HK1200857B - Methods of treating cancer using pd-1 axis binding antagonists and vegf antagonists - Google Patents

Description

Methods for treating cancer using a PD-1 axis binding antagonist and a VEGF antagonist

Cross-reference to related applications

This application claims the benefit of U.S. Provisional Application No. 61/653,861, filed May 31, 2012, which is incorporated herein by reference in its entirety.

Sequence Listing

This application contains a Sequence Listing, which has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. Said ASCII copy, created on May 28, 2013, is named P4926R1WO.txt and is 14,285 bytes in size.

Background of the Invention

Providing two distinct signals to T cells is a widely accepted model for lymphocyte activation of resting T lymphocytes by antigen-presenting cells (APCs). Lafferty et al., Aust. J. Exp. Biol. Med. ScL 53:27-42 (1975). This model further provides for self- versus non-self discrimination and immune tolerance. Bretscher et al., Science 169:1042-1049 (1970); Bretscher, P.A., P.N.A.S. USA 96:185-190 (1999); Jenkins et al., J. Exp. Med. 165:302-319 (1987). Following recognition of a foreign antigenic peptide presented in the context of the major histocompatibility complex (MHC), the first signal, or antigen-specific signal, is transduced via the T cell receptor (TCR). The second or co-stimulatory signal is delivered to T cells through co-stimulatory molecules expressed on antigen presenting cells (APCs), and induces T cells to promote clonal expansion, cytokine secretion, and effector function. Lenschow et al., Ann. Rev. Immunol. 14: 233 (1996). In the absence of co-stimulation, T cells can become refractory to antigen stimulation, fail to initiate an effective immune response, and further can lead to tolerance or exhaustion to foreign antigens.

In the two-signal model, T cells receive both positive and negative second co-stimulatory signals. The regulation of such positive and negative signals is crucial for maximizing the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. Negative second signals seem to be necessary for inducing T cell tolerance, and positive signals promote T cell activation. Although the simple two-signal model still provides an effective explanation for non-immune lymphocytes, the host's immune response is a dynamic process, and co-stimulatory signals can also be provided to antigen-exposed T cells. The mechanism of co-stimulation is of interest in therapeutics because the operation of co-stimulatory signals has been shown to provide means for enhancing or terminating cell-based immune responses. Recently, it has been found that T cell dysfunction or anergy occurs in parallel with the induction and sustained expression of inhibitory receptors (programmed death 1 polypeptide (PD-1)). Therefore, PD-1 and other molecules signaled via interactions with PD-1, such as the therapeutic targeting of programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) are areas of strong interest.

PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al., Intern. Immun. 2007, 19(7):813; Thompson RH et al., Cancer Res 2006, 66(7):3381). Interestingly, in contrast to T lymphocytes in normal tissues and peripheral blood T lymphocytes, most tumor-infiltrating T lymphocytes primarily express PD-1, indicating that upregulation of PD-1 on tumor-reactive T cells can contribute to impaired anti-tumor immune responses (Blood 2009114(8):1537). This may be due to the use of PD-L1 signaling mediated by PD-L1-expressing tumor cells interacting with PD-1-expressing T cells to reduce T cell activation and evade immune surveillance (Sharpe et al., Nat Rev 2002) (Keir ME et al., 2008 Annu. Rev. Immunol. 26:677). Therefore, inhibition of the PD-L1/PD-1 interaction can enhance CD8 ⁺ T cell-mediated tumor killing.

It has been proposed that inhibition of PD-1 axis signaling via its direct ligands (e.g., PD-L1, PD-L2) is a means of enhancing T cell immunity to treat cancer (e.g., tumor immunity). In addition, similar enhancement of T cell immunity has been observed by inhibiting the binding of PD-L1 to its binding partner B7-1. Optimal therapeutic treatments may combine blocking of PD-1 receptor/ligand interactions with other anticancer agents. There remains a need for such optimal therapies for treating various cancers, stabilizing various cancers, preventing various cancers, and/or delaying the formation of various cancers.

All references, publications, and patent applications disclosed herein are hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention describes combination therapies comprising oxaliplatin, folinic acid and 5-FU with or without a VEGF antagonist and a PD-1 axis binding antagonist.

Provided herein are methods of treating or delaying cancer progression in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and oxaliplatin, folinic acid, and 5-FU. In some aspects, the method further comprises administering a VEGF antagonist.

The cancer can be melanoma, colorectal cancer, non-small cell lung cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, hematological malignancy or renal cell carcinoma. The cancer can be in the early stage or in the late stage. In some embodiments, the subject being treated is a human.

In some embodiments, treatment results in a response that is sustained in the individual after cessation of treatment. In some embodiments, treatment produces a complete response, a partial response, or stable disease in the subject.

In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist, a PD-L1 binding antagonist or a PD-L2 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody (e.g., antibodies MDX-1106, CT-011, and Merck 3745 described herein), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody (e.g., antibodies YW243.55.S70 and MDX-1105 described herein), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, the PD-L2 binding antagonist is an antibody, an antigen-binding fragment thereof, an immunoadhesin (except AMP-224 described herein), a fusion protein, or an oligopeptide.

In some embodiments, the VEGF antagonist is an antibody, such as a monoclonal antibody. In some embodiments, the anti-VEGF antibody binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709. The anti-VEGF antibody can be a humanized antibody or a human antibody. In some embodiments, the anti-VEGF antibody is bevacizumab. In some embodiments, the anti-VEGF antibody has a heavy chain variable region comprising the following amino acid sequence:

EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEIVVGW

INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP

HYYGSSHWYF DVWGQGTLVT VSS(SEQ ID NO: 22)

and a light chain variable region comprising the following amino acid sequence:

DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF

TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ

GTKVEIKR (SEQ ID NO: 23).

In another aspect, a kit comprising a PD-1 axis binding antagonist, oxaliplatin, folinic acid, and 5-FU, with or without a VEGF antagonist, is provided for treating cancer or delaying cancer progression in an individual or enhancing immune function in an individual having cancer. The kit may comprise a PD-1 axis binding antagonist and a package insert containing instructions for use of the PD-1 axis binding antagonist in combination with oxaliplatin, folinic acid, and 5-FU, with or without a VEGF antagonist, for treating cancer or delaying cancer progression in an individual or enhancing immune function in an individual having cancer. The kit may comprise a VEGF antagonist and a package insert containing instructions for use of the VEGF antagonist in combination with a PD-1 axis binding antagonist and oxaliplatin, folinic acid, and 5-FU, for treating cancer or delaying cancer progression in an individual or enhancing immune function in an individual having cancer. The kit may comprise a PD-1 axis binding antagonist and a VEGF antagonist, and a package insert comprising instructions for using the PD-1 axis binding antagonist and the VEGF antagonist to treat cancer or delay cancer progression in an individual or to enhance immune function in an individual having cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph depicting changes in tumor volume in the case of co-treatment with anti-PD-L1 antibody and FOLFOX. The data show significant tumor growth reduction and sustained anti-tumor effect compared to anti-PD-L1 antibody or FOLFOX treatment alone.

FIG2 is a graph showing changes in body weight of the treatment groups shown in FIG1.

Figure 3 is a graph depicting changes in tumor volume in the case of anti-PD-L1 antibody in combination with FOLFOX compared to the combination of anti-PD-L1 antibody with FOLFOX and anti-VEGF antibody. The data demonstrate that the additional administration of anti-VEGF antibody significantly reduced tumor growth and resulted in a durable anti-tumor effect compared to the combination of anti-PD-L1 antibody and FOLFOX.

FIG. 4 is a graph showing changes in body weight of the treatment groups shown in FIG. 3 .

Detailed Description of the Invention

I. General Technology

The techniques and procedures described or referred to herein are generally well understood and commonly employed by those skilled in the art and are performed using conventional methods, such as, for example, the widely utilized methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor, eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J.E. Cellis, 1998) Academic Press; Animal Cell Culture (edited by R.I. Freshney), 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A.Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); CurrentProtocols in Immunology (J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical (ed. D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (ed. P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (ed. M. Zanetti and J. D. Capra, Harwood Academic Approach) Publishers,1995); and Cancer: Principles and Practice of Oncology (V.T.DeVita et al., eds., J.B.Lippincott Company, 1993).

II. Definitions

The term "PD-1 axis binding antagonist" is a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, thereby eliminating T cell dysfunction resulting from signaling on the PD-1 signaling axis (resulting in restoration or enhancement of T cell function). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

The term "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction derived from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, eliminate or interfere with signal transduction derived from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative co-stimulatory signals, thereby making dysfunctional T cells less non-dysfunctional, the negative co-stimulatory signals being mediated by signal transduction mediated by PD-1 or by cell surface proteins expressed on T lymphocytes. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In one specific aspect, the PD-1 binding antagonist is MDX-1106 described herein. In another specific aspect, the PD-1 binding antagonist is Merck 3745 described herein. In another specific aspect, the PD-1 binding antagonist is CT-011 described herein.

The term "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction derived from the interaction of PD-L1 with one or more of its binding partners, such as PD-1, B7-1. In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonist includes anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, eliminate or interfere with signal transduction derived from the interaction of PD-L1 with one or more of its binding partners, such as PD-1, B7-1. In one embodiment, the PD-L1 binding antagonist reduces the negative co-stimulatory signal, thereby making the dysfunctional T cells less non-dysfunctional, the negative co-stimulatory signal being mediated by signal transduction mediated by or via cell surface proteins expressed on T lymphocytes via PD-L1. 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 described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1105 described herein.

The term "PD-L2 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction derived from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partners. In a specific aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, eliminate or interfere with signal transduction derived from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, a PD-L2 binding antagonist reduces negative co-stimulatory signals mediated by signaling mediated by PD-L2 or by cell surface proteins expressed on T lymphocytes, thereby making dysfunctional T cells less non-dysfunctional. In some embodiments, a PD-L2 binding antagonist is a PD-L2 immunoadhesin. In a specific aspect, the PD-L2 immunoadhesin is AMP-224 described herein.

"VEGF antagonists" refer to molecules that neutralize, block, inhibit, eliminate, reduce, or interfere with VEGF activity, including its binding to one or more VEGF receptors. VEGF antagonists include anti-VEGF antibodies and antigen-binding fragments thereof, receptor molecules and derivatives that specifically bind to VEGF, thereby isolating it from binding to one or more receptors, anti-VEGF receptor antibodies, and VEGF receptor antagonists such as small molecule inhibitors of VEGFR tyrosine kinase.

The term "VEGF" or "VEGF-A" is used to refer to the 165 amino acid human vascular endothelial growth factor and the related 121, 145, 189, and 206 amino acid human vascular endothelial growth factors, as described, for example, in Leung et al. Science, 246:1306 (1989); and Houck et al. Mol. Endocrin., 5:1806 (1991), as well as naturally occurring allelic forms and processed forms thereof. VEGF-A is part of a gene family that includes VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF. VEGF-A primarily binds to two high-affinity receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), the latter being the primary transmitter of the VEGF-A mitogenic signal in vascular endothelial cells. In addition, neuropilin-1 has been identified as a receptor for the heparin-binding VEGF-A isoform and may play a role in vascular development. The terms "VEGF" or "VEGF-A" also refer to VEGF from non-human species such as mouse, rat, or primate. VEGF from a particular species is sometimes indicated as hVEGF for human VEGF and mVEGF for murine VEGF. The term "VEGF" is also used to refer to a truncated form of the polypeptide comprising amino acids 8-109 or 1-109 of the 165-amino acid human vascular endothelial growth factor. Any of these forms of VEGF may be identified herein by, for example, "VEGF (8-109),""VEGF(1-109)," or "VEGF _165. " The amino acid positions of "truncated" native VEGF are numbered as indicated in the native VEGF sequence. For example, amino acid position 17 (methionine) in a truncated native VEGF is also position 17 (methionine) in native VEGF. The truncated native VEGF has binding affinity to KDR and Flt-1 receptors comparable to that of native VEGF.

An "anti-VEGF antibody" refers to an antibody that binds to VEGF with sufficient affinity and specificity. The selected antibody will typically have a binding affinity for VEGF; for example, the antibody may bind hVEGF with a _Kd value between 100 nM and 1 pM. Antibody affinity can be determined, for example, by surface plasmon resonance-based assays (such as the BIAcore assay described in PCT Application Publication No. WO2005/012359); enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA). In certain embodiments, the anti-VEGF antibodies of the invention can be used as therapeutic agents to target and interfere with diseases or conditions in which VEGF activity is implicated. Additionally, the antibodies can be subjected to other biological activity assays, for example, to assess their efficacy as therapeutic agents. Such assays are known in the art and depend on the target antigen and intended use of the antibody. Examples include HUVEC inhibition assays; tumor cell growth inhibition assays (as described, for example, in WO 89/06692); antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and agonist activity or hematopoietic assays (see WO 95/27062). Anti-VEGF antibodies generally do not bind to other VEGF homologs, such as VEGF-B or VEGF-C, nor to other growth factors, such as PlGF, PDGF, or bFGF.

A "chimeric VEGF receptor protein" refers to a VEGF receptor molecule having an amino acid sequence derived from at least two different proteins (at least one of which is a VEGF receptor protein). In certain embodiments, the chimeric VEGF receptor protein is capable of binding to VEGF and inhibiting the biological activity of VEGF.

"Anti-angiogenic agent" or "angiogenesis inhibitor" refers to a small molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody, or conjugate or fusion protein thereof that directly or indirectly inhibits angiogenesis, vasculogenesis, or unwanted vascular permeability. It should be understood that anti-angiogenic agents include those that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, anti-angiogenic agents are antibodies or other antagonists of angiogenic agents as defined above, such as antibodies to VEGF-A, antibodies to VEGF-A receptors (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec ^™ (Imatinib Mesylate). Anti-angiogenic agents also include natural angiogenesis inhibitors, such as angiostatin, endostatin, etc. See, e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol. 53:217-39 (1991); Streit and Detmar, Oncogene 22:3172-3179 (2003) (e.g., Table 3 listing antiangiogenic therapies in malignant melanoma); Ferrara & Alitalo, Nature Medicine 5:1359-1364 (1999); Tonini et al., Oncogene 22:6549-6556 (2003) (e.g., Table 2 listing known antiangiogenic factors); Sato, Int. J. Clin. Oncol. 8:200-206 (2003) (e.g., Table 1 listing antiangiogenic agents used in clinical trials).

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

"Enhancing T cell function" means inducing, causing or stimulating T cells to have a sustained or amplified biological function, or restoring or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include: elevated secretion of gamma interferon from CD8 ⁺ T cells, elevated proliferation, elevated antigen responsiveness (e.g., virus or pathogen clearance) relative to such levels before intervention. In one embodiment, the enhanced level is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to those of ordinary skill in the art.

A "T cell dysfunction disorder" is a T cell disorder or condition characterized by decreased responsiveness to antigenic stimulation. In a specific embodiment, a T cell dysfunction disorder is a disorder specifically associated with inappropriately elevated signaling through PD-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 a specific aspect, the decreased responsiveness results in ineffective control of pathogens or tumors expressing the immunogen. Examples of T cell dysfunction disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity.

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

"Immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity facilitates the elimination of tumor cells through an immune response. Examples of enhancing tumor immunogenicity include treatment with anti-PD-L antibodies, oxaliplatin, folinic acid, and 5-FU, with or without VEGF antagonists.

A "sustained response" refers to a persistent effect on reducing tumor growth after treatment has ceased. For example, the tumor size can remain the same or smaller than its 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 1.5 times, 2.0 times, 2.5 times, or 3.0 times the duration of treatment.

The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-chain molecules, and antibody fragments (e.g., Fab, F(ab') ₂ , and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies are composed of five basic heterotetrameric units plus an additional polypeptide called a J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2-5 basic four-chain units that, combined with J chains, can polymerize to form multivalent assemblies. In the case of IgG, the four-chain unit is typically approximately 150,000 daltons. Each light chain is linked to a heavy chain by a single covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the heavy chain isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain ( _VH ) at the N-terminus, followed by three (for α and γ chains) or four (for μ and ε isotypes) constant domains ( _CH ). Each light chain has a variable domain ( _VL ) at the N-terminus, followed by a constant domain ( _CL ) at its other end. _{The VL} is aligned with the _VH , while the _CL is aligned with the first heavy chain constant domain ( _CH1 ). Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. A _VH and a _VL pair together to form an antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th ed., Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Based on the amino acid sequence of their constant domains, L chains from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ). Based on the amino acid sequence of their heavy chain constant domain ( _CH ), immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on minor differences in C _H sequence and function; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of an antibody heavy or light chain. The heavy chain variable domain and light chain variable domain may be referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

The term "variable" refers to the fact that certain segments of the variable domain differ widely in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed across the entire span of the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions, which mostly adopt a β-pleated sheet conformation and are connected by three HVRs that form loops connecting and, in some cases, forming part of the β-pleated sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerizations, amidations) that may be present in very small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Unlike typical polyclonal antibody preparations that contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Beyond their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be generated by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004); Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and techniques for producing human or human-like antibodies in animals that have part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or a radiolabel.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably to refer to an antibody in its substantially complete form, as distinguished from an antibody fragment. Specifically, intact antibodies include those having heavy and light chains (including an Fc region). The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding and/or variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies (see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, the name reflecting its ability to crystallize readily. The Fab fragment consists of an intact L chain and H chain variable region ( _VH ) and the first constant domain ( _CH1 ) of the heavy chain. Each Fab fragment is monovalent for antigen binding, i.e., it has one antigen-binding site. Pepsin treatment of an antibody produces a larger F(ab') ₂ fragment, which is roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen-binding activities and still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the _CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residues of the constant domains bear a free thiol group. F(ab') ₂ antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment contains the carboxyl-terminal portions of two H chains held together by disulfide bonds. The effector functions of an antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. From the folding of these two domains arise six hypervariable loops (three loops each from the heavy and light chains) that contribute the amino acid residues required for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

"Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising _VH and _VL antibody domains linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315, 1994.

The "functional fragment" of an antibody of the present invention comprises a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or an Fc region of an antibody that retains or has improved FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The term "diabody" refers to small antibody fragments prepared by constructing sFv fragments (see the previous paragraph) using a short linker (about 5-10 residues) between the _VH and _VL domains. Due to the short linker, the V domains are paired between chains rather than within chains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the _VH and _VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

Monoclonal antibodies are specifically included herein as "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies in which the antigen-binding region of the antibody is derived from an antibody generated by immunizing a macaque monkey with, for example, an antigen of interest. As used herein, "humanized antibodies" are a subset of "chimeric antibodies".

"Humanized" forms of non-human (e.g., mouse) antibodies refer to chimeric antibodies that minimally comprise sequences derived from non-human immunoglobulins. In one embodiment, a humanized antibody refers to an immunoglobulin in which the HVR residues (defined below) in a human immunoglobulin (recipient antibody) are replaced with HVR residues from a non-human species (donor antibody) (such as mouse, rat, rabbit, or non-human primate) with desired specificity, affinity, and/or ability. In some cases, the framework ("FR") residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may be included in residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further improve the performance of the antibody, such as binding affinity. In general, a humanized antibody will comprise at least one, typically two, substantially entire variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may contain one or more individual FR residue substitutions that improve antibody performance (such as binding affinity, isomerization, immunogenicity, etc.). The number of these amino acid substitutions in the FRs is generally no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see, for example, Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

"Human antibody" refers to an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or is produced using any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991); Marks et al., J. Mol. Biol. 222: 581 (1991)). Also useful for preparing human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol. 147 (1): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal, such as an immunized xenogeneic mouse, that has been modified to produce human antibodies in response to antigenic stimulation but whose endogenous loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE ^™ technology). See also, e.g., Li et al., Proc. Natl. Ascad. Sci. USA, 103:3557-3562 (2006), for human antibodies generated via human B-cell hybridoma technology.

The terms "hypervariable region", "HVR" or "HV" as used herein refer to regions in an antibody variable domain that are highly variable in sequence and/or form structurally defined loops. Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 display the greatest diversity of the six HVRs, and it is believed that H3 in particular plays a unique role in conferring precise specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in: Methods in Molecular Biology 248:1-25 (Lo ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

Many HVR descriptions are used and covered herein. The Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead to the positions of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on analysis of available complex crystal structures. The residues in each of these HVRs are recorded below.

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

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

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

A "human consensus framework" or "acceptor human framework" refers to a framework that represents the most common amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the sequence subset is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest , 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for _VL , the subgroup can be subgroup κI, κII, κIII, or κIV, as in Kabat et al., supra. Additionally, for VH, the subgroup can be subgroup I, subgroup II, or subgroup III, as in Kabat et al., supra. Alternatively, a human consensus framework can be derived from the above frameworks, wherein specific residues, such as human framework residues, are selected based on their homology to the donor framework by aligning the donor framework sequence with a collection of various human framework sequences. An acceptor human framework or human consensus framework "derived" from a human immunoglobulin framework can comprise the same amino acid sequence thereof, or it can contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

A "VH subgroup III consensus framework" comprises a consensus sequence obtained from the amino acid sequences in variable heavy chain subgroup III of Kabat et al., supra. In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences:

EVQLVESGGGLVQPGGSLRLSCAAS(HC-FR1)(SEQ ID NO:30),

WVRQAPGKGLEWV(HC-FR2)(SEQ ID NO:31),

RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(HC-FR3)(SEQ ID NO:32),

WGQGTLVTVSA(HC-FR4) (SEQ ID NO:33).

The "VLκI consensus framework" comprises a consensus sequence obtained from the amino acid sequences in variable light chain kappa subgroup I of Kabat et al., supra. In one embodiment, the VL subgroup I consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences:

DIQMTQSPSSSLSASVGDRVTITC(LC-FR1)(SEQ ID NO:34),

WYQQKPGKAPKLLIY(LC-FR2)(SEQ ID NO:35)、

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(LC-FR3)(SEQ ID NO:36),

FGQGTKVEIKR(LC-FR4) (SEQ ID NO:37).

"Amino acid modification" at a designated position (e.g., an Fc region) refers to the replacement or deletion of a designated residue, or the insertion of at least one amino acid residue near a designated residue. Insertion "near" a designated residue means insertion within one to two residues thereof. Insertion can be at the N-terminus or C-terminus of a designated residue. Preferred amino acid modifications herein are substitutions.

An "affinity matured" antibody is one that has one or more alterations in one or more HVRs that result in an improvement in the affinity of the antibody for the antigen compared to a parent antibody that does not have those alterations. In one embodiment, affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies can be generated by procedures known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and/or framework residues is described in, for example, Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

As used herein, the term "specific binding" or "specific for..." refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules (including biological molecules). For example, an antibody that specifically binds to a target (which may be an epitope) is one that binds to the target with greater affinity, avidity, more readily, and/or for a greater duration than it binds to other targets. In one embodiment, the extent to which the antibody binds to an unrelated target is less than about 10% of the extent to which the antibody binds to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that specifically binds to the target has a dissociation constant (Kd) of ≤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 between proteins from different species. In another embodiment, specific binding can include but does not require exclusive binding.

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 function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence having a desired binding specificity different from the antigen recognition and binding site of an antibody (i.e., "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 IgG-1, IgG-2 (including IgG2A and IgG2B), IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. Preferably, the Ig fusion comprises a substitution of at least one variable region in the Ig molecule with a domain of a polypeptide or antibody as described herein. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2, and CH3, or the hinge, CH1, CH2, and CH3 regions, of an IgG1 molecule. See also U.S. Patent No. 5,428,130, issued June 27, 1995, for the generation of immunoglobulin fusions. For example, useful immunoadhesins as second agents for use in combination therapies herein include polypeptides comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2, or an extracellular or PD-L1 or PD-L2 binding portion of PD-1, fused to a constant domain of an immunoglobulin sequence, such as PD-L1 ECD–Fc, PD-L2 ECD–Fc, and PD-1 ECD-Fc, respectively. Immunoadhesin combinations of IgFc and cell surface receptor ECD are sometimes referred to as soluble receptors.

"Fusion protein" and "fusion polypeptide" refer to a polypeptide having two covalently linked parts, 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 linked via a peptide linker, but in-frame with each other.

"PD-1 oligopeptide", "PD-L1 oligopeptide", or "PD-L2 oligopeptide" is an oligopeptide that binds (preferably specifically binds) to a PD-1, PD-L1 or PD-L2 negative co-stimulatory polypeptide, respectively, including a receptor, ligand or signaling component, respectively, as described herein. Such oligopeptides can be chemically synthesized using known oligopeptide synthesis techniques or can be prepared and purified using recombinant technology. Such oligopeptides are typically at least about 5 amino acids in length, or at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 98, 99, or 100 amino acids or more. Such oligopeptides can be identified using well-known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO 84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides asAntigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102: 259-274 (1987); Schoofs et al., J. Immunol., 140: 611-616 (1988), Cwirla, S.E. et al., Proc. Natl. Acad. Sci. USA, 87: 6378 (1990); Lowman, H.B. et al. , Biochemistry, 30: 10832 (1991); Clackson, T. et al., Nature, 352: 624 (1991); Marks, J.D. et al., J. Mol. Biol., 222: 581 (1991); Kang, A.S. et al., Proc. Natl. Acad. Sci. USA, 88: 8363 (1991), and Smith, G.P., Current Opin. Biotechnol., 2: 668 (1991).

A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. For example, a VEGF-specific antagonist antibody binds to VEGF and inhibits VEGF's ability to induce vascular endothelial cell proliferation or induce vascular permeability. The anti-PD-L1 antibodies of the present invention block signaling through PD-1, thereby restoring the functional response of T cells to antigen stimulation from a dysfunctional state.

An "agonist" or activating antibody is an antibody that enhances or initiates signaling through the antigen to which it binds. In some embodiments, an agonist antibody causes or activates signaling in the absence of a natural ligand.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is generally defined as the segment from the amino acid residue at its Cys226 or Pro230 position to the carboxyl terminus. The C-terminal lysine (residue 447, according to the EU numbering system) in the Fc region can be eliminated, for example, during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, the composition of the intact antibody can include antibody groups in which all K447 residues are eliminated, antibody groups in which no K447 residue is eliminated, and antibody groups with and without K447 residues mixed. Suitable native sequence Fc regions for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is an FcR (gamma receptor) that binds to an IgG antibody, including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see M.Annu.Rev.Immunol.15:203-234 (1997)). For review of FcRs, see Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses additional FcRs, including those identified in the future.

The term "Fc receptor" or "FcR" also includes the neonatal 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)). Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol Today 18(12):592-8 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-40 (1997); Hinton et al., J. Biol. Chem 279(8):6213-6 (2004); and WO2004/92219 (Hinton et al.)). The in vivo binding and serum half-life of high-affinity human FcRn binding polypeptides can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with variant Fc regions. WO 2000/42072 (Presta) describes antibody variants with improved or abolished FcR binding. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

The phrase "substantially reduced" or "substantially different" as used herein refers to a sufficiently high degree of difference between two values (typically one associated with a molecule and the other associated with a reference/comparator molecule) that one skilled in the art would consider the difference between the two values to be statistically significant within the context of the biological property being measured by the value (e.g., Kd value). For example, the difference between the two values is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value of the reference/comparator molecule.

The phrase "substantially similar" or "substantially identical" as used herein refers to a sufficiently high degree of similarity between two values (e.g., one relating to an antibody of the invention and the other relating to a reference/comparator antibody) 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 within the context of the biological property measured by the value (e.g., Kd value). The difference between the two values as a function of the reference/comparator value 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%.

"Carrier" as used herein includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cells or mammals to which they are exposed at the dosages and concentrations employed. Typically, a physiologically acceptable carrier is a pH-buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN ^™ , polyethylene glycol (PEG), and PLURONICS ^™ .

"Package insert" refers to instructions customarily included in commercial packages of drugs, that contain information about the indications, usage, dosage, administration, contraindications, other drugs used in combination with the packaged product and/or warnings concerning the use of such drug.

As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the clinical pathological process. Desired effects of treatment include a reduction in the rate of disease progression, an improvement or alleviation of the disease state, and a regression or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are alleviated or eliminated, including but not limited to a reduction in the proliferation (or destruction) of cancerous cells, a reduction in symptoms from the disease, an improvement in the quality of life of those suffering from the disease, a reduction in the dosage of other drugs needed to treat the disease, a delay in the progression of the disease, and/or a prolonged survival of the individual.

As used herein, "delaying the progression of a disease" means postponing, hindering, slowing down, delaying, stabilizing, and/or slowing the development of a disease (such as cancer). Depending on the individual's medical history and/or treatment, this delay can be of varying lengths of time. As will be apparent to one skilled in the art, sufficient or significant delay can substantially encompass prevention, as the individual does not develop the disease. For example, the development of advanced stage cancers, such as metastases, can be delayed.

"Effective amount" is at least the minimum concentration required to achieve a measurable improvement or prevention of a particular condition. The effective amount herein can vary with factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit a desired response in an individual. An effective amount is also the amount at which the therapeutic beneficial effect exceeds any toxicity or adverse effect of the treatment. For preventive use, the beneficial or desired result includes the following result, such as eliminating or reducing risk, alleviating severity, or delaying the onset of the disease, including the biochemistry of the disease, histology and/or behavioral symptoms, the intermediate pathological phenotype presented during its complication and disease formation. For therapeutic use, the beneficial or desired result includes clinical results, such as reducing one or more symptoms from the disease, improving the quality of life of those subjects suffering from the disease, reducing the dosage of other drugs needed for treating the disease, enhancing the effect of another drug (such as via targeting), delaying the progression of the disease, and/or prolonging survival. In the case of cancer or tumors, the effective amount of the drug can have an effect in reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., slowing down or desirably stopping) cancer cell infiltration into peripheral organs; inhibiting (i.e., slowing down and desirably stopping) tumor metastasis; inhibiting tumor growth to a certain extent; and/or alleviating one or more symptoms associated with the disease to a certain extent. The effective amount can be administered in one or more administrations. For the purposes of the present invention, the effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a preventive or therapeutic treatment. As understood in the clinical context, the effective amount of a drug, compound, or pharmaceutical composition can be achieved with or without another drug, compound, or pharmaceutical composition. Thus, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and if, together with one or more other agents, the desired result can be achieved or the desired result is achieved, then a single agent can be considered to be given in an effective amount.

As used herein, "in conjunction with" refers to administering a therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" refers to administering another therapeutic modality before, during, or after administering one therapeutic modality to a subject.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included within this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (hepatic carcinoma), bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer (kidney or renal cancer), prostate cancer, vulvar cancer, thyroid cancer, and various types of head and neck cancer, as well as B-cell lymphomas (including low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky disease), NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

"Metastasis" refers to the spread of cancer from its primary site to other locations in the body. Cancer cells can break away from the primary tumor, infiltrate into lymph and blood vessels, circulate via the bloodstream, and grow in distant lesions (metastasis) in normal tissues elsewhere in the body. Metastasis can be local or distal. Metastasis is a continuous process that depends on whether tumor cells fall off from the primary tumor, spread via the bloodstream, and stop at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Stimulating and inhibitory molecular pathways within the tumor cells regulate this behavior, and the interaction between the tumor cells and the host cells in the distant site is also important.

"Subject" refers to a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or cat. Preferably, the subject refers to a human. In this article, a patient is also a subject.

As used herein, "complete response" or "CR" refers to the disappearance of all target lesions; "partial response" or "PR" refers to a decrease of at least 30% in the sum of the longest diameters of target lesions (SLD), taking the baseline SLD as a reference; and "stable disease" or "SD" refers to neither sufficient shrinkage of target lesions to qualify as a PR nor sufficient increase to qualify as a PD, taking the minimum SLD since the start of treatment as a reference.

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

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, and the amount of time a patient has experienced stable disease.

As used herein, "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

As used herein, "overall survival" refers to the percentage of individuals in a group who are likely to survive after a specified duration.

"Chemotherapeutic agent" refers to a chemical compound that can be 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; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphamide, and dapoxetine; phosphoramide), triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including its synthetic analogs topotecan CPT-11 (irinotecan), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; pemetrexed; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral alpha-4 integrin inhibitor; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, and chlorambucil. hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediynes (e.g., calicheamicins, especially calicheamicin gamma and calicheamicin omega (see, e.g., Nicolaou et al., Angew, Chem. Intl. Ed. Engl. 33:183-186 (1994)); anthracyclines (dynemicins), including dynemicins A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine amino acid, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolidoxorubicin, doxorubicin hydrochloride liposome injection (and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin) , streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine, tegafur, capecitabine, epothilone; 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, and imatinib (a 2-phenylaminopyrimidine derivative), as well as other c-Kit inhibitors; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, 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 acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");thiotepa; taxoids, such as paclitaxel; albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANE ^™) . ), and doxetaxel; chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (platinum); etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (oxaliplatin); leucovovin; vinorelbine (novantrone); edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS. 2000; difluoromethylornithine (DMFO); retinoids, such as tretinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP (an abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy) and FOLFOX (an abbreviation for the treatment regimen of oxaliplatin (ELOXATIN ^™ ) combined with 5-FU and folinic acid).

This definition also includes antihormonal agents that act to modulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or whole-body treatments. They can themselves be hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; antiprogestins; estrogen receptor downregulators (ERDs); estrogen receptor antagonists, such as fulvestrant; agents that function to suppress or shut down the ovaries, such as luteinizing hormone-releasing hormone (LHRH) agonists, such as leuprolide acetate (and leuprolide), goserelin acetate, buserelin acetate, and leuprolide. acetate) and triptorelin; antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole. In addition, this definition of chemotherapeutic agents includes bisphosphonates such as clodronate (e.g., or clodronate), etidronate NE-58095, zoledronic acid/zoledronic acid. acid/zoledronate) alendronate, pamidronate, tiludronate, or risedronate, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly antisense oligonucleotides that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as vaccines and gene therapy vaccines , such as vaccines, vaccines and vaccines; topoisomerase 1 inhibitors (for example); antiestrogens, such as fulvestrant; Kit inhibitors, such as imatinib or EXEL-0862 (a tyrosine kinase inhibitor); EGFR inhibitors, such as erlotinib or cetuximab; anti-VEGF inhibitors, such as bevacizumab; irinotecan; rmRH (for example); lapatinib and lapatinib ditosylate (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases, also known as GW572016); 17AAG (geldanamycin derivative, which is a heat shock protein (Hsp) 90 poison); and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

As used herein, the term "cytokine" generally refers to a protein released by one cell population that acts as an intercellular mediator on another cell or has an autocrine effect on the cell that produces the protein. Examples of such cytokines include lymphokines, monokines; interleukins ("IL"), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including rIL-2; tumor necrosis factors, such as TNF-α or TNF-β, TGF-β1-3; and other polypeptide factors, including leukemia inhibitory factor ("LIF"), ciliary ganglion neurotrophic factor ("CNTF"), CNTF-like cytokine ("CLC"), cardiotrophin ("CT"), and kit ligand ("KL").

As used herein, the term "chemokine" refers to a soluble factor (e.g., cytokine) that has the ability to selectively induce chemotaxis and activation of leukocytes. They also trigger processes such as angiogenesis, inflammation, wound healing, and tumorigenesis. Examples of chemokines include IL-8, the human homolog of mouse keratinocyte chemoattractant (KC).

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Reference herein to "about" a value or parameter includes (and describes) variations involving the value or parameter itself. For example, a description referring to "about X" includes a description of "X."

As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the invention. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, sugarates, formates, benzoates, glutamates, methanesulfonates (mesylate), ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthoic acid)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as acetate ions, succinate ions, or other counterions. The counterion can be any organic or inorganic module that stabilizes the charge of the parent compound. Additionally, pharmaceutically acceptable salts may have more than one charged atom in their structure. In the case where multiple charged atoms are components of a pharmaceutically acceptable salt, multiple counterions may be present. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

If the compound of the present invention is a base, the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, by treating the free base with an inorganic acid (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, and the like) or with an organic acid (such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid/fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid (such as glucuronic acid or galacturonic acid), an alpha hydroxy acid (such as citric acid or tartaric acid), an amino acid (such as aspartic acid or glutamic acid), an aromatic acid (such as benzoic acid or cinnamic acid), a sulfonic acid (such as p-toluenesulfonic acid or ethanesulfonic acid), and the like.

If the compound of the present invention is an acid, the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or an alkaline earth metal hydroxide, etc. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids (such as glycine and arginine), ammonium, primary/secondary/tertiary amines, and cyclic amines (such as piperidine, morpholine and piperazine), and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and/or toxicologically with the other ingredients comprising the formulation and/or the mammal to be treated therewith.

It should be understood that aspects and variations of the invention described herein include "consisting of" and/or consisting essentially of the aspects and variations.

III. Methods

The methods of the present invention may find application in treating conditions where enhanced immunogenicity is desired, such as increasing tumor immunogenicity to treat cancer.A variety of cancers may be treated, or their progression may be delayed.

In some embodiments, the individual has melanoma. The melanoma can be in the early stage or in the late stage. In some embodiments, the individual has colorectal cancer. The colorectal cancer can be in the early stage or in the late stage. In some embodiments, the individual has non-small cell lung cancer. The non-small cell lung cancer can be in the early stage or in the late stage. In some embodiments, the individual has pancreatic cancer. The pancreatic cancer can be in the early stage or in the late stage. In some embodiments, the individual has a hematologic malignancy. The hematologic malignancy can be in the early stage or in the late stage. In some embodiments, the individual has ovarian cancer. The ovarian cancer can be in the early stage or in the late stage. In some embodiments, the individual has breast cancer. The breast cancer can be in the early stage or in the late stage. In some embodiments, the individual has renal cell carcinoma. The renal cell carcinoma can be in the early stage or in the late stage.

In some embodiments, the subject treated is a human.

The combination therapy of the present invention comprises administering a PD-1 axis binding antagonist and oxaliplatin, folinic acid, and 5-FU. In another aspect, the present invention provides a combination therapy comprising administering a PD-1 axis binding antagonist, a VEGF antagonist, and oxaliplatin, folinic acid, and 5-FU. The PD-1 axis binding antagonist and the VEGF antagonist can be administered in any suitable manner known in the art. For example, the PD-1 axis binding antagonist and the VEGF antagonist can be administered sequentially (at different times) or simultaneously (at the same time).

In some embodiments, the method of the present invention may further include administering another therapy. Another therapy may be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. Another therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, another therapy is the administration of a small molecule enzymatic inhibitor or an anti-metastatic agent. In some embodiments, another therapy is the administration of a side effect limiter (e.g., an agent intended to reduce the occurrence and/or severity of treatment side effects, such as an anti-nausea agent, etc.). In some embodiments, another therapy is radiotherapy. In some embodiments, another therapy is surgery. In some embodiments, another therapy is a combination of radiotherapy and surgery. Another therapy may be one or more chemotherapeutic agents described above.

Any of the PD-1 axis binding antagonists and VEGF antagonists described below can be used in the methods of the invention.

PD-1 axis binding antagonists

Provided herein are methods for treating cancer or delaying cancer progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist in combination with oxaliplatin, folinic acid, and 5-FU, with or without administration of a VEGF antagonist. For example, the PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.

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 partner. 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 binding partner. In a specific aspect, the PD-L2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

In some embodiments, the PD-1 binding antagonist is selected from the group consisting of MDX-1106, Merck 3475, and CT-011. In some embodiments, the PD-1 binding antagonist is selected from the group consisting of YW243.55.S70 and MDX-1105. In some embodiments, the PD-L2 binding antagonist is AMP-224. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007/005874. Antibody YW243.55.S70 (SEQ ID No. 20) is an anti-PD-L1 antibody described in WO2010/077634A1. MDX-1106, also known as MDX-1106-04, ONO-4538, or BMS-936558, is an anti-PD-1 antibody described in WO2006/121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a soluble PD-L2-Fc fusion receptor described in WO2010/027827 and WO2011/066342.

Examples of anti-PD-L1 antibodies and methods for their production that can be used in the methods of the invention are described in PCT patent application WO 2010/077634 A1, which is incorporated herein by reference.

In some embodiments, the PD-1 axis 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.

Anti-PD-L1 antibodies useful in the present invention (including compositions containing such antibodies), such as those described in WO2010/077634A1, can be used in combination with oxaliplatin, folinic acid, or 5-FU, with or without a VEGF antagonist, to treat cancer.

In one embodiment, the anti-PD-L1 antibody comprises a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

(a) HVR-H1 sequence is GFTFSX ₁ SWIH (SEQ ID NO: 1);

(b) HVR-H2 sequence is AWIX ₂ PYGGSX ₃ YYADSVKG (SEQ ID NO: 2);

(c) HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 3);

Further wherein: _X1 is D or G; _X2 is S or L; _X3 is T or S.

In a specific aspect, _X1 is D; _X2 is S and _X3 is T. In another aspect, the polypeptide further comprises a variable region heavy chain framework sequence juxtaposed between the HVRs according to the following formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In another aspect, the framework sequence is a VH subgroup III consensus framework. In yet another aspect, at least one framework sequence is as follows:

HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 5)

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:6)

HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 7).

In yet another aspect, the heavy chain polypeptide is further combined with a variable region light chain comprising HVR-L1, HVR-L2 and HVR-L3, wherein:

(a) HVR-L1 sequence is RASQX ₄ X ₅ X ₆ TX ₇ X ₈ A (SEQ ID NO: 8);

(b) HVR-L2 sequence is SASX ₉ LX ₁₀ S (SEQ ID NO: 9);

(c) HVR-L3 sequence is QQX ₁₁ X ₁₂ X ₁₃ X ₁₄ PX ₁₅ T (SEQ ID NO: 10);

Further wherein: _X4 is D or V; _X5 is V or I; _X6 is S or N; _X7 is A or F; _X8 is V or L; _X9 is F or T; _X10 is Y or A; _X11 is Y, G, F, or S; _X12 is L, Y, F, or W; _X13 is Y, N, A, T, G, F, or I; _X14 is H, V, P, T, or I; _X15 is A, W, R, P, or T.

In yet another aspect, _X4 is D; _X5 is V; _X6 is S; _X7 is A; X8 is V; _X9 is F; _X10 is Y; _X11 is Y; _X12 is L; _X13 is Y; _X14 is H; _X15 is A. In yet another aspect, the light chain further comprises a variable region light chain framework sequence juxtaposed between the HVRs according to the following formula: (LC-FR1)-(HVR-L1)-(LC- _FR2 )-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the framework sequence is a VLκI consensus framework. In yet another aspect, at least one framework sequence is as follows:

LC-FR1 is DIQMTQSPSSLSSASVGDRVTITC(SEQ ID NO:11)

LC-FR2 is WYQQKPGKAPKLLIY(SEQ ID NO:12)

LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)

LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).

In another embodiment, an isolated anti-PD-L1 antibody or antigen-binding fragment is provided, comprising heavy and light chain variable region sequences, wherein:

(a) The heavy chain comprises HVR-H1, HVR-H2 and HVR-H3, wherein further:

(i) HVR-H1 sequence is GFTFSX ₁ SWIH (SEQ ID NO: 1),

(ii) HVR-H2 sequence is AWIX ₂ PYGGSX ₃ YYADSVKG (SEQ ID NO: 2)

(iii) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 3), and

(b) a light chain comprising HVR-L1, HVR-L2 and HVR-L3, wherein further:

(i) HVR-L1 sequence is RASQX ₄ X ₅ X ₆ TX ₇ X ₈ A (SEQ ID NO: 8),

(ii) the HVR-L2 sequence is SASX ₉ LX ₁₀ S (SEQ ID NO: 9), and

(iii) HVR-L3 sequence is QQX ₁₁ X ₁₂ X ₁₃ X ₁₄ PX ₁₅ T (SEQ ID NO: 10)

Further wherein: _X1 is D or G; _X2 is S or L; _X3 is T or S; _X4 is D or V; _X5 is V or I; _X6 is S or N; _X7 is A or F; _X8 is V or L; _X9 is F or T; _X10 is Y or A; _X11 is Y, G, F, or S; _X12 is L, Y, F, or W; _X13 is Y, N, A, T, G, F, or I; _X14 is H, V, P, T, or I; _X15 is A, W, R, P, or T.

In one specific aspect, _X1 is D; _X2 is S and _X3 is T. In another aspect, _X4 is D; _X5 is V; _X6 is S; _X7 is A; X8 is V; _X9 is F; _X10 is Y; _X11 is Y; _X12 is L; _X13 is _{Y; X14 is} H; _X15 is A. In yet another aspect, _X1 is D; _X2 is S and X3 is T, _X4 is D; _X5 is V; _X6 is S; _X7 is A; _X8 is V; _X9 is F; _X10 is Y; _X11 is Y; _X12 is L; _X13 is Y; _X14 is H and ^X15 _is A.

In other aspects, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another aspect, the heavy chain framework sequences are VH subgroup III consensus frameworks. In yet another aspect, the one or more heavy chain framework sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)

HC-FR4 WGQGTLVTVSA (SEQ ID NO:7).

In yet another aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more light chain framework sequences are as follows:

LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:11)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)

LC-FR4 FGQGTKVEIKR (SEQ ID NO: 14).

In another specific aspect, the antibody further comprises a human or mouse constant region. In another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In another specific aspect, the human constant region is IgG1. In another aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In another aspect, the mouse constant region is IgG2A. In another specific aspect, the antibody has reduced or minimal effector function. In another specific aspect, minimal effector function is derived from an "effector-less Fc mutation" or aglycosylation. In another embodiment, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, an anti-PD-L1 antibody is provided, comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain further comprises HVR-H1, HVR-H2 and HVR-H3 sequences that have at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 15), AWISPYGGSTYYADSVKG (SEQ ID NO: 16) and RHWPGGFDY (SEQ ID NO: 3), respectively, or

(b) the light chain further comprises HVR-L1, HVR-L2 and HVR-L3 sequences that have at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 17), SASFLYS (SEQ ID NO: 18) and QQYLYHPAT (SEQ ID NO: 19), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the following HVRs: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the following HVRs: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, the one or more heavy chain framework sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)

HC-FR4 WGQGTLVTVSA (SEQ ID NO:7).

In yet another aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more light chain framework sequences are as follows:

LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:11)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)

LC-FR4FGQGTKVEIKR (SEQ ID NO:14).

In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function results from an "effector-minor Fc mutation" or aglycosylation. In yet another embodiment, the effector-minor Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, an isolated anti-PD-L1 antibody is provided, comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity with the following heavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWIS

PYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFD

YWGQGTLVTVSA(SEQ ID NO:20),

or

(b) the light chain sequence has at least 85% sequence identity with the following light chain sequence:

DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF

LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR

(SEQ ID NO: 21).

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the following HVRs: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the following HVRs: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, the one or more heavy chain framework sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)

HC-FR4 WGQGTLVTVSA (SEQ ID NO:7).

In yet another aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more light chain framework sequences are as follows:

LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:11)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)

LC-FR4 FGQGTKVEIKR (SEQ ID NO: 14).

In yet another specific aspect, the antibody further comprises a human or mouse constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the mouse constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function is derived from production in prokaryotic cells. In yet another specific aspect, minimal effector function is derived from an "effect-minor Fc mutation" or aglycosylation. In yet another embodiment, the effect-minor Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, the present invention provides a composition comprising any one of the anti-PD-L1 antibodies described above in combination with at least one pharmaceutically acceptable carrier.

In yet another embodiment, an isolated nucleic acid is provided that encodes a light chain or heavy chain variable region sequence of an anti-PD-L1 antibody, wherein:

(a) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences that have at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 15), AWISPYGGSTYYADSVKG (SEQ ID NO: 16), and RHWPGGFDY (SEQ ID NO: 3), respectively, and

(b) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences that have at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 17), SASFLYS (SEQ ID NO: 18), and QQYLYHPAT (SEQ ID NO: 19), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In various aspects, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In other aspects, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, the one or more heavy chain framework sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:5)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:6)

HC-FR4 WGQGTLVTVSA (SEQ ID NO:7).

In yet another aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more light chain framework sequences are as follows:

LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:11)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:12)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:13)

LC-FR4 FGQGTKVEIKR (SEQ ID NO: 14).

In yet another specific aspect, the antibody further comprises a human or mouse constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the mouse constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function is derived from production in prokaryotic cells. In yet another specific aspect, minimal effector function is derived from an "effect-minor Fc mutation" or aglycosylation. In yet another embodiment, the effect-minor Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another aspect, the nucleic acid further comprises a vector suitable for expressing the nucleic acid encoding any of the anti-PD-L1 antibodies described previously. In yet another specific aspect, the vector further comprises a host cell suitable for expressing the nucleic acid. 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.

Anti-PD-L1 antibodies or antigen-binding fragments thereof can be produced using methods known in the art, for example, by a method comprising culturing a host cell containing a nucleic acid encoding any of the previously described 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.

In yet another embodiment, the present invention provides a composition comprising an anti-PD-L1 antibody or antigen-binding fragment thereof as provided herein and at least one pharmaceutically acceptable carrier.

VEGF antagonists

The present invention provides a method for treating cancer or slowing the progression of cancer in an individual comprising administering an effective amount of a PD-1 pathway antagonist and a VEGF antagonist in combination with oxaliplatin, folinic acid, and 5-FU. Any known VEGF antagonist is contemplated.

(i) VEGF antigen

The VEGF antigen to be used to generate antibodies can be, for example, the VEGF ₁₆₅ molecule as well as other isoforms of VEGF or fragments thereof containing the desired epitope. Other forms of VEGF that can be used to generate the anti-VEGF antibodies of the invention will be apparent to those skilled in the art.

Human VEGF was first obtained by screening a cDNA library prepared from human cells using bovine VEGF cDNA as a hybridization probe. Leung et al. (1989) Science, 246:1306. One cDNA thus identified encodes a 165-amino acid protein with greater than 95% homology to bovine VEGF; this 165-amino acid protein is often referred to as human VEGF (hVEGF) or _VEGF165 . The mitogenic activity of human VEGF was confirmed by expressing the human VEGF cDNA in mammalian host cells. Culture medium conditioned by cells transfected with the human VEGF cDNA promoted the proliferation of capillary endothelial cells, whereas control cells did not. Leung et al. (1989) Science, supra.

Although vascular endothelial growth factor can be isolated and purified from natural sources and subsequently used for therapeutic purposes, the relatively low concentration of the protein in follicular cells and the high cost, both in terms of effort and expense, required to recover VEGF have proven commercially unprofitable. Consequently, further efforts were made to clone and express VEGF via recombinant DNA technology. (See, e.g., Ferrara, Laboratory Investigation 72:615-618 (1995) and references cited therein).

Originating from alternative RNA splicing, VEGF is expressed in various tissues as a variety of homodimeric forms (121, 145, 165, 189, and 206 amino acids per monomer). VEGF ₁₂₁ is a soluble mitogen that does not bind heparin; longer forms of VEGF bind heparin with increasing affinity. The heparin-bound form of VEGF can be cleaved at the carboxyl terminus by plasmin to release a diffusible form of VEGF. The amino acid sequence of the carboxyl-terminal peptide identified after plasmin cleavage is Arg ₁₁₀ -Ala _111. VEGF (1-110), the amino-terminal "core" protein isolated as a homodimer, binds to neutralizing monoclonal antibodies (such as those designated 4.6.1 and 3.2E3.1.1) and soluble forms of the VEGF receptor with similar affinity compared to the intact VEGF ₁₆₅ homodimer.

Several molecules structurally related to VEGF have also been identified, including placental growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D, and VEGF-E. Ferrara and Davis-Smyth (1987) Endocr. Rev., supra; Ogawa et al. J. Biological Chem. 273:31273-31281 (1998); Meyer et al. EMBO J., 18:363-374 (1999). The receptor tyrosine kinase Flt-4 (VEGFR-3) has been identified as a receptor for VEGF-C and VEGF-D. Joukov et al. EMBO. J. 15: 1751 (1996); Lee et al. Proc. Natl. Acad. Sci. USA 93: 1988-1992 (1996); Achen et al. (1998) Proc. Natl. Acad. Sci. USA 95: 548-553. VEGF-C has been shown to be involved in the regulation of lymphangiogenesis. Jeltsch et al. Science 276: 1423-1425 (1997).

Two VEGF receptors have been identified, Flt-1 (also known as VEGFR-1) and KDR (also known as VEGFR-2). Shibuya et al. (1990) Oncogene 8:519-527; de Vries et al. (1992) Science 255:989-991; Terman et al. (1992) Biochem. Biophys. Res. Commun. 187:1579-1586. Neuropilin-1 has been shown to be a selective VEGF receptor, capable of binding heparin-binding VEGF isoforms (Soker et al. (1998) Cell 92:735-45). Both Flt-1 and KDR belong to the receptor tyrosine kinase (RTK) family. RTKs comprise a large family of transmembrane receptors with diverse biological activities. Currently, at least nineteen (19) unique RTK subfamilies have been identified. The receptor tyrosine kinase (RTK) family includes receptors that are crucial for the growth and differentiation of a variety of cell types (Yarden and Ullrich (1988) Ann. Rev. Biochem. 57: 433-478; Ullrich and Schlessinger (1990) Cell 61: 243-254). The intrinsic function of RTKs is activated upon ligand binding, leading to phosphorylation of the receptor and various cellular substrates, which in turn leads to a variety of cellular responses (Ullrich and Schlessinger (1990) Cell 61: 203-212). Thus, receptor tyrosine kinase-mediated signal transduction is initiated by extracellular interaction with specific growth factors (ligands), which is generally followed by receptor dimerization, stimulation of intrinsic protein tyrosine kinase activity, and receptor transphosphorylation. This creates a binding site for intracellular signal transduction molecules and leads to the formation of complexes with a range of cytoplasmic signaling molecules, driving appropriate cellular responses. (e.g., cell division, differentiation, metabolic effects, changes in the extracellular microenvironment) See Schlessinger and Ullrich (1992) Neuron 9:1-20. Structurally, both Flt-1 and KDR have seven immunoglobulin-like domains in the extracellular domain, a single transmembrane region, and a consensus tyrosine kinase sequence interrupted by a kinase insert domain. Matthews et al. (1991) Proc. Natl. Acad. Sci. USA 88:9026-9030; Terman et al. (1991) Oncogene 6:1677-1683.

(ii) Anti-VEGF antibodies

Anti-VEGF antibodies useful in the methods of the present invention include any antibody or antigen-binding fragment thereof that binds to VEGF with sufficient affinity and specificity and is capable of reducing or inhibiting the biological activity of VEGF. Anti-VEGF antibodies generally do not bind to other VEGF homologs, such as VEGF-B or VEGF-C, or other growth factors, such as PlGF, PDGF, or bFGF.

In certain embodiments of the present invention, anti-VEGF antibodies include, but are not limited to, monoclonal antibodies that bind to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB10709; the recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al. (1997) Cancer Res. 57:4593-4599. In one embodiment, the anti-VEGF antibody is "bevacizumab (BV)", also known as "rhuMAb VEGF" or "bevacizumab." It comprises mutated human IgG1 framework regions and antigen-binding complementarity-determining regions from mouse anti-hVEGF monoclonal antibody A.4.6.1, blocking human VEGF from binding to its receptor. Approximately 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgG1, and approximately 7% of the sequence is derived from mouse antibody A4.6.1.

Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. Patent No. 6,884,879, issued February 26, 2005. Additional antibodies include the G6 or B20 series antibodies (e.g., G6-31, B20-4.1), as described in PCT Publication No. WO 2005/012359, PCT Publication No. WO 2005/044853, and U.S. Patent Application No. 60/991,302, the contents of which are expressly incorporated herein by reference. For additional antibodies, see U.S. Patent Nos. 7,060,269, 6,582,959, 6,703,020; 6,054,297; WO 98/45332; WO 96/30046; WO 94/10202; EP 0666868 Bl; U.S. Patent Application Publication Nos. 2006009360, 20050186208, 20030206899, 20030190317, 20030203409, and 20050112126; and Popkov et al., Journal of Immunological Methods 288: 149-164 (2004). Other antibodies include those that bind to a functional epitope on human VEGF comprising residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and C104 or comprising residues F17, Y21, Q22, Y25, D63, I83, and Q89.

In one embodiment of the invention, the anti-VEGF antibody comprises a heavy chain variable region comprising the following amino acid sequence:

EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW

INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP

HYYGSSHWYF DVWGQGTLVT VSS(SEQ ID NO: 22)

and a light chain variable region comprising the following amino acid sequence:

DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF

TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ

GTKVEIKR (SEQ ID NO: 23).

In some embodiments, the anti-VEGF antibody comprises: a CDRH1 comprising the amino acid sequence, GYTFTNYGMN (SEQ ID NO:24); a CDRH2 comprising the amino acid sequence, WINTYTGEPTYAADFKR (SEQ ID NO:25); a CDRH3 comprising the amino acid sequence, YPHYYGSSHWYFDV (SEQ ID NO:26); a CDRL1 comprising the amino acid sequence, SASQDISNYLN (SEQ ID NO:27); a CDRL2 comprising the amino acid sequence, FTSSLHS (SEQ ID NO:28); and a CDRL3 comprising the amino acid sequence, QQYSTVPWT (SEQ ID NO:29).

According to the present invention, a "G6 series antibody" refers to an anti-VEGF antibody derived from the sequence of the G6 antibody or a G6-derived antibody according to any of Figures 7, 24-26, and 34-35 of PCT Publication No. WO2005/012359, the entire disclosure of which is incorporated herein by reference. See also PCT Publication No. WO2005/044853, the contents of which are expressly incorporated herein by reference. In one embodiment, the G6 series antibody binds to a functional epitope on human VEGF comprising residues F17, Y21, Q22, Y25, D63, I83, and Q89.

According to the present invention, a "B20 series antibody" refers to an anti-VEGF antibody derived from the sequence of the B20 antibody or a B20-derived antibody according to any of Figures 27-29 of PCT Publication No. WO2005/012359, the entire disclosure of which is incorporated herein by reference. See also PCT Publication No. WO2005/044853 and U.S. Patent Application 60/991,302, the contents of which are expressly incorporated herein by reference. In one embodiment, the B20 series antibody binds to a functional epitope on human VEGF comprising residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and C104.

According to the present invention, a "functional epitope" refers to an amino acid residue in an antigen that strongly contributes to antibody binding. Mutation of any of the contributing residues in the antigen (e.g., alanine or homolog mutation to wild-type VEGF) will disrupt antibody binding, resulting in a relative affinity ratio (IC50 mutant VEGF/IC50 wild-type VEGF) greater than 5 (see Example 2 of WO2005/012359). In one embodiment, the relative affinity ratio is determined by solution-bound phage display ELISA. Briefly, 96-well Maxisorp immunoplates (NUNC) are coated with the test antibody in Fab format at a concentration of 2 μg/ml in PBS overnight at 4°C and blocked with PBS, 0.5% BSA, and 0.05% Tween 20 (PBT) for 2 hours at room temperature. Serial dilutions of phage displaying hVEGF alanine point mutants (residues 8-109) or wild-type hVEGF (8-109) in PBT were first incubated on Fab-coated plates at room temperature for 15 minutes. The plates were then washed with PBS, 0.05% Tween 20 (PBST). Bound phage were detected with an anti-M13 monoclonal antibody-horseradish peroxidase conjugate (Amersham Pharmacia) diluted 1:5000 in PBT. Color was developed with 3,3',5,5'-tetramethylbenzidine (TMB, Kirkegaard & Perry Labs, Gaithersburg, MD) substrate for approximately 5 minutes, quenched with 1.0 M H3PO4, and read spectrophotometrically at 450 nm. The ratio of IC50 values (IC50,ala/IC50,wt) represents the fold reduction in binding affinity (relative binding affinity).

(iii) VEGF receptor molecules

The two most comprehensively characterized VEGF receptors are VEGFR1 (also known as Flt-1) and VEGFR2 (the mouse homologs are also known as KDR and FLK-1). Each receptor has different specificities for each VEGF family member, but VEGF-A binds to both Flt-1 and KDR. The full-length Flt-1 receptor consists of an extracellular domain with seven Ig domains, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. The extracellular domain is involved in VEGF binding, while the intracellular domain is involved in signal transduction.

VEGF receptor molecules or fragments thereof that specifically bind to VEGF can be used in the methods of the present invention to bind to and sequester the VEGF protein, thereby preventing it from signaling. In certain embodiments, the VEGF receptor molecule or VEGF-binding fragment thereof is a soluble form, such as sFlt-1. The soluble form of the receptor exerts an inhibitory effect on the biological activity of the VEGF protein by binding to VEGF, thereby preventing it from binding to its native receptor present on the surface of target cells. VEGF receptor fusion proteins are also included, examples of which are described below.

A chimeric VEGF receptor protein refers to a receptor molecule that has amino acid sequences derived from at least two different proteins (at least one of which is a VEGF receptor protein, such as flt-1 or KDR receptor) and is capable of binding to and inhibiting the biological activity of VEGF. In certain embodiments, the chimeric VEGF receptor protein of the present invention is composed of amino acid sequences derived from only two different VEGF receptor molecules; however, an amino acid sequence comprising one, two, three, four, five, six, or all seven Ig-like domains from the extracellular ligand-binding region of the flt-1 and/or KDR receptor can be linked to an amino acid sequence from another unrelated protein, such as an immunoglobulin sequence. Other amino acid sequences that can be combined with Ig-like domains will be apparent to those of ordinary skill in the art. Examples of chimeric VEGF receptor proteins include, for example, soluble Flt-1/Fc, KDR/Fc, or Flt-1/KDR/Fc (also known as VEGF Trap) (see, for example, PCT Application Publication No. WO 97/44453).

The soluble VEGF receptor proteins or chimeric VEGF receptor proteins of the present invention include VEGF receptor proteins that are not anchored to the cell surface via a transmembrane domain. Thus, soluble forms of VEGF receptors (including chimeric receptor proteins), while capable of binding and inactivating VEGF, do not contain a transmembrane domain and thus generally do not become associated with the cell membrane of cells in which the molecule is expressed.

IV. Kit

In another aspect, a kit comprising a PD-L1 axis binding antagonist and/or a VEGF antagonist is provided for treating cancer or delaying the progression of cancer in an individual or for enhancing immune function in an individual with cancer. In some embodiments, the kit comprises a PD-1 axis binding antagonist and a package insert containing instructions for use of the PD-1 axis binding antagonist in combination with oxaliplatin, folinic acid, 5-FU with or without a VEGF antagonist to treat cancer or delay the progression of cancer in an individual or to enhance immune function in an individual with cancer. In some embodiments, the kit comprises oxaliplatin, folinic acid, 5-FU with or without a VEGF antagonist and a package insert containing instructions for use of the PD-1 axis binding antagonist in combination with oxaliplatin, folinic acid, 5-FU with or without a VEGF antagonist to treat cancer or delay the progression of cancer in an individual or to enhance immune function in an individual with cancer. In some embodiments, the kit comprises a PD-1 axis binding antagonist and oxaliplatin, folinic acid, 5-FU, with or without a VEGF antagonist, and a package insert containing instructions for using the PD-1 axis binding antagonist and oxaliplatin, folinic acid, 5-FU, with or without a VEGF antagonist, to treat or delay cancer progression in an individual, or to enhance immune function in an individual with cancer. Any PD-1 axis binding antagonist and/or VEGF antagonist described herein can be included in the kit.

Example

The present invention can be further understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

Example 1: FOLFOX enhances the anti-tumor activity of anti-PD-L1 with or without anti-VEGF antibody

To determine whether FOLFOX (oxaliplatin, folinic acid, and 5-FU) enhances the anti-tumor activity of anti-PD-L1 in the presence or absence of anti-VEGF antibodies, a mouse model of colorectal cancer was treated with the combined therapy. Briefly, female C57BL/6 mice were subcutaneously inoculated with 100 μL of HBSS: 100,000 MC38 murine colorectal cells in Matrigel in the unilateral chest area. When the mice reached a mean tumor volume of 220 mm ³ , they were randomly assigned to one of the treatment groups outlined below on experimental day 0. Treatment was initiated on experimental day 1. Mice were weighed and tumors were measured 2 to 3 times per week for the entire study.

Experimental group:

1) Control (isotype control antibody (anti-gp120 antibody)), 10 mg/kg ip, 100 μL, three times a week for three weeks, n=10

2) Anti-PD-L1 antibody, 10 mg/kg ip, 100 μL, three times a week for three weeks, n=10

3) FOLFOX (see below), administered once a week for two weeks, n=10

4) FOLFOX (see below), once a week for two weeks + anti-PD-L1 antibody, 10 mg/kg ip, 100 μL, three times a week for three weeks, n=10

5) FOLFOX (see below), once a week for two weeks + anti-VEGF antibody, 5 mg/kg ip, 100 μL, twice a week for three weeks, n=10

6) FOLFOX (see below), once a week for two weeks + anti-VEGF antibody, 5 mg/kg ip, 100 μL, twice a week for three weeks + anti-PD-L1 antibody, 10 mg/kg ip, 100 μL, three times a week for three weeks, n=10

ip = intraperitoneal

sc = subcutaneous

For these studies, FOLFOX dosing was performed as follows: On experimental days 1 and 8, mice were given oxaliplatin (5 mg/kg, ip, 50 μL water) followed by folinic acid (100 mg/kg, ip, 250 μL water) (administration time = 0 hours) and 5-FU (25 mg/kg, ip) followed by 5-FU (25 mg/kg, sc) (administration time = 2 hours). Anti-PD-L1 antibody and anti-gp120 antibody were administered on experimental days 1, 3, 5, 8, 10, 12, 15, 17, and 19 (administration time = 4 hours). Anti-VEGF antibody was administered on experimental days 1, 4, 8, 11, 15, and 18 (administration time = 6 hours).

Mice were monitored for tumor growth and weight changes. Tumor volume was measured using an UltraCal-IV caliper (model 54-10-111; Fred V. Fowler Company; Newton, MA). Tumor volume was calculated using the following equation:

Tumor volume (mm ³ )=(length×width ² )×0.5.

The length and width were measured perpendicular to each other. Animal weight was measured using an Adventura Pro AV812 balance (Ohaus Corporation; Pine Brook, NJ). Percent body weight change was calculated using the following equation:

Body weight change (%)=[(weight _{day new} −weight _{day 0} )/weight _{day 0} ]×100.

Data were analyzed using R, version 2.9.2 (R Development Core Team 2008; R Foundation for Statistical Computing; Vienna, Austria), and mixed models were fitted using the nlme package, version 3.1-96 (Pinheiro et al., 2009) within R. Graphs were performed in Prism, version 5.0b for Mac (GraphPad Software, Inc.; La Jolla, CA).

Repeated measurements of tumor volume from the same animal over time were analyzed using a mixed modeling approach (Pinheiro and Bates 2010). This approach addressed both repeated measurements and moderate dropouts before the end of the study (for reasons that could be statistically classified as missing at random (MAR)). The fixed effect change in _log2 (volume) by time and dose was modeled as the sum of the main effects using a natural cubic regression spline basis for time and an automatically determined natural spline basis for dose as an interaction. The intercept and growth rate (slope) were assumed to vary randomly across animals. Tumor growth inhibition (%TGI) as a percentage of the control-treated group was calculated using the following equation as the percentage of the fitted area under the curve (AUC) for the corresponding treated group relative to the control for each day that the control-treated mice remained on study:

%TGI = 100 x (1 - _AUCdose / _AUCvehicle ).

For these studies, a complete response (CR) was defined as an individual animal whose tumor volume decreased below the limit of detection (LOD) at any time during the study. A partial response (PR) was defined as an individual animal whose tumor volume decreased by 50% of its initial tumor volume at any time during the study. The overall response rate (ORR) was defined as the sum of complete and partial responses.

The time before progression 5X (TTP5X) is defined as the time in days that the fitted tumor volume of the group (based on the mixed modeling analysis described above) exceeds 5 times the starting volume, rounded to the nearest half day, and reported as TTP5X for the group. Repeated measurements of body weight changes from the same animal over time were also analyzed using linear mixed effects analysis.

Blocking the PD-1 axis using anti-PD-L1 antibodies is effective as a single-agent therapy in preventing tumor growth. Combination therapy of anti-PD-L1 antibodies with oxaliplatin, folinic acid, and 5-FU (FOLFOX) significantly inhibited tumor growth, indicating that this chemotherapy combination enhanced the anti-tumor activity of anti-PD-L1 antibodies (Figure 1). Adding anti-VEGF to this combination therapy further enhanced this anti-tumor activity and the durability of the anti-tumor response, even after treatment cessation (Figure 4).

Example 2: Phase 1b study of MPDL3280A and bevacizumab with or without modified FOLFOX-6

The primary objective of the study was to evaluate the safety, pharmacology, and preliminary efficacy of MPDL3280A administered with bevacizumab (arm A) and with bevacizumab plus FOLFOX (specifically, modified FOLFOX-6 or mFOLFOX-6) (arm B) in patients with solid tumors, including metastatic colorectal cancer (mCRC). Arm A evaluated 10 mg/kg (or a selected dose level that did not exceed the single-agent MTD or MAD) of MPDL3280A with bevacizumab (15 mg/kg) every 3 weeks (q3w) for up to one year. Patients who had not received oxaliplatin for metastatic disease were enrolled in arm B and received MPDL3280A with bevacizumab and FOLFOX every 2 weeks (q2w). The mFOLFOX-6 regimen consists of concurrent intravenous (IV) oxaliplatin (85 mg/m ² ) and IV folinic acid (400 mg/m ² ) administered over approximately 120 minutes, followed by 5-FU (400 mg/m ² ) administered as an IV bolus, followed by 2400 mg/m ² administered by continuous IV infusion over approximately 46 hours. Oxaliplatin is administered for up to 8 cycles. Treatment continues for up to 1 year.

Claims (14)

1. Use of an anti-PD-L1 antibody in the preparation of a medicament or kit for use in a method of treating cancer or delaying cancer progression in an individual, wherein said method comprises administering an effective amount of an anti-PD-L1 antibody, oxaliplatin, leucovorin, 5-FU, and an anti-VEGF antibody to said individual. The anti-PD-L1 antibody comprises heavy chain and light chain variable region sequences, wherein: (a) The heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein (i) The HVR-H1 is as shown in GFTFSDSWIH (SEQ ID NO:15). (ii) The HVR-H2 is as shown in AWISPYGGSTYYADSVKG (SEQ ID NO:16). (iii) The HVR-H3 is as shown in RHWPGGFDY (SEQ ID NO:3); and (b) The light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein (i) The HVR-L1 is as shown in RASQDVSTAVA (SEQ ID NO:17). (ii) The HVR-L2 is as shown in SASFLYS (SEQ ID NO:18). (iii) The HVR-L3 is as shown in QQYLYHPAT (SEQ ID NO:19). The anti-PD-L1 antibody inhibits the binding of PD-L1 to both PD-1 and B7-1, and the anti-VEGF antibody binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 generated from hybridoma ATCC HB 10709. 2. The use of claim 1, wherein the anti-PD-L1 antibody comprises: a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 20 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 21. 3. The use of claim 1, wherein the anti-PD-L1 antibody is YW243.55.S70. 4. The use of any one of claims 1-3, wherein the anti-PD-L1 antibody is a monoclonal antibody. 5. Use according to any one of claims 1-4, wherein the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of: Fab, Fab'-SH, Fv, scFv, and (Fab') ₂ fragments. 6. Use according to any one of claims 1-5, wherein the anti-PD-L1 antibody is a humanized antibody. 7. The use of any one of claims 1-5, wherein the anti-PD-L1 antibody is a human antibody. 8. The use of any one of claims 1-7, wherein the anti-VEGF antibody is a humanized antibody. 9. Use according to any one of claims 1-8, wherein the anti-VEGF antibody is bevacizumab. 10. Use according to any one of claims 1-8, wherein the anti-VEGF antibody has a heavy chain variable region as shown in the following amino acid sequence: and the light chain variable region as shown in the following amino acid sequence: 11. Use according to any one of claims 1-10, wherein the treatment results in a persistent response in the individual after the treatment is stopped. 12. Use according to any one of claims 1-10, wherein the subject has colorectal cancer. 13. A kit comprising an anti-PD-L1 antibody and a packaging insert containing instructions for use of the anti-PD-L1 antibody in combination with oxaliplatin, leucovorin, 5-FU, and an anti-VEGF antibody to treat cancer or delay cancer progression in an individual. The anti-PD-L1 antibody comprises heavy chain and light chain variable region sequences, wherein: (a) The heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein (i) The HVR-H1 is as shown in GFTFSDSWIH (SEQ ID NO:15). (ii) The HVR-H2 is as shown in AWISPYGGSTYYADSVKG (SEQ ID NO:16). (iii) The HVR-H3 is as shown in RHWPGGFDY (SEQ ID NO:3); and (b) The light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein (i) The HVR-L1 is as shown in RASQDVSTAVA (SEQ ID NO:17). (ii) The HVR-L2 is as shown in SASFLYS (SEQ ID NO:18). (iii) The HVR-L3 is as shown in QQYLYHPAT (SEQ ID NO:19). The anti-PD-L1 antibody inhibits the binding of PD-L1 to both PD-1 and B7-1, and the anti-VEGF antibody binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 generated from hybridoma ATCC HB 10709. 14. A kit comprising an anti-PD-L1 antibody that inhibits the binding of PD-L1 to both PD-1 and B7-1, oxaliplatin, leucovorin, 5-FU, and an anti-VEGF antibody, and a packaging insert containing instructions for use of the anti-PD-L1 antibody and the oxaliplatin, leucovorin, 5-FU, and anti-VEGF antibody in an individual to treat cancer or delay cancer progression. The anti-PD-L1 antibody comprises heavy chain and light chain variable region sequences, wherein: (a) The heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein (i) The HVR-H1 is as shown in GFTFSDSWIH (SEQ ID NO:15). (ii) The HVR-H2 is as shown in AWISPYGGSTYYADSVKG (SEQ ID NO:16). (iii) The HVR-H3 is as shown in RHWPGGFDY (SEQ ID NO:3); and (b) The light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein (i) The HVR-L1 is as shown in RASQDVSTAVA (SEQ ID NO:17). (ii) The HVR-L2 is as shown in SASFLYS (SEQ ID NO:18). (iii) The HVR-L3 is as shown in QQYLYHPAT (SEQ ID NO:19). The anti-VEGF antibody described therein binds to the same epitope as monoclonal anti-VEGF antibody A4.6.1 generated from hybridoma ATCC HB 10709.