HK1230525A1 - Combination therapy comprising ox40 binding agonists and pd-1 axis binding antagonists - Google Patents

Description

Combination therapy comprising an OX40 binding agonist and a PD-1 axis binding antagonist

Cross reference to related applications

This application claims priority to U.S. provisional application serial No. 61/917,264 filed on 12/17/2013 and U.S. provisional application serial No. 62/080,991 filed on 11/17/2014, each of which is incorporated herein by reference in its entirety.

Submission of sequence Listing on an ASCII text File

The contents of the following submissions on an ASCII text file are incorporated herein in their entirety by reference: sequence listing in Computer Readable Form (CRF) (file name: 146392030640seqlist. txt, recording date: 2014, 12, 16 days, size: 72 KB).

Technical Field

The present invention relates to methods of treating cancer by administering a PD-1 axis binding antagonist and an OX40 binding agonist.

Background

Providing two distinct signals to T cells is a widely accepted model for lymphocyte activation by Antigen Presenting Cells (APCs) on resting T lymphocytes. Lafferty et al, Aust.J.Exp.biol.Med.Sci 53:27-42 (1975). This model further provides self-to-non-self discrimination and immune tolerance. Bretscher et al, Science 169: 1042-; bretscher, P.A., Proc.Nat.Acad.Sci.USA 96:185-190 (1999); jenkingset al., J.exp.Med.165:302-319 (1987). Upon recognition of foreign antigenic peptides presented in the context of the Major Histocompatibility Complex (MHC), a first signal or antigen-specific signal is transduced via the T Cell Receptor (TCR). A second or co-stimulatory signal is delivered to T cells via a co-stimulatory molecule expressed on Antigen Presenting Cells (APCs), inducing the T cells to facilitate 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, do not mount an effective immune response, and further can lead to tolerance or depletion to foreign antigens.

In the two-signal model, T cells receive both positive and negative second costimulatory signals. Modulation of such positive and negative signals is crucial to maximizing the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. Negative secondary signals appear to be necessary for inducing T cell tolerance, while positive signals promote T cell activation. While the simple two-signal model still provides a valid explanation for naive lymphocytes, the host immune response is a dynamic process and can also provide co-stimulatory signals to antigen-exposed T cells. The mechanism of co-stimulation is of therapeutic interest, as manipulation of the co-stimulatory signal has been shown to provide a means of enhancing or terminating the cell-based immune response. Recently, it has been found that T cell dysfunction or anergy occurs in parallel with the induced and sustained expression of the inhibitory receptor, the apoptosis 1 polypeptide (PD-1). Thus, therapeutic agents that target PD-1 and other molecules that signal via interaction with PD-1, such as apoptosis ligand 1(PD-Ll) and apoptosis ligand 2(PD-L2), are a field 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, most tumor-infiltrating T-lymphocytes predominantly express PD-1, in contrast to T-lymphocytes in normal tissues and peripheral Blood T-lymphocytes, indicating that upregulation of PD-1 on tumor-reactive T-cells can contribute to an impaired anti-tumor immune response (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 result in attenuation of T cell activation and escape immune surveillance (sharp et al, Nat Rev 2002) (Keir ME et al, 2008annu. Thus, inhibition of the PD-L1/PD-1 interaction may enhance CD8+ T cell mediated tumor killing.

Therapeutic agents that target PD-1 and other molecules that signal via interaction with PD-1, such as apoptosis ligand 1(PD-L1) and apoptosis ligand 2(PD-L2), are a field of strong interest. Inhibition of PD-L1 signaling has been proposed as a means of enhancing T cell immunity to treat cancer (e.g., tumor immunity) and infections, including both acute and chronic (e.g., persistent) infections. An optimal therapeutic treatment would combine the blocking of the PD-1 receptor/ligand interaction with an agent that directly inhibits tumor growth. There remains a need for optimal therapies for treating, stabilizing, preventing, and/or delaying the development of various cancers.

The mechanism of co-stimulation is of therapeutic interest, as manipulation of co-stimulatory signals has been shown to provide a means of enhancing or terminating cell-based immune responses. OX40 (also known as CD34, TNFRSF4, or ACT35 antigen), a member of the tumor necrosis factor receptor superfamily, provides costimulatory signals to CD4+ and CD8+ T cells, resulting in enhanced cell proliferation, survival, effector function, and migration. OX40 signaling also enhances memory T cell development and function. OX40 is not constitutively expressed on naive T cells, but is induced after T Cell Receptor (TCR) involvement. The ligand OX40L of OX40 is expressed predominantly on antigen presenting cells. OX40 is highly expressed by activated CD4+ T cells, activated CD8+ T cells, memory T cells, and regulatory T (treg) cells.

Combining OX40 signaling with other signaling pathways that are deregulated in tumor cells can further enhance therapeutic efficacy. Thus, there remains a need for such optimal therapies for treating, or delaying the development of, various cancers, immune-related diseases, and T cell dysfunctional disorders.

All references, including patent applications, patent publications, and UniProtKB/Swiss-Prot accession numbers, cited herein are hereby incorporated by reference in their entirety as if each individual reference were specifically and individually indicated to be incorporated by reference.

Summary of The Invention

In one aspect, provided herein is a method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody).

In another aspect, provided herein is a method of enhancing immune function in an individual having cancer comprising administering an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody).

In yet other aspects, provided herein are methods of treating an infection (e.g., of a bacterium or virus or other pathogen). In some embodiments, the infection is viral and/or bacterial. In some embodiments, the infection is pathogenic. In some embodiments, the infection is a chronic infection.

In another aspect, provided herein is the use of a human PD-1 axis binding antagonist in the manufacture of a medicament for treating or delaying progression of cancer (or, in some embodiments, treating infection) in an individual, wherein the medicament comprises the human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administering the medicament in combination with a composition comprising an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) and an optional pharmaceutically acceptable carrier.

In another aspect, provided herein is a use of an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) in the manufacture of a medicament for treating or delaying progression of cancer (or, in some embodiments, treating infection) in an individual, wherein the medicament comprises the OX40 binding agonist and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administering the medicament in combination with a composition comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier.

In another aspect, provided herein are compositions comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for use in treating or delaying progression of cancer (or, in some embodiments, treating an infection) in an individual, wherein the treatment comprises administering the composition in combination with a second composition, wherein the second composition comprises an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) and an optional pharmaceutically acceptable carrier.

In another aspect, provided herein are compositions comprising an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) and an optional pharmaceutically acceptable carrier for use in treating or delaying progression of cancer (or, in some embodiments, treating an infection) in an individual, wherein the treatment comprises administering the composition in combination with a second composition, wherein the second composition comprises a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier.

In another aspect, provided herein is a kit comprising a medicament comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament in combination with a composition comprising an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer (or, in some embodiments, treating an infection) in an individual.

In another aspect, provided herein is a kit comprising a first drug comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and a second drug comprising an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) and an optional pharmaceutically acceptable carrier. In some embodiments, the kit further comprises a package insert comprising instructions for administering the first medicament and the second medicament for treating or delaying progression of cancer (or, in some embodiments, treating an infection) in an individual.

In another aspect, provided herein is a kit comprising a medicament comprising an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament in combination with a composition comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer (or, in some embodiments, treating an infection) in an individual.

In some embodiments, the cancer is breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, renal cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, or hepatocellular carcinoma.

In some embodiments, the individual has cancer or has been diagnosed with cancer.

In some embodiments, the cancer cells (in a sample of cancer from the individual) do not express PD-L1. In some embodiments, the PD-L1 biomarker is deleted from the sample when it comprises 0% of the sample. In some embodiments, PD-L1 biomarker expression is determined by protein expression (e.g., by Immunohistochemical (IHC) methods).

In some embodiments, the cancer cells (in a sample of cancer from an individual) express PD-L1. In some embodiments, the PD-L1 biomarker is present in the sample when it constitutes more than 0% of the sample. In some embodiments, the PD-L1 biomarker is detected in the sample by protein expression. In some embodiments, protein expression is determined by Immunohistochemistry (IHC). In some embodiments, an anti-PD-L1 antibody is used to detect a PD-L1 biomarker. In some embodiments, the PD-L1 biomarker is detected as weak staining intensity by IHC, moderate staining intensity by IHC, or strong staining intensity by IHC. In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 antibody, and wherein the PD-L1 biomarker is detected as moderate staining intensity by IHC, or strong staining intensity by IHC.

In some embodiments, the individual has cancer that is resistant to a PD-1 axis binding antagonist. In some embodiments, the individual is refractory to a PD-1 axis binding antagonist. In some embodiments, the patient does not respond effectively to a PD-1 axis binding antagonist.

In some embodiments, the individual has cancer with high T cell infiltrates (e.g., as determined using a diagnostic test). In some embodiments, the individual has cancer with low or substantially undetectable T cell infiltrates (e.g., as determined using a diagnostic test).

In some embodiments of the methods, uses, compositions, and kits described above and herein, the treatment or administration of the human PD-1 axis binding antagonist and the OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) results in a sustained response in the individual after cessation of the treatment.

In some embodiments, the combination treatment of an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) and a PD-1 axis binding antagonist (e.g., an anti-PD-1 or anti-PDL 1 antibody) is synergistic, whereby the effective dose of an OX40 binding agent (e.g., an anti-human OX40 agonist antibody) in the combination is reduced relative to the effective dose of the OX40 binding agent (e.g., an anti-human OX40 agonist antibody) as a single agent.

In some embodiments, the OX40 binding agonist is administered prior to the PD-1 axis binding antagonist, simultaneously with the PD-1 axis binding antagonist, or after the PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist and the OX40 binding agonist are in the same composition.

In some embodiments, the PD-1 axis binding antagonist and the OX40 binding agonist are in separate compositions. In some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of: PD-1 binding antagonists, PDL1 binding antagonists and PDL2 binding antagonists. In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some cases In embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to its ligand binding partner. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to PDL 1. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to PDL 2. In some embodiments, the PD-1 binding antagonist inhibits PD-1 from binding to both PDL1 and PDL 2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 binding antagonist is nivolumab. In some embodiments, the PD-1 binding antagonist is pembrolizumab. In some embodiments, the PD-1 binding antagonist is CT-011. In some embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments, the PD-1 axis binding antagonist is a PDL1 binding antagonist. In some embodiments, the PDL1 binding antagonist inhibits PDL1 from binding PD-1. In some embodiments, the PDL1 binding antagonist inhibits binding of PDL1 to B7-1. In some embodiments, the PDL1 binding antagonist inhibits PDL1 from binding both PD-1 and B7-1. In some embodiments, the PDL1 binding antagonist is an anti-PDL 1 antibody. In some embodiments, the anti-PDL 1 antibody is a monoclonal antibody. In some embodiments, the anti-PDL 1 antibody is an antibody fragment selected from the group consisting of: fab, Fab '-SH, Fv, scFv, and (Fab') ₂And (3) fragment. In some embodiments, the anti-PDL 1 antibody is a humanized or human antibody. In some embodiments, the PDL1 binding antagonist is selected from the group consisting of: YW243.55.S70, MPDL3280A, MDX-1105, and MEDI 4736. In some embodiments, the antibody comprises a heavy chain comprising the HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO: 1), the HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO: 2), and the HVR-H3 sequence of RHWPGGFDY (SEQ ID NO: 3), and a light chain comprising the HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO: 4), the HVR-L2 sequence of SASFLYS (SEQ ID NO: 5), and the HVR-L3 sequence of QQYLYHPAT (SEQ ID NO: 6). In some embodiments, the antibody comprises a heavy chain variable region comprising the following amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 8),

the light chain variable region comprises the following amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO：9)。

in some embodiments, the PD-1 axis binding antagonist is a PDL2 binding antagonist. In some embodiments, the PDL2 binding antagonist is an antibody. In some embodiments, the PDL2 binding antagonist is an immunoadhesin. In some embodiments, the PD-1 axis binding antagonist is an antibody (e.g., an anti-PD 1 antibody, an anti-PDL 1 antibody, or an anti-PDL 2 antibody) comprising one or more aglycosylation site mutations (e.g., substitutions). In some embodiments, the substitution mutation comprises one or more substitutions at amino acid positions N297, L234, L235, and D265(EU numbering). In some embodiments, the substitution mutation is selected from the group consisting of: N297G, N297A, L234A, L235A, and D265A (EU numbering). In some embodiments, the antibody is human IgG 1.

In some embodiments, the OX40 binding agonist is selected from the group consisting of: OX40 agonist antibodies, OX40L agonist fragments, OX40 oligoreceptor, and OX40 immunoadhesin. In some embodiments, the OX40 agonist antibody binds to human OX 40. In some embodiments, the OX40 agonist antibody is any of the anti-human OX40 agonist antibodies disclosed herein (e.g., in paragraphs 198-226). In some embodiments, the OX40 agonist antibody is MEDI6469, MEDI0562, or MEDI 6383. In some embodiments, the OX40 agonist antibody is a full length IgG1 antibody. In some embodiments, the OX40 binding agonist is a trimeric OX40L-Fc protein. In some embodiments, the OX40 binding agonist is a trimeric OX40L fusion protein described in U.S. patent No.7,959,925. In some embodiments, the OX40 binding agonist comprises one or more OX40L extracellular domains. In some embodiments that may be combined with any other embodiment, the OX40 binding agonist (e.g., OX40 agonist antibody) is not MEDI 6383. In some embodiments that may be combined with any other embodiment, the OX40 binding agonist (e.g., OX40 agonist antibody) is not MEDI 0562. In some embodiments, the OX40 binding agonist (e.g., an OX40 agonist antibody) is a human and/or humanized antibody. In some embodiments, the OX40 binding agonist (e.g., OX40 agonist antibody) is a subtractive anti-human OX40 antibody (e.g., depletes cells expressing human OX 40). In some embodiments, the cell expressing human OX40 is a CD4+ effector T cell. In some embodiments, the human OX 40-expressing cell is a Treg cell. In some embodiments, the depleting is by ADCC and/or phagocytosis. In some embodiments, the antibody mediates ADCC by binding to Fc γ rs expressed by human effector cells and activating the human effector cell function. In some embodiments, the antibody mediates phagocytosis by binding to Fc γ rs expressed by human effector cells and activating the human effector cell function. In some embodiments, the human effector cell is selected from the group consisting of a macrophage, a Natural Killer (NK) cell, a monocyte, and a neutrophil. In some embodiments, the human effector cell is a macrophage. In some embodiments, the OX40 binding agonist (e.g., OX40 agonist antibody) has a functional Fc region. In some embodiments, the effector function of the functional Fc region is ADCC. In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are ADCC and phagocytosis. In some embodiments, the Fc region is human IgG 1. In some embodiments, the Fc region is human IgG 4.

In some embodiments of the methods, uses, compositions, and kits described above and herein, the PD-1 axis binding antagonist and/or the OX40 binding agonist (e.g., anti-human OX40 agonist antibody) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intracamerally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments of the methods, uses, compositions, and kits described above and herein, the treatment further comprises administering a chemotherapeutic agent for treating or delaying progression of cancer in the individual. In some embodiments, the individual has been treated with a chemotherapeutic agent prior to the combination treatment of the PD-1 axis binding antagonist and the OX40 binding agonist. In some embodiments, an individual treated with the combination of the PD-1 axis binding antagonist and/or the OX40 binding agonist is refractory to treatment with a chemotherapeutic agent. Some embodiments of the methods, uses, compositions, and kits described throughout this application further comprise administering a chemotherapeutic agent for treating or delaying progression of cancer.

In some embodiments of the methods, uses, compositions, and kits described above and herein, the CD8T cells in the individual have enhanced priming, activation, proliferation, and/or lysis activity relative to prior to administration of the combination. In some embodiments, the number of CD8T cells is increased relative to prior to administration of the combination. In some embodiments, the CD8T cell is an antigen-specific CD8T cell. In some embodiments, Treg function is arrested relative to prior to administration of the combination. In some embodiments, T cell depletion is reduced relative to prior to administration of the combination. In some embodiments, the number of Treg cells is reduced relative to prior to administration of the combination. In some embodiments, the plasma interferon gamma is elevated relative to prior to administration of the combination. In some embodiments, the number of memory T effector cells is increased relative to prior to administration of the combination. In some embodiments, memory T effector cell activation and/or proliferation is increased relative to prior to administration of the combination. In some embodiments, memory T effector cells are detected in peripheral blood. In some embodiments, detection of memory T effector cells is by detecting CXCR 3.

Provided herein are methods for monitoring the pharmacodynamic activity of an OX40 agonist treatment by measuring the level of expression of one or more marker genes, proteins (e.g., cytokines, e.g., gamma interferon) and/or cellular composition (e.g., percentage of tregs and/or absolute number of tregs; e.g., number of CD8+ effector T cells) in a leukocyte-containing sample (e.g., peripheral blood) obtained from a subject, wherein the subject has been treated with a PD-1 axis binding antagonist and an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody), and wherein the one or more marker genes are selected from a T cell marker gene, or a memory T cell marker gene (e.g., a marker of T-effect memory cells); and determining that the treatment exhibits pharmacodynamic activity based on the expression level of the one or more marker genes, proteins and/or cellular constituents in the sample obtained from the subject as compared to the reference, wherein an increased expression level of the one or more marker genes as compared to the reference is indicative of the pharmacodynamic activity of the OX40 agonist treatment. The expression level of a marker gene, protein and/or cellular composition can be measured by one or more of the methods described herein. In some embodiments, provided herein are methods of monitoring pharmacodynamic activity for OX40 agonist therapy and PD-1 axis binding antagonist combination therapy, comprising measuring the level of proliferating CD8+ T cells (e.g., Ki67 +/percent of total CD8+ T cells) in a sample (e.g., a peripheral blood sample) from an individual, wherein an increase in the level of proliferating CD8+ T cells in the sample as compared to a reference (e.g., a level prior to combination therapy) is indicative of the pharmacodynamic activity of the combination therapy. In some embodiments, provided herein are methods for monitoring the pharmacodynamic activity of an OX40 agonist therapy and a PD-1 axis binding antagonist combination therapy, comprising measuring the level of activated CD8+ T cells (e.g., the percentage of CXCR3 +/total CD8+ T cells) in a sample (e.g., a peripheral blood sample) from an individual, wherein an increase in the level of activated CD8+ T cells in the sample as compared to a reference (e.g., the level prior to the combination therapy) is indicative of the pharmacodynamic activity of the combination therapy.

Provided herein are methods for monitoring responsiveness of a subject to an OX40 agonist treatment by measuring the level of expression of one or more marker genes, proteins (e.g., cytokines, e.g., gamma interferon) and/or cellular composition (e.g., percentage of tregs and/or absolute number of tregs; e.g., number of CD8+ effector T cells, in a peripheral blood sample) in a leukocyte-containing sample (e.g., peripheral blood) obtained from the subject, wherein the subject has been treated with a PD-1 axis binding antagonist and an OX40 binding agonist (e.g., anti-human OX40 agonist antibody), and wherein the one or more marker genes are selected from a T cell marker gene, or a memory T cell marker gene (e.g., a marker of T-effector memory cells); and classifying the subject as responsive or non-responsive to the treatment based on the expression level of the one or more marker genes, proteins and/or cellular constituents in the sample obtained from the subject as compared to a reference, wherein an increased expression level of the one or more marker genes as compared to the reference is indicative of responsiveness or lack of responsiveness to OX40 agonist treatment. The expression level of a marker gene, protein and/or cellular composition can be measured by one or more of the methods described herein. In some embodiments, provided herein are methods for monitoring responsiveness to a combination treatment of an OX40 agonist treatment and a PD-1 axis binding antagonist, comprising measuring in a sample (e.g., a peripheral blood sample) from an individual the level of proliferating CD8+ T cells (e.g., the percentage of Ki67 +/total CD8+ T cells), wherein an increase in the level of proliferating CD8+ T cells in the sample as compared to a reference (e.g., the level prior to the combination treatment) is indicative of responsiveness to the combination treatment. In some embodiments, provided herein are methods for monitoring responsiveness to a combination treatment of an OX40 agonist treatment and a PD-1 axis binding antagonist, comprising measuring in a sample (e.g., a peripheral blood sample) from an individual the level of activated CD8+ T cells (e.g., the percentage of CXCR3 +/total CD8+ T cells), wherein an increase in the level of activated CD8+ T cells in the sample as compared to a reference (e.g., the level prior to the combination treatment) is indicative of responsiveness to the combination treatment.

It is to be understood that one, some, or all of the features of the various embodiments described herein may be combined to form further embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description.

Brief Description of Drawings

FIG. 1: tumor infiltrating CD8+ T cells expressed high levels of PD-1 inhibitory receptors in the CT26 colorectal syngeneic tumor model (control treated mice). Approximately half of CD8+ TIL expressing PD-1 also expressed OX 40. Representative flow cytometry dot plots from one mouse out of 5, day 2 after the start of treatment with control antibody.

FIGS. 2A and B: (figure 2A) treatment with anti-OX 40 agonist antibody alone and anti-OX 40 agonist antibody in combination with anti-PDL 1 antagonist antibody significantly reduced the proportion of Foxp3+ T regulatory cells (relative to the total number of CD45+ cells) within the tumor. (figure 2B) treatment with anti-OX 40 agonist antibody alone and anti-OX 40 agonist antibody in combination with anti-PDL 1 antagonist antibody significantly reduced the absolute number of Foxp3+ T regulatory cells within the tumor in the CT26 colorectal tumor model. For both (fig. 2A) and (fig. 2B): data are from day 9 after treatment initiation, each symbol representing one individual mouse. Mice were given either a control antibody or anti-PDL 1 antibody, first dose of 10mg/kg IV on day 1, followed by BIW (twice a week) 5mg/kg IP. anti-OX 40 agonist antibodies were administered as a first dose on day 1 of 0.1mg/kg IV followed by TIW (thrice a week) of 0.1mg/kg IP.

Fig. 3A and B: treatment with anti-OX 40 agonist antibody increased PDL1 expression on tumor myeloid (CD11B + Gr-1 low/medium) cells and (fig. 3B) tumor cells in a CT26 colorectal syngeneic tumor model (fig. 3A). Data were from day 9 after starting treatment. Each dot/square represents an individual mouse. PDL1 expression was measured by flow cytometry by geometric mean fluorescence intensity (geo MFI). P <0.01, p <0.05 as calculated by unpaired t-test. For the control antibody, the dose in this experiment was administered as the first dose on day 1 of 10mg/kg IV followed by BIW 5mg/kg IP. anti-OX 40 agonist antibodies were administered at a first dose of 0.1mg/kg IV followed by TIW 0.1mg/kg IP on day 1.

Fig. 4A, 4B: treatment with anti-OX 40 agonist antibody and anti-PDL 1 antagonist antibody exhibited synergistic combination efficacy in the MC38 colorectal cancer isogenic tumor model in C57BL/6 mice. (FIG. 4A) mean tumor volume (mm) measured over time (days) according to treatment groups³) And n is 10/group. (drawing)4B) Individual tumor volumes were measured over time according to treatment groups. The black line indicates the mean of the group. The blue dashed line indicates the average of the control group. The grey lines are individual animals. The red line indicates individual animals that exited the study due to ulcerative tumors or excessive tumor size. Control antibody, anti-PDL 1 antibody, or anti-OX 40 agonist antibody was administered as a first dose of 10mg/kg IV on day 1, followed by 3 weeks of TIW 10mg/kg IP.

Fig. 5A, 5B: treatment with anti-OX 40 agonist antibody and anti-PDL 1 antagonist antibody exhibited synergistic combination efficacy in a CT26 colorectal syngeneic tumor model in Balb/c mice. (FIG. 5A) mean tumor volume (mm) measured over time (days) according to treatment groups³) And n is 10/group. (FIG. 5B) individual tumor volumes were measured over time according to treatment groups. The control antibody or anti-PDL 1 was administered as a first dose of 10mg/kg IV on day 1, followed by 3 weeks TIW 5mg/kg IP. The anti-OX 40 agonist antibody was administered as a single dose at 1mg/kg IV on day 1. The black line indicates the mean of the group. The blue dashed line indicates the average of the control group. The grey lines are individual animals. The red line indicates individual animals that exited the study due to ulcerative tumors or excessive tumor size.

Fig. 6A, 6B: anti-OX 40 agonist antibody single agent treatment showed dose responsiveness in a CT26 colorectal cancer isogenic tumor model in Balb/c mice. (FIG. 6A) mean tumor volume (mm) measured over time (days) according to treatment groups³) And n is 10/group. (FIG. 6B) individual tumor volumes were measured over time according to treatment groups. The black line indicates the mean of the group. The blue dashed line indicates the average of the control group. The grey lines are individual animals. The red line indicates individual animals that exited the study due to ulcerative tumors or excessive tumor size. Control antibody was administered as a first dose of 1mg/kg IV on day 1, followed by 3 weeks TIW1mg/kg IP. anti-OX 40 agonist antibodies were administered as a first dose on day 1 of 0.01mg/kg, 0.1mg/kg, or 1mg/kg IV, followed by 3 weeks of TIW IP.

Fig. 7A, 7B: combination of sub-maximal dose of anti-OX 40 agonist antibody plus anti-PDL 1 antagonist antibody in CT26 colorectal cancer isogenic tumor model in Balb/c miceThe treatment exhibited a synergistic combination efficacy. (FIG. 7A) mean tumor volume (mm) measured over time (days) according to treatment groups³) And n is 10/group. (FIG. 7B) individual tumor volumes were measured over time according to treatment groups. The black line indicates the mean of the group. The blue dashed line indicates the average of the control group. The grey lines are individual animals. The red line indicates individual animals that exited the study due to ulcerative tumors or excessive tumor size. The control antibody or anti-PDL 1 was administered as a first dose of 10mg/kg IV on day 1, followed by 3 weeks TIW 10mg/kg IP. anti-OX 40 agonist antibodies were administered at a first dose of 0.1mg/kg IV on day 1, followed by 3 weeks TIW of 0.1mg/kg IP.

Fig. 8A, 8B: in a separate experiment, anti-OX 40 agonist antibody plus anti-PDL 1 administered at a single 0.1mg/kg IV injection of sub-maximal effective dose exhibited synergistic combination efficacy in a CT26 colorectal syngeneic tumor model in Balb/c mice. (FIG. 8A) mean tumor volume (mm) measured over time (days) according to treatment groups³) And n is 10/group. (FIG. 8B) individual tumor volumes were measured over time according to treatment groups. The black line indicates the mean of the group. The blue dashed line indicates the average of the control group. The grey lines are individual animals. The red line indicates individual animals that exited the study due to ulcerative tumors or excessive tumor size. The control antibody or anti-PDL 1 was administered as a first dose of 10mg/kg IV on day 1, followed by 3 weeks of TIW 5 mg/kgIP. anti-OX 40 antibody was administered as a first or single dose 0.1mg/kg IV on day 1, followed by 3 weeks TIW 0.1 mg/kgIP.

FIGS. 9A, B, C and D: effects of combined treatment of OX40 agonist antibodies and PDL1 antagonists (anti-PDL 1 antagonist antibodies) on the levels of proliferating T cells, Treg cells, plasma interferon-gamma, and activated T cells in peripheral blood. Analysis of peripheral blood collected from combination-treated CT26 mice revealed an increase in effector cell proliferation and inflammatory T cell markers. CD8+ T cell proliferation (fig. 9A), Treg cells (fig. 9B), plasma interferon gamma levels (fig. 9C) and levels of activated T cells (fig. 9D) were measured. (figure 9A) the level of proliferating CD8+ T cells (expressed as a percentage of ki67 +/total CD8+ T cells) in animals treated with a combination of an OX40 agonist antibody and a PDL1 antagonist was significantly increased compared to treatment with either an OX40 agonist antibody or a PDL1 antagonist antibody alone. (figure 9B) reduced peripheral blood tregs were observed for OX40 agonist antibody single agent treatment and treatment with OX40 agonist antibody and PDL1 antagonist combination treatment. (fig. 9C) elevated plasma interferon gamma (IFNg) was observed for combined treatment of OX40 agonist and PDL1 antagonist. (figure 9D) the levels of activated T cells (specifically, activated memory Teff cells) in animals treated with a combination of OX40 agonist antibody and PDL1 antagonist were significantly increased compared to treatment with OX40 agonist or PDL1 antagonist alone.

Figure 10 shows the correlation of OX40 expression with PDL1 diagnostic status in cancer samples from human patients with Urothelial Bladder Cancer (UBC) and non-small cell lung cancer (NSCLC). Tissue samples were from patients who participated in phase 1 clinical trials of anti-PDL 1 antibody MPDL 3280A. As disclosed herein, the PD-L1 biomarker status of tumor infiltrating Immune Cells (IC) was determined using IHC. OX40 expression levels were determined using rtPCR analysis (Fluidigm). Triangles indicate that the patient has a partial or complete clinical response; the circle indicates that the patient shows stable disease; the squares indicate that the patient has progressive disease.

FIGS. 11A, B, C, D, E, and F: an exemplary IHC analysis of a control cell sample is shown. (fig. 11A) negative control IHC staining of parental HEK-293 cells; (FIG. 11B) IHC staining of HEK-293 cells transfected with recombinant human PD-L1 with weak staining intensity; (FIG. 11C) IHC staining of HEK-293 cells transfected with recombinant human PD-L1 with moderate staining intensity; (FIG. 11D) IHC staining of HEK-293 cells transfected with recombinant human PD-L1 with strong staining intensity; (fig. 11E) positive tissue control IHC staining of placental tissue samples; (FIG. 11F) positive tissue control IHC staining of tonsil tissue samples. All IHC staining was performed using a proprietary anti-PDL 1 antibody.

Fig. 12A, B and C: shows results from (fig. 12A) triple negative breast cancer; (fig. 12B) malignant melanoma; (fig. 12C) exemplary PDL1 positive IHC staining of tumor samples of NSCLC, adenocarcinoma.

Detailed Description

The inventors of the present application demonstrated that the combination of anti-human OX40 agonist antibodies with anti-PDL 1 immunotherapy resulted in synergistic inhibition of tumor growth, and an increased response rate.

In one aspect, provided herein are methods, compositions and uses for treating or delaying progression of cancer in an individual comprising administering an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist.

In another aspect, provided herein are methods, compositions and uses for enhancing immune function in an individual having cancer comprising administering an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist.

In another aspect, provided herein are methods, compositions and uses for treating an infection (e.g., of a bacterium or virus or other pathogen) in an individual having cancer comprising administering an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist.

I. Definition of

Before the present invention is described in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

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

It is understood that the various aspects and embodiments of the invention described herein include aspects and embodiments that "comprise," consist of … …, "and" consist essentially of … ….

As used herein, the term "OX 40" refers to any native OX40 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed OX40 as well as any form of OX40 that results from processing in a cell. The term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants. An exemplary amino acid sequence of human OX40 lacking a signal peptide is shown in SEQ ID NO: 60

(LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI)。

"OX 40 activation" refers to the activation of the OX40 receptor. Generally, OX40 activation results in signal transduction.

The terms "anti-OX 40 antibody" and "antibody that binds OX 40" refer to an antibody that is capable of binding OX40 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting OX 40. In one embodiment, the anti-OX 40 antibody binds to an unrelated non-OX 40 protein to less than about 10% of the binding of the antibody to OX40 as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that binds OX40 has a concentration of ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (Kd). In certain embodiments, the anti-OX 40 antibody binds to an OX40 epitope that is conserved among OX40 from different species.

The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of the PD-1 axis binding partner with one or more of its binding partners, thereby removing T cell dysfunction resulting from signaling on the PD-1 signaling axis-one outcome is restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2). In some embodiments, the PD-1 binding antagonist is a molecule that inhibits binding of PD-1 to one or more of its binding partners. In a particular aspect, the PD-1 binding antagonist inhibits PD-1 from binding 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 resulting 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 (mediated signaling via PD-1) mediated by or via cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a particular embodiment, the PD-1 binding antagonist is MDX-1106(nivolumab) as described herein. In another specific embodiment, the PD-1 binding antagonist is MK-3475(pembrolizumab) as described herein. In another specific embodiment, the PD-1 binding antagonist is CT-011(pidilizumab) as described herein. In another particular aspect, the PD-1 binding antagonist is AMP-224 as described herein.

The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits binding of PD-L1 to its binding partner. In a particular aspect, the PD-L1 binding antagonist inhibits PD-L1 from binding to PD-1 and/or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting 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 negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes (signaling is mediated via PD-L1), thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a particular aspect, the anti-PD-L1 antibody is yw243.55.s70 as described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1105 as described herein. In yet another specific aspect, the anti-PD-L1 antibody is MPDL3280A described herein. In yet another specific aspect, the anti-PD-L1 antibody is MEDI4736 described herein.

The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits PD-L2 from binding to one or more of its binding partners. In a particular aspect, the PD-L2 binding antagonist inhibits PD-L2 from binding 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 resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In one embodiment, the PD-L2 binding antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes (signaling is mediated via PD-L2), thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. The term includes the common requirement that antigen recognition can occur, but that the subsequent immune response is exhausted and/or anergic, which is ineffective in controlling infection or tumor growth.

As used herein, the term "dysfunction" also includes an inability to sense or respond to antigen recognition, in particular, an impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2), and/or target cell killing.

The term "anergy" refers to incomplete or inadequate signal resulting from delivery via a T cell receptor (e.g., intracellular Ca in the absence of ras activation⁺²Increased) unresponsive to an antigenic stimulus. T cell anergy can also occur following stimulation with antigen in the absence of co-stimulation, generating cells that become insensitive to subsequent activation of the antigen even in the context of co-stimulation. The non-responsive state can often be overridden by the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and/or gain effector function.

The term "depletion" refers to the depletion of T cells as a state of T cell dysfunction resulting from sustained TCR signaling that occurs during many chronic infections and cancers. It is distinguished from anergy in that it does not occur via incomplete or absent signaling, but rather occurs as a result of sustained signaling. It is defined by poor effector function, sustained inhibitory receptor expression and a transcriptional state that is distinct from the transcriptional state of functional effector or memory T cells. Depletion prevents optimal control of infection and tumors. Depletion may result from both extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) and cell intrinsic negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).

By "enhancing T cell function" is meant inducing, causing or stimulating a T cell to have sustained or amplified biological function, or restoring or reactivating exhausted or reactivatedInactive T cells. Examples of enhancing T cell function include: elevated levels from CD8 relative to pre-intervention such levels⁺Gamma-interferon secretion by T cells, increased proliferation, increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance). In one embodiment, the level of enhancement 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 dysfunctional disorder" is a T cell disorder or condition characterized by decreased responsiveness to antigenic stimuli. In a specific embodiment, the T cell dysfunctional disorder is a disorder specifically associated with inappropriately elevated signaling via PD-1. In another embodiment, a T cell dysfunctional disorder is one in which the T cell is anergic or has reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. In a particular aspect, the reduced responsiveness results in ineffective control of the pathogen or tumor expressing the immunogen. Examples of T cell dysfunctional disorders characterized by T cell dysfunction include unresolved acute infection, chronic infection and tumor immunity.

"tumor immunity" refers to the process by which a tumor evades immune recognition and clearance. As such, as a therapeutic concept, tumor immunity is "treated" when such evasion is diminished, and the tumor is 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 aids in the elimination of tumor cells by immune response. Examples of enhancing tumor immunogenicity include treatment with PD-1 axis binding antagonists and OX40 binding agonists.

"sustained response" refers to a sustained effect on reducing tumor growth after treatment is stopped. For example, the tumor size may remain the same or smaller than the size at the beginning of the administration phase. In some embodiments, the sustained response has a duration 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 length of the duration of treatment.

The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and free of other ingredients that would have unacceptable toxicity to the subject to whom the formulation will be administered. Such formulations are sterile. "pharmaceutically acceptable" excipients (vehicles, additives) are those active ingredients that are employed so as to provide an effective dosage for proper administration to the subject mammal.

As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the treated individual or cell during the course of clinical pathology. Desirable effects of treatment include reducing the rate of disease progression, ameliorating or alleviating the disease state, and regression or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated, including but not limited to reduced proliferation (or destruction) of cancerous cells, reduced symptoms resulting from the disease, increased quality of life of those individuals with the disease, reduced dosages of other drugs required to treat the disease, and/or prolonged survival of the individual.

As used herein, "delaying the progression of a disease" means delaying, hindering, slowing, delaying, stabilizing, and/or delaying the development of a disease (such as cancer). This delay can be of varying lengths of time depending on the disease history and/or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay may essentially encompass prevention, as the individual does not develop disease. For example, the formation of late stage cancer, such as metastasis, may be delayed.

An "effective amount" is at least the minimum amount necessary to achieve a measurable improvement or prevention of a particular condition. An effective amount herein may vary with factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also an amount where the therapeutically beneficial effect exceeds any toxic or adverse effects of the treatment. For prophylactic use, beneficial or desired results include results such as elimination or reduction of risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presented during disease development. For therapeutic use, beneficial or desired results include clinical results, such as reducing one or more symptoms resulting from the disease, increasing the quality of life of those subjects with the disease, decreasing the dosage of another drug needed to treat 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 a cancer or tumor, the effective amount of the drug is to reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and desirably stop) tumor metastasis; inhibit tumor growth to some extent; and/or may have an effect in alleviating to some extent one or more symptoms associated with the condition. An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to effect prophylactic or therapeutic treatment, either directly or indirectly. As understood in the clinical setting, an effective amount of a drug, compound, or pharmaceutical composition can be achieved with or without another drug, compound, or pharmaceutical composition. As such, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if, together with one or more other agents, the desired result or results are achieved.

As used herein, "associated with/combined with/together with … …" refers to the administration of one therapeutic modality over another. Thus, "associated with/combined with/together with … …" refers to the administration of one therapeutic modality before, during, or after the administration of another therapeutic modality to an individual.

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

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

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

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial diffusible melanoma, lentigo malignant melanoma, acromelanoma, nodular melanoma, multiple myeloma, and B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunocytogenic NHL, high grade lymphoblastic NHL, high grade small non-nucleated NHL, storage disease (bulk disease) NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's (Waldenstrom) 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 nevus (phakomatoess), edema (such as associated with brain tumors) and megger's (Meigs) syndrome, brain tumors and brain cancers, as well as head and neck cancers, and associated metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma (carcinoid carcinosa), head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Also, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast cancer, including metastatic forms of those cancers.

As used herein, the term "cytotoxic agent" refers to any agent that is detrimental to a cell (e.g., causes cell death, inhibits proliferation, or otherwise impedes cell function). Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At)²¹¹，I¹³¹，I¹²⁵，Y⁹⁰，Re¹⁸⁶，Re¹⁸⁸，Sm¹⁵³，Bi²¹²，P³²，Pb²¹²And radioactive isotopes of Lu); a chemotherapeutic agent; a growth inhibitor; enzymes and fragments thereof such as nucleolytic enzymes; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Exemplary cytotoxic agents may be selected from the group consisting of antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitorsAgents, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutics, pro-apoptotic agents, inhibitors of LDH-a, inhibitors of fatty acid biosynthesis, inhibitors of cell cycle signaling, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism. In one embodiment, the cytotoxic agent is a taxane (taxane). In one embodiment, the taxane is paclitaxel (paclitaxel) or docetaxel (docetaxel). In one embodiment, the cytotoxic agent is a platinum agent. In one embodiment, the cytotoxic agent is an antagonist of EGFR. In one embodiment, the antagonist of EGFR is N- (3-ethynylphenyl) -6, 7-bis (2-methoxyethoxy) quinazolin-4-amine (e.g., erlotinib). In one embodiment, the cytotoxic agent is a RAF inhibitor. In one embodiment, the RAF inhibitor is a BRAF and/or CRAF inhibitor. In one embodiment, the RAF inhibitor is vemurafenib (vemurafenib). In one embodiment, the cytotoxic agent is a PI3K inhibitor.

"chemotherapeutic agents" include compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (erlotinib) (ll:)Genentech/OSI Pharm), bortezomib (bortezomib), (b), (dMillennium Pharm), disulfiram (disulphiram), epigallocatechin gallate (epigallocatechin gallate), salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol (radicol), lactate dehydrogenase A (LDH-A), fulvestrant (fulvestrant) ((r))AstraZeneca), sunitinib (sunitinb), (sunitinibPfizer/Sugen), letrozole (letrozole), (L-Toxole)Novartis), imatinib mesylate (imatinib mesylate), (I) a salt of N, NNovartis)，finasunate(Novartis), oxaliplatin (oxaliplatin) ((oxaliplatin)Sanofi), 5-FU (5-fluorouracil), leucovorin (leucovorin), Rapamycin (Rapamycin) (Sirolimus),wyeth), Lapatinib (Lapatinib), (Lapatinib)GSK572016, Glaxo Smith Kline), Lonafami (SCH66336), Sorafenib (sorafenib) (S) (G) (Bayer Labs), gefitinib (gefitinib) ((E) gefitinibAstraZeneca), AG1478, alkylating agents (alkylating agents), such as thiotepa and thiotepaCyclophosphamide (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodopa), carboquone (carboquone), metoclopramide (meteredopa), and uretepa (uredpa); ethyleneimines and methylmelamines, including altretamine, triethylenemelamine (triethyleneamine, triethylenephosphoramide (triethylenephosphoramide), triethylenethiophosphoramide (triethylenephosphoramide) and trimethymetamine (trimethymethamine), annonaceous acetogenins (acetogenins) (especially bullatacin and bullatacin), camptothecins (camptothecins) (including topotecan and irinotecan), bryostatin (britin), calalystatins (CC-1065 (including adozelesin), camphol (carzinan) and bizelesin (bizelesin) synthetic analogues), cryptophycins (cryptophycins) (especially phycoerythrin 1 and phycoerythrin 8), camphol (carmustine) and bizelesin (bizelesin), cystatin (carmustine, estramustin (acidine, carmustine (indole, carmustine (indole, carmustine (indole, carmustine), carmustine, indole, carmustine, indole, carmustine (indole, carmustine, carmusti; Epothilones (esperamicins); and neocarzinostatin (neocarzinostatin) chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin (acarinomysins), actinomycin (actinomycin), anthranilic (authramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), carbacetin, carminomycin (caminomycin), carcinomycin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditorelbumin (detorubicin), 6-diaza-5-oxo-L-norleucine,(doxorubicin)), morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcelomycin), mitomycin (mitomycin) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (polyplomycin), pofiomycin (porfiromycin), puromycin (puromycin), triiron doxorubicin (quelamycin), rodobicin (rodorubicin), streptonigrin (strepatorigrin), streptozocin (streptazocin), tubercidin (tubicin), wudimethicin (zionexen), zocin (zorubicin), zorubicin (zoxidin), zoxitin (zoxitin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinol (calusterone), dromostanolone propionate, Epiepitioandrostanol (epitiostanol), mepiquitane (mepithiostane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (frilic acid); acetoglucurolactone (acegultone); an aldophosphamide glycoside (aldophosphamideglycoside); aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defofamine); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfosmithine; ammonium etitanium acetate; an epothilone; etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidainine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidamnol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine); Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane (rizoxane); rhizomycin (rhizoxin); sisofilan (sizofuran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin (verrucin) A, bacillocin (roridin) A and snakes (anguidine); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); cyclophosphamide (cyclophosphamide); thiotepa (thiotepa); taxols (taxoids), such as TAXOL (TAXOL) (paclitaxel; Bristol-Mye)rs Squibb Oncology,Princeton,N.J.)，(Cremophor-free), Albumin-engineered nanoparticle dosage forms of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and taxotere(docetaxel, doxetaxel); Sanofi-Aventis); chlorambucil (chlorembucil);(gemcitabine); 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs such as cisplatin (cissplatin) and carboplatin (carboplatin); vinblastine (vinblastine); etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); vincristine (vincristine); (vinorelbine); oncostatin (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); capecitabine (capecitabine)Ibandronate (ibandronate); CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids (retinoids), such as retinoic acid (retinoic acid); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Chemotherapeutic agents also include (i) anti-hormonal agents that act to modulate or inhibit the action of hormones on tumors, such as anti-estrogens and Selective Estrogen Receptor Modulators (SERMs), including, for example, tamoxifen (includingTamoxifen citrate), raloxifene (raloxifene), droloxifene (droloxifene), iodoxyfene, 4-hydroxytamoxifene, trioxifene (trioxifene), naloxifene (keoxifene), LY117018, onapristone (onapristone), and(toremifene citrate); (ii) aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal gland, such as, for example, 4(5) -imidazole, aminoglutethimide,(megestrol acetate), (exemestane); Pfizer), formestane (formestanie), fadrozole (fadrozole),(vorozole),(letrozole; Novartis), and(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprolide (leuprolide), and goserelin (goserelin); buserelin (buserelin), triptorelin (tripterelin), medroxyprogesterone acetate (medroxyprogesterone acetate), diethylstilbestrol (diethylstilbestrol), bemese (premarin), fluoxymesterone (fluoroxymesterone), all-trans retinoic acid, fenretinide (fenretinide), and troxacitabine (troxacitabine) (1, 3-dioxolane nucleoside cytosine analogues); (iv) protein kinase inhibitors; (v) a lipid kinase inhibitor; (vi) antisense oligonucleotides, particularly for inhibiting signaling involving abnormal cell proliferationAntisense oligonucleotides that direct gene expression in a pathway, such as, for example, PKC- α, Ralf, and H-Ras, (vii) ribozymes, such as VEGF expression inhibitors (e.g., as) And inhibitors of HER2 expression; (viii) vaccines, such as gene therapy vaccines, e.g. AndrIL-2; topoisomerase 1 inhibitors, such asrmRH; and (ix) and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

Chemotherapeutic agents also include antibodies, such as alemtuzumab (Campath), bevacizumab (bevacizumab)Genentech); cetuximab (cetuximab) (C)Imclone); panitumumab (panitumumab)Amgen), rituximab (rituximab), (b)Genentech/Biogen Idec), pertuzumab (pertuzumab) ((ii) Genentech/Biogen Idec), (ii) and (iii) pharmaceutically acceptable salts thereof2C4, Genentech), trastuzumab (trastuzumab) ((R) 2C4, Genentech)Genentech), tositumomab (tosit)umomab (Bexxar, Corixia), and antibody drug conjugates, gemtuzumab ozogamicin (gemtuzumab ozogamicin) ((r))Wyeth). Additional humanized monoclonal antibodies that have therapeutic potential as agents in combination with the compounds of the invention include: aprezumab (apilizumab), aselizumab (aselizumab), atlizumab (atlizumab), palivizumab (bapineuzumab), bivatuzumab mertansine, mocratizumab (cantuzumab mertansine), sijilizumab (cedelizumab), pemirolizumab (certolizumab), pemphilizumab (certolizumab), cidfutuzumab (ciduzumab), ciduzumab (ciduzumab), daclizumab (daclizumab), eculizumab (eculizumab), efuzumab (efuzumab), epratuzumab (epratuzumab), erlizumab (erlipizumab), polvivizumab (feluzumab), phentuzumab (fontuzumab), fonuzumab (fontizumab), geminuzumab (gemtuzumab ozogamicin), trastuzumab (zezumab), trastuzumab (fonuzumab), trastuzumab (matuzumab), trastuzumab (gemtuzumab), trastuzumab (gemtuzumab (geminizumab), trastuzumab (metuzumab), trastuzumab (matuzumab), trastuzumab (gemtuzumab (matuzumab), trastuzumab (gemtuzumab (matuzumab), trastuzumab (gemtuzumab), trastuzumab (matuzumab), trastuzumab (mat, paclobutrazumab (paclobutrazumab), pecuutizumab, petuuzumab, pexizumab (pexizumab), rilizumab, ranibizumab (ranibizumab), relivizumab, rayleigh mab (relizumab), resyvizumab (reslizumab), rovizumab (rovelzumab), luluzumab (ruplizumab), siruzumab (sibutrumab), sibuzumab (sibilizumab), solizumab (siplizumab), solituzumab (sontuzumab), sontezumab (sontuzumab), tacatuzumab, taduzumab (taluzumab), tefilzumab (tebuczumab), tolizumab (tocuzumab), tollizumab (toralizumab), interleukin (tuzumab), interleukin (interleukin), wumocukin (ulizumab), cujuzumab (ilezumab), interferon (abcuzumab), and (abcuizumab/wuvizumab), rabucizumab (tuzumab/wubix), rabizumab (tuzumab), tuzumab (tuvukiuk), tuzumab (tuvuzumab (tuvukiyukiyukiyu (tuvu), tuzumab (tachizu), and (rabuk), rabuk (abyutsutuzumab, beuk (rabuikovit), tachizu (abcuikovit), beuk (rab), beuk (rab), be Recombinant human specific sequence full-length IgG genetically modified to recognize interleukin-12 p40 protein₁Lambda antibodies.

Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, which are otherwise also referred to as "EGFR antagonists. Examples of such agents include antibodies and small molecules that bind EGFR. Examples of EGFR-binding antibodies include MAb 579(ATCC CRL HB8506), MAb455(ATCC CRL HB8507), MAb 225(ATCC CRL 8508), MAb 528(ATCC CRL 8509) (see U.S. patent No.4,943,533, Mendelsohn et al) and variants thereof, such as chimeric 225(C225 or cetuximab;) And reconstituted human 225(H225) (see WO96/40210, Imclone Systems Inc.; IMC-11F8, a fully human EGFR-targeting antibody (Imclone); antibodies that bind to type II mutant EGFR (U.S. Pat. No.5,212,290); humanized and chimeric antibodies that bind to EGFR as described in U.S. Pat. No.5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or Panitumumab (Panitumumab) (see WO98/50433, Abgenix/Amgen); EMD55900 (Straglitoto et al, Eur.J. Cancer 32A:636-640(1996)), EMD7200(matuzumab), a humanized antibody that is directed against EGFR and competes for binding with both EGFR and EGF (EMD/Merck), human antibody, Huzhu-121, GFR 201, GmG 73,201, and EGF), and humanized antibodies that bind to EGFR (EMD/Merck) and that bind to EGFR, such as EGFR, E5,120, 35, 120, 120,120, 35, 120,120, 120,120,120,120, 120,120, 120,120,120, 120, 120,120,120,120,150, 120,120,344,150, 7,150, 97, and similar to EGFR, 7,120,120,150, 23,150, 35,150, 23,150, such as described in U.7,150, 6,150, 35,150, 23,150, 35,150, and similar to EGFR, 23,150, 35,150, similar to EGFR, 23,150, similar to EGFR, such as described in U.E, and similar antibodies, such as described in U.S. Pat. No. 3,120,120,120,120,150, 7,120,120,120,120,150, 7,120,120,150, 7,120,120,120,120,150, 7,150, and similar to EGFR, 7,150, 7,120,150, 7,150, and similar to EGFR, 7,120,120,150, and similar to Compounds described in WO98/14451, WO98/50038, WO99/09016, and WO 99/24037. Specific small molecule EGFR antagonists include OSI-774(CP-358774, erlotinib,Genentech/OSI Pharmaceuticals); PD 183805(CI 1033, 2-propenamide, N- [4- [ (3-chloro-4-fluorophenyl) amino)]-7- [3- (4-morpholinyl) propoxy]-6-quinazolinyl]Dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (gefitinib)4- (3 '-chloro-4' -fluoroanilino) -7-methoxy-6- (3-morpholinopropoxy) quinazoline, AstraZeneca); ZM 105180 ((6-amino-4- (3-methylphenyl-amino) -quinazoline, Zeneca); BIBX-1382(N8- (3-chloro-4-fluoro-phenyl) -N2- (1-methyl-piperidin-4-yl) -pyrimido [5, 4-d)]Pyrimidine-2, 8-diamine, Boehringer Ingelheim); PKI-166((R) -4- [4- [ (1-phenylethyl) amino)]-1H-pyrrolo [2,3-d]Pyrimidin-6-yl]-phenol); (R) -6- (4-hydroxyphenyl) -4- [ (1-phenylethyl) amino group]-7H-pyrrolo [2,3-d]Pyrimidines); CL-387785(N- [4- [ (3-bromophenyl) amino)]-6-quinazolinyl]-2-butynylamide); EKB-569(N- [4- [ (3-chloro-4-fluorophenyl) amino group]-3-cyano-7-ethoxy-6-quinolinyl]-4- (dimethylamino) -2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571(SU 5271; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors, such as lapatinib (lapatinib) ((R)) GSK572016 or N- [ 3-chloro-4- [ (3-fluorophenyl) methoxy group]Phenyl radical]-6[5[ [ [ 2-methylsulfonyl) ethyl group]Amino group]Methyl radical]-2-furyl radical]-4-quinazolinamines).

Chemotherapeutic agents also include "tyrosine kinase inhibitors" including the EGFR-targeting drugs mentioned in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724,714, an oral ErbB2 receptor tyrosine kinase selective inhibitor (Pfizer and OSI); dual HER inhibitors that preferentially bind EGFR but inhibit both HER2 and EGFR overexpressing cells,such as EKB-569 (available from Wyeth); lapatinib (GSK 572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); raf-1 inhibitors, such as antisense agents ISIS-5132, available from ISIS pharmaceutical 1s, that inhibit Raf-1 signaling; non-HER targeted TK inhibitors, such as imatinib mesylate (i: (ii)Available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors, such as sunitinib (sunitinib) ((iii))Available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035, 4- (3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2,3-d ]Pyrimidines; curcumin (diferuloylmethane, 4, 5-bis (4-fluoroanilino) -phthalimide); tyrphostine containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to a nucleic acid encoding HER); quinoxalines (U.S. patent No.5,804,396); trypostins (U.S. Pat. No.5,804,396); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); pan HER inhibitors such as CI-1033 (Pfizer); affinitac (ISIS 3521; ISIS/Lilly); imatinib mesylatePKI166 (Novartis); GW2016(Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (pfizer); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1C11(Imclone), rapamycin (sirolimus,) (ii) a Or any of the following patent publications: U.S. patent nos. 5,804,396; WO1999/09016(American Cyanamid); WO1998/43960(American cyanamid); WO1997/38983(Warner Lambert); WO1999/06378(Warner Lambert); WO1999/06396 (WarnerLambert); WO1996/30347(Pfizer, Inc); WO1996/33978 (Zeneca); WO1996/3397(Zeneca) and WO1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone (dexamethasone), interferon, colchicine (colchicine), chlorphenamine (methaprine), cyclosporine (cyclosporine), amphotericin (amphotericin), metronidazole (metronidazole), alemtuzumab (alemtuzumab), alitretinoin (alitretinoin), allopurinol (allopurinol), amifostine (amifostine), arsenic trioxide (arsenical trioxide), asparaginase (asparagase), live BCG, bevacizumab (bevacizumab), bexarotene (bexarotene), cladribine (cladribine), ritaline (clofanabine), darbevacetin alfa, denbuzin (dexrazine), dexrazoxane (dexamectin), interferon alpha (interferon alpha-2), interferon alpha (interferon alpha, 2-acetate), nelarabine (nellabine), norflumumab (nofetumumab), opril (oprefletekin), palifermin (pamidronate), pemetrexed (peganurate), pegamphenzyme (pegademase), pemetrexed (pegaspartase), PEG filgrastim (pegfilgrastim), pemetrexed disodium (pemetrexed disodium), plicamycin (plicamycin), porfimer sodium (porfimer sodium), quinacrine (quinacrine), rasburiase (rasburicase), sargrastim (sargramostim), temozolomide (temozolomide), VM-26, 6-TG, toremifene (toremifene), tretinoin, ATRA, valrubicin (valrubicin), zoledronate (letronate), and zoledronate (zoledronate), and pharmaceutically acceptable salts thereof.

The chemotherapeutic agent further comprises hydrocortisone (hydrocortisone)) Hydrocortisone acetate (hydrocortisone acetate), cortisone acetate (cortisone acetate), tixocortol pivalate (tixocortol pivalate), triamcinolone acetonide (triamcinolone acetonide), triamcinolone alcohol (triamcinolone alcohol), mometasone (mometasone), amcinolone acetonide), budesonide (budesonide), desonide (desonide), fluocinolone acetonide acetate (betamethasone), betamethasone sodium phosphate (betamethasone phosphate), dexamethasone (dexamethosone), dexamethasone sodium phosphate (dexamethosone phosphate), fluocinolone acetonide (flumethasone), hydrocortisone (hydrocortisone), hydrocortisone-17-butyrate (hydrocortisone-17-tyrosone propionate), interferon acetate (corticotropin-17-acetate), interferon (interferon-citrate), interferon (interferon-2), interferon-2-acetate (interferon-citrate), interferon-2-citrate (interferon-2-acetate), interferon-citrate-2, interferon-2-citrate, interferon-2-acetate, interferon-2-citrate, interferon-2-citrate, and a-2-citrate, a-2, a-2-a, a-a Interleukin-13 (IL-13) blockers, such as lebrikizumab, interferon α (IFN) blockers, such as Rontalizumab, β7-integrin blockers such as rhuMAb Beta7, IgE pathway blockers such as anti-M1 prime, secretory homotrimeric LTa3 and membrane-bound heterotrimeric LTa1/β 2 blockers such as anti-lymphotoxin α (LTa), radioisotopes, e.g., At²¹¹，I¹³¹，I¹²⁵，Y⁹⁰，Re¹⁸⁶，Re¹⁸⁸，Sm¹⁵³，Bi²¹²，P³²，Pb²¹²And a Lu radioisotope; miscellaneous investigational agents, such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH₃Or farnesyltransferase inhibitors (L-739749, L-744832; polyphenols, such as quercetin (quercetin), resveratrol (resveratrol), piceatannol, epigallocatechin gallate (epigallocatechin gallate), theaflavin (theaflavin), flavanols (flavanols), procyanidins (procyanidins), betulinic acid (betulinic acid) and derivatives thereof; autophagy inhibitors, such as chloroquine, -9-tetrahydrocannabinol (dronol),) β -lapachone (lapachone), lapachol (lapachol), colchicines (colchicines), betulinic acid (betulinic acid), acetyl camptothecin, scopoletin (scopolectin), and 9-aminocamptothecin), podophyllotoxin (podophyllotoxin), tegafur (tegafur) Bexarotene (bexarotee)Diphosphonates (bisphosphates), such as clodronate (e.g. clodronate)Or) Etidronate sodium (etidronate)NE-58095, zoledronic acid/zoledronateAlendronate (alendronate)Pamidronate (pamidronate)Tiludronate (tirudronate)Or risedronate (risedronate)And epidermal growth factor receptor (EGF-R); vaccines, e.g.A vaccine; perifosine (perifosine), COX-2 inhibitors (e.g., celecoxib (celecoxib) or etoricoxib (etoricoxib)), proteosome inhibitors (e.g., PS 341); CCI-779; tipifarnib (R11577); orafenaib, ABT 510; bcl-2 inhibitors, such as oblimersen sodiumpixantrone; farnesyl transferase inhibitors, such as lonafarnib (SCH 6636, SARASAR)^TM) (ii) a And pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine, and prednisolone combination therapy) and FOLFOX (oxaliplatin)^TM) Abbreviation for treatment regimen combining 5-FU and folinic acid).

Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs having analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin (aspirin), propionic acid derivatives such as ibuprofen (ibuprolofen), fenoprofen (fenoprofen), ketoprofen (ketoprofen), flurbiprofen (flurbiprofen), oxaprozin (oxazin) and naproxen (naproxen), acetic acid derivatives such as indomethacin (indomethacin), sulindac (sulindac), etodolac (etodolac), diclofenac (difofenac), enolic acid derivatives such as piroxicam (piroxicam), meloxicam (meloxicam), tenoxicam (tenoxicam), oxaxicam (droxicam), lornoxicam (lornoxicam) and isoxicam (isoxicam), fenamic acid derivatives such as mefenamic acid (mefenamic acid), meclofenamic acid (clofenamic acid), flufenamic acid (COX), fenamic acid (fenamic acid), fenamic acid (COX), fenamic acid (2-loxb), etoricoxib (etoricoxib), etoricoxib (loxacin (loxb), etoricoxib (loxacin), etoricoxib), rofecoxib (rofecoxib), and valdecoxib (valdecoxib). NSAIDs may be indicated for symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory joint disease, ankylosing spondylitis, psoriatic arthritis, reiter's syndrome, acute gout, dysmenorrhea, bone metastasis pain, headache and migraine, post-operative pain, mild to moderate pain due to inflammation and tissue injury, fever, ileus, and renal colic.

"growth inhibitory agent" as used herein refers to a compound or composition that inhibits cell growth in vitro or in vivo. In one embodiment, the growth inhibitory agent is a growth inhibitory antibody that prevents or reduces proliferation of cells expressing the antigen to which the antibody binds. In another embodiment, the growth inhibitory agent may be an agent that significantly reduces the percentage of cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a position outside the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M-phase blockers include the vincas (vincristine) and vinblastine (vinblastine)), taxanes (taxanes), and topoisomerase II inhibitors such as doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide), and bleomycin (bleomycin). Those retardationAgents of G1 also spill over into S-phase arrest, for example DNA alkylating agents such as tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), mechlorethamine (mechloroethylamine), cisplatin (cissplatin), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil) and ara-C. For more information see, e.g., The eds of Mendelsohn and Israel, The Molecular basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and anticancer drugs", Murakami et al, WB Saunders, Philadelphia, 1995, e.g., page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel derived from taxus baccata (c) Rhone-Poulenc Rorer is Parietai (R) ((R))Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.

"radiotherapy" or "radiotherapy" refers to the use of directed gamma rays or beta rays to induce sufficient damage to cells to limit their ability to function normally or to destroy them altogether. It will be appreciated that many ways are known in the art to determine the dosage and duration of treatment. Typical treatments are given as one administration, while typical doses range from 10-200 units per day (Gray).

"subject" or "individual" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and livestock animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cattle, etc. Preferably, the mammal is a human.

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

An "isolated" antibody refers to an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to materials that would interfere with the research, diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified to (1) greater than 95% by weight, and in some embodiments greater than 99% by weight, as determined by, for example, the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using, for example, a rotor cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions and staining with, for example, Coomassie blue or silver. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, an isolated antibody will typically be prepared by at least one purification step.

"native antibody" refers to a heterotetrameric glycoprotein of about 150,000 daltons, typically composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V) at one end _H) Followed by a plurality of constant domains. Each light chain has a variable domain (V) at one end_L) And the other end is a constant domain; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form the interface between the light and heavy chain variable domains.

The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to the other portions of the immunoglobulin, i.e., the variable domains that contain the antigen binding site.Constant domain heavy chain-containing C_H1，C_H2 and C_H3 domains (collectively CH) and the CHL (or CL) domain of the light chain.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "V_H". The variable domain of the light chain may be referred to as "V_L". These domains are generally the most variable parts of an antibody and contain an antigen binding site.

The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of the antibodies. It is concentrated in three segments called hypervariable regions (HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β -sheet conformation, connected by three HVRs that form loops connecting, and in some cases forming part of, the β -sheet structure. The HVRs in each chain are held together in close proximity by the FR region and, together with the HVRs of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

The "light chain" of an antibody (immunoglobulin) from any mammalian species can be classified into one of two distinct types, called kappa ("κ") and lambda ("λ"), depending on the amino acid sequence of its constant domains.

As used herein, the term IgG "isotype" or "subclass" means any immunoglobulin subclass defined by the chemical and antigenic characteristics of its constant regions.

Antibodies based on the amino acid sequence of the constant domain of their heavy chains(immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which may be further divided into subclasses (isotypes), e.g. IgG₁，IgG₂，IgG₃，IgG₄，IgA₁And IgA₂The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al, Cellular and mol.

The terms "full-length antibody" and "intact antibody" are used interchangeably herein to refer to the antibody in substantially intact form, rather than the antibody fragment defined below. The term specifically refers to antibodies in which the heavy chain comprises an Fc region.

"naked antibody" (naked antibody) "for the purposes of the present invention refers to an antibody which is not conjugated to a cytotoxic moiety or a radiolabel.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. In some embodiments, the antibody fragment described herein is an antigen binding fragment. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having an antigen-binding site, and a remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produced an F (ab')₂A fragment which has two antigen binding sites and is still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, the two-chain Fv species consists of a tightly, non-covalently bound oneA dimer of one heavy chain variable domain and one light chain variable domain. In the single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain may be covalently linked by a flexible peptide linker, such that the light and heavy chains may associate in a "dimeric" structure analogous to that of a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact at V _H-V_LAn antigen binding site is defined on the surface of the dimer. Together, the six HVRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, with only a lower affinity than the entire binding site.

The Fab fragment comprises the heavy and light chain variable domains, and further comprises the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain 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 domain carry a free thiol group. F (ab')₂Antibody fragments were originally generated as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for binding to an antigen. For reviews of scFv see for example Pl ü ckthun, in The Pharmacologyof Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, 1994, p.269-315.

The term "diabodies" refers to antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) linked in the same polypeptide chain (VH-VL). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain, thereby creating two antigen binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Tri-antibodies (Triabody) and tetra-antibodies (tetrabody) are also described in Hudson et al, nat. Med.9:129-134 (2003).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates that the antibody is not characteristic of a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies typically comprise an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones such as hybridoma clones, phage clones or recombinant DNA clones. It will be appreciated that the target binding sequence selected may be further altered, for example to improve affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins.

The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, Monoclonal Antibodies used In accordance with the present invention may be generated 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, 2 nd edition 1988); Hammerling et al, In: Monoclonal Antibodies and T-Cell hybrids: 681(Elsevier, N.Y.,1981)), the recombinant DNA method (see, for example, U.S. Pat. No.4,816,567), the phage display technique (see, for example, Clackson et al., 352: 624: 628 (1991); J.Acad.1247, Biokl et al, WO 32: 96; 20. J.31, Biokl et al., 2000: 96; (Lellson et al., USA) 340. 99.72. J.55, Biokl et al: 96; (Legend et al., 3. 31, USA) and No. 32, methods 284(1-2):119-132(2004)), and techniques for producing human or human-like antibodies in animals having part or all of a human immunoglobulin locus or a gene encoding a human immunoglobulin sequence (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, Yeast in Immuno.7:33 (1993); U.S. patent nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; and 5,661,016; marks et al, Bio/Technology10:779-783 (1992); lonberg et al, Nature 368:856-859 (1994); morrison, Nature 368: 812-; fishwild et al, Nature Biotechnol.14: 845-; neuberger, Nature Biotechnol.14:826 (1996); lonberg and Huszar, Intern.Rev.Immunol.13:65-93 (1995)).

Monoclonal antibodies specifically include "chimeric" antibodies wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include "primatized" antibodies in which the antigen binding region of the antibody is derived from an antibody produced, for example, by immunizing macaques with an antigen of interest.

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In one embodiment, a humanized antibody is one in which HVR residues 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 having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see, e.g., Jones et al, Nature321:522-525 (1986); riechmann et al, Nature 332: 323-; and Presta, curr, Op, Structure, biol.2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol.1:105-115 (1998); harris, biochem. Soc. Transactions23: 1035-; hurle and Gross, curr. Op. Biotech.5: 428-; and U.S. patent nos. 6,982,321 and 7,087,409.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries and Winter (J.mol.biol.227: 381 (1991); Marks et al, J.mol.biol.222:581 (1)991)). Also useful for the preparation of human monoclonal antibodies are the methods described in the following references: 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 deWinkel, curr, opin, pharmacol, 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal, such as an immunized XENOMOUSE (xenomice), that has been modified to produce human antibodies in response to antigenic stimuli but whose endogenous genome has been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for Xenomose^TMA technique). See also, e.g., Li et al, Proc. Natl.Acad.Sci.USA,103:3557-3562(2006), for human antibodies generated via human B-cell hybridoma technology.

"species-dependent antibody" refers to an antibody that has a stronger binding affinity for an antigen from a first mammalian species than for a homolog of the antigen from a second mammalian species. Typically, a species-dependent antibody "specifically binds" to a human antigen (e.g., binding affinity (K)_d) A value of no more than about 1x 10^-7M, preferably not more than about 1x 10^-8M, and preferably no more than about 1x 10^-9M), but has a binding affinity for a homolog of the antigen from the second non-human mammalian species that is at least about 50-fold weaker than the binding affinity for a human antigen, or at least about 500-fold weaker than the binding affinity for the human antigen, or at least about 1000-fold weaker than the binding affinity for the human antigen. The species-dependent antibody may be any of the various types of antibodies defined above, but is preferably a humanized or human antibody.

The terms "hypervariable region", "HVR" or "HV", when used herein, refer to regions of an antibody variable domain which are hypervariable 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). Among natural antibodies, H3 and L3 show the greatest diversity of these six HVRs, and H3 in particular is thought to play a unique role in conferring precise specificity to antibodies. See, e.g., 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 of only heavy chains, are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al Nature 363:446 + 448 (1993); sheffet al Nature Structure biol.3:733-736, (1996).

A description of many HVRs is used and is contemplated herein. 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 instead refers to the position of the structural loops (Chothia and Lesk J.mol.biol.196:901-917 (1987)). The AbM HVR represents a compromise between the Kabat HVR and Chothia structural loops, and results in the use of oxford molecular's AbM antibody modeling software. The "contact" HVRs are based on analysis of the available complex crystal structure. The residues for each of these HVRs are recorded below.

The HVRs can include the following "extended HVRs": 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.

"framework" or "FR" residues refer to those residues in the variable domain other than the HVR residues as defined herein.

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

The Kabat numbering system is generally used when referring to residues in the variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al, Sequences of Immunological interest.5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in an immunoglobulin heavy chain constant region (e.g., Kabat et al, see EU index reported above). "EU index as in Kabat" refers to the residue numbering of the human IgG1EU antibody.

The expression "linear antibody" refers to the antibodies described in Zapata et al (1995) Protein Eng,8(10): 1057-1062. Briefly, these antibodies comprise a pair of Fd segments (VH-CH1-VH-CH1) in tandem that form, with a complementary light chain polypeptide, a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

As used herein, the terms "bind," "specific binding," or "specific for …" refer 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 binds or specifically binds a target (which may be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In one embodiment, the extent to which the antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds a target has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins from different species. In another embodiment, specific binding may include, but need not be exclusive binding.

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

As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, e.g., a predictive, diagnostic, and/or prognostic indicator. Biomarkers can serve as indicators of particular disease or disorder (e.g., cancer) subtypes characterized by particular molecular, pathological, histological, and/or clinical characteristics. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polynucleotide copy number alterations (e.g., DNA copy number), polypeptides and polynucleotide modifications (e.g., post-translational modifications), carbohydrate and/or glycolipid-based molecular markers.

The terms "biomarker signature", "biomarker expression signature" or "expression signature" are used interchangeably herein to refer to one or a set of biomarkers whose expression is an indicator, e.g., a predictive, diagnostic and/or prognostic indicator. Biomarker signatures can serve as indicators of particular disease or disorder (e.g., cancer) subtypes characterized by particular molecular, pathological, histological, and/or clinical features. In some embodiments, the biomarker signature is a "gene signature". The term "gene signature" is used interchangeably with "gene expression signature" and refers to a polynucleotide or set of polynucleotides whose expression is an indicator, e.g., a predictive, diagnostic and/or prognostic indicator. In some embodiments, the biomarker signature is a "protein signature". The terms "protein signature" and "protein expression signature" are used interchangeably to refer to a polypeptide or set of polypeptides whose expression is an indicator, e.g., a predictive, diagnostic and/or prognostic indicator.

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

The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a biological sample. "expression" generally refers to the process by which information (e.g., gene coding and/or epigenetic) is converted into structures present and operating in a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Transcribed polynucleotides, translated polypeptides, or fragments of a polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered expressed, whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of a polypeptide (e.g., by proteolysis). "expressed gene" includes genes that are transcribed into a polynucleotide (e.g., mRNA) and then translated into a polypeptide, as well as genes that are transcribed into RNA but not translated into a polypeptide (e.g., transport and ribosomal RNA).

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

"reduced expression," "reduced expression level," or "reduced level" refers to reduced expression or reduced level of a biomarker in an individual relative to a control, such as an individual not suffering from a disease or disorder (e.g., cancer) or an internal control (e.g., a housekeeping biomarker). In some embodiments, the reduced expression is little or no expression.

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

As used herein, "amplification" generally refers to the process of generating multiple copies of a desired sequence. "multicopy" means at least 2 copies. "copy" does not necessarily mean complete sequence complementarity or identity to the template sequence. For example, the copies may comprise nucleotide analogs such as deoxyinosine, intentional sequence changes (such as sequence changes introduced via primers comprising sequences that are hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.

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

The "stringency" of the hybridization reaction can be readily determined by one of ordinary skill in the art, and is generally calculated empirically based on probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it can be concluded that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For additional details and explanations of the stringency of hybridization reactions, see Ausubel et al, Current protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

As defined herein, "stringent conditions" or "high stringency conditions" can be identified by: (1) low ionic strength and high temperature are used for washing, e.g. 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate, 50 ℃; (2) denaturing agents such as formamide, e.g., 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH 6.5 and 750mM sodium chloride, 75mM sodium citrate, 42 ℃; or (3) hybridization overnight at 42 ℃ in a solution using 50% formamide, 5 XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 XDenhart (Denhardt) solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS and 10% dextran sulfate, washing at 42 ℃ for 10 minutes in 0.2 XSSC (sodium chloride/sodium citrate), followed by a 10 minute high stringency wash consisting of EDTA-containing 0.1 XSSC at 55 ℃.

"moderately stringent conditions" can be defined as described in Sambrook et al, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Press,1989, and includes the use of less stringent wash solutions and hybridization conditions (e.g., temperature, ionic strength and% SDS) than those described above. An example of moderately stringent conditions is those comprising: 20% formamide, 5 XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 XDenHart's solution, 10% dextran sulfate and 20mg/ml denatured, sheared salmon sperm DNA solution at 37 degrees C temperature in the temperature of overnight incubation, then at about 37-50 ℃ in 1 XSSC washing filter. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc., as needed to accommodate factors such as probe length.

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

"quantitative real-time polymerase chain reaction" or "qRT-PCR" refers to a form of PCR in which the amount of PCR product is measured at each step of the PCR reaction. This technique has been described in a number of publications including Cronin et al, am.j.pathol.164(1):35-42 (2004); ma et al, Cancer cell.5: 607-.

The term "microarray" refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.

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

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

The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer). For example, "diagnosis" may refer to the identification of a particular cancer type. "diagnosis" may also refer to the classification of a particular cancer subtype, e.g., a subtype characterized by histopathological criteria or molecular features, e.g., expression of one or a set of biomarkers, e.g., a particular gene or protein encoded by the gene.

The term "aiding diagnosis" is used herein to refer to a method of aiding in the clinical determination of the presence or nature of a symptom or condition associated with a particular type of disease or disorder (e.g., cancer). For example, a method of aiding in making a diagnosis of a disease or condition (e.g., cancer) may include measuring a particular biomarker in a biological sample from an individual.

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

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

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

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

"correlating" or "correlating" means comparing the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol in any manner. For example, the results of a first analysis or protocol may be used to implement a second protocol, and/or the results of a first analysis or protocol may be used to decide whether a second analysis or protocol should be implemented. For embodiments of polypeptide analysis or protocols, the results of a polypeptide expression analysis or protocol can be used to determine whether a particular treatment regimen should be implemented. For embodiments of polynucleotide analysis or protocols, the results of the polynucleotide expression analysis or protocol can be used to determine whether a particular treatment protocol should be implemented.

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

"effective response" or "responsiveness" of a patient to drug treatment and the like refer to a clinical or therapeutic benefit administered to a patient at risk for or suffering from a disease or condition, such as cancer. In one embodiment, such benefits include any one or more of the following: extended survival (including overall survival and progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating signs or symptoms of cancer.

Patients who "do not have an effective response" to treatment refer to patients who do not have extended survival (including overall survival and progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating any of the signs or symptoms of cancer.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted immunoglobulins that bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. The main cell mediating ADCC, NK cells, expresses only Fc γ RIII, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravech and Kinet, annu.rev.immunol.9:457-92(1991) page 464 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. Pat. No.5,500,362 or 5,821,337 or U.S. Pat. No.6,737,056 (Presta). Effector cells useful in such assays include PBMC and NK cells. Alternatively/additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models such as those disclosed in Clynes et al, PNAS (USA)95: 652-. An exemplary assay for assessing ADCC activity is provided in the examples herein.

"complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that has bound to its cognate antigen. To assess complement activation, CDC assays can be performed, for example as described in Gazzano-Santoro et al, J.Immunol.methods 202:163 (1996). Polypeptide variants having altered Fc region amino acid sequences (polypeptides having variant Fc regions) and increased or decreased C1q binding ability are described, for example, in U.S. patent No.6,194,551B1 and WO 1999/51642. See also, e.g., Idusogene et al, J.Immunol.164: 4178-.

"depleting anti-OX 40 antibody" refers to an anti-OX 40 antibody that kills or depletes OX40 expressing cells. Depleting OX 40-expressing cells can be accomplished by a variety of mechanisms, such as antibody-dependent cell-mediated cytotoxicity and/or phagocytosis. Depletion of OX 40-expressing cells can be assayed in vitro, and exemplary methods for in vitro ADCC and phagocytosis assays are provided herein. In some embodiments, the OX 40-expressing cell is a human CD4+ effector T cell. In some embodiments, the OX 40-expressing cell is a transgenic BT474 cell expressing human OX 40.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds an IgG antibody (gamma receptor), including receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of those receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). Inhibiting the receptor Fc γ RIIB comprises in its cytoplasmic domain an immunoreceptorTyrosine-based inhibitory motifs (ITIMs) (see, e.g.Annu.Rev.Immunol.15:203-234 (1997)). For reviews of FcRs see, for example, ravechand Kinet, Annu. Rev. Immunol.9:457-492 (1991); capel et al, immunolmethods 4:25-34 (1994); and de Haas et al, J.Lab.Clin.Med.126:330-41 (1995). The term "FcR" encompasses other fcrs herein, including those that will be 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)) and for the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology,15(7):637-640 (1997); Hinton et al., J.biol. chem.279(8):6213-6216 (2004); WO 2004/92219(Hinton et al.)). The in vivo binding and serum half-life of human FcRn high affinity binding polypeptides to human FcRn can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having variant Fc regions. WO 2000/42072(Presta) describes antibody variants with increased or decreased binding to FcR. See also, for example, Shield et al, J.biol.chem.9(2):6591-6604 (2001).

A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q combinations; CDC; fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions generally require that the Fc region be associated with a binding domain (e.g., an antibody variable domain) and can be evaluated using a variety of assays, such as those disclosed in the definitions herein.

"human effector cells" refer to leukocytes which express one or more fcrs and which exert effector function. In certain embodiments, the cell expresses at least Fc γ RIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. The effector cells may be isolated from their natural source, e.g., blood.

A cancer or biological sample "having human effector cells" is a cancer or biological sample in which human effector cells (e.g., infiltrating human effector cells) are present in the sample in a diagnostic test.

A cancer or biological sample "having cells that express FcR" is a cancer or biological sample in which FcR expressing cells (e.g., cells that infiltrate FcR expressing cells) are present in the sample in a diagnostic test. In some embodiments, the FcR is an Fc γ R. In some embodiments, the FcR is an activating Fc γ R.

PD-1 axis binding antagonists

Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist. Also provided herein are methods for enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist.

For example, PD-1 axis binding antagonists include PD-1 binding antagonists, PDL1 binding antagonists and PDL2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB 2. Alternative names for "PDL 1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL 2" include B7-DC, Btdc, and CD 273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL 2.

In some embodiments, the PD-1 binding antagonist is a molecule that inhibits binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PDL1 and/or PDL 2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a particular aspect, the PDL1 binding partner is PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a particular aspect, the PDL2 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 an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of: nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, andis an anti-PD-1 antibody described in WO 2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab,and SCH-900475, an anti-PD-1 antibody described in WO 2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO 2011/066342.

In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registry number 946414-94-4). In yet another embodiment, an isolated anti-PD-1 antibody is provided, comprising a heavy chain variable region sequence from SEQ ID NO: 10 and/or a light chain variable region comprising a light chain variable region amino acid sequence from SEQ ID NO: 11, and a light chain variable region of the light chain variable region amino acid sequence of seq id no. In yet another embodiment, there is provided an isolated anti-PD-1 antibody comprising heavy and/or light chain sequences, wherein:

(a) The heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a heavy chain sequence of seq id no:

QVQLVESGGGVGRSLRLDKASGITFSNSGMHWVRQAGPGLEWvAvIWYDGSKRYYAVKGRFDNSKLFLQMLNADTAVYYCATYDDYWGQGTLVTVSSASTKGPSVFPLAPCSSTSTAALGCLVKDPEVPVLVTVSVVSGALTVHSGQSQQSSGLSVTVTSSVTSSVTSSTKTYTTCDHKPSNKRVSSKKPSKKPCPPCPAPEPHPEPHPEPFLGGPSVFLFPPKPKPVLTPRTPEVTCVVVQEDVVWVDGVGVTGVGVGAKQPVQVQVQVQVQVQVQVSKLVGLSGVGLSVTLSVTLSCKQVPLSGQPSDVSVQVQVQVQVQVSVKSVHVSKLVGVLKSVTLSVTLSVTLSVTLSVTLSGQVQVQVSVQVSVKGFKGQVVLKSVTLSVTLSVTLSVTLSVTLSVTLSVTLSGQVQVQVQVQVQVQVQVVLKSVTLSVTLSVTLSVTLSVTLSVTLSVTLSVTLSVTLSVTLSVTLSVTLSVTLSVTLSVTLSVT

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

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：11)。

in some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS registry number 1374853-91-4). In yet another embodiment, an isolated anti-PD-1 antibody is provided, comprising a heavy chain variable region sequence from SEQ ID NO: 12 and/or a light chain variable region comprising a light chain variable region amino acid sequence from SEQ ID NO: 13, or a light chain variable region of the light chain variable region amino acid sequence of 13. In yet another embodiment, there is provided an isolated anti-PD-1 antibody comprising heavy and/or light chain sequences, wherein:

(a) The heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a heavy chain sequence of seq id no:

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEDLGGPSVFLFPPKPKDTLMISRTPEYTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 12), or

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

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：13)。

in some embodiments, the PDL1 binding antagonist is an anti-PDL 1 antibody. In some embodiments, the anti-PDL 1 binding antagonist is selected from the group consisting of: YW243.55.S70, MPDL3280A, MDX-1105, and MEDI 4736. MDX-1105, also known as BMS-936559, is an anti-PDL 1 antibody described in WO 2007/005874. The antibody YW243.55.S70 (heavy and light chain variable region sequences shown in SEQ ID Nos. 20 and 21, respectively) is an anti-PDL 1 antibody described in WO 2010/077634A 1. MEDI4736 is an anti-PDL 1 antibody described in WO2011/066389 and US 2013/034559.

Examples of anti-PDL 1 antibodies useful in the methods of the invention and methods of their production are described in PCT patent application WO2010/077634 a1 and U.S. patent No.8,217,149, which are incorporated herein by reference.

In some embodiments, the PD-1 axis binding antagonist is an anti-PDL 1 antibody. In some embodiments, the anti-PDL 1 antibody is capable of inhibiting one of PDL1 and PD-1And/or between PDL1 and B7-1. In some embodiments, the anti-PDL 1 antibody is a monoclonal antibody. In some embodiments, the anti-PDL 1 antibody is an antibody fragment selected from the group consisting of: fab, Fab '-SH, Fv, scFv, and (Fab')₂And (3) fragment. In some embodiments, the anti-PDL 1 antibody is a humanized antibody. In some embodiments, the anti-PDL 1 antibody is a human antibody.

anti-PDL 1 antibodies useful in the invention (including compositions comprising such antibodies), such as those described in WO2010/077634 a1, may be used in combination with OX40 binding agonists to treat cancer. In some embodiments, the anti-PDL 1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 7 or 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 9.

In one embodiment, an anti-PDL 1 antibody comprises a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2 and HVR-H3 sequences, wherein:

(a) The HVR-H1 sequence is GFTFSX₁SWIH(SEQ ID NO:14)；

(b) The HVR-H2 sequence is AWIX₂PYGGSX₃YYADSVKG(SEQ ID NO:15)；

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

further wherein: x₁Is D or G; x₂Is S or L; x₃Is T or S.

In a particular aspect, X₁Is D; x₂Is S and X₃Is T. In another aspect, the polypeptide further comprises a variable region heavy chain framework sequence juxtaposed between HVRs according to the formula: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR 4). 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:16)

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO:17)

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:18)

HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 19).

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) the HVR-L1 sequence is RASQX₄X₅X₆TX₇X₈A(SEQ ID NO:20)；

(b) The HVR-L2 sequence is SASX₉LX₁₀S(SEQ ID NO:21)；

(c) The HVR-L3 sequence is QQX₁₁X₁₂X₁₃X₁₄PX₁₅T(SEQ ID NO:22)；

Further wherein: x₄Is D or V; x₅Is V or I; x₆Is S or N; x₇Is A or F; x₈Is V or L; x₉Is F or T; x ₁₀Is Y or A; x₁₁Is Y, G, F, or S; x₁₂Is L, Y, F or W; x₁₃Is Y, N, A, T, G, F or I; x₁₄Is H, V, P, T or I; x₁₅Is A, W, R, P or T.

In yet another aspect, X₄Is D; x₅Is V; x₆Is S; x₇Is A; x₈Is V; x₉Is F; x₁₀Is Y; x₁₁Is Y; x₁₂Is L; x₁₃Is Y; x₁₄Is H; x₁₅Is A. In yet another aspect, the light chain further comprises a variable region light chain framework sequence juxtaposed between HVRs according to the following formula: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the framework sequence is a VL κ I consensus framework. In yet another aspect, at least one framework sequence is as follows:

LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:23)

LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO:24)

LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:25)

LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 26).

In another embodiment, an isolated anti-PDL 1 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) the HVR-H1 sequence is GFTFSX₁SWIH(SEQ ID NO:14)，

(ii) The HVR-H2 sequence is AWIX ₂PYGGSX₃YYADSVKG(SEQ ID NO:15)

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

(b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, wherein further:

(i) the HVR-L1 sequence is RASQX₄X₅X₆TX₇X₈A(SEQ ID NO:20)，

(ii) The HVR-L2 sequence is SASX₉LX₁₀S (SEQ ID NO:21), and

(iii) the HVR-L3 sequence is QQX₁₁X₁₂X₁₃X₁₄PX₁₅T(SEQ ID NO:22)

Further wherein: x₁Is D or G; x₂Is S or L; x₃Is T or S; x₄Is D or V; x₅Is V or I; x₆Is S or N; x₇Is A or F; x₈Is V or L; x₉Is F or T; x₁₀Is Y or A; x₁₁Is Y, G, F, or S; x₁₂Is L, Y, F or W; x₁₃Is Y, N, A, T, G, F or I; x₁₄Is H, V, P, T or I; x₁₅Is A, W, R, P or T.

In a particular aspect, X₁Is D; x₂Is S and X₃Is T. In another aspect, X₄Is D; x₅Is V; x₆Is S; x₇Is A; x₈Is V; x₉Is F; x₁₀Is Y; x₁₁Is Y; x₁₂Is L; x₁₃Is Y; x₁₄Is H; x₁₅Is A. In yet another aspect, X₁Is D; x₂Is S and X₃Is T, X₄Is D; x₅Is V; x₆Is S; x₇Is A; x₈Is V; x₉Is F; x₁₀Is Y; x₁₁Is Y; x₁₂Is L; x₁₃Is Y; x₁₄Is H and X¹⁵Is A.

In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between 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 HVRs as follows: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In a further aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more heavy chain framework sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:16)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:17)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:18)

HC-FR4 WGQGTLVTVSA(SEQ ID NO:19)。

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

LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:23)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:24)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:25)

LC-FR4 FGQGTKVEIKR(SEQ ID NO:26)。

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, IgG 4. In yet another specific aspect, the human constant region is IgG 1. In yet another aspect, the murine constant region is selected from the group consisting of: IgG1, IgG2A, IgG2B, IgG 3. In yet another aspect, the murine constant region is IgG 2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from "effector-less Fc mutation" or aglycosylation. In yet another embodiment, the effector smaller Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, an anti-PDL 1 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 having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO:1), AWISPYGGSTYYADSVKG (SEQ ID NO:2) and RHWPGGFDY (SEQ ID NO:3), respectively, or

(b) The light chain further comprises HVR-L1, HVR-L2 and HVR-L3 sequences having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO:4), SASFLYS (SEQ ID NO:5) and QQYLYHPAT (SEQ ID NO:6), respectively.

In a particular 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 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 HVRs as follows: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In a further aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more heavy chain framework sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO：16)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO：17)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO：18)

HC-FR4 WGQGTLVTVSA(SEQ ID NO：19)。

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

LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO：23)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO：24)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：25)

LC-FR4 FGQGTKVEIKR(SEQ ID NO：26)。

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, IgG 4. In yet another specific aspect, the human constant region is IgG 1. In yet another aspect, the murine constant region is selected from the group consisting of: IgG1, IgG2A, IgG2B, IgG 3. In yet another aspect, the murine constant region is IgG 2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from "effector-less Fc mutations" or aglycosylation. In yet another embodiment, the effector smaller Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, an isolated anti-PDL 1 antibody is provided, comprising heavy and light chain variable region sequences, wherein:

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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 28), or

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

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO9)。

in a particular 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 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 HVRs as follows: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In a further aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more heavy chain framework sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:16)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:17)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:18)

HC-FR4 WGQGTLVTVSA(SEQ ID NO:19)。

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

LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:23)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:24)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:25)

LC-FR4 FGQGTKVEIKR(SEQ ID NO:26)。

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, IgG 4. In yet another specific aspect, the human constant region is IgG 1. In yet another aspect, the murine constant region is selected from the group consisting of: IgG1, IgG2A, IgG2B, IgG 3. In yet another aspect, the murine constant region is IgG 2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function results from production in prokaryotic cells. In yet another specific aspect, minimal effector function results from "effector-less Fc mutations" or aglycosylation. In yet another embodiment, the effector smaller Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In another further embodiment, there is provided an isolated anti-PDL 1 antibody comprising heavy and light chain variable region sequences, wherein:

(a) The heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7), or

(b) The light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO：9)。

in yet another embodiment, there is provided an isolated anti-PDL 1 antibody comprising heavy and light chain variable region sequences, wherein:

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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI

SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 8), or

(b) The light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO：9)。

in a particular 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 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 HVRs as follows: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In another aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more heavy chain framework sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO：16)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO：17)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO：18)

HC-FR4 WGQGTLVTVSS(SEQ ID NO：27)。

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

LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO：23)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO：24)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：25)

LC-FR4 FGQGTKVEIKR(SEQ ID NO：26)。

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, IgG 4. In yet another specific aspect, the human constant region is IgG 1. In yet another aspect, the murine constant region is selected from the group consisting of: IgG1, IgG2A, IgG2B, IgG 3. In yet another aspect, the murine constant region is IgG 2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function results from production in prokaryotic cells. In yet another specific aspect, minimal effector function results from "effector-less Fc mutations" or aglycosylation. In yet another embodiment, the effector smaller Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, the anti-PDL 1 antibody is MPDL3280A (CAS registry number: 1422185-06-5). In yet another embodiment, an isolated anti-PDL 1 antibody is provided, which comprises a heavy chain variable region comprising a heavy chain variable region amino acid sequence from:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLFWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 8),

the light chain variable region comprises the following amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO：9)。

in yet another embodiment, an isolated anti-PDL 1 antibody is provided, comprising a heavy chain and/or light chain sequence, wherein:

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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVTLFPPKPKDTLMI5RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO：29),

and/or

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

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFCQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：30)。

in yet another embodiment, the present invention provides a composition comprising any one of the anti-PDL 1 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-PDL 1 antibody, wherein:

(a) the heavy chain further comprises HVR-H1, HVR-H2 and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 1), AWISPYGGSTYYADSVKG (SEQ ID NO: 2) and RHWPGGFDY (SEQ ID NO: 3), respectively, and

(b) the light chain further comprises HVR-L1, HVR-L2 and HVR-L3 sequences having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 4), SASFLYS (SEQ ID NO: 5) and QQYLYHPAT (SEQ ID NO: 6), respectively.

In a particular 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 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 HVRs as follows: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In another aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more heavy chain framework sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:16)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:17)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:18)

HC-FR4 WGQGTLVTVSA(SEQ ID NO:19)。

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

LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:23)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:24)

LC-FR3GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:25)

LC-FR4 FGQGTKVEIKR(SEQ ID NO:26)。

in yet another specific aspect, an antibody described herein (such as an anti-PD-1 antibody, an anti-PDL 1 antibody, or an anti-PDL 2 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, IgG 4. In yet another specific aspect, the human constant region is IgG 1. In yet another aspect, the murine constant region is selected from the group consisting of: IgG1, IgG2A, IgG2B, IgG 3. In yet another aspect, the murine constant region is IgG 2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function results from production in prokaryotic cells. In yet another specific aspect, minimal effector function results from "effector-less Fc mutations" or aglycosylation. In yet another aspect, the effector minor Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another aspect, provided herein is a nucleic acid encoding any of the antibodies described herein. In some embodiments, the nucleic acid further comprises a vector suitable for expressing a nucleic acid encoding any one of the previously described anti-PDL 1, anti-PD-1, or anti-PDL 2 antibodies. In yet another specific aspect, the vector further comprises a host cell suitable for expression of 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 Chinese Hamster Ovary (CHO).

The antibodies or antigen-binding fragments thereof may 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-PDL 1, anti-PD-1, or anti-PDL 2 antibodies or antigen-binding fragments in a form suitable for expression under conditions suitable for the production of such antibodies or fragments, and recovering the antibodies or fragments.

In some embodiments, the isolated anti-PDL 1 antibody is aglycosylated. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are the recognition sequences for enzymatic attachment of the carbohydrate module to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of the sugar N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of glycosylation sites from the antibody is conveniently achieved by altering the amino acid sequence such that one of the tripeptide sequences (for N-linked glycosylation sites) described above is removed. Changes can be made by substituting an asparagine, serine or threonine residue within the glycosylation site with another amino acid residue (e.g., a glycine, alanine or conservative amino acid substitution).

In any of the embodiments herein, the isolated anti-PDL 1 antibody is capable of binding to human PDL1 (e.g., human PDL1 shown in UniProtKB/Swiss-Prot accession No. Q9NZQ7.1), or a variant thereof.

In yet another embodiment, the present invention provides a composition comprising an anti-PDL 1, anti-PD-1, or anti-PDL 2 antibody or antigen-binding fragment thereof as provided herein and at least one pharmaceutically acceptable carrier. In some embodiments, the anti-PDL 1, anti-PD-1, or anti-PDL 2 antibody or antigen-binding fragment thereof administered to the individual is a composition comprising one or more pharmaceutically acceptable carriers. Any pharmaceutically acceptable carrier described herein or known in the art may be used.

In some embodiments, the anti-PDL 1 antibody described herein is in a formulation comprising the antibody in an amount of about 60mg/mL, histidine acetate at a concentration of about 20mM, sucrose at a concentration of about 120mM, and polysorbate (e.g., polysorbate 20) at a concentration of 0.04% (w/v), and the formulation has a pH of about 5.8. In some embodiments, the anti-PDL 1 antibody described herein is in a formulation comprising the antibody in an amount of about 125mg/mL, histidine acetate at a concentration of about 20mM, sucrose at a concentration of about 240mM, and polysorbate (e.g., polysorbate 20) at a concentration of 0.02% (w/v), and the formulation has a pH of about 5.5.

OX40 binding agonists

Provided herein is a method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist. Also provided herein is a method of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist.

OX40 binding agonists include, for example, OX40 agonist antibodies (e.g., anti-human OX40 agonist antibodies), OX40L agonist fragments, OX40 oligomeric receptors, and OX40 immunoadhesins.

In some embodiments, the OX40 agonist antibody increases CD4+ effector T cell proliferation and/or increases cytokine production of the CD4+ effector T cell as compared to proliferation and/or cytokine production prior to treatment with the OX40 agonist antibody. In some embodiments, the cytokine is IFN- γ.

In some embodiments, the OX40 agonist antibody increases memory T cell proliferation and/or increases cytokine production by the memory cell. In some embodiments, the cytokine is IFN- γ.

In some embodiments, the OX40 agonist antibody inhibits Treg suppression of effector T cell function. In some embodiments, the effector T cell function is effector T cell proliferation and/or cytokine production. In some embodiments, the effector T cell is a CD4+ effector T cell.

In some embodiments, the OX40 agonist antibody increases OX40 signaling in a target cell expressing OX 40. In some embodiments, OX40 signaling is detected by monitoring NFkB downstream signaling.

In some embodiments, the anti-human OX40 agonist antibody is a subtractive anti-human OX40 antibody (e.g., depletes cells expressing human OX 40). In some embodiments, the cell expressing human OX40 is a CD4+ effector T cell. In some embodiments, the human OX 40-expressing cell is a Treg cell. In some embodiments, the depleting is by ADCC and/or phagocytosis. In some embodiments, the antibody mediates ADCC by binding to Fc γ rs expressed by human effector cells and activating the human effector cell function. In some embodiments, the antibody mediates phagocytosis by binding to Fc γ rs expressed by human effector cells and activating the human effector cell function. Exemplary human effector cells include, for example, macrophages, Natural Killer (NK) cells, monocytes, neutrophils. In some embodiments, the human effector cell is a macrophage.

In some embodiments, the anti-human OX40 agonist antibody has a functional Fc region. In some embodiments, the effector function of the functional Fc region is ADCC. In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are ADCC and phagocytosis. In some embodiments, the Fc region is human IgG 1. In some embodiments, the Fc region is human IgG 4.

In some embodiments, the anti-human OX40 agonist antibody is a human or humanized antibody. In some embodiments, the OX40 binding agonist (e.g., OX40 agonist antibody) is not MEDI 6383. In some embodiments, the OX40 binding agonist (e.g., OX40 agonist antibody) is not MEDI 0562.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in U.S. patent No.7,550,140, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain comprising the sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYTMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRYSQVHYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQvSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：31)

and/or a light chain comprising the sequence:

DIVMTQSPDSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKAGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNHPTTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSrLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：32)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody 008, which is described in U.S. patent No.7,550,140. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody 008 as described in U.S. patent No.7,550,140.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in U.S. patent No.7,550,140. In some embodiments, the anti-human OX40 agonist antibody comprises the following sequence:

DIQMTQSPDSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKAGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNHPTTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEvTHQGLSSPvTKSFNRGEC(SEQ ID NO：33)。

In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody SC02008 described in U.S. patent No.7,550,140. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody SC02008 described in U.S. patent No.7,550,140.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in U.S. patent No.7,550,140. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain comprising the sequence:

EVQLVESGGGLVHPGGSLRLSCAGSGFTFSSYAMHWVRQAPGKGLEWVSAIGTGGGTYYADSVMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYDNVMGLYWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：34)

and/or a light chain comprising the sequence:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：35)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody 023 described in U.S. patent No.7,550, 140. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody 023 described in U.S. patent No.7,550,140.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in U.S. patent No.7,960,515, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

EVQLvESGGGLvQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIDYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARESGWYLFDYWGQGTLVTVSS(SEQ ID NO：36)

And/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGGGTKVEIK(SEQ ID NO：37)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody 11D4 described in U.S. patent No.7,960,515. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody 11D4 described in U.S. patent No.7,960, 515.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in U.S. patent No.7,960,515. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDQSTADYYFYYGMDVWGQGTTVTVSS(SEQ ID NO：38)

and/or a light chain variable region comprising the sequence:

EIVVTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK(SEQ ID NO：39)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody 18D8 described in U.S. patent No.7,960,515. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody 18D8 described in U.S. patent No.7,960,515.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2012/027328, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLKWMGWINTETGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCANPYYDYVSYYAMDYWGQGTTVTVSS(SEQ ID NO：40)

And/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYLYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPRTFGQGTKLEIK(SEQ ID NO：41)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody hu106-222 described in WO 2012/027328. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody hu106-222 described in WO 2012/027328.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2012/027328. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

EVQLVESGGGLVQPGGSLRLSCAASEYEFPSHDMSWVRQAPGKGLELVAAINSDGGSTYYPDTMERRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHYDDYYAWFAYWGQGTMVTVSS(SEQ ID NO：42)

and/or a light chain variable region comprising the sequence:

EIVLTQSPATLSLSPGERATLSCRASKSVSTSGYSYMHWYQQKPGQAPRLLIYLASNLFSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRELPLTFGGGTKVEIK(SEQ ID NO：43)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody Hu119-122 described in WO 2012/027328. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody Hu119-122 described in WO 2012/027328.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2013/028231, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain comprising the sequence:

MYLGLNYVFIVFLLNGVQSEVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVNGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCTWGEVFYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYITCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：44)

And/or a light chain comprising the sequence:

MRPSIQFLGLLFWLHGAQCDIQMTQSPSSLSLASLGGKVTITCKSSQDINKYIAWYQHQPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：45)。

in some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

MYLGLNYVFIVFLLNGVQSEvKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVNGRFTISRDDSKSSVYLQMNSLRAEDTGlYYCTWGEVFYFDYWGQGTTLTVSS(SEQ ID NO：61)

and/or a light chain variable region comprising the sequence:

MRPSIQFLGLLLFWLHGAQCDIQMTQSPSSLSASLGGKVTITCKSSQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLTFGAGTKLELK(SEQ ID NO：62)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody Mab CH 119-43-1 described in WO 2013/028231. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody Mab CH 119-43-1 described in WO 2013/028231.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2013/038191, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

EVQLQQSGPELVKPGASVKMSCKASGYTFTSYvMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCANYYGSLSMDYWGQGTSVTTVSS(SEQ ID NO：46)

and/or a light chain variable region comprising the sequence:

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKR(SEQ ID NO：47)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO 2013/038191. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 20E5 described in WO 2013/038191.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2013/038191. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

EVQLQQSGPELVKPGASVKISCKTSGYTFKDYTMHWVKQSHGKSLEWIGGIYPNNGGSTYNQNFKDKATLTVDKSSSTAYMEFRSLTSEDSAVYYCARMGYHGPHLDFDVWGAGTTVTVSP(SEQ ID NO：48)

and/or a light chain variable region comprising the sequence:

DIVMTQSHKFMSTSLGDRVSITCKASQDVGAAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGGGSGTDFTLTISNVQSEDLTDYFCQQYINYPLTFGGGTKLEIKR(SEQ ID NO：49)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO 2013/038191. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 12H3 described in WO 2013/038191.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a1, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGYINPYNDGTKYNEKFKGRVTITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS(SEQ ID NO：50)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR(SEQ ID NO：51)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 20E5 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGYINPYNDGTKYNEKFKGRVTITSDTSSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS(SEQ ID NO：50)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFArYFCQQGNTLPWTFGQGTKVEIKR(SEQ ID NO：52)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 20E5 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDGTKYNEKFKGRATITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS(SEQ ID NO：53)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR(SEQ ID NO：51)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 20E5 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDGTKYNEKFKGRATITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS(SEQ ID NO：53)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR(SEQ ID NO：52)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 20E5 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEvKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCANYYFSSLSMDYWGQGTLVTVSS(SEQ ID NO：54)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR(SEQ ID NO：51)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of somatic clone 20E5 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLVTVSS

(SEQ ID NO：54)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR(SEQ ID NO：52)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 20E5 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEVKKPGSSvKvSCKASGYTFKDYTMHWVRQAPGQGLEWMGGIYPNNGGSTYNQNFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTVSS(SEQ ID NO：55)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR(SEQ ID NO：56)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 12H3 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWMGGIYPNNGGSTYNQNFKDRVTITADKSTSTAYMELSSLRSRDTAVYYCARMGYHGPHLDFDVWGQGTTVTVSS(SEQ ID NO：55)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRvTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR(SEQ ID NO：57)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 12H3 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QvQLVQSGAEvKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGGSTYNQNFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTVSS(SEQ ID NO：58)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRvTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR(SEQ ID NO：56)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 12H3 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEvKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGGSTYNQNFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTVSS(SEQ ID NO：58)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR(SEQ ID NO：57)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 12H3 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEvKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGGSTYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTVSS(SEQ ID NO：59)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR(SEQ ID NO：56)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 12H3 described in WO2014/148895a 1.

In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2014/148895a 1. In some embodiments, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence:

QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGGSTYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQGTTVTVSS(SEQ ID NO：59)

and/or a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR(SEQ ID NO：57)。

in some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014/148895a 1. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody clone 12H3 described in WO2014/148895a 1.

In some embodiments, the agonist anti-human OX40 antibody is L106BD (Pharmingen product No. 340420). In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody L106(BD Pharmingen product number 340420). In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody L106(BD Pharmingen product No. 340420).

In some embodiments, the agonist anti-human OX40 antibody is ACT35(Santa cruz biotechnology, catalog No. 20073). In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody ACT35(Santa cruz biotechnology, catalog number 20073). In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody ACT35(Santa Cruz Biotechnology, catalog No. 20073).

In some embodiments, the OX40 agonist antibody is MEDI 6469. In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody MEDI 6469. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody MEDI 6469.

In some embodiments, the OX40 agonist antibody is MEDI 0562. In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody MEDI 0562. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody MEDI 0562.

In some embodiments, the OX40 agonist antibody is an agonist antibody that binds to the same epitope as any of the OX40 agonist antibodies listed above.

In some embodiments, the anti-human OX40 agonist antibody has a functional Fc region. In some embodiments, the Fc region is human IgG 1. In some embodiments, the Fc region is human IgG 4. In some embodiments, the anti-human OX40 agonist antibody is engineered to increase effector function (e.g., as compared to effector function in wild-type IgG 1). In some embodiments, the antibody has increased binding to an Fc γ receptor. In some embodiments, the antibody lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In some embodiments, the Fc region comprises bisected oligosaccharides, for example wherein biantennary oligosaccharides attached to the antibody Fc region are bisected by GlcNAc. In some embodiments, the antibody comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334(EU residue numbering) of the Fc region.

OX40 agonists useful for the methods described herein are in no way intended to be limited to antibodies. Non-antibody OX40 agonists are contemplated and are well known in the art.

As noted above, OX40L (also referred to as CD134L) acts as a ligand for OX 40. Thus, agonists that exhibit partial or complete OX40L may act as OX40 agonists. In some embodiments, an OX40 agonist can include one or more OX40L extracellular domains. Examples of extracellular domains of OX40L may include the OX40 binding domain. In some embodiments, an OX40 agonist may be a soluble form of OX40L that includes one or more extracellular domains of OX40L but lacks other, insoluble domains of the protein, such as a transmembrane domain. In some embodiments, the OX40 agonist is a soluble protein comprising one or more extracellular domains of OX40L capable of binding OX 40L. In some embodiments, an OX40 agonist may be linked to another protein domain, for example, to increase its effectiveness, half-life, or other desirable characteristic. In some embodiments, an OX40 agonist can include one or more OX40L extracellular domains linked to an immunoglobulin Fc domain.

In some embodiments, the OX40 agonist can be any of the OX40 agonists described in U.S. patent No.7,696,175.

In some embodiments, the OX40 agonist can be an oligomeric or polymeric molecule. For example, an OX40 agonist may contain one or more domains (e.g., leucine zipper domains) that allow for protein oligomerization. In some embodiments, an OX40 agonist can include one or more OX40L extracellular domains linked to one or more leucine zipper domains.

In some embodiments, the OX40 agonist may be any of the OX40 agonists described in european patent No. ep0672141b1.

In some embodiments, the OX40 agonist can be a trimeric OX40L fusion protein. For example, an OX40 agonist may include one or more OX40L extracellular domains linked to an immunoglobulin Fc domain and a trimerization domain (including, but not limited to, isoleucine zipper domain).

In some embodiments, the OX40 agonist may be any one of the OX40 agonists described in international publication No. wo2006/121810, such as OX40 immunoadhesin. In some embodiments, the OX40 immunoadhesin can be a trimeric OX40-Fc protein. In some embodiments, the OX40 agonist is MEDI 6383.

Preparation of antibodies

Antibodies described herein are prepared using techniques available in the art for generating antibodies, exemplary methods of which are described in more detail in the sections below.

The antibody is directed against an antigen of interest (i.e., PD-L1 (such as human PD-L1), OX40 (such as human OX 40)). Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to a mammal suffering from a disorder results in a therapeutic benefit in the mammal.

In certain embodiments, an antibody provided herein has ≦ 1 μ M ≦ 150nM, ≦ 100nM, ≦ 50nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (Kd).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with Fab versions of the antibody of interest and its antigen as described in the assays described below. By using the minimum concentration of (in the presence of unlabeled antigen in the titration series¹²⁵I) The Fab is equilibrated with labeled antigen and then the solution binding affinity of the Fab for the antigen is measured by capturing the bound antigen with an anti-Fab antibody coated plate (see, e.g., Chen et al, J.mol.biol.293: 865-. To establish the assay conditions, theMulti-well plates (Thermo Scientific) were coated with 5. mu.g/ml capture anti-Fab antibodies (Cappel Labs) in 50mM sodium carbonate (pH 9.6) overnight, followed by blocking with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23 ℃). In the non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ] ¹²⁵I]Mixing the antigen with serial dilutions of the Fab of interest. The Fab of interest was then incubated overnight; however, incubation may continue for a longer period of time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution was then removed and treated with 0.1% polysorbate 20(TWEEN-) The plate was washed 8 times. After drying the plates, 150. mu.l/well scintillation fluid (MICROSCINT-20) was added^TM(ii) a Packard) and then the plate is counted for 10 minutes on a topcount tm gamma counter (Packard). Selection of each Fab gives less than or equal to maximal knotsThe 20% concentration of total was used in the competitive binding assay.

According to another embodiment, Kd is determined using a surface plasmon resonance assay-2000 or-3000(BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH 4.8 and then injected at a flow rate of 5. mu.l/min to obtain approximately 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78nM to 500nM) in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20. TM.) surfactant were injected at 25 ℃ at a flow rate of approximately 25. mu.l/min. Using a simple one-to-one Langmuir (Langmuir) binding model ( Evaluation software version 3.2) calculate the binding rate (k) by fitting both the binding and dissociation sensorgrams simultaneously_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay⁶M^-1s^-1The rate of binding can then be determined using fluorescence quenching techniques, i.e.measuring the binding rate in PBS pH 7.2 in the presence of increasing concentrations of antigen, according to measurements performed with a stirred cuvette in a spectrometer such as a spectrophotometer equipped with a flow cut-off device (Aviv Instruments) or a 8000 series SLM-AMINOTM spectrophotometer (ThermoSpectronic)An increase or decrease in the fluorescence emission intensity (excitation 295 nM; emission 340nM, 16nM bandpass) of 20nM anti-antigen antibody (Fab format) at 25 ℃.

(i) Antigen preparation

Soluble antigens or fragments thereof (optionally conjugated with other molecules) can be used as immunogens for generating antibodies. For transmembrane molecules, such as receptors, fragments of these (e.g., the extracellular domain of the receptor) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells may be derived from natural sources (e.g., cancer cell lines), or may be cells transformed by recombinant techniques to express the transmembrane molecule. Other antigens and forms thereof useful for making antibodies will be apparent to those skilled in the art.

(ii) Certain antibody-based methods

Polyclonal antibodies are preferably generated by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant in the animal. Using bifunctional or derivatizing reagents, e.g. maleimidobenzoyl sulphosuccinimide ester (conjugated via a cysteine residue), N-hydroxysuccinimide (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N ═ C ═ NR, where R and R¹Being different hydrocarbon groups, it may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor.

Animals are immunized against an antigen, immunogenic conjugate or derivative by mixing, for example, 100 μ g or 5 μ g of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals were boosted with an initial amount of 1/5-1/10 of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, blood was collected from the animals, and the antibody titer of the serum was determined. Animals were boosted until the titer reached a plateau (pateau). Preferably, the animal is boosted with the same antigen but conjugated to a different protein and/or by a different cross-linking agent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Also, a coagulant such as alum is suitably used to enhance the immune response.

Monoclonal Antibodies of the invention can be generated using Hybridoma methods, which are first described in Kohler et al, Nature,256:495(1975), and further described in, for example, 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 Cell hybrids 563-. Other methods include those described, for example, in U.S. Pat. No.7,189,826 for the production of monoclonal human native IgM antibodies from hybridoma cell lines. The human hybridoma technique (Trioma technology) is described in Vollmers and Brandlein, Histology and Histopathology,20(3):927-937(2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology,27(3):185-91 (2005).

For various other hybridoma techniques, see, e.g., US 2006/258841; US 2006/183887 (fully human antibody); US 2006/059575; US 2005/287149; US 2005/100546; US 2005/026229; and U.S. Pat. nos.7,078,492 and 7,153,507. An exemplary protocol for generating monoclonal antibodies using the hybridoma method is described below. In one embodiment, a mouse or other suitable host animal (such as a hamster) is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies are produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of the invention or a fragment thereof and an adjuvant such as monophosphoryl lipid a (mpl)/trehalose two-stick mycolate (TDM) (Ribi immunochem. The polypeptides (e.g., antigens) of the invention or fragments thereof can be prepared using methods well known in the art, such as recombinant methods, some of which are further described herein. Sera from immunized animals are assayed for anti-antigen antibodies and optionally administered for booster immunizations. Lymphocytes are isolated from an animal that produces anti-antigen antibodies. Alternatively, lymphocytes are immunized in vitro.

The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells. See, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986). Myeloma cells that support stable high-level antibody production by selected antibody-producing cells and are sensitive to a medium such as HAT medium can be used with efficient fusion. Exemplary myeloma cells include, but are not limited to, murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors (available from the cell distribution center of the institute of Soleck (Salk), San Diego, Calif. USA), and SP-2 or X63-Ag8-653 cells (available from the American type culture Collection, Rockville, Md. USA). Human myeloma and mouse-human heteromyeloma cell lines are also described for the production of human Monoclonal antibodies (Kozbor, J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal antibody production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells so prepared are seeded and cultured in a suitable medium, such as one containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent HGPRT-deficient cells from growing. Preferably, serum-free hybridoma cell culture methods are used to reduce the use of animal-derived serum, such as fetal bovine serum, as described, for example, in Evenet al, Trends in Biotechnology,24(3),105-108 (2006).

Oligopeptides that are tools for increasing productivity of hybridoma cell cultures are described in Franek, Trends in monoclonal Antibody Research,111-122 (2005). In particular, standard media are rich in certain amino acids (alanine, serine, asparagine, proline) or protein hydrolysate fractions and apoptosis can be significantly suppressed by synthetic oligopeptides consisting of 3-6 amino acid residues. The peptide is present in millimolar or higher concentrations.

The culture medium in which the hybridoma cells are growing can be assayed for production of monoclonal antibodies that bind to the antigens of the invention. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined by, for example, Scatchard analysis. See, e.g., Munson et al, anal. biochem.107:220 (1980).

After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (see, e.g., Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo in animals as ascites tumors. Monoclonal antibodies secreted by the subclones can be suitably separated from the culture fluid, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. One procedure for isolating proteins from hybridoma cells is described in US 2005/176122 and U.S. Pat. No.6,919,436. The method involves the use of minimally salts, such as lyotropic salts, during the binding process, and preferably also small amounts of organic solvents during the elution process.

(iii) Library-derived antibodies

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art, such as the method described in example 3. Other Methods are reviewed, for example, in Hoogenboom et al, in Methods in Molecular Biology 178:1-37 (O' Brien et al, ed., Human Press, Totowa, NJ,2001), and further described, for example, in McCafferty et al, Nature 348: 552-; clackson et al, Nature 352: 624-; marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, in Methods in Molecular Biology 248:161-175(Lo, ed., Human Press, Totowa, NJ, 2003); sidhu et al, J.mol.biol.338(2): 299-; leeet al, j.mol.biol.340(5): 1073-; fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-; and Lee et al, J.Immunol.methods 284(1-2):119-132 (2004).

In some phage display methods, the repertoire of VH and VL genes, respectively, are cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al, Ann.Rev.Immunol.12:433-455 (1994). Phage typically display antibody fragments either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the non-immune repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a large panel of non-self and also self-antigens in the absence of any immunization, as described by Griffiths et al, EMBO J,12: 725-. Finally, non-rearranged V gene segments can also be synthesized by cloning non-rearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter, J.mol.biol.227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No.5,750,373 and U.S. patent publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

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

(iv) Chimeric, humanized and human antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature 332:323-329 (1988); queen et al, Proc.Nat' l Acad.Sci.USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; kashmiri et al, Methods36:25-34(2005) (record SDR (a-CDR) grafting); padlan, mol.Immunol.28:489-498(1991) (described as "resurfacing"); dall' Acqua et al, Methods36: 43-60(2005) (record "FR shuffling"); and Osbournet al, Methods36: 61-68(2005) and Klimka et al, Br.J. cancer 83:252-260(2000) (describing the "guided selection" approach to FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al, j. immunol.151:2296 (1993)); framework regions derived from consensus sequences of a specific subset of human antibodies of the light or heavy chain variable regions (see, e.g., Carter et al, proc.Natl.Acad.Sci.USA 89:4285 (1992); and Presta et al, J.Immunol.151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front.biosci.13:1619-1633 (2008)); and by screening FR library derived framework region (see for example Baca et al, J.biol.chem.272: 10678-.

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Generally, human antibodies are described in van Dijk and van de Winkel, Curr, Opin, Pharmacol.5:368-74(2001), and Lonberg, Curr, Opin, Immunol.20: 450-.

Human antibodies can be made by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which exists extrachromosomally or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For an overview of the method for obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XeNOMOUSE^TMA technique; U.S. Pat. No.5,770,429, which describesA technique; U.S. Pat. No.7,041,870, which describes K-MTechnology, and U.S. patent application Publication No. us 2007/0061900, which describesProvided is a technique. The human variable regions from the whole antibodies generated by such animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the Production of human Monoclonal antibodies have been described (see, e.g., Kozbor, J.Immunol.133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York,1987) and Boerner et al, J.Immunol.147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al, proc.natl.acad.sci.usa 103:3557-3562 (2006). Other methods include those described in, for example, U.S. Pat. No.7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines, and Ni, Xiandai Mianyixue26(4):265-268(2006), which describes human-human hybridomas. The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandrein, Histology and Histopathlogy 20(3): 927-.

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

(v) Antibody fragments

Antibody fragments may be generated by conventional means, such as enzymatic digestion, or by recombinant techniques. In some cases, it may be advantageous to use antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and may result in easier access to solid tumors. For a review of certain antibody fragments see Hudson et al (2003) nat. Med.9: 129-.

Various techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and biophysical methods 24:107-117 (1992); and Brennan et al, Science 229:81 (1985)). However, these fragments can now be produced directly from recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted by E.coli, thus allowing easy production of large quantities of these fragments. Antibody fragments can be isolated from the phage antibody libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab') ₂Fragments (Carteret et al, Bio/Technology 10: 163-. According to another method, F (ab') can be isolated directly from recombinant host cell cultures₂And (3) fragment. Fab and F (ab') with extended in vivo half-life comprising salvage receptor binding epitope residues₂Fragments are described in U.S. Pat. No.5,869,046. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. patent nos. 5,571,894; and 5,587,458. Fv and scFv are the only types with intact binding sites, lacking constant regions; as such, they may be suitable for reducing non-specific binding when used in vivo. scFv fusion proteins can be constructed to generate fusion of the effector protein at the amino or carboxy terminus of the scFv. See, for example, Antibody Engineering, eds. Borebaeck, supra. Antibody fragments may also be "linear antibodies," for example as described in U.S. Pat. No.5,641,870. Such linear antibodies may be monospecific or bispecific.

(vi) Multispecific antibodies

Multispecific antibodies have binding specificities for at least two different epitopes, wherein the epitopes are typically derived from different antigens. Although such molecules typically bind only two different epitopes (i.e., bispecific antibodies, BsAb), this expression, as used herein, encompasses antibodies with additional specificity, such as trispecific antibodies. Can be used for Bispecific antibodies are prepared as full length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies). In one aspect, bispecific antibodies that bind OX40 and PD-1 are provided. In one aspect, bispecific antibodies that bind to OX40 and PDL1 are provided.

Methods for constructing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature,305:537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is rather cumbersome and the product yield is low. Similar procedures are disclosed in WO 93/08829 and Travecker et al, EMBO J.,10:3655-3659 (1991).

One approach known in the art for generating bispecific antibodies is the "node-in-pocket" or "bump-in-cavity" approach (see, e.g., U.S. Pat. No.5,731,168). In this approach, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each comprise an interface. The interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. These interfaces can be engineered such that a "knob" or "bump" (which terms are used interchangeably herein) located in the interface of one immunoglobulin polypeptide corresponds to a "hole" or "cavity" (which terms are used interchangeably herein) located in the interface of another immunoglobulin polypeptide. In some embodiments, the pockets are the same or similar size as the segments and are positioned so that when two interfaces interact, the segments of one interface can be positioned in the corresponding pockets of the other interface. Without wishing to be bound by theory, it is believed that this stabilizes the heterodimer and favors heteromultimer formation over other species, such as homomultimers. In some embodiments, this approach can be used to facilitate heteromultimerization of two different immunoglobulin polypeptides, creating a bispecific antibody comprising two immunoglobulin polypeptides having binding specificities for different epitopes.

In some embodiments, a segment may be constructed by replacing a small amino acid side chain with a larger side chain. In some embodiments, a pocket may be constructed by replacing a large amino acid side chain with a smaller side chain. The knots or holes may be present in the original interface or may be synthetically introduced. For example, an introduced segment or pocket can be synthesized by altering the nucleic acid sequence encoding the interface to replace at least one "original" amino acid residue with at least one "import" amino acid residue. Methods for altering nucleic acid sequences may include standard molecular biology techniques well known in the art. The side chain volumes of the various amino acid residues are shown in the table below. In some embodiments, the original residue has a smaller side chain volume (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), while the import residues for segment formation are naturally occurring amino acids and can include arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the original residues have a larger side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan), while the import residues for hole formation are naturally occurring amino acids and may include alanine, serine, threonine, and valine.

Table 1: characterization of amino acid residues

^aThe molecular weight of the amino acid minus the molecular weight of water. The values are from Handbook of Chemistry and Physics,43^rded.,Cleveland,Chemical Rubber Publishing Co.,1961。

^bValues are from A.A.Zamyytnin, prog.Biophys.mol.biol.24:107-123, 1972.

^cValues are from C.Chothia, J.mol.biol.105:1-14,1975. Accessible surface areas are defined in FIGS. 6-20 of this reference.

In some embodiments, the original residues used to form the nodes or holes are identified based on the three-dimensional structure of the heteromultimer. Techniques known in the art for obtaining three-dimensional structures may include X-ray crystallography and NMR. In some embodiments, the interface is a CH3 domain of an immunoglobulin constant domain. In these embodiments, the CH3/CH3 interface of human IgG1 involves 16 residues on the four antiparallel beta chains per domain. Without wishing to be bound by theory, the mutated residues are preferably located on the two central antiparallel beta strands to minimize the risk that the knob will be held by the surrounding solvent, rather than the complementary pocket in the partner CH3 domain. In some embodiments, the mutations in the two immunoglobulin polypeptides that form the corresponding nodes and cavities correspond to one or more pairs provided in the tables below.

Table 2: exemplary kits of corresponding node and pocket forming mutations

Mutations are indicated by the original residue, followed by the position using the Kabat numbering system, and then the input residue (all residues are given in the one letter amino acid code). Multiple mutations are separated by colons.

In some embodiments, the immunoglobulin polypeptide comprises a CH3 domain comprising one or more of the amino acid substitutions listed in table 2 above. In some embodiments, the bispecific antibody comprises a first immunoglobulin polypeptide comprising a CH3 domain comprising one or more amino acid substitutions listed in the left column of table 2, and a second immunoglobulin polypeptide comprising a CH3 domain comprising one or more corresponding amino acid substitutions listed in the right column of table 2.

Following mutation of the DNA as described above, polynucleotides encoding modified immunoglobulin polypeptides having one or more corresponding nodal or pocket forming mutations can be expressed and purified using standard recombinant techniques and cell systems known in the art. See, e.g., U.S. Pat. Nos. 5,731,168; 5,807,706, respectively; 5,821,333, respectively; 7,642,228, respectively; 7,695,936, respectively; 8,216,805, respectively; U.S. publication No. 2013/0089553; and Spiess et al, Nature Biotechnology 31: 753-. The modified immunoglobulin polypeptides may be produced using prokaryotic host cells, such as e.coli, or eukaryotic host cells, such as CHO cells. The immunoglobulin polypeptides carrying the corresponding nodes and cavities may be expressed together as heteromultimers in a host cell and purified, or may be expressed in multiple single cultures, purified separately, and assembled in vitro. In some embodiments, two bacterial host cells (one expressing the immunoglobulin polypeptide with a knob and the other expressing the immunoglobulin polypeptide with a hole) are co-cultured using standard bacterial culture techniques known in the art. In some embodiments, the two strains may be mixed in a specific ratio, for example to achieve equal expression levels in culture. In some embodiments, the two strains may be mixed in a 50:50, 60:40, or 70:30 ratio. After expression of the polypeptide, the cells can be lysed together and the protein can be extracted. Standard techniques known in the art that allow measurement of the abundance of homomultimer versus heteromultimeric species may include size exclusion chromatography. In some embodiments, each modified immunoglobulin polypeptide is expressed separately using standard recombinant techniques, and may be assembled together in vitro. Assembly can be achieved, for example, by purifying each modified immunoglobulin polypeptide, mixing and incubating them together in equal mass, reducing the disulfide (e.g., by treatment with dithiothreitol), concentrating, and reoxidizing the polypeptide. The bispecific antibody formed can be purified using standard techniques, including cation exchange chromatography, and measured using standard techniques, including size exclusion chromatography. For a more detailed description of these methods, see Speiss et al, Nat Biotechnol 31: 753-. In some embodiments, the modified immunoglobulin polypeptide may be expressed separately in CHO cells and assembled in vitro using the methods described above.

According to a different approach, antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is to an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Typically, a first heavy chain constant region (CH1) is present in at least one of the fusions that includes the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields, this provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when expression of at least two polypeptide chains in the same ratio leads to high yields or when the ratio is of no particular significance.

In one embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. Since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient separation route, it was found that this asymmetric structure facilitates the separation of the desired bispecific compound from the unwanted immunoglobulin chain combinations. The method is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology,121:210 (1986).

According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. One interface comprises at least part of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size to the large side chains are created at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heterologous conjugate may be coupled to avidin, while the other antibody is coupled to biotin. Such antibodies have been suggested for use, for example, in targeting immune system cells to unwanted cells (U.S. Pat. No.4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugated antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art, as well as a number of crosslinking techniques, and are disclosed in U.S. Pat. No.4,676,980.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science 229:81(1985) describes proteolytic cleavage of intact antibodies to F (ab')₂Protocol for fragmentation. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reverted back to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immobilization reagents for enzymes.

Recent advances have facilitated the direct recovery of Fab' -SH fragments from E.coli, which fragments can be chemically coupled to formBispecific antibodies. A fully humanized bispecific antibody F (ab') is described in Shalaby et al, J.Exp.Med.,175:217-225(1992)₂And (4) generation of molecules. Each Fab' fragment was secreted separately from E.coli and subjected to directed chemical coupling in vitro to form bispecific antibodies.

Various techniques for the direct production and isolation of bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies have been generated using leucine zippers. Kostelny et al, J.Immunol.,148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The "diabody" technique described by Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-. The fragment comprises heavy chain variable domains (V) connected by a linker_H) And a light chain variable domain (V)_L) The linker is too short to allow pairing between the two domains on the same strand. Thus, V on a segment is forced_HAnd V_LDomain and complementary V on another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for constructing bispecific antibody fragments by using single chain fv (sfv) dimers has also been reported. See Gruber et al, J.Immunol.,152:5368 (1994).

Antibodies with more than two titers are contemplated. For example, trispecific antibodies can be prepared. Tuft et al, j.immunol.,147:60 (1991).

(vii) Single domain antibodies

In some embodiments, the antibodies of the invention are single-domain antibodies. Single domain antibodies are single polypeptide chains that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No.6,248,516B1). In one embodiment, the single domain antibody consists of all or part of the heavy chain variable domain of the antibody.

(viii) Antibody variants

In some embodiments, amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can be introduced into the amino acid sequence of a subject antibody at the time of sequence preparation.

(ix) Substitution, insertion, and deletion variants

In certain embodiments, antibody variants are provided having one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table 1 under the heading of "conservative substitutions". More substantial variations are provided in table 1 under the heading of "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Table 3: exemplary substitutions

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

a. hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

b. neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

c. acidic: asp, Glu;

d. basic: his, Lys, Arg;

e. residues that influence chain orientation: gly, Pro;

f. aromatic: trp, Tyr, Phe.

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

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

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

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

One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" and is described in Cunningham and Wells, Science,244:1081-1085 (1989). In this method, a residue or set of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that indicate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. As alternative candidates, such contact and adjacent residues may be targeted or eliminated. Variants can be screened to determine if they contain the desired property.

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

(x) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.

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

In one embodiment, antibody variants are provided comprising an Fc region wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function. In particular, antibodies having reduced amounts of fucose relative to the amount of fucose on the same antibody produced in wild-type CHO cells are contemplated herein. That is, they are characterized as having a reduced amount of fucose than they would have if produced by native CHO cells (e.g., CHO cells that produce native glycosylation patterns, such as CHO cells containing native FUT8 genes). In certain embodiments, the antibody is an antibody on which less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans comprise fucose. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In certain embodiments, the antibody is an antibody in which none of the N-linked glycans comprises fucose, i.e., wherein the antibody is completely free of fucose, or is free of fucose or is afucosylated. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all sugar structures (e.g. complexed, heterozygous and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g. as described in WO 2008/077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. patent publication No. us 2003/0157108(Presta, L.); US 2004/0093621(Kyowa hakko kogyo co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); Yamane-Ohnuki et al, Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13CHO cells (Ripka et al, Arch. biochem. Biophys.249: 533. 545 (1986); U.S. patent application No. US 2003/0157108A1, Presta, L; and WO 2004/056312A1, Adams et al, especially in example 11), and knock-out cell lines such as alpha-1, 6-fucosyltransferase gene FUT8 knock-out CHO cells (see, e.g., Yamane-Ohnuki et al, Biotech. Bioeng.87:614 (2004); Kanda, Y.et al, Biotechnol. Bioeng.94(4): 680. 688 (2006); and WO 2003/085107).

Antibody variants having bisected oligosaccharides are further provided, for example, where the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878(Jean-Mairet et al); U.S. Pat. No.6,602,684(Umana et al); US 2005/0123546(Umana et al); and Ferrara et al, Biotechnology and Biotechnology, 93(5), 851-861 (2006). Also provided are antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997/30087(Patel et al); WO 1998/58964(Raju, S.); and WO 1999/22764(Raju, S.).

In certain embodiments, an antibody variant comprising an Fc region described herein is capable of binding to Fc γ RIII. In certain embodiments, antibody variants comprising an Fc region described herein have ADCC activity in the presence of human effector cells or increased ADCC activity in the presence of human effector cells as compared to an otherwise identical antibody comprising a human wild type IgG1Fc region.

(xi) Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

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

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

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

In certain embodiments, an antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334(EU residue numbering) of the Fc region. In an exemplary embodiment, the antibody comprises the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.

In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. nos. 6,194,551; WO 99/51642; and Idusogene et al, J.Immunol.164: 4178-.

Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn), 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)), are described in US2005/0014934A1(Hinton et al). Those antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 of the Fc region (e.g., substitution of residue 434 of the Fc region) (U.S. patent No.7,371,826). See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260; U.S. Pat. Nos. 5,624,821; and WO 94/29351, which concerns other examples of Fc region variants.

(xii) Antibody derivatives

The antibodies of the invention can be further modified to include additional non-proteinaceous moieties known in the art and readily available. In certain embodiments, the moiety suitable for derivatization of the antibody is a water-soluble polymer. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the specific properties or function of the antibody to be improved, whether the antibody derivative is to be used in a treatment under specified conditions, and the like.

(xiii) Vectors, host cells, and recombinant methods

Antibodies can also be generated using recombinant methods. For recombinant production of anti-antigen antibodies, nucleic acids encoding the antibodies are isolated and inserted into replicable vectors for further cloning (DNA amplification) or expression. DNA encoding the antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). Many vectors are available. Carrier members typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

(a) Signal sequence component

The antibodies of the invention can be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, preferably a signal sequence at the N-terminus of the mature protein or polypeptide or other polypeptide having a specific cleavage site. The heterologous signal sequence of choice is preferably one which is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is replaced with a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the native signal sequence may be replaced by, for example, a yeast invertase leader, an alpha factor leader (including Saccharomyces and Kluyveromyces alpha factor leaders), an acid phosphatase leader, a Candida albicans glucoamylase leader, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, can be utilized.

(b) Origin of replication

Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, in cloning vectors, such sequences are those which enable the vector to replicate independently of the host chromosomal DNA, including origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeasts and viruses. The origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the 2. mu. plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) can be used for cloning vectors in mammalian cells. In general, mammalian expression vectors do not require an origin of replication component (the SV40 origin may generally be used simply because it contains an early promoter).

(c) Selection gene components

Expression and cloning vectors may comprise a selection gene, also referred to as a selectable marker. Typical selection genes encode the following proteins: (a) conferring resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) supplementing the nutritional deficiency; or (c) provide key nutrients not available from complex media, such as a gene encoding a bacillus D-alanine racemase.

One example of a selection scheme utilizes drugs to retard the growth of host cells. Those cells successfully transformed with the heterologous gene produce proteins that confer drug resistance and thus survive the selection protocol. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of a suitable selectable marker for mammalian cells is one that can identify cells competent to take up antibody-encoding nucleic acids, such as DHFR, Glutamine Synthase (GS), thymidine kinase, metallothionein-I and-II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

For example, cells transformed with the DHFR gene are identified by culturing the transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified along with any other co-transformed nucleic acids. A Chinese Hamster Ovary (CHO) cell line deficient in endogenous DHFR activity (e.g., ATCC CRL-9096) can be used.

Alternatively, GS gene-transformed cells were identified by culturing the transformants in a medium containing L-methionine sulfoximine (Msx), an inhibitor of GS. Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acids. The GS selection/amplification system may be used in combination with the DHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts comprising endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody of interest, a wild-type DHFR gene, and another selectable marker such as aminoglycoside 3' -phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No.4,965,199.

A suitable selection gene for yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al, Nature 282:39 (1979)). the trp1 gene provides a selectable marker for yeast mutants lacking the ability to grow in tryptophan, such as ATCC No.44076 or PEP 4-1. The presence of a trp1 lesion in the genome of a yeast host cell then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2 deficient yeast strains (ATCC 20,622 or 38,626) were complemented with known plasmids carrying the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used to transform Kluyveromyces yeast. Alternatively, expression systems for large-scale production of recombinant calf chymosin in Kluyveromyces lactis have been reported. Van den berg, Bio/Technology 8:135 (1990). Also disclosed are stable multi-copy expression vectors suitable for secretion of mature recombinant human serum albumin by industrial strains of the genus Kluyveromyces. Fleer et al, Bio/Technology 9: 968-.

(d) Promoter component

Expression and cloning vectors typically contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding an antibody. Promoters suitable for use in prokaryotic hosts include the phoA promoter, the beta-lactamase and lactose promoter systems, the alkaline phosphatase promoter, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Promoters for use in bacterial systems will also comprise Shine-Dalgarno (s.d.) sequences operably linked to DNA encoding the antibody.

Promoter sequences for eukaryotic cells are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is the CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes is the AATAAA sequence, which may be the signal to add a poly A tail to the 3' end of the coding sequence. All these sequences are suitably inserted into eukaryotic expression vectors.

Examples of promoter sequences suitable for use in a yeast host include the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters which are inducible promoters having the additional advantage of controlling transcription by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Vectors and promoters suitable for yeast expression are further described in EP 73,657. Yeast enhancers may also be advantageously used with yeast promoters.

Transcription of antibodies from vectors in mammalian host cells can be controlled by, for example, promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis b virus, simian virus 40(SV40), or from heterologous mammalian promoters such as the actin promoter or an immunoglobulin promoter, and from heat shock promoters, provided such promoters are compatible with the host cell system.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. U.S. Pat. No.4,419,446 discloses a system for expressing DNA in a mammalian host using bovine papilloma virus as a vector. An improvement of this system is described in U.S. Pat. No.4,601,978. For the expression of human interferon-beta cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus, see also Reyes et al, Nature 297: 598-. Alternatively, the rous sarcoma virus long terminal repeat can be used as a promoter.

(e) Enhancer element component

Transcription of DNA encoding the antibodies of the invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein and insulin). However, typically an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. For enhanced elements for activation of eukaryotic promoters see also Yaniv, Nature 297:17-18 (1982). Enhancers may be spliced into the vector at positions 5' or 3' to the antibody coding sequence, but are preferably located at sites 5' to the promoter.

(f) Transcription termination component

Expression vectors for eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are typically available from the 5 'and occasionally 3' ends of untranslated regions of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors disclosed therein.

(g) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast or higher eukaryotes as described above. Prokaryotes suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example enterobacteriaceae, such as Escherichia (Escherichia) e.g. Escherichia coli or Escherichia coli (e.coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) e.g. Salmonella typhimurium, Serratia (Serratia) e.g. Serratia marcescens (Serratia marcans), Shigella (Shigella), and bacillus (bacillus) e.g. bacillus subtilis (b.licheniformis) and bacillus (b.heliformis) (e.g. bacillus licheniformis 41P disclosed in DD 266,710 published 4.12.1989), Pseudomonas (Pseudomonas) such as Pseudomonas aeruginosa, Pseudomonas aeruginosa. A preferred E.coli cloning host is E.coli 294(ATCC 31,446), although other strains such as E.coli B, E.coli X1776(ATCC 31,537) and E.coli W3110(ATCC 27,325) are also suitable. These examples are illustrative and not restrictive.

Full-length antibodies, antibody fusion proteins, and antibody fragments can be produced in bacteria, particularly when glycosylation and Fc effector function are not required, such as when a therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) that itself exhibits efficacy in tumor cell destruction. Full-length antibodies have a longer half-life in circulation. Production in E.coli is faster and more cost effective. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237(Carter et al.), U.S. Pat. No.5,789,199 (Joly et al.), U.S. Pat. No.5,840,523(Simmons et al.), which describes a Translation Initiation Region (TIR) and signal sequences that optimize expression and secretion. See also Charlton, Methods in Molecular Biology, volume 248(B.K.C.Lo, eds., Humana Press, Totowa, NJ,2003), pp.245-254, which describes the expression of antibody fragments in E.coli. After expression, the antibody can be isolated from the E.coli cell slurry in a soluble fraction and purified by, for example, a protein A or G column (depending on the isotype). A final purification can be performed similar to the process used for purifying antibodies expressed in e.g. CHO cells.

In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or commonly used baker's yeast are among the most commonly used lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and useful in the present invention, such as Schizosaccharomyces pombe (Schizosaccharomyces pombe); kluyveromyces hosts such as, for example, Kluyveromyces lactis (k.lactis), Kluyveromyces fragilis (k.fragilis) (ATCC 12,424), Kluyveromyces bulgaricus (k.bulgaricus) (ATCC 16,045), Kluyveromyces williamsii (k.wickraimi) (ATCC 24,178), k.wallidi (ATCC 56,500), Kluyveromyces drosophilus (k.drosophilarium) (ATCC 36,906), Kluyveromyces thermotolerans (k.thermotolerans), and Kluyveromyces marxianus (k.marxianus); yarrowia (EP 402,226); pichia pastoris (Pichia pastoris) (EP 183,070); candida genus (Candida); trichoderma reesei (Trichoderma reesei) (EP244,234); neurospora crassa (Neurospora crassa); schwanniomyces (Schwanniomyces), such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora (Neurospora), Penicillium (Penicillium), torticollis (Tolypocladium), and Aspergillus (Aspergillus) hosts such as Aspergillus nidulans (a. nidulans) and Aspergillus niger (a. niger). For a review discussing the use of yeasts and filamentous fungi for the production of therapeutic proteins see, e.g., Gerngross, nat. Biotech.22:1409-1414 (2004).

Certain fungal and yeast strains may be selected in which the glycosylation pathway has been "humanized" resulting in the production of antibodies having a partially or fully human glycosylation pattern. See, e.g., Li et al, nat. Biotech.24:210-215(2006) (which describes humanization of the glycosylation pathway in Pichia pastoris); and Gerngross et al, supra.

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants and corresponding permissive insect host cells have been identified which are derived from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori (Bombyx mori). A variety of viral strains are publicly available for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used in accordance with the present invention as viruses herein, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, lemna (Leninaceae), alfalfa (m.truncatula), and tobacco may also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177; 6,040,498; 6,420,548, respectively; 7,125,978, respectively; and 6,417,429 (PLANTIBODIIES described for antibody production in transgenic plants^TMA technique).

Vertebrate cells can be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7, ATCCRL 1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli (sertoli) cells (TM4, Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); vero cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); bovine murine (buffalo rat) hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCCCCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982); MRC5 cells; FS4 cells; and human hepatoma line (Hep G2) The cell line comprises Chinese Hamster Ovary (CHO) cells including DHFR^-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 and Sp 2/0. For reviews of certain mammalian host cell lines suitable for antibody production see, e.g., yazakian Wu, Methods in Molecular Biology, volume 248(b.k.c.lo, eds., Humana Press, Totowa, NJ,2003), pp.255-268.

Host cells are transformed with the expression or cloning vectors described above for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying the genes encoding the desired sequences.

(h) Culturing host cells

Host cells for producing the antibodies of the invention can be cultured in a variety of media. Commercial culture media such as Ham's F10(Sigma), minimal essential medium (MEM, Sigma), RPMI-1640(Sigma), and Dulbecco's modified Eagle's medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media described in the following documents can be used as the medium for the host cells: ham et al, meth.enz.58:44 (1979); barnes et al, anal. biochem.102:255 (1980); U.S. patent nos. 4,767,704; 4,657,866, respectively; 4,927,762, respectively; 4,560,655, respectively; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN) ^TMDrugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included in suitable concentrations. It will be apparent to the skilled person that the culture conditions (such as temperature, pH, etc.) are those previously selected for the host cell for expression.

(xiv) Purification of antibodies

When recombinant techniques are used, the antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris of the host cells or lysed fragments is removed, for example, by centrifugation or ultrafiltration. Carter et al, Bio/Technology10: 163-. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant from such an expression system is typically first concentrated using a commercial protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. In any of the above steps, a protease inhibitor such as PMSF may be included to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being one of the generally preferred purification steps. The suitability of protein a as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein a can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, j. immunol. meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma 3(Guss et al, EMBO J.5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices may be used. Physically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene enable faster flow rates and shorter processing times than agarose. If the antibody comprises C_H3 Domain, then Bakerbond ABX can be used^TMPurification was performed on resin (j.t. baker, phillips burg, NJ). Depending on the antibody to be recovered, other protein purification techniques may also be used, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, heparin Sepharose^TMChromatography on, anions Or chromatography on a cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

In general, various methodologies for preparing antibodies for research, testing, and clinical use are well established in the art, are consistent with the methodologies described above, and/or are deemed appropriate for a particular antibody of interest by one of skill in the art.

C. Selection of biologically active antibodies

One or more "biological activity" assays may be performed on the antibodies generated as described above to select antibodies with beneficial properties from a therapeutic perspective or to select formulations and conditions that retain the biological activity of the antibody. Antibodies can be tested for their ability to bind to the antigen against which they are generated. For example, methods known in the art (such as ELISA, Western Blot, etc.) can be used.

For example, for an anti-PDL 1 antibody, the antigen binding properties of the antibody may be assessed in an assay that detects the ability to bind to PDL 1. In some embodiments, for example, binding may be by saturation; ELISA (enzyme-Linked immuno sorbent assay); and/or competition assays (e.g., RIA) to determine binding of the antibody. Also, the antibodies may be subjected to other biological activity assays, for example, to assess their effectiveness as therapeutics. Such assays are known in the art and depend on the intended use of the target antigen and antibody. For example, the biological effects of blocking PDL1 by antibodies can be assessed in CD8+ T cells, a mouse model of lymphocytic choriomeningitis virus (LCMV), and/or a syngeneic tumor model, e.g., as described in us patent 8,217,149.

To screen for Antibodies that bind to a particular epitope on the antigen of interest (e.g., those that block binding of the anti-PDL 1 antibody of the examples to PDL 1), a conventional cross-blocking assay can be performed, such as described in Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping can be performed to determine whether an antibody binds to an epitope of interest, e.g., as described in Champe et al, J.biol.chem.270:1388-1394 (1995).

In one aspect, assays are provided for identifying anti-OX 40 antibodies that have biological activity. Biological activities can include, for example, binding to OX40 (e.g., binding to human and/or cynomolgus monkey OX40), increasing OX 40-mediated signal transduction (e.g., increasing NFkB-mediated transcription), depleting cells (e.g., T cells) expressing human OX40, enhancing T effector function (e.g., CD4+ effector T cells, CD8+ effector T cells) (e.g., by increasing effector T cell proliferation and/or increasing cytokine production by effector T cells (e.g., gamma interferon)), enhancing memory T cell function (e.g., CD4+ memory T cells) (e.g., by increasing memory T cell proliferation and/or increasing cytokine production by memory T cells (e.g., gamma interferon)), inhibiting Treg suppression that modulates T cell function (e.g., by decreasing effector T cell function (e.g., CD4+ effector T cell function, CD8+ effector T cell function)). Also provided are antibodies having such biological activity in vivo and/or in vitro.

In certain embodiments, antibodies of the invention are tested for such biological activity.

T cell co-stimulation can be determined using methods known in the art, and exemplary methods are disclosed herein. For example, T cells (e.g., memory or effector T cells) can be obtained from peripheral white blood cells (e.g., isolated from human whole blood using Ficoll gradient centrifugation). Memory T cells (e.g., CD4+ memory T cells) or effector T cells (e.g., CD4+ Teff cells) can be isolated from PBMCs using methods known in the art. For example, a Miltenyi CD4+ memory T cell isolation kit or a Miltenyi naive CD4+ T cell isolation kit can be used. Isolated T cells are cultured in the presence of antigen presenting cells (e.g., irradiated L cells expressing CD32 and CD 80) and activated by the addition of anti-CD 3 antibody in the presence or absence of OX40 agonist antibody. The effect of an agonist OX40 antibody on T cell proliferation can be measured using methods well known in the art. For example, the CellTiter Glo kit (Promega) can be used and the results read on a multi-marker reader (Perkin Elmer). The effect of agonist OX40 antibodies on T cell function can also be determined by analyzing cytokines produced by T cells. In one embodiment, CD4+ T cells are assayed for interferon gamma production, for example by measuring interferon gamma in the cell culture supernatant. Methods for measuring interferon gamma are well known in the art.

Treg cell function can be determined using methods known in the art, and exemplary methods are disclosed herein. In one example, the ability of tregs to suppress the proliferation of effector T cells is determined. T cells are isolated from human whole blood (e.g., memory T cells or naive T cells) using methods known in the art. The purified CD4+ naive T cells were labeled (e.g., with CFSE) and the purified Treg cells were labeled with different reagents. Irradiated antigen presenting cells (e.g., L cells expressing CD32 and CD 80) are co-cultured with labeled purified naive CD4+ T cells and purified tregs. Cocultures were activated using anti-CD 3 antibody and tested in the presence or absence of agonist OX40 antibody. After a suitable time (e.g., 6 days of co-culture), FACS analysis is used to track the level of CD4+ naive T cell proliferation by dye dilution in reduced marker staining (e.g., reduced CFSE marker staining).

OX40 signaling can be determined using methods well known in the art, and exemplary methods are disclosed herein. In one embodiment, transgenic cells are generated that express human OX40 and a reporter gene comprising an NFkB promoter fused to a reporter gene (e.g., β luciferase). Addition of OX40 agonist antibodies to cells resulted in increased transcription of NFkB, which was detected using an assay against a reporter gene.

Phagocytosis can be determined, for example, by using monocyte-derived macrophages or U937 cells, a human histiocytic lymphoma cell line with the morphology and characteristics of mature macrophages. OX40 expressing cells were added to monocyte derived macrophages or U937 cells in the presence or absence of anti-OX 40 agonist antibodies. After culturing the cells for a suitable period of time, the percent phagocytosis is determined by examining the percentage of cells double stained for the marker of 1) macrophages or U937 cells and 2) cells expressing OX40, and dividing this by the total number of cells displaying the marker of cells expressing OX40 (e.g., GFP). Analysis can be performed by flow cytometry. In another embodiment, the analysis may be performed by fluorescence microscopy analysis.

ADCC can be determined, for example, using methods well known in the art. Exemplary methods are described in the definitions section, and exemplary assays are disclosed in the examples. In some embodiments, the level of OX40 on OX40 expressing cells used for testing in an ADCC assay is characterized. Cells were stained with a detectably labeled anti-OX 40 antibody (e.g., PE labeled) and then fluorescence levels were determined using flow cytometry, with results presented as Median Fluorescence Intensity (MFI). In another embodiment, ADCC can be analyzed by the CellTiter Glo assay kit and cell viability/cytotoxicity can be determined by chemiluminescence.

The binding affinity of each antibody to Fc γ RIA, Fc γ RIIA, Fc γ RIIB, and two allotypes of Fc γ RIIIA (F158 and V158) can be measured in an ELISA-based ligand binding assay using the corresponding recombinant Fc γ receptor. The purified human Fc γ receptor was expressed as a fusion protein containing the extracellular domain of the gamma chain of the receptor linked to a C-terminal Gly/6 xHis/glutathione S-transferase (GST) polypeptide tag. The binding affinity of the antibodies to those human Fc γ receptors was determined as follows. For the low affinity receptors, two allotypes, F-158 and V-158, of Fc γ RIIA (CD32A), Fc γ RIIB (CD32B), and Fc γ RIIIA (CD16), two allotypes, F-158 and V-158, can be raised against human kappa chain F (ab')₂Fragment (ICN biomedicalal; Irvine, CA) Cross-Linked (antibodies: cross-Linked with F (ab') in approximate molar ratio 1: 3)₂) Antibodies were tested as multimers. Plates were coated with anti-GST antibody (Genentech) and blocked with Bovine Serum Albumin (BSA). Using Phosphate Buffered Saline (PBS) containing 0.05% Tween-20 and ELx405^TMAfter washing with a plate washer (Biotek Instruments; Winooski, VT), Fc γ receptor was added to the plate at 25 ng/well and incubated for 1 hour at room temperature. After washing the plates, serial dilutions of the test antibody were added as multimeric complexes, and the plates were incubated for 2 hours at room temperature. Cleaning the plate to remove After binding of the antibody, goat anti-human F (ab') conjugated with horseradish peroxidase (HRP)₂F (ab')₂Fragments (Jackson ImmunoResearch Laboratories; WestGrove, Pa.) detect antibodies that bind to Fc γ receptors, followed by addition of the substrate, Tetramethylbenzidine (TMB) (Kirkegaardand Perry Laboratories; Gaithersburg, Md.). Depending on the Fc γ receptor tested, the plates were incubated at room temperature for 5-20 minutes to allow for color development. With 1M H₃PO₄The reaction is terminated and a microplate reader is used (190, Molecular Devices; sunnyvale, CA) measures the absorbance at 450 nm. Dose-response binding curves were generated by plotting the mean absorbance values from duplicate antibody dilutions against antibody concentration. Values for effective antibody concentration (EC) at which 50% of the maximal response from binding to Fc gamma receptor was detected were determined after fitting the binding curve with a four-parameter equation using SoftMax Pro (Molecular Devices)₅₀)。

Cells for use in any of the above in vitro assays include cells or cell lines that naturally express OX40 or that are engineered to express OX 40. Such cells include activated T cells that naturally express OX40, Treg cells, and activated memory T cells. Such cells also include cell lines that express OX40 and cell lines that do not normally express OX40 but have been transfected with a nucleic acid encoding OX 40. Exemplary cell lines provided herein for use in any of the above in vitro assays include transgenic BT474 cells expressing human OX40 (a human breast cancer cell line).

It is understood that any of the above assays may be performed using the immunoconjugates of the invention in place of or in addition to an anti-OX 40 antibody.

It is understood that any of the above assays can be performed using an anti-OX 40 antibody and another therapeutic agent, such as a PD-1 axis binding agent (e.g., an anti-PD-1 or anti-PDL 1 antibody).

D. Pharmaceutical compositions and formulations

Also provided herein are pharmaceutical compositions and formulations comprising a PD-1 axis binding antagonist and/or antibody described herein, such as an anti-PDL 1 antibody, or an anti-human OX40 agonist antibody, and a pharmaceutically acceptable carrier.

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

An exemplary lyophilized antibody formulation is described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulation comprising a histidine-acetate buffer.

The compositions and formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Suitably, such active ingredients are present in combination in an amount effective for the intended purpose.

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

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

Methods of treatment

Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody). In some embodiments, the treatment results in a sustained response in the individual after cessation of the treatment. The methods described herein are useful for treating conditions where enhanced immunogenicity is desired, such as for the treatment of cancer to increase tumor immunogenicity. Also provided herein are methods of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody). In yet other aspects, provided herein are methods of treating an infection (e.g., a bacterial or viral or other pathogen infection). In some embodiments, the infection is a viral and/or bacterial infection. In some embodiments, the infection is a pathogen infection. In some embodiments, the infection is an acute infection. In some embodiments, the infection is a chronic infection.

Any PD-1 axis binding antagonist and OX40 binding agonist known in the art or described herein can be used in the methods.

In some embodiments, the subject is a human.

In some embodiments, the individual has been treated with an OX40 binding agonist therapy prior to the PD-1 axis binding antagonist and OX40 binding agonist (e.g., anti-human OX40 agonist antibody) combination treatment.

In some embodiments, the individual has a (proven to be resistant) cancer that is resistant to one or more PD-1 axis antagonists. In some embodiments, resistance to a PD-1 axis antagonist comprises cancer relapse or refractory cancer. Recurrence may refer to the reoccurrence of the cancer at the original site or new site after treatment. In some embodiments, resistance to a PD-1 axis antagonist comprises progression of cancer during treatment with a PD-1 axis antagonist. In some embodiments, resistance to a PD-1 axis antagonist comprises a cancer that does not respond to treatment. The cancer may be resistant at the start of treatment, or may become resistant during treatment. In some embodiments, the cancer is in an early stage or an advanced stage.

In another aspect, the individual has a cancer that expresses (has been shown to express, e.g., in a diagnostic test) a PD-L1 biomarker. In some embodiments, the patient's cancer expresses a low PD-L1 biomarker. In some embodiments, the patient's cancer expresses a high PD-L1 biomarker. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is deleted from the sample when it comprises 0% of the sample.

In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is present in the sample when it comprises more than 0% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 1% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 5% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 10% of the sample.

In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is detected in the sample using a method selected from the group consisting of: FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY ay techniques, and FISH, and combinations thereof.

In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is detected in a sample by protein expression. In some embodiments, protein expression is determined by Immunohistochemistry (IHC). In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 antibody. In some embodiments, the PD-L1 biomarker is detected by IHC as weak staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC as moderate staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC as strong staining intensity. In some embodiments, the PD-L1 biomarker is detected on tumor cells, tumor-infiltrating immune cells, stromal cells, and any combination thereof. In some embodiments, the staining is membrane staining, cytoplasmic staining or a combination thereof.

In some embodiments of any of the methods, assays, and/or kits, the absence of the PD-L1 biomarker is detected as absence or absence of staining in the sample. In some embodiments of any of the methods, assays, and/or kits, the presence of the PD-L1 biomarker is detected as any staining in the sample.

In some embodiments, the combination therapies of the invention comprise administering a PD-1 axis binding antagonist and an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody). The PD-1 axis binding antagonist and OX40 binding agonist can be administered in any suitable manner known in the art. For example, the PD-1 axis binding antagonist and OX40 binding agonist can be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the PD-1 axis binding antagonist is in a separate composition from the OX40 binding agonist. In some embodiments, the PD-1 axis binding antagonist is in the same composition as the OX40 binding agonist.

The PD-1 axis binding antagonist and OX40 binding agonist (e.g., anti-human OX40 agonist antibodies) can be administered by the same route of administration or by different routes of administration. In some embodiments, the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intracamerally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the OX40 binding agonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intracamerally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist can be administered to prevent or treat disease. Appropriate dosages of the PD-1 axis binding antagonist and/or OX40 binding agonist (e.g., anti-human OX40 agonist antibody) can be determined based on the type of disease being treated, the type of PD-1 axis binding antagonist and OX40 binding agonist, the severity and course of the disease, the clinical condition of the individual, the clinical history and response to treatment of the individual, and the discretion of the attending physician. In some embodiments, the combination treatment of an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) and a PD-1 axis binding antagonist (e.g., an anti-PD-1 or anti-PD-L1 antibody) is synergistic, whereby the effective dose of an OX40 binding agent (e.g., an anti-human OX40 agonist antibody) in the combination is reduced relative to the effective dose of an OX40 binding agent (e.g., an anti-human OX40 agonist antibody) as a single agent.

As a general recommendation, the antibody administered to humans will be in a therapeutically effective amount in the range of about 0.01 to about 50mg/kg of patient body weight, whether by one or more administrations. In some embodiments, the antibody used is, for example, administered daily at about 0.01 to about 45mg/kg, about 0.01 to about 40mg/kg, about 0.01 to about 35mg/kg, about 0.01 to about 30mg/kg, about 0.01 to about 25mg/kg, about 0.01 to about 20mg/kg, about 0.01 to about 15mg/kg, about 0.01 to about 10mg/kg, about 0.01 to about 5mg/kg, or about 0.01 to about 1 mg/kg. In some embodiments, the antibody is administered at 15 mg/kg. However, other dosage regimens may be useful. In one embodiment, the anti-PDL 1 antibody described herein is administered to a human at a dose of about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, or about 1400mg on day 1 of a 21-day cycle. The dose may be administered as a single dose or as multiple doses (e.g. 2 or 3 doses), such as infusion. The dose of antibody administered in the combination therapy may be reduced compared to monotherapy. The progress of this therapy is readily monitored by conventional techniques.

In some embodiments, the method may further comprise additional therapies. The additional therapy can be radiation therapy, 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. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an antimetabolite. In some embodiments, the additional therapy is administration of a side-effect limiting agent (e.g., an agent intended to reduce the incidence of and/or reduce the severity of a treatment side-effect, such as a cardiac depressant or the like). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some cases In embodiments, the other therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PI3K/AKT/mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and/or a chemopreventive agent. In some embodiments, the additional therapy is CTLA-4 (also known as CD152), e.g., a blocking antibody, ipilimumab (also known as MDX-010, MDX-101, or) Tremelelmumab (also known as ticilimumab or CP-675,206), antagonists against B7-H3 (also known as CD276), e.g. blocking antibodies, MGA271, antagonists against TGF β, e.g. metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299, therapies comprising adoptive transfer of T cells expressing a Chimeric Antigen Receptor (CAR), e.g. cytotoxic T cells or CTLs, therapies comprising adoptive transfer of T cells comprising a dominant-negative TGF β receptor, e.g. a dominant-negative TGF β type II receptor, therapies comprising HERCREEM regimen (see e.g. clinicaltrials. gov idifier NCT 889954), agonists against CD137 (also known as TNFRSF9, 4-1BB, or ILA), e.g. activating antibodies, urelimumab (also known as CD BMS-663513), agonists against CD 7, e.g. dnrsf 367, 4-1BB, or ilae.g. agonist antibodies against mmasian receptor (also known as monoclonal antibody), e-T-2-T-agonist (also known as monoclonal antibody), e.g. agonist antibody, CD-T-2, or agonist antibody(s) in combination with an agonist (e.g. agonist), e.g. agonist antibody, mmasian-T-2, CD-T-2, e-agonist, e.g. agonist, e-agonist, e.g. agonist, antibody(s) or agonist), e.g. agonist antibody (s-agonist), e.g. agonist antibody (mmtamicine-agonist), e.g. agonist), antibody (s-agonist), e.g. agonist), antibody (also known as agonist), antibody (s-agonist), e.g. agonist), antibody (s-T-7-agonist), e.g. agonist), e.g Genentech), DMUC5754A, antibody-drug conjugates targeting endothelin B receptor (EDNBR), e.g., antibodies against EDNBR conjugated to MMAE, angiogenesisInhibitors, antibodies against VEGF, e.g. VEGF-A, bevacizumab (also known asGenentech), antibodies against angiopoietin 2 (also known as Ang2), MEDI3617, antineoplastic agents, agents targeting CSF-1R (also known as M-CSFR or CD115), anti-CSF-1R (also known as IMC-CS4), interferons, such as interferon α or interferon gamma, Roferon-a, GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargrastim (sargramostim), or) IL-2 (also known as aldesleukin) or) IL-12, an antibody targeting CD20 (in some embodiments, the antibody targeting CD20 is obinutuzumab (also known as GA101 or GA 101)) Or rituximab (rituximab)), an antibody targeting GITR (in some embodiments, the antibody targeting GITR is TRX518), and a cancer vaccine (in some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine; in some embodiments, the peptide Cancer vaccine is a multivalent long peptide, a multiple peptide, a mixture of peptides, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci,104:14-21,2013)), in combination with an adjuvant, a TLR agonist, e.g., Poly-ICLC (also known as Poly-ICLC) ) LPS, MPL, or CpG ODN, Tumor Necrosis Factor (TNF) α -1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, HVEM antagonists, ICOS agonists, for example by administering ICOS-L, or an agonistic antibody to ICOS, CX3CL1 targeted therapy, CXCL10 targeted therapy, CCL5 targeted therapy, LFA-1 or ICAM1 agonists, selectin agonists,targeted therapy, an inhibitor of B-Raf, vemurafenib (also known asdabrafenib (also known as dabrafenib)) Erlotinib (also known as erlotinib)) Inhibitors of MEK, such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2), cobimetinib (also known as GDC-0973 or XL-518), trametinib (also known as GDC-0973 or XL-518)) Inhibitors of K-Ras, inhibitors of c-Met, onartuzumab (also known as MetMAb), inhibitors of Alk, AF802 (also known as CH5424802 or alectinib), inhibitors of phosphatidylinositol 3-kinase (PI3K), BKM120, idelalisib (also known as GS-1101 or CAL-101), perifosine (also known as KRX-0401), Akt, MK2206, GSK690693, GDC-0941, inhibitors of mTOR, sirolimus (also known as rapamycin), temsirolimus (also known as CCI-779 or Temsirolimus 779) ) Everolimus (also known as RAD001), ridaforolimus (also known as AP-23573, MK-8669, or deforolimus), OSI-027, AZD8055, INK128, dual PI3K/mTOR inhibitors, XL765, GDC-0980, BEZ235 (also known as NVP-BEZ235), BGT226, GSK2126458, PF-04691502, PF-05212384 (also known as PKI-587). The additional therapy may be one or more chemotherapeutic agents described herein.

The efficacy of any of the methods described herein (e.g., combination therapy comprising administering an effective amount of a combination of a PD-1 axis binding antagonist and an OX40 binding agonist) can be tested in various models known in the art, such as clinical or preclinical models. Suitable preclinical models are exemplified herein, and may further include, but are not limited to, the ID8 ovarian cancer, GEM model, B16 melanoma, RENCA renal cell carcinoma, CT26 colorectal cancer, MC38 colorectal cancer, and Cloudman melanoma cancer models.

The efficacy of any of the methods described herein (e.g., a combination therapy comprising administering an effective amount of a combination of a PD-1 axis binding antagonist and an OX40 binding agonist) can be tested in a GEM model of tumor formation (including, but not limited to, a GEM model of non-small cell lung cancer, pancreatic ductal adenocarcinoma, or melanoma). For example, such as Jackson, E.L., et al (2001) Genes Dev.15(24):3243-8 (described as Kras) ^G12D) And Lee, C.L., et al (2012) Dis. model Mech.5(3):397-402 (FRT-mediated p 53)^{Deficiency of}Alleles), after treatment with adenovirus recombinase at p53^{Deficiency of}Expression of Kras in background^G12DAs a preclinical model of non-small cell lung cancer. Also for example, such as Jackson, E.L., et al (2001) Genes Dev.15(24):3243-8 (described as Kras)^G12D) And Aguirre, A.J., et al (2003) Genes Dev.17(24):3112-26(p16/p 19)^{Deficiency of}Alleles), described in p16/p19^{Deficiency of}Expression of Kras in background^G12DAs a preclinical model of Pancreatic Ductal Adenocarcinoma (PDAC). As another example, Genes Dev.21(4):379-84 (described Braf) may be used, e.g., Dankort, D., et al (2007)^V600E) And Trotman, L.C., et al (2003) PLoS biol.1(3): E59 (PTEN)^{Deficiency of}Alleles), with PTEN specific for melanocytes after treatment with inducible (e.g. 4-OHT treatment) recombinase^{Deficiency of}Expression of Braf in the background^V600EAs a preclinical model of melanoma. For any of these exemplary models, after tumor formation, mice were randomly recruited into treatment groups receiving treatment with a combination anti-PDL 1 and OX40 binding agonist (e.g., anti-human OX40 agonist antibody) or control treatment. Tumor size (e.g., tumor volume) is measured during the course of treatment, and overall survival is also monitored.

In another aspect, provided herein are methods for enhancing immune function in an individual having cancer comprising administering an effective amount of a combination of a PD-1 axis binding antagonist and an OX40 binding agonist.

In some embodiments of the methods of the present disclosure, the cancer (in some embodiments, a sample of the cancer of a patient examined using a diagnostic test) has an elevated level of T cell infiltration. As used herein, T cell infiltration of a cancer may refer to the presence of T cells, such as Tumor Infiltrating Lymphocytes (TILs), within the cancer tissue or other relevant sites. It is known in the art that T cell infiltration may be associated with improved clinical outcome in certain cancers (see e.g. Zhang et al,N.Engl.J.Med.348(3):203-213(2003))。

however, T cell depletion is also a major immunological feature of cancer, in which many Tumor Infiltrating Lymphocytes (TILs) express high levels of inhibitory co-receptors and lack the ability to produce effector cytokines (whery, E.J).Nature immunology12:492-499(2011)；Rabinovich,G.A.,et al.,Annual review of immunology25:267-296(2007)). In some embodiments of the methods of the present disclosure, the subject has a T cell dysfunctional disorder. In some embodiments of the methods of the present disclosure, the T cell dysfunctional disorder is characterized by T cell anergy or secretion of cytokines, decreased ability to proliferate or execute lytic activity. In some embodiments of the methods of the present disclosure, the T cell dysfunctional disorder is characterized by T cell depletion. In some embodiments of the methods of the present disclosure, the T cells are CD4+ and CD8+ T cells. Without being bound by theory, OX40 binding agonist therapy may increase T cell (e.g., CD4+ T cells, CD8+ T cells, memory T cells) priming, activation, and/or proliferation relative to prior to administration of the combination. In some embodiments, the T cell is a CD4+ and/or CD8+ T cell.

In some embodiments of the methods of the present disclosure, the cancer (in some embodiments, a sample of the patient's cancer is examined using a diagnostic test) has a low level of T cell infiltration. In some embodiments, the cancer (in some embodiments, a sample from which the patient is examined for cancer using a diagnostic test) is free of detectable T cell infiltrates. In some embodiments, the cancer is a non-immunogenic cancer (e.g., a non-immunogenic colorectal cancer and/or ovarian cancer). Without being bound by theory, OX40 binding agonist therapy may increase T cell (e.g., CD4+ T cells, CD8+ T cells, memory T cells) priming, activation, and/or proliferation relative to prior to administration of the combination.

In some embodiments of the methods of the present disclosure, the CD4 and/or CD8T cells after activation in an individual are characterized relative to γ -IFN prior to administration of the combination⁺Producing CD4 and/or CD8T cells and/or enhanced lytic activity. Gamma-IFN may be measured by any means known in the art⁺Including, for example, Intracellular Cytokine Staining (ICS), which involves cell fixation, permeabilization, and staining with antibodies to γ -IFN. Lysis activity can be measured by any means known in the art, for example using a cell killing assay with mixed effects and target cells.

In some embodiments, CD8+ T cells are characterized by, for example, the presence of CD8b expression (e.g., by rtPCR using, for example, Fluidigm) (CD8b is also known as the T cell surface glycoprotein CD8 β chain; CD8 antigen, α polypeptide p 37; accession No. NM _ 172213). In some embodiments, the CD8+ T cells are from peripheral blood. In some embodiments, the CD8+ T cells are from a tumor.

In some embodiments, the Treg cells are characterized, for example, by the presence of Fox3P expression (e.g., by rtPCR, e.g., using Fluidigm) (Foxp3 also known as forkhead box protein P3; scurfin; Foxp 37; immunodeficiency, polyendocrine, enteropathy, X-linked; accession No. NM — 014009). In some embodiments, the tregs are from peripheral blood. In some embodiments, the Treg cells are from a tumor.

In some embodiments, inflammatory T cells are characterized by, for example, the presence of Tbet and/or CXCR3 expression (e.g., by rtPCR using, for example, Fluidigm). In some embodiments, the inflammatory T cells are from peripheral blood. In some embodiments, the inflammatory T cell is from a tumor.

In some embodiments of the methods of the present disclosure, the CD4 and/or CD8T cells exhibit increased release of a cytokine selected from the group consisting of: IFN-gamma, TNF-alpha and interleukins. Cytokine release may be measured by any means known in the art, for example, by detecting the presence of released cytokines in samples containing CD4 and/or CD8T cells using Western blotting, ELISA, or immunohistochemical assays.

In some embodiments of the methods of the present disclosure, the CD4 and/or CD8T cells are effector memory T cells. In some embodiments of the methods of the present disclosure, the CD4 and/or CD8 effector memory T cells are characterized as having CD44^{Height of}CD62L^{Is low in}Expression of (2). CD44 may be detected by any means known in the art^{Height of}CD62L^{Is low in}E.g., by preparing single cell suspensions of tissues (e.g., cancer tissues) and performing surface staining and flow cytometry using commercial antibodies against CD44 and CD 62L. In some embodiments of the methods of the present disclosure, CD4 and/or CD8 effector memory T cells are characterized by having expression of CXCR3 (also known as C-X-C chemokine receptor type 3; Mig receptor; IP10 receptor; G protein coupled receptor 9; interferon inducible protein 10 receptor; accession number NM-001504). In some embodiments, the CD4 and/or CD8 effector memory T cells are from peripheral blood. In some embodiments, the CD4 and/or CD8 effector memory T cells are from a tumor.

In some embodiments of the methods of the present disclosure, administering to the individual an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist is characterized by an elevated inflammatory marker of CD8T cells (e.g., CXCR3) relative to prior to administration of the human PD-1 axis binding antagonist and OX40 binding agonist. CXCR3/CD8T cells can be measured by any means known in the art and the methods described in the examples. In some embodiments, CXCR3/CD8T cells are from peripheral blood. In some embodiments, the CXCR3/CD8T cell is from a tumor.

In some embodiments of the methods of the invention, Treg function is suppressed relative to prior to administration of the combination. In some embodiments, T cell depletion is reduced relative to prior to administration of the combination.

In some embodiments, the number of tregs is reduced relative to prior to administration of the combination. In some embodiments, the plasma interferon gamma is elevated relative to prior to administration of the combination. Treg numbers can be assessed, for example, by determining the percentage of CD4+ Fox3p + CD45+ cells (e.g., by FACS analysis). In some embodiments, the absolute number of tregs in, for example, a sample is determined. In some embodiments, the tregs are from peripheral blood. In some embodiments, the tregs are from a tumor.

In some embodiments, T cells priming, activation, and/or proliferation is increased relative to prior to administration of the combination. In some embodiments, the T cell is CD4⁺And/or CD8⁺T cells. In some embodiments, by determining Ki67⁺CD8⁺T cell proliferation is detected by percentage of T cells (e.g., by FACS analysis). In some embodiments, by determining Ki67⁺CD4⁺T cell proliferation is detected by percentage of T cells (e.g., by FACS analysis). In some embodiments, the T cells are from peripheral blood. In some embodiments, the T cell is from a tumor.

Any PD-1 axis binding antagonist and OX40 binding agonist known in the art or described herein can be used in the methods of the present disclosure.

Detection and diagnostic method

In some embodiments, the sample is obtained prior to PDL1 axis binding antagonist treatment (in some embodiments, prior to OX40 binding agonist, e.g., anti-human OX40 agonist antibody treatment, e.g., combination treatment with PD-1 axis binding antagonist treatment). In some embodiments, the tissue sample is formalin fixed and paraffin embedded, archived, fresh or frozen.

In some embodiments, the sample is whole blood. In some embodiments, the whole blood comprises immune cells, circulating tumor cells, and any combination thereof.

The presence and/or expression level/amount of a biomarker (e.g., PD-L1) can be determined qualitatively and/or quantitatively based on any suitable standard known in the art, including but not limited to DNA, mRNA, cDNA, protein fragment, and/or gene copy number. In certain embodiments, the presence and/or expression level/amount of the biomarker in the first sample is increased or elevated compared to the presence/absence and/or expression level/amount in the second sample. In certain embodiments, the presence/absence and/or expression level/amount of the biomarker in the first sample is reduced or decreased compared to the presence and/or expression level/amount in the second sample. In certain embodiments, the second sample is a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue. Additional disclosures for determining the presence/absence and/or expression level/amount of a gene are described herein.

In some embodiments of any of the methods, increased expression refers to an overall increase in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of any of the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as measured by standard art-known methods such as those described herein. In certain embodiments, elevated expression refers to an increase in the expression level/amount of a biomarker in a sample, wherein the increase is at least about any of 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold greater than the expression level/amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression refers to an overall increase that is about 1.5 fold, about 1.75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 2.75 fold, about 3.0 fold, or about 3.25 fold greater compared to a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).

In some embodiments of any of the methods, reduced expression refers to an overall reduction in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of any of the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as compared to the reference sample, as detected by standard art-known methods such as those described herein. In certain embodiments, reduced expression refers to a reduction in the expression level/amount of a biomarker in a sample, wherein the reduction is any of at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold of the expression level/amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

The presence and/or expression levels/amounts of various biomarkers in a sample can be analyzed by a number of methodologies, many of which are known in the art and understood by those skilled in the art, including but not limited to immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY ay, proteomics, blood-based quantitative assays (such as, for example, serum ELISA), biochemical enzyme activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction ("PCR") (including quantitative real-time PCR ("qRT-PCR") and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA, and the like), RNA-Seq, FISH, microarray analysis, gene expression profiling, and/or gene expression series analysis ("SAGE"), and any of a wide variety of assays that can be performed by protein, gene, and/or tissue array analysis. Typical Protocols for assessing the status of genes and gene products are found, for example, in Ausubel et al, eds.,1995, Current Protocols in molecular Biology, Units 2(Northern Blotting),4(Southern Blotting),15(Immunoblotting) and 18(PCR Analysis). Multiplex immunoassays such as those available from rules based Medicine or Meso Scale Discovery ("MSD") may also be used.

In some embodiments, the presence and/or expression level/amount of a biomarker is determined using a method comprising: (a) performing gene expression profiling, PCR (such as rtPCR or qRT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH on a sample (e.g., a subject cancer sample); and (b) determining the presence and/or expression level/amount of the biomarker in the sample. In some embodiments, microarray methods include the use of microarray chips having one or more nucleic acid molecules capable of hybridizing under stringent conditions to nucleic acid molecules encoding the genes described above or having one or more polypeptides (such as peptides or antibodies) capable of binding to one or more proteins encoded by the genes described above. In one embodiment, the PCR method is qRT-PCR. In one embodiment, the PCR method is multiplex PCR. In some embodiments, gene expression is measured by microarray. In some embodiments, gene expression is measured by qRT-PCR. In some embodiments, expression is measured by multiplex PCR.

Methods for assessing mRNA in a cell are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled ribonucleic acid probes specific for one or more genes, Northern blotting and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for one or more genes, and other amplification-type detection methods such as, for example, branched DNA, SISBA, TMA, and the like).

mRNA can be conveniently determined from a sample from a mammal using Northern, dot blot or PCR analysis. In addition, such methods can include one or more steps that allow for the determination of the level of a target mRNA in a biological sample (e.g., by simultaneously examining the level of a comparative control mRNA sequence for a "housekeeping" gene, such as an actin family member). Optionally, the sequence of the amplified target cDNA can be determined.

Optional methods include protocols for examining or detecting mRNA, such as a target mRNA, in a tissue or cell sample by microarray technology. Test and control mRNA samples from the test and control tissue samples were reverse transcribed and labeled using a nucleic acid microarray to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and location of each member of the array is known. For example, a selection set of genes whose expression correlates with increased or decreased clinical benefit of anti-angiogenic therapy can be arrayed on a solid support. Hybridization of a labeled probe to a particular array member indicates that the sample from which the probe was obtained expresses the gene.

According to some embodiments, the presence and/or expression level/amount is measured by observing the protein expression level of the aforementioned genes. In certain embodiments, the methods comprise contacting the biological sample with an antibody against a biomarker described herein (e.g., an anti-PD-L1 antibody) under conditions that allow binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such methods may be in vitro or in vivo. In one embodiment, the antibody is used to select subjects eligible for treatment with a PD-L1 axis binding antagonist, e.g., for selection of biomarkers for patients.

In certain embodiments, IHC and staining protocols are used to examine the presence and/or expression level/amount of biomarker proteins in a sample. IHC staining of tissue sections has been shown to be a reliable method of determining or detecting the presence of proteins in a sample. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is PD-L1. In some embodiments, PD-L1 is detected by immunohistochemistry. In some embodiments, increased expression of the PD-L1 biomarker in a sample from an individual is increased protein expression, and in still other embodiments, is determined using IHC. In one embodiment, the expression level of the biomarker is determined using a method comprising: (a) performing an IHC analysis on a sample (such as a subject cancer sample) with an antibody; and (b) determining the level of expression of the biomarker in the sample. In some embodiments, IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control cell line stained sample or a tissue sample from a non-cancer patient).

IHC may be performed in combination with additional techniques such as morphological staining and/or fluorescence in situ hybridization. There are two general approaches to IHC; direct and indirect assays. According to the first assay, the binding of an antibody to a target antigen is determined directly. The direct assay uses a labeled reagent, such as a fluorescent label or an enzyme-labeled primary antibody, which can be visualized without additional antibody interaction. In a typical indirect assay, unconjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. In the case of secondary antibodies conjugated to enzyme labels, chromogenic or fluorogenic substrates are added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody.

The primary and/or secondary antibodies used in IHC are typically labeled with a detectable moiety. There are a large number of markers which can generally be grouped into the following categories: (a) radioisotopes, e.g.³⁵S，¹⁴C，¹²⁵I，³H and¹³¹(ii) colloidal gold particles, (c) fluorescent labels including, but not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, phycoerythrin (phytorytherin), phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more of the foregoing, (d) the presence of various enzyme-substrate labels, and U.S. Pat. No.4,275,149 provides a review of some of them examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.4,737,456), luciferin, 2, 3-dihydrophthalazinedione (dihydrophthalazinedione), malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), phosphatase, β -galactosidase, alkaline amylase oxidase, lysozyme (e.g., glucose-phosphate oxidase, glucose-phosphate dehydrogenase, glucose-6-degluco-glucose-dehydrogenase, and derivatives of any one of the foregoing Catalase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase (lactoperoxidase), microperoxidase (microperoxidase), and the like.

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

In some embodiments of any of the methods, PD-L1 is detected by immunohistochemistry using an anti-PD-L1 diagnostic antibody (i.e., primary antibody). In some embodiments, the PD-L1 diagnostic antibody specifically binds to human PD-L1. In some embodiments, the PD-L1 diagnostic antibody is a non-human antibody. In some embodiments, the PD-L1 diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the PD-L1 diagnostic antibody is a monoclonal antibody. In some embodiments, the PD-L1 diagnostic antibody is directly labeled.

The specimen thus prepared may be mounted and covered with a cover slip. Slide evaluation is then determined, for example using a microscope, and staining intensity criteria routinely used in the art can be employed. In one embodiment, it is understood that when cells and/or tissues from a tumor are examined using IHC, staining (relative to the stroma or surrounding tissue that may be present in the sample) is typically determined or assessed in the tumor cells and/or tissues. In some embodiments, it is understood that when IHC is used to examine cells and/or tissues from a tumor, staining includes assays or assessments in tumor-infiltrating immune cells, including immune cells within or around the tumor. In some embodiments, the presence of the PD-L1 biomarker is detected by IHC in > 0% of the samples, in at least 1% of the samples, in at least 5% of the samples, or in at least 10% of the samples, as described in table 4 below. In some embodiments, the presence of the PD-L1 biomarker is detected by IHC in < 5% of the cells. In some embodiments, the presence of the PD-L1 biomarker is detected by IHC in < 1% of cells. In some embodiments, the presence of the PD-L1 biomarker is detected by IHC in 0% of the cells.

In some embodiments of any of the methods, assays, and/or kits, the presence of a PD-L1 biomarker is detected by IHC with PD-L1 staining at any intensity. In some embodiments, the PD-L1 biomarker is detected by IHC with weak staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC at moderate staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC with strong staining intensity.

In some embodiments, the PD-L1 biomarker is detected by IHC in tumor cells, tumor-infiltrating immune cells, and combinations thereof.

anti-PD-L1 antibodies suitable for use in IHC are well known in the art. The ordinarily skilled artisan understands that other suitable anti-PD-L1 antibodies can be identified and characterized by comparison to anti-PD-L1 antibodies using, for example, the IHC protocol disclosed herein.

Placenta and tonsil tissues (strong PD-L1 staining intensity) were used; HEK-293 cells transfected with recombinant human PD-L1 (varying degrees of PD-L1 staining intensity, weak, medium and strong intensity) exemplify positive tissue controls. For an exemplary PD-L1IHC standard, see below.

TABLE 4

In some embodiments, PD-L1 status is diagnosed according to the guidelines provided in table 4 above.

In some embodiments, the criteria for diagnostic evaluation of PD-L1IHC are provided below:

TABLE 5

In some embodiments, the PDL1 status is diagnosed in accordance with the guidelines provided in table 5 above. In some embodiments, a sample scored as IHC 0 and/or IHC 1 may be considered PDL1 biomarker negative. In some embodiments, a sample scored as IHC 2 and/or IHC 3 may be considered positive for the PDL1 biomarker. In some embodiments, the sample is diagnosed as IHC 0, IHC 0 and/or 1, IHC 1 and/or 2, IHC 2 and/or 3, or IHC 3.

In some embodiments, PDL1 expression is assessed for a tumor or tumor sample. As used herein, a tumor or tumor sample can encompass a region of the tumor that is partially or entirely occupied by tumor cells. In some embodiments, a tumor or tumor sample can further encompass a region of the tumor occupied by cells within a tumor-associated tumor and/or a tumor-associated matrix (e.g., an adjacent peritumoral desmoplastic matrix). The tumor-associated intratumoral cells and/or tumor-associated stroma may comprise a region of immune infiltrant (e.g., tumor-infiltrating immune cells as described herein) immediately adjacent and/or contiguous with the main tumor mass. In some embodiments, tumor cells are evaluated for PDL1 expression. In some embodiments, PDL1 expression is assessed for immune cells within the tumor region (such as tumor-infiltrating immune cells) as described above.

In an alternative method, the sample may be contacted with an antibody specific for the biomarker under conditions sufficient to form an antibody-biomarker complex, and the complex detected. The presence of biomarkers can be detected in a number of ways, such as by Western blot and ELISA procedures for assaying a wide variety of tissues and samples, including plasma or serum. There are a number of immunoassay techniques that use such assay formats, see, for example, U.S. Pat. nos. 4,016,043, 4,424,279 and 4,018,653. These include both single and two-site or "sandwich" assays of the non-competitive type, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to the target biomarkers.

The presence and/or expression level/amount of a selected biomarker in a tissue or cell sample may also be examined via a functional or activity-based assay. For example, if the biomarker is an enzyme, assays known in the art can be performed to determine or detect the presence of a given enzyme activity in a tissue or cell sample.

In certain embodiments, the sample is normalized for both the difference in the amount of biomarker determined and the variability in the quality of the sample used, as well as the variability between assay runs. Such normalization can be achieved by detecting and incorporating the expression of specific normalization biomarkers, including well-known housekeeping genes. Alternatively, normalization can be based on the mean or median signal of all assayed genes or a larger subset thereof (global normalization approach). The measured normalized amount of subject tumor mRNA or protein is compared to the amount found in the reference set on a gene-by-gene basis. The normalized expression level of each mRNA or protein per test tumor per subject can be expressed as a percentage of the expression level measured in the reference set. The presence and/or expression level/amount measured in a particular subject sample to be analyzed will fall within a certain percentage of this range, which can be determined by methods well known in the art.

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

In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined multiplex of samples from the same subject or individual obtained at one or more time points different from when the test sample was obtained. For example, a reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from the same subject or individual at a time point prior to the time at which the test sample is obtained. Such a reference sample, reference cell, reference tissue, control sample, control cell or control tissue may be useful if the reference sample is obtained during the initial diagnosis of the cancer and the test sample is obtained later when the cancer becomes metastatic.

In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a multiplexed sample from a combination of one or more healthy individuals that are not the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a multiplex sample from a combination of one or more individuals who have a disease or disorder (e.g., cancer) and are not the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a pooled RNA sample or pooled plasma or serum sample from normal tissue from one or more individuals other than the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a pooled RNA sample or pooled plasma or serum sample from tumor tissue of one or more individuals who have a disease or disorder (e.g., cancer) and are not the subject or individual.

In some embodiments, the sample is a tissue sample from an individual. In some embodiments, the tissue sample is a tumor tissue sample (e.g., biopsy). In some embodiments, the tissue sample is lung tissue. In some embodiments, the tissue sample is kidney tissue. In some embodiments, the tissue sample is skin tissue. In some embodiments, the tissue sample is pancreatic tissue. In some embodiments, the tissue sample is stomach tissue. In some embodiments, the tissue sample is bladder tissue. In some embodiments, the tissue sample is esophageal tissue. In some embodiments, the tissue sample is mesothelial tissue. In some embodiments, the tissue sample is breast tissue. In some embodiments, the tissue sample is thyroid tissue. In some embodiments, the tissue sample is colorectal tissue. In some embodiments, the tissue sample is head and neck tissue. In some embodiments, the tissue sample is osteosarcoma tissue. In some embodiments, the tissue sample is prostate tissue. In some embodiments, the tissue sample is ovarian tissue, HCC (liver), blood cells, lymph nodes, and/or bone/bone marrow tissue. In some embodiments, the tissue sample is colon tissue. In some embodiments, the tissue sample is an endometrial sample. In some embodiments, the tissue sample is brain tissue (e.g., glioblastoma, neuroblastoma, and the like).

In some embodiments, a tumor tissue sample (the terms "tumor sample" are used interchangeably herein) may encompass a tumor region that is partially or entirely occupied by tumor cells. In some embodiments, a tumor or tumor sample can further encompass a region of the tumor occupied by cells within a tumor-associated tumor and/or a tumor-associated matrix (e.g., an adjacent peritumoral desmoplastic matrix). The tumor-associated intratumoral cells and/or tumor-associated stroma may comprise a region of immune infiltrant (e.g., tumor-infiltrating immune cells as described herein) immediately adjacent and/or contiguous with the main tumor mass.

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

In some embodiments of any of the methods, the cancer displays human effector cells (e.g., is infiltrated by human effector cells). Methods for detecting human effector cells are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of human effector cells. In some embodiments, the human effector cell is one or more of an NK cell, a macrophage, a monocyte. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

In some embodiments of any of the methods, the cancer displays FcR expressing cells (e.g., is infiltrated by FcR expressing cells). Methods for detecting FcR are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of FcR expressing cells. In some embodiments, the FcR is an Fc γ R. In some embodiments, the FcR is an activating Fc γ R. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

In some embodiments, the PD-L1 biomarker is detected in the sample using a method selected from the group consisting of: FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY ay techniques, and FISH, and combinations thereof. In some embodiments, the PD-L1 biomarker is detected using FACS analysis. In some embodiments, the PD-L1 biomarker is PD-L1. In some embodiments, PD-L1 expression is detected in a blood sample. In some embodiments, PD-L1 expression is detected on circulating immune cells in a blood sample. In some embodiments, the circulating immune cells are CD3+/CD8+ T cells. In some embodiments, the immune cells are isolated from the blood sample prior to analysis. Any suitable method of isolating/enriching such cell populations may be used, including but not limited to cell sorting. In some embodiments, PD-L1 expression in a sample from the subject is elevated in response to treatment with an inhibitor of the PD-L1/PD-1 axis pathway (such as an anti-PD-L1 antibody). In some embodiments, PD-L1 expression is elevated on circulating immune cells (such as CD3+/CD8+ T cells) in the blood sample.

Provided herein are methods for monitoring the pharmacodynamic activity of an OX40 agonist treatment by measuring the expression level of one or more marker genes, proteins (e.g., cytokines, e.g., gamma interferon) and/or cellular composition (e.g., percentage of tregs and/or absolute number of tregs; e.g., number of CD8+ effector T cells) in a leukocyte-containing sample obtained from a subject, wherein the subject has been treated with a PD-1 axis binding antagonist and an OX40 binding agonist (e.g., an OX anti-human 40 agonist antibody), and wherein the one or more marker genes are selected from a T cell marker gene, or a memory T cell marker gene (e.g., a marker of T-effector memory cells); and determining that the treatment exhibits pharmacodynamic activity based on the expression level of the one or more marker genes, proteins and/or cellular constituents in the sample obtained from the subject as compared to the reference, wherein an increased expression level of the one or more marker genes as compared to the reference is indicative of the pharmacodynamic activity of the OX40 agonist treatment. The expression level of a marker gene, protein and/or cellular composition can be measured by one or more of the methods described herein.

As used herein, "Pharmacodynamic (PD) activity" may refer to the effect of a treatment (e.g., an OX40 agonist in combination with a PD-1 axis antagonist treatment) on a subject. An example of PD activity can include modulating the expression level of one or more genes. Without wishing to be bound by theory, it is believed that monitoring PD activity (such as by measuring expression of gene markers) may be advantageous during clinical trials examining OX40 agonists and PD-1 axis antagonists. For example, monitoring PD activity can be used to monitor response to therapy, toxicity, and the like.

In some embodiments, the expression level of one or more marker genes, proteins, and/or cellular constituents can be compared to a reference, which can include a sample from a subject not receiving treatment (e.g., OX40 agonist treatment in combination with a PD-1 axis binding antagonist). In some embodiments, a reference may comprise a sample from the same subject prior to receiving treatment (e.g., OX40 agonist treatment in combination with a PD-1 axis binding antagonist). In some embodiments, a reference may comprise reference values from one or more samples from other subjects receiving treatment (e.g., OX40 agonist treatment in combination with a PD-1 axis antagonist). For example, a population of patients may be treated and a mean, average, or median value of the expression levels of one or more genes may be generated from the population as a whole. A set of samples obtained from cancers with shared characteristics (e.g., the same cancer type and/or stage, or exposure to a common treatment, such as an OX40 agonist in combination with a PD-1 axis binding antagonist) from a population may be studied, such as in a clinical outcome study. This kit can be used to derive a reference, such as a reference number that can be compared to a sample of a subject. Any reference described herein can be used as a reference for monitoring PD activity.

Provided herein are methods for monitoring responsiveness of a subject to an OX40 agonist treatment by measuring the level of expression of one or more marker genes, proteins (e.g., cytokines, e.g., gamma interferon) and/or cellular composition (e.g., percentage of tregs and/or absolute number of tregs; e.g., number of CD8+ effector T cells, in a peripheral blood sample) in a leukocyte-containing sample obtained from the subject, wherein the subject has been treated with a PD-1 axis binding antagonist and an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody), and wherein the one or more marker genes are selected from a T cell marker gene, or a memory T cell marker gene (e.g., a marker of T-effector memory cells); and classifying the subject as responsive or non-responsive to the treatment based on the expression level of the one or more marker genes, proteins and/or cellular constituents in the sample obtained from the subject as compared to a reference, wherein an increased expression level of the one or more marker genes as compared to the reference is indicative of responsiveness or lack of responsiveness to OX40 agonist treatment. The expression level of a marker gene, protein and/or cellular composition can be measured by one or more of the methods described herein.

In some embodiments, a reference for monitoring responsiveness can include a sample from a subject not receiving treatment (e.g., OX40 agonist treatment in combination with a PD-1 axis binding antagonist). In some embodiments, a reference for monitoring responsiveness may comprise a sample from the same subject prior to receiving treatment (e.g., OX40 agonist treatment in combination with a PD-1 axis binding antagonist). In some embodiments, a reference for monitoring responsiveness may include reference values from one or more samples from other patients receiving treatment (e.g., OX40 agonist treatment in combination with a PD-1 axis binding antagonist). For example, a population of patients may be treated and a mean, average, or median value of the expression levels of one or more genes may be generated from the population as a whole. A set of samples obtained from cancers with shared characteristics (e.g., the same cancer type and/or stage, or exposure to a common treatment, such as an OX40 agonist) from a population may be studied, such as in a clinical outcome study. This kit can be used to derive a reference, such as a reference number that can be compared to a sample of a subject. Any reference described herein can be used as a reference for monitoring PD activity.

Certain aspects of the present disclosure relate to measuring the expression level of one or more genes or one or more proteins in a sample. In some embodiments, the sample may comprise leukocytes. In some embodiments, the sample may be a peripheral blood sample (e.g., from a patient having a tumor). In some embodiments, the sample is a tumor sample. Tumor samples may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal lamina (basal lamina), and any other cell type associated with a tumor. In some embodiments, the sample is a tumor tissue sample containing tumor infiltrating leukocytes. In some embodiments, the sample can be processed to separate or isolate one or more cell types (e.g., leukocytes). In some embodiments, the sample can be used without separating or isolating the cell type.

The tumor sample may be obtained from the subject by any method known in the art, including but not limited to biopsy, endoscopy, or surgical procedures. In some embodiments, tumor samples can be prepared by methods such as freezing, fixing (e.g., by using formalin or similar fixative), and/or embedding in paraffin. In some embodiments, the tumor sample may be sectioned. In some embodiments, a fresh tumor sample (i.e., not yet prepared by the methods described above) may be used. In some embodiments, tumor samples can be prepared by incubation in solution to maintain mRNA and/or protein integrity.

In some embodiments, the sample may be a peripheral blood sample. Peripheral blood samples may include white blood cells, PBMCs, and the like. Leukocytes can be isolated from a peripheral blood sample using any technique known in the art. For example, a blood sample may be collected, red blood cells may be lysed, and white blood cell pellets may be isolated for use as a sample. In another example, density gradient separation is used to separate leukocytes (e.g., PBMCs) from erythrocytes. In some embodiments, a fresh peripheral blood sample (i.e., not yet prepared by the methods described above) may be used. In some embodiments, peripheral blood samples can be prepared by incubation in solution to maintain mRNA and/or protein integrity.

In some embodiments, responsiveness to treatment may refer to any one or more of: extended survival (including overall survival and progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating the signs or symptoms of cancer. In some embodiments, responsiveness may refer to an improvement in one or more factors of a published set used to determine the status (i.e., response, stability, or progression) of a tumor in a cancer patient according to RECIST guidelines. For a more detailed discussion of these guidelines, see Eisenhauer et al, Eur J Cancer 2009,45: 228-47; topalian et al, N Engl J Med 2012,366: 2443-54; wolchok et al, Clin Can Res 2009,15: 7412-20; and therase, p.et al, j.natl.cancer inst.92:205-16 (2000). Responsive subjects can refer to subjects whose cancer shows improvement, e.g., in accordance with one or more factors based on RECIST criteria. A non-responsive subject may refer to a subject whose cancer does not show improvement, e.g., in accordance with one or more factors based on RECIST criteria.

Conventional response criteria may not be suitable for characterizing the anti-tumor activity of immunotherapeutics that can produce a delayed response that may have previously had an initial significant radiological progression, including the appearance of new lesions. Thus, improved response criteria have been developed that take into account the potential for new lesions to appear and allow confirmation of radiologic progression at the time of subsequent evaluation. Thus, in some embodiments, responsiveness may refer to an improvement in one or more factors according to immune-related response criteria 2 (irRC). See, e.g., Wolchok et al, Clin Can Res 2009,15: 7412-20. In some embodiments, new lesions are added to a defined tumor burden and, for example, radiologic progression is tracked at the time of subsequent assessment. In some embodiments, the presence of non-target lesions is included in the assessment of complete response, and not in the assessment of radiologic progression. In some embodiments, radiologic progression may be determined only on the basis of measurable disease, and/or may be confirmed by a consistent assessment of ≧ 4 weeks from the first recording day.

In some embodiments, responsiveness may include immune activation. In some embodiments, responsiveness may include treatment efficacy. In some embodiments, responsiveness may include immune activation and therapeutic efficacy.

Article of manufacture or kit

In another embodiment of the invention, an article of manufacture or kit is provided comprising a PD-1 axis binding antagonist and/or an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody). In some embodiments, the article of manufacture or kit further comprises a package insert comprising instructions for using the PD-1 axis binding antagonist in combination with an OX40 binding agonist to treat or delay progression of cancer or enhance immune function in an individual having cancer. Any of the PD-1 axis binding antagonists and/or OX40 binding agonists described herein may be included in the article of manufacture or kit.

In some embodiments, the PD-1 axis binding antagonist and OX40 binding agonist (e.g., anti-human OX40 agonist antibody) are in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be made of various materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloys (such as stainless steel or hastelloy). In some embodiments, the container contains the formulation and a label on or associated with the container may indicate instructions for use. The article of manufacture or kit may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with printed instructions for use. In some embodiments, the article of manufacture further comprises one or more additional pharmaceutical agents (e.g., chemotherapeutic agents and antineoplastic agents). Suitable containers for the one or more additional medicaments include, for example, bottles, vials, bags and syringes.

This description is deemed to be sufficient to enable those skilled in the art to practice the invention. Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Examples

The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications and changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Materials and methods

In vivo tumor models: CT26 and MC38 colorectal cell lines were maintained in Genentech. For the CT26 study, 8-10 week old female Balb/c mice (Charles River Laboratories; Hollister, Calif.) were inoculated subcutaneously 0.1X 10 on one side in the right flank⁶And CT26 cells. For the MC38 study, 8-10 week old female C57BL/6 mice (Charles river laboratories) were unilaterally vaccinated subcutaneously 0.1X 10 in the right flank ⁶MC38 cells. When the tumor reaches about 150mm³At mean tumor volume, mice were recruited, randomized into treatment groups, and antibody treatment was started on the following day 1. All animal studies were conducted in accordance with guidelines and regulations set forth in the animal welfare act and the guidelines for care and use of laboratory animals and the IACUC guidelines. The treatment groups were as follows: 1) control antibody, 10mg/kg IV first dose followed by 5mg/kg IP, BIWx2, n-5; 2) a murine anti-mouse OX40 agonist monoclonal antibody (a chimeric antibody having rat anti-mouse OX40 variable regions derived from OX86 antibody and mouse IgG2a Fc. Thus, this murine antibody is capable of performing effector functions including but not limited to ADCC), 0.1mg/kg IV first dose followed by IP, TIWx2, n-5; 3) murine anti-PD-L1, 10mg/kg IV first dose followed by 5mg/kg IP, TIWx2, n-5; and 4) murine anti-mouse OX40 monoclonal antibody, 0.1mg/kg IV first dose followed by IP, TIWx2 and murine anti-PD-L1, 10mg/kg IV first dose followed by 5mg/kg IP TIWx2, n-5. Mice were sacrificed on day 9 after the first dose and harvested ex vivoAnd (5) peripheral blood.

Antibody: all treatment antibodies were generated at Genentech. The control antibody was anti-gp 120 mouse IgG1, clone 10 E7.1D2. The anti-OX 40 antibody was cloned OX-86 mouse-IgG 2a (generated by cloning the rat anti-mouse OX40 agonist antibody OX-86 onto the mouse IgG2a backbone), while anti-PD-L1 was clone 6E11.1.9 mouse IgG 1. Dose administration schedule as indicated on the legend, the first or single dose was administered Intravenously (IV) and subsequent doses were administered Intraperitoneally (IP). The antibody was diluted in PBS or 20mM histidine acetate, 240mM sucrose, 0.02% polysorbate 20, pH 5.5. TIW indicates three administrations per week and BIW indicates two administrations per week.

Tumor processing and flow cytometry: tumors were harvested and minced with razor blades, after which they were digested IN RPMI-1640 medium containing 5% fetal bovine serum plus 0.25mg/ml collagenase D (Roche; Indianapolis, IN) and 0.1mg/ml DNase I (Roche) IN C tubes (Miltenyi Biotec; San Diego, Calif.) on a shaking platform at 37 ℃ for 15 minutes. Following incubation, tumors were processed on gentlemecs (miltenyi biotec), filtered, and washed to generate single cell suspensions. Cells were counted on a Vi-Cell counter (Beckman Coulter; Brea, CA).

Peripheral blood was assessed for T cell activation and proliferation by flow cytometry. 50uL blood was stained with commercial antibodies against CD45, CD3, CD4, CD8, CXCR3 (all BD Biosciences) and Ki67(eBiosciences) following the manufacturer's instructions. Cells were first stained with live/dead near-infrared viability dye (Life Technologies; GrandIsland, N.Y.) in PBS on ice for 30 minutes and then washed. The cells were then blocked for Fc receptor with purified anti-CD 16/-CD32 (BDbiosciences; San Jose, Calif.) followed by a 30 min surface staining on ice in PBS + 0.5% BSA +2mM EDTA buffer. To assess PD-1 and OX40 expression of T cells, cells were stained as follows: PD1-FITC, CD3-percp. cy5.5, CD4-PE-Cy7, CD8 pacifiic Blue, CD45v500, (BD Biosciences); OX40Alexa Fluor 647(Genentech, clone 1H 1). To assess PD-L1 expression for various cell types, staining was as follows: CD11b-FITC, Gr-1PE-Cy7, CD8Alexa 700, CD45v500, CD4-PerCp.Cy5.5 (BDbiosciences); PDL 1-Biotin (Genentech, clone 6F8.2.5) followed by streptavidin-PE (BDbioscience). To evaluate the Foxp3+ T regulatory cell population, the cell surface was first stained with CD45-PE-Cy7, CD4PerCp-Cy5.5(BDbiosciences) and then fixed overnight at 4 ℃ in 1 XFoxp 3 fixation/permeabilization buffer (ebiosciences; SanDiego, Calif.). Cells were then permeabilized in 1 × Foxp3 permeabilization buffer (eBioscience) and stained with Foxp3-fitc (eBioscience). Stained cells were obtained using FACS Diva software on Fortessa or FACS Canto II (BD Biosciences) and then analyzed on FlowJo software.

Tumor dissection and Fluidigm expression analysis: RNA was extracted from FFPE-derived archived tumors of UBC or NSCLC as described (Powles, T.et al (2014) Nature515: 558-62; Herbst, R.S.et al (2014) Nature515: 563-7). Briefly, tumor FFPE sections were macro dissected to enrich for neoplastic tissue and the tissue was lysed using tumor lysis buffer and proteinase K to allow complete digestion and release of nucleic acids. RNA was isolated using a high purity FFPE RNA minikit (Roche applied Sciences, Indianapolis, Ind.) according to the manufacturer's protocol.

Gene expression analysis was performed using the BioMark HD real-time PCR platform (Fluidigm) as previously described (Shames, D.S.et al (2013) PLoS ONE 8: e 56765). All Taq-man assays in the expression panel were FAM-MGB and were custom made via Life Technologies, either order-made or customer-designed, including four reference genes: SP2, GUSB, TMEM55B and VPS 33B. The geometric median of Ct values for the four reference genes (SP2, GUSB, TMEM55B and VPS33B) was calculated for each sample and the expression levels were determined using the Δ Ct (dct) method as follows: ct (target gene) 2 geometric median Ct (reference gene). The high-to-low expression classification was derived using the median mRNA expression level (as determined by an immuno chip (iChip)) between patients in the study as cut-off. The P value was determined by t-test.

PD-L1 Immunohistochemistry (IHC): cancer cell lines or tumor samples were analyzed for formalin-fixed, paraffin-embedded (FFPE) sections.

Prior to antigen retrieval, formalin-fixed, paraffin-embedded tissue sections were deparaffinized, blocked, and incubated with anti-PD-L1 antibody. After incubation with secondary antibody and enzymatic development, sections were counterstained and dehydrated in serial alcohols and xylene before coverslipping.

IHC was performed using the following protocol. For formalin-fixed, paraffin-embedded (FFPE) tissue sections 4mm thick, PD-L1 was stained with anti-human PD-L1 rabbit monoclonal antibody using a concentration of 4.3mg/ml on an automated staining platform, and signals were visualized by diaminobenzidine; sections were counterstained with hematoxylin. PD-L1 expression on tumor-infiltrating immune cells was evaluated using the following scoring protocol:

PD-L1IHC staining was performed using either the Ventana Benchmark XT or Benchmark Ultra system using the following reagents and materials:

a first antibody: anti-PD-L1 rabbit monoclonal primary antibody

Specimen type: formalin-fixed, paraffin-embedded (FFPE) sections of tissue samples and control cell pellets of different staining intensities

Protocol species: human being

The instrument comprises the following steps: BenchMark XT or BenchMark Ultra

Epitope recovery conditions: cell conditioning, Standard 1(CC1, Ventana, Cat # 950-: 1/100, 6.5. mu.g/ml/36 ℃ for 16 minutes

Diluent agent: antibody dilution buffer (Tris buffered saline containing carrier protein and Brig-35)

Negative control: non-immuneRabbit IgG, 6.5. mu.g/ml (cell signaling) or aloneDiluent

And (3) detection: the Optiview or Ultraview universal DAB detection kit (Ventana) and amplification kit (if applicable) were used according to the manufacturer's instructions (Ventana).

Secondary dyeing: ventana hematoxylin II (catalog #790 and 2208)/reagent with blueing (catalog #760 and 2037) (4 min and 4 min, respectively)

The Benchmark protocol is as follows:

1. paraffin (selection)

2. Paraffin removal (selection)

3. Cell conditioning (selection)

4. Regulator #1 (option)

5. Standard CC1 (selection)

6. Antibody incubation temperature (selection)

7.36 ℃ antibody incubation (selection)

8. Titration (selection)

9. Automatic dispensing (Primary antibody), and incubation (16 minutes)

10. Counterstain (selection)

11. One drop (hematoxylin II) was applied (counterstain), coverslipped and incubated (4 min)

12. Afterdyeing (selection)

13. One drop (bluing reagent) was applied (post-counterstaining), coverslipped and incubated (4 min)

14. Wash slides in soapy water to remove oil

15. Rinsing the slide with water

16. Dehydration of slides through 95% ethanol, 100% ethanol to xylene (Leica auto stain machine program #9)

17. Cover with a cover glass.

Results

OX40 is known to be a costimulatory molecule expressed on activated CD4T effector (Teff) cells and T regulatory (Treg) cells. OX40 is not constitutively expressed on naive T cells, but is induced after T Cell Receptor (TCR) involvement. The linkage of OX40 in the presence of TCR stimulation is known to enhance T effector function via a dual mechanism of potentiating Teff cell activation and suppressing Treg cells. anti-OX 40 treatment was found to reduce Treg activity in an in vitro Treg suppression assay. These results indicate that OX40 agonist treatment is able to modulate several key T cell functions.

Inhibition of PD-L1 signaling has been proposed as a means of enhancing T cell immunity for the treatment of cancer (e.g., tumor immunity) and infections, including both acute and chronic (e.g., persistent) infections.

We examined whether T cells in tumors express PD-1 and OX 40. As shown in figure 1, intratumoral CD8+ T cells expressed inhibitory receptors such as PD-1, but a larger proportion of these cells also expressed OX 40. This result suggests that OX40 stimulation of Teff cells may counteract the effects of PD-1 and other inhibitory receptors expressed on T cells.

Treatment with anti-OX 40 agonist antibody (single agent) significantly reduced the proportion of intratumoral Foxp3+ regulatory T cells relative to the total number of CD45+ cells (CD45 defines all hematopoietic cells such as leukocytes; fig. 2A) and significantly reduced the absolute number of intratumoral Foxp3+ tregs (fig. 2B). In addition, treatment with a combination of anti-OX 40 agonist antibody and anti-PD-L1 antagonist antibody significantly reduced the proportion of intratumoral Foxp3+ regulatory T cells relative to the total number of CD45+ cells (fig. 2A) and the absolute number of intratumoral Foxp3+ tregs (fig. 2B). These results indicate that OX40 agonist-mediated reduction of intratumoral Foxp3+ tregs is maintained when OX40 agonist is administered in combination with an anti-PD-L1 antagonist.

We examined the effect of OX40 agonist treatment on PD-L1 expression. anti-OX 40 agonist treatment significantly increased PD-L1 expression in tumor cells and intratumoral myeloid cells, suggesting that PD-L1 can limit anti-OX 40 efficacy in a negative feedback manner (fig. 3A and B). Without being bound by theory, these results suggest that OX40 agonist treatment may enhance PD-1 axis inhibitor treatment because OX40 agonist treatment increases PD-L1 expression. Clinical data correlate elevated PD-L1 expression as a potentiation of response to PD1 axis antagonists (e.g., anti-PD-L1 antagonist antibodies).

anti-OX 40 agonist antibody and anti-PD-L1 antagonist antibody treatment exhibited a synergistic combination of efficacy in CT26 and MC38 colorectal cancer syngeneic tumor models (fig. 4A and B, 5A and B). Analysis of individual tumor volume measurements (individual mice from each experiment; fig. 4B, 5B) revealed that the animals treated in combination showed significant tumor size reduction at a higher frequency than the animals treated with either agent (OX40 agonist, PD-L1 antagonist) alone. In other words, the frequency of animals with partial and complete responses in combination treated animals was significantly higher compared to animals treated with either agent alone.

Analysis of peripheral blood collected from combination-treated CT26 mice revealed an increase in effector cell proliferation and inflammatory T cell markers (fig. 9A, B, C, and D). CD8+ T cell proliferation (fig. 9A), Treg cells (fig. 9B), plasma interferon gamma levels (fig. 9C) and levels of activated T cells (fig. 9D) were examined. Elevation of proliferation (Ki67), plasma interferon gamma, and inflammatory markers (Tbet, CXCR3) in the combined arm (relative to either single agent arm) revealed a synergistic effect of anti-PD-L1 (checkpoint blockade) and anti-OX 40 (co-stimulatory) actions.

In particular, the levels of proliferating CD8+ T cells (expressed as a percentage of ki67 +/total CD8+ T cells) were significantly increased in animals treated with a combination of an OX40 agonist and a PD-L1 antagonist compared to treatment with an OX40 agonist or a PD-L1 antagonist alone (fig. 9A). The level of CD8+ T cells in proliferation in the combination treated animals was greater than the additive effect of the single agent treatment group, indicating that the synergistic effect of the OX40 agonist treatment in combination with PD-1 axis inhibition could be detected by analysis of peripheral blood markers and cells.

In addition, reduced peripheral blood tregs were observed for OX40 agonist single agent treatment and the reduction in peripheral blood tregs was maintained in the (OX40 agonist and PD-L1 antagonist) combination therapy arm (fig. 9B). Elevated plasma gamma interferon was observed for OX40 agonist and PD-L1 antagonist combinations (fig. 9C).

Chemokine receptor CXCR3 is a G α i protein-coupled receptor in the CXC chemokine receptor family. There are two different variants of CXCR 3: CXCR3-A binds to CXC chemokines CXCL9(MIG), CXCL10(IP-10), and CXCL11(I-TAC), while CXCR3-B binds to CXCL4 in addition to CXCL9, CXCL10, and CXCL11 (Clark-Lewis, I.et al. (2003) J.biol.chem.278(1): 289-95). CXCR3 is expressed primarily on activated T lymphocytes and NK cells, and on some epithelial cells. CXCR3 and CCR5 are preferentially expressed on Th1 cells and are upregulated on effector memory CD8T cells (Groom, j.r. and Luster, A.D (2011) exp.cell res.317(5): 620-31). CXCR3 is capable of modulating leukocyte trafficking. Chemokine binding to CXCR3 induces a variety of cellular responses, most notably integrin activation, cytoskeletal changes and chemotactic migration of inflammatory cells (Groom, j.r.and Luster, A.D (2011) exp.cell res.317(5): 620-31).

The levels of activated T cells (specifically, activated memory Teff cells, determined using CXCR3 marker) in animals treated with a combination of an OX40 agonist and a PD-L1 antagonist were significantly elevated compared to treatment with OX40 agonist or PD-L1 antagonist alone (fig. 9D). The levels of T memory effector cells (CXCR3+) in the combination treated animals were greater than the additive effect of the single agent treatment group, indicating that the synergistic effect of the OX40 agonist treatment in combination with PD-1 axis inhibition could be detected by analysis of peripheral blood markers and cells. An increase in proliferation (Ki67) and inflammatory markers (CXCR3) on CD8T cells in the combined treatment arm may suggest that cytotoxicity is enhanced through a synergistic effect of anti-PD-L1 (checkpoint blockade) and anti-OX 40 (co-stimulatory) actions.

In addition, the effect of the combined treatment is detected by the increase in the effect and inflammatory T cell markers (e.g., by rtpcr (fluidigm)) in the combined treatment tumor sample analyzed as compared to samples treated with either agent alone. For example, markers for tregs (Fox3p), CD8+ Teffs (CD8b), and activated T cells (e.g., Tbet, CXCR3, such as interferon gamma response-related genes) can be analyzed.

Experiments were conducted to examine the dose-response effects of OX40 agonist antibody treatment in a CT26 colorectal cancer isogenic tumor model. Single agent treatment with anti-OX 40 agonist antibody showed dose responsiveness (figure 6A, B). The 0.1mg/ml dose showed sub-maximal efficacy, which was selected for further combination treatment experiments.

Figures 7A and B show the results of treatment with sub-therapeutic doses of anti-OX 40 agonist antibody in combination with anti-PD-L1 antagonist antibody, compared to treatment with either agent alone. Synergistic combination efficacy was observed suggesting that the maximum effective dose of the OX40 agonist antibody may be lower when treated in combination with PD-1 axis antagonists.

Figures 8A and B show the results of treatment with a single sub-therapeutic level of an anti-OX 40 agonist antibody in combination with an anti-PD-L1 antagonist antibody, compared to treatment with either agent alone. Synergistic combination efficacy was observed suggesting that the maximum effective dose of the OX40 agonist antibody may be lower when combined with PD-1 axis antagonists to provide OX40 agonist antibodies.

FIG. 10 shows the correlation of OX40 expression with the diagnostic status of PD-L1 in cancer samples from human patients with Urothelial Bladder Cancer (UBC) and non-small cell lung cancer (NSCLC). Tissue samples were from patients who participated in a phase 1 clinical trial of the anti-PD-L1 antibody MPDL 3280A. As disclosed herein, the PD-L1 biomarker status of tumor infiltrating Immune Cells (IC) was determined using IHC. OX40 expression levels were determined using rtPCR analysis (Fluidigm). In UBC, OX40 expression was observed in patients with PD-L1IHC status of 0 or 1. The level of OX40 expression is correlated with PD-L1IHC status, wherein elevated PD-L1 expression is correlated with elevated OX40 expression. In NSCLC, OX40 expression was observed in patients with low or no PD-L1 expression (by IHC) (as well as in samples with PD-L1IHC status of 2 and 3). These results suggest (a) the potential for improved response of PD-1 axis binding antagonists in combination with OX40 binding agonists (e.g., anti-human OX40 agonist antibodies) in patients with PD-L1IHC 0 and/or 1 status; (b) the potential for improved response of a PD-1 axis binding antagonist and OX40 binding agonist (e.g., anti-human OX40 agonist antibody) combination treatment in patients not responding to prior PD-1 axis binding antagonist treatment; and (c) the potential for improved response of a PD-1 axis binding antagonist in combination with an OX40 binding agonist (e.g., an anti-human OX40 agonist antibody) in patients with PD-L1IHC 2 and/or 3 status.

The results of a clinical study evaluating the anti-PD-L1 antibody MPDL3280A for the treatment of cancer suggest that PD-L1 expression correlates with clinical response to MPDL 3280A. It was found that the correlation of tumor-infiltrating immune cell PD-L1 expression with treatment response appeared to be stronger than the correlation of tumor cell PD-L1 expression. Tumor-infiltrating immune cells may be more sensitive to IFNg expression and may preferentially act to suppress pre-treatment pre-existing T cell responses (Herbst, r.s.et al (2014) Nature 515: 563-7). Without wishing to be bound by theory, it is believed that OX40 agonist treatment may increase IFNg expression, leading to enhanced PD-L1 expression in tumor-infiltrating immune cells and concomitant increased responsiveness to PD-1 axis binding antagonist treatment. Combination treatments comprising an OX40 binding agonist and a PD-1 axis binding antagonist may therefore be useful in treating patients with lower PD-L1 biomarker states.

PD-L1 expression was scored by IHC: the presence or absence of PD-L1 expression in tumor specimens was assessed by IHC in human formalin-fixed, paraffin-embedded (FFPE) tissue using an anti-PD-L1 specific antibody that detects PD-L1. To measure and quantify the relative expression of PD-L1 in tumor samples, a PD-L1IHC scoring system was developed to measure PD-L1 specific signals in tumor cells and tumor-infiltrating immune cells. Immune cells are defined as cells with lymphoid and/or macrophage/histiocyte morphology.

Tumor cell staining was expressed as the percentage of all tumor cells showing membrane staining of any intensity. Invasive immune cell staining is defined as the percentage of the total tumor area occupied by immune cells showing any intensity of staining. The total tumor area encompasses malignant cells as well as tumor-associated stroma, including the area of immune infiltrates immediately adjacent and bordering the main tumor mass. In addition, invasive immune cell staining was defined as the percentage of all tumor-invasive immune cells.

The PD-L1 staining intensity has a wide dynamic range in tumor tissues. Regardless of subcellular localization, signals were also classified as strong, moderate, weak, or negative staining.

As shown in fig. 11, negative signal intensity was characterized by the absence of any detectable signal, as exemplified using HEK-293 cells (fig. 11A). In comparison, positive signal intensity was characterized by gold to dark brown membrane staining, as exemplified using HEK-293 cells transfected with recombinant human PD-L1 (fig. 11B-D). Finally, positive signal intensity was also exemplified by staining of the placental trophoblasts (fig. 11E) and strong staining in the crypt tonsil region (fig. 11F), and was often in the form of a membrane, characterized by gold to dark brown staining. In tumor tissue, PD-L1 negative samples were defined as having no detectable signal or only weak cytoplasmic background staining when evaluated using a 20-fold objective lens. In contrast, PD-L1 positive samples exhibited predominantly membrane staining in tumor cells and/or infiltrating immune cells. The staining of PD-L1 was observed at different intensities, ranging from weak (thin, light brown film) to strong (dark brown thick film, easily recognizable at low magnification).

Figure 12 shows three representative PD-L1 positive tumor samples: for triple negative breast cancer, most tumor cells were observed to be strongly positive for PD-L1, showing a combination of membrane and cytoplasmic staining (100-fold magnification) (fig. 12A). For malignant melanoma, a cluster of immune cells was observed, some of them with PD-L1 membrane staining, and rare tumor cells (arrows) with PD-L1 membrane staining (400 x magnification) (fig. 12B). For NSCLC, adenocarcinoma, a cluster of immune cells with strong PD-L1 staining and several tumor cells (arrows) with membrane and/or cytoplasmic PD-L1 staining (400-fold magnification) were observed (fig. 12C).

Staining in positive cases tends to be concentrated in terms of spatial distribution and intensity. The percentage of stained tumor or immune cells showing any intensity was visually estimated and used to determine PD-L1 status. An isotype negative control was used to assess the presence of background in the test samples.

Staining requires a series of tissue sections for H & E, a second series of tissue sections for anti-PD-L1, and a third series of tissue sections for isotype negative control antibody. HEK-293 cell line controls transfected with PD-L1 or tonsil slides were used as assay-specific reference and running controls. PDL-1 status Standard

The table shown above describes one embodiment of determining the PD-L1 status using the PD-L1 staining standard. In another embodiment, a sample with an IHC score of IHC 0 and/or 1 may be considered negative for PD-L1, while a sample with an IHC score of IHC 2 and/or 3 may be considered positive for PD-L1. In some embodiments, the tumor itself is assessed for PD-L1 expression (e.g., PD-L1 staining).

In some cases, a PD-L1 positive status may comprise the presence of any intensity of discernible PD-L1 staining in tumor cells or tumor-infiltrating immune cells, up to 50% of the tumor area occupied by tumor cells, desmoplastic stroma within the associated tumor, and around the continuous tumor. As such, a positive staining of PD-L1 includes staining of up to 50% of tumor cells or tumor-infiltrating immune cells that exhibit any intensity.

Evaluable slides stained with anti-PD-L1 were evaluated as described above. Negative staining intensity is characterized by the absence of any detectable signal or signal appearing grayish to blue (rather than brown or brown) and the absence of membrane enhancement. If there is no (e.g. missing) membrane staining, then the case is negative.

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

Claims (91)

1. A method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist.

2. The method of claim 1, wherein the PD-1 axis binding antagonist is selected from the group consisting of: PD-1 binding antagonists, PDL1 binding antagonists and PDL2 binding antagonists.

3. The method of claim 2, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

4. The method of claim 3, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner.

5. The method of claim 3, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PDL 1.

6. The method of claim 3, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PDL 2.

7. The method of claim 3, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to both PDL1 and PDL 2.

8. The method of any one of claims 3-7, wherein the PD-1 binding antagonist is an antibody.

9. The method of claim 3, wherein the PD-1 binding antagonist is nivolumab.

10. The method of claim 3, wherein the PD-1 binding antagonist is pembrolizumab.

11. The method of claim 3, wherein the PD-1 binding antagonist is CT-011.

12. The method of claim 3, wherein the PD-1 binding antagonist is AMP-224.

13. The method of claim 2, wherein the PD-1 axis binding antagonist is a PDL1 binding antagonist.

14. The method of claim 13, wherein the PDL1 binding antagonist inhibits the binding of PDL1 to PD-1.

15. The method of claim 13, wherein the PDL1 binding antagonist inhibits the binding of PDL1 to B7-1.

16. The method of claim 13, wherein the PDL1 binding antagonist inhibits the binding of PDL1 to both PD-1 and B7-1.

17. The method of any one of claims 13-16, wherein the PDL1 binding antagonist is an anti-PDL 1 antibody.

18. The method of claim 17, wherein the anti-PDL 1 antibody is a monoclonal antibody.

19. The method of claim 17, wherein the anti-PDL 1 antibody is an antibody fragment selected from the group consisting of: fab, Fab '-SH, Fv, scFv, and (Fab')₂And (3) fragment.

20. The method of claim 17, wherein the anti-PDL 1 antibody is a humanized or human antibody.

21. The method of claim 13, wherein the PDL1 binding antagonist is selected from the group consisting of: YW243.55.S70, MPDL3280A, MDX-1105, and MEDI 4736.

22. The method of claim 17, wherein the antibody comprises a heavy chain comprising the HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO: 1), the HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO: 2), and the HVR-H3 sequence of RHWPGGFDY (SEQ ID NO: 3), and a light chain comprising the HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO: 4), the HVR-L2 sequence of SALYSFS (SEQ ID NO: 5), and the HVR-L3 sequence of QQYLYHPAT (SEQ ID NO: 6).

23. The method of claim 17, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 8),

the light chain variable region comprises the following amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO：9)。

24. the method of claim 2, wherein the PD-1 axis binding antagonist is a PDL2 binding antagonist.

25. The method of claim 24, wherein the PDL2 binding antagonist is an antibody.

26. The method of claim 24, wherein the PDL2 binding antagonist is an immunoadhesin.

27. The method of any one of claims 8, 17-23, and 25, wherein the antibody is a human IgGl having an Asn to Ala substitution at position 297 according to EU numbering.

28. The method of any one of claims 1-27, wherein the OX40 binding agonist is selected from the group consisting of: OX40 agonist antibodies, OX40L agonist fragments, OX40 oligoreceptor, and OX40 immunoadhesin.

29. The method of any one of claims 1-28, wherein the OX40 binding agonist is an OX40 agonist antibody that binds human OX 40.

30. The method of claim 29, wherein the OX40 agonist antibody is MEDI6469, MEDI0562, or MEDI 6383.

31. The method of claim 29, wherein the OX40 agonist antibody is a full length human IgGl antibody.

32. The method of any one of claims 1-28, wherein the OX40 binding agonist is a trimeric OX40L-Fc protein.

33. The method of any one of claims 1-28, wherein the OX40 binding agonist is an OX40L agonist fragment comprising one or more extracellular domains of OX 40L.

34. The method of any one of claims 1-33, wherein the cancer is breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, renal cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, or hepatocellular carcinoma.

35. The method of any one of claims 1-34, wherein the individual has cancer or has been diagnosed with cancer.

36. The method of any one of claims 1-35, wherein the treatment results in a sustained response in the individual after cessation of the treatment.

37. The method of any one of claims 1-36, wherein the OX40 binding agonist is administered prior to the PD-1 axis binding antagonist, simultaneously with the PD-1 axis binding antagonist, or after the PD-1 axis binding antagonist.

38. The method of any one of claims 1-37, wherein the individual is a human.

39. A method of enhancing immune function in an individual having cancer comprising administering an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist.

40. The method of claim 39, wherein the CD8T cells in the individual have enhanced priming, activation, proliferation and/or lytic activity relative to prior to administration of the PD-1 axis binding antagonist and the OX40 binding agonist.

41. The method of claim 39, wherein the number of CD8T cells is increased relative to prior to administration of the combination.

42. The method of claim 41, wherein the CD8T cells are antigen-specific CD8T cells.

43. The method of claim 39, wherein Treg function is suppressed relative to prior to administration of the combination.

44. The method of claim 39, wherein T cell depletion is reduced relative to prior to administration of the combination.

45. The method of any one of claims 39-44, wherein the PD-1 axis binding antagonist is selected from the group consisting of: PD-1 binding antagonists, PDL1 binding antagonists and PDL2 binding antagonists.

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

47. The method of claim 46, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner.

48. The method of claim 46, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PDL 1.

49. The method of claim 46, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PDL 2.

50. The method of claim 46, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to both PDL1 and PDL 2.

51. The method of any one of claims 46-50, wherein the PD-1 binding antagonist is an antibody.

52. The method of claim 46, wherein the PD-1 binding antagonist is nivolumab.

53. The method of claim 46, wherein the PD-1 binding antagonist is pembrolizumab.

54. The method of claim 46, wherein the PD-1 binding antagonist is CT-011.

55. The method of claim 46, wherein the PD-1 binding antagonist is AMP-224.

56. The method of claim 45, wherein the PD-1 axis binding antagonist is a PDL1 binding antagonist.

57. The method of claim 56, wherein the PDL1 binding antagonist inhibits the binding of PDL1 to PD-1.

58. The method of claim 56, wherein the PDL1 binding antagonist inhibits the binding of PDL1 to B7-1.

59. The method of claim 56, wherein the PDL1 binding antagonist inhibits the binding of PDL1 to both PD-1 and B7-1.

60. The method of any one of claims 56-59, wherein the PDL1 binding antagonist is an anti-PDL 1 antibody.

61. The method of claim 60, wherein the anti-PDL 1 antibody is a monoclonal antibody.

62. The method of claim 60, wherein the anti-PDL 1 antibody is an antibody fragment selected from the group consisting of: fab, Fab '-SH, Fv, scFv, and (Fab')₂And (3) fragment.

63. The method of claim 60, wherein the anti-PDL 1 antibody is a humanized or human antibody.

64. The method of claim 56, wherein the PDL1 binding antagonist is selected from the group consisting of: YW243.55.S70, MPDL3280A, MDX-1105, and MEDI 4736.

65. The method of claim 60, wherein the anti-PDL 1 antibody comprises a heavy chain comprising the HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO: 1), the HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO: 2), and the HVR-H3 sequence of RHWPGGFDY (SEQ ID NO: 3), and a light chain comprising the HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO: 4), the HVR-L2 sequence of SASFLYS (SEQ ID NO: 5), and the HVR-L3 sequence of QQYLYHPAT (SEQ ID NO: 6).

66. The method of claim 60, wherein the anti-PDL 1 antibody comprises a heavy chain variable region comprising the amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 8),

The light chain variable region comprises the following amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO：9)。

67. the method of any one of claims 51, 60-63, 65, and 66, wherein the antibody is a human IgGl having an Asn to Ala substitution at position 297 according to EU numbering.

68. The method of claim 45, wherein the PD-1 axis binding antagonist is a PDL2 binding antagonist.

69. The method of claim 68, wherein the PDL2 binding antagonist is an antibody.

70. The method of claim 68, wherein the PDL2 binding antagonist is an immunoadhesin.

71. The method of any one of claims 39-70, wherein the OX40 binding agonist is selected from the group consisting of: OX40 agonist antibodies, OX40L agonist fragments, OX40 oligoreceptor, and OX40 immunoadhesin.

72. The method of claim 71, wherein the OX40 binding agonist is an OX40 agonist antibody that binds human OX 40.

73. The method of claim 72, wherein the OX40 agonist antibody is MEDI6469, MEDI0562, or MEDI 6383.

74. The method of claim 72, wherein the OX40 agonist antibody is a full length IgG1 antibody.

75. The method of any one of claims 39-70, wherein the OX40 binding agonist is a trimeric OX40L-Fc protein.

76. The method of any one of claims 39-70, wherein the OX40 binding agonist is an OX40L agonist fragment comprising one or more extracellular domains of OX 40L.

77. The method of any one of claims 39-76, wherein the cancer is breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, renal cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, or hepatocellular carcinoma.

78. The method of any one of claims 39-77, wherein the individual has been diagnosed with cancer.

79. The method of any one of claims 39-78, wherein the treatment results in a sustained response in the individual after cessation of the treatment.

80. The method of any one of claims 39-79, wherein the OX40 binding agonist is administered prior to the PD-1 axis binding antagonist, simultaneously with the PD-1 axis binding antagonist, or after the PD-1 axis binding antagonist.

81. The method of any one of claims 39-80, wherein the individual is a human.

82. The method of any one of claims 1-81, wherein the PD-1 axis binding antagonist and/or the OX40 binding agonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intracamerally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

83. The method of any one of claims 1-82, further comprising administering a chemotherapeutic agent to treat or delay progression of cancer.

84. Use of a human PD-1 axis binding antagonist in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administering the medicament in combination with a composition comprising an OX40 binding agonist and an optional pharmaceutically acceptable carrier.

85. use of an OX40 binding agonist in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the OX40 binding agonist and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administering the medicament in combination with a composition comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier.

86. A composition comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises administering the composition in combination with a second composition, wherein the second composition comprises an OX40 binding agonist and an optional pharmaceutically acceptable carrier.

87. A composition comprising an OX40 binding agonist and an optional pharmaceutically acceptable carrier for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises administering the composition in combination with a second composition, wherein the second composition comprises a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier.

88. A kit comprising a medicament comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament in combination with a composition comprising an OX40 binding agonist and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer in an individual.

89. A kit comprising a first drug comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and a second drug comprising an OX40 binding agonist and an optional pharmaceutically acceptable carrier.

90. The kit of claim 89, wherein the kit further comprises a package insert comprising instructions for administering the first medicament and the second medicament for treating or delaying progression of cancer in an individual.

91. A kit comprising a medicament comprising an OX40 binding agonist and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament in combination with a composition comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer in an individual.