system (BenchMark ULTRA, Roche) or a semi-automated staining system (Autostainer, Thermo Scientific). Table 2 shows the protocol for the BenchMark ULTRA system.

Table 2. IHC Protocol for the BenchMark ULTRA System Using SP142

Solution Selection BenchMark ULTRA Software Tools NexES v10.6 Staining procedure U OptiView DAB IHC v4 Dewaxing Selected Cell conditioning 48 minutes CC1 Preprimary peroxidase Selected Primary incubation 16 minutes, 36°C OptiView HQ Connector 8 minutes OptiView HRP Polymer 8 minutes OptiView Amplification Selected Amplifier and amplified H2O2 8 minutes Amplified Polymers 8 minutes Hematoxylin II 4 minutes Bluing reagent 4 minutes

Example 5. Determination of PD-L1 Positivity in IC, TC, and Their Combinations

The presence of PD-L1-positive IC, TC, or a combination of IC and TC in tissue stained with an anti-PD-L1 antibody (e.g., any anti-PD-L1 antibody of the present invention, such as SP142) can be scored using different cutoff values (e.g., 1%, 5%, 10%, etc.). These different cutoff values can be used for patient stratification, for example, for selecting patients who are likely to respond to a particular anti-cancer therapy (e.g., an anti-cancer therapy comprising a PD-1 axis binding antagonist).

Immune cell scoring method

The PD-L1 expression of ICs can be scored based on the percentage of tumor area covered by PD-L1 positive ICs of any intensity (referred to herein as "IC%"). Tumor area, as used herein, refers to the area occupied by tumor cells and their associated intratumoral and adjacent peritumoral stroma (e.g., the area of a tumor section) (Figures 12A-12C). Tumor area was chosen as the denominator in this scoring method because ICs are not only present within the stroma, but in some cases exist as single cells or have diffuse spread within tumor cells. As described below, in some cases, ICs are observed as aggregates within tumor sections, while in other cases, ICs are observed as foci of one or a few cells that spread across tumor sections.

An exemplary workflow for determining the IC% of a tumor sample is shown in Figure 13. In some cases, serial sections of the tumor sample are stained with H&E or anti-PD-L1 antibodies (e.g., SP142) as described above. The first step in the workflow is to examine the H&E stained slides to determine the presence of tumors, necrosis, and/or ICs. Next, the corresponding PD-L1 stained slides are examined, for example, using a microscope at low magnification (e.g., 2x or 4x objective lens; 20x-40x total magnification), and the overall PD-L1 staining pattern is assessed. For example, it can be determined whether the tumor tissue section exhibits PD-L1 staining in both IC, TC, IC, and TC, or whether the tumor tissue section does not exhibit substantial PD-L1 staining. Next, the PD-L1 stained slides are examined at higher magnifications (e.g., 10x or 20x objective lens, 100x-200x magnification) to examine IC staining of stroma and tumor cell groups. The use of higher magnifications can confirm weakly stained ICs, as well as distinguish ICs in strong TC staining. Finally, examine PD-L1-stained slides at lower magnification (e.g., 2x or 4x objective; 20-40x total magnification) to determine or estimate the percentage of PD-L1-positive ICs (the percentage of tumor area covered by PD-L1-positive ICs of any intensity).

For example, as shown in Figure 14, after checking and confirming staining at a higher magnification, the percentage of tumor area covered by PD-L1-positive ICs with any PD-L1 staining intensity can be determined at a lower magnification (e.g., 2x or 4x objective; 20-40x total magnification). In cases where the IC staining pattern is characterized by single-cell spread, reference images for a specific range of IC staining (e.g., 1%, 5%, 10%) can be used for comparison purposes to estimate or determine the percentage of tumor area covered by PD-L1-positive ICs with any intensity of PD-L1 staining (e.g., see Figures 15A and 15B and 16A-16H).

Tumor cell scoring method

The PD-L1 expression of tumor cells (TC) can be scored based on the total percentage of tumor cells that show any identifiable membrane PD-L1 staining of any intensity (also referred to herein as "TC%"). TCs are easily distinguished from ICs based on the morphology of the cells in H&E and IHC stained slides. See, for example, Figures 17A-17C. Even with respect to particle mass, membrane staining should be viewed as linear staining (i.e., arranged along the contours of the cell membrane). Figures 18A-18F show examples of PD-L1-stained NSCLC tumor tissue sections exhibiting a specific range of TC PD-L1 staining (i.e., TC% <5%, ≥5 to <50%, or ≥50%).

Other implementation plans

Although the present invention has been described in detail by way of illustration and example for the purpose of clear understanding, the description and examples should not be construed as limiting the scope of the present invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

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Claims (79)

1. A specific PD-L1 binding isolation antibody, wherein the antibody comprises the following hypervariable region (HVR): (a) HVR-H1, whose amino acid sequence is shown in SNGLT (SEQ ID NO:2); (b) HVR-H2, whose amino acid sequence is shown in TINKDASAYYASWAKG (SEQ ID NO:3); (c)HVR-H3, whose amino acid sequence is shown in IAFKTGTSI (SEQ ID NO:4); (d)HVR-L1, whose amino acid sequence is shown in QASESVYSNNYLS (SEQ ID NO:9); (e) HVR-L2, whose amino acid sequence is shown in LASTLAS (SEQ ID NO:10); and (f)HVR-L3, whose amino acid sequence is shown in IGGKSSSTDGNA (SEQ ID NO:11). 2. The antibody of claim 1, further comprising the following heavy chain variable domain (VH) framework region (FR): (a)FR-H1, whose amino acid sequence is shown in QSLEESGGRLVKPDETLTITCTVSGIDLS (SEQ ID NO:5); (b)FR-H2, whose amino acid sequence is shown in WVRQAPGEGLEWIG (SEQ ID NO:6); (c)FR-H3, whose amino acid sequence is shown in RLTISKPSSTKVDLKITSPTTEDTATYFCGR (SEQ ID NO:7); and (d)FR-H4, whose amino acid sequence is shown in WGPGTLVTVSS (SEQ ID NO:8). 3. The antibody of claim 2, comprising the VH sequence of SEQ ID NO:16. 4. The antibody of claim 1, further comprising the following light chain variable domain (VL)FR: (a)FR-L1, whose amino acid sequence is shown in AIVMTQTPSPVSAAVGGTVTINC (SEQ ID NO:12); (b)FR-L2, whose amino acid sequence is shown in WFQQKPGQPPKLLIY (SEQ ID NO:13); (c)FR-L3, whose amino acid sequence is shown in GVPSRFKGSGSGTQFTLTISGVQCDDAATYYC (SEQ ID NO:14); and (d)FR-L4, whose amino acid sequence is shown in FGGGTEVVVR (SEQ ID NO:15). 5. The antibody of claim 4, comprising the VL sequence of SEQ ID NO:17. 6. The antibody of claim 1, comprising: (a) a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:16; (b) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:17; or (c) the VH sequence as in (a) and the VL sequence as in (b). 7. The antibody of claim 1, wherein the antibody comprises the VH sequence of SEQ ID NO:16 and the VL sequence of SEQ ID NO:17. 8. The antibody of claim 1, wherein the antibody binds to an epitope comprising amino acid residues 279-290 of the human PD-L1 polypeptide as shown in SEQ ID NO:1. 9. The antibody of claim 1, wherein the antibody is a monoclonal antibody. 10. The antibody of claim 9, wherein the monoclonal antibody is a rabbit monoclonal antibody. 11. The antibody of claim 1, wherein the antibody is an IgG antibody. 12. The antibody of claim 1, wherein the antibody is an antibody fragment that specifically binds to PD-L1, wherein the antibody fragment is Fab, a single-chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab') ₂ , or a biantibody. 13. An isolated nucleic acid encoding an isolated antibody as described in any one of claims 1-12. 14. A vector comprising the nucleic acid as described in claim 13. 15. A host cell comprising the vector as described in claim 14. 16. An immunoconjugate comprising the antibody as claimed in any one of claims 1-12. 17. The antibody according to any one of claims 1-12, used for detecting the presence or expression level of PD-L1 in a biological sample. 18. The antibody for the use of claim 17, wherein the detection is performed by immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, enzyme-linked immunosorbent assay (ELISA), or Western blotting. 19. The antibody for the use of claim 18, wherein the detection is performed by IHC. 20. The antibody for the use of claim 17, wherein the sample comprises fixed tissue. 21. The antibody for the use of claim 20, wherein the fixed tissue is formalin-fixed paraffin-embedded (FFPE) tissue. 22. The antibody for the use of claim 17, wherein the sample is derived from a subject who has cancer or an immune dysfunction or is at risk of such a disease. 23. The antibody for the use of claim 22, wherein the immune dysfunction is a T-cell dysfunction disorder. 24. The antibody for the use of claim 23, wherein the T-cell dysfunction is an unresolved acute infection, chronic infection, or tumor immune disorder. 25. Use of the antibody manufacturing reagent or kit of any one of claims 1-12 for a method of detecting the presence or expression level of PD-L1 in a biological sample from a subject, wherein the method comprises contacting the biological sample with the antibody of any one of claims 1-12 and detecting the presence of the bound antibody. 26. The use as described in claim 25, wherein the detection is performed by IHC, IF, flow cytometry, ELISA, or immunoblotting. 27. The use as described in claim 26, wherein the detection is performed by IHC. 28. The use as claimed in claim 25, wherein the sample comprises fixed tissue. 29. The use as claimed in claim 28, wherein the fixed tissue is an FFPE tissue. 30. The use as described in claim 25, wherein the sample is derived from a subject suffering from cancer or immune dysfunction or at risk thereof. 31. The use as described in claim 30, wherein the immune dysfunction is a T-cell dysfunction disorder. 32. The use as described in claim 31, wherein the T-cell dysfunction is an unresolved acute infection, chronic infection, or tumor immune disorder. 33. The use as described in claim 30, wherein the sample is derived from a subject suffering from cancer. 34. The use as claimed in claim 25, wherein the presence or expression level of PD-L1 in the sample indicates that the subject may be responding to treatment with anticancer therapy. 35. The use as claimed in claim 25, wherein the presence or expression level of PD-L1 in the sample indicates that the subject is more likely to respond to treatment with anticancer therapy. 36. The use as claimed in claim 25, wherein the presence or expression level of PD-L1 in the sample indicates the likelihood that the subject will exhibit benefits from treatment with anticancer therapy. 37. The use as described in claim 33, wherein the method further comprises selecting an anticancer therapy for the subject based on the presence or expression level of PD-L1 in the sample. 38. The use as described in claim 33, wherein the method further comprises administering a therapeutically effective amount of anticancer therapy to the subject. 39. The use as described in claim 33, wherein the cancer is non-small cell lung cancer, squamous cell carcinoma, small cell lung cancer, peritoneal cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, leukemia, or head and neck cancer. 40. The use as described in claim 39, wherein the cancer is non-small cell lung cancer. 41. The use as described in claim 40, wherein the non-small cell lung cancer is adenocarcinoma or squamous cell carcinoma of the lung. 42. The use as described in claim 39, wherein the liver cancer is hepatocellular carcinoma. 43. The use as described in claim 39, wherein the liver cancer is hepatocellular carcinoma. 44. The use as described in claim 39, wherein the liver cancer is a cancerous tumor of the liver. 45. The use as described in claim 39, wherein the gastrointestinal cancer is colon cancer. 46. The use as described in claim 39, wherein the gastrointestinal cancer is colorectal cancer. 47. The use as claimed in claim 39, wherein the uterine cancer is endometrial cancer. 48. The use as described in claim 33, wherein the sample is a tumor sample. 49. The use as claimed in claim 48, wherein the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, or any combination thereof. 50. The use as claimed in claim 49, wherein the tumor sample has a detectable PD-L1 expression level in tumor-infiltrating immune cells, and the area of the tumor-infiltrating immune cells accounts for about 1% or more of the tumor sample. 51. The use as claimed in claim 50, wherein the tumor sample has a detectable PD-L1 expression level in tumor-infiltrating immune cells, and the area of the tumor-infiltrating immune cells accounts for about 5% or more of the tumor sample. 52. The use as claimed in claim 51, wherein the tumor sample has a detectable PD-L1 expression level in tumor-infiltrating immune cells, and the area of the tumor-infiltrating immune cells accounts for about 10% or more of the tumor sample. 53. The use as claimed in claim 48, wherein the tumor sample has a detectable PD-L1 expression level in about 1% or more of the tumor cells in the tumor sample. 54. The use as claimed in claim 53, wherein the tumor sample has a detectable PD-L1 expression level in about 5% or more of the tumor cells in the tumor sample. 55. The use as claimed in claim 54, wherein the tumor sample has a detectable PD-L1 expression level in about 10% or more of the tumor cells in the tumor sample. 56. The use as claimed in claim 55, wherein the tumor sample has a detectable PD-L1 expression level in about 50% or more of the tumor cells in the tumor sample. 57. The use as claimed in claim 38, wherein the anticancer therapy comprises a PD-1 axis binding antagonist. 58. The use as described in claim 57, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist, a PD-1 binding antagonist, or a PD-L2 binding antagonist. 59. The use as described in claim 58, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist. 60. The use as claimed in claim 59, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. 61. The use as described in claim 60, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. 62. The use as claimed in claim 60, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. 63. The use as claimed in claim 60, wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. 64. The use as described in claim 59, wherein the PD-L1 binding antagonist is an antibody. 65. The use as described in claim 64, wherein the antibody is atezolizumab, MDX-1105 and MEDI4736 (dvorumab) or MSB0010718C (avelumab). 66. The use as described in claim 58, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist. 67. The use as claimed in claim 66, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners. 68. The use as claimed in claim 67, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. 69. The use as claimed in claim 67, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. 70. The use as claimed in claim 67, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. 71. The use as described in claim 66, wherein the PD-1 binding antagonist is an antibody. 72. The use as described in claim 71, wherein the antibody is MDX 1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108. 73. The use as described in claim 66, wherein the PD-1 binding antagonist is an Fc fusion protein. 74. The use as described in claim 73, wherein the Fc fusion protein is AMP-224. 75. The use as claimed in claim 38, wherein the method further comprises administering an effective amount of a second therapeutic agent to the patient. 76. The use as described in claim 75, wherein the second therapeutic agent is a cytotoxic agent, a growth inhibitor, a radiation therapy agent, an anti-angiogenic agent, or a combination thereof. 77. The use as described in claim 25, wherein the subject is a human being. 78. Use of a reagent or kit for antibody manufacturing according to any one of claims 1-12 for a method of predicting the responsiveness of a subject with cancer to treatment with an anticancer therapy comprising a PD-1 axis binding antagonist, wherein the method comprises: The presence or expression level of PD-L1 in a tumor sample obtained from a subject is determined by contacting the tumor sample with an anti-PD-L1 antibody and detecting the presence of the bound antibody, wherein the presence or expression level of PD-L1 in the sample indicates that the subject is more likely to respond to treatment with an anticancer therapy including a PD-1 axis binding antagonist, and wherein said anti-PD-L1 antibody comprises the following HVR: (a) HVR-H1, whose amino acid sequence is shown in SNGLT (SEQ ID NO:2); (b) HVR-H2, whose amino acid sequence is shown in TINKDASAYYASWAKG (SEQ ID NO:3); (c)HVR-H3, whose amino acid sequence is shown in IAFKTGTSI (SEQ ID NO:4); (d)HVR-L1, whose amino acid sequence is shown in QASESVYSNNYLS (SEQ ID NO:9); (e) HVR-L2, whose amino acid sequence is shown in LASTLAS (SEQ ID NO:10); and (f)HVR-L3, whose amino acid sequence is shown in IGGKSSSTDGNA (SEQ ID NO:11). 79. Use of a reagent or kit for antibody manufacturing according to any one of claims 1-12 for a method of selecting an anticancer therapy comprising a PD-1 axis binding antagonist for a subject suffering from cancer, wherein the method comprises: The presence or expression level of PD-L1 in a tumor sample obtained from a subject is determined by contacting the tumor sample with an anti-PD-L1 antibody and detecting the presence of the bound antibody, wherein the anti-PD-L1 antibody comprises the following HVR: (a) HVR-H1, whose amino acid sequence is shown in SNGLT (SEQ ID NO:2); (b) HVR-H2, whose amino acid sequence is shown in TINKDASAYYASWAKG (SEQ ID NO:3); (c)HVR-H3, whose amino acid sequence is shown in IAFKTGTSI (SEQ ID NO:4); (d)HVR-L1, whose amino acid sequence is shown in QASESVYSNNYLS (SEQ ID NO:9); (e) HVR-L2, whose amino acid sequence is shown in LASTLAS (SEQ ID NO:10); and (f) HVR-L3, whose amino acid sequence is shown in IGGKSSSTDGNA (SEQ ID NO:11); and The selection of anticancer therapies, including PD-1 axis binding antagonists, for subjects is based on the presence or expression level of PD-L1 in tumor samples.