Methods of treating cancers using PD-1 axis binding antagonists and taxanes

Abstract

To provide methods and compositions for treating cancers and for enhancing an immune function in an individual having cancer.SOLUTION: The present invention provides 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 a taxane.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 61 / 917,264, filed December 17, 2013.

[0002] The present invention relates to methods of treating cancer and enhancing immune function in an individual by administering a PD-1 axis binding antagonist and a taxane. [Background technology]

[0003] Providing two distinct signals to T cells is a widely accepted model for lymphocyte activation of resting T lymphocytes by antigen-presenting cells (APCs). This model also provides discrimination between non-self and self tolerance. The primary or antigen-specific signal is delivered through the T cell receptor (TCR) after recognition of foreign antigenic peptides presented in the context of the major histocompatibility complex (MHC). The second or costimulatory signal is delivered to T cells by costimulatory molecules expressed on APCs, inducing T cells to promote clonal expansion, cytokine secretion and effector functions. In the absence of costimulation, T cells can become refractory to antigenic stimulation, resulting in a tolerogenic response to either foreign or endogenous antigens.

[0004] In the two-signal model, T cells receive both positive and negative secondary costimulatory signals. Regulation of such positive and negative signals is crucial for maximizing the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. Negative secondary signals appear to be required for the induction of T cell tolerance, while positive signals promote T cell activation. Although the simple two-signal model still provides a valid description for naive lymphocytes, the host immune response is a dynamic process, and costimulatory signals can also be provided to antigen-exposed T cells.

[0005] The mechanism of costimulation is of therapeutic interest because manipulation of costimulatory signals provides a means to enhance or terminate cell-based immune responses. T cell dysfunction or anergy occurs concomitantly with induced and persistent expression of programmed death 1 polypeptide (PD-1), an inhibitory receptor that binds to ligands including PD-L1 and PD-L2. PD-L1 is overexpressed in many cancers and is often associated with poor prognosis. The majority of tumor-infiltrating T lymphocytes express PD-1 predominantly in contrast to T lymphocytes in normal tissues and peripheral blood T lymphocytes, indicating that upregulation of PD-1 on tumor-reactive T cells may contribute to the dysfunction of antitumor immune responses. This may be due to the exploitation of PD-L1 signaling mediated by PD-L1-expressing tumor cells interacting with PD-1-expressing T cells, resulting in attenuation of T cell activity and evasion of immunological surveillance. Thus, inhibition of PD-L1 / PD-L2 interaction may enhance CD8+ T cell-mediated killing of tumors. Summary of the Invention [Problem to be solved by the invention]

[0006] An optimal therapeutic treatment would combine blockade of the PD-1 receptor / ligand interaction with agents that directly inhibit tumor growth. There is a need for optimal therapies for the treatment, stabilization, prevention and / or delay of progression of various cancers. [Means for solving the problem]

[0007] The present invention relates to methods for treating cancer and enhancing immune function in individuals with cancer by administering a PD-1 axis binding antagonist and a taxane.

[0008] In one aspect, the invention features a method for treating or delaying the progression of cancer in an individual, the method including administering to the individual effective amounts of a human PD-1 axis binding antagonist and a taxane, in some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.

[0009] In some embodiments of the above aspects, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to its ligand binding partners. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 binding antagonist is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (lambrolizumab), CT-011 (pidilizumab), and AMP-224.

[0010] In other embodiments of the above aspects, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. In some embodiments, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 19, the HVR-H2 sequence of SEQ ID NO: 20, and the HVR-H3 sequence of SEQ ID NO: 21; and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 22, the HVR-L2 sequence of SEQ ID NO: 23, and the HVR-L3 sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0011] In some embodiments of the foregoing aspects, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2 binding antagonist is an antibody. In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0012] In any of the preceding embodiments of the above aspects, the cancer may be, but is not limited to, breast cancer (including triple negative breast cancer (TNBC)), bladder cancer (including urothelial bladder cancer (UBC), muscle invasive bladder cancer, and BCG refractory non-muscle invasive bladder cancer), colorectal cancer, rectal cancer, lung cancer (including non-small cell lung cancer, which may be squamous or non-squamous), glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma (including renal cell carcinoma), prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, gastric cancer, esophageal cancer, prostate cancer, endometrial cancer, renal cancer, ovarian cancer, mesothelioma, and hemo malignancies (including myelodysplastic syndromes (MDS) and multiple myeloma). In certain embodiments, the cancer may be lung cancer (including non-small cell lung cancer, which may be squamous or non-squamous), bladder cancer (including UBC), breast cancer (including TNBC), renal cell carcinoma, melanoma, colorectal cancer, and heme malignancies (including myelodysplastic syndromes (MDS) and multiple myeloma). In some embodiments, the lung cancer is non-small cell lung cancer, which may be squamous or non-squamous. In some embodiments, the bladder cancer is UBC. In some embodiments, the breast cancer is TNBC. In some embodiments, the heme malignancies are MDS or multiple myeloma.

[0013] In any of the preceding embodiments of the above aspects, the individual has or has been diagnosed with cancer. In some embodiments, the cancer cells in the individual express PD-L1.

[0014] In any of the preceding embodiments of the above aspects, the treatment may result in a response in the individual. In some embodiments, the response is a complete remission. In some embodiments, the response is a sustained remission after cessation of treatment. In some embodiments, the response is a complete remission that persists after cessation of treatment.

[0015] In any of the preceding embodiments of the above aspects, the taxane is administered prior to, concurrently with, or after the PD-1 axis binding antagonist. In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®), paclitaxel, or docetaxel. In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®). In some embodiments, the taxane is paclitaxel.

[0016] In another aspect, the invention features a method of enhancing immune function in an individual with cancer, comprising administering an effective amount of a PD-1 axis binding antagonist and a taxane. In some embodiments, CD8+ T cells in the individual have enhanced priming, activation, proliferation and / or cytolytic activity compared to before administration of the PD-1 axis binding antagonist and the taxane. In some embodiments, the number of CD8+ T cells is increased compared to before administration of the combination. In some embodiments, the CD8+ T cells are antigen-specific CD8+ T cells. In some embodiments, Treg function is suppressed compared to before administration of the combination. In some embodiments, T cell exhaustion is decreased compared to before administration of the combination. In some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.

[0017] In some embodiments of the above aspects, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to its ligand binding partners. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 binding antagonist is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (lambrolizumab), CT-011 (pidilizumab), and AMP-224.

[0018] In other embodiments of the above aspects, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. In some embodiments, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 19, the HVR-H2 sequence of SEQ ID NO: 20, and the HVR-H3 sequence of SEQ ID NO: 21; and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 22, the HVR-L2 sequence of SEQ ID NO: 23, and the HVR-L3 sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0019] In other embodiments of the foregoing aspects, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2 binding antagonist is an antibody. In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0020] In any of the preceding embodiments of the above aspects, the cancer may be breast cancer (including triple negative breast cancer (TNBC)), bladder cancer (including urothelial bladder cancer (UBC), muscle invasive bladder cancer and BCG refractory non-muscle invasive bladder cancer), colorectal cancer, rectal cancer, lung cancer (including non-small cell lung cancer, which may be squamous or non-squamous), glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma (including renal cell carcinoma), prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, gastric cancer, esophageal cancer, prostate cancer, endometrial cancer, renal cancer, ovarian cancer, mesothelioma, and hemo malignancies (including myelodysplastic syndromes and multiple myeloma). In certain embodiments, the cancer may be lung cancer (including non-small cell lung cancer, which may be squamous or non-squamous), bladder cancer (including UBC), breast cancer (including TNBC), renal cell carcinoma, melanoma, colorectal cancer, and heme malignancies (e.g., myelodysplastic syndrome (MDS) and multiple myeloma). In some embodiments, the lung cancer is non-small cell lung cancer, which may be squamous or non-squamous. In some embodiments, the bladder cancer is UBC. In some embodiments, the breast cancer is TNBC. In some embodiments, the heme malignancy is MDS or multiple myeloma.

[0021] In some embodiments, the cancer cells in the individual express PD-L1. In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®), paclitaxel or docetaxel. In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®). In some embodiments, the taxane is paclitaxel.

[0022] In some embodiments of any one of the above aspects, the PD-1 axis binding antagonist and / or taxane are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intracerebroventricularly or intranasally.

[0023] In some embodiments of any one of the above aspects, the method may further include administering an effective amount of a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a platinum-based chemotherapeutic agent. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin.

[0024] In another aspect, the invention features the use of a human PD-1 axis binding antagonist in the manufacture of a medicament for treating or delaying the progression of cancer in an individual, the medicament comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and the treatment comprising administering the medicament in combination with a composition comprising a taxane and an optional pharmaceutically acceptable carrier.

[0025] In another aspect, the invention features the use of a taxane in the manufacture of a medicament for treating or delaying the progression of cancer in an individual, where the medicament comprises a taxane and an optional pharmaceutically acceptable carrier, and 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.

[0026] In another aspect, the invention features a composition comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for use in treating or delaying the progression of cancer in an individual, wherein the treatment includes administration of the composition in combination with a second composition, the second composition comprising a taxane and an optional pharmaceutically acceptable carrier.

[0027] In another aspect, the invention features a composition comprising a taxane and optional pharmaceutically acceptable carrier for use in treating or delaying the progression of cancer in an individual, where the treatment includes administering the composition in combination with a second composition, the second composition comprising a human PD-1 axis binding antagonist and optional pharmaceutically acceptable carrier.

[0028] In another aspect, the invention features a kit including a pharmaceutical agent comprising a PD-1 axis binding antagonist and an optional pharma- ceutically acceptable carrier; and a package insert including instructions for administering the pharmaceutical agent in combination with a composition comprising a taxane and an optional pharma- ceutically acceptable carrier to treat or delay the progression of cancer in an individual.

[0029] In another aspect, the invention features a kit that includes a first agent comprising a PD-1 axis binding antagonist and an optional pharma- ceutically acceptable carrier, and a second agent comprising a taxane and an optional pharma- ceutically acceptable carrier, hi some embodiments, the kit further includes a package insert that includes instructions for administering the first agent and the second agent to treat or delay the progression of cancer in an individual.

[0030] In another aspect, the invention features a kit including a pharmaceutical agent comprising a taxane and an optional pharma- ceutically acceptable carrier; and a package insert including instructions for administering the pharmaceutical agent in combination with a composition comprising a PD-1 axis binding antagonist and an optional pharma- ceutically acceptable carrier to treat or delay the progression of cancer in an individual.

[0031] In any one of the preceding aspects, the PD-1 axis binding antagonist may be selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some 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 PD-L1. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 binding antagonist is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (lambrolizumab), CT-011 (pidilizumab) and AMP-224. In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody. In some embodiments, the antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105 and MEDI4736. In some embodiments, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 19, the HVR-H2 sequence of SEQ ID NO: 20, and the HVR-H3 sequence of SEQ ID NO: 21; and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 22, the HVR-L2 sequence of SEQ ID NO: 23, and the HVR-L3 sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0032] In any one of the preceding aspects, the taxane can be nab-paclitaxel (ABRAXANE®), paclitaxel or docetaxel. In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®). In some embodiments, the taxane is paclitaxel.

[0033] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other embodiments of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows. [Brief description of the drawings]

[0034] [Figure 1] 1 shows that combination therapy of anti-PD-L1 antibody and paclitaxel + carboplatin demonstrates synergistic anti-tumor efficacy compared to control antibody or paclitaxel + carboplatin alone in a syngeneic MC38 colorectal tumor model in vivo in C57BL / 6 mice. The graph shows a cubic spline fit of the tumor volume for each treatment group as a function of time. A cubic spline fit is a mathematical algorithm that selects the best smooth curve that fits all the data per treatment group. Mice bearing established subcutaneous MC38 tumors of approximately 100-200 mm3 were treated with a single dose of carboplatin at 80 mg / kg by intraperitoneal (IP) injection plus 10 mg / kg paclitaxel by intravenous (IV) injection, and 10 mg / kg of anti-gp120 antibody or anti-PD-L1 (clone 25A1 mIgG2a.DANA) dosed three times weekly for three weeks. N=10 mice per group. [Figure 2A]2A-2B are graphs showing that dexamethasone (Dex) inhibits the efficacy of anti-PD-L1 antibody (αPD-L1) monotherapy in a syngeneic MC38 colorectal tumor model in C57BL / 6 mice. FIG. 2A shows a cubic spline fit of the tumor volumes for each treatment group, while FIG. 2B shows plots (trellis plots) for individual mice (black curves show cubic spline fits of tumor volumes for each treatment group). Each graph in FIG. 2B includes a dashed line representing the cubic spline fit of the control group. For FIG. 2B, the horizontal dashed line at approximately 300 mm3 is one measure for progression volume (2x the initial tumor volume). Tumor volumes below 32 mm3 (shown by the horizontal dashed line in FIG. 2B) are visible but too small to be measured accurately. Mice bearing established subcutaneous MC38 tumors of approximately 100-200 mm3 were treated with either saline or dexamethasone at a single dose of 4 mg / kg IV, followed 12 hours later by treatment with either control anti-gp120 antibody or anti-PD-L1 (clone 25A1.mIgG2a.DANA) at 10 mg / kg IP three times per week for three weeks. N=10 mice per group. [Figure 2B]2A-2B are graphs showing that dexamethasone (Dex) inhibits the efficacy of anti-PD-L1 antibody (αPD-L1) monotherapy in a syngeneic MC38 colorectal tumor model in C57BL / 6 mice. FIG. 2A shows a cubic spline fit of the tumor volumes for each treatment group, while FIG. 2B shows plots (trellis plots) for individual mice (black curves show cubic spline fits of tumor volumes for each treatment group). Each graph in FIG. 2B includes a dashed line representing the cubic spline fit of the control group. For FIG. 2B, the horizontal dashed line at approximately 300 mm3 is one measure for progression volume (2x the initial tumor volume). Tumor volumes below 32 mm3 (shown by the horizontal dashed line in FIG. 2B) are visible but too small to be measured accurately. Mice bearing established subcutaneous MC38 tumors of approximately 100-200 mm3 were treated with either saline or dexamethasone at a single dose of 4 mg / kg IV, followed 12 hours later by treatment with either control anti-gp120 antibody or anti-PD-L1 (clone 25A1.mIgG2a.DANA) at 10 mg / kg IP three times per week for three weeks. N=10 mice per group. [Diagram 3] Graph showing that dexamethasone inhibits antigen-specific T cell responses in an OTI adoptive T cell transfer and vaccination model. CD8+ T cells were purified from OTI Thy1.1 mice and injected IV at 2.5 million cells per mouse. The next day, recipient mice were vaccinated IP with 250 ng of anti-DEC205 fused to full-length ovalbumin plus a single IV dose of either 4 mg / kg saline or dexamethasone. Two days later, mice were euthanized and total OTICD8+ cells were counted from the pancreas by flow cytometry. N=5 mice per group. Each symbol represents an individual mouse. P values ​​were calculated by two-tailed unpaired t-test. [Figure 4A]FIG. 4B is a graph showing that combination therapy of anti-PD-L1 antibody and nab-paclitaxel (ABRAXANE®, Abx) + carboplatin (Carbo) resulted in a strong synergistic anti-tumor effect, achieving durable complete remission (4 out of 8 mice) that lasted for more than 90 days in a syngeneic MC38 colorectal tumor model in C57BL / 6 mice. The graphs show tumor volume as a function of time. FIG. 4A shows a cubic spline fit of the tumor volumes for each treatment group, while FIG. 4B shows a trellis plot for individual mice (black curves show cubic spline fits of the tumor volumes for each treatment group). Each graph in FIG. 4B includes a dashed line that represents the cubic spline fit of the control group. For FIG. 4B, the horizontal dashed line at approximately 600 mm3 is one measure for progression volume (2x the initial tumor volume). Tumor volumes below 32 mm3 (indicated by horizontal dashed lines in Figure 4B) were visible but too small to be accurately measured. Mice bearing established subcutaneous MC38 tumors of approximately 300 mm3 were treated with anti-gp120 control antibody or anti-PD-L1 antibody (clone YW243.55.S70 mIgG2a.DANA) administered by IP injection at 10 mg / kg three times per week for three weeks, plus saline or carboplatin plus 75 mg / kg IP weekly for three weeks, ABRAXANE® at 15 mg / kg IV weekly for three weeks as indicated. N=8 mice per group. [Figure 4B]FIG. 4B is a graph showing that combination therapy of anti-PD-L1 antibody and nab-paclitaxel (ABRAXANE®, Abx) + carboplatin (Carbo) resulted in a strong synergistic anti-tumor effect, achieving durable complete remission (4 out of 8 mice) that lasted for more than 90 days in a syngeneic MC38 colorectal tumor model in C57BL / 6 mice. The graphs show tumor volume as a function of time. FIG. 4A shows a cubic spline fit of the tumor volumes for each treatment group, while FIG. 4B shows a trellis plot for individual mice (black curves show cubic spline fits of the tumor volumes for each treatment group). Each graph in FIG. 4B includes a dashed line that represents the cubic spline fit of the control group. For FIG. 4B, the horizontal dashed line at approximately 600 mm3 is one measure for progression volume (2x the initial tumor volume). Tumor volumes below 32 mm3 (indicated by horizontal dashed lines in Figure 4B) were visible but too small to be accurately measured. Mice bearing established subcutaneous MC38 tumors of approximately 300 mm3 were treated with anti-gp120 control antibody or anti-PD-L1 antibody (clone YW243.55.S70 mIgG2a.DANA) administered by IP injection at 10 mg / kg three times per week for three weeks, plus saline or carboplatin plus 75 mg / kg IP weekly for three weeks, ABRAXANE® at 15 mg / kg IV weekly for three weeks as indicated. N=8 mice per group. [Figure 5A]5A-5C are graphs showing that mice previously cured of primary MC38 tumors with anti-PD-L1 antibody and nab-paclitaxel (ABRAXANE®) + carboplatin therapy (mice achieving complete remission as described in FIG. 1A) generate anti-tumor T cell memory responses. Upon secondary re-immunization with fresh MC38 tumor cells, tumors failed to grow in 100% (4 / 4) cured mice. FIG. 5A shows that splenocytes harvested 7 days after secondary challenge have comparable numbers of CD4+ and CD8+ T cells to primary challenged naive mice. FIG. 5B shows the results of flow cytometry analysis showing that upon in vitro stimulation with PMA plus ionomycin, T cells from cured mice have enhanced interferon-γ (IFN-γ) production as assessed by intracellular cytokine staining compared to primary challenged mice. Error bars represent standard deviation for n = 5 (primary challenged mice) or n = 4 (cured mice, secondary challenge), and flow cytometry dot plots represent one mouse from each group. P values ​​were calculated by two-tailed, unpaired t-test. [Figure 5B]5A-5C are graphs showing that mice previously cured of primary MC38 tumors with anti-PD-L1 antibody and nab-paclitaxel (ABRAXANE®) + carboplatin therapy (mice achieving complete remission as described in FIG. 1A) generate anti-tumor T cell memory responses. Upon secondary re-immunization with fresh MC38 tumor cells, tumors failed to grow in 100% (4 / 4) cured mice. FIG. 5A shows that splenocytes harvested 7 days after secondary challenge have comparable numbers of CD4+ and CD8+ T cells to primary challenged naive mice. FIG. 5B shows the results of flow cytometry analysis showing that upon in vitro stimulation with PMA plus ionomycin, T cells from cured mice have enhanced interferon-γ (IFN-γ) production as assessed by intracellular cytokine staining compared to primary challenged mice. Error bars represent standard deviation for n = 5 (primary challenged mice) or n = 4 (cured mice, secondary challenge), and flow cytometry dot plots represent one mouse from each group. P values ​​were calculated by two-tailed, unpaired t-test. [Figure 6A] Graphs showing results from a Phase 1b clinical trial testing the efficacy of combination therapy of anti-PD-L1 antibody (MPDL3280A) with taxane and carboplatin. FIG. 6A is a graph showing the change in tumor size over time after treatment with MPDL3280A, nab-paclitaxel (ABRAXANE®) and carboplatin. The objective response rate (ORR) was 9 / 14 patients with 3 complete responses (CR) and 6 partial responses (PR). FIG. 6B is a graph showing the change in tumor size over time after treatment with MPDL3280A with paclitaxel + carboplatin. The ORR was 2 / 6 (33%) patients with 2 partial responses. [Figure 6B]Graphs showing results from a Phase 1b clinical trial testing the efficacy of combination therapy of anti-PD-L1 antibody (MPDL3280A) with taxane and carboplatin. FIG. 6A is a graph showing the change in tumor size over time after treatment with MPDL3280A, nab-paclitaxel (ABRAXANE®) and carboplatin. The objective response rate (ORR) was 9 / 14 patients with 3 complete responses (CR) and 6 partial responses (PR). FIG. 6B is a graph showing the change in tumor size over time after treatment with MPDL3280A with paclitaxel + carboplatin. The ORR was 2 / 6 (33%) patients with 2 partial responses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] I. Definition Before describing the present invention 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.

[0036] As used in this specification and the accompanying drawings, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates to the contrary. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0037] The term "approximately" as used herein refers to the normal range of error for each value that is readily known to one of ordinary skill in the art. Reference herein to a value or parameter with "approximately" includes (and describes) embodiments directed to the value or parameter itself.

[0038] It is understood that aspects and embodiments of the invention described herein include "comprising", "consisting" and "consisting essentially of" these aspects and embodiments.

[0039] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis partner with any one or more of its binding partners, thereby eliminating T cell dysfunction resulting from signaling on the PD-1 signaling axis, thereby restoring or enhancing T cell function (e.g., proliferation, cytokine production, and / or target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0040] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses or prevents signal transduction that results from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and / or PL-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In certain aspects, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress or prevent signal transduction that results from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed upon T lymphocyte-mediated signaling through PD-1, thereby attenuating the dysfunctional T cell's mechanism of dysfunction (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a particular aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab), as described herein. In another particular aspect, the PD-1 binding antagonist is MK-3475 (lambrolizumab), as described herein. In another particular aspect, 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.

[0041] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses or prevents signaling that results from the interaction of PD-1 with any one or more of its binding partners, such as PD-1 and / or B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In particular aspects, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress or prevent signaling that results from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In one embodiment, the PD-L1 binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed upon T lymphocyte-mediated signaling through PD-L1, thereby attenuating the dysfunctional T cell's mechanism of dysfunction (e.g., enhancing effector responses 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 particular aspect, the anti-PD-L1 antibody is MDX-1105, as described herein. In yet another particular aspect, the anti-PD-L1 antibody is MPDL3280A, as described herein. In yet another particular aspect, the anti-PD-L1 antibody is MEDI4736, as described herein.

[0042] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses or prevents signaling that results from the interaction of PD-L2 with any one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In certain aspects, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress or prevent signaling that results from the interaction of PD-L2 with any one or more of its binding partners, such as PD-1. In one embodiment, the PD-L2 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed upon T lymphocyte-mediated signaling through PD-L2, thereby attenuating the mechanical dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0043] As used herein, a "taxane" is a diperthene that can bind to tubulin to promote microtubule assembly and stabilization and / or prevent microtubule depolymerization. Taxanes included herein include the taxoid 10-deacetylbaccatin III and / or its derivatives. Exemplary taxanes include, but are not limited to, paclitaxel (i.e., TAXOL®, CAS #33069-62-4), docetaxel (i.e., TAXOTERE®, CAS #114977-28-5), larotaxel, carbazitaxel, mirataxel, tesetaxel, and / or orataxel. In some embodiments, the taxane is an albumin-coated nanoparticle (e.g., nanoalbumin-bound (nab)-paclitaxel, i.e., ABRAXANE® and / or nab-docetaxel ABI-008). In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®). In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®). In some embodiments, the taxane is formulated in CREMAPHOR® (e.g., TAXOL®) and / or Tween, such as polysorbate 80 (e.g., TAXOTERE®). In some embodiments, the taxane is a liposome-encapsulated taxane. In some embodiments, the taxane is a prodrug and / or conjugated form of taxane (e.g., DHA covalently conjugated to paclitaxel, paclitaxel poliglumex, and / or linoleyl carbonate-paclitaxel). In some embodiments, the paclitaxel is formulated substantially free of any surfactant (eg, in the absence of CREMAPHOR and / or Tween--eg, TOCOSOL® paclitaxel).

[0044] The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. This term includes the common elements of both "exhaustion" and / or anergy, where antigen recognition may occur but the subsequent immune response is ineffective to control infection or tumor growth.

[0045] As used herein, the term "dysfunctional" also includes refractory or unresponsiveness to antigen recognition, specifically, 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.

[0046] The term "anergy" refers to the incomplete or insufficient signal delivered through the T cell receptor (e.g., intracellular Ca in the absence of ras activation). +2 Anergy refers to a state of unresponsiveness to antigenic stimulation resulting from increased T cell proliferation (increase in T cell proliferation and proliferation rate). T cell anergy also results upon stimulation with antigen in the absence of costimulation, such that the cells become refractory to subsequent activation by antigen even in the context of costimulation. The unresponsive state is often overcome by the presence of interleukin 2. Anergic T cells do not undergo clonal expansion and / or acquire effector functions.

[0047] The term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction resulting from persistent TCR signaling that occurs during many chronic infections and cancers. It is distinct from anergy in that it arises from persistent signaling, not through defective or insufficient signaling. It is defined by low effector function, sustained expression of inhibitory receptors, and a transcriptional state quite different from that of functional effector or memory T cells. Exhaustion prevents optimal control of infections and tumors. Exhaustion can be brought about by both extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) as well as cell-intrinsic negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).

[0048] "Enhancing T cell function" means inducing, influencing, or stimulating T cells to sustain or amplify biological function, or to regenerate or reactivate exhausted or inactive T cells. Examples of enhancing T cell function include increased secretion of gamma interferon from CD8+ T cells, increased proliferation, and increased antigen responsiveness (e.g., clearance of viruses or pathogens or tumors) compared to pre-intervention levels. In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. Methods for measuring such enhancement are known to those skilled in the art.

[0049] "T cell dysfunction disorder" is a disorder or condition of T cells characterized by reduced responsiveness to antigenic stimulation. In certain embodiments, the T cell dysfunction disorder is a disorder specifically associated with increased inappropriate signaling through PD-1. In another embodiment, the T cell dysfunction disorder is a disorder in which T cells are in an anergic state or have a reduced ability to secrete cytokines, proliferate, or exert cytolytic activity. In certain aspects, the reduced responsiveness results in ineffective control of pathogens or tumors expressing immunogens. Examples of T cell dysfunction disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity.

[0050] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated and dealt with, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0051] "Immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic, and enhancing tumorigenicity aids in the clearance of tumor cells by the immune response. Examples of enhancing tumorigenicity include treatment with PD-1 axis binding antagonists and taxanes.

[0052] "Sustained remission" refers to a sustained effect on the reduction of tumor growth after the cessation of treatment. 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 remission is at least as long as the duration of treatment, for example, 1.5 times, 2.0 times, 2.5 times, or 3.0 times the duration of treatment.

[0053] As used herein, "reducing or inhibiting cancer recurrence" means reducing or inhibiting tumor or cancer recurrence or tumor or cancer progression. As disclosed herein, cancer recurrence and / or cancer progression includes, but is not limited to, cancer metastasis.

[0054] As used herein, "complete response" or "CR" refers to the disappearance of all target lesions.

[0055] As used herein, "partial response" or "PR" means at least a 30% reduction in the sum of longitudes (SLD) of a target lesion, based on the baseline SLD.

[0056] As used herein, "stable disease" or "SD" means that target lesions have not shrunk sufficiently to qualify for PR or increased sufficiently to qualify for PD, based on the smallest SLD since treatment began.

[0057] As used herein, "disease progression" or "PD" means at least a 20% increase in the SLD of a target lesion or the presence of one or more new lesions, based on the smallest SLD recorded since the start of treatment.

[0058] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not worsen. Progression-free survival can include the amount of time a patient has a complete or partial response, as well as the amount of time a patient experiences stable disease.

[0059] As used herein, "overall response rate" or "objective response rate" (ORR) means the sum of the complete response (CR) rate and the partial response (PR) rate.

[0060] As used herein, "overall survival" (OS) refers to the percentage of individuals within a group who have a high probability of being alive after a particular duration of time.

[0061] By "pharmaceutical formulation" is meant a preparation having a form that allows the biological activity of the active ingredient to be effective and that does not contain any additional components that are unacceptably toxic to the subject to which it is to be administered. Such formulations are sterile. A pharmaceutical acceptable excipient (vehicle, additive) is an excipient that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient utilized.

[0062] As used herein, "treatment" refers to a clinical intervention intended to modify the natural course of the individual or cells being treated during the clinical pathology course. Desired effects of treatment include slowing the rate of disease progression, improving or mitigating the disease state, and remission or improved prognosis. For example, an individual has been successfully "treated" if one or more symptoms associated with cancer are alleviated or eliminated, including, but not limited to, reducing (or destroying) the proliferation of cancer cells, reducing symptoms as a result of the disease, improving the quality of life of those suffering from the disease, reducing the dose of other drugs required to treat the disease, and / or prolonging the life of the individual.

[0063] As used herein, "delaying progression" of a disease means to prolong, prevent, slow, retard, stabilize, and / or postpone the onset of a disease (e.g., cancer). The length of time of this delay may vary depending on the medical history and / or history of the individual being treated. As will be appreciated by those skilled in the art, a sufficient or significant delay effectively encompasses prevention, in the sense that the individual does not develop the disease. For example, the onset of terminal cancer, such as metastasis, may be delayed.

[0064] An "effective amount" is the minimum amount required to achieve at least a measurable improvement or prevention of a particular disorder. As used herein, an effective amount may vary depending on factors such as the patient's condition, age, sex, weight, and the ability of the agent to elicit a desired response in an individual. An effective amount is also an amount in which any toxic or adverse effects of the treatment are outweighed by the therapeutically beneficial effects. In the case of prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, reducing the severity, or delaying the onset of a disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease. In the case of therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms as a result of the disease, improving the quality of life of the person suffering from the disease, reducing the dose of other drugs required to treat the disease, and enhancing the effect of another drug, such as by targeting, slowing the progression of the disease, and / or prolonging life. In the case of cancer or tumors, an effective amount of an agent may be effective in reducing the number of cancer cells; reducing the size of the tumor; inhibiting (i.e., slowing to some extent, preferably stopping) the invasion of cancer cells into surrounding organs; inhibiting (i.e., slowing to some extent, preferably stopping) the metastasis of the tumor; inhibiting to some extent the growth of the tumor; and / or alleviating to some extent one or more symptoms associated with the disorder. An effective amount may be administered in one or more administrations. For purposes of the present invention, an effective amount of an agent, compound, or pharmaceutical composition is an amount sufficient to achieve, directly or indirectly, a prophylactic or therapeutic treatment. As will be understood from a clinical perspective, an effective amount of an agent, compound, or pharmaceutical composition may or may not be achieved in conjunction with another agent, compound, or pharmaceutical composition. Thus, 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 given in an effective amount if a desired result can be achieved or is achieved in conjunction with one or more other agents.

[0065] As used herein, "in conjunction with" refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" refers to the administration of one therapeutic modality before, during, or after the administration of the other therapeutic modality to an individual.

[0066] A "disorder" is any physical condition that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0067] The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

[0068] The term "tumor" as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor," as referred to herein, are not mutually exclusive.

[0069] The terms "cancer" and "cancerous" refer to or describe the 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 malignant lymphoma. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer (e.g., urothelial bladder cancer (UBC), muscle invasive bladder cancer (MIBC), and BCG-refractory non-muscle invasive bladder cancer (NMIBC)), urinary tract cancer, liver cancer, , breast cancer (e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC) that is estrogen receptor (ER-), progesterone receptor (PR-) and HER2 (HER2-) negative), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer (e.g., renal cell carcinoma (RCC), prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal cancer, penile carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, melanoma, nodular melanoma, multiple myeloma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleavable cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); Acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); and post-transplant lymphoproliferative disorder (PTLD), myelodysplastic syndromes (MDS), as well as abnormal vascular proliferation associated with phacomatosis, edema (e.g., associated with brain tumors), Meigs syndrome, brain and head and neck cancers and associated metastases.In certain embodiments, the cancers amenable to treatment with the methods and compositions of the present invention may be breast cancer (e.g., triple-negative breast cancer), bladder cancer (e.g., UBC, MIBC, and NMIBC), colorectal cancer, rectal cancer, lung cancer (including non-small cell lung cancer, which may be squamous or non-squamous), glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma (e.g., RCC), prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and hemo malignancies (e.g., MDS and multiple myeloma). In some embodiments, the cancer is selected from small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. In other embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, glioblastoma, and breast carcinoma (including metastatic forms of these cancers). In certain embodiments, the cancer is selected from lung cancer (e.g., non-small cell lung cancer, which may be squamous or non-squamous), bladder cancer (e.g., UBC), breast cancer (e.g., TNBC), RCC, melanoma, colorectal cancer, and hemolytic malignancies (e.g., MDS and multiple myeloma). In some embodiments, the lung cancer is non-small cell lung cancer, which may be squamous or non-squamous. In some embodiments, the bladder cancer is UBC. In some embodiments, the breast cancer is TNBC. In some embodiments, the hemolytic malignancies are MDS or multiple myeloma.

[0070] As used herein, the term "cytotoxic agent" refers to any agent that is detrimental to cells (e.g., causing cell death, inhibiting proliferation, or otherwise interfering with the function of cells). Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212and radioisotopes of Lu; chemotherapeutic agents; growth inhibitors; enzymes and fragments thereof, such as nucleases; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. Exemplary cytotoxic agents can be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A, fatty acid biosynthesis inhibitors, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and cancer metabolism inhibitors. In one embodiment, the cytotoxic agent is a platinum-based chemotherapeutic agent. In one embodiment, the cytotoxic agent is an antagonist of EGFR. In one embodiment, the cytotoxic agent is N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g., erlotinib, TARCEVA™). 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. In one embodiment, the cytotoxic agent is a PI3K inhibitor.

[0071] As used herein, a "chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer. Examples of chemotherapy drugs include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer / Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU, 5-FU-1, 5-FU-2 ... (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafamib (SCH 66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelanamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelanine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (especially cryptophycin 1 and cryptophycin 8);Adenocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5a-reductase; including finasteride and dutasteride; vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat; dolastatins; aldesleukin, talc; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongiostatins; nitrogen mustards, such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifostatin; sufamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 33:183-186 (1994); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, ausramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norrho Icin, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements. , such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diazicon; elfomitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamunol; nitraelin; pentostatin; fenameth; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); Razoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitrol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("A ra-C");cyclophosphamide;thiotepa;taxanes;chlorambucil;GEMZAR® (gemcitabine);6-thioguanine;mercaptopurine;methotrexate;vinblastine;etoposide (VP-16);ifosfamide;mitoxantrone;vincristine;NAVELBINE® (vinorelbine);novantrone;teniposide;edatrexate;daunomycin;aminopterin;Capecitabine (XELODA®); ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid and pharma- ceutically acceptable salts, acids and derivatives of any of the above;

[0072] Chemotherapeutic agents also include "platinum-based" chemotherapeutic agents, which include organic compounds that contain platinum as an integral part of the molecule. Typically, platinum-based chemotherapeutic agents are coordination complexes of platinum. Platinum-based chemotherapeutic agents are sometimes referred to in the art as "platins." Examples of platinum-based chemotherapeutic agents include, but are not limited to, carboplatin, cisplatin, and oxaliplatin.

[0073] Chemotherapeutic agents also include (i) antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY117018, onapristone, and FARESTON® (toremifene citrate); (ii) anti-inflammatory agents that act to regulate or inhibit hormone action on tumors, such as anti-inflammatory agents, such as 4(5)-isopropyl tert-butyl esterase inhibitors (ITBs), and (iii) anti-inflammatory agents that act to regulate or inhibit hormone action on tumors, such as, for example, 4(5)-isopropyl tert-butyl esterase inhibitors (ITBs), and (iv) anti-inflammatory agents that act to regulate or inhibit hormone action on tumors, such as, for example, 4(5)-isopropyl tert-butyl esterase inhibitors (ITBs), and (v) anti-inflammatory agents that act to regulate or inhibit hormone action on tumors, such as, for example, 4(5)-isopropyl tert-butyl esterase inhibitors (ITBs), and (vi ... (iii) aromatase inhibitors, which block the enzyme aromatase that regulates estrogen production in the adrenal glands, such as midazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestany, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) flutamide, nilutamide, bicalutamide, leu (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Ralf and H-Ras. (vii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, such as ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; topoisomerase 1 inhibitors, such as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceutical acceptable salts, acids, and derivatives of the above.

[0074] Chemotherapeutic drugs also include antibodies, such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia) and the antibody drug conjugates, gemtuzumab, ozogamicin (MYLOTARG®, Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include apolizumab, acelizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidofusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motavizumab, and motavizumab. Mab, natalizumab, nimotuzumab, norobizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tuccituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and interleukin-12 and anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), a recombinant exclusively human sequence full-length IgG1λ antibody genetically modified to recognize the p40 protein.

[0075] Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise directly interact with EGFR and prevent or reduce its signaling activity, and are alternatively referred to as "EGFR antagonists." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb579 (ATCC CRL HB8506), MAb455 (ATCC CRL HB8507), MAb225 (ATCC CRL 8508), MAb528 (ATCC CRL 8509) (see U.S. Pat. No. 4,943,533) and variants thereof, such as chimeric 225 (C225 or cetuximab: ERBUTIX®) and reconstituted human 225 (H225) (see, e.g., WO 96 / 40210, Imclone Systems, Inc.,). Inc.); IMC-11F8 (Imclone), a fully human EGFR-targeting antibody; antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,252,290); 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 manitumumab (WO 98 / 50433, Abgenix / Amgen); EMD 55900 (Stragliotto et al.,. Eur. J. Cancer 32A:636-640 (1996); EMD7200 (matuzumab), a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-alpha for EGFR binding (EMD / Merck); the human EGFR antibody, HuMax-EGFR (GenMab); the fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3 and described in US 6,235,883; MDX-447 (Medarex Inc); and mAb806 or humanized mAb806 (Johns et al. al., J. Biol. Chem. 279(29):30375-30384 (2004)). Anti-EGFR antibodies may be conjugated to cytotoxic agents, thus generating immunoconjugates (see, e.g., EP659439A2, Merck Patent GmbH).EGFR antagonists include small molecules, e.g., those disclosed in U.S. Patents: 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,33 2, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008 and 5,747,498, and compounds described in PCT publications WO98 / 14451, WO98 / 50038, WO99 / 09016 and WO99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech / OSI Pharmaceuticals); PD 183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 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]-7H-pyrrolo[2,3-d]pyrimidine;CL-387785(N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butanamide);EKB-569(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butanamide)(Wyeth);AG1478(Pfizer);AG1571(SU 5271; Pfizer; dual EGFR / HER2 tyrosine kinase inhibitors, such as lapatinib (TYKERB®, GSK572016 or N-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

[0076] Chemotherapeutic agents also include "tyrosine kinase inhibitors," including the EGFR-targeted agents noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724, 714 (Pfizer and OSI), which are oral selective inhibitors of the ErbB2 receptor tyrosine kinase; dual HER inhibitors, such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; lapatinib (GSK572016; 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), which inhibit Raf-1 signaling; and ISIS inhibitors, which inhibit Raf-1 signaling. Raf-1 inhibitors, such as the antisense agent ISIS-5132 available from Rharmaceuticals; non-HER targeted tyrosine kinase inhibitors, such as imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors, such as sunitinib (SUTENT®, available from Pfizer); VEFG receptor tyrosine kinase inhibitors, for example vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); the MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, for example PD 153035, 4-(3-chloroanilino)quinazoline;pyridopyrimidines;pyrimidopyrimidines;pyrrolopyrimidines, such as CGP59326, CGP60261 and CGP62706;pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines;curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide);tyrphostins containing a nitrothiophene moiety;PD-0183805 (Warner-Lamber);antisense molecules (e.g., nucleic acids encoding HER);quinoxalines (U.S. Pat. No. 5,804,396);tryphostins (U.S. Pat. No. 5,804,396);ZD6474 (Astra Zeneca);PTK-787 (Novartis / Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Afinitac (ISIS 3521; Isis / Lilly); Imatinib mesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®); or U.S. Pat. No. 5,804,396; WO1999 / 09016 (American Cyanamid); WO1998 / 43960 (American Cyanamid); WO1997 / 38983 (Warner Lambert); WO1999 / 06378 (Warner Lambert); WO1999 / 06396 (Warner Lambert); WO1996 / 30347 (Pfizer, Inc); WO1996 / 33978 (Zeneca); WO1996 / 3397 (Zeneca) and WO1996 / 33980 (Zeneca).

[0077] Chemotherapy drugs also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, live BCG, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, interferon alfa-2c, interferon alfa-2d, interferon alfa-2e, interferon alfa-2f, interferon alfa-2g, interferon alfa-2h ... Also included are alpha-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutical acceptable salts thereof.

[0078] Chemotherapy drugs also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate ... thizone-17-valerate, aclomethasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN BioTherapeutics, LLC; antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide minocycline, sulfasalazine; tumor necrosis factor alpha (TNFα) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi ), interleukin 1 (IL-1) blockers such as anakinra (Kineret), T cell costimulatory blockers such as abatacept (Orencia), interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®); interleukin 13 (IL-13) blockers such as lebrikizumab, interferon alpha (IFN) blockers such as rontalizumab; beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers such as anti-lymphotoxin alpha (LTa); radioisotopes (e.g., At 211 , I 131、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212and radioisotopes of Lu); various investigational drugs such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832), polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid and its derivatives; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin; podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAM®), AX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOOPE® vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium ( Also included are GENASENSE®; pixantrone; farnesyltransferase inhibitors, such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen using oxaliplatin in combination with 5-Fu and leucovorin (ELOXATIN™).

[0079] Chemotherapeutic drugs also include nonsteroidal anti-inflammatory drugs that have analgesic, antipyretic and anti-inflammatory effects.NSAIDs include nonselective inhibitors of the enzyme cyclooxygenase.Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib and valdecoxib. Possible indications for NSAIDs include symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathy, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headaches and migraines, post-operative pain, mild to moderate pain due to inflammation and tissue injury, fever, ileus, and renal colic.

[0080] As used herein, "growth inhibitor" refers to a compound or composition that inhibits cell growth either in vitro or in vivo. In one embodiment, the growth inhibitor 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 inhibitor may significantly reduce the percentage of cells in S phase. Examples of growth inhibitors include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. These agents that block G1 also extend to S-phase arrest, e.g., DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antitineoplastic drugs" by Murakami et al. (WB Saunders, Philadelphia, 1995), e.g., p. 13.

[0081] "Radiation therapy" refers to the use of directed gamma or beta radiation to induce sufficient damage to cells to limit the cells' ability to function normally or to destroy the cells altogether. It will be appreciated in the art that there are many methods for determining the dose and duration of treatment. A typical treatment is administered as a single dose, with typical doses in the range of 10-200 units (Grays) per day.

[0082] By "subject" or "individual" for purposes of treatment is meant 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. A subject or individual may be a patient.

[0083] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

[0084] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that would interfere with the antibody's research, diagnostic or therapeutic uses, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified, for example, (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody as determined by the method of Lowry; (2) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues, for example, using a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, for example, using Coomassie blue or silver staining. Since at least one component of the antibody's natural environment is not present, an isolated antibody includes the antibody in situ inside a recombinant cell. Ordinarily, however, an isolated antibody will have been prepared by at least one purification step.

[0085] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, 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, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains a variable domain (V) at one end. H ) followed by several constant domains. Each light chain has a variable domain (V L ) at its other end and 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. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.

[0086] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain is the C H 1. C H 2 and C H It contains 3 domains (collectively CH) and the light chain CHL (or CL).

[0087] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain is referred to as "V H The light chain variable domain is sometimes referred to as "V L These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0088] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the specificity and binding of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. The variability is concentrated within three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions, which for the most part adopt a beta-sheet configuration, connected by three HVRs that form loops that connect and in some cases are part of the beta-sheet structure. The HVRs within each chain are held in close proximity to each other by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0089] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0090] The term IgG "isotype" or "subclass" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions.

[0091] Antibodies (immunoglobulins) can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different immunoglobulin classes are well known and are generally described in Abbas et al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000). An antibody may be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0092] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment, as defined below. These terms refer in particular to an antibody with its heavy chains, including the Fc region.

[0093] For purposes herein, a "naked antibody" is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0094] An "antibody fragment" preferably comprises a portion of an intact antigen, including its antigen-binding region. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed with antibody fragments.

[0095] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment (the name reflects its ability to crystallize readily). Pepsin treatment produces an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0096] An "Fv" is the minimum antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species is composed of a dimer of one heavy and one light chain variable domain in tight non-covalent association. In a single-chain Fv (scFv) species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker, thus allowing the light and heavy chains to associate in a "dimeric" structure similar to that of a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only three HVRs specific for one antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the complete binding site.

[0097] Fab fragments contain the heavy and light chain variable domains, and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a 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 ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0098] A "single-chain Fv" or "scFv" antibody fragment comprises 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 which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0099] By "diabody" is meant an antibody fragment having two antigen-binding sites, comprising a heavy chain variable domain (VH) attached to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0100] 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" refers to the nature of the antibody as not being a mixture of individual antibodies. In certain embodiments, such monoclonal antibodies typically comprise an antibody comprising a polypeptide sequence that binds to a single target, where the target-binding polypeptide sequence was obtained by a process comprising the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that the selected target-binding sequence can be further modified, e.g., to improve affinity for the target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, and the like, and that an antibody comprising a modified target-binding sequence is also a monoclonal antibody of the present invention. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0101] 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, the monoclonal antibodies to be used in accordance with the present invention can be produced using, for example, hybridoma methods (see, e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage spray techniques (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 222:581-597 (1992)), and the like. et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al. al.,Proc.Natl.Acad.Sci.USA 90:2551(1993);Jakobovits et al.,Nature 362:255~258(1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg et al. The antibodies may be produced by a variety of techniques, including those described herein, including those disclosed herein (see, for example, U.S. Pat. No. 6,313,131, 1995; see ...).

[0102] As used herein, monoclonal antibodies specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of one chain(s) are identical or homologous to corresponding sequences in antibodies 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; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATTZED® antibodies in which the antigen-binding region of the antibody is derived from an antibody produced, such as by immunizing macaque monkeys with the antigen of interest.

[0103] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of 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 cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. In general, a 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 human immunoglobulin sequences. A humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0104] A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or has been produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to immunized xenomouse, transgenic animals that have been modified to produce human antibodies in response to antigen challenge but have their endogenous loci disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via human B cell hybridoma technology.

[0105] A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Typically, a species-dependent antibody "specifically binds" to a human antigen (e.g., about 1×10 -7 M or less, preferably about 1×10 -8 M or less and preferably about 1×10-9 The species-dependent antibody has a binding affinity (Kd) value of M or less for a homologue of the antigen from a second non-human mammalian species that is at least 50-fold lower or at least about 500-fold lower or at least about 1000-fold lower than its binding affinity for the human antigen. The species-dependent antibody may be any of the various types of antibodies as defined above, but is preferably a humanized or human antibody.

[0106] The term "hypervariable region", "HVR" or "HV" as used herein refers to a region of an antibody variable domain that is sequence hypervariable and / or forms structurally defined loops. Generally, antibodies contain six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In natural antibodies, H3 and L3 show the greatest diversity of the six HVRs, and H3 in particular is thought to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies composed only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0107] Several HVR delineations are in use and are encompassed herein. The Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs are a compromise between the Kabat HVRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on analysis of available complex crystal structures. The residues derived from each of these HVRs are listed below: JPEG2025020147000001.jpg55158

[0108] The HVR may comprise an "extended HVR" of 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. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0109] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0110] "Numbering of variable domain residues as in Kabat" or "numbering of amino acid positions as in Kabat" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b and 82c, etc. according to Kabat). The Kabat residue numbering can be determined for a given antibody by alignment of the "standard" Kabat numbered sequence with the antibody sequence at the regions of homology.

[0111] The Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues within an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0112] 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 tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigen-binding regions with complementary light chain polypeptides. Linear antibodies can be bispecific or monospecific.

[0113] The terms "bind", "specifically bind to" or "specific for" as used herein refer to a measurable and reproducible interaction, such as binding between an antibody and a target, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily and / or for a longer period of time than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on one protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0114] II. PD-1 axis binding antagonists Provided herein is a method for treating or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and a taxane. Also provided herein is a method for enhancing immune function in an individual with cancer, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and a taxane. For example, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. PD-1 (Programmed Death 1) is also referred to in the art as "Programmed Cell Death 1", "PDCD1", "CD279" and "SLEB2". An exemplary human PD-1 is set forth in niProtKB / Swiss-Prot Accession No. Q15116. PD-L1 (programmed death ligand-1) is also referred to in the art as "programmed cell death 1 ligand 1," "PDCD1LG1," "CD274," "B7-H," and "PDL1." An exemplary human PD-L1 is set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7.1. PD-L2 (programmed death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2," "PDCD1LG2," "CD273," "B7-DC," "Btdc," and "PDL2." An exemplary human PD-L2 is set forth in UniProtKB / Swiss-Prot Accession No. Q9BQ51. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[0115] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain embodiments, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its ligand binding partner. In certain embodiments, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In certain embodiments, the PD-L2 binding partner is PD-1. The antagonist may be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide.

[0116] 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 MDX-1106 (nivolumab), MK-3475 (lambrolizumab), and CT-011 (pidilizumab). In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO2010 / 077634. ​​MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007 / 005874. MEDI4736 is an anti-PD-L1 monoclonal antibody described in WO2011 / 066389 and US2013 / 034559. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558 or nivolumab, is an anti-PD-1 antibody described in WO2006 / 121168. MK-3475, also known as lambrolizumab, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT, hBAT-1 or pidilizumab, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0117] In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody can inhibit the binding between PD-L1 and PD-1 and / or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the anti-PD-L1 antibody is a humanized antibody. In some embodiments, the anti-PD-1 antibody is a human antibody.

[0118] Examples of anti-PD-L1 antibodies that are useful in the methods of the invention and methods for making them are described in PCT patent applications WO2010 / 077634, WO2007 / 005874, WO2011 / 066389 and US2013 / 034559, which are incorporated herein by reference. The anti-PD-L1 antibodies that are useful in the invention (including compositions comprising such antibodies) may be used in combination with taxanes to treat cancer.

[0119] Anti-PD-1 antibody In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternative names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558, or nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). In still further embodiments, provided is an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising a heavy chain variable region amino acid sequence from SEQ ID NO: 1 and / or a light chain variable region comprising a light chain variable region amino acid sequence from SEQ ID NO: 2. In still further embodiments, provided is an isolated anti-PD-1 antibody comprising a heavy chain and / or light chain sequence, wherein (a) The heavy chain sequence: QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1); and (b) the light chain sequence: It 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 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

[0120] Anti-PD-L1 antibody In some embodiments, the antibody in the formulation comprises at least one (e.g., at least two, at least three, or at least four) tryptophan in the heavy and / or light chain sequence. In some embodiments, the amino acid tryptophan is in the HVR region, framework region, and / or constant region of the antibody. In some embodiments, the antibody comprises two or three tryptophan residues in the HVR region. In some embodiments, the antibody in the formulation is an anti-PD-L1 antibody. PD-L1 (Programmed Death Ligand 1), also known as PDL1, B7-H1, B7-4, CD274, and B7-H, is a transmembrane protein whose interaction with PD-1 inhibits T cell activation and cytokine production. In some embodiments, the anti-PD-L1 antibodies described herein bind to human PD-L1. Examples of anti-PD-L1 antibodies that can be used within the methods described herein are described in PCT patent application WO2010 / 077634A1 and US8,217,149, which are incorporated herein by reference in their entireties.

[0121] In some embodiments, the anti-PD-L1 antibody is capable of inhibiting the binding between PD-L1 and PD-1 and / or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the anti-PD-L1 antibody is a humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody.

[0122] The anti-PD-L1 antibodies described in WO2010 / 077634A1 and US8,217,149 may be used in the methods described herein. In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region sequence of SEQ ID NO: 3 and / or a light chain variable region sequence of SEQ ID NO: 4. In still further embodiments, provided is an isolated anti-PD-L1 antibody comprising a heavy chain variable region and / or a light chain variable region sequence, wherein: (a) The heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 3), and (b) the light chain sequence: It 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 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

[0123] In one embodiment, the anti-PD-L1 antibody comprises a heavy chain variable region comprising HVR-H1, HVR-H2 and HVR-H3 sequences, wherein: (a) the HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO:5); (b) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 6); (c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 7); Further, wherein X1 is D or G; X2 is S or L; and X3 is T or S. In one particular embodiment, X1 is D; X2 is S and X3 is T.

[0124] In another embodiment, the polypeptide further comprises variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another embodiment, the framework sequences are derived from a human consensus framework sequence. In a further embodiment, the framework sequence is a VH subgroup III consensus framework. In yet a further embodiment, at least one of the framework sequences is as follows: HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8). HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 9) HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 10). HC-FR4 is WGQGTLVTVSA (sequence number: 11).

[0125] In yet a further embodiment, the heavy chain polypeptide is further combined with a light chain variable region comprising HVR-L1, HVR-L2 and HVR-L3, (a) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 12); (b) HVR-L2 sequence is SASX9LX 10 S (SEQ ID NO: 13); (c) HVR-L3 sequence, QQX 11 X 12 X 13 X 14 PX 15 T (SEQ ID NO: 14); where X4 is D or V;X5 is V or I;X6 is S or N;X7 is A or F;X8 is V or L;X9 is F or T;X 10 is Y or A;X 11 is Y, G, F, or S;X 12 is L, Y, F or W;X 13 is Y, N, A, T, G, F or I;X 14 is H, V, P, T or I;X 15is A, W, R, P or T. In still further embodiments, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X 10 is Y;X 11 is Y;X 12 L;X 13 is Y;X 14 H;X 15 is A.

[0126] In still further embodiments, the light chain further comprises a variable region light chain framework sequence juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In still further embodiments, the framework sequence is derived from a human consensus framework sequence. In still further embodiments, the framework sequence is a VL kappa I consensus framework. In still further embodiments, at least one of the framework sequences is as follows: LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15). LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 16) LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYY (SEQ ID NO: 17) LC-FR4 is FGQGTKVEIKR (sequence number 18).

[0127] In another embodiment, provided is an isolated anti-PD-L1 antibody or antigen-binding fragment comprising a heavy chain and a light chain variable region sequence, (a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, further comprising: (i) The HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO: 5) (ii) The HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 6). (iii) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 7), and (b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, further comprising: (i) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 12) (ii) The HVR-L2 sequence is SASX9LX 10 S (SEQ ID NO: 13); and (iii) The HVR-L3 sequence is QQX 11 X 12 X 13 X 14 PX 15 X1 is D or G; X2 is S or L; X3 is T or S; X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; X8 is V or L; X9 is F or T; 10 is Y or A;X 11 is Y, G, F or S;X 12 is L, Y, F or W;X 13 is Y, N, A, T, G, F or I;X 14 is H, V, P, T or I;X 15 is A, W, R, P or T. In one particular embodiment, X1 is D; X2 is S and X3 is T. In another embodiment, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X 10 is Y;X 11 is Y;X 12 L;X 13 is Y;X 14 H;X 15 is A. In yet another embodiment, X1 is D; X2 is S and X3 is T, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X 10 is Y;X 11 is Y;X 12 L;X 13 is Y;X 14 H and X 15 is A.

[0128] In a further embodiment, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs such as (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4) and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs such as (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet a further embodiment, the framework sequences are derived from a human consensus framework sequence. In yet a further embodiment, the heavy chain framework sequence is derived from a Kabat subgroup I, II or III sequence. In yet a further embodiment, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet a further embodiment, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs: 8, 9, 10 and 11. In yet further aspects, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In yet further aspects, the light chain framework sequences are VL kappa I consensus framework. In yet further aspects, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18.

[0129] In yet further particular embodiments, the antibody further comprises a human or mouse constant region. In yet further aspects, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet further particular aspects, the human constant region is IgG1. In yet further aspects, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet further aspects, the mouse constant region is IgG2A. In yet further aspects, the antibody has low or minimal effector function. In yet further aspects, the minimal effector function is the result of an "effectorless Fc mutation" or deglycosylation. In yet further embodiments, the effectorless Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0130] In yet another embodiment, provided is an anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, 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: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20), and RHWPGGFDY (SEQ ID NO: 21), respectively; or (b) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity to RASQDVSTAVA (sequence number 22), SASFLYS (sequence number 23), and QQYLYHPAT (sequence number 24), respectively.

[0131] In certain embodiments, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0132] In another embodiment, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs, such as (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs, such as (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another embodiment, the framework sequences are derived from a human consensus framework sequence. In yet a further embodiment, the heavy chain framework sequence is derived from a Kabat subgroup I, II or III sequence. In yet a further embodiment, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet a further embodiment, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs: 8, 9, 10 and 11. In yet further aspects, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In yet further aspects, the light chain framework sequences are VL kappa I consensus framework. In yet further aspects, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18.

[0133] In yet further particular embodiments, the antibody further comprises a human or mouse constant region. In yet further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet further particular embodiments, the human constant region is IgG1. In yet further embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet further embodiments, the mouse constant region is IgG2A. In yet further particular embodiments, the antibody has low or minimal effector function. In yet further particular embodiments, the minimal effector function is the result of an "effectorless Fc mutation" or deglycosylation. In yet further embodiments, the effectorless Fc mutation is the substitution N297A or D265A / N297A substitution in the constant region.

[0134] In another further embodiment, provided is an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein: (a) The heavy chain sequence: and / or has at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 25); and / or (b) the light chain sequence: It has at least 85% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

[0135] In certain embodiments, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another embodiment, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs, such as (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs, such as (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another embodiment, the framework sequences are derived from human consensus framework sequences. In a further aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet a further aspect, the heavy chain framework sequences are VH subgroup III consensus frameworks. In still a further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NO:8, 9, 10, and WGQGTLVTVSS (SEQ ID NO:27).

[0136] In yet further aspects, the light chain framework sequences are derived from a Kabat kappa I, II, III or IV subgroup sequence. In yet further aspects, the light chain framework sequences are VL kappa I consensus framework. In yet further aspects, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18.

[0137] In yet further particular embodiments, the antibody further comprises a human or mouse constant region. In yet further particular embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet further particular embodiments, the human constant region is IgG1. In yet further particular embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet further particular embodiments, the mouse constant region is IgG2A. In yet further particular embodiments, the antibody has low or minimal effector function. In yet further particular embodiments, the minimal effector function results from production in a prokaryotic cell. In yet further particular embodiments, the minimal effector function results from an "effectorless Fc mutation" or deglycosylation. In yet further embodiments, the effectorless Fc mutation is the substitution N297A or D265A / N297A substitution in the constant region.

[0138] In a further embodiment, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs, such as (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs, such as (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet a further embodiment, the framework sequences are derived from a human consensus framework sequence. In yet a further embodiment, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet a further embodiment, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet a further embodiment, one or more of the heavy chain framework sequences are as follows: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 29) HC-FR2 WVRQAPGKGLEWVA (SEQ ID NO: 30) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 10) HC-FR4 WGQGTLVTVSS (sequence number 27).

[0139] In still further embodiments, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In still further embodiments, the light chain framework sequences are VL kappa I consensus framework. In still further embodiments, one or more of the light chain framework sequences are as follows: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 16) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17) LC-FR4 FGQGTKVEIK (sequence number 28).

[0140] In yet further particular embodiments, the antibody further comprises a human or mouse constant region. In yet further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet further particular embodiments, the human constant region is IgG1. In yet further embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet further embodiments, the mouse constant region is IgG2A. In yet further particular embodiments, the antibody has low or minimal effector function. In yet further particular embodiments, the minimal effector function is the result of an "effectorless Fc mutation" or deglycosylation. In yet further embodiments, the effectorless Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0141] In yet another embodiment, provided is an anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein: (c) the heavy chain further comprises HVR-H1, HVR-H2 and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20), and RHWPGGFDY (SEQ ID NO: 21), respectively; and / or (d) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity to RASQDVSTAVA (sequence number 22), SASFLYS (sequence number 23), and QQYLYHPAT (sequence number 24), respectively.

[0142] In certain embodiments, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0143] In another embodiment, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs, such as (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs, such as (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another embodiment, the framework sequences are derived from a human consensus framework sequence. In yet a further embodiment, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In yet a further embodiment, the heavy chain framework sequences are VH subgroup III consensus frameworks. In yet a further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10, and WGQGTLVTVSSASTK (SEQ ID NO:31).

[0144] In yet further embodiments, the light chain framework sequence is derived from a Kabat kappa I, II, II or IV subgroup sequence. In yet further embodiments, the light chain framework sequence is a VL kappa I consensus framework. In yet further embodiments, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18. In yet further particular embodiments, the antibody further comprises a human or mouse constant region. In yet further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet further particular embodiments, the human constant region is IgG1. In yet further embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet further embodiments, the mouse constant region is IgG2A. In yet further particular embodiments, the antibody has low or minimal effector function. In yet further particular embodiments, the minimal effector function is the result of an "effectorless Fc mutation" or deglycosylation. In yet a further embodiment, the effectorless Fc mutation is the substitution N297A or a D265A / N297A substitution in the constant region.

[0145] In still a further embodiment, provided is an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein: (a) The heavy chain sequence: Has at least 85% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 26); or (b) the light chain sequence: It has at least 85% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

[0146] In some embodiments, provided is an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 amino acid sequence of SEQ ID NO:4. In some embodiments, provided is an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 amino acid sequence of SEQ ID NO:26. In some embodiments, provided is an isolated anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 amino acid sequence of SEQ ID NO:4, and the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 amino acid sequence of SEQ ID NO:26. In some embodiments, 1, 2, 3, 4 or 5 amino acid residues at the N-terminus of the heavy and / or light chain may be deleted, substituted or modified.

[0147] In yet a further embodiment, provided is a polypeptide comprising a heavy chain and a light chain, (a) the heavy chain sequence is EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV and / or has at least 85% sequence identity to FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32); and / or (b) the light chain sequence is the light chain sequence: An isolated anti-PD-L1 antibody, which has at least 85% sequence identity to: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

[0148] In some embodiments, provided is an isolated anti-PD-L1 antibody comprising heavy and light chain sequences, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, provided is an isolated anti-PD-L1 antibody comprising heavy and light chain sequences, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, provided is an isolated anti-PD-L1 antibody comprising heavy and light chain sequences, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:33, and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:32.

[0149] In some embodiments, the isolated anti-PD-L1 antibody is deglycosylated. Glycosylation of the antibody 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 moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of the sugars N-acetylgalactosamine, galactose or xylose to one 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 accomplished by altering the amino acid sequence such that one of the above tripeptide sequences (for N-linked glycosylation sites) is removed. The alteration may be made by substituting an asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (eg, glycine, alanine, or a conservative substitution).

[0150] In any of the embodiments herein, the isolated anti-PD-L1 antibody may bind to human PD-L1, such as the human PD-L1 set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof.

[0151] In yet further embodiments, provided is an isolated nucleic acid encoding any of the antibodies described herein. In some embodiments, the nucleic acid further comprises a vector suitable for expressing the nucleic acid encoding any of the aforementioned anti-PD-L1 antibodies. In yet further particular embodiments, the vector is in a host cell suitable for expressing the nucleic acid. In yet further particular embodiments, the host cell is a eukaryotic or prokaryotic cell. In yet further particular embodiments, the eukaryotic cell is a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell.

[0152] Antibodies or antigen-binding fragments thereof may be produced using methods known in the art, such as by a process which includes culturing a host cell containing nucleic acid encoding any of the above-mentioned anti-PD-L1 antibodies or antigen-binding fragments in a form suitable for expression, under conditions suitable for producing such antibodies or fragments, and recovering the antibody or fragment.

[0153] III. Antibody Preparation The antibodies described herein are prepared using techniques available in the art for antibody production, exemplary methods of which are described in more detail in the following sections.

[0154] The antibody is directed against an antigen of interest (e.g., PD-L1 (e.g., human PD-L1), PD1 (e.g., human PD-L1), PD-L2 (e.g., human PD-L2), etc.). Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to a mammal suffering from a disorder can result in a therapeutic benefit in the mammal.

[0155] In certain embodiments, the antibodies provided herein have a concentration of 1 μM or less, 150 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, 10-9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0156] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the antibody of interest and a Fab version of its antigen, as described by the following assay: The solution binding affinity of the Fab for the antigen is determined by the minimal concentration of ( 125 I) Fab is equilibrated with labeled antigen and then measured by capturing bound antigen using a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multi-well plates (Thermo Scientific) are coated with 5 μg / ml of capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23° C.). In non-adsorbent plates (Nunc #269620), either 100 pM or 26 pMa[ 125 I]-antigen is mixed with serial dilutions of the Fab of interest. The Fab of interest is then incubated overnight, although incubation may continue for longer (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate is dry, 150 μl / well of scintillant (MICROSCINT-20™: Packard) is added and the plate is counted on a TOPCOUNT™ gamma counter (Packard) for 10 minutes. Each Fab that provides a concentration of 20% or less of maximum binding is selected for use in competitive binding assays.

[0157] According to another embodiment, the Kd is measured at 25° C. using a surface plasmon resonance assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip at approximately 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. with a flow rate of approximately 25 μl / min. Association rates (K) were calculated using a single one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. on ) and dissociation rate (K off ) is calculated. The equilibrium dissociation constant (Kd) is K off / K on The association rate is calculated as a ratio of 10 to 10. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). The surface plasmon resonance assay described above demonstrated that the association rate was 10 6 M -1 s -1Above this rate, the association rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured in a spectrometer such as a spectrophotometer equipped with stopped flow (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (Thermo Spectronic) with a stirred cuvette.

[0158] (i) Preparation of antigen Soluble antigens or fragments, optionally conjugated to 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 the transmembrane molecule can be used as immunogens. Such cells can be from natural sources (e.g., cancer cell lines) or can be cells transformed by recombinant techniques to express the transmembrane molecule. Other antigens and forms thereof that are useful for preparing antibodies will be apparent to those skilled in the art.

[0159] (ii) Certain antibody-based methods Polyclonal antibodies are raised in animals, preferably by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant, such as maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR, where R and R 1It 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, using a bifunctional or derivatizing agent such as (wherein x is a different alkyl group).

[0160] Animals are immunized against the antigen, immunogenic conjugate or derivative by combining 3 volumes of complete Freund's adjuvant with, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) and injecting the solution intradermally at multiple sites. One month later, animals are boosted subcutaneously at multiple sites with 1 / 5 to 1 / 10 the original amount of peptide or conjugate in complete Freund's adjuvant. Seven to 14 days later, animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, animals are boosted with a conjugate of the same antigen but conjugated to a different protein and / or through a different cross-linking reagent. Conjugates can also be made in recombinant cell culture as protein fusions. It is also suitable to use an aggregating agent such as alum to enhance the immune response.

[0161] The monoclonal antibodies of the invention can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975) and further described for human-human hybridomas 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 T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 for the production of monoclonal human native IgM antibodies from hybridoma cell lines. Human hybridoma technology (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).

[0162] For various hybridoma technologies, see, for example, U.S. Patent Application Publication Nos. 2006 / 258841; 2006 / 183887 (fully human antibodies), 2006 / 059575; 2005 / 287149; 2005 / 100546; and 2005 / 026229; and U.S. Patent Nos. 7,078,492 and 7,153,507. An exemplary protocol for producing monoclonal antibodies using hybridoma technology is described as follows. In one embodiment, a mouse or other suitable host animal, such as a hamster, is immunized to elicit lymphocytes that produce or have the capacity to produce antibodies that specifically bind to the protein used for immunization. Antibodies are produced in animals by multiple subcutaneous (SC) or intraperitoneal (IP) injections of the polypeptide of the invention or a fragment thereof and an adjuvant such as monophosphoryl lipid A (MPL) / trehalose diclinomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT). The polypeptide (e.g., antigen) of the invention or a fragment thereof may be prepared by methods well known in the art, such as recombinant methods, some of which are further described herein. Serum from the immunized animal is assayed for anti-antigen antibodies, and optional booster immunizations are performed. Lymphocytes from the animal producing anti-antigen antibodies are isolated. Alternatively, lymphocytes may be immunized in vitro.

[0163] The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. See, e.g., Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma cells can be used that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium, such as HAT medium. Exemplary myeloma cells include, but are not limited to, mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, 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 have also been 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)).

[0164] The hybridoma cells thus prepared are seeded and grown in a suitable medium, e.g., a medium 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 the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells. Preferably, a serum-free hybridoma cell culture method is used to reduce the use of animal-derived serum, such as fetal bovine serum, as described, e.g., in Even et al., Trends in Biotechnology, 24(3), 105-108 (2006).

[0165] Oligopeptides as a means to improve the productivity of hybridoma cell cultures are described in Franek, Trends in Monoclonal Antibody Research, 111-122 (2005). Specifically, standard media can be enriched with certain amino acids (alanine, serine, asparagine, proline) or protein hydrolysate fractions, and apoptosis can be significantly inhibited with synthetic oligopeptides consisting of 3-6 amino acid residues. The peptides are present at millimolar or higher concentrations.

[0166] The culture medium in which the hybridoma cells are growing may be assayed for the production of monoclonal antibodies that bind to the antibody of the present invention. The binding specificity of the monoclonal antibodies produced by the hybridoma cells may be determined by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) or by immunoprecipitation. The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis. See, e.g., Munson et al., Anal. Biochem., 107:220 (1980).

[0167] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity and / or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. See, e.g., Goding, supra. Suitable media for this purpose include, e.g., D-MEM or RPMI-1640 medium. Additionally, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, e.g., protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. One procedure for isolation of proteins from hybridoma cells is described in US2005 / 176122 and US Patent No. 6,919,436. The method includes using minimal salts, such as lyotropic salts, in the binding process, and preferably also using small amounts of organic solvents in the elution process.

[0168] (iii) Library-derived antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Additional methods are reviewed, e.g., in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, e.g., McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0169] In one particular phage display method, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The phage display antibody fragments either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single antibody source against a wide range of non-self antigens, as well as self antigens, without any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode highly variable CDR3 regions, and performing 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 Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0170] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0171] (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. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or a non-human primate, such as a monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. The chimeric antibody includes an antigen-binding fragment thereof.

[0172] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. A humanized antibody generally comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. A humanized antibody optionally also comprises 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 the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0173] Humanized antibodies and methods for their production are reviewed in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0174] Human framework regions that may be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al. al., J. Biol. Chem. 271:22611-22618 (1996)).

[0175] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0176] Human antibodies may be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus or is extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally already inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. US2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example by combining with a different human constant region.

[0177] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See 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). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005)).

[0178] Human antibodies may be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences may then be combined with the desired human constant regions. Techniques for human antibody library selection are described below.

[0179] (v) antibody fragment Antibody fragments may be produced by conventional means such as enzymatic digestion or by recombinant techniques. In certain circumstances, it is advantageous to use antibody fragments rather than whole antibodies. The relatively small size of fragments may allow for rapid clearance and lead to improved access to solid tumors. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

[0180] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via 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 by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted by E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from the host cell culture. Fab and F(ab')2 fragments with increased in vivo half-life containing salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See, e.g., WO 93 / 16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv are the only species with intact combining sites that are devoid of constant regions; therefore, they may be suitable for low non-specific binding during in vivo use. scFv fusion proteins can be constructed to produce fusion of effector proteins at either the amino or carboxy terminus of the scFv. See Antibody Engineering, ed. Borrebaeck, supra. An antibody fragment may also be a "linear antibody," for example, as described in U.S. Patent No. 5,641,870. Such linear antibodies may be monospecific or bispecific.

[0181] (vi) Multispecific antibodies Multispecific antibodies have binding specificities for at least two different epitopes, where the epitopes usually originate from different antigens. Such molecules usually bind to two different epitopes (i.e., bispecific antibodies, BsAbs), although as used herein, the term also encompasses antibodies with additional specificities, such as trispecific antibodies. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0182] Methods for making bispecific antibodies are known in the art. 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 (see, for example, Millstein et al., Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually performed by affinity chromatography steps, is quite laborious and the product yield is low. Similar procedures are disclosed in WO93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0183] One approach known in the art for making bispecific antibodies is the "knob-into-hole" or "protuberance-into-cavity" approach (see, e.g., U.S. Pat. No. 5,731,168). In this approach, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each contain an interface. An interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide, thus allowing the two immunoglobulin polypeptides to associate. These interfaces may be engineered such that a "knob" or "protuberance" (these terms may be used interchangeably herein) in the interface of one immunoglobulin polypeptide corresponds to a "hole" or "cavity" (these terms may be used interchangeably herein) in the interface of the other immunoglobulin polypeptide. In some embodiments, the holes are of the same or similar size as the knobs and are suitably positioned such that when the two interfaces interact, a knob of one interface can be positioned within a corresponding hole of the other interface. Without wishing to be bound by theory, it is believed that this favors stabilizing heteromultimers and favoring their formation over other species, such as homomultimers. In some embodiments, this approach may be used to promote heteromultimerization of two different immunoglobulin polypeptides to create bispecific antibodies comprising two immunoglobulin polypeptides with different epitope binding specificities.

[0184] In some embodiments, knobs may be constructed by replacing small amino acid side chains with larger side chains. In some embodiments, holes may be constructed by replacing large amino acid side chains with smaller side chains. The knobs or holes may be present in the original interface or may be synthetically introduced. For example, knobs or holes may be synthetically introduced by modifying 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 modifying nucleic acid sequences may include standard molecular biology techniques well known in the art. The side chain volumes of various amino acid residues are shown in the table below. In some embodiments, the original residues have small side chain volumes (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and the import residues to form the knobs are naturally occurring amino acids and may include arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the original residue has a large side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan) and the import residue to form the hole may be a naturally occurring amino acid that includes alanine, serine, threonine, and valine.

[0185] Table 1: Characteristics of amino acid residues TIFF2025020147000002.tif176170 a The molecular weight of the amino acid minus the molecular weight of water. The source of the value is Handbook of Chemistry and Physic, 43 th ed.Cleveland,Chemical Rubber Publishing Co.,1961. b Figures are from AAZamyatnin, Prog. Biophys. Mol. Biol. 24:107~123, 1972. cValues ​​are taken from C. Chothia, J. Mol. Biol. 105:1-14, 1975. Accessible surface area is defined in Figures 6-20 of this reference.

[0186] In some embodiments, the original residues for forming the knob or hole are identified based on the three-dimensional structure of the heteromultimer. Techniques known in the art for obtaining the three-dimensional structure may include X-ray crystallography and NMR. In some embodiments, the interface is the CH3 domain of an immunoglobulin constant domain. In these embodiments, the CH3 / CH3 interface of human IgG1 involves 16 residues on each domain located on four antiparallel β-strands. Without wishing to be bound by theory, the mutated residues are preferably located on the two central antiparallel β-strands to minimize the risk that the knob may be accommodated by the surrounding solvent rather than a compensatory hole in the partner CH3 domain. In some embodiments, the corresponding knob- and hole-forming mutations in the two immunoglobulin polypeptides correspond to one or more pairs shown in the table below.

[0187] Table 2. Exemplary sets of corresponding knob- and hole-forming mutations JPEG2025020147000003.jpg66154Mutations are represented by the original residue, followed by its position using the Kabat numbering system, and then the import residue (all residues are provided in single-letter amino acid codes). Multiple mutations are separated by colons.

[0188] In some embodiments, the immunoglobulin polypeptide comprises a CH3 domain that comprises one or more amino acid substitutions listed above in Table 2. In some embodiments, the bispecific antibody comprises a first immunoglobulin polypeptide that comprises a CH3 domain that comprises one or more amino acid substitutions listed in the left column of Table 2, and a second immunoglobulin polypeptide that comprises a CH3 domain that comprises one or more corresponding amino acid substitutions listed in the right column of Table 2.

[0189] After DNA mutation as described above, polynucleotides encoding modified immunoglobulin polypeptides with one or more corresponding knob- or hole-forming mutations can be expressed and purified using standard recombinant techniques and cell systems known in the art. See, e.g., U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; U.S. Patent Application Publication No. 2013 / 0089553; and Spiess et al., Nature Biotechnology 31:753-758, 2013. Modified immunoglobulin polypeptides are produced using prokaryotic host cells such as E. coli or eukaryotic host cells such as CHO cells. Corresponding knob- and hole-bearing immunoglobulin polypeptides may be expressed and co-purified as heteromultimers in host cells in co-culture, or they may be expressed in a single culture, purified separately, and assembled in vitro. In some embodiments, two strains of bacterial host cells, one expressing an immunoglobulin polypeptide with a knob and one expressing an 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 the culture. In some embodiments, the two strains may be mixed in a ratio of 50:50, 60:40, or 70:30. After polypeptide expression, the cells may be lysed together and proteins may be extracted. Standard techniques known in the art that allow for measuring the abundance of homomultimeric versus heteromultimeric species may include size exclusion chromatography. In some embodiments, each modified immunoglobulin polypeptide may be expressed separately using standard recombinant techniques and assembled together in vitro, for example by purifying each modified immunoglobulin polypeptide, mixing and incubating them in equal masses, reducing disulfides (e.g., by treatment with dithiothreitol), concentrating, and re-oxidizing the polypeptides.The bispecific antibodies formed may 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. 31:753-8, 2013. In some embodiments, the modified immunoglobulin polypeptides may be expressed separately in CHO cells and assembled in vitro using the methods described above.

[0190] According to a different approach, antibody variable domains (antibody-antigen combining sites) with the desired binding specificities are fused to immunoglobulin constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain, including at least a portion of the hinge, CH2 and CH3 regions. It is typical to have the first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one fusion. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This allows great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields. 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 equal ratios results in higher yields or when the ratio is of no particular importance.

[0191] In one embodiment of this approach, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate separation of the desired bispecific compound from undesired immunoglobulin chain combinations, since the presence of immunoglobulin light chains in only one half of the bispecific molecule provides a facile separation method. This approach is disclosed in WO94 / 04690. For further details of the generation of bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology, 121:210 (1986).

[0192] 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 which are recovered from recombinant cell culture. One interface is the C H The antibody comprises at least a portion of the three domains. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain(s) is created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other undesired end products such as homodimers.

[0193] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (WO91 / 00360, WO92 / 200373 and EP03089). Heteroconjugate antibodies may be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0194] Techniques for generating bispecific antibodies from antibody fragments are also disclosed in the literature. For example, bispecific antibodies can be prepared using chemical coupling. Brennan et al., Science, 229:81 (1985) describe a procedure in which intact antibodies are proteolytically split to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then converted back to a Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The generated bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0195] Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe the production of fully humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment is separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

[0196] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were attached to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provided an alternative mechanism for making bispecific antibody fragments. The fragments consist of a light chain variable domain (V) separated by a linker that is too short to allow pairing between the two domains on the same chain. L ) linked to a heavy chain variable domain (V H ) in one fragment. H and V L The domain is the complementary V of another fragment. L and V H The domains are forced to pair to form two antigen-binding sites. Another strategy for making bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152:5368 (1994).

[0197] Another technique for making bispecific antibody fragments is the "bispecific T cell engager" or BiTE® approach (see, e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261 and WO2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, the single polypeptide chain may comprise a variable heavy chain (VLC) and a variable heavy chain (VH) separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. H ) and variable light chain (V L The single polypeptide further comprises two single chain Fv (scFv) fragments each having a CD4+ domain. The single polypeptide further comprises a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, which may be specific for a different cell type, and thus cells of two different cell types are brought into close proximity or tethered when each scFv is engaged with its cognate epitope. One particular embodiment of this approach includes an scFv that recognizes a cell surface antigen expressed by an immune cell, such as a CD3 polypeptide on a T cell, bound to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell.

[0198] Being a single polypeptide, the bispecific T cell engager may be expressed using any prokaryotic or eukaryotic expression system known in the art, such as, for example, CHO cell lines. However, specific purification techniques (see, for example, EP1691833) may be necessary to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted using an imidazole concentration gradient. The eluate is further purified using anion exchange chromatography, and the polypeptide is eluted using a sodium chloride concentration gradient. Finally, the eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species.

[0199] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147:60 (1991).

[0200] (vii) single domain antibody In some embodiments, the antibody of the present invention is a single domain antibody. A single domain antibody is a single polypeptide chain that includes all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In one embodiment, a single domain antibody is composed of all or a portion of the heavy chain domain of an antibody.

[0201] (viii) Antibody variants In some embodiments, modification(s) of the amino acid sequence of the antibodies described herein are contemplated. For example, it may be desired to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may 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 into, 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 properties. Amino acid alterations may be introduced into the antibody amino acid sequence of interest as the sequence is generated.

[0202] (ix) Substitution, insertion and deletion mutants In certain embodiments, antibody variants are provided that have 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 changes are provided in Table 1 under the heading of "exemplary substitutions" and as further described below in relation to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0203] Table 3. Example substitutions JPEG2025020147000004.jpg77127JPEG2025020147000005.jpg30127

[0204] Amino acids may be grouped according to common side chain properties: 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 affect chain orientation: Gly, Pro; f. Aromatics: Trp, Tyr, Phe.

[0205] Non-conservative substitutions involve the exchange of a member of one of these classes for another class.

[0206] One type of substitutional variant involves the substitution of one or more hypervariable regions of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study are those that have a modification (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or that substantially retain certain biological properties of the parent antibody. One exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated 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).

[0207] For example, to improve antibody affinity, modifications (e.g., substitutions) may be made in HVRs, i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or within the SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. (Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves a HVR-directed approach, in which multiple HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often specifically targeted.

[0208] Substitutions, insertions or deletions may be made within one or more HVRs, so long as, in certain embodiments, the modifications do not substantially reduce the ability of the antibody to bind antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made within the HVRs. Such modifications may be outside of HVR "hot spots" or SDRs. In certain embodiments of the above-mentioned variant VH and VL sequences, each HVR is either unmodified or contains no more than one, two or three amino acid substitutions.

[0209] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of targeted residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether they result in antigen-antibody interaction. Further substitutions may be introduced at amino acid locations that show functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of an antigen-antibody complex identifies contact points between the antibody and the antigen. Such contact and adjacent residues are targeted or removed as substitution candidates. The mutants may be screened to determine whether they contain the desired properties.

[0210] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0211] (x) Glycosylation mutants In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. 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 removed.

[0212] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells generally comprise branched, biantennary oligosaccharides attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may comprise a variety of carbohydrates, such as mannose, N-acetylglucosamide (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention may be made to create antibody variants with certain improved properties.

[0213] In one embodiment, antibody variants comprising an Fc region are provided that have reduced or absent fucose in the carbohydrate structure attached to the Fc region, which may improve ADCC function. Specifically contemplated herein are antibodies with reduced fucose compared to the amount of fucose on the same antibody produced in wild-type CHO cells. That is, they are characterized as having a lower amount of fucose than would be present if produced by a native CHO cell (e.g., a CHO cell that produces a native glycosylation pattern, e.g., a CHO cell that contains a native FUT8 gene). In certain embodiments, the antibody is one in which less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans thereon contain fucose. For example, the amount of fucose in such antibodies may be between 1% and 80%, between 1% and 65%, between 5% and 65%, or between 20% and 40%. In certain embodiments, the antibody is one in which none of the N-linked glycans thereon contain fucose, i.e., the antibody is completely fucose-free, does not contain any fucose at all, or is afucosylated. The amount of fucose is determined by calculating the average amount of fucose within the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. By Asn297 is meant the asparagine residue at about position 297 (EU numbering of Fc region residues) within the Fc region, although due to minor sequence variations within antibodies, Asn297 may also be located about ±3 amino acid positions upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. US2003 / 0157108 (Presta, L.); U.S. Patent Application Publication No. US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 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 Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108 A1; and WO2004 / 056312 A1, especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout 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 WO2003 / 085107).

[0214] Further provided are antibody variants with bisected oligosaccharides, e.g., where the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucose content and / or improved ADCC function. Examples of such antibody variants are described, e.g., in WO2003 / 011878; U.S. Pat. No. 6,602,684; US2005 / 0123546, and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO1997 / 30087; WO1998 / 58964; and WO1999 / 22764.

[0215] In certain embodiments, the antibody variants comprising the Fc region described herein are capable of binding to FcγRIII. In certain embodiments, the antibody variants comprising the Fc region described herein have ADCC activity in the presence of human effector cells and have increased ADCC activity in the presence of human effector cells compared to an otherwise identical antibody comprising a human wild-type IgG1 Fc region.

[0216] (xi) Fc domain mutant In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0217] In certain embodiments, the present invention contemplates antibody variants that have some, but not all, effector functions, making them desirable candidates for applications where the half-life of the antibody in vivo is important, but where some effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcyR binding (and thus likely lacks ADCC activity) but retains FcRn binding activity. NK cells, the primary cells for mediating ADCC, express only FcyRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be utilized (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® 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 or additionally, the ADCC activity of the molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be performed to confirm that the antibody cannot bind to C1q and thus lacks CDC activity. See, for example, C1q and C3C binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg et al., Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can be performed using methods known in the art (see, e.g., Petkova et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0218] Antibodies with reduced effector function include antibodies with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at one or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0219] Certain antibody variants have been described with improved or diminished binding to FcRs (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)).

[0220] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at Fc region positions 298, 333 and / or 334 (EU numbering of residues). In an exemplary embodiment, the antibody comprises the following amino acid substitutions in the Fc region: S298A, E333A and K334A.

[0221] In some embodiments, modifications are made within the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0222] Antibodies with increased half-lives 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)), have been described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0223] (xii) Antibody derivatives The antibodies of the present invention may be further modified to include additional non-proteinaceous moieties that are known and readily available in the art. In certain embodiments, the moieties suitable for derivatization of the antibodies are water-soluble polymers. 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 (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used under defined conditions in a therapy, etc.

[0224] (xiii) Vectors, host cells and recombination methods Antibodies can also be produced using recombinant methods. For recombinant production of anti-antigen antibodies, nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding the antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0225] (a) Signal sequence component The antibodies of the invention may be produced recombinantly directly, but also as fusion polypeptides with heterologous polypeptides, preferably other polypeptides having a signal sequence or a specific cleavage at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed by the host cell (e.g., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeast secretion, the native signal sequence can be replaced, for example, by the yeast invertase leader, factor leader (including the Saccharomyces and Kluyveromyses α-factor leaders) or acid phosphatase leader, the C. albicans glucoamylase leader, or a 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, are available.

[0226] (b) Origin of replication Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are suitable for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).

[0227] (c) Selected Gene Constituents Expression and cloning vectors may contain a selection gene, also called a selectable marker. Typical selection genes (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline, (b) complement an auxotrophic deficiency, or (c) encode a protein that supplies an essential nutrient not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

[0228] One example of a selection scheme utilizes drugs to arrest the growth of host cells. Those cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

[0229] Further examples of suitable selectable markers for mammalian cells are those that allow the identification of cells competent to take up the antibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein I and II, preferably a primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, and the like.

[0230] For example, cells transformed with the DHFR gene are identified by culturing the transformants in 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) may be used.

[0231] Alternatively, cells transformed with the GS gene are identified by culturing the transformants in medium containing the GS inhibitor L-methionine sulfoximine (Msx). Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acids. The GS selection / amplification system may also be used in combination with the DHFR selection / amplification system described above.

[0232] Alternatively, host cells transformed or co-transformed with DNA sequences encoding the antibody of interest, a wild-type DHFR gene and another selectable marker, such as an aminoglycoside 3'-phosphotransferase (APH), (particularly wild-type hosts containing endogenous DHFR), can be selected by growing the cells in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

[0233] A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trp1 gene provides a selection marker for mutant yeast strains lacking the ability to grow in tryptophan, e.g., ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1 lesion in the yeast host cell genome 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) are complemented by known plasmids carrying the Leu2 gene.

[0234] Additionally, vectors derived from the 1.6 μm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale production of recombinant calf chymosin for K. lactis has been reported. Van den Berg, Bio / Technology, 8:135 (1990). Stable multicopy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio / Technology, 9:968-975 (1991).

[0235] (d) Promoter components Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the antibody. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoter, 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 also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the antibody.

[0236] For eukaryotes, promoter sequences are known. Virtually all eukaryotes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated. Another sequence found 70-80 bases upstream from the transcription start site of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be a signal for addition of a poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

[0237] Examples of suitable promoter sequences for use with yeast hosts include promoters for 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, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0238] Other yeast promoters that are inducible promoters with the added advantage of transcription controlled 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 use in yeast expression are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.

[0239] Antibody transcription from a vector in a mammalian host cell can be controlled by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, Simian Virus 40 (SV40), or from heterologous mammalian promoters, such as the actin promoter or immunoglobulin promoters, heat shock promoters, provided that such promoters are compatible with the host cell system.

[0240] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,44. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) on expression of human β-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

[0241] (e) Components of enhancer elements Transcription of DNA encoding the antibody 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, α-fetoprotein, and insulin). Typically, however, enhancers from eukaryotic viruses are 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. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced ​​into the vector at a position 5' or 3' to the antibody coding sequence, but is preferably located at a site 5' from the promoter.

[0242] (f) Transcription termination component Expression vectors used in eukaryotic host cells (nucleated cells from yeast, fungi, insects, plants, animals, humans or other multicellular organisms) also contain sequences necessary for the termination of transcription and stabilization of the mRNA. Such sequences are commonly available from the 5' and, occasionally, 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain 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 WO94 / 11026 and the expression vectors disclosed therein.

[0243] (g) Selection and transformation of host cells Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotes, yeast or higher eukaryotes described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive microorganisms, such as Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescans, and Shigella, as well as Bacillus, for example, B. subtilis and B. licheniformis (e.g., B. licheniformis 41P, disclosed in DD 266,710 published April 12, 1989), Pseudomonas, for example, P. aeruginosa and Streptomyces. One 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 rather than limiting.

[0244] Full-length antibodies, antibody fusion proteins and antibody fragments can be produced in bacteria, especially when glycosylation and Fc effector functions are not required, for example when the therapeutic antibody is conjugated to a cytotoxic agent (such as a toxin) that is itself effective in destroying tumor cells. 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, for example, 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 describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion. See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes expression of antibody fragments in E. coli. After expression, the antibody may be isolated from the E. coli cell paste in a soluble fraction and can be purified, for example, through a protein A or G column depending on the isotype. Final purification can be carried out similarly to the process for purifying antibodies expressed, for example, in CHO cells.

[0245] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species and strains are also known to be effective against Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, e.g. Schwanniomyces and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger, are commonly available and useful herein. For a review discussing the use of yeast and filamentous fungi for the production of therapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004).

[0246] Certain fungal and yeast strains may be selected in which the glycosylation pathway has been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describing humanization of the human glycosylation pathway in Pichia pastoris); and Gerngross et al., supra.

[0247] Suitable host cells for the 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 have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (snake moth, larvae), Aedes aegypti (Aedes aegypti, mosquito), Aedes albopictus (Aedes albopictus, mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Various virus strains for transfection are publicly available, for example Autographa californica NPV and Bombyx mori NPV Bm-5 strain, and such viruses may be used as viruses herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0248] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0249] As hosts, vertebrate cells may be used, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); human embryonic kidney line (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 CCL10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2). Other useful mammalian host cell lines include DHFR - These include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268.

[0250] Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0251] (h) Cultivation of host cells The host cells used to produce the antibody of the present invention may be cultured in a variety of media. For the culture of the host cells, commercially available 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. In addition, the culture media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Any of the media described in U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. Reissue No. 30,985 may be used as the culture medium for the host cells. Any of these media may optionally contain hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride), and / or other growth factors (e.g., sodium chloride). The culture medium may be supplemented with salts of various nutrients (e.g., thorium, calcium, magnesium and phosphates), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™ drug), trace elements (defined as inorganic compounds normally present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements that would be known to one of skill in the art may also be included. Culture conditions such as temperature, pH, etc. are those conventionally used with the host cell selected for expression and will be apparent to one of skill in the art.

[0252] (xiv) Purification of antibodies When using recombinant techniques, antibodies can be produced intracellularly in the periplasmic space or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cellular debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0253] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography typically being one of the preferred purification steps. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. To purify antibodies that are based on human γ1, γ2, or γ4 heavy chains, Protein A can be used (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). For all mouse isotypes and human γ3, Protein G is recommended (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody is C H If three domains are involved, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE™ chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the antibody to be recovered.

[0254] Generally, a variety of methods for preparing antibodies in research, testing and clinical use are well established in the art, consistent with the methods described above and / or as deemed appropriate by the skilled artisan for a particular antibody of interest.

[0255] (xv) Selection of biologically active antibodies The antibodies produced as described above can be subjected to one or more "biological activity" assays to select antibodies with beneficial properties from a therapeutic standpoint, or to select formulations and conditions that preserve the biological activity of the antibody. Antibodies can be tested for their ability to bind the antigen for which they were produced. For example, methods known in the art (e.g., ELISA, Western Blot, etc.) may be used.

[0256] For example, for anti-PD-L1 antibodies, the antigen-binding properties of the antibody can be assessed in assays that detect its ability to bind to PD-L1. In some embodiments, antibody binding may be determined, for example, by saturation binding; ELISA; and / or competitive assays (e.g., RIA). The antibody may also be subjected to other biological activity assays, for example to evaluate its effectiveness as a therapeutic agent. Such assays are known in the art and depend on the target antigen and intended use of the antibody. For example, the biological effects of antibody-mediated PD-L1 blockade can be assessed in CD8+ T cells, lymphocytic chorioperiostitis virus (LCMV) mouse models, and / or syngeneic tumor models, as described in U.S. Pat. No. 8,217,149.

[0257] To screen for antibodies that bind to a particular epitope on the antigen of interest (e.g., that block the binding of an example anti-PD-L1 antibody to PD-L1), one can perform routine cross-blocking assays, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping as described in Champe et al., J. Biol. Chem. 270:1388-1394 (1995) can be performed to determine whether an antibody binds to an epitope of interest.

[0258] IV. Pharmaceutical Compositions and Formulations Also provided herein are pharmaceutical compositions and formulations comprising a PD-1 axis binding antagonist and / or antibody (e.g., an anti-PD-L1 antibody) described herein and a pharma- ceutical acceptable carrier. The invention also provides pharmaceutical compositions and formulations comprising a taxane, such as nab-paclitaxel (ABRAXANE®), paclitaxel, or docetaxel.

[0259] The pharmaceutical compositions and formulations described herein can be prepared by mixing the active ingredient (e.g., a PD-1 axis binding antagonist and / or a taxane) having the desired purity in the form of a lyophilized formulation or an aqueous solution with one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers, such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of suitable pharmacopoeitic carriers include, but are not limited to, glycines, 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 non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacopoeitic carriers herein further include interstitial drug dispersion agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0260] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, which formulations include a histidine-acetate buffer.

[0261] The compositions and formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. Such active ingredients are preferably present in a combination in amounts that are effective for the intended purpose.

[0262] The active ingredient may be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in microcapsules prepared, for example, by droplet formation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0263] Sustained release preparations can also be prepared.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibody, which matrices are in the form of shaped articles, such as films or microcapsules.The preparations to be used for in vivo administration are generally sterile.Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

[0264] IV. Treatment method Provided herein are methods of treating cancer or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and a taxane (e.g., nab-paclitaxel (ABRAXANE®) or paclitaxel). In some embodiments, the treatment results in a response in the individual following treatment. In some embodiments, the response is a complete remission. In some embodiments, the treatment results in a sustained remission in the individual following cessation of treatment. The methods described herein can be used in treating conditions where enhanced immunogenicity is desired, such as increasing tumor immunogenicity for cancer treatment. Also provided herein are methods of enhancing immune function in an individual with cancer, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and a taxane (e.g., nab-paclitaxel (ABRAXANE®) or paclitaxel). Any of the PD-1 axis binding antagonists and taxanes known in the art and described herein may be used within the methods. In some embodiments, the methods further include administering a platinum-based chemotherapeutic agent. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin.

[0265] In some embodiments, the individual is a human. In some embodiments, the individual suffers from cancer. In some embodiments, the cancer is breast cancer (e.g., triple-negative breast cancer), bladder cancer (e.g., UBC, MIBC, and NMIBC), colorectal cancer, rectal cancer, lung cancer (non-small cell lung cancer, which may be squamous or non-squamous), glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma (e.g., RCC), prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, gastric cancer, esophageal cancer, prostate cancer, endometrial cancer, renal cancer, ovarian cancer, mesothelioma, and hemo malignancies (e.g., MDS and multiple myeloma). In some embodiments, the cancer is selected from small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma. In certain embodiments, the cancer is selected from among lung cancer (including non-small cell lung cancer, which may be squamous or non-squamous), bladder cancer (e.g., UBC), breast cancer (e.g., TNBC), RCC, melanoma, colorectal cancer, and hemolytic malignancies (e.g., MDS) and multiple myeloma. In some embodiments, the lung cancer is non-small cell lung cancer, which may be squamous or non-squamous. In some embodiments, the bladder cancer is UBC. In some embodiments, the breast cancer is TNBC. In some embodiments, the hemolytic malignancies are MDS or multiple myeloma.

[0266] In some embodiments, the individual has been treated with a cancer therapy prior to combination therapy with a PD-1 axis binding antagonist and a taxane. In some embodiments, the individual has a cancer that is resistant to one or more cancer therapies. In some embodiments, resistance to cancer therapy includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer at the original site or at a new site after treatment. In some embodiments, resistance to cancer therapy includes progression of cancer during treatment with an anti-cancer therapy. In some embodiments, resistance to cancer therapy includes cancer that does not respond to treatment. The cancer may be resistant at the beginning of treatment or may acquire resistance during treatment. In some embodiments, the cancer is an early stage cancer or a late stage cancer.

[0267] In some embodiments, the combination therapy of the invention comprises administration of a PD-1 axis binding antagonist and a taxane. The PD-1 axis binding antagonist and the taxane (e.g., nab-paclitaxel (ABRAXANE®) or paclitaxel) may be administered in any suitable manner known in the art. For example, the PD-1 axis binding antagonist and the taxane may be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the PD-1 axis binding antagonist is in a separate composition from the taxane. In some embodiments, the PD-1 axis binding antagonist is in the same composition as the taxane.

[0268] The PD-1 axis binding antagonist and the taxane may be administered by the same or different routes of administration. In some embodiments, the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the taxane is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intracerebroventricularly, or intranasally. An effective amount of the PD-1 axis binding antagonist and the taxane may be administered to prevent or treat a disease. The appropriate dosage of the PD-1 axis binding antagonist and / or the taxane may be determined based on the type of disease to be treated, the type of PD-1 axis binding antagonist and the taxane, the severity and course of the disease, the individual's clinical condition, the individual's clinical history, and response to treatment, and the discretion of the attending physician.

[0269] As a general proposition, a therapeutically effective amount of an antibody (e.g., an anti-PD-L1 antibody) administered to a human is in the range of about 0.01 to about 50 mg / kg of patient body weight, whether administered once or more times. In some embodiments, the antibody used is about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg, for example, administered once a day. In some embodiments, the antibody is administered at a rate of 15 mg / kg. However, other dosing regimens may be useful. In one embodiment, the anti-PD-L1 antibodies described herein are administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg on day 1 of a 21-day cycle. In some embodiments, the anti-PD-L1 antibody MPDL3280A is administered IV (intravenously) at a rate of 1200 mg once every three weeks (q3w). This dose may be administered as a single dose or as multiple doses (e.g., two or three doses), such as by infusion. The dose of the antibody administered in the combination therapy may be reduced compared to a single therapy. The progress of this therapy is easily monitored by conventional techniques.

[0270] As a general proposition, a therapeutically effective amount of a taxane (e.g., nab-paclitaxel (ABRAXANE®) or paclitaxel) administered to a human is about 25 to about 300 mg / m, regardless of whether it is administered once or more than once. 2 (For example, about 25 mg / m 2 , about 50mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 125mg / m 2 , about 150mg / m 2, about 175mg / m 2 , about 200mg / m 2 , about 225mg / m 2 , about 250mg / m 2 , about 275mg / m 2 , or about 300 mg / m 2 For example, in some embodiments, the range is about 100 mg / m 2 of nab-paclitaxel (ABRAXANE®) is administered once weekly (q1w). 2 Nab-paclitaxel (ABRAXANE®) is administered IV at a rate of about 200 mg / m 2 In some embodiments, 200 mg / m baclitaxel is administered every three weeks. 2 Paclitaxel is administered IV at a rate of: In some embodiments, a taxane (e.g., nab-paclitaxel (ABRAXANE®) or paclitaxel) may be administered weekly, every two weeks, every three weeks, or every four weeks on days 1, 8, and 15 of each 21 day cycle, or on days 1, 8, and 15 of each 28 day cycle.

[0271] In some embodiments, the method may further include an additional therapy. The additional therapy may 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 above. The additional therapy may be in the form of an adjuvant or nanoadjuvant therapy. In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (an agent intended to reduce the occurrence and / or severity of side effects of the treatment, such as an antiemetic agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PI3K / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy may be one or more of the chemotherapeutic agents described herein.

[0272] In some embodiments, the method further comprises administering a platinum-based chemotherapy agent together with the PD-1 axis binding antagonist and the taxane. In some embodiments, the platinum-based chemotherapy agent is carboplatin. The dosage and administration of carboplatin are well known in the art. An exemplary dosage of carboplatin is administered at a target area under the curve (AUC) of 6mg / ml. In some embodiments, carboplatin is administered intravenously once every three weeks.

[0273] In some embodiments, the methods include administering the anti-PD-L1 antibody MPDL3280A at a rate of 1200 mg IV once every three weeks (q3w) and nab-paclitaxel (ABRAXANE®) at 100 mg / m once a week (q1w). 2and carboplatin administered IV every three weeks (q3w) at a target AUC of 6 mg / ml. In some embodiments, the methods include the anti-PD-L1 antibody MPDL3280A administered IV at a rate of 1200 mg once every three weeks (q3w) and paclitaxel administered IV at 200 mg / ml once every three weeks. 2 and carboplatin given IV once every 3 weeks (q3w) with a target AUC of 6 mg / ml.

[0274] V. Other Combination Therapies Also provided herein are methods for treating or delaying the progression of cancer in an individual, comprising administering to the individual a human PD-1 axis binding antagonist and a taxane in combination with another anti-cancer agent or cancer therapy, hi some embodiments, the methods comprise administering to the individual a human PD-1 axis binding antagonist, a taxane, and a platinum-based chemotherapeutic agent in combination with another anti-cancer agent or cancer therapy.

[0275] In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with chemotherapy or a chemotherapeutic agent. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with radiation therapy or a radiation therapy agent. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with a targeted therapy or a targeted therapy agent. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with immunotherapy or an immunotherapy agent, such as a monoclonal antibody.

[0276] Without wishing to be bound by theory, it is believed that enhancing T cell stimulation by activating costimulatory molecules or inhibiting negative costimulatory molecules promotes tumor cell death, thus treating or slowing the progression of cancer. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an agonist directed against an activating costimulatory molecule. In some embodiments, the activating costimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist directed against an activating costimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an antagonist directed against an inhibitory costimulatory molecule. In some embodiments, inhibitory costimulatory molecules may include CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase. In some embodiments, antagonists directed against inhibitory costimulatory molecules are antagonist antibodies that bind to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase.

[0277] In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an antagonist, such as a blocking antibody, directed against CTLA-4 (also known as CD152). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with ipilimumab (also known as MDX-010, MDX-101, or YERVOY®). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with tremelimumab (also known as ticilimumab or CP-675,206). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an antagonist, such as a blocking antibody, directed against B7-H3 (also known as CD276). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with MGA271. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an antagonist directed against TGF-beta, such as meterimumab (also known as CAT-192), fresolimumab (also known as GC1008) or LY2157299.

[0278] In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with a treatment comprising adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with a treatment comprising adoptive transfer of T cells comprising a dominant negative TGF beta receptor, e.g., a dominant-negative TGF beta type II receptor. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with a treatment comprising the HERCREEM protocol (see, e.g., ClinicalTrials.gov Identifier NCT00889954).

[0279] In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an agonist, such as an activating antibody, directed against CD137 (also known as TNFRSF9, 4-1BB or ILA). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with urelumab (known as BMS-663513). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an agonist, such as an activating antibody, directed against CD40. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with CP-870893. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an agonist, such as an activating antibody, directed against OX40 (also known as CD-134). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an anti-OX40 antibody (e.g., AgonOX). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an agonist, such as an activating antibody, directed against CD27. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with CDX-1127. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an antagonist directed against indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).

[0280] In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with the antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE). In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599). In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with trastuzumab emtansine (also known as T-DM1, ado-transtuzumab emtansine, or KADCYLA®, Genentech). In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with DMUC5754A. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an antibody-drug conjugate that targets the endothelin B receptor (EDNBR), such as an antibody directed against EDNBR conjugated with MMAE.

[0281] In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an angiogenesis inhibitor. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an antibody directed against VEGF, such as VEGF-A. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with bevacizumab (also known as AVASTIN®, Genetech). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an antibody directed against angiopoietin 2 (also known as Ang2). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with MEDI3617.

[0282] In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an anti-tumor drug. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an agent that targets CSF-1R (also known as M-CSFR or CD115). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with anti-CSF-1R (also known as IMC-CS4). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an interferon, such as interferon alpha or interferon gamma. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with Roferon A (also known as recombinant interferon alpha 2a). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with GM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor, rhu GM-CSF, also known as sargramostim or LEUKINE®). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with IL-2 (also known as aldesleukin or PROLEUKIN®). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with IL-12. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an antibody that targets CD20. In some embodiments, the antibody that targets CD20 is obinutuzumab (also known as GA101 or GAZYVA®) or rituximab. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an antibody that targets GITR, hi some embodiments, the antibody that targets GITR is TRX518.

[0283] In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a custom-made cancer vaccine. In some embodiments, the peptide cancer vaccine is a multivalent long peptide, a multipeptide, a peptide cocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an adjuvant. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with a treatment comprising a TLR agonist, such as poly-ICIC (also known as HILTONOL®), LPS, MPL, or CpG ODN. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with tumor necrosis factor (TNF) alpha. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with IL-1. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with HMGB1. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with an IL-10 antagonist. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with an IL-4 antagonist. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with an IL-13 antagonist. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with an HVEM antagonist. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an ICOS agonist, for example by administering ICOS-L or an agonist antibody directed against ICOS. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with a therapy that targets CX3CL1.In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with a therapy that targets CXCL9. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with a therapy that targets CXCL10. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with a therapy that targets CCL5. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with an LFA-1 or ICAM1 agonist. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with a selectin agonist.

[0284] In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with a targeted therapy. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an inhibitor of B-Raf. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with vemurafenib (also known as ZELBORAF®). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with dabrafenib (also known as TAFINLAR®). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with erlotinib (also known as TARCEVA®). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an inhibitor of MEK, such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with cobimetinib (also known as GDC-0973 or XL-518). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with trametinib (also known as MEKINIST®). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an inhibitor of K-Ras. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an inhibitor of c-Met. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with onartuzumab (also known as MetMAb). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an inhibitor of Alk. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with AF802 (also known as CH5424802 or alectinib). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an inhibitor of phosphatidylinositol 3-kinase (PI3K).In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with BKM120. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with perifosine (also known as KRX-0401). In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with an inhibitor of Akt. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with MK2206. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with GSK690693. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with GDC-0941. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with an inhibitor of mTOR. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with sirolimus (also known as rapamycin). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with temsirolimus (also known as CCI-779 or TORISEL®). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with everolimus (also known as RAD001). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with ridaforolimus (also known as AP-23573, MK-8669 or deforolimus). In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with OSI-027. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with AZD8055. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with INK128. In some embodiments, the PD-1 axis binding antagonist and taxane may be administered in combination with a dual PI3k / mTOR inhibitor.In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with XL765. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with GDC-0980. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with BEZ235 (also known as NVP-BEZ235). In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with BGT226. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with GSK2126458. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with PF-04691502. In some embodiments, a PD-1 axis binding antagonist and a taxane may be administered in combination with PF-05212384 (also known as PKI-587).

[0285] VI. Manufactured Articles or Kits In another embodiment of the invention, an article of manufacture and / or kit is provided that includes a PD-1 axis binding antagonist and / or a taxane. In some embodiments, the article of manufacture or kit further includes a package insert that includes instructions for using the PD-1 axis binding antagonist in combination with a taxane to treat or delay the progression of cancer in an individual or to enhance immune function in an individual with cancer. Any of the PD-1 axis binding antagonists and / or taxanes described herein may be included in the article of manufacture or kit.

[0286] In some embodiments, the PD-1 axis binding antagonist and the taxane are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on or associated with the container may indicate instructions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions. In some embodiments, the article of manufacture further includes one or more additional agents (e.g., chemotherapeutic agents and antitumor agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0287] The specification is considered to be sufficient to enable one 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. EXAMPLES

[0288] The present invention will be more fully understood by reference to the following examples. However, these examples should not be considered as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are merely for illustrative purposes, and that various modifications and changes in light thereof will be suggested to those skilled in the art, which should be included within the spirit and scope of this application and the scope of the appended claims.

[0289] Example 1: Combination treatment with anti-PD-L1 antibody and nab-paclitaxel (ABRAXANE®) + carboplatin achieved durable complete response in an MC38 colorectal tumor model. Materials and Methods In vivo tumor models The MC38 colorectal tumor cell line was maintained at Genentech. Seven to ten week old C57BL / 6 female mice (Charles River Laboratories; Hollister, CA) were inoculated subcutaneously with 100,000 MC38 cells into the right flank. Tumors grew to approximately 100-300 mm. 3 Once a mean tumor volume of 100 mg / kg was reached, mice were recruited and randomized into treatment groups, and antibody and / or chemotherapy treatments were initiated on the following day.

[0290] To analyze repeated measurements of tumor volume from the same animal over time, a mixed modeling approach was used (Pinheiro et al. nmle: Linear and Nonlinear Mixed Effects Models. R. package version 3.1-108 (2013)). This approach accounts for both repeated measurements and a small amount of dropout before the end of the study. Cubic regression splines were used to fit a nonlinear profile to the time course of log2(tumor volume) in the different treatments. Fits were performed via linear mixed-effects models within R, version 2.15, 2, using the nlme package, version 3.1 108 (R Foundation for Statistical Computing; Vienna, Austria).

[0291] For the MC38 re-immunization study shown in Figures 5A-5B, cured mice previously treated with the combination of anti-PD-L1 + ABRAXANE® + carboplatin were subcutaneously inoculated with 100,000 MC38 cells in the flank opposite the primary tumor. In parallel, naïve female C57BL / 6 mice were also inoculated with 100,000 MC38 cells. After 7 days, all mice were euthanized and spleens were harvested for flow cytometry analysis. All animal experiments were performed in accordance with the guidelines and regulations set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee (IACUC) guidelines.

[0292] In vitro stimulation of splenocyte cultures from reimmunized mice Splenocytes were cultured in triplicate at 1,000,000 cells per well in 96-well U-bottom plates with 10 ng / ml phorbol 12-myristate 13-acetate (PMA) and 1 μg / ml ionomycin (Sigma-Alrich; St. Louis, MO) plus GOLGIPLUG™ (Brefeldin A) (BD Biosciences; San Jose, CA) for 4 hours at 37° C. Cells were harvested, stained with surface markers CD4 FITC (fluorescein isothiocyanate), CD3 PE (phycoerythrin), and CD8 PerCp-Cy5.5 (BD Biosciences), and fixed with 4% paraformaldehyde for 30 minutes on ice. Cells were permeabilized with 1x permeabilization buffer (BD Biosciences) and stained with rat anti-mouse anti-interferon-γ (IFN-γ)-allophycocyanin (APC)-conjugated antibody or rat IgG1-APC isotype control antibody (BD Biosciences) and run on a BD Biosciences LSRII using FACSDIVA™ software. Flow cytometric analysis was performed using FlowJo software (TreeStar).

[0293] Antibodies and Treatments All therapeutic antibodies were generated at Genentech. The control antibody was anti-gp120 mouse IgG1 (mIgG1), clone 10E7.1D2. Anti-PD-L1 was either a human / mouse reverse chimera, clone YW243.55.S70 mIgG2a.DANA or fully mouse clone 25A1 mIgG2a.DANA. ABRAXANE® was obtained from Abraxis Bioscience, Inc. (owned by Celgene; Summit, NJ). Carboplatin was obtained from Hospira, Inc. (Lake Forest, IL). Dexamethasone was obtained from West-Ward Pharmaceuticals (Eatontown, NJ). Dosing schedules and routes of administration are indicated in the figure brief. Antibodies were diluted in either PBS or 20 mM histidine acetate, 240 mM sucrose, 0.02% polysorbate 20, pH 5.5. Chemotherapy drugs and dexamethasone were diluted in saline.

[0294] In vivo vaccination studies OTI Thy1.1 CD8+ T cells were isolated by negative selection from the spleen and mesenteric lymph nodes of donor OTI Thy1.1 females (Genentcch colony) using a MACS CD8 isolation kit (Miltenyi Biotec). Purified CD8+ cells were labeled with CFSE (Life Technologies; Grand Island, NY) and cultured at 2.5 × 10 6The cells were injected intravenously (IV) into female C57BL / 6 recipients (Charles River Laboratories). The next day, mice were vaccinated with 250 ng of anti-DEC205 fused to full-length ovalbumin (produced by Genentech) intraperitoneally (IP) plus 4 mg of saline or dexamethasone IV per kg body weight. Two days after vaccination, mice were put to rest and spleens were harvested for analysis. Total cell counts of splenic cell suspensions were determined by flow cytometry using the ratio of live cell events to a fixed number of known concentrations of fluorescent beads (catalog number 9003-53-6, Polysciences, Inc.; Warrington, PA). OTI CD8+ T cells were identified by flow cytometry by staining with Thy1.1 PE-Cy7 and CD8 Pacific Blue (BD Biosciences) and run on a BD Biosciences LSRII using FACSDIVA™ software. Flow cytometric analysis was performed using FlowJo software (TreeStar).

[0295] result This study evaluated the efficacy of anti-PD-L1 antibodies in the context of cancer therapy in preclinical mouse tumor models. Combination treatment with anti-PD-L1 antibody (clone 25A1 mIgG2a.DANA) and paclitaxel + carboplatin resulted in a synergistic anti-tumor response when compared to control antibody or paclitaxel + carboplatin treatment alone in a syngeneic MC38 colorectal tumor model (Figure 1). Ten percent of mice (1 / 10) had a partial response to the combination of anti-PD-L1 and paclitaxel + carboplatin compared to no mice in the control antibody or paclitaxel + carboplatin alone groups (Figure 1). Strong anti-tumor responses that resulted in a reduction in tumor size were tracked as partial responses (PR), defined in this example as a greater than 50% but less than 100% reduction from the initial tumor volume, or complete responses, defined in this example as a 100% reduction in tumor volume. Combination treatment with anti-PD-L1 antibody and paclitaxel + carboplatin also extended the time to progression: the time to progression (TTP) for the control antibody (defined in this example as 5 times the initial tumor volume) was 11 days, for paclitaxel + carboplatin it was 15.5 days, and for the combination treatment of anti-PD-L1 antibody and paclitaxel + carboplatin it was 25 days.

[0296] In clinical settings, treatment with paclitaxel (formulated in a potentially toxic solvent) typically involves premedication with corticosteroids such as dexamethasone to reduce the likelihood of hypersensitivity reactions. However, corticosteroids such as dexamethasone have immunosuppressive effects and can inhibit T cell responses, which in turn reduces the activity of PD-1 axis binding antagonists such as anti-PD-L1 agents. Consistently, administration of dexamethasone abolished the efficacy of single-agent anti-PD-L1 treatment in a syngeneic MC38 colorectal tumor model (Figures 2A and 2B). Furthermore, dexamethasone inhibited antigen-specific T cell responses in OTI adoptive T cell transfer and vaccination models (Figure 3). Thus, without wishing to be bound by theory, treatment with corticosteroids such as dexamethasone may attenuate or offset some of the benefits of PD-1 axis binding antagonists, such as anti-PD-L1 therapy, reducing T cell function and the enhanced ability of T cells to promote anti-tumor responses, such as CD8+ T cell-mediated killing of tumors.

[0297] Combination treatment with anti-PD-L1 antibody (chimeric YW243.55.S70.mIgG2a.DANA) and nab-paclitaxel (ABRAXANE®) + carboplatin resulted in unexpectedly strong synergistic anti-tumor efficacy compared to treatment with control antibody, single-agent anti-PD-L1 antibody, or ABRAXANE® + carboplatin alone in a syngeneic MC38 colorectal tumor model (Figures 4A and 4B). Combination anti-PD-L1 antibody and ABRAXANE®) + carboplatin therapy achieved durable complete responses lasting more than 90 days in 4 / 8 mice (Figures 4A and 4B). This synergy is even stronger than that observed as a result of anti-PD-L1 and paclitaxel + carboplatin combination treatment. The TTP (5x the initial tumor volume) was 11.5 days for the control antibody alone, 9 days for the anti-PD-L1 antibody alone, 13.5 days for ABRAXANE® + carboplatin, and none for the combination therapy of anti-PD-L1 antibody and ABRAXANE® + carboplatin, where 4 / 8 mice showed complete regression. This indicates that the combination therapy of anti-PD-L1 antibody and ABRAXANE® + carboplatin significantly prolonged the progression-free period at a higher level than the combination therapy of anti-PD-L1 antibody and paclitaxel + carboplatin. Furthermore, all cured mice (i.e., mice showing complete remission) from the combination therapy of anti-PD-L1 and ABRAXANE® + carboplatin treatment were able to completely reject a secondary challenge with the same MC38 tumor cell line, indicating that this therapy generates a T cell memory response (Figures 5A-5B). In vitro restimulation of splenocytes from these cured mice demonstrated increased T cell effector function, as observed by enhanced production of interferon-gamma (IFN-γ) from both CD4+ and CD8+ T cells compared to naive primary antigen-challenged mice (Figures 5A-5B).

[0298] The unexpectedly strong antitumor synergistic activity and the unexpected ability to obtain complete remissions and generate T cell memory responses represent therapeutic advantages of combination therapy with a PD-1 axis binding antagonist and a taxane, such as nab-paclitaxel (ABRAXANE®). Furthermore, unlike paclitaxel therapy, nab-paclitaxel (ABRAXANE®) treatment typically does not involve premedication with corticosteroids such as dexamethasone. The results presented here indicate that combination therapy with a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody) and nab-paclitaxel (ABRAXANE®) also allows for a simpler treatment regimen that can avoid the use of corticosteroids, thus reducing the likelihood of potential adverse effects.

[0299] Example 2: Combination treatment of nab-paclitaxel (ABRAXANE®) and carboplatin with an anti-PD-L1 antibody achieved complete remission in a Phase 1b clinical trial for patients with non-small cell lung cancer A Phase 1b clinical study was conducted to evaluate the efficacy of combination therapy using an anti-PD-L1 antibody (MPDL3280A) in combination with a taxane (nab-paclitaxel (ABRAXANE®) or paclitaxel) and carboplatin in patients with non-small cell lung cancer (NSCLS).

[0300] The dosing protocol for this clinical study was as follows: 1) MPDL3280A / ABRAXANE® / carboplatin combination therapy: (a) MPDL3280A administered IV at 1200 mg once every 3 weeks (q3w); (b) 100 mg / m once a week (q1w). 2 and (c) carboplatin administered IV once every three weeks (q3w) at a target area under the curve (AUC) of 6 mg / ml. 2) MPDL3280A / paclitaxel / carboplatin combination therapy: (a) MPDL3280A administered IV at 1200 mg once every 3 weeks (q3w); (b) MPDL3280A administered IV at 220 mg / m once every 3 weeks (q3w) 2 (c) carboplatin given IV once every three weeks (q3w) with a target AUC of 6 mg / ml.

[0301] Table 4 shows the results of a study of 14 patients treated with MPDL3280A in combination with ABRAXANE® and carboplatin. Table 5 shows the results of a study of 6 patients treated with MPDL3280A in combination with paclitaxel plus carboplatin.

[0302] Table 4: Efficacy of MPDL3280A / ABRAXANE® / Carboplatin combination therapy TIFF2025020147000006.tif42170

[0303] Table 5: Efficacy of MPDL3280A / paclitaxel / carboplatin combination therapy TIFF2025020147000007.tif41170

[0304] As shown in Table 4 and Figure 6A, the combination treatment with MPDL3280A and nab-paclitaxel (ABRAXANE®) + carboplatin resulted in unexpectedly strong antitumor efficacy with an objective response rate (ORR, CR+PR) of 64.3%. Unexpectedly, 21.4% (3 / 14) of patients treated with the combination therapy of MDPL3280A and nab-paclitaxel (ABRAXANE®) + carboplatin achieved a complete response (i.e., the complete absence of detectable tumor mass). 42.9% (6 / 14) of patients experienced a partial response.

[0305] Combination treatment with MPDL3280A and paclitaxel + carboplatin also resulted in antitumor efficacy, although it was somewhat less robust than the combination of MPDL3280A / nab-paclitaxel (ABRAXANE®) + carboplatin in the relatively small sample size of study subjects. The ORR for the combination of MPDL3280A and paclitaxel + carboplatin was 33.3%, with both responders experiencing partial responses (Table 5 and Figure 6B).

[0306] Consistent with the preclinical studies shown in Example 1, the unexpectedly strong anti-tumor activity and the unexpected ability to obtain sustained complete responses represent a major therapeutic advantage of combination therapy with a PD-1 axis-binding antagonist (e.g., an anti-PD-L1 antibody) and a taxane, such as nab-paclitaxel (ABRAXANE®).

Claims

1. A medicament comprising MPDL3280A for treating or delaying the progression of non-small cell lung cancer (NSCLC) in an individual, the medicament is used in combination with (i) nab-paclitaxel or paclitaxel and (ii) carboplatin; MPDL3280A is formulated for intravenous administration once every two weeks; Medicine.

2. A medicament comprising MPDL3280A for treating or delaying the progression of non-small cell lung cancer (NSCLC) in an individual, the medicament is used in combination with (i) nab-paclitaxel or paclitaxel, (ii) carboplatin, and (iii) an anti-VEGF antibody, wherein the anti-VEGF antibody is bevacizumab; MPDL3280A is formulated for intravenous administration once every two weeks; Medicine.

3. The pharmaceutical agent described in claim 1 or 2, wherein the NSCLC is non-squamous NSCLC.

4. The pharmaceutical agent described in claim 1 or 2, wherein the NSCLC is squamous cell NSCLC.

5. The pharmaceutical agent described in any one of claims 1 to 4, wherein the NSCLC is metastatic.

6. The pharmaceutical agent described in any one of claims 1 to 5, wherein the individual has NSCLC or has been diagnosed with NSCLC.

7. The pharmaceutical described in claim 6, wherein cancer cells in an individual express PD-L1.

8. A pharmaceutical described in any one of claims 1 to 7, wherein the treatment results in a response in an individual.

9. The pharmaceutical agent described in claim 8, wherein the response is complete remission.

10. The pharmaceutical agent described in claim 8 or claim 9, wherein the response is sustained remission after cessation of treatment.

11. The pharmaceutical agent of any one of claims 1 to 10, wherein nab-paclitaxel or paclitaxel and / or carboplatin is formulated for administration prior to, simultaneous with, or following MPDL3280A.

12. The pharmaceutical composition of claim 11, wherein nab-paclitaxel or paclitaxel and / or carboplatin is formulated for administration prior to MPDL3280A.

13. The method of claim 11, wherein nab-paclitaxel or paclitaxel and / or carboplatin is formulated for simultaneous administration with MPDL3280A.

14. The pharmaceutical composition of claim 11, wherein nab-paclitaxel or paclitaxel and / or carboplatin is formulated for administration after MPDL3280A.

15. A pharmaceutical agent described in any one of claims 1 and 3 to 14, used in combination with nab-paclitaxel.

16. A pharmaceutical composition according to any one of claims 2 to 14, wherein the pharmaceutical composition is used in combination with paclitaxel.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein MPDL3280A is formulated for administration at a dose of 840 mg.

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