Cancer immunotherapy by disrupting PD-1 / PD-l1 signaling

Abstract

To provide methods for treating subjects afflicted with cancer.SOLUTION: Provided herein is a method for immunotherapy of a cancer patient comprising administering to the patient an Ab that inhibits signaling from a PD-1 / PD-L1 signaling pathway, or a combination of such an Ab and an anti-CTLA-4 Ab. Also provided is a method of immunotherapy for a subject afflicted with cancer comprising selecting a patient as a suitable candidate for immunotherapy on the basis of an assessment that the proportion of cells expressing PD-L1 on the cell surface in a test tissue sample derived from the patient exceeds a predetermined threshold level, and administering the anti-PD-1 Ab to the selected subject. Further provided are rabbit mAbs that bind specifically to a cell surface-expressed PD-L1 antigen in a FFPE tissue sample, and an automated IHC method for assessing cell surface expression in FFPE tissues using the provided anti-PD-L1 Abs.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 647,442, filed May 15, 2012, and U.S. Provisional Patent Application No. 61 / 790,747, filed March 15, 2013, the contents of which are incorporated herein by reference in their entireties.

[0002] Throughout this application, various other publications are referred to in parentheses by author name and date, or by patent or patent publication number. Full citations for these publications may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety in order to more fully describe the state of the art known to those skilled in the art as of the date of the invention described and claimed in this application. However, citation of a document should not be construed as an admission that such document is prior art to the present invention.

[0003] Technical Field The present invention relates to methods for immunotherapy of cancer patients comprising administering to the patient antibodies that disrupt the PD-1 / PD-L1 signaling pathway. Biomarkers can be used as part of the treatment to identify suitable patients for immunotherapy and to predict the effectiveness of anti-PD-1 treatment. [Background technology]

[0004] 2. Background of the Invention Human cancers have numerous genetic and epigenetic alterations that produce neoantigens potentially recognizable by the immune system (Sjoblom et al., 2006). The adaptive immune system, composed of T and B lymphocytes, has a strong anticancer potential with a broad ability to respond to various tumor antigens and fine specificity. Furthermore, the immune system has demonstrated considerable plasticity and memory components. The successful exploitation of all these properties of the adaptive immune system makes immunotherapy unique among all cancer treatment modalities. However, although an endogenous immune response against cancer is observed in preclinical models and patients, this response is ineffective and established cancers are considered "self" and tolerated by the immune system. This state of tolerance allows tumors to utilize several different mechanisms to actively subvert antitumor immunity. These mechanisms include dysfunctional T-cell signaling (Mizoguchi et al., 1992), inhibitory regulatory cells (Facciabene et al., 2012), and the co-opting of endogenous “immune checkpoints” that serve to down-regulate the strength of the adaptive immune response by tumors to avoid immune destruction and protect normal tissues from collateral damage (Topalian et al., 2011; Mellman et al., 2011).

[0005] Until recently, cancer immunotherapy has focused substantial efforts on approaches to enhance antitumor immune responses by adoptive transfer of activated effector cells, immunization against relevant antigens, or provision of nonspecific immune stimulants, such as cytokines. However, in the past decade, intensive efforts to develop specific immune checkpoint pathway inhibitors have been launched to provide novel immunotherapeutic approaches to treat cancer, including the development of an antibody (Ab), ipilimumab (YERVOY®), that binds and inhibits cytotoxic T-lymphocyte antigen-4 (CTLA-4) for the treatment of patients with advanced melanoma (Hodi et al., 2010), and the development of Abs that block the inhibitory PD-1 pathway, as described herein.

[0006] Programmed death-1 (PD-1) is an important immune checkpoint receptor expressed by activated T and B cells that mediates immune suppression. PD-1 is a member of the CD28 family of receptors that includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified, programmed death-ligand-1 (PD-L1) and programmed death-ligand-2 (PD-L2), which are expressed on antigen-presenting cells and in a number of human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al., 2000; Latchman et al., 2001). Unlike CTLA-4, PD-1 functions primarily in peripheral tissues where activated T-cells can encounter the immunosuppressive PD-L1 (B7-H1) and PD-L2 (B7-DC) ligands expressed by tumor and / or stromal cells (Flies et al., 2011; Topalian et al., 2012a). Inhibition of the PD-1 / PD-L1 interaction mediates potent antitumor activity in preclinical models (U.S. Patent Nos. 8,008,449 and 7,943,743), and the use of Ab inhibitors of the PD-1 / PD-L1 interaction to treat cancer has entered clinical trials (Brahmer et al., 2010; Topalian et al., 2012b; Brahmer et al., 2012; Flies et al., 2011; Pardoll, 2012; Hamid and Carvajal, 2013).

[0007] The promise of the new field of personalized medicine is that advances in pharmacogenomics will be increasingly used to tailor treatment to defined subpopulations and ultimately individual patients to enhance efficacy and minimize side effects. Recent successes include, for example, the development of imatinib mesylate (GLEEVEC®), a protein tyrosine kinase inhibitor that inhibits bcr-abl tyrosine kinase to treat Philadelphia chromosome positive chronic myeloid leukemia (CML); crizotinib (XALKORI®), which treats 5% of patients with late-stage non-small cell lung cancer expressing a mutated anaplastic lymphoma kinase (ALK) gene; and vemurafenib (ZELBORAF®), an inhibitor of the mutated B-RAF protein (V600E-BRAF) expressed in approximately half of melanoma tumors. However, unlike the clinical development of small molecule agents that target discrete activating mutations found in select cancer populations, a particular challenge in cancer immunotherapy has been the identification of mechanism-based predictive biomarkers to enable patient selection and guide treatment management. Progress in validating PD-L1 expression as a biomarker to screen patients for anti-PD-1 immunotherapy is described herein. Summary of the Invention

[0008] Summary of the Invention The present disclosure provides a method for immunotherapy of a subject suffering from cancer, comprising administering to the subject a composition comprising a therapeutically effective amount of an agent that disrupts, reduces, or suppresses signaling from an inhibitory immune regulator. In a preferred embodiment, the agent is an Ab. In another preferred embodiment, the inhibitory immune regulator is a component of the PD-1 / PD-L1 signaling pathway. In a further preferred embodiment, the Ab disrupts the interaction between PD-1 and PD-L1. In one embodiment, the Ab is an anti-PD-1 Ab of the present invention or an anti-PD-L1 Ab of the present invention. In a preferred embodiment, the anti-PD-1 Ab of the present invention is nivolumab (BMS-936558) and the anti-PD-L1 Ab of the present invention is BMS-936559. In one embodiment, the subject has been pretreated for the cancer. In another embodiment, the cancer is an advanced, metastatic, and / or refractory cancer. In a preferred embodiment, administration of the Ab, or antigen-binding portion thereof, to a subject induces a durable clinical response in the subject.

[0009] The application also provides a method for immunotherapy of a subject suffering from cancer comprising: (a) selecting a subject who is a suitable candidate for immunotherapy comprising: (i) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells, (ii) assessing the percentage of cells in the test tissue sample that express PD-L1 on their cell surface, and (iii) selecting the subject as a suitable candidate based on assessing that the percentage of cells in the test tissue sample that express PD-L1 on their cell surface exceeds a predetermined threshold level; and (b) administering to the selected subject a composition comprising a therapeutically effective amount of an agent that disrupts signaling from the PD-1 / PD-L1 pathway, e.g., an anti-PD-1 or anti-PD-L1 Ab.

[0010] The application further provides a method for treating a subject suffering from cancer comprising: (a) selecting a subject not suitable for immunotherapy with an agent that disrupts signaling from the PD-1 / PD-L1 pathway, e.g., anti-PD-1 or anti-PD-L1 Ab, comprising: (i) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells; (ii) assessing the percentage of cells in the test tissue sample that express PD-L1 on their cell surface; and (iii) selecting the subject as not suitable for immunotherapy based on assessing that the percentage of cells in the test tissue sample that express PD-L1 on their cell surface is below a predetermined threshold level; and (b) administering to the selected subject a standard of care therapy other than an agent that disrupts signaling from the PD-1 / PD-L1 pathway.

[0011] In addition, the application provides methods for selecting a cancer patient for immunotherapy with an agent that disrupts signaling from the PD-1 / PD-L1 pathway, e.g., an anti-PD-1 or anti-PD-L1 Ab, comprising: (a) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, where the test tissue sample contains tumor cells and tumor infiltrating inflammatory cells; (b) assaying the test tissue sample to determine the percentage of cells that express PD-L1 on their cell surface; (c) comparing the percentage of cells that express PD-L1 on their cell surface to a predetermined threshold percentage; and (d) selecting the patient for immunotherapy based on the assessment that PD-L1 is expressed in cells of the test tissue sample.

[0012] The application further provides a method for predicting the therapeutic efficacy of an agent that disrupts signaling from the PD-1 / PD-L1 pathway, e.g., an anti-PD-1 or anti-PD-L1 Ab, to treat a cancer patient, comprising: (a) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, where the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells; (b) assaying the test tissue sample to determine the percentage of cells that express PD-L1 on their cell surface; (c) comparing the percentage of cells that express PD-L1 on their cell surface to a predetermined threshold; and (d) predicting the therapeutic efficacy of the agent, wherein when the percentage of cells that express PD-L1 on their cell surface is above a threshold percentage, the agent is predicted to be effective in treating the patient, and when the percentage of cells that express PD-L1 on their cell surface is below a threshold percentage, the agent is predicted to be ineffective in treating the patient.

[0013] The present application also provides methods for determining an immunotherapeutic regimen comprising an agent that disrupts signaling from the PD-1 / PD-L1 pathway, e.g., an anti-PD-1 or anti-PD-L1 Ab, for treating a cancer patient, comprising: (a) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, where the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells; (b) assaying the test tissue sample to determine the percentage of cells that express PD-L1 on their cell surface; (c) comparing the percentage of cells that express PD-L1 on their cell surface to a predetermined threshold percentage; and (d) determining the immunotherapeutic regimen comprising the agent based on a determination that the percentage of cells that express PD-L1 on their cell surface exceeds the predetermined threshold percentage.

[0014] In one embodiment of the methods described herein, the test tissue sample is a formalin-fixed paraffin-embedded (FFPE) sample. In another embodiment, assessing the proportion of cells expressing PD-L1 on the cell surface in the test tissue sample is accomplished by immunohistochemical (IHC) staining of the FFPE sample. In a preferred embodiment, mAb28-8 or 5H1 is used in an automated IHC assay to bind to PD-L1 on the surface of cells in the test tissue sample. In preferred embodiments of any of the methods described herein, the cancer is melanoma (MEL), renal cell carcinoma (RCC), squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer (CRC), castration-resistant prostate cancer (CRPC), hepatocellular carcinoma (HCC), squamous cell carcinoma of the head and neck, carcinoma of the esophagus, ovary, gastrointestinal tract and breast, or a hematological malignancy, such as multiple myeloma, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, and chronic myelogenous leukemia.

[0015] The present invention further provides a mAb or antigen-binding portion thereof that specifically binds to human PD-L1 antigen expressed on the cell surface in FFPE tissue samples. In a preferred embodiment, the mAb or antigen-binding portion thereof does not bind to cytoplasmic PD-L1 antigen in FFPE tissue samples. In another preferred embodiment, the monoclonal Ab (mAb) is a rabbit mAb designated 28-8, 28-1, 28-12, 29-8 or 20-12, or a mouse mAb designated 5H1.

[0016] Other features and advantages of the present invention will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all cited references, including scientific articles, journal reports, GenBank entries, patents, and patent applications, cited throughout this application are expressly incorporated herein by reference. [Brief description of the drawings]

[0017] [Figure 1A]Cross-competition between 5C4 and other HuMabs' anti-PD-1 mAbs for binding to human PD-1 (hPD-1) expressed on CHO cells. A, 5C4 Fab fragments substantially blocked binding of mAb 5C4 itself, as well as 2D3 and 7D3; B, 5C4 Fab fragments substantially blocked binding of mAb 4H1; C, 5C4 mAb substantially blocked binding of mAb 17D8. [Figure 1B] Cross-competition between 5C4 and other HuMabs' anti-PD-1 mAbs for binding to human PD-1 (hPD-1) expressed on CHO cells. A, 5C4 Fab fragments substantially blocked binding of mAb 5C4 itself, as well as 2D3 and 7D3; B, 5C4 Fab fragments substantially blocked binding of mAb 4H1; C, 5C4 mAb substantially blocked binding of mAb 17D8. [Figure 1C] Cross-competition between 5C4 and other HuMabs' anti-PD-1 mAbs for binding to human PD-1 (hPD-1) expressed on CHO cells. A, 5C4 Fab fragments substantially blocked binding of mAb 5C4 itself, as well as 2D3 and 7D3; B, 5C4 Fab fragments substantially blocked binding of mAb 4H1; C, 5C4 mAb substantially blocked binding of mAb 17D8.

[0018] [Figure 2A]Cross-competition of human anti-hPD-L1 mAbs conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6, and 13G4, and significantly blocked by itself; B, binding of labeled 3G10 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; C, binding of labeled 10A5 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; D, binding of labeled 11E6 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and E, binding of labeled 12A4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and F, binding of labeled 13G4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10. [Figure 2B] Cross-competition of human anti-hPD-L1 mAbs conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6, and 13G4, and significantly blocked by itself; B, binding of labeled 3G10 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; C, binding of labeled 10A5 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; D, binding of labeled 11E6 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and E, binding of labeled 12A4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and F, binding of labeled 13G4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10. [Figure 2C]Cross-competition of human anti-hPD-L1 mAbs conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6, and 13G4, and significantly blocked by itself; B, binding of labeled 3G10 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; C, binding of labeled 10A5 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; D, binding of labeled 11E6 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and E, binding of labeled 12A4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and F, binding of labeled 13G4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10. [Figure 2D] Cross-competition of human anti-hPD-L1 mAbs conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6, and 13G4, and significantly blocked by itself; B, binding of labeled 3G10 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; C, binding of labeled 10A5 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; D, binding of labeled 11E6 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and E, binding of labeled 12A4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and F, binding of labeled 13G4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10. [Figure 2E]Cross-competition of human anti-hPD-L1 mAbs conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6, and 13G4, and significantly blocked by itself; B, binding of labeled 3G10 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; C, binding of labeled 10A5 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; D, binding of labeled 11E6 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and E, binding of labeled 12A4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and F, binding of labeled 13G4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10. [Figure 2F] Cross-competition of human anti-hPD-L1 mAbs conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6, and 13G4, and significantly blocked by itself; B, binding of labeled 3G10 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; C, binding of labeled 10A5 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; D, binding of labeled 11E6 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and E, binding of labeled 12A4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10; and F, binding of labeled 13G4 was significantly blocked by each of the anti-PD-L1 Abs tested, except for 10H10.

[0019] [Diagram 3]Cross-competitive inhibition of biotinylated mAb 12A4 binding to ES-2 cells by human anti-hPD-L1 mAbs. Fluorescence of bound biotin-12A4 is plotted against the concentration of unlabeled hPD-L1 HuMab.

[0020] [Figure 4] Spider plot showing activity of anti-PD-1 mAb in patients with treatment refractory melanoma (MEL). A representative plot of change in tumor burden over time demonstrates the time course of change in the sum of the longest diameters of target lesions compared to baseline in 27 MEL patients treated with 5C4 at a dose of 1.0 mg / kg. In the majority of patients who achieved an objective response (OR), the response was sustained and evident by the end of cycle 3 of treatment (6 months) (vertical dashed line). Tumor regression, e.g., prolonged reduction in tumor burden in the presence of new lesions, followed the conventional and "immune-related" pattern of response.

[0021] [Diagram 5] Activity of anti-PD-1 mAb in patients with metastatic RCC. Partial recurrence of metastatic RCC in a 57-year-old patient treated with 5C4 at 1 mg / kg is described. The patient had previously undergone curative surgery and developed progressive disease after receiving sunitinib, temsirolimus, sorafenib, and pazopanib. The arrow indicates regression of the recurrent tumor in the surgical field.

[0022] [Figure 6]Activity of anti-PD-1 mAb in patients with metastatic MEL. A complete response of metastatic MEL is described in a 62-year-old patient treated with 5C4 at 3 mg / kg associated with vitiligo. (i) Pretreatment CT scan, inguinal lymph node metastasis (arrow); (ii) 13 months after treatment. Numerous metastases in the subcutaneous tissue and retroperitoneum also completely regressed (not shown). Vitiligo developed 6 months after treatment; photographs were taken in September under visible light (iii) and ultraviolet light (iv). Skin biopsy with immunohistochemistry for microphthalmia-associated transcription factor (MITF) shows melanocytes (arrow) at the epidermal-dermal junction in normal skin (v) and few (vi) or absent (vii) melanocytes in skin partially or completely affected by vitiligo.

[0023] [Figure 7] Activity of anti-PD-1 mAb in patients with metastatic NSCLC. A partial response is described in a patient with metastatic NSCLC (non-squamous tissue) treated with 5C4 at 10 mg / kg. Arrows indicate early progression, then regression in lung lesions (an "immune-related" pattern of response).

[0024] [Figure 8A]Correlation between tumor PD-L1 expression and anti-PD-1 clinical response. Pretreatment tumor cell surface expression of PD-L1 correlates with OR to PD-1 blockade as determined by IHC on formalin-fixed paraffin-embedded specimens. 42 subjects with advanced cancers including melanoma, non-small cell lung cancer, colorectal cancer, renal cell carcinoma and castration-resistant prostate cancer (n=18, 10, 7, 5 and 2, respectively) were studied. A, There was a significant correlation between objective clinical response and tumor cell surface PD-L1 expression. Patients with PD-L1 negative tumors did not experience OR. B, Examples of IHC analysis with anti-PD-L1 mAb 5H1 are shown in a melanoma lymph node metastasis (top row), a renal cell carcinoma nephrectomy specimen (middle row), and a lung adenocarcinoma brain metastasis (bottom row). All at 400X original magnification. Arrows indicate one of many tumor cells in each specimen with surface membrane staining for PD-L1. Asterisks indicate normal glomeruli in nephrectomy specimens that were negative for PD-L1 staining. [Figure 8B] Correlation between tumor PD-L1 expression and anti-PD-1 clinical response. Pretreatment tumor cell surface expression of PD-L1 correlates with OR to PD-1 blockade as determined by IHC on formalin-fixed paraffin-embedded specimens. 42 subjects with advanced cancers including melanoma, non-small cell lung cancer, colorectal cancer, renal cell carcinoma and castration-resistant prostate cancer (n=18, 10, 7, 5 and 2, respectively) were studied. A, There was a significant correlation between objective clinical response and tumor cell surface PD-L1 expression. Patients with PD-L1 negative tumors did not experience OR. B, Examples of IHC analysis with anti-PD-L1 mAb 5H1 are shown in a melanoma lymph node metastasis (top row), a renal cell carcinoma nephrectomy specimen (middle row), and a lung adenocarcinoma brain metastasis (bottom row). All at 400X original magnification. Arrows indicate one of many tumor cells in each specimen with surface membrane staining for PD-L1. Asterisks indicate normal glomeruli in nephrectomy specimens that were negative for PD-L1 staining.

[0025] [Figure 9]Graphical comparison of mAb28-8 and 5H1 binding to PD-L1 antigen in tumor tissues by histoscore analysis. Rabbit mAb28-8 showed higher histoscores in 7 out of 10 samples tested.

[0026] [Figure 10A] Spider plots showing activity of anti-PD-L1 mAbs in patients with treatment refractory MEL and NSCLC. Representative plots demonstrate the time course of target lesion tumor burden in patients with MEL treated with BMS-936559 at doses of 1 (A), 3 (B), 10 mg / kg (C), and in patients with NSCLC treated at 10 mg / kg (D). In the majority of patients who achieved OR, responses were durable and evident by the end of cycle 2 of treatment (3 months), regardless of dose or tumor type. Tumor regression followed the conventional and "immune-related" pattern of response. [Figure 10B] Spider plots showing activity of anti-PD-L1 mAbs in patients with treatment refractory MEL and NSCLC. Representative plots demonstrate the time course of target lesion tumor burden in patients with MEL treated with BMS-936559 at doses of 1 (A), 3 (B), 10 mg / kg (C), and in patients with NSCLC treated at 10 mg / kg (D). In the majority of patients who achieved OR, responses were durable and evident by the end of cycle 2 of treatment (3 months), regardless of dose or tumor type. Tumor regression followed the conventional and "immune-related" pattern of response. [Figure 10C]Spider plots showing activity of anti-PD-L1 mAbs in patients with treatment refractory MEL and NSCLC. Representative plots demonstrate the time course of target lesion tumor burden in patients with MEL treated with BMS-936559 at doses of 1 (A), 3 (B), 10 mg / kg (C), and in patients with NSCLC treated at 10 mg / kg (D). In the majority of patients who achieved OR, responses were durable and evident by the end of cycle 2 of treatment (3 months), regardless of dose or tumor type. Tumor regression followed the conventional and "immune-related" pattern of response. [Figure 10D] Spider plots showing activity of anti-PD-L1 mAbs in patients with treatment refractory MEL and NSCLC. Representative plots demonstrate the time course of target lesion tumor burden in patients with MEL treated with BMS-936559 at doses of 1 (A), 3 (B), 10 mg / kg (C), and in patients with NSCLC treated at 10 mg / kg (D). In the majority of patients who achieved OR, responses were durable and evident by the end of cycle 2 of treatment (3 months), regardless of dose or tumor type. Tumor regression followed the conventional and "immune-related" pattern of response.

[0027] [Figure 11] Complete response in a patient with melanoma treated with BMS-936559 at 3 mg / kg. The circles show an initial increase in size of the lung nodule at 6 weeks and 3 months, followed by complete regression at 10 months (an "immune-related" pattern of response).

[0028] [Figure 12] Complete response in a patient with melanoma treated with BMS-936559 at 1 mg / kg. The patient developed a solitary brain metastasis 3 months after the start of treatment, which was successfully treated with stereotactic radiosurgery. A partial response (circled) was noted in the abdominal disease at 8 months, with no evidence of disease at 15 months.

[0029] [Figure 13]Partial response in a patient with NSCLC (non-squamous histology) treated with BMS-936559 at 10 mg / kg, showing response in disease in the right lung pleura and liver.

[0030] [Figure 14A] Clinical activity in MEL patients receiving concurrent nivolumab and ipilimumab regimens. A, A typical spider plot shows the change from baseline in tumor burden, measured as the sum of the products of the perpendicular diameters of all target lesions, in patients receiving concurrent 1 mg / kg nivolumab + 3 mg / kg ipilimumab regimens at the MTD. Triangles indicate the first occurrence of new lesions. B, A typical waterfall plot shows the maximum response rate in baseline target lesions in patients receiving concurrent regimens. [Figure 14B] Clinical activity in MEL patients receiving concurrent nivolumab and ipilimumab regimens. A, A typical spider plot shows the change from baseline in tumor burden, measured as the sum of the products of the perpendicular diameters of all target lesions, in patients receiving concurrent 1 mg / kg nivolumab + 3 mg / kg ipilimumab regimens at the MTD. Triangles indicate the first occurrence of new lesions. B, A typical waterfall plot shows the maximum response rate in baseline target lesions in patients receiving concurrent regimens.

[0031] [Figure 15] Tumor regression in a 52-year-old MEL patient receiving a concomitant regimen of 1 mg / kg nivolumab + 3 mg / kg ipilimumab. The patient presented with extensive neck, mediastinal, axillary, abdominal and pelvic lymphadenopathy, bilateral pulmonary nodules, small bowel metastases, peritoneal implants and widespread subcutaneous nodules. Baseline lactate dehydrogenase (LDH) was 2.25x upper limit of normal, hemoglobin was 9.7 g / dL and symptoms included nausea and vomiting. Within 4 weeks of treatment, LDH normalized, symptoms improved (increased appetite, decreased vomiting) and skin lesions had regressed. At the 12-week scan, there was a marked reduction in all areas of disease. Arrows indicate location of metastatic disease.

[0032] [Figure 16A] Clinical activity in MEL patients receiving various concurrent regimens of nivolumab and ipilimumab. Representative spider plots show the change from baseline in tumor burden, measured as the sum of the products of the perpendicular diameters of all target lesions, in patients receiving concurrent regimens of 0.3 mg / kg nivolumab + 3 mg / kg ipilimumab (A), 3 mg / kg nivolumab + 1 mg / kg ipilimumab (B), or 3 mg / kg nivolumab + 3 mg / kg ipilimumab (C). Triangles indicate the first occurrence of new lesions. [Figure 16B] Clinical activity in MEL patients receiving various concurrent regimens of nivolumab and ipilimumab. Representative spider plots show the change from baseline in tumor burden, measured as the sum of the products of the perpendicular diameters of all target lesions, in patients receiving concurrent regimens of 0.3 mg / kg nivolumab + 3 mg / kg ipilimumab (A), 3 mg / kg nivolumab + 1 mg / kg ipilimumab (B), or 3 mg / kg nivolumab + 3 mg / kg ipilimumab (C). Triangles indicate the first occurrence of new lesions. [Figure 16C] Clinical activity in MEL patients receiving various concurrent regimens of nivolumab and ipilimumab. Representative spider plots show the change from baseline in tumor burden, measured as the sum of the products of the perpendicular diameters of all target lesions, in patients receiving concurrent regimens of 0.3 mg / kg nivolumab + 3 mg / kg ipilimumab (A), 3 mg / kg nivolumab + 1 mg / kg ipilimumab (B), or 3 mg / kg nivolumab + 3 mg / kg ipilimumab (C). Triangles indicate the first occurrence of new lesions.

[0033] [Figure 17]Tumor regression in a 61-year-old MEL patient receiving a concomitant regimen of 0.3 mg / kg nivolumab + 3 mg / kg ipilimumab. The patient presented with stage IV (M1c) MEL of unknown primary site with metastases to the stomach and mesentery. After a recent transfusion, lactate dehydrogenase was 225 and hemoglobin was 9.6 g / dL. Twelve weeks after the start of study treatment, a CT scan showed an 86% reduction in bulk disease burden.

[0034] [Figure 18A] Clinical activity in MEL patients receiving sequential regimens of nivolumab and ipilimumab. Typical spider plots show the change from baseline in tumor burden, measured as the sum of the products of the perpendicular diameters of all target lesions, in patients receiving sequential regimens of 1 mg / kg nivolumab (A) or 3 mg / kg nivolumab (B) before and after ipilimumab treatment. Triangles indicate the first occurrence of new lesions. C, Typical waterfall plot shows the maximum response rate in baseline target lesions in patients receiving sequential regimens. "*" indicates patients who had radiological progression on previous ipilimumab treatment. [Figure 18B] Clinical activity in MEL patients receiving sequential regimens of nivolumab and ipilimumab. Typical spider plots show the change from baseline in tumor burden, measured as the sum of the products of the perpendicular diameters of all target lesions, in patients receiving sequential regimens of 1 mg / kg nivolumab (A) or 3 mg / kg nivolumab (B) before and after ipilimumab treatment. Triangles indicate the first occurrence of new lesions. C, Typical waterfall plot shows the maximum response rate in baseline target lesions in patients receiving sequential regimens. "*" indicates patients who had radiological progression on previous ipilimumab treatment. [Figure 18C]Clinical activity in MEL patients receiving sequential regimens of nivolumab and ipilimumab. Typical spider plots show the change from baseline in tumor burden, measured as the sum of the products of the perpendicular diameters of all target lesions, in patients receiving sequential regimens of 1 mg / kg nivolumab (A) or 3 mg / kg nivolumab (B) before and after ipilimumab treatment. Triangles indicate the first occurrence of new lesions. C, Typical waterfall plot shows the maximum response rate in baseline target lesions in patients receiving sequential regimens. "*" indicates patients who had radiological progression on previous ipilimumab treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description of the Invention The present invention relates to a method for immunotherapy of a subject suffering from a disease, e.g., cancer or an infectious disease, comprising administering to the subject a composition comprising a therapeutically effective amount of a compound or agent that enhances an endogenous immune response, stimulating activation of an endogenous response or inhibiting suppression of an endogenous response. More specifically, the present application provides a method for enhancing an endogenous immune response in a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of an agent, e.g., an Ab or antigen-binding portion thereof, that disrupts or inhibits signaling from an inhibitory immune regulator, thereby treating the patient. In one embodiment, the inhibitory immune regulator is a component of the PD-1 / PD-L1 signaling pathway. Thus, one embodiment of the present invention provides a method for immunotherapy of a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of an Ab or antigen-binding portion thereof that disrupts the interaction between the PD-1 receptor and its ligand, PD-L1. In one preferred embodiment, the Ab or antigen-binding portion thereof specifically binds to PD-1. In another preferred embodiment, the Ab or antigen-binding portion thereof specifically binds to PD-L1. One embodiment involves the use of an anti-PD-1 Ab in combination with another anti-cancer agent, preferably an anti-CTLA-4 Ab, to treat cancer. In one other embodiment, a subject is selected as suitable for immunotherapy in a method that includes measuring surface expression of PD-L1 in a test tissue sample obtained from a patient with cancer of the tissue, e.g., determining the percentage of cells in the test tissue sample that express PD-L1 on the surface of the cells, and selecting the patient for immunotherapy based on the assessment that PD-L1 is expressed on the surface of cells in the test tissue sample.

[0036] term In order that this specification may be more readily understood, certain terms are first defined. As used herein, unless expressly provided otherwise herein, each of the following terms has the meaning set forth below. Further definitions are set forth herein.

[0037] "Administration" refers to the physical introduction of a composition containing a therapeutic agent into a subject, using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration for the Abs of the invention include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the Abs of the invention can be administered via non-parenteral routes, for example, topical, epidermal or mucosal routes of administration, for example, intranasally, orally, intravaginally, rectally, sublingually, or topically. Administration can also be, for example, once, multiple times, and / or one or more times over an extended period of time.

[0038] As used herein, an "adverse event" (AE) is any unfavourable and generally unintended or undesirable sign (including laboratory abnormalities), symptom, or disease associated with the use of a medical treatment. For example, an adverse event may be associated with the activation of the immune system or the expansion of immune system cells (e.g., T cells) in response to the treatment. A medical treatment may have one or more associated AEs, each of which may have the same or different levels of severity. Reference to a method that can "alter adverse events" refers to a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0039] An "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, inter-connected by disulfide bonds. Each H chain comprises a heavy chain variable region (herein referred to as V H The heavy chain constant region includes three constant domains, C H1, C H2 and C H3 Each light chain comprises a light chain variable region (referred to herein as V L The light chain constant region comprises one constant domain, C L Includes: V H and V L The regions can be further divided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L contains three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0040] Antibodies are generally -5 From 10 -11 M -1 The following dissociation constant (K D ) specifically binds to its cognate antigen with high affinity reflected by approximately 10 -4 M -1 Any K above D However, Abs that specifically bind to an antigen are generally considered to have a specific binding activity. -7 M or less, preferably 10 -8 M or less, even more preferably 5x10 -9 M or less, and more preferably 10 -8 M to 10 -10 K below M D"Substantially identical" to a given antigen when it exhibits a high degree of sequence identity with the given antigen, e.g., at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99% sequence identity with the sequence of the given antigen. As an example, an Ab that specifically binds human PD-1 may also have cross-reactivity with PD-1 antigens from certain primate species, but may not cross-react with PD-1 antigens from certain rodent species or antigens other than PD-1, e.g., human PD-L1 antigen.

[0041] Immunoglobulins may be from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, including, but not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both natural and non-natural Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully synthetic Abs; and single-chain Abs. Non-human Abs may be humanized by recombinant methods to reduce their immunogenicity in humans. Except where expressly stated, and unless the context indicates otherwise, the term "antibody" also includes any antigen-binding fragment or portion of said immunoglobulins, including monovalent and bivalent fragments or portions, and single-chain Abs.

[0042] An "isolated antibody" refers to an Ab that is substantially free of other Abs with different antigen specificities (e.g., an isolated Ab that specifically binds to PD-1 is substantially free of Abs that specifically bind to antigens other than PD-1). However, an isolated Ab that specifically binds to PD-1 may have cross-reactivity to other antigens, e.g., PD-1 molecules from different species. Furthermore, an isolated Ab may be substantially free of other cellular material and / or chemicals. In comparison, an "isolated" nucleic acid refers to a nucleic acid composition of matter that is significantly different from the nucleic acid as it occurs in nature, i.e., has a unique chemical identity, properties, and utility. For example, isolated DNA, unlike naturally occurring DNA, is a free-standing piece of naturally occurring DNA and is not an integral part of the larger structural complexes found in nature, chromosomes. Furthermore, isolated DNA, unlike naturally occurring genomic DNA, may generally be used in applications or methods in which naturally occurring genomic DNA is inappropriate, e.g., as a PCR primer or hybridization probe for measuring gene expression and detecting biomarker genes or mutations, among others, to diagnose disease or evaluate the effectiveness of treatment. Isolated nucleic acids can be purified using standard techniques well known in the art to be substantially free of other cellular components or other contaminants, such as other cellular nucleic acids or proteins. Examples of isolated nucleic acids include genomic DNA, PCR-amplified DNA, cDNA, and RNA fragments.

[0043] The term "monoclonal antibody" ("mAb") refers to a preparation of Ab molecules of single molecular composition, i.e., Ab molecules which are essentially identical in primary sequence, and which display a single binding specificity and affinity for a particular epitope. mAbs are an example of isolated Abs. MAbs may be produced by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art.

[0044] "Human" antibody (HuMAb) refers to an Ab having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, when the Ab contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human Ab of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, the term "human antibody" as used herein is not intended to include Abs in which CDR sequences derived from the germline of another mammalian species, e.g., mouse, are grafted onto human framework sequences. The terms "human" Ab and "fully human" Ab are used interchangeably.

[0045] A "humanized" antibody refers to an Ab in which some, most, or all of the amino acids outside the CDR domain of a non-human Ab are replaced with the corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an Ab, some, most, or all of the amino acids outside the CDR domain are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Some additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not destroy the Ab's ability to bind to a particular antigen. A "humanized" Ab retains the same antigen specificity as the original Ab.

[0046] A "chimeric antibody" refers to an Ab in which the variable region is derived from one species and the constant region is derived from another species, e.g., the variable region is derived from a murine Ab and the constant region is derived from a human Ab.

[0047] An "antigen-binding portion" (also called an "antigen-binding fragment") of an Ab refers to one or more fragments of an Ab that retain the ability to specifically bind to the antigen bound by the intact Ab.

[0048] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and proliferation leads to the formation of malignant tumors that can invade adjacent tissues and even metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0049] "Immune response" refers to the actions of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells in the liver, including Abs, cytokines, and complement, that result in the selective targeting, binding, damaging, destroying, and / or removing from the vertebrate body, invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0050] "Immune regulator" refers to a substance, agent, signaling pathway, or component thereof that modulates the immune response. "Control," "modulation," or "regulation" of the immune response refers to any change in the cells of the immune system or the activity of such cells. Such regulation includes promotion or suppression of the immune system, which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur within the immune system. Both inhibitory and stimulatory immune regulators have been identified, some of which may have enhanced function in the cancer microenvironment.

[0051] The term "immunotherapy" refers to the treatment of a subject suffering from, or at risk of suffering from, a disease, or having a recurrence of a disease, by methods involving inducing, enhancing, suppressing, or modifying an immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to the subject, with the purpose of reversing, mitigating, ameliorating, inhibiting, delaying, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical signs associated with a disease.

[0052] "Enhancing an endogenous immune response" means increasing the efficacy or potency of an immune response present in a subject. This increase in efficacy and potency can be accomplished, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0053] The "predetermined threshold" for cell surface PD-L1 expression indicates the percentage of cells in a test tissue sample, including tumor cells and tumor infiltrating inflammatory cells, at which the sample is scored as positive for cell surface PD-L1 expression. The predetermined threshold for cells expressing PD-L1 on the cell surface ranges from at least about 0.01% to at least about 20% of the total number of cells, as assayed by IHC with mAb28-8. In a preferred embodiment, the predetermined threshold for cells expressing PD-L1 on the cell surface ranges from at least about 0.1% to at least about 10% of the total number of cells. More preferably, the predetermined threshold is at least about 5%. Even more preferably, the predetermined threshold is at least about 1%, or in the range of 1-5%.

[0054] "Programmed Death-1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is mainly expressed on T cells previously activated in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms and species homologs of hPD-1, and analogs that share at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank accession number U64863.

[0055] "Programmed death-ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2) that downregulates T cell activation and cytokine secretion when bound to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and analogs that share at least one epitope in common with hPD-L1. The complete hPD-L1 sequence can be found under GenBank Accession No. Q9NZQ7.

[0056] "Signal transduction pathway" or "signal transduction pathway" refers to the biochemical relationship between various signal transduction molecules that play a role in transmitting a signal from one part of a cell to another part of a cell. "Cell surface receptor" includes, for example, molecules and complexes of molecules located on the surface of a cell that can receive a signal and transmit such a signal through the cell membrane of the cell. An example of a cell surface receptor of the present invention is the PD-1 receptor, which is located on the surface of activated T cells, activated B cells and myeloid cells and transmits a signal that causes a decrease in tumor-infiltrating lymphocytes and a decrease in T cell proliferation. "Inhibitor" of signal transduction refers to a compound or agent that antagonizes or reduces the initiation, reception or transmission of any component of a signal transduction pathway, for example, a signal transmitted by a receptor or its ligand, either stimulatory or inhibitory.

[0057] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, cats, rabbits and ferrets, rodents, such as mice, rats and guinea pigs, birds, such as chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a mammal, such as a non-human primate, sheep, dog, cat, rabbit, ferret or rodent. In a more preferred embodiment, the subject is a human. The terms "subject", "patient" and "individual" are used interchangeably herein.

[0058] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent, e.g., an Ab of the invention, is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject against onset of disease or promotes regression of disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of damage or disability due to disease affliction. The ability of a therapeutic agent to promote regression of disease can be assessed, for example, in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, using a variety of methods known to those of skill in the art, or by assaying the activity of the agent in in vitro assays.

[0059] As an example, an anti-cancer drug promotes the regression of cancer in a subject. In a preferred embodiment, a therapeutically effective amount of the drug promotes the regression of cancer to the point of eliminating the cancer. "Promoting the regression of cancer" means that the administration of an effective amount of the drug, alone or in combination with an anti-tumor drug, causes a decrease in tumor growth or tumor size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease symptom-free periods, or prevention of damage or disability due to disease affliction. In addition, the terms "effective" and "effectiveness" in the context of treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote the regression of cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (side effects) at the cell, organ and / or organism level due to the administration of the drug.

[0060] As an example for the treatment of tumors, a therapeutically effective amount of the drug preferably inhibits cell proliferation or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, compared to untreated subjects. In other preferred embodiments of the present invention, tumor regression can be observed and sustained for at least about 20 days, more preferably at least about 40 days, and even more preferably at least about 60 days. Despite these ultimate measurements of therapeutic efficacy, the evaluation of immunotherapeutic drugs must also take into account "immune-related" response patterns.

[0061] "Immune-related" response pattern refers to the clinical response pattern often observed in cancer patients treated with immunotherapeutic agents that induce cancer-specific immune responses or produce antitumor effects by modifying natural immune processes. This response pattern is characterized by an initial increase in tumor burden or the appearance of new lesions, which is classified as disease progression and is synonymous with drug failure in the evaluation of conventional chemotherapeutic agents, followed by a beneficial therapeutic effect. Therefore, proper evaluation of immunotherapeutic agents may require long-term monitoring of the effects of these agents on target diseases.

[0062] A therapeutically effective amount of a drug includes a "prophylactically effective amount," which is any amount of drug that inhibits the onset or recurrence of cancer when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or a subject with a recurrence of cancer. In a preferred embodiment, a prophylactically effective amount completely prevents the onset or recurrence of cancer. "Inhibiting" the onset or recurrence of cancer means either reducing the likelihood of the onset or recurrence of cancer, or completely preventing the onset or recurrence of cancer.

[0063] "Tumor-infiltrating inflammatory cells" are any type of cell that typically participates in an inflammatory response in a subject and infiltrates tumor tissue. Such cells include tumor-infiltrating lymphocytes (TILs), macrophages, monocytes, eosinophils, histiocytes, and dendritic cells.

[0064] The use of the alternative (e.g., "or") should be understood to mean either one, both, or a combination thereof of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to indicate "one or more" of any described or listed components.

[0065] The term "about" or "essentially comprises" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by a person skilled in the art, and that depends in part on the method by which the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprises" can mean within 1 or more standard deviations through the practice in the art. Alternatively, "about" or "essentially comprises" can mean a range of up to 20%. Furthermore, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprises" should be considered to be within an acceptable error range for the particular value or composition.

[0066] As described herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise stated, should be understood to include every integer value within the stated range, and, where appropriate, fractions thereof (e.g., integer tenths and percentiles).

[0067] Various aspects of the invention are further described in the following subsections.

[0068] Antibodies of the Invention The Abs of the present invention include various Abs having the structural and functional properties described herein, including high affinity binding to PD-1 or PD-L1, respectively. These Abs can be used, for example, as therapeutic Abs to treat subjects suffering from disease, or as reagents in diagnostic assays to detect their cognate antigens. Human mAbs (HuMAbs) that specifically bind to PD-1 with high affinity (e.g., bind to human PD-1 and can cross-react with PD-1 from other species, e.g., cynomolgus monkeys) are described in U.S. Patent No. 8,008,449, and HuMAbs that specifically bind to PD-L1 with high affinity are described in U.S. Patent No. 7,943,743. The Abs of the present invention include, but are not limited to, all of the anti-PD-1 and anti-PD-L1 Abs described in U.S. Patent Nos. 8,008,449 and 7,943,743, respectively. Other anti-PD-1 mAbs are described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, and 8,168,757, and PCT Publication No. WO 2012 / 145493, and anti-PD-L1 mAbs are described, for example, in U.S. Patent Nos. 7,635,757 and 8,217,149, U.S. Publication No. 2009 / 0317368, and PCT Publication Nos. WO 2011 / 066389 and WO 2012 / 145493. To the extent these anti-PD-1 and anti-PD-L1 mAbs exhibit the structural and functional properties described herein for the antibodies of the invention, they are also included as antibodies of the invention.

[0069] Anti-PD-1 Antibodies of the Invention Each of the anti-PD-1 HuMAbs described in U.S. Pat. No. 8,008,449 has one or more of the following properties: (a) a cytoplasmic affinity of 1×10 or more as determined by surface plasmon resonance using a Biacore biosensor system; -7 K below M D(b) binds to human PD-1 in a mixed lymphocyte reaction (MLR) assay; (c) does not substantially bind to human CD28, CTLA-4, or ICOS; (d) increases T-cell proliferation in an MLR assay; (e) increases IL-2 secretion in an MLR assay; (f) binds to human PD-1 and cynomolgus PD-1; (g) inhibits binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates an antigen-specific memory response; (i) stimulates Ab responses; and (j) inhibits tumor cell proliferation in vivo. Anti-PD-1 Abs of the present invention include mAbs that specifically bind human PD-1 and exhibit at least one, and preferably at least five, of the above properties.

[0070] US Patent No. 8,008,449 illustrates seven anti-PD-1 HuMAbs: 17D8, 2D3, 4H1, 5C4 (also referred to herein as nivolumab or BMS-936558), 4A11, 7D3 and 5F4. Isolated DNA molecules encoding the heavy and light chain variable regions of these Abs have been sequenced and the amino acid sequences of the variable regions have been deduced. The V of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 H The amino acid sequences are provided herein as SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7, respectively. L The amino acid sequences are provided herein as SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14, respectively.

[0071] Preferred anti-PD-1 Abs of the invention include anti-PD-1 HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, and 5F4. These preferred Abs specifically bind human PD-1 and are characterized by the following: (a) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:1 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:8; (b) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:2 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:9; (c) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:3 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:10; (d) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:4 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:5; (e) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:5 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:12; (f) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:6 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:13; or (g) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:7 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:14.

[0072] When each of these Abs can bind to PD-1, V H and V LSequences can be "mixed and matched" to prepare other anti-PD-1 Abs of the invention. PD-1 binding of such "mixed and matched" Abs can be tested using binding assays well known in the art, such as enzyme-linked immunosorbent assays (ELISAs), Western blots, radioimmunoassays, and Biacore analysis (see, e.g., U.S. Patent No. 8,008,449). Preferably, V H and V L When chains are mixed and matched, a particular V H / V L V from pair H The sequence is structurally similar to V H Similarly, a particular V H / V L V from pair L The sequence is structurally similar to V L Accordingly, an anti-PD-1 Ab of the invention is an isolated mAb, or antigen-binding portion thereof, comprising: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14, wherein the Ab comprises a mAb or antigen-binding portion thereof that specifically binds to PD-1, preferably human PD-1.

[0073] The CDR domains of the above Abs have been depicted using the Kabat system, and these Abs can also be defined by the combination of their three heavy and three light chain CDRs (see U.S. Pat. No. 8,008,449). Each of these Abs can bind to PD-1, and the antigen-binding specificity is primarily provided by the CDR1, CDR2, and CDR3 regions, thus making the V H CDR1, CDR2 and CDR3 sequences and V κ CDR1, CDR2 and CDR3 sequences can be "mixed and matched" (i.e., CDRs from different Abs can be mixed and matched, but each Ab has the same V HCDR1, CDR2 and CDR3 and V κ (It must contain CDR1, CDR2 and CDR3.) PD-1 binding of such "mixed and matched" Abs can be tested using the binding assays described above (e.g., ELISAs, Western blots, radioimmunoassays and Biacore analysis).

[0074] The Abs of the invention also include isolated Abs that specifically bind to PD-1 and comprise a heavy chain variable region derived from a particular germline heavy chain immunoglobulin and / or a light chain variable region derived from a particular germline light chain immunoglobulin. Specifically, in one embodiment, the Abs of the invention comprise: (a) a human V H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 3-33 or 4-39 germline sequence, and / or a human V κ The present invention also includes an isolated Ab comprising a light chain variable region comprising contiguously linked amino acids having a sequence derived from an L6 or L15 germline sequence. H 3-33, V H 4-39, V κ L6 and V κ V encoded by the L15 germline gene H and V κ The amino acid sequence of the region is provided in US Pat. No. 8,008,449.

[0075] As used herein, an Ab can be identified as comprising a heavy or light chain variable region "derived from" a particular human germline immunoglobulin by comparing the amino acid sequence of the human Ab with the amino acid sequence encoded by a human germline immunoglobulin gene and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., the best percent sequence identity) to the sequence of the human Ab. A human Ab "derived from" a particular human germline immunoglobulin may contain amino acid differences compared to the germline sequence, for example, by naturally occurring somatic mutations or deliberate introduction of site-specific mutations. However, the selected human Ab will generally be at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene and contain amino acid residues that identify the human Ab as human when compared to germline immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences). In certain cases, a human Ab can be at least 95%, or even at least 96%, 97%, 98% or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.

[0076] In one embodiment, the sequence of a human Ab derived from a particular human germline sequence exhibits up to 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene, in other embodiments, the human Ab may exhibit up to 5, or even up to 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0077] Preferred Abs of the present invention also include (a) human V H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 3-33 germline sequence, and a human V κ (b) a light chain variable region comprising contiguously linked amino acids having a sequence derived from a human V HA heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 4-39 germline sequence, and a human V κ The invention also includes an isolated Ab or antigen-binding portion thereof, comprising a light chain variable region comprising contiguously linked amino acids having a sequence derived from the L15 germline sequence. H 3-33 and V κ V from each of the L6 germline sequences H and V κ Examples of Abs having the V include 17D8, 2D3, 4H1, 5C4 and 7D3. H 4-39 and V κ V from each of the L15 germline sequences H and V κ Examples of Abs with regions include 4A11 and 5F4.

[0078] In yet other embodiments, the anti-PD-1 Abs of the invention comprise heavy and light chain variable regions having amino acid sequences highly similar or homologous to the amino acid sequences of the preferred anti-PD-1 Abs described herein, wherein the Abs retain the functional properties of the preferred anti-PD-1 Abs of the invention. For example, the Abs of the invention include mAbs comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises contiguously linked amino acids having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, and the light chain variable region comprises contiguously linked amino acids having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14. In other embodiments, the V H and / or V L The amino acid sequence may exhibit at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the above sequences.

[0079] As used herein, the percent sequence identity (also referred to as percent sequence homology) between two sequences (amino acid or nucleotide sequences) is a function of the number of identical positions shared by the sequences relative to the length of the sequences compared, taking into account the number of gaps and the length of each gap, to maximize the degree of sequence identity between the two sequences (i.e., % identity=number of identical positions / total number of positions compared×100).Comparing sequences and determining the percent identity between two sequences can be accomplished using mathematical algorithms well known to those skilled in the art (see, for example, U.S. Patent No. 8,008,449).

[0080] Antibodies with very similar amino acid sequences may have essentially the same functional properties if the difference in sequence is a conservative modification. As used herein, "conservative sequence modification" refers to an amino acid modification that does not significantly affect the binding properties of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Thus, for example, one or more amino acid residues in the CDR region of the Ab of the present invention can be replaced with other amino acid residues from the same side chain family, and the modified Ab can be tested for retained function using functional assays that are well known in the art. Thus, one embodiment of the anti-PD-1 Abs of the invention comprises heavy and light chain variable regions comprising CDR1, CDR2, and CDR3 domains, respectively, where one or more of these CDR domains comprise contiguously linked amino acids having the same sequence as, or conservative modifications of, the CDR sequences of the preferred anti-PD-1 Abs described herein (e.g., 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, or 5F4), and the Abs retain the desired functional properties of the preferred anti-PD-1 Abs of the invention.

[0081] Furthermore, it is well known in the art that the heavy chain CDR3 is the primary determinant of Ab binding specificity and affinity, and that multiple Abs with the same binding properties based on a common CDR3 sequence can be predictably generated (see, for example, Klimka et al., 2000; Beiboer et al., 2000; Rader et al., 1998; Barbas et al., 1994; Barbas et al., 1995; Ditzel et al., 1996; Berezov et al., 2001; Igarashi et al., 1995; Bourgeois et al., 1998; Levi et al., 1993; Polymenis and Stoller, 1994; and Xu and Davis, 2000). The literature generally demonstrates that when the heavy chain CDR3 sequence of a given Ab is defined, variations in the other five CDR sequences do not significantly affect the Ab's binding specificity. Thus, an Ab of the invention containing six CDRs can be defined by specifying the sequence of the heavy chain CDR3 domain.

[0082] The anti-PD-1 Abs of the invention also include isolated Abs that specifically bind human PD-1 and cross-compete with any of HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, and 5F4 for binding to human PD-1. Thus, the anti-PD-1 Abs of the invention include Abs that specifically bind human PD-1 and cross-compete with any of HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, and 5F4 for binding to human PD-1. The anti-PD-1 Abs of the invention include Abs that specifically bind human PD-1 and cross-compete with any of HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, and 5F4 for binding to human PD-1. Thus, the anti-PD-1 Abs of the invention include Abs that specifically bind human PD-1 and cross-compete with any of HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, and 5F4 for binding to human PD-1. (e) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:5 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:12; (f) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:6 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:13; or (g) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:7 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:14.

[0083] The ability of Abs to cross-compete for binding to an antigen indicates that these Abs bind to the same epitope region of the antigen (i.e., the same or overlapping or adjacent epitopes) and sterically hinder the binding of other cross-competing Abs to a particular epitope region. Thus, the ability of a test Ab to competitively inhibit the binding of, for example, 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 to human PD-1 demonstrates that the test Ab binds to the same epitope region of human PD-1 as each of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4. All isolated Abs that bind to the same epitope region of human PD-1 as HuMAb 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 are included in the Abs of the present invention. These cross-competing Abs are expected to have very similar functional properties due to the binding of PD-1 to the same epitope region. For example, cross-competing anti-PD-1 mAbs 5C4, 2D3, 7D3, 4H1 and 17D8 have been shown to have similar functional properties (see U.S. Pat. No. 8,008,449, Examples 3-7). The higher the degree of cross-competition, the more similar the functional properties. Furthermore, cross-competing Abs can be easily identified based on their ability to cross-compete with 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 in standard PD-1 binding assays. For example, Biacore analysis, ELISA assay or flow cytometry can be used to demonstrate cross-competition with the Abs of the present invention (see, for example, Examples 1 and 2).

[0084] In one embodiment, the Abs that cross-compete with 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, or 5F4 for binding to human PD-1, or that bind to the same epitope region of human PD-1 as 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, or 5F4, are mAbs. For administration to human patients, these cross-competing Abs are preferably chimeric Abs, or more preferably humanized or human Abs. Such human mAbs can be prepared and isolated as described in U.S. Patent No. 8,008,449. The data provided in Example 1 show that 5C4, or a Fab fragment thereof, cross-competes with each of 2D3, 7D3, 4H1, or 17D8 for binding to hPD-1 expressed on the surface of cells, indicating that all five anti-PD-1 mAbs bind to the same epitope region of hPD-1 (Figures 1A-1C).

[0085] The anti-PD-1 Abs of the invention may further comprise one or more of the VL1A-specific Abs described herein as starting materials, to engineer modified Abs that may have altered properties from the starting Ab. H and / or V L Abs having the sequence: H and / or V L ), for example, by modifying one or more residues in one or more CDR regions and / or in one or more framework regions. Additionally or alternatively, Abs can be engineered by modifying residues in the constant region, for example, to alter the effector function of the Ab. Particular modifications to Abs include CDR grafting, V H and / or V κ Site-specific mutations of amino acid residues within the CDR1, CDR2 and / or CDR3 regions to reduce the immunogenicity of Abs. H and / or V κThese include site-specific mutations of amino acid residues within the framework regions, generally modifications within the Fc region to alter one or more functional properties of the Ab, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity, and chemical modifications, such as pegylation or modifications in glycosylation patterns, to increase or decrease the biological (e.g., serum) half-life of the Ab. Specific examples of such modifications and methods of engineering Abs are described in detail in U.S. Patent No. 8,008,449. The anti-PD-1 Abs of the present invention specifically bind human PD-1 and include all such engineered Abs obtained by modification of any of the above-described anti-PD-1 Abs.

[0086] The anti-PD-1 Abs of the invention also include antigen-binding portions of said Abs. It has been extensively demonstrated that the antigen-binding function of an Ab can be performed by fragments of a full-length Ab. Examples of binding fragments encompassed within the term "antigen-binding portion" of an Ab include: (i) V L , V H , C L and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by disulfide bridges at the hinge region; (iii) a V H and C H1 (iv) an Fd fragment consisting of the V domain of a single arm of an Ab; L and V H It contains an Fv fragment consisting of domains.

[0087] These fragments, first obtained via proteolysis with enzymes such as papain and pepsin, have then been engineered into monovalent and multivalent antigen-binding fragments. For example, the V of the two domains of the Fv fragment L and V H are encoded by separate genes, L and V HUsing recombinant methods, the domains can be linked by a synthetic linker peptide that allows them to be made as a single protein chain that pairs to form a monovalent molecule known as a single chain variable fragment (scFv). Divalent or bivalent scFvs (di-scFv or bi-scFv) are composed of two V H and two V's L By linking two scFvs in a single peptide chain known as tandem scFvs, which contain the scFv variable regions, ScFv dimers and higher multimers can also be made using a linker peptide of less than 10 amino acids that is too short for the two variable regions to fold together, allowing the scFvs to dimerize and produce diabodies or form other multimers. Diabodies are characterized by the K D It has been shown that scFv-C binds to cognate antigens with much higher affinity than the corresponding scFvs, with dissociation constants up to 40-fold lower than the alpha-linked alpha-linker. Very short linkers (≦3 amino acids) have resulted in the formation of trivalent triabodies or tetravalent tetrabodies that exhibit even higher affinity for antigen than diabodies. Other variants include scFv-C H3 Minibodies, which are dimers, and larger scFv-Fc fragments (scFv-C H2 -C H3 dimers), and further, isolated CDRs may exhibit antigen-binding function. These Ab fragments are engineered using conventional recombinant techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact Abs. All of the above proteolytic and engineered fragments of Abs and related variants (for further details, see Hollinger and Hudson, 2005; Olafsen and Wu, 2010) are intended to be encompassed within the term "antigen-binding portion" of an Ab.

[0088] Anti-PD-L1 Antibodies of the Invention Each of the anti-PD-L1 HuMAbs described in U.S. Patent No. 7,943,743 has one or more of the following properties: -7 K below M D Anti-PD-L1 Abs of the invention have been shown to (a) bind to human PD-L1 in a mixed lymphocyte reaction (MLR) assay; (b) increase T-cell proliferation in a mixed lymphocyte reaction (MLR) assay; (c) increase interferon-γ production in an MLR assay; (d) increase IL-2 secretion in an MLR assay; (e) stimulate Ab responses; (f) inhibit the binding of PD-L1 to PD-1; and (g) reverse the suppressive effects of T regulatory cells on T cell effector cells and / or dendritic cells. Anti-PD-L1 Abs of the invention include mAbs that specifically bind human PD-L1 and exhibit at least one, and preferably at least four, of the above properties.

[0089] U.S. Patent No. 7,943,743 exemplifies ten anti-PD-1 HuMAbs: 3G10, 12A4 (also referred to herein as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4. Isolated DNA molecules encoding the heavy and light chain variable regions of these Abs have been sequenced, and the amino acid sequences of the variable regions have been deduced. The V of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 H The amino acid sequences are shown in SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24, respectively, and their V L The amino acid sequences are shown in SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34, respectively.

[0090] Preferred anti-PD-L1 Abs of the invention include the anti-PD-L1 HuMAbs 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4. These preferred Abs specifically bind human PD-L1 and are selected from the group consisting of: (a) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 15 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 25; (b) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 16 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 26; (c) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 17 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 27; (d) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 18 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 28; (e) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 19. (f) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:20 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:30; (g) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:21 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:31; (h) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:22 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:32; (i) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:23 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:33;or (j) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:24 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:34;

[0091] When each of these Abs can bind to PD-L1, V H and V L Sequences can be "mixed and matched" to prepare other anti-PD-L1 Abs of the invention. PD-L1 binding of such "mixed and matched" Abs can be tested using binding assays well known in the art, such as ELISAs, Western blots, radioimmunoassays, and Biacore analysis (see, e.g., U.S. Patent No. 7,943,743). Preferably, V H and V L When chains are mixed and matched, a particular V H / V L V from pair H The sequence is structurally similar to V H Similarly, a particular V H / V L V from pair L The sequence is structurally similar to V L Accordingly, the Ab of the invention also includes a mAb, or antigen-binding portion thereof, that specifically binds to PD-L1, preferably human PD-L1, comprising a heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in any of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, and a light chain variable region comprising contiguously linked amino acids having the sequence set forth in any of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34.

[0092] The CDR domains of the above anti-PD-L1 HuMAbs were depicted using the Kabat system, and these Abs can also be defined by the combination of their three heavy and three light chain CDRs (see U.S. Patent No. 7,943,743). Since each of these Abs can bind to PD-L1, and the antigen-binding specificity is primarily provided by the CDR1, CDR2, and CDR3 regions, the V H CDR1, CDR2 and CDR3 sequences and V κ CDR1, CDR2 and CDR3 sequences can be "mixed and matched" (i.e., CDRs from different Abs can be mixed and matched, but each Ab has the same V H CDR1, CDR2 and CDR3 and V κ (It must contain CDR1, CDR2 and CDR3.) PD-L1 binding of such "mixed and matched" Abs can be tested using, for example, ELISAs, Western blots, radioimmunoassays and Biacore analysis.

[0093] The antibodies of the invention also include isolated Abs that specifically bind to PD-L1 and comprise a heavy chain variable region derived from a particular germline heavy chain immunoglobulin and / or a light chain variable region derived from a particular germline light chain immunoglobulin. Specifically, in one embodiment, the Abs of the invention comprise: (a) a human V H heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 1-18, 1-69, 1-3 or 3-9 germline sequence, and / or a human V κ Abs comprising a light chain variable region comprising contiguously linked amino acids having a sequence derived from an L6, L15, A27, or L18 germline sequence. H 1-18, V H 1-3, V H 1-69, V H 3-9, V κ L6, V κ L15 and V κ V encoded by the A27 germline gene H and V κThe amino acid sequence of the region is provided in US Pat. No. 7,943,743.

[0094] Preferred Abs of the present invention are: (a) human V H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 1-18 germline sequence, and a human V κ (b) a light chain variable region comprising contiguously linked amino acids having a sequence derived from the L6 germline sequence; H A heavy chain variable region comprising contiguous amino acids having a sequence derived from the 1-69 germline sequence, and a human V κ (c) a light chain variable region comprising contiguously linked amino acids having a sequence derived from the L6 germline sequence; H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 1-3 germline sequence, and a human V κ (d) a light chain variable region comprising contiguously linked amino acids having a sequence derived from the L15 germline sequence; H A heavy chain variable region comprising contiguous amino acids having a sequence derived from the 1-69 germline sequence, and a human V κ (e) a light chain variable region comprising contiguously linked amino acids having a sequence derived from the A27 germline sequence; H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 3-9 germline sequence, and a human V κ (f) a light chain variable region comprising contiguously linked amino acids having a sequence derived from a human V15 germline sequence; or H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 3-9 germline sequence, and a human V κ The invention includes an isolated Ab or antigen-binding portion thereof comprising a light chain variable region comprising contiguously linked amino acids having a sequence derived from the L18 germline sequence.

[0095] V H 1-18 and V κ V from each of the L6 germline sequences H and V κ An example of an Ab having the VH 1-69 and V κ V from each of the L6 germline sequences H and V κ Examples of Abs with regions include 12A4, 1B12, 7H1 and 12B7. H 1-3 and V κ V from each of the L15 germline sequences H and V κ An example of an Ab having the following structure is 10A5. H 1-69 and V κ V from each of the A27 germline sequences H and V κ Examples of Abs with regions include 5F8, 11E6, and 11E6a. H 3-9 and V κ V from each of the L15 germline sequences H and V κ An example of an Ab having the sequence is 10H10. H 1-3 and V κ V from each of the L15 germline sequences H and V κ An example of an Ab having the following structure is 10A5. H 3-9 and V κ V from each of the L18 germline sequences H and V κ An example of an Ab having this structure is 13G4.

[0096] In one embodiment, the anti-PD-L1 Abs of the invention comprise heavy and light chain variable regions having amino acid sequences highly similar or homologous to the amino acid sequences of the preferred anti-PD-L1 Abs described herein, wherein the Abs retain the functional properties of said anti-PD-L1 Abs of the invention. For example, the Abs of the invention include mAbs comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises contiguously linked amino acids having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, and the light chain variable region comprises contiguously linked amino acids having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34. In another embodiment, the Abs of the invention comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises contiguously linked amino acids having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, and the light chain variable region comprises contiguously linked amino acids having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34. H and / or V L The amino acid sequence may exhibit at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the above sequences.

[0097] One embodiment of the anti-PD-L1 Abs of the invention comprises heavy and light chain variable regions comprising CDR1, CDR2 and CDR3 domains, respectively, and one or more of these CDR domains comprise contiguously linked amino acids having the same sequence as, or conservative modifications of, the CDR sequences of the preferred anti-PD-L1 Abs described herein (e.g., 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4), and such Abs retain the desired functional properties of the preferred anti-PD-L1 Abs of the invention.

[0098] Based on evidence that the heavy chain CDR3 is the primary determinant of Ab binding specificity and affinity, it has been demonstrated that, generally, when the heavy chain CDR3 sequence of a given Ab is defined, variability in the sequences of the other five CDRs does not significantly affect the binding specificity of the Ab. Thus, the anti-PD-L1 Abs of the invention include isolated Abs that contain six CDRs and are defined by specifying the sequence of the heavy chain CDR3 domain.

[0099] The anti-PD-L1 Abs of the invention also include isolated Abs that specifically bind human PD-L1 and cross-compete with any of HuMAbs 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 for binding to human PD-L1. Thus, the anti-PD-L1 Abs of the invention The Abs exhibit, for binding to PD-L1, (a) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 15 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 25; (b) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 16 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 26; (c) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 17 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 27; (d) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 18 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 28. a human light chain variable region; (e) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:19 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:29; (f) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:20 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:30; (g) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:21 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:31; (h) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:22 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:32;(i) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:23 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:33; or (j) an isolated Ab or antigen-binding portion thereof that cross-competes with a reference Ab or a reference antigen-binding portion thereof, the Ab or antigen-binding portion thereof comprising a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:24 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:34;

[0100] The ability of an Ab to cross-compete with any of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 for binding to human PD-L1 demonstrates that such Abs bind to the same epitope region of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, respectively. All isolated Abs that bind to the same epitope region of human PD-L1 as HuMAb 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, or 13G4 are included in the Abs of the invention. These cross-competing Abs are expected to have very similar functional properties by virtue of their binding of PD-L1 to the same epitope region. For example, cross-competing anti-PD-L1 mAbs 3G10, 1B12, 13G4, 12A4 (BMS-936559), 10A5, 12B7, 11E6 and 5F8 have been shown to have similar functional properties (see US Pat. No. 7,943,743, Examples 3-11), while mAb 10H10, which binds to a different epitope region, functions differently (US Pat. No. 7,943,743, Example 11). The higher the degree of cross-competition, the more similar the functional properties. Furthermore, cross-competing Abs can be identified in standard PD-L1 binding assays familiar to those skilled in the art, such as Biacore analysis, ELISA assays or flow cytometry. In a preferred embodiment, the Ab that cross-competes with 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4 for binding to human PD-1, or binds to the same epitope region of human PD-L1 as 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4, is a mAb, preferably a chimeric Ab, or more preferably a humanized or human Ab. Such human mAbs can be prepared and isolated as described in U.S. Patent No. 7,943,743.

[0101] The data provided in Example 2 show that each of the anti-PD-L1 HuMAbs 5F8, 7H1, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7, and 13G4, i.e., all of the HuMAbs tested except for 10H10, substantially blocked the binding of mAbs 3G10, 10A5, 11E6, 12A4, and 13G4 to Chinese hamster ovary (CHO) cells expressing PD-L1 cells. HuMAb 10H10 only substantially blocked its own binding to CHO / PD-L1 cells. These data show that 3G10, 10A5, 11E6, 12A4, and 13G4 cross-compete with all of the HuMAbs tested except for 10H10 for binding to the same epitope region of human PD-L1 (Figure 2A-F).

[0102] The data provided in Example 3 show that binding of HuMAb 12A4 to PD-L1-expressing ES-2 ovarian carcinoma cells was substantially blocked by 12A4 itself or by 1B12 and 12B7, and moderately to significantly blocked by mAbs 5F8, 10A5, 13G4 and 3G10, but not by mAb 10H10. These data, which are highly consistent with the data in Example 2, show that 12A4 itself, as well as two other HuMabs, 12B7 and 1B12, substantially cross-compete with 12A4 for binding to the same epitope region of human PD-L1, presumably the same epitope; 5F8, 10A5, 13G4 and 3G10 showed lower but significant levels of cross-competition with 12A4, suggesting that these mAbs may bind to epitopes that overlap with the 12A4 epitope; whereas 10H10 did not cross-compete with 12A4 at all (Figure 3), suggesting that this mAb binds to a different epitope region than 12A4.

[0103] The anti-PD-L1 Abs of the invention may also be administered in combination with one or more of the V H and / or V LThis includes Abs engineered starting from an Ab having a sequence, which may have altered properties from the starting Ab. Anti-PD-L1 Abs can be engineered with the various modifications described above to engineer modified anti-PD-1 Abs of the invention.

[0104] The anti-PD-L1 Abs of the present invention also include isolated Abs selected for their ability to bind to PD-L1 in formalin-fixed paraffin-embedded (FFPE) tissue specimens. The use of FFPE samples is important for long-term follow-up analysis of the correlation between PD-L1 expression in tumors and disease prognosis or progression. In addition, studies in measuring PD-L1 expression are often performed in frozen specimens due to the difficulty in isolating anti-human PD-L1 Abs that can be used to stain PD-L1 in FFPE specimens by IHC (Hamanishi et al., 2007) and, in particular, Abs that specifically bind to membrane PD-L1 in these tissues. The use of different Abs to stain PD-L1 in frozen versus FFPE tissues, and the ability of certain Abs to distinguish between membrane and / or cytoplasmic forms of PD-L1, may explain some of the differing data reported in the literature in which PD-L1 expression is associated with disease prognosis (Hamanishi et al., 2007; Gadiot et al., 2011). This application provides several rabbit mAbs that specifically bind with high affinity to membrane human PD-L1 in FFPE tissue samples containing tumor cells and tumor-infiltrating inflammatory cells.

[0105] Rabbit and mouse anti-hPD-L1 mAbs were produced as described in Example 9. Of approximately 200 Ab multiclones screened, only 10 rabbit multiclone Abs were found to specifically detect the membrane form of PD-L1, and the top five multiclones (designated numbers 13, 20, 28, 29, and 49) were subsequently subcloned. The clone that yielded the most potent specific detection of membrane PD-L1, rabbit clone 28-8, was selected for the IHC assay. The sequences of the variable regions of mAb28-8 are set forth in SEQ ID NOs: 35 and 36, respectively. As expressed using the Kabat system, the sequences of the heavy and light chain CDR domains of mAb28-8 are set forth in SEQ ID NOs: 37-42. Rabbit clones 28-1, 28-12, 29-8, and 20-12 were the next best mAbs for potent detection of membrane PD-L1 in FFPE tissues.

[0106] The anti-PD-L1 Abs of the invention also include antigen-binding portions of the above Abs, including Fab, F(ab')2, Fd, Fv, and scFv, di-scFv or bi-scFv, and scFv-Fc fragments, diabodies, triabodies, tetrabodies, and isolated CDRs (see Hollinger and Hudson, 2005; Olafsen and Wu, 2010 for further details).

[0107] Nucleic acid molecules encoding antibodies of the invention Another aspect of the present specification relates to an isolated nucleic acid molecule encoding any of the Abs of the present invention.These nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure form.The nucleic acids of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences.In a preferred embodiment, the nucleic acid is cDNA.

[0108] The nucleic acids of the present invention can be obtained using standard molecular biology techniques. For Abs expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the Abs produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. Nucleic acids encoding Abs obtained from immunoglobulin gene libraries (e.g., using phage display technology) can be retrieved from the library.

[0109] Preferred nucleic acid molecules of the invention include the V of the anti-PD-1 HuMAbs, 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4. H and V κ No. 8,008,449), and the V H and V κ The sequences encoding the VL1 gene are described in U.S. Pat. No. 7,943,743. H The isolated DNA encoding the region is H The DNA encoding the heavy chain constant region (C), a sequence known in the art and which can be obtained by standard PCR amplification, was then synthesized. H1 , C H2 and C H3 The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is preferably an IgG1 or IgG4 constant region. L The isolated DNA encoding the region is V L The DNA encoding the light chain constant region (C), a sequence known in the art and which can be obtained by standard PCR amplification, was then synthesized. LThe light chain constant region can be converted to a full-length light chain gene by operably linking it to another DNA molecule encoding the light chain constant region. The light chain constant region can be a kappa or lambda constant region, but is more preferably a kappa constant region.

[0110] Pharmaceutical Compositions The antibody of the present invention may be constituted into a composition, e.g., a pharmaceutical composition, comprising one or a combination of Abs, or an antigen-binding portion thereof, and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants, e.g., preservatives, wetting agents, emulsifying agents and dispersing agents.

[0111] Dosing regimens are adjusted to provide the optimum desired response, e.g., therapeutic response or minimal side effects. In administering anti-PD-1 or anti-PD-L1 Abs, the dosage ranges from about 0.0001 to about 100 mg / kg of subject's body weight, usually from about 0.001 to about 20 mg / kg of subject's body weight, more usually from about 0.01 to about 10 mg / kg of subject's body weight. Preferably, the dosage is within the range of 0.1-10 mg / kg of body weight. For example, the dosage can be 0.1, 0.3, 1, 3, 5 or 10 mg / kg of body weight, more preferably 0.3, 1, 3 or 10 mg / kg of body weight. The dosing schedule is generally designed to achieve an exposure that results in sustained receptor occupancy (RO) based on the typical pharmacokinetic properties of the Ab. Typical treatment regimens require administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once a month, or once a month from three to six months. Dosage and scheduling may be varied during the course of treatment. For example, dosing schedules may include administering Abs (i) every 2 weeks in a 6 week cycle; (ii) every 4 weeks for 6 doses, then every 3 months; (iii) every 3 weeks; (iv) 3-10 mg / kg body weight once, then 1 mg / kg body weight every 2-3 weeks. Considering that IgG4 Abs generally have a half-life of 2-3 weeks, preferred dosing regimens for anti-PD-1 or anti-PD-L1 Abs of the invention include 0.3-10 mg / kg body weight, preferably 3-10 mg / kg body weight, more preferably 3 mg / kg body weight via intravenous administration, with Abs given every 14 days in a 6 or 12 week cycle until complete response or established progressive disease.

[0112] In some methods, two or more mAbs with different binding specificities are administered simultaneously, provided that the dose of each Ab administered is within a specified range. Antibodies are usually administered multiple times. The interval between single doses can be, for example, weekly, biweekly, triweekly, monthly, trimonthly, or yearly. Intervals can also be irregular, as indicated by measuring blood levels of Ab against the target antigen in the patient. In some methods, doses are adjusted to achieve a plasma Ab concentration of about 1-1000 μg / ml, and in some methods, about 25-300 μg / ml.

[0113] Alternatively, Abs can be administered as sustained release formulations in cases where less frequent administration is required. The dose and frequency vary depending on the half-life of Abs in patients. Generally, human Abs show the longest half-life, followed by humanized Abs, chimeric Abs, and non-human Abs. The dose and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, generally, relatively low doses are administered at relatively infrequent intervals for a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses are sometimes required at relatively short intervals until the progression of the disease is reduced or terminated, preferably until the patient shows partial or complete improvement of the symptoms of the disease. The patient can then be administered a prophylactic regimen.

[0114] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without causing undue toxicity to the patient.The selected dosage level depends on various pharmacokinetic factors, such as the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or substances used in combination with the particular composition used, the age, sex, weight, condition, general health and past medical history of the patient being treated, and factors well known in the medical field.The composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods well known in the art.As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results.

[0115] Uses and Methods of the Invention The Abs, Ab compositions, nucleic acids and methods of the invention have numerous in vitro and in vivo utilities, such as methods for determining and quantifying expression of PD-1 or PD-L1, including binding of Abs to target polypeptides or measuring the amount of nucleic acids encoding these polypeptides, and methods for immunotherapy of subjects suffering from disease, including administering to the subject a composition comprising a therapeutically effective amount of a therapeutic agent that inhibits signaling from an inhibitory immune regulator. In a preferred embodiment of the latter method, the inhibitory immune regulator is a component of the PD-1 / PD-L1 signaling pathway, and the therapeutic agent disrupts signaling of this pathway. More preferably, the therapeutic agent is an Ab that interferes with the interaction between PD-1 and PD-L1. In one preferred embodiment of this method, the Ab specifically binds to PD-1 and blocks the interaction of PD-1 with PD-L1 and / or PD-L2. In another preferred embodiment, the therapeutic agent is an Ab that specifically binds to PD-L1 and blocks the interaction of PD-L1 with PD-1 and / or B7-1 (CD80). Thus, the present application provides a method for enhancing an immune response in a subject, comprising administering anti-PD-1 and / or anti-PD-L1 Abs to disrupt the interaction between PD-1 and PD-L1, and a method for treating a disease mediated by such an enhancement of the immune response. When Abs to PD-1 and PD-L1 are administered together, the two can be administered sequentially in any order or simultaneously. In one aspect, the present application provides a method for modifying an immune response in a subject, comprising administering to the subject anti-PD-1 and / or anti-PD-L1 Abs of the present invention, or antigen-binding portions thereof, such that the immune response in the subject is modified. Preferably, the immune response is enhanced, augmented, stimulated, or upregulated. In a preferred embodiment, the Abs of the present invention are human Abs.

[0116] Preferred subjects include human patients who need to increase immune response.The immunotherapy method described herein is particularly suitable for treating human patients with disorders that can be treated by enhancing T cell-mediated immune response.In one embodiment, the method is used for treating subjects suffering from diseases caused by infectious agents.In a preferred embodiment, the method is used for treating subjects suffering from or at risk of suffering from cancer.

[0117] cancer immunotherapy Blockade of PD-1 / PD-L1 interactions has been shown to enhance immune responses in vitro (U.S. Patent Nos. 8,008,449 and 7,943,743; Fife et al., 2009) and mediate preclinical antitumor activity (Dong et al., 2002; Iwai et al., 2002). However, the molecular interactions that can be blocked by these two Abs are not identical: the anti-PD-1 Abs of the present invention disrupt PD-1 / PD-L1 and can disrupt PD-1 / PD-L2 interactions; in contrast, the anti-PD-L1 Abs of the present invention also disrupt PD-1 / PD-L1 interactions, whereas they do not block PD-1 / PD-L2 interactions but can instead disrupt PD-1-independent PD-L1 / CD80 interactions, and have been shown to downregulate T-cell responses in vitro and in vivo (Park et al., 2010; Paterson et al., 2011; Yang et al., 2011; Butte et al., 2007; Butte et al., 2008). Thus, among these various ligand-receptor pairings, different interactions may predominate in different cancer types and contribute to different activity profiles for the two Abs.

[0118] Disruption of PD-1 / PD-L1 interaction by antagonistic Abs can increase immune responses against cancerous cells in patients. PD-L1 is not expressed in normal human cells, but is abundant in various human cancers (Dong et al., 2002). The interaction between PD-1 and PD-L1 weakens T cell responses as manifested in a decrease in tumor-infiltrating lymphocytes (TILs) and an increase in T cell receptor-mediated proliferation, leading to T cell anergy, exhaustion or apoptosis, and immune evasion by cancerous cells (Zou and Chen, 2008; Blank et al., 2005; Konishi et al., 2004; Dong et al., 2003; Iwai et al., 2002). Immune suppression can be reversed by inhibiting the local interaction between PD-L1 and PD-1 using anti-PD-1 and / or anti-PD-L1 Abs. These Abs can be used alone or in combination to inhibit the growth of cancerous tumors. In addition, either or both of these Abs may be used in conjunction with other immunogenic and / or anti-cancer agents, such as cytokines, standard cancer chemotherapy, vaccines, radiation therapy, surgical procedures, or other Abs.

[0119] Immunotherapy for cancer patients using anti-PD-1 antibodies The present application provides a method for immunotherapy of a subject suffering from cancer, comprising administering to the subject a composition comprising a therapeutically effective amount of an Ab or antigen-binding portion thereof that disrupts the interaction of PD-1 with PD-L1 and / or PD-L2. The present application also provides a method for inhibiting the proliferation of tumor cells in a subject, comprising administering to the subject an Ab or antigen-binding portion thereof that disrupts the interaction of PD-1 with PD-L1 and / or PD-L2 in an amount effective to inhibit the proliferation of tumor cells. In a preferred embodiment, the subject is a human. In another preferred embodiment, the Ab or antigen-binding portion thereof is an anti-PD-1 Ab or antigen-binding portion thereof of the present invention. In one embodiment, the Ab or antigen-binding portion thereof is an IgG1 or IgG4 isotype. In another embodiment, the Ab or antigen-binding portion thereof is a mAb or antigen-binding portion thereof. In a further embodiment, the Ab or antigen-binding portion thereof is a chimeric, humanized or human Ab or antigen-binding portion thereof. In a preferred embodiment for treating a human patient, the Ab or antigen-binding portion thereof is a human Ab or antigen-binding portion thereof.

[0120] The clinical trial described in the Examples used an anti-PD-1 HuMA, nivolumab (referred to as 5C4 in U.S. Patent No. 8,008,449), to treat cancer. Although 5C4 was selected as the lead Ab to participate in the clinical trial, it is noteworthy that several of the anti-PD-1 Abs of the present invention share the 5C4 functional properties that are important for the therapeutic activity of 5C4, such as binding specifically to human PD-1 with high affinity, increasing T-cell proliferation, IL-2 secretion and interferon-γ production in MLR assays, inhibiting the binding of PD-L1 and / or PD-L2 to PD-1, and inhibiting tumor cell proliferation in vivo. Furthermore, certain anti-PD-1 Abs of the present invention, 17D8, 2D3, 4H1 and 7D3, each show a V H 3-33 and V κ V with sequences derived from the L6 germline sequence H and V κIt is structurally related to 5C4, which contains the 5C4-antibody region. In addition, 5C4, 2D3, 7D3, 4H1, and 17D8 all cross-compete for binding to the same epitope region of hPD-1 (Example 1). Thus, the preclinical characterization of nivolumab and other anti-PD-1 HuMabs indicates that the methods of treating cancer provided herein can be performed using a variety of Abs selected from the broad genus of anti-PD-1 Abs of the invention.

[0121] Thus, one embodiment of the immunotherapy methods described herein includes: (a) administering to a patient a human V H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 3-33 germline sequence, and a human V κ (b) a light chain variable region comprising contiguously linked amino acids having a sequence derived from a human V H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 4-39 germline sequence, and a human V κ administering to the patient an anti-PD-1 Ab, or an antigen-binding portion thereof, comprising a light chain variable region comprising contiguously linked amino acids having a sequence derived from the L15 germline sequence.

[0122] In another embodiment, the Ab or antigen-binding portion thereof administered to the patient has the following binding activity for binding to PD-1: (a) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:1 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:8; (b) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:2 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:9; (c) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:3 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:10; (d) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:4 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:5; (e) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:5 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:12; (f) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:6 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:13; or (g) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:7 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:14. In a preferred embodiment, the Ab or antigen-binding portion thereof cross-competes with nivolumab for binding to PD-1.

[0123] In one embodiment of the immunotherapy methods described herein, the anti-PD-1 Ab, or antigen-binding portion thereof, administered to the patient comprises: (a) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:1, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:8; (b) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:2, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:9; (c) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:3, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:10; (d) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:4, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:5; (e) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:5 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:12; (f) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:6 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:13; or (g) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:7 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:14. In a preferred embodiment, the anti-PD-1 Ab or antigen binding portion comprises a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:4 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:11. In another preferred embodiment, the anti-PD-1 Ab is nivolumab.

[0124] In one embodiment of the method of the present application, the anti-PD-1 Ab is formulated for intravenous administration. In a preferred embodiment, the Ab is administered intravenously at a dose of 3 mg / kg over 60 minutes every 2 weeks. Generally, treatment is continued as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

[0125] In clinical trials of anti-PD-1 immunotherapy described in the Examples below, interesting ORs with durable clinical responses, even in heavily pretreated patients, were observed across multiple tumor types, including a significant proportion of NSCLC, MEL, and RCC patients, and at various sites of metastasis, including liver, lung, lymph nodes, and bone. MEL and RCC are considered immunogenic neoplasms that have previously been shown to respond to cancer immunotherapy, for example, interferon-alpha and interleukin-2 in both MEL and RCC (Eton et al., 2002; Coppin et al., 2005; McDermott and Atkins, 2006) and anti-CTLA-4 Ab in MEL (Hodi et al., 2010). In MEL patients, significant ORR was observed with nivolumab across different doses of 0.1, 0.3, 1, 3 or 10 mg / kg with an overall response rate of about 31%, and similarly, a comparable ORR of about 30% was observed in RCC patients treated with nivolumab doses of 1 or 10 mg / kg (see Example 7). In contrast, immunotherapy has historically had a very low success rate in lung cancer. This failure has been attributed to NSCLC being "non-immunogenic" (see, e.g., Holt and Disis, 2008; Holt et al., 2011). The ability of lung cancer to thwart the immune system is due to multiple factors, including secretion of immunosuppressive cytokines, loss of major histocompatibility complex antigen expression, and co-opting of T cell inhibitory pathways (Dasanu et al., 2012; Marincola et al., 2000; Brahmer et al., 2012).

[0126] Despite the limited efficacy of immunotherapy historically seen in lung cancer and the various mechanisms used by lung cancer cells to evade immune system attack, various tumor cell vaccines (e.g., belagenpumatucel-L, a whole cell-based vaccine that blocks the action of TGF-β2) and antigen-based vaccines that enhance antigen-specific antitumor immunity (e.g., humoral EGF vaccines and vaccines that include MAGE-A3 fusion proteins or parts of tumor-associated MUC1 antigens) have been evaluated in clinical trials (Holt et al., 2011; Shepherd et al., 2011; Dasanu et al., 2012; Brahmer et al., 2012). However, while such antigen-specific vaccines have shown some promise for the treatment of NSCLC, Holt et al. (2011) described that trials of non-specific immunotherapeutic interventions have failed to improve outcomes in NSCLC, suggesting the need to combine them with antigen-specific vaccines. These authors opined that truly effective immunotherapy of NSCLC can only result from the implementation of strategies that both enhance antitumor immunity and neutralize tumor-mediated immunosuppression, and concluded that the absent or weak initial immune response in patients with NSCLC makes it unlikely that nonspecific stimulation of the immune system or removal of immunosuppression would produce a change in clinical outcome.

[0127] Therefore, the results reported herein in NSCLC are particularly remarkable, unexpected and surprising. In NSCLC patients, OR was observed with nivolumab doses of 1, 3 or 10 mg / kg, with response rates of 3%, 24% and 20%, respectively (see Example 7). OR was observed across NSCLC tissues: 9 responders (17%) out of 54 squamous and 13 (18%) out of 74 non-squamous. This level of activity seen with nivolumab in NSCLC patients with significant prior therapy (54% with 3 lines of prior therapy) and across tissues is unprecedented, especially in squamous tissue patients (see Gridelli et al., 2008; Miller, 2006), and offers a highly favorable benefit / risk dynamic in terms of efficacy and safety, compared to existing standard treatments.

[0128] In one embodiment of the immunotherapy of the present application, anti-PD-1 Abs are indicated as monotherapy locally to progressive or metastatic squamous or non-squamous NSCLC after platinum-based therapy (whether maintenance) and one other treatment. In another embodiment, anti-PD-1 Abs are indicated as monotherapy locally to progressive or metastatic squamous or non-squamous NSCLC after failure of platinum-based therapy and one other previous chemotherapy regimen. In a further embodiment, anti-PD-1 Abs are indicated as monotherapy locally to progressive or metastatic squamous or non-squamous NSCLC after failure of two lines of therapy, one of which must include a platinum-based regimen. In yet a further embodiment, anti-PD-1 Abs are indicated as monotherapy locally to progressive or metastatic squamous or non-squamous NSCLC after at least one previous chemotherapy, after failure of at least one previous platinum-based therapy, or after progression on at least one platinum doublet therapy. In all of the immunotherapy methods described herein, treatment may be continued as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

[0129] Durability of clinical responses to anti-PD-1 in heavily pretreated cancer patients The critical role of the PD-1 pathway in suppressing antitumor immunity, first shown in experimental models, is now being confirmed in clinical trials. As described herein, monotherapy with drugs that block PD-1 (nivolumab) or its main ligand PD-L1 (BMS-936559) can mediate regression in patients with treatment-refractory advanced cancer. The objective response rate (ORR) in heavily pretreated NSCLC patients receiving anti-PD-1 Ab, including those with squamous histology, is surprising and unexpected, since standard salvage therapy has historically shown little benefit in these patients (Scagliotti et al., 2011). The OR was long-term, with a median response duration of approximately 18 months in 22 of 129 responders in this study (see Example 7). In addition, a pattern of tumor regression was observed that was consistent with an immune-related pattern of response.

[0130] These findings establish the PD-1 pathway as a novel therapeutic focus in oncology (Pardoll, 2012; Topalian et al., 2012c; Hamid and Carvajal, 2013). In the current study, where 54% of patients had progressive disease after 3 or more previous systemic regimens, a preliminary analysis was performed up to March 2013. This latest analysis supports and strengthens the data obtained and conclusions reached from the previous February 2012 analysis. Thus, conventional OR or long-term disease stabilization was demonstrated in patients with NSCLC (17% and 10%, respectively), MEL (31%, 7%) and RCC (29%, 27%) across all doses tested (see Example 7). Furthermore, 13 patients (4%) showed an unusual "immune-related" response pattern as previously described with anti-CTLA-4 treatment, some of which were sustained (Sharma et al., 2011).

[0131] The persistence of OR across multiple cancer types in patients treated with anti-PD-1 Ab is particularly remarkable. The latest analysis again highlighted the persistence of clinical activity in nivolumab-treated patients, which has not been observed with chemotherapy or small molecule inhibitors in general to date, but has been observed in some patients with advanced melanoma receiving immunotherapy including ipilimumab and high doses of interleukin-2 (Topalian et al., 2011; Hodi et al., 2010). The persistence of partial tumor regression after drug cessation suggests that PD-1 blockade resets the immune equilibrium between tumor and host and that OS benefit may ultimately prove significantly longer than has been measured so far. Further follow-up is required to determine the ultimate persistence of tumor regression and disease stabilization in patients on these clinical trials.

[0132] Remarkably, the sustained objective tumor regression and disease stabilization induced by nivolumab in well-pretreated patients with advanced NSCLC, MEL and RCC translates into survival outcomes that are superior to historical data for these patient populations treated with conventional chemotherapy and / or tyrosine kinase inhibitor (TKI) treatment. In NSCLC, 1- and 2-year survival rates were 42% and 14%, respectively, with median OS of 9.2 and 10.1 months in patients with squamous and non-squamous cell carcinomas, respectively (see Example 7). This high level of efficacy is particularly impressive, since 54% of these patients had received three or more previous therapies. Furthermore, follow-up of many lung cancer patients was relatively limited, so these figures may change as data matures. Historically, 2L chemotherapy for lung cancer (i.e., docetaxel and pemetrexed) has achieved a median OS of 7.5-8.3 months, and a 1-year survival rate of approximately 30% (Shepherd et al., 2000; Hanna et al., 2004). In the 2L / 3L population, erlotinib-treated patients had a median survival of 6.7 months compared with 4.7 months in placebo-treated patients (Shepherd et al., 2005). No treatment is currently approved for use in lung cancer beyond the 3L setting, and minimal data exists to assess survival in this patient population, except for retrospective reviews that reported a median survival of 5.8-6.5 months and a 1-year survival of 25% (Girard et al., 2009; Scartozi et al., 2010).

[0133] In nivolumab-treated MEL patients, a median OS of 16.8 months was achieved with breakthrough survival rates of 62% (1 year) and 43% (2 years) (see Example 7). Survival outcomes in pretreated melanoma patients supported the recent FDA approval of ipilimumab and vemurafenib. In a recent Phase 3 study involving melanoma patients with at least one previous treatment for metastatic disease, ipilimumab increased median OS from 6.4 to 10.1 months compared with gp100 peptide vaccine (Hodi et al., 2010). In a Phase 2 study of ipilimumab in previously treated patients, 2-year survival rates ranged from 24.2-32.8% (Lebbe et al., 2012). In previously treated patients with BRAF mutant melanoma enrolled in a large phase 2 trial of vemurafenib, the median OS was 15.9 months, with 1-year survival of 58% ( Sosman et al., 2012 ).

[0134] Thus, in one embodiment, immunotherapy with anti-PD-1 Ab is indicated locally in progressive or metastatic MEL as monotherapy after treatment with dacarbazine (whether maintenance) and one other treatment. In other embodiments, anti-PD-1 Ab is indicated locally in progressive or metastatic MEL as monotherapy after failure of dacarbazine-based therapy. In further embodiments, anti-PD-1 Ab is indicated locally in progressive or metastatic MEL as monotherapy after failure of two lines of therapy, one of which must include a dacarbazine-based regimen. In yet further embodiments, anti-PD-1 Ab is indicated locally in progressive or metastatic MEL as monotherapy after at least one prior chemotherapy, after failure of at least one prior dacarbazine-based therapy, or after progression on at least dacarbazine therapy. In all of the immunotherapy methods described herein, treatment may be continued as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

[0135] In nivolumab-treated patients with RCC, the median OS was reached at 22 months (March 2013, date of analysis) in 45% who had received 3 or more previous therapies and 71% who had received previous antiangiogenic therapy. Breakthrough survival rates of 70% (1 year) and 50% (2 years) were observed (see Example 7). In a recent phase 3 study involving kidney cancer patients whose disease had progressed after antiangiogenic therapy, everolimus was compared to placebo: median OS was 14.8 vs. 14.4 months, respectively (Motzer et al., 2008; Motzer et al., 2010). A recent phase 3 study comparing sorafenib and temsirolimus in a sunitinib-refractory kidney cancer population resulted in a median OS of 16.6 and 12.3 months, respectively (Hutson et al., 2012). Thus, for NSCLC and MEL, treatment of heavily pretreated RCC patient populations with nivolumab resulted in a significantly longer median OS (>22 months) than treatment of poorly refractory populations with standard therapy. Controlled phase 3 trials with promising survival endpoints are ongoing in NSCLC, MEL and RCC (NCT01673867, NCT01721772, NCT01642004, NCT01668784 and NCT01721746 (Clinical Trials Website, http: / / www.clinicaltrials.gov Results from these trials are expected to further demonstrate the high efficacy and durability of response to nivolumab in these cancers compared with standard of care.

[0136] In one embodiment, immunotherapy with anti-PD-1 Ab is indicated locally in advanced or metastatic RCC as monotherapy after treatment (whether maintenance) with antiangiogenic TKI or mTOR inhibitor and one other treatment. In another embodiment, anti-PD-1 Ab is indicated locally in advanced or metastatic RCC as monotherapy after failure of treatment with antiangiogenic TKI or mTOR inhibitor. In a further embodiment, anti-PD-1 Ab is indicated locally in advanced or metastatic RCC as monotherapy after failure of two lines of treatment, one of which must include antiangiogenic TKI or mTOR inhibitor. In yet a further embodiment, anti-PD-1 Ab is indicated locally in advanced or metastatic RCC as monotherapy after at least one previous chemotherapy, after failure of at least one previous antiangiogenic TKI or mTOR inhibitor-based treatment, or after progression on at least antiangiogenic TKI or mTOR inhibitor treatment. Anti-PD-1 immunotherapy may be continued as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

[0137] Strikingly, OS was significantly longer than PFS in lung cancer, melanoma, and kidney cancer patients receiving nivolumab. These results mirror those reported for ipilimumab (Hodi et al., 2010) and reflect the observation that early tumor growth or development of new lesions can progress to disease stabilization or regression in some patients receiving immune checkpoint blockade. These findings suggest that progression-free survival may not be the optimal endpoint for determining the efficacy of nivolumab and other agents in this class.

[0138] The data described herein that demonstrates the high efficacy, durability and broad applicability of anti-PD-1 immunotherapy for treating cancer has led to nivolumab being tested against additional types of cancer.For example, increased PD-L1 expression has been reported in various hematological malignancies, and based on the fact that it can prevent host immune response from exerting beneficial effects on malignant cells, a study has been started to confirm the ability of nivolumab to mediate antitumor activity in patients with hematological malignancies (multiple myeloma, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, and chronic myelogenous leukemia) (NCT01592370).Nivolumab is also tested as a monotherapy in advanced hepatocellular carcinoma (NCT01658878).

[0139] In summary, the results of the anti-PD-1 immunotherapy described herein are remarkable in at least three ways: First, the efficacy of anti-PD-1 has been shown to surpass historical efficacy data for patients in standard of care treatment for cancer. Remarkably, this efficacy has been demonstrated in patients in a heavily pretreated population, where approximately half of the patients had progressive disease after three or more prior systemic regimens. Such patients suffering from progressive, metastatic and / or refractory cancers are notoriously difficult to treat. Thus, the present application provides a method for immunotherapy of patients suffering from progressive, metastatic and / or refractory cancers, comprising administering to the patient a therapeutically effective amount of an Ab or antigen-binding portion thereof that disrupts the interaction of PD-1 with PD-L1 and / or PD-L2. In any one embodiment of the therapeutic methods described herein, the subject has been pretreated for cancer; for example, the subject has received at least one, two, or three prior lines of therapy for cancer.

[0140] Second, the therapeutic methods of the present application have been shown to be applicable to a wide variety of cancers of various genera. Based on the surprising finding that even "non-immunogenic" cancers, such as NSCLC (Holt et al., 2011) and difficult to treat cancers, such as ovarian and gastric cancer (as well as other cancers that have been treated, including MEL, RCC, and CRC), are amenable to treatment with anti-PD-1 and / or anti-PD-L1 (see Examples 7 and 14), the present application generally provides methods for the immunotherapy of patients substantially afflicted with any of a very wide range of cancers.

[0141] Third, treatment with anti-PD-1 or anti-PD-L1 Abs has been shown to produce significant and sustained clinical activity in cancer patients. Thus, the present application provides an immunotherapeutic method for inducing a sustained clinical response in cancer patients, comprising administering to the patient a therapeutically effective amount of an Ab or antigen-binding portion thereof that disrupts the interaction of PD-1 with PD-L1 and / or PD-L2. In a preferred embodiment of any of the therapeutic methods described herein, the clinical response is a sustained response.

[0142] As used herein, a "durable" response is a therapeutic or clinical response that exceeds the expected median OS rate in a patient population. The expected median OS rate varies in different cancers and different patient populations. In one embodiment, a durable response exceeds the expected median OS rate in the relevant patient population by at least 10%, preferably at least 20%, more preferably at least 30%, and even more preferably at least 50%. The main benefit of the immunotherapeutic approach based on PD-1 pathway blockade may be the functional recovery of exhausted T cells with the long-term production of memory T cells that can maintain antitumor immune surveillance and inhibit tumor growth for a long period of time that extends to many years even in the absence of sustained treatment (Kim and Ahmed, 2010). Indeed, long-term follow-up studies of patients after cessation of nivolumab treatment have confirmed that a patient with CRC experienced a complete response that was ongoing after 3 years; a patient with RCC experienced a partial response that was ongoing for 3 treatment-free years, which converted to a complete response that was ongoing at 12 months; and a patient with melanoma achieved a partial response that was stable for 16 treatment-free months, and recurrent disease was successfully treated with re-induction anti-PD-1 therapy (Lipson et al., 2013).

[0143] Immune-related clinical responses It has become evident that conventional response criteria may not adequately evaluate the activity of immunotherapeutic agents, since progressive disease (by initial radiological assessment) does not necessarily reflect treatment failure. For example, treatment with the anti-CTLA-4 Ab ipilimumab has been shown to produce four distinct response patterns, all of which were associated with good survival: (a) shrinkage in baseline lesions without new lesions; (b) persistent stable disease (in some patients, then a slow and steady decrease in total tumor burden); (c) response after an increase in total tumor burden; and (d) response in the presence of new lesions. Thus, to adequately evaluate immunotherapeutic agents, the long-term effect on the target disease must also be captured. In this regard, systematic immune-related response criteria (irRC) have been proposed, which take into account the initial increase in tumor burden and / or the occurrence of new lesions and seek to enhance the characterization of immune-related response patterns (Wolchok et al., 2009). Although the full impact of these unconventional response patterns remains to be determined in randomized trials of nivolumab on survival endpoints, the present observations are reminiscent of findings with ipilimumab, where a significant prolongation of OS was observed in treated patients (Hodi et al., 2010; Robert et al., 2011).

[0144] The overall risk / benefit profile of anti-PD-1 immunotherapy is also favorable, with a low incidence of more severe drug-related adverse events (AEs; >grade 3), a particular event consistently observed with other immunotherapeutic agents to date, suggesting that anti-PD-1 immunotherapy could be delivered in an outpatient setting with minimal supportive care.

[0145] A wide range of cancers treatable with anti-PD-1 immunotherapy The clinical data presented herein demonstrates that immunotherapy based on PD-1 blockade is not limited to "immunogenic" tumor types, such as MEL and RCC, but extends to tumor types not generally considered to be immune-responsive, including NSCLC. The unexpected success with treatment-refractory metastatic NSCLC highlights the possibility that any neoplasm may be "immunogenic" in the context of appropriate immune modulation, and suggests that PD-1 blockade as an immunotherapeutic approach may be broadly applicable across a wide variety of tumor types. Thus, cancers that may be treated using the anti-PD-1 Abs of the present invention also include cancers that are generally responsive to immunotherapy, as well as cancers that are traditionally considered non-immunogenic. Non-limiting examples of cancers that are preferred for treatment include NSCLC, MEL, RCC, CRC, CRPC, HCC, squamous cell carcinoma of the head and neck, carcinomas of the esophagus, ovary, gastrointestinal tract and breast, and hematological malignancies. Although NSCLC is not considered to be generally responsive to immunotherapy, the data described herein unexpectedly demonstrate that both squamous and non-squamous NSCLC respond to treatment with anti-PD-1 Ab. Additionally, the present application provides treatment of refractory or recurrent malignancies whose growth can be inhibited using the anti-PD-1 Abs of the invention.

[0146] Based on the broad applicability of the anti-PD-1 immunotherapy provided herein, the method of the present invention is directed to the use of anti-PD-1 Examples of cancers that may be treated using Abs include liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, transrectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, childhood solid tumors, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, tumors of the spinal axis, tumors of the brain stem nervous system ... The present invention includes cancers including hematologic malignancies such as glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including asbestos-induced cancers, hematological malignancies such as multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combination of the above cancers. The present invention is also applicable to the treatment of metastatic cancers.

[0147] Medical Uses of Anti-PD-1 Ab One aspect of the present invention is the use of any of the anti-PD-1 Abs or antigen-binding portions thereof of the present invention for the manufacture of a medicament for inhibiting signaling from the PD-1 / PD-L1 pathway, thereby enhancing an endogenous immune response in a subject suffering from cancer. Another aspect is the use of any of the anti-PD-1 Abs or antigen-binding portions thereof of the present invention for the manufacture of a medicament for immunotherapy of a subject suffering from cancer, comprising disrupting the interaction between PD-1 and PD-L1. These uses for the manufacture of medicaments are broadly applicable to the full range of cancers described herein. In preferred embodiments of these uses, the cancers include squamous NSCLC, non-squamous NSCLC, MEL, RCC, CRC, CRPC, HCC, squamous cell carcinoma of the head and neck, and carcinomas of the esophagus, ovary, gastrointestinal tract and breast, and hematological malignancies. The present application also provides medical uses of any of the anti-PD-1 Abs or antigen-binding portions thereof of the present invention corresponding to all embodiments of the methods of treatment using the anti-PD-1 Abs described herein.

[0148] The present application also provides the anti-PD-1 Abs or antigen-binding portions thereof of the present invention for use in treating a subject suffering from cancer, comprising enhancing an endogenous immune response in the subject by inhibiting signaling from the PD-1 / PD-L1 pathway. The present application further provides the anti-PD-1 Abs or antigen-binding portions thereof of the present invention for use in immunotherapy of a subject suffering from cancer, comprising disrupting the interaction between PD-1 and PD-L1. These Abs may be used in enhancing an endogenous immune response against or in immunotherapy of the full range of cancers described herein. In preferred embodiments, the cancers include squamous NSCLC, non-squamous NSCLC, MEL (e.g., metastatic malignant MEL), RCC, CRC, CRPC, HCC, squamous cell carcinoma of the head and neck, and carcinomas of the esophagus, ovary, gastrointestinal tract and breast, and hematological malignancies.

[0149] Combination therapy including anti-PD-1 antibody Although monotherapy with anti-PD-1 and anti-PD-L1 Abs is shown herein to significantly increase the survival of patients with lung cancer, melanoma, renal cancer, and potentially other malignancies, preclinical data indicates that synergistic treatment combinations based on PD-1 pathway blockade can have even more potent effects. Clinical evaluation of nivolumab combined with ipilimumab (anti-CTLA-4), whose mechanism of action is different yet similar to nivolumab (Parry et al., 2005; Mellman et al., 2011; Topalian et al., 2012c), continues, and results from Phase 1 trials are provided herein (see also NCT01024231; NCT01844505; NCT01783938; Wolchok et al., 2013a; Wolchok et al., 2013b; Hodi et al., 2013). Clinical trials have also been initiated combining administration of nivolumab with melanoma vaccines (NCT01176461, NCT01176474; Weber et al., 2013), ipilimumab (BMS-986015), human IgG4 anti-KIR Ab in patients with advanced solid tumors (NCT01714739; Sanborn et al., 2013), cytokines, e.g., IL-21 in patients with advanced or metastatic solid tumors (NCT01629758; Chow et al., 2013), chemotherapeutic agents, e.g., platinum-based doublet chemotherapy in chemotherapy-naïve NSCLC patients (NCT01454102; Rizvi et al., 2013), and small molecule targeted therapy in patients with metastatic RCC (NCT01472081; Amin et al., 2013).

[0150] In one aspect, the present application relates to the combination of anti-PD-1 Abs with different cancer treatments, including chemotherapy regimens, radiation therapy, surgery, hormone deprivation, and angiogenesis inhibitors, for the treatment of various cancers. PD-1 blockade can also be effectively combined with immunogens, such as preparations of cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), antigen-presenting cells, such as dendritic cells bearing tumor-associated antigens, cells transfected with genes encoding immune-stimulating cytokines (He et al., 2004), and / or other immunotherapeutic Abs (e.g., anti-CTLA-4, anti-PD-L1, and / or anti-LAG-3 Abs). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1, and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.

[0151] Combination of anti-PD-1 and anti-CTLA-4 Abs for treating advanced melanoma Considering that immunological checkpoints are non-redundant and can inhibit T cell activation, proliferation and effector function in lymph nodes and / or the intratumor microenvironment, and based on preclinical data that a combination of anti-CTLA-4 and anti-PD-1 had a stronger antitumor effect in mouse tumor models than either Ab alone (see U.S. Pat. No. 8,008,449), the hypothesis that combined blockade of CTLA-4 and PD-1 could result in better antitumor activity than single agents was tested in a clinical trial in MEL patients (Example 15).

[0152] Although not formally compared in this study, the concurrent nivolumab / ipilimumab regimen achieved an ORR greater than that achieved by either nivolumab (Example 7) or ipilimumab alone (Hodi et al., 2010). Of great importance, rapid and deep responses were achieved in a significant proportion of treated patients, with a "deep" tumor response indicating a response in target lesions characterized by a reduction of 80% or more from baseline criteria by radiological assessment. In this study, the majority of responding patients, including those with extensive and bulky tumor burden, achieved >80% tumor regression at the time of first tumor evaluation. Particularly striking was the observation that 31% of response-evaluable patients treated with a concurrent regimen (comprising (i) an induction dosing schedule with combined administration of anti-PD-1 and anti-CTLA-4 Abs, followed by administration of anti-PD-1 Ab alone, and (ii) a maintenance dosing schedule with less frequent combined administration of anti-PD-1 and anti-CTLA-4 Abs) demonstrated >80% tumor regression by week 12. At the MTD for the concurrent regimen, all nine responding patients demonstrated >80% tumor regression, with three CRs. In contrast, in clinical experience to date, <3% of MEL patients receiving nivolumab or ipilimumab at 3 mg / kg achieved a CR (Example 7; Hodi et al., 2010). Thus, the overall activity of this immunotherapy combination in this preliminary Phase 1 trial compares favorably with other agents approved or in development for advanced melanoma, including targeted inhibitors of activated kinases (Chapman et al., 2011).An additional advantage of this combination is the durability of response, as demonstrated in this trial with nivolumab (as described herein) and ipilimumab, as well as in longer-term immunotherapy trials (Wolchok et al., 2013d).

[0153] These initial data suggest that rapid responses of better magnitude may be achieved in patients treated with the nivolumab / ipilimumab combination compared to the historical experience with either agent alone. Responses were generally durable and were observed even in patients in whom treatment was terminated early due to toxicity. Responding patients included patients with elevated LDH, M1c disease, and bulky multifocal tumor burden. As with previous reports on ipilimumab or nivolumab monotherapy, conventional ORRs do not fully capture the extent of clinical activity and possible benefit in patients treated with the concomitant nivolumab / ipilimumab regimen in that many patients experienced either prolonged SD or unconventional immune-related patterns of response. Indeed, of seven patients in the concomitant regimen with SD ≥ 24 weeks or irSD ≥ 24 weeks as the best OR, six demonstrated meaningful tumor regression of at least 19%, and the seventh patient had a reduction in tumor burden after prolonged SD. Previous experience with checkpoint blockade monotherapy supports the observation that some patients may survive long periods with SD as the best OR, providing evidence for the hypothesis that restoration of the equilibrium phase of immune surveillance is the desired outcome ( Screiber et al., 2011 ).

[0154] The observation that patients can achieve OR when treated sequentially with nivolumab after prior ipilimumab indicates that non-response to CTLA-4 blockade does not prevent clinical benefit from PD-1 blockade, further supporting the non-redundant nature of these co-inhibitory pathways. Remarkably, the data described herein (Example 8) suggests an association between the occurrence of response and tumor PD-L1 expression in patients receiving nivolumab, and previous data shows a correlation between increased OS and peripheral ALC in patients treated with ipilimumab (Berman et al., 2009; Ku et al., 2010; Postow et al., 2012; Delyon et al., 2013). In this study of the nivolumab / ipilimumab combination, clinical responses were observed in patients regardless of lymphocyte count or baseline tumor PD-L1 expression (Example 17), suggesting that the immune response generated by the combination therapy has unique properties compared to either monotherapy. The data suggest that baseline tumor PD-L1 expression and lymphocyte counts may be less relevant in the setting of active combination regimens that can induce rapid and significant tumor regression, but it is also noteworthy that different anti-PD-L1 Abs (rabbit mAb28-8 vs. mouse 5H1mAb) were used in different IHC assays to measure PD-L1 expression in combination therapy studies compared to nivolumab monotherapy studies. In addition to the changes in IHC assays and Abs, the different results may also reflect differences in biopsy samples and tumor heterogeneity. The utility of PD-L1 expression as a biomarker for anti-PD-1 efficacy will be further prospectively evaluated in randomized phase 3 studies (see, for example, NCT01721772, NCT01668784, and NCT01721746).

[0155] The range of adverse events observed in patients treated with the concurrent regimen was qualitatively similar to the experience with nivolumab or ipilimumab monotherapy, with the rate of AEs being increased in patients treated with the combination. Grade 3-4 treatment-related AEs were observed in 53% of patients treated with the concurrent nivolumab / ipilimumab regimen, compared with the historical rate of 20% in patients treated with ipilimumab monotherapy at a dose of 3 mg / kg (Hodi et al., 2010) and 17% in patients treated with nivolumab alone (Example 5). In the sequential regimen cohort, 18% of patients experienced grade 3-4 treatment-related AEs. AEs experienced by patients treated with the concurrent and sequential regimens were manageable and / or generally reversible using existing treatment algorithms.

[0156] Collectively, these results suggest that nivolumab and ipilimumab can be administered simultaneously with a manageable safety profile and induce durable clinical responses. More rapid and deeper clinical tumor responses were observed in patients treated with the combination compared to responses obtained with either single agent.

[0157] At the clinical cutoff date in February 2013 for the study described in Example 15, of 52 subjects on concurrent regimens who were evaluable for response, 21 (40%) had an OR by modified World Health Organization (mWHO) criteria (Wolchok et al., 2009). An additional 2 subjects (4%) had an unconfirmed OR. In cohort 1 (0.3 mg / kg nivolumab and 3 mg / kg ipilimumab), 3 of 14 evaluable subjects had an OR by mWHO (ORR: 21%, including 1 CR and 2 PR). In cohort 2 (1 mg / kg nivolumab and 3 mg / kg ipilimumab), 9 of 17 evaluable subjects had an OR by mWHO (ORR: 53%, including 3 CR and 6 PR). In cohort 2a (nivolumab 3 mg / kg and ipilimumab 1 mg / kg), 6 of 15 evaluable subjects had an OR by mWHO (ORR: 40%; including 1 CR and 5 PR). In cohort 3 (nivolumab 3 mg / kg and ipilimumab 3 mg / kg), 3 of 6 evaluable subjects had an objective response by mWHO (ORR: 50%; including 3 PR).Based on these data, the invention described herein provides a method of treating a subject suffering from cancer comprising administering to the subject (a) an Ab, or antigen-binding portion thereof, that specifically binds and inhibits PD-1; and (b) an Ab, or antigen-binding portion thereof, that specifically binds and inhibits CTLA-4, wherein each Ab is administered at a dose ranging from 0.1 to 20.0 mg / kg body weight in a concurrent regimen comprising (i) administering at least 2, 4, 6, 8, or 10 doses of a combination of anti-PD-1 and anti-CTLA-4 Abs, with a dosing frequency of at least once every 2, 3, or 4 weeks, or at least once a month, followed by (ii) administering at least 2, 4, 6, 8, or 10 doses of a combination of anti-PD-1 and anti-CTLA-4 Abs, with a dosing frequency of at least once every 2, 3, or 4 weeks, or at least once a month. and (ii) a maintenance dosing schedule comprising at least 4, 6, 8, 10, 12 or 16 doses of a combination of anti-PD-1 and anti-CTLA-4 Abs, at a dosing frequency of at least once every 8, 12 or 16 weeks, or at least once quarterly, or for as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

[0158] In one embodiment of this method, the maintenance dosing schedule comprises up to 4, 6, 8, 10, 12 or 16 doses of the combination of anti-PD-1 and anti-CTLA-4 Ab. In other embodiments, the concomitant regimen comprises (i) an induction dosing schedule comprising 2, 4, 6 or 8 doses of the combination of anti-PD-1 and anti-CTLA-4 Ab at a dosing frequency of once every 2, 3 or 4 weeks, or once a month, followed by 2, 4, 6, 8 or 12 doses of the anti-PD-1 Ab alone at a dosing frequency of once every 2, 3 or 4 weeks, or once a month; and then (ii) a maintenance dosing schedule comprising 4, 6, 8, 10, 12 or 16 doses of the combination of anti-PD-1 and anti-CTLA-4 Ab at a dosing frequency of once every 8, 12 or 16 weeks, or once a quarter, or for as long as clinical benefit is observed, or until unmanageable toxicity or disease progression occurs. In another embodiment, each anti-PD-1 and anti-CTLA-4 Ab is individually administered at a dose of 0.1, 0.3, 0.5, 1, 3, 5, 10 or 20 mg / kg. In a further embodiment, the dose of each anti-PD-1 and anti-CTLA-4 Ab is kept constant during the induction and maintenance dosing schedules.In yet other embodiments, the anti-PD-1 and anti-CTLA-4 Abs are at the following doses: (a) 0.1 mg / kg of anti-PD-1 Ab and 3 mg / kg of anti-CTLA-4 Ab; (b) 0.3 mg / kg of anti-PD-1 Ab and 3 mg / kg of anti-CTLA-4 Ab; (c) 1 mg / kg of anti-PD-1 Ab and 3 mg / kg of anti-CTLA-4 Ab; (d) 3 mg / kg of anti-PD-1 Ab and 3 mg / kg of anti-CTLA-4 Ab; (e) 5 mg / kg of anti-PD-1 Ab and 3 mg / kg of anti-CTLA-4 Ab; (f) 10 mg / kg of anti-PD-1 Ab and 3 mg / kg of anti-CTLA-4 Ab; (g) 0.1 mg / kg of anti-PD-1 Ab and 1 mg / kg of anti-CTLA-4 Ab; (h) 0.3 mg / kg of anti-PD-1 Ab and 1 mg / kg of anti-CTLA-4 Ab; (i) 1 mg / kg of anti-PD-1 Ab and 1 mg / kg of anti-CTLA-4 Ab; (j) 3 mg / kg of anti-PD-1 Ab and 1 mg / kg of anti-CTLA-4 Ab; (k) 5 mg / kg of anti-PD-1 Ab and 1 mg / kg of anti-CTLA-4 Ab; or (l) 10 mg / kg of anti-PD-1 Ab and 1 mg / kg of anti-CTLA-4 Ab.

[0159] In the protocol described in Example 15, the dosing regimen of 3 mg / kg nivolumab and 3 mg / kg ipilimumab exceeded the MTD (although combinations of ipilimumab and other anti-PD-1 Abs may have higher or lower MTDs), and both cohort 2 (1 mg / kg nivolumab and 3 mg / kg ipilimumab) and cohort 2a (3 mg / kg nivolumab and 1 mg / kg ipilimumab) had similar clinical activity. In addition, the majority of responses to the combination of nivolumab and ipilimumab occurred in the first 12 weeks. Considering the uncertainty of whether ipilimumab administered for 12 weeks contributes to clinical benefit, and the fact that the schedule approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for ipilimumab is once every 3 weeks for a total of 4 times, in a preferred embodiment, anti-CTLA-4 Ab is administered during the induction dosing schedule once every 3 weeks for a total of 4 times. Monotherapy treatment with nivolumab at 3 mg / kg every 2 weeks until progression has been shown to be associated with sustained responses (Examples 4-7), and a maintenance dosing schedule including administration of nivolumab every 12 weeks has been shown to be effective (Example 15). Thus, starting at week 12, which is after the completion of 4 combined nivolumab and ipilimumab, nivolumab at 3 mg / kg can be administered every 2 to at least 12 weeks until progression. Thus, in a preferred embodiment of the concomitant regimen method, the anti-PD-1 and anti-CTLA-4 Abs are administered at doses of (a) 1 mg / kg anti-PD-1 Ab and 3 mg / kg anti-CTLA-4 Ab; or (b) 3 mg / kg anti-PD-1 Ab and 1 mg / kg anti-CTLA-4 Ab, and the concomitant regimen further comprises (i) an induction dosing schedule comprising four doses of the combination of anti-PD-1 and anti-CTLA-4 Ab at a dosing frequency of once every three weeks, followed by four doses of anti-PD-1 alone at a dosing frequency of once every three weeks; and then (ii) a maintenance dosing schedule comprising up to eight doses of the combination of anti-PD-1 and anti-CTLA-4 antibodies at a dosing frequency of 2 to 12 or more weeks, or for as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

[0160] Exposure-response analysis of nivolumab monotherapy over a dose range of 1 mg / kg to 10 mg / kg shows similar clinical activity (Example 7), while exposure-response analysis of ipilimumab monotherapy at 0.3 mg / kg, 3 mg / kg and 10 mg / kg demonstrates increasing activity with increasing dose in Phase 2 studies (Wolchok et al., 2010). Thus, the 3 mg / kg ipilimumab dose (cohort 2) may be more clinically impactful than the choice of 3 mg / kg nivolumab (cohort 2a). Thus, in a more preferred embodiment of the simultaneous regimen method, anti-PD-1 and anti-CTLA-4 Ab are administered at doses of 1 mg / kg anti-PD-1 Ab and 3 mg / kg anti-CTLA-4 Ab.

[0161] In one embodiment of the method of the present application, the anti-PD-1 and anti-CTLA-4 Abs are formulated for intravenous administration. In one other embodiment, when the anti-PD-1 and anti-CTLA-4 Abs are administered in combination, they are administered within 30 minutes of each other. Either of the Abs may be administered first, i.e., in one embodiment, the anti-PD-1 Ab is administered before the anti-CTLA-4 Ab, while in another embodiment, the anti-CTLA-4 Ab is administered before the anti-PD-1 Ab. Generally, either of the Abs is administered intravenously over a period of 60 minutes. In a further embodiment, the anti-PD-1 and anti-CTLA-4 Abs are administered either simultaneously mixed as a single composition in a pharma- ceutically acceptable formulation for simultaneous administration, or simultaneously as a separate composition with either of the Abs in a pharma- ceutically acceptable formulation.

[0162] The data described in Example 7 demonstrate that immunotherapy with nivolumab produced significant clinical activity in MEL patients who did not respond to previous ipilimumab treatment. Thus, the present application provides a continuous regimen method for treating a subject suffering from cancer who has been previously treated with anti-CTLA-4 Ab, comprising administering to the subject up to 6 to up to 72 doses of an Ab that specifically binds and inhibits PD-1, or an antigen-binding portion thereof, at a dose ranging from 0.1 to 20.0 mg / kg body weight and at a dosing frequency of at least once a week, at least once every 2, 3 or 4 weeks, or at least once a month, or as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs. In one embodiment of this method, administration of anti-PD-1 Ab to the subject is initiated within 1-24 weeks after the last treatment with anti-CTLA-4 Ab. In other embodiments, administration of the anti-PD-1 Ab to the subject is initiated within 1, 2, 4, 8, 12, 16, 20, or 24 weeks after the subject's last treatment with an anti-CTLA-4 Ab. In a preferred embodiment, administration of the anti-PD-1 Ab is initiated within 4, 8, or 12 weeks after the subject's last treatment with an anti-CTLA-4 Ab. One embodiment of the method includes administering the anti-PD-1 Ab at a dose of 0.1-20 mg / kg, e.g., 0.1, 0.3, 0.5, 1, 3, 5, 10, or 20 mg / kg. In a preferred embodiment, the anti-PD-1 Ab is administered at a dose of 1 or 3 mg / kg. In one embodiment, the continuous regimen includes administering the anti-PD-1 Ab to the subject 6 to 72 times at a dosing frequency of once per week, once every 2, 3, or 4 weeks, or once per month, or as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs. In a preferred embodiment, anti-PD-1 is administered at a dose of 1 or 3 mg / kg at a dosing frequency of once every two weeks for up to 48 doses. In other preferred embodiments, the anti-PD-1 Ab is formulated for intravenous administration.

[0163] In one aspect of any of the simultaneous or sequential regimen methods of the present application, the treatment results in at least one therapeutic effect selected from a reduction in tumor size and / or growth, tumor elimination, a reduction in the number of metastatic lesions over time, a complete response, a partial response, and stable disease. Based on the wide range of cancers for which nivolumab has shown clinical responses, the combination therapy method of the present application can also be applied to a variety of cancers. Examples of cancers that can be treated by these methods include liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, transrectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, childhood solid tumors, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the CNS, primary CNS lymphoma, tumor angiogenesis, tumors of the spinal axis, brain stem glioma, pituitary adenoma, Kaposi's The present invention includes sarcomas, epidermoid carcinomas, squamous cell carcinomas, environmentally induced cancers including asbestos induced cancers, hematological malignancies such as multiple myeloma, B cell lymphoma, Hodgkin's lymphoma / primary mediastinal B cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, follicular lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combination of the above cancers. The present invention is also applicable to the treatment of metastatic, refractory or recurrent cancers. In a preferred embodiment, the cancer to be treated is selected from MEL, RCC, squamous NSCLC, non-squamous NSCLC, CRC, CRPC, OV, GC, HCC, PC, squamous cell carcinoma of the head and neck, carcinoma of the esophagus, gastrointestinal tract and breast, and hematological malignancies. In a more preferred embodiment, the cancer is MEL.

[0164] In one embodiment, the subject is pretreated for cancer. For example, the patient may have been treated with one or two or more previous systemic regimens of the type described herein as standard treatment. In one other embodiment, the cancer is progressive, recurrent, metastatic and / or refractory cancer. In a preferred embodiment, the simultaneous or sequential regimen treatment induces a sustained clinical response in the subject. In a preferred embodiment, the subject is a human, and the anti-PD-1 Ab inhibits human PD-1 and the anti-CTLA-4 Ab inhibits human CTLA-4.

[0165] The anti-PD-1 Ab used in the present methods can be any of the therapeutic anti-PD-1 Abs of the present invention. In a preferred embodiment, the anti-PD-1 Ab is a mAb, which can be a chimeric, humanized or human Ab. In one embodiment, the anti-PD-1 Ab comprises the CDR1, CDR2 and CDR3 domains in the heavy chain variable region and the CDR1, CDR2 and CDR3 domains in the light chain variable region of 17D8, 2D3, 4H1, 5C4 (nivolumab), 4A11, 7D3 or 5F4, respectively, as described and characterized in U.S. Patent No. 8,008,449. In a further embodiment, the anti-PD-1 Ab comprises the heavy and light chain variable regions of 17D8, 2D3, 4H1, 5C4 (nivolumab), 4A11, 7D3 or 5F4, respectively. In further embodiments, the anti-PD-1 Ab is 17D8, 2D3, 4H1, 5C4 (nivolumab), 4A11, 7D3, or 5F4. In preferred embodiments, the anti-PD-1 Ab is nivolumab.

[0166] The anti-CTLA-4 antibody of the present invention binds to human CTLA-4 so as to disrupt the interaction between CTLA-4 and human B7 receptor. Because the interaction between CTLA-4 and B7 transduces a signal that causes the inactivation of T cells that have CTLA-4 receptors, disrupting the interaction effectively induces, enhances or prolongs the activation of such T cells, thereby inducing, enhances or prolongs an immune response. Anti-CTLA-4 Abs are described, for example, in U.S. Patent Nos. 6,051,227, 7,034,121, in PCT Publications WO00 / 37504 and WO01 / 14424. A typical clinical anti-CTLA-4 Ab is human mAb 10D1 (now known as ipilimumab and commercially available as YERVOY®), as described in U.S. Patent No. 6,984,720. In one aspect of any of the methods of the present application, the anti-CTLA-4 Ab is a mAb. In one other embodiment, the anti-CTLA-4 antibody is a chimeric, humanized or human antibody. In a preferred embodiment, the anti-CTLA-4 antibody is ipilimumab.

[0167] The present application also relates to the use of an anti-PD-1 Ab, or an antigen-binding portion thereof, in combination with an anti-CTLA-4 Ab, or an antigen-binding portion thereof, for the manufacture of a co-administered medicament for treating a subject suffering from cancer in a concomitant regimen, wherein the anti-PD-1 and anti-CTLA-4 Abs are each administered at a dose ranging from 0.1 to 20.0 mg / kg body weight, and further wherein the concomitant regimen comprises (i) administering a combination of anti-PD-1 and anti-CTLA-4 Abs at least 2, 4, 6, 8, or 12 times, with a dosing frequency of at least once every 2, 3, or 4 weeks, or at least once a month, followed by (ii) administering a combination of anti-PD-1 and anti-CTLA-4 Abs at least 2, 3, or 4 weeks, or at least once a month, (ii) an induction dosing schedule comprising at least 2, 4, 6, 8, or 10 administrations of the Ab alone; then (iii) a maintenance dosing schedule comprising up to 4, 6, 8, 10, 12, or 16 administrations of the combination of anti-PD-1 and anti-CTLA-4 Abs, at a dosing frequency of at least once every 8, 12, or 16 weeks, or at least once a quarter, or for as long as clinical benefit is observed, or until unmanageable toxicity or disease progression occurs. The present application provides for the use of a combination of anti-PD-1 and anti-CTLA-4 Abs for the manufacture of a co-administered medicament corresponding to all aspects of the methods of treatment using these Abs described herein, and is broadly applicable to the full range of cancers described herein.

[0168] The present application also provides an anti-PD-1 Ab, or antigen-binding portion thereof, for use in combination with an anti-CTLA-4 Ab, or antigen-binding portion thereof, to treat a subject suffering from cancer in a concomitant regimen, comprising anti-PD-1 and anti-CTLA-4 Abs, or antigen-binding portions thereof. Each Ab is administered at a dose ranging from 0.1 to 20.0 mg / kg body weight, and the concomitant regimen further comprises: (i) an induction dosing schedule comprising at least 2, 4, 6, 8, or 12 doses of the combination of anti-PD-1 and anti-CTLA-4 antibodies, at a dosing frequency of at least once every 2, 3, or 4 weeks, or at least once a month, followed by at least 2, 4, 6, 8, or 10 doses of anti-PD-1 alone, at a dosing frequency of at least once every 2, 3, or 4 weeks, or at least once a month; and then (ii) a maintenance dosing schedule comprising up to 4, 6, 8, 10, 12, or 16 doses of the combination of anti-PD-1 and anti-CTLA-4 antibodies, at a dosing frequency of at least once every 8, 12, or 16 weeks, or at least once a quarter, or for as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

[0169] Methods for screening patient populations and selecting patients as suitable for immunotherapy with combined anti-PD-1 and anti-CTLA-4 using simultaneous or sequential regimens based on the PD-L1 biomarker assay, and methods for predicting efficacy of Ab combinations, are performed as described for anti-PD-1 monotherapy, to the extent that this biomarker is applicable to combination therapy with anti-PD-1 and anti-CTLA-4 Abs.

[0170] The present application further provides a kit for treating a subject suffering from cancer, comprising: (a) a dose of an Ab or antigen-binding portion thereof that specifically binds and inhibits PD-1, in the range of 0.1 to 20.0 mg / kg body weight; (b) a dose of an Ab or antigen-binding portion thereof that specifically binds and inhibits CTLA-4, in the range of 0.1 to 20.0 mg / kg body weight; and (c) instructions for using a combination of anti-PD-1 and anti-CTLA-4 antibodies in any of the co-regimens. In one embodiment, several doses of anti-PD-1 and anti-CTLA-4 Abs are mixed within a single pharmaceutical formulation for co-administration. In another embodiment, the doses of anti-PD-1 and anti-CTLA-4 Abs are formulated as separate compositions with either of the Abs in a pharma-ceutically acceptable formulation.

[0171] The application further provides kits for treating a subject suffering from cancer comprising: (a) a dose ranging from 0.1 to 20.0 mg / kg body weight of an Ab or antigen-binding portion thereof that specifically binds and inhibits PD-1; and (b) instructions for using the anti-PD-1 Ab in any of the sequential regimen methods.

[0172] The combined PD-1 and CTLA-4 blockade can also be further combined with standard cancer treatment. For example, the combined PD-1 and CTLA-4 blockade can be effectively combined with chemotherapy regimens for the treatment of MEL, for example, in further combination with dacarbazine or IL-2. In these examples, it can be possible to reduce the dose of chemotherapy drugs. The scientific rationale is that after the combined use of PD-1 and CTLA-4 blockade and chemotherapy, cell death, which is the result of the cytotoxic action of many chemotherapy compounds, should result in increased levels of tumor antigens in the antigen presentation pathway. Other combination treatments that can result in synergy with the combined PD-1 and CTLA-4 blockade through cell death include radiation therapy, surgery, or hormone deprivation. Each of these protocols creates a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with the combined PD-1 and CTLA-4 blockade. Inhibition of angiogenesis causes tumor cell death, which can also be a source of tumor antigens fed into the host antigen presentation pathway.

[0173] Immunotherapy for cancer patients using anti-PD-L1 antibodies PD-L1, PD-1-positive, tumor-infiltrating CD4 + and CD8 +PD-L1 is the predominant PD-1 ligand upregulated in solid tumors where it can inhibit the respective cytokine production and cytolytic activity of T cells (Dong et al., 2002; Hino et al., 2010; Taube et al., 2012). These properties make PD-L1 a promising target for cancer immunotherapy. The clinical trial of anti-PD-L1 immunotherapy described in the Examples demonstrates for the first time that mAb blockade of the immunoinhibitory ligand PD-L1 results in both durable tumor regression and long-term (≧24 weeks) disease stabilization in patients with metastatic NSCLC, MEL, RCC and OV, including patients with extensive prior therapy. BMS-936559, a human anti-PD-L1 HuMAb, had a favorable safety profile across doses up to 10 mg / kg, as evidenced by the low (9%) incidence of grade 3-4 drug-related AEs. These findings support the conclusion that PD-L1 is a promising target for cancer immunotherapy. - / - Mild autoimmune phenotype seen in mice (Dong et al., 2004) and PD-1 - / - Compared with mice, CTLA-4 - / - This is consistent with the more severe hyperproliferation seen in mice (Phan et al., 2003; Tivol et al., 1995; Nishimura et al., 1999). Many of the toxicities associated with anti-PD-L1 treatment in patients were immune-related, suggesting a favorable response. The range and frequency of adverse events of special interest (AEOSI) differed slightly between anti-PD-L1 and anti-CTLA-4, highlighting the different biology of these pathways (Ribas et al., 2005). Although infusion reactions were observed with BMS-936559, they were mild in most patients. Severe colitis, a drug-related AE observed in ipilimumab-treated patients (Beck et al., 2006), was rarely seen with anti-PD-L1.

[0174] As noted above for anti-PD-1 immunotherapy, another important attribute of anti-PD-L1 treatment is the durability of responses across multiple tumor types. This is particularly notable given the advanced disease and prior treatment of the patients in the current study. Although not a direct comparison, this durability appears to be better than that observed with most chemotherapies and kinase inhibitors used to treat these cancers.

[0175] Because peripheral blood T-cells express PD-L1, in vivo RO with BMS-963559 can be assessed as a pharmacokinetic criterion. Median RO was 65.8%, 66.2%, and 72.4% for the doses tested. Although these studies provide direct assessment and evidence of target engagement in patients treated with BMS-936559, the relationship between RO in peripheral blood and the tumor microenvironment remains poorly understood.

[0176] Based on the clinical data described herein, the application provides a method for immunotherapy of a subject suffering from cancer, comprising administering to the subject a composition comprising a therapeutically effective amount of an anti-PD-L1 Ab or antigen-binding portion thereof of the invention. The application also provides a method for inhibiting tumor cell growth in a subject, comprising administering to the subject an anti-PD-L1 Ab or antigen-binding portion thereof of the invention. In a preferred embodiment, the subject is a human. In one embodiment, the Ab or antigen-binding portion thereof is an IgG1 or IgG4 isotype. In other embodiments, the Ab or antigen-binding portion thereof is a mAb or antigen-binding portion thereof. In a further embodiment, the Ab or antigen-binding portion thereof is a chimeric, humanized or human Ab or antigen-binding portion thereof. In a preferred embodiment for treating a human patient, the Ab or antigen-binding portion thereof is a human Ab or antigen-binding portion thereof.

[0177] The clinical trial described in the Examples used anti-PD-L1 HuMAb BMS-936559 to treat cancer. While BMS-936559 (referred to as HuMAb 12A4 in U.S. Patent No. 7,943,743) was selected as the lead anti-PD-L1 Ab to enter the clinical trial, it is notable that several of the anti-PD-L1 Abs of the present invention share 12A4 functional properties that are important for the therapeutic activity of 12A4, such as binding specifically to human PD-L1 with high affinity, increasing T-cell proliferation, IL-2 secretion and interferon-γ production in an MLR assay, inhibiting the binding of PD-L1 to PD-1, and reversing the suppressive effect of T regulatory cells on T cell effector cells and / or dendritic cells. Furthermore, certain of the anti-PD-L1 Abs of the present invention, namely 1B12, 7H1 and 12B7, have been shown to inhibit V-cell proliferation, V-cell proliferation, V-cell secretion and V-cell cytotoxicity, respectively. H 1-69 and V κ V with sequences derived from the L6 germline sequence H and V κ 12A4 is structurally related to 12A4, including the PD-L1 region. In addition, at least 12B7, 3G10, 1B12, and 13G4 cross-compete with 12A4 for binding to the same epitope region of hPD-L1, while 5F8 and 10A5 can bind to the same or overlapping epitope region as 12A4 (Examples 2 and 3). Thus, the preclinical characterization of 12A4 and other anti-PD-L1 HuMabs indicates that the methods of treating cancer provided herein may be practiced using any of the broad genus of anti-PD-L1 Abs of the invention.

[0178] Accordingly, the present application provides: (a) a human V H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 1-18 germline sequence, and a human V κ (b) a light chain variable region comprising contiguously linked amino acids having a sequence derived from the L6 germline sequence; H A heavy chain variable region comprising contiguous amino acids having a sequence derived from the 1-69 germline sequence, and a human V κ(c) a light chain variable region comprising contiguously linked amino acids having a sequence derived from the L6 germline sequence; H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 1-3 germline sequence, and a human V κ (d) a light chain variable region comprising contiguously linked amino acids having a sequence derived from the L15 germline sequence; H A heavy chain variable region comprising contiguous amino acids having a sequence derived from the 1-69 germline sequence, and a human V κ (e) a light chain variable region comprising contiguously linked amino acids having a sequence derived from the A27 germline sequence; H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 3-9 germline sequence, and a human V κ (f) a light chain variable region comprising contiguously linked amino acids having a sequence derived from a human V15 germline sequence; or H A heavy chain variable region comprising contiguously linked amino acids having a sequence derived from the 3-9 germline sequence, and a human V κ The present invention provides a method of immunotherapy comprising administering to a patient an anti-PD-L1 Ab, or an antigen-binding portion thereof, comprising a light chain variable region comprising contiguously linked amino acids having a sequence derived from the L18 germline sequence.

[0179] In one embodiment, the anti-PD-L1 Ab, or antigen-binding portion thereof, administered to the patient has a binding activity against PD-L1 that is: (a) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 15, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 25; (b) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 16, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 26; (c) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 17, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 27; (d) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 18, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 28; (e) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 19, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 20; (f) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:20 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:30; (g) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:21 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:31; (h) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:22 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:32; (i) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:23 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:33;or (j) cross-competes with a reference Ab, or a reference antigen-binding portion thereof, comprising a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:24 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:34. In a preferred embodiment, the Ab, or antigen-binding portion thereof, cross-competes for binding to PD-1 with a reference Ab, or a reference antigen-binding portion thereof, comprising a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:16 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:26;

[0180] In one preferred embodiment of the immunotherapy methods described herein, the anti-PD-L1 Ab, or antigen-binding portion thereof, administered to the subject comprises: (a) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 15, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 25; (b) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 16, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 26; (c) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 17, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 27; (d) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 18, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 28; (e) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 19. (f) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:20 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:30; (g) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:21 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:31; (h) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:22 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:32; (i) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:23 and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:33;or (j) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:24, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:34. In a more preferred embodiment, the anti-PD-L1 Ab or antigen binding portion comprises a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:16, and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:26;

[0181] A wide range of cancers treatable with anti-PD-L1 immunotherapy The clinical activity of anti-PD-L1 in patients with advanced NSCLC was surprising and unexpected, as was the activity of anti-PD-1 in these patients, since NSCLC is thought to respond poorly to immune-based therapy (Holt and Disis, 2008; Holt et al., 2011). The present clinical data obtained with the inventive anti-PD-L1 Ab, BMS-936559, validates and extends the evidence obtained using anti-PD-1 Abs that immunotherapy based on PD-1 blockade is not only applicable to "immunogenic" tumor types, such as MEL and RCC, but is also effective in a broad range of cancers, including treatment-refractory metastatic NSCLC, which is not generally considered to be responsive. Preferred cancers that may be treated using the anti-PD-L1 Abs of the invention include MEL (e.g., metastatic melanoma), RCC, squamous NSCLC, non-squamous NSCLC, CRC, ovarian cancer (OV), gastric cancer (GC), breast cancer (BC), pancreatic carcinoma (PC) and esophageal carcinoma. Additionally, the invention includes refractory or recurrent malignancies whose growth may be inhibited using the anti-PD-L1 Abs of the invention.

[0182] Thus, based on the indications provided herein with a very broad range of applicability of anti-PD-L1 immunotherapy, examples of cancers that may be treated using anti-PD-L1 Abs in the methods of the invention include bone cancer, skin cancer, cancer of the head and neck, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, kidney cancer, uterine cancer, castration-resistant prostate cancer, colon cancer, transrectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, carcinoma of the ovaries, gastrointestinal tract and breast, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, acute myeloid leukemia, and the like. The present invention includes chronic or acute leukemias including leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphomas, tumor angiogenesis, tumors of the spinal axis, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, multiple myeloma, environmentally induced cancers including asbestos induced cancers, metastatic cancers, and any combination of the above cancers. The present invention is also applicable to the treatment of metastatic cancers.

[0183] Combination therapy with anti-PD-L1 Ab Optionally, Abs against PD-L1 can be combined with immunogens, such as preparations of cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), antigen-presenting cells, such as dendritic cells bearing tumor-associated antigens, and cells transfected with genes encoding immune-stimulating cytokines (He et al., 2004). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. PD-L1 blockade can also be effectively combined with standard cancer treatments, including chemotherapy regimens, radiation therapy, surgery, hormone deprivation, and angiogenesis inhibitors, as well as other immunotherapeutic Abs (e.g., anti-PD-1, anti-CTLA-4, or anti-LAG-3 Abs).

[0184] Use of anti-PD-L1 Ab The application provides the use of any anti-PD-L1 Ab, or antigen-binding portion thereof, of the invention for the manufacture of a medicament for inhibiting signaling from the PD-1 / PD-L1 pathway, thereby enhancing an endogenous immune response in a subject suffering from cancer. The application also provides the use of any anti-PD-L1 Ab, or antigen-binding portion thereof, of the invention for the manufacture of a medicament for immunotherapy of a subject suffering from cancer, comprising disrupting the interaction between PD-1 and PD-L1. The application provides medical uses of any anti-PD-L1 Ab, or antigen-binding portion thereof, of the invention, corresponding to all embodiments of the methods of treatment using the anti-PD-L1 Ab described herein.

[0185] The present application also provides the anti-PD-L1 Abs or antigen-binding portions thereof of the present invention for use in treating a subject suffering from cancer, including enhancing an endogenous immune response in a subject suffering from cancer by inhibiting signaling from the PD-1 / PD-L1 pathway. The present application further provides the anti-PD-L1 Abs or antigen-binding portions thereof of the present invention for use in immunotherapy of a subject suffering from cancer, including disrupting the interaction between PD-1 and PD-L1. These Abs may be used in enhancing an endogenous immune response against or in immunotherapy of the full range of cancers described herein. In preferred embodiments, the cancers include MEL (e.g., metastatic malignant MEL), RCC, squamous NSCLC, non-squamous NSCLC, CRC, ovarian cancer (OV), gastric cancer (GC), breast cancer (BC), pancreatic carcinoma (PC) and esophageal carcinoma.

[0186] Validation of cancer immunotherapy by immune checkpoint blockade A significant consequence of the clinical activity of immune checkpoint blockade is that significant endogenous immune responses against tumor antigens are generated, and these responses can be therapeutically exploited to mediate clinical tumor regression upon checkpoint inhibition. Indeed, there is evidence of a mechanism termed adaptive resistance, in which inhibitory ligands, such as PD-L1, are induced in response to immune attack (Gajewski et al., 2010; Taube et al., 2012). This possible mechanism of tumor-induced immune resistance suggests that PD-1 / PD-L1-directed therapy may synergize with other treatments that augment endogenous antitumor immunity. Follow-up studies have demonstrated that patients continue to show tumor control after cessation of PD-1 / PD-L1 pathway blockade (Lipson et al., 2013). Such tumor control may be reflected in the generation of an effective immune memory that allows for sustained antitumor immune responses and sustained control of tumor growth.

[0187] The data described herein in clinical trials of Abs blocking the immunomodulatory receptor PD-1 and one of its cognate ligands, PD-L1, are unprecedented. These data constitute the largest clinical experience to date with PD-1 pathway-directed cancer immunotherapy and the first report to specifically describe the safety, tolerability, and initial clinical activity of anti-PD-L1-directed agents. These findings indicate that both anti-PD-1 and anti-PD-L1 have a favorable overall safety profile and provide clear evidence of clinical activity across a variety of cancers, including NSCLC, tumors not historically considered to respond to immunotherapy, as well as tumors known to respond to immunotherapy, including MEL, RCC, and OV. Thus, these data strongly validate the PD-1 / PD-L1 pathway as an important target for therapeutic intervention in cancer.

[0188] The striking similarities observed between the patterns of clinical activity observed with anti-PD-1 and anti-PD-L1 mAbs in the tumor types analyzed to date demonstrate the general importance of the PD-1 / PD-L1 signaling pathway in tumor immune resistance and as a target for therapeutic intervention. Although the molecular interactions blocked by these two Abs are not identical, regardless of the mechanistic details, it is clearly demonstrated herein that both the anti-PD-1 and anti-PD-L1 Abs of the present invention are useful in treating patients suffering from a wide variety of cancers.

[0189] infectious disease Other methods of the invention are used to treat patients who have been exposed to a particular toxin or pathogen. For example, another aspect of the application provides a method of treating an infectious disease in a subject, comprising administering to the subject an anti-PD1 or anti-PD-L1 Ab of the invention, or an antigen-binding portion thereof, such that the subject is treated for the infectious disease. Preferably, the Ab is a human anti-human PD-1 or PD-L1 Ab (e.g., any human Ab described herein). Alternatively, the Ab is a chimeric or humanized Ab.

[0190] Similar to tumor applications as described above, Ab-mediated PD-1 or PD-L1 blockade can be used alone or in combination with a vaccine as an adjuvant to enhance immune responses against pathogens, toxins, and / or self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful include those for which there is currently no effective vaccine or for which conventional vaccines are not fully effective. These include, but are not limited to, HIV, hepatitis (A, B, and C), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa. PD-1 and / or PD-L1 blockade is particularly useful against established infections, such as HIV, with agents that present antigens that have changed during infection. The novel epitopes on these antigens are recognized as foreign upon anti-human PD-1 or PD-L1 administration, thus inducing a strong T cell response that is not suppressed by negative signals via the PD-1 / PD-L1 pathway.

[0191] In the above methods, PD-1 or PD-L1 blockade can be combined with other forms of immunotherapy, such as cytokine treatment (e.g., administration of interferon, GM-CSF, G-CSF or IL-2).

[0192] kit Also within the scope of the present invention are pharmaceutical kits comprising a combination of anti-PD-1 and / or anti-PD-L1 Abs of the present invention, or anti-PD-1 and anti-CTLA-4 Abs for therapeutic use, and diagnostic kits comprising anti-PD-L1 Abs of the present invention for assaying membrane PD-L1 expression as a biomarker for screening patients for immunotherapy or to predict the efficacy of immunotherapeutic agents. The kits will generally include a label that supports the intended use of the contents of the kit and instructions for use. The term label includes any writing, or other written material provided on or with the kit, or otherwise associated with the kit. In one embodiment of the pharmaceutical kit, the anti-PD1 and / or anti-PD-L1 Abs may be packaged with other therapeutic agents in unit dosage form. In one embodiment of the diagnostic kit, the anti-PD-L1 Abs may be packaged with other reagents for performing an assay to detect and / or quantify PD-L1 expression.

[0193] In one preferred embodiment, the pharmaceutical kit comprises the anti-human PD-1 HuMAb Nivolumab. In another preferred embodiment, the pharmaceutical kit comprises the anti-human PD-L1 HuMAb BMS-936559. In yet another preferred embodiment, the pharmaceutical kit comprises the anti-human CTLA-4 HuMAb Ipilimumab. In one preferred embodiment, the diagnostic kit comprises the V-cell fusion protein VIII, the amino acid sequences of which are set forth in SEQ ID NOs: 35 and 36, respectively. H and V κ In another preferred embodiment, the diagnostic kit comprises the mouse anti-human PD-L1 mAb, 5H1 (Dong et al., 2002).

[0194] PD-L1 biomarkers for predicting anti-PD-1 efficacy A particular challenge in cancer immunotherapy is the identification of mechanism-based predictive biomarkers that can guide patient selection and treatment management. The data described in the Examples below show that cell surface PD-L1 expression in tumors is a useful molecular marker for predicting the efficacy of, or selecting patients for, immunotherapy with anti-PD-1 and potentially other immune checkpoint inhibitors.

[0195] There are conflicting reports in the literature about the clinical significance of PD-L1 expressed in tumors. Several studies have concluded that PD-L1 expression in tumors correlates with poor prognosis for patients. See, for example, Hino et al. (2010) (MEL); Hamanishi et al. (2007) (OV); Thompson et al. (2006) (RCC). These findings can be rationalized on the basis that the interaction of PD-L1 on tumor cells and PD-1 on T cells helps to subvert the immune response against tumors and causes immune evasion from tumor-specific T cells (Blank et al., 2005). However, in contrast to the above studies, Gadiot et al. (2011) and Taube et al. (2012) have recently reported that PD-L1 expression in melanoma tumors correlates with a tendency for better survival. These seemingly contradictory data may reflect the relatively small number of patients analyzed, the different histological subtypes tested, or the different methodologies used, e.g., the use of different Abs to stain PD-L1, the use of frozen versus paraffin-embedded material for IHC, and the detection of membrane and / or cytoplasmic staining of PD-L1. Taube et al. (2012) describe PD-L1 as a type I transmembrane molecule and hypothesize that the cytoplasmic presence of PD-L1 may indicate an intracellular store of this polypeptide that can be located at the cell surface upon appropriate stimulation, but that it is cell surface PD-L1 expression that is biologically relevant as a potential biomarker for predicting clinical response to PD-1 blockade. See also Brahmer et al. (2010), who describe preliminary evidence of a correlation between membrane PD-L1 expression and anti-PD-1 efficacy, obtained in a small sample size of only nine patients. The data obtained from the analysis of large samples and described in the Examples below on the use of membrane PD-L1 expression as a biomarker for anti-PD-1 efficacy support the hypothesis that PD-L1 expression can be used as a biomarker to predict anti-PD-1 clinical response and to screen patients to identify suitable candidates for immunotherapy with anti-PD-1 Abs.Although the utility of this biomarker for screening patients for or predicting clinical response to anti-PD-1 immunotherapy has been demonstrated, PD-L1 expression may also potentially be applied more broadly as a concomitant biomarker for other types of inhibitors of inhibitory immune regulators.

[0196] Specifically, membrane PD-L1 expression was assayed using an automated IHC protocol and rabbit anti-hPD-L1 Ab. Strikingly, in the first set of analyzed data (see Example 8), patients with cell surface PD-L1-negative tumors (MEL, NSCLC, CRC, RCC, and CRPC) did not experience ORs after treatment with the anti-PD-1 Ab, nivolumab. In contrast, cell surface expression of PD-L1 on tumor cells in pretreatment biopsies may be associated with an increased rate of ORs in patients treated with nivolumab. Although tumor cell expression of PD-L1 may be driven by a constitutive oncogenic pathway, it may reflect "adaptive immune resistance" in response to an intrinsic anti-tumor immune response that is part of the host inflammatory response, which may be maintained unless relieved by blockade of the PD-1 / PD-L1 pathway (Taube et al., 2012). This new concept of adaptive immune resistance in cancer immunology suggests that inhibitory ligands, such as PD-L1, are induced in response to immune attack (Gajewski et al., 2010; Taube et al., 2012). A significant consequence of the clinical activity of immune checkpoint blockade described herein is that significant endogenous immune responses to tumor antigens are generated, and these responses can be therapeutically exploited to mediate clinical tumor regression upon checkpoint inhibition. This possible mechanism of tumor-induced immune resistance suggests that PD-1 / PD-L1-directed therapy may synergize with other treatments that augment endogenous antitumor immunity.

[0197] Assaying cell surface PD-L1 expression by automated IHC As described in the Examples, an automated IHC method has been developed to assay the expression of PD-L1 on the surface of cells in FFPE tissue specimens. The present application provides a method for detecting the presence of human PD-L1 antigen in a test tissue sample or quantifying the level of human PD-L1 antigen or the percentage of cells in the sample expressing the antigen, comprising contacting the test sample and a negative control sample with a mAb that specifically binds to human PD-L1 under conditions that allow the formation of a complex between the Ab or a portion thereof and human PD-L1. Preferably, the test and control tissue samples are FFPE samples. The formation of the complex is then detected, and a difference in the complex formation between the test sample and the negative control sample indicates the presence of human PD-L1 antigen in the sample. Various methods can be used to quantify PD-L1 expression.

[0198] In certain embodiments, the automated IHC method includes: (a) deparaffinizing and rehydrating mounted tissue sections in an autostainer; (b) retrieving antigens using a decloaking chamber heated to 110° C. for 10 minutes and a pH 6 buffer; (c) loading reagents onto the autostainer; and (d) running the autostainer to include a step of neutralizing endogenous peroxidase in the tissue specimen; blocking nonspecific protein binding sites on the slide; incubating the slide with primary Ab; incubating with post-primary blocking agent; incubating with NovoLink Polymer; adding chromogenic substrate and developing; and counterstaining with hematoxylin.

[0199] To assess PD-L1 expression in tumor tissue samples, pathologists performed membrane PD-L1 screening in each region under a microscope. +The number of tumor cells is observed and the percentage of cells that are positive is mentally estimated and then averaged to arrive at the final percentage. Various staining intensities are defined as 0 / negative, 1+ / weak, 2+ / moderate, and 3+ / strong. Generally, percentage values ​​are first assigned to 0 and 3+ buckets, and then the intermediate 1+ and 2+ intensities are considered. In highly heterogeneous tissues, the specimen is divided into zones, and each zone is scored separately and then combined into a single set of percentage values. The percentage of negative and positive cells for the various staining intensities is determined from each area, and the median value is assigned to each zone. Final percentage values ​​are given to the tissue for each staining intensity category: negative, 1+, 2+, and 3+. The sum of all staining intensities should equal 100%.

[0200] Staining is also evaluated in tumor-infiltrating inflammatory cells, such as macrophages and lymphocytes. In most cases, macrophages serve as an internal positive control, since staining is observed in the majority of macrophages. Although it is not necessary to stain with 3+ intensity, the absence of staining in macrophages should be considered to exclude any technical failure. Macrophages and lymphocytes are only evaluated for cell membrane staining and scored for all samples as positive or negative for the respective cell category. Staining is also characterized according to an external / internal tumor immune cell designation. "Internal" means that the immune cells are within the tumor tissue and / or on the border of the tumor area, without being physically inserted into the tumor cells. "External" means that there is no physical association with the tumor, and the immune cells are found in the periphery, either in conjunction with or associated with any relevant adjacent tissue.

[0201] In one embodiment of these scoring methods, samples are scored by two pathologists working independently and then the scores are combined. In another embodiment, the identification of positive and negative cells is scored using appropriate software.

[0202] The Histoscore is used as an additional quantitative measure of the IHC data. The Histoscore is calculated as follows: Histoscore = [(%Tumor x1 (low intensity)) + (%Tumor x2 (medium intensity)) + (%Tumor x3 (high intensity)]

[0203] To determine the HistoScore, a pathologist estimates the percentage of staining cells in each intensity category within a specimen. Because expression of many biomarkers is heterogeneous, the HistoScore is a more accurate representation of total expression. Final HistoScore ranges from 0 (no expression) to 300 (maximal expression).

[0204] An alternative means of quantifying PD-L1 expression in test tissue samples IHC is to determine the adjusted inflammation score (AIS), defined as multiplying the density of inflammation with the percentage of PD-L1 expression by tumor-infiltrating inflammatory cells ( Taube et al., 2012 ).

[0205] Anti-PD-1 cancer immunotherapy including patient selection process The application also provides a method for immunotherapy of a subject suffering from cancer comprising: (a) selecting a subject who is a suitable candidate for immunotherapy, e.g., administration of an anti-PD-1 Ab, comprising: (i) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells, (ii) assessing the percentage of cells in the test tissue sample that express PD-L1 on their cell surface, and (iii) selecting the subject as a suitable candidate based on assessing that the percentage of cells in the test tissue sample that express PD-L1 on their cell surface exceeds a predetermined threshold level; and (b) administering to the selected subject a therapeutically effective amount of an agent that inhibits signaling from an inhibitory immune regulator, e.g., a composition comprising an anti-PD-1 Ab.

[0206] There is evidence that membrane PD-L1 expression is a surrogate for the endogenous antitumor immune response that is part of the host inflammatory response (Gajewski et al., 2010; Taube et al., 2012). Thus, cell surface expression of PD-L1 on tumor and / or inflammatory cells in the tumor microenvironment may be a marker for selecting cancer patients who will benefit not only from treatment with anti-PD-1 Abs, but also from treatment with anti-PD-L1 Abs as well as treatments targeting inhibitory immunoregulatory pathways other than the PD-1 / PD-L1 pathway. For example, cell surface expression of PD-L1 on tumors and / or tumor-infiltrating inflammatory cells can be used as a marker to identify or select suitable cancer patients who would benefit from immunotherapy with agents, e.g., Abs, that target and disrupt or inhibit signaling from immune checkpoints, e.g., PD-L1, cytotoxic T-lymphocyte antigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), killer immunoglobulin-like receptors (KIR), killer cell lectin-like receptor G1 (KLRG-1), natural killer cell receptor 2B4 (CD244), and CD160 (Pardoll, 2012; Baitsch et al., 2012). In one preferred embodiment, the inhibitory immune regulator is a component of the PD-1 / PD-L1 signaling pathway. In other preferred embodiments, the inhibitory immune regulator is an anti-PD-1 Ab of the present invention. In yet other preferred embodiments, the inhibitory immune regulator is an anti-PD-L1 Ab of the present invention.

[0207] Where any immunotherapy method involving assaying PD-L1 expression, i.e., using PD-L1 expression biomarkers, is described below as involving the selection of patients suitable or unsuitable for anti-PD-1 immunotherapy, or as involving the administration of anti-PD-1 Abs for immunotherapeutic purposes, it should be understood that these methods also have broader use in the selection of patients suitable or unsuitable for immunotherapy with, or the administration of inhibitors of, inhibitory immune regulators (e.g., CTLA-4, BTLA, TIM3, LAG3 or KIR) or components or ligands thereof. Furthermore, it should be understood that in any method involving the measurement of PD-L1 expression in a test tissue sample, the step involving providing a test tissue sample obtained from the patient is an optional step; i.e., in one embodiment the method includes this step, and in another embodiment the method does not include this step. It should be understood that in a preferred embodiment, the "assessing" step to identify or determine the number or percentage of cells expressing PD-L1 on their cell surface in the test tissue sample is performed by a transformative method that assays for PD-L1 expression, for example, by performing a reverse transcriptase-polymerase chain reaction (RT-PCR) assay or an IHC assay. In another embodiment, no transformative step is included, and PD-L1 expression is assessed, for example, by reviewing a test result report from a laboratory. In one embodiment, the steps of the method of assessing PD-L1 expression or the method including assessing PD-L1 expression provide intermediate results that can be provided to a physician or other medical practitioner for use in selecting appropriate candidates for immunotherapy and / or administering an immunotherapy agent to a patient. In one embodiment, the steps of providing intermediate results can be performed by a physician or a person acting under the guidance of a physician. In other embodiments, these steps are performed by an independent laboratory or by an independent person, for example, a laboratory assistant.

[0208] The application also provides methods for treating a subject suffering from cancer comprising: (a) selecting a subject not suitable for treatment with an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab immunotherapy, including: (i) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells; (ii) assessing a percentage of cells in the test tissue sample that express PD-L1 on their cell surface; and (iii) selecting the subject as not suitable for immunotherapy with an inhibitor of an inhibitory immune regulator, e.g., an anti-PD-1 Ab, based on assessing that the percentage of cells in the test tissue sample that express PD-L1 on their cell surface is below a predetermined threshold level; and (b) administering to the selected subject a standard of care therapy other than an inhibitor of an inhibitory immune regulator, e.g., an anti-PD-1 Ab.

[0209] Measurement of PD-L1 expression In one embodiment of any of the methods of the present application, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 RNA. In a further embodiment, the presence of PD-L1 RNA is determined by RT-PCR, in situ hybridization, or RNase protection. In other embodiments, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of a PD-L1 polypeptide. In a further embodiment, the presence of a PD-L1 polypeptide is determined by immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In a preferred embodiment, PD-L1 expression is assayed by IHC. Flow cytometry may be particularly suitable for assaying PD-L1 expression in cells of hematological tumors. In preferred embodiments of all these methods, cell surface expression of PD-L1 is assayed using, for example, IHC or in vivo imaging.

[0210] Imaging technologies have provided important tools in cancer research and treatment. Recent developments in molecular imaging systems, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), fluorescence reflectance imaging (FRI), fluorescence-mediated tomography (FMT), bioluminescence imaging (BLI), laser scanning confocal microscopy (LSCM) and multiphoton microscopy (MPM), will usher in even better use of these technologies in cancer research. Some of these molecular imaging systems allow clinicians not only to see where tumors are in the body, but also to visualize the expression and activity of specific molecules, cells, and biological processes that affect tumor dynamics and / or responsiveness to therapeutic agents (Condeelis and Weissleder, 2010). The sensitivity and resolution of PET, plus Ab specificity, make immunoPET imaging particularly attractive for monitoring and assaying antigen expression in tissue samples (McCabe and Wu, 2010; Olafsen et al., 2010). In one embodiment of any of the methods of the present application, PD-L1 expression is assayed by immunoPET imaging.

[0211] In one embodiment of any of the methods of the present application, the percentage of cells in the test tissue sample that express PD-L1 is assessed by performing an assay to determine the presence of PD-L1 polypeptide on the surface of cells in the test tissue sample. In one embodiment, the test tissue sample is a FFPE tissue sample. In a preferred embodiment, the presence of PD-L1 polypeptide is determined by an IHC assay. In a further embodiment, the IHC assay is performed using an automated process. In a further embodiment, the IHC assay is performed using an anti-PD-L1 mAb that binds to a PD-L1 polypeptide.

[0212] Abs that specifically bind to PD-L1 expressed on the cell surface in FFPE tissues Abs can bind to antigens in fresh tissues, but fail to fully recognize antigens in FFPE tissue samples. This phenomenon, well known in the art, is believed to be mainly due to intra- and intermolecular cross-linking of polypeptides induced by formalin fixation, which changes the epitopes recognized by Abs (Sompuram et al., 2006). In addition, several factors known to affect staining in FFPE tissues, such as variable time to fixation, insufficient fixation duration, differences in fixatives used, tissue processing, Ab clones and dilutions, antigen retrieval, detection systems, and interpretation of results using different threshold points, are important variables that can affect tissue antigenicity and IHC measurements (Bordeaux et al., 2010). In particular, the lack of anti-human PD-L1 Abs that stain PD-L1 in FFPE specimens has been noticed in the art (Hamanishi et al., 2007). Taube et al. (2012) and Gadiot et al. (2011) also reported difficulties in identifying anti-PD-L1 Abs that specifically bind to PD-L1 in FFPE tissues, and conflicting test results for the same Abs. Our own analysis of five commercially available anti-hPD-L1 Abs shows that these Abs were unable to distinguish between FFPE cells that express PD-L1 and cells that did not express PD-L1 (see Example 9, Table 7). Thus, the conflicting results reported by different groups in the association of PD-L1 expression to tumor prognosis may in part reflect the unique ability of the anti-PD-L1 Abs used to detect PD-L1 polypeptide in FFPE tissue samples. Thus, there is a need for anti-hPD-L1 Abs that specifically bind to PD-L1 expressed on the cell surface in FFPE tissue samples in order to detect hPD-L1 on the cell surface using an IHC assay in FFPE tissues.

[0213] The present application provides a mAb or antigen-binding portion thereof that specifically binds to PD-L1 antigen expressed on the cell surface in FFPE tissue samples. In a preferred embodiment, the mAb or antigen-binding portion thereof does not bind to cytoplasmic PD-L1 polypeptide in FFPE tissue samples or exhibits very low levels of background binding. In one other embodiment, the presence or absence of specific binding to cell surface expressed or cytoplasmic PD-L1 polypeptide is detected by immunohistochemical staining. In one preferred aspect of the invention, the mAb or antigen-binding portion is a rabbit Ab or portion thereof. In another preferred embodiment, the mAb is the rabbit mAb designated 28-8, 28-1, 28-12, 29-8 or 20-12. In a more preferred embodiment, the mAb is the rabbit mAb designated 28-8 or antigen-binding portion thereof. In a further embodiment, the mAb comprises a heavy chain variable region (V) comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO:35. H ) and a light chain variable region (V) comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:36. κ In other embodiments, the mAb is an Ab comprising V having the sequence set forth in SEQ ID NO:35. H and V having the sequence set forth in SEQ ID NO: 36. κ The CDR1, CDR2 and CDR3 domains are included in the

[0214] It is known in the art that rabbit Abs have certain advantages over mouse Abs. For example, rabbit Abs generally show more diverse epitope recognition, improved immune response to small epitopes, and higher specificity and affinity compared to mouse Abs (see, e.g., Fischer et al., 2008; Cheang et al., 2006; Rossi et al., 2005). For example, rabbit's lower immunodominance and larger B cell repertoire cause better epitope recognition compared to mouse Abs. Furthermore, the high specificity and novel epitope recognition of rabbit antibodies leads to successful recognition of post-translational modifications (Epitomics, 2013). In addition, many protein targets related to signal transduction and disease are highly conserved between mice, rats and humans and therefore may be recognized as self-antigens by mouse or rat hosts, making them less immunogenic. This problem is circumvented by producing Abs in rabbits. Moreover, in applications where two antigen-specific Abs are required, it is more convenient to have the Abs derived from two different species. Thus, for example, it is easy to multiplex a rabbit Ab, e.g., 28-8, with other Abs (which may be mouse Abs, since the best immune marking Abs are mouse Abs) that can mark immune cells (e.g., macrophages and lymphocytes) that also express PD-L1. Thus, a rabbit anti-hPD-L1 mAb, e.g., 28-8, is particularly suitable for IHC assays to detect surface-expressed PD-L1 in FFPE tissue samples, potentially with distinct advantages over mouse Abs, e.g., 5H1.

[0215] As described in Example 9, a large number (185) of Ab multiclones from both rabbit and mouse immunizations were screened, and only 10 rabbit Ab multiclones, but no mouse Abs, specifically detected the membrane form of hPD-L1. After more extensive screening by multiple rounds of IHC, 15 purified rabbit subclones were selected based on their staining specificity and intensity (see Table 5). After screening by IHC on FFPE tissues and further characterization of the antibodies to determine binding affinity and cross-competition by surface plasmon resonance, mAb28-8 was selected as the Ab with the best combination of high affinity and specificity binding to membrane PDF-L1 and low background staining.

[0216] In one aspect of the invention, the mAbs or antigen-binding portions thereof cross-compete with murine mAb 5H1 for binding to PD-L1, indicating that these antibodies bind to the same epitope region of PD-L1. In one other aspect, the mAbs or antigen-binding portions thereof do not cross-compete with murine mAb 5H1 for binding to PD-L1, indicating that they do not bind to the same epitope region of PD-L1.

[0217] The application also provides nucleic acids encoding any of the rabbit anti-hPD-L1 Abs, or portions thereof, described herein.

[0218] Immunotherapy methods including measurement of cell surface PD-L1 expression The availability of rabbit Abs that specifically bind with high affinity to membrane PD-L1 in FFPE tissue specimens facilitates methods that include detecting PD-L1 polypeptides on the surface of cells in FFPE tissue samples. Accordingly, the present application also provides a method for immunotherapy of a subject suffering from cancer, comprising: (a) selecting a subject who is a suitable candidate for immunotherapy, comprising: (i) optionally providing a test tissue sample obtained from a patient with cancer in FFPE tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (ii) assessing the percentage of cells in the test tissue sample that express PD-L1 on their cell surface by IHC using a rabbit anti-human PD-L1 Ab, e.g., mAb 28-8, which binds PD-L1; and (iii) selecting the subject as a suitable candidate based on assessing that the percentage of cells in the test tissue sample that express PD-L1 on their cell surface exceeds a predetermined threshold level; and (b) administering to the selected subject a therapeutically effective amount of an agent that inhibits an inhibitory immune regulator, e.g., a composition comprising an anti-PD-1 Ab.

[0219] In one embodiment of the method of using IHC to assay PD-L1 expression in FFPE tissues, an automated IHC assay is used. The automated IHC process is performed in an automated stainer and includes: (a) deparaffinizing the FFPE samples with xylene and rehydrating the samples; (b) activating the antigens using an antigen retrieval device; (c) blocking non-specific protein binding sites by incubation with Protein Block; (d) incubating the samples with a primary anti-PD-L1 Ab; (e) adding a secondary Ab conjugated with polymeric horseradish peroxidase (HRP); (f) detecting the bound secondary Ab, including staining with 3,3'-diaminobenzidine (DAB) chromogen; and / or (g) counterstaining with hematoxylin. This automated IHC process is optimized by minimizing the number of steps, optimizing incubation times, and selecting primary Ab, blocking, and detection reagents that produce strong specific staining with low levels of background staining. In a preferred embodiment of this automated IHC assay, the primary anti-PD-L1 Ab is rabbit mAb28-8 or mouse mAb5H1. In one embodiment of the invention, this IHC assay, and any other IHC assay described herein for measuring PD-L1 expression, may be used as part of an immunotherapy method. In other embodiments, any IHC method described herein is used independently of any therapeutic process requiring administration of a therapeutic agent, i.e., solely as a diagnostic method for assaying PD-L1 expression.

[0220] In one embodiment of any of the immunotherapy methods described herein, the Ab administered to the selected subject is any of the anti-PD-1 or anti-PD-L1 Abs, or antigen-binding portions thereof, of the present invention. In one preferred embodiment, the subject is a human. In another preferred embodiment, the Ab is a human Ab, or antigen-binding portion thereof. In a more preferred embodiment, the anti-PD-1 Ab is nivolumab and the anti-PD-L1 Ab is BMS-936559. In one other embodiment, the anti-PD-1 Ab is an Ab, or antigen-binding portion thereof, that cross-competes with nivolumab for binding to PD-1 and the anti-PD-L1 Ab is an Ab, or antigen-binding portion thereof, that cross-competes with BMS-936559 for binding to PD-L1. In a preferred embodiment, the cancer to be treated is selected from the group consisting of MEL, RCC, squamous NSCLC, non-squamous NSCLC, CRC, castration-resistant prostate cancer, CRPC, HCC, squamous cell carcinoma of the head and neck, carcinoma of the esophagus, ovary, gastrointestinal tract and breast, and hematological malignancies.

[0221] In one embodiment of the described method, the predetermined threshold is based on the percentage of (a) tumor cells, (b) tumor infiltrating inflammatory cells, (c) specific tumor infiltrating inflammatory cells, e.g., TILs or macrophages, or (d) a combination of tumor cells and tumor infiltrating inflammatory cells in the test tissue sample that express PD-L1 on the cell surface. In one embodiment, the predetermined threshold is at least 0.001% of tumor cells expressing membrane PD-L1 as determined by IHC. In another embodiment, the predetermined threshold is at least 0.01%, preferably at least 0.1%, and more preferably at least 1% of tumor cells expressing membrane PD-L1 as determined by IHC. In one embodiment, the predetermined threshold is at least 5% of tumor cells expressing membrane PD-L1 as determined by IHC. In one embodiment, the predetermined threshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5% of tumor cells expressing membrane PD-L1 as determined by IHC and / or a single tumor infiltrating inflammatory cell expressing membrane PD-L1 as determined by IHC. In one other embodiment, the predetermined threshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5% of tumor infiltrating inflammatory cells expressing membrane PD-L1 as determined by IHC. In one other embodiment, the predetermined threshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5% of tumor infiltrating lymphocytes expressing membrane PD-L1 as determined by IHC. In one other embodiment, the predetermined threshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5% of tumor infiltrating macrophages expressing membrane PD-L1 as determined by IHC. In yet another embodiment, the predetermined threshold is a single tumor cell or a single tumor infiltrating inflammatory cell expressing at least membrane PD-L1 as determined by IHC. Preferably, PD-L1 expression is assayed by automated IHC using mAb 28-8 or 5H1 as the primary Ab.

[0222] The application also provides methods for treating subjects suffering from cancer comprising: (a) screening a plurality of subjects to identify subjects who are not suitable candidates for immunotherapy comprising administering to the subjects an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, comprising: (i) optionally providing test tissue samples from a plurality of subjects, where the test tissue samples include tumor cells and tumor infiltrating inflammatory cells; (ii) assessing the percentage of cells in the test tissue samples that express PD-L1 on their cell surface; and (iii) selecting the subjects as not suitable candidates for immunotherapy based on assessing that the percentage of cells in the test tissue sample of the subjects that express PD-L1 on their cell surface is below a predetermined threshold level; and (b) administering to the selected subjects a standard of care therapy other than an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab.

[0223] The application further provides a method for immunotherapy of a subject suffering from cancer comprising: (a) screening a plurality of subjects to identify subjects who are suitable candidates for immunotherapy comprising: (i) optionally providing test tissue samples from a plurality of subjects, where the test tissue samples comprise tumor cells and tumor-infiltrating inflammatory cells; (ii) assessing the percentage of cells in the test tissue samples that express PD-L1 on their cell surface; and (iii) selecting the subjects as candidates who are suitable for immunotherapy with an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, based on an assessment that the percentage of cells in the test tissue samples that express PD-L1 on their cell surface exceeds a predetermined threshold level; and (b) administering to the selected subjects a composition comprising a therapeutically effective amount of the agent.

[0224] The application provides a method for screening a plurality of subjects to identify subjects who are suitable candidates for a treatment comprising: (a) (i) optionally providing test tissue samples from a plurality of subjects, wherein the test tissue samples include tumor cells and tumor infiltrating inflammatory cells; (ii) assessing the percentage of cells in the test tissue samples that express PD-L1 on their cell surface, wherein the subject is identified as a suitable candidate for anti-PD-1 Ab immunotherapy when the percentage of cells in the tissue sample that express PD-L1 on their cell surface exceeds a predetermined threshold level, and the subject is identified as a candidate who is not a suitable candidate for anti-PD-1 Ab immunotherapy when the percentage of cells in the test tissue sample that express PD-L1 on their cell surface is below a predetermined threshold level; and (b) administering a therapeutically effective amount of an agent that inhibits an inhibitory immune regulator, e.g., a composition comprising an anti-PD-1 Ab, to the subjects identified as suitable candidates for anti-PD-1 Ab immunotherapy, or (c) administering a standard of care treatment other than the agent to the subjects identified as suitable candidates for anti-PD-1 Ab immunotherapy. Further provided is a method for treating a subject suffering from cancer comprising administering to a subject identified as not being a suitable candidate for Ab immunotherapy.

[0225] One aspect of the invention is a method for immunotherapy of a subject suffering from cancer comprising: (a) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (b) determining that the percentage of cells in the test tissue sample that express PD-L1 on their cell surface is above a predetermined threshold level; and (c) based on the determination, administering to the subject a therapeutically effective amount of an agent that inhibits an inhibitory immune regulator, e.g., a composition comprising an anti-PD-1 Ab. Another aspect of the invention is a method for treatment of a subject suffering from cancer comprising: (a) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (b) determining that the percentage of cells in the test tissue sample that express PD-L1 on their cell surface is below a predetermined threshold level; and (c) based on the determination, administering to the subject an agent that inhibits an inhibitory immune regulator, e.g., a standard of care therapy other than an anti-PD-1 Ab.

[0226] Yet another aspect of the invention is a method for immunotherapy of a subject suffering from cancer, comprising: (a) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells; (b) determining the percentage of cells in the test tissue sample that express PD-L1 on their cell surface; (c) selecting an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, as a treatment for the subject based on the recognition that the anti-PD-1 Ab is effective in patients whose test tissue sample comprises a percentage of cells that express PD-L1 on their cell surface above a predetermined threshold level; and (d) administering to the subject a composition comprising a therapeutically effective amount of the agent. In another aspect, the application provides a method for immunotherapy of a subject suffering from cancer, comprising: (a) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells; (b) determining the percentage of cells in the test tissue sample that express PD-L1 on their cell surface; (c) selecting a standard of care therapy other than an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, as a treatment for the subject based on the recognition that the agent is not effective in patients in which the percentage of cells in the test tissue sample that express PD-L1 on their cell surface is below a predetermined threshold level; and (d) administering the standard of care therapy to the subject.

[0227] The application also provides a method of selecting an immunotherapy for a subject suffering from cancer, comprising: (a) assaying cells of a test tissue sample, including tumor cells and tumor-infiltrating inflammatory cells, to assess the percentage of cells in the test tissue sample that express PD-L1; and (b) selecting an immunotherapy comprising a therapeutically effective amount of an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, for the subject based on an assessment that the percentage of cells expressing membrane PD-L1 is above a predetermined threshold level.The application further provides a method of selecting a treatment for a subject suffering from cancer, comprising: (a) assaying cells of a test tissue sample, including tumor cells and tumor-infiltrating inflammatory cells, to assess the percentage of cells in the test tissue sample that express PD-L1; and (b) selecting a standard of care treatment other than an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, for the subject based on an assessment that the percentage of cells expressing membrane PD-L1 is below a predetermined threshold level.

[0228] In addition, the application provides a method for treating a subject suffering from cancer, comprising administering to the subject a composition comprising a therapeutically effective amount of an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, wherein the subject has been selected based on the percentage of cells in a test tissue sample from the subject that express PD-L1 being determined to be above a predetermined threshold level, the test tissue sample comprising tumor cells and tumor-infiltrating inflammatory cells. The application also provides a method for treating a subject suffering from cancer, comprising administering to the subject a standard of care treatment other than an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, wherein the subject has been selected based on the percentage of cells in a test tissue sample from the subject that express PD-L1 being determined to be below a predetermined threshold level, the test tissue sample comprising tumor cells and tumor-infiltrating inflammatory cells.

[0229] The application further provides methods for selecting a cancer patient for immunotherapy with an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, comprising: (a) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, where the test tissue sample contains tumor cells and tumor infiltrating inflammatory cells; (b) assaying the test tissue sample to determine the percentage of cells that express PD-L1 on their cell surface; (c) comparing the percentage of cells that express PD-L1 on their cell surface to a predetermined threshold percentage; and (d) selecting the patient for immunotherapy based on an assessment that the percentage of cells in the test tissue sample that express surface PD-L1 exceeds a predetermined threshold level.

[0230] In any of the methods described herein that include a step for assessing PD-L1 expression, the test tissue sample may be a FFPE tissue sample, and PD-L1 polypeptide on the cell surface is detected by IHC using an anti-PD-L1 Ab, for example, mAb28-8 or 5H1.

[0231] In addition, in any method where an immunotherapy is selected or administered based on an assessment that a proportion of cells in a test tissue sample from a subject express PD-L1 at a level above a predetermined threshold level, a complementary method of treatment may be performed where a standard of care treatment other than immunotherapy is selected or administered based on an assessment that a proportion of cells in a test tissue sample from a subject express PD-L1 at a level below a predetermined threshold level.

[0232] The application further provides a method for predicting the therapeutic efficacy of an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, for treating a cancer patient, comprising: (a) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells; (b) assaying the test tissue sample to determine the percentage of cells that express PD-L1 on their cell surface; (c) comparing the percentage of cells that express PD-L1 on their cell surface to a predetermined threshold; and (d) predicting the therapeutic efficacy of the agent, wherein when the percentage of cells that express PD-L1 on their cell surface exceeds the threshold percentage, the agent is predicted to be effective in treating the patient, and when the percentage of cells that express PD-L1 on their cell surface is below the threshold percentage, the agent is predicted to be ineffective in treating the patient.

[0233] The application also provides methods for determining an immunotherapeutic regimen comprising an agent that inhibits an inhibitory immune regulator, e.g., an anti-PD-1 Ab, for treating a cancer patient, comprising: (a) optionally providing a test tissue sample obtained from a patient having cancer of the tissue, where the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells; (b) assaying the test tissue sample to determine the percentage of cells that express PD-L1 on their cell surface; (c) comparing the percentage of cells that express PD-L1 on their cell surface to a predetermined threshold percentage; and (d) determining an immunotherapeutic regimen comprising the agent based on a determination that the percentage of cells that express PD-L1 on their cell surface exceeds the predetermined threshold percentage.

[0234] Standard treatment drugs Some methods of treatment described herein include administration of standard of care treatment to the patient. As used herein, a "standard of care treatment" is a treatment process that includes a drug or combination of drugs, radiation therapy (RT), surgery, or other medical interventions that are recognized, accepted, and / or widely used by physicians when appropriate for one type of patient, disease, or clinical situation. Standard of care treatments for various types of cancer are well known to those skilled in the art. For example, the National Comprehensive Cancer Network (NCCN), a collaboration of 21 major cancer centers in the USA, has published the NCCN Standard of Care Guidelines in Oncology (NCCN GUIDELINES®), which provides detailed, up-to-date information on standard of care treatments for a wide variety of cancers (see NCCN GUIDELINES®, 2013). As an example, standard of care treatments for MEL, RCC, and NSCLC are summarized below.

[0235] melanoma MEL is a malignant tumor of melanocytes, the melanin-producing cells found primarily in the skin. Although less common than other skin cancers, it is the most dangerous skin cancer, leading to death in the majority (75%) of skin cancers if not diagnosed early. The incidence of MEL is increasing worldwide in Caucasian populations, especially in those with small amounts of skin pigmentation who receive excessive UV exposure from the sun. Incidence rates are <10-20 per 100,000 populations in Europe; 20-30 per 100,000 in the USA; and 50-60 per 100,000 in Australia, where the highest incidence rates are observed (Garbe et al., 2012). MEL accounts for approximately 5% of all new cases of cancer in the United States (US), and incidence rates continue to increase by approximately 3% per year. This represents an estimated 76,690 new cases in the US in 2013, with 9,480 associated deaths (Siegel et al. (2013)).

[0236] In in situ (stage 0) or early stage MEL (stage I-II), surgical resection is the primary treatment. In general, prognosis is good with a 5-year survival rate of over 90% for patients with localized disease and tumor thickness of 1.0 mm or less (NCCN GUIDELINES®, 2013-Melanoma). When surgical resection is not feasible for in situ melanoma due to comorbidities or cosmetically sensitive tumor site, topical imiquimod and radiation therapy are emerging as treatments, especially for lentigo maligna. Chemotherapeutic agents for treating MEL include dacarbazine, temozolomide and imatinib for melanoma with c-KIT mutations, high-dose interleukin-2, and paclitaxel with or without carboplatin. However, these treatments are only marginally successful, with response rates of less than 20% in the first-line (1L) and second-line (2L) settings.

[0237] In patients with localized melanoma ≥1.0 mm thick, survival rates range from 50-90%. The likelihood of regional lymph node involvement increases with increasing tumor thickness. In stage III MEL (clinically positive nodes and / or in-transit disease), 5-year survival rates range from 20-70%. By far the most lethal is stage IV MEL, with long-term survival of less than 10% in patients with distant metastatic melanoma (NCCN GUIDELINES®, 2013-Melanoma).

[0238] Although various treatments including resection to distant sites, intralesional injections, laser ablation, radiation therapy and biochemotherapy (combination of chemotherapy and biological agents such as interferon-alpha and IL-2) have been investigated, no specific treatment has been identified for metastatic MEL. The therapeutic landscape for metastatic MEL has recently seen dramatic improvements with the development of novel drugs such as vemurafenib and ipilimumab. Vemurafenib specifically inhibits signaling by BRAF, a mutated intracellular kinase present in approximately 50% of patients with metastatic MEL. In clinical trials, vemurafenib confers an estimated progression-free survival (PFS) of 5.3 months in BRAF mutation-positive patients compared to only 1.6 months with dacarbazine, and a median OS of 15.9 months with vemurafenib compared to 5.6-7.8 months with dacarbazine (Chapman et al., 2011; Sosman et al., 2012). Ipilimumab is a HuMAb that inhibits the immune checkpoint receptor CTLA-4, thereby stimulating T-cell immune responses. Ipilimumab with or without glycoprotein 100 (gp100) peptide vaccine improved median OS to 10.0-10.1 months in patients with previously treated metastatic MEL, compared with a median OS of 6.4 months in patients receiving gp100 alone (Hodi et al., 2010). Besides these two agents, no other agents have demonstrated OS benefit in phase 3 randomized trials. Dacarbazine has been approved by the FDA and EMA for the treatment of metastatic MEL with a reported objective response rate of 5-20% and a median OS of about 6.4 months, but these responses are short-lived. Other drugs, such as temozolomide and fotemustine, did not cause a significant improvement in survival compared to dacarbazine. IL-2 has also been approved by the FDA for the treatment of metastatic MEL, as it is associated with a 15-20% response rate, including a 4-6% complete response that can be durable, but is associated with significant toxicities, including hypotension, cardiac arrhythmias, and pulmonary edema.Further details of standard of care treatment for melanoma are provided by Garbe et al. (2012) and NCCN GUIDELINES®, 2013-Melanoma. Despite the recent approval of ipilimumab and vemurafenib for advanced MEL, there is still a large unmet need for patients who have progressed on anti-CTLA-4 therapy and BRAF inhibitors (depending on BRAF status) or who have previously untreated, unresectable, or metastatic BRAF wild-type MEL. The 5-year survival rate for late-stage MEL is currently only 15%.

[0239] renal cell carcinoma RCC is the most common type of kidney cancer in adults, responsible for approximately 90% of kidney tumors, of which 80-90% are clear cell tumors (NCCN GUIDELINES®, 2013-Kidney Cancer). It is also the most lethal of all genitourinary tumors. An estimated 65,150 patients will be diagnosed with kidney cancer and 13,680 will die from the disease in the United States in 2013 (Siegel et al. (2013)).

[0240] In clinically localized RCC (stages IA and IB), surgical resection, including radical nephrectomy and nephron-preserving surgery, is an effective treatment. Partial nephrectomy is generally not appropriate for patients with locally advanced tumors (stages II and III), in which case radical nephrectomy is preferred. If the tumor is confined to the renal parenchyma, the 5-year survival rate is 60-70%, but this is considerably lower in stage IV disease when metastases have spread. Stage IV RCC is relatively resistant to RT and chemotherapy, and patients can benefit from surgical procedures, although cytoreductive nephrectomy prior to systemic therapy is recommended for patients with a possible surgically resectable primary and multiple resectable metastases.

[0241] Until recently, the cytokines IL-2 and IFNα were the only active systemic treatments for advanced or metastatic RCC, both providing a reported ORR of 5-27%. However, due to the limited clinical benefit and substantial toxicity profile of each of these agents, recent targeted agents have been widely replaced by cytokines in the treatment of advanced or metastatic renal cell carcinoma. The recognition of the importance of hypoxia-inducible factor alpha (HIFα) signaling in the pathogenesis of clear cell RCC has triggered extensive testing of two classes of targeted therapies, antiangiogenic tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors, in 1L and 2L treatments (Mulders, 2009). Targeting angiogenesis is rational because constitutive HIFα activation leads to the upregulation or activation of several proteins, including vascular endothelial growth factor (VEGF), which can subsequently lead to tumor growth and neovascularization. Targeting the mTOR pathway is important because activation of the upstream PI3K / Akt / mTOR signaling pathway is one way that constitutive HIFα activation or upregulation occurs (Mulders, 2009). Agents that target angiogenesis include VEGF-receptor (VEGFr) TKIs (e.g., sorafenib, sunitinib, pazopanib, axitinib, and tivozanib) and VEGF-binding mAbs (e.g., bevacizumab), while agents that target the mTOR pathway include mTOR inhibitors (e.g., everolimus and temsirolimus) (Mulders, 2009; NCCN GUIDELINES®, 2013-Kidney Cancer). However, many patients develop resistance and OS improvement has only been shown in one phase 3 trial in high-risk patients: temsirolimus showed a statistically significant benefit on OS in patients with advanced RCC compared with IFNα (10.9 vs. 7.3 months) (Hudes et al., 2007). Everolimus has also demonstrated a 2.1 month improvement in median PFS, but no OS improvement, versus placebo (Motzer et al., 2008).Of the five approved antiangiogenic agents (sorafenib, sunitinib, bevacizumab, pazopanib and axitinib) and two approved mTOR inhibitors (temsirolimus, everolimus), only everolimus is specifically approved for use after treatment failure with antiangiogenic therapy. In the United States, everolimus is indicated for the treatment of advanced RCC after treatment failure with sunitinib or sorafenib, and in Europe, everolimus is more broadly indicated for patients with advanced RCC whose disease progresses during or after treatment with VEGF-targeted therapy.

[0242] non-small cell lung cancer NSCLC is the leading cause of cancer death in the United States and worldwide, surpassing breast, colon, and prostate cancers combined. An estimated 228,190 new cases of lung and bronchial cancer will be diagnosed in the United States, with approximately 159,480 deaths from the disease (Siegel et al., 2013). The majority of patients (approximately 78%) will be diagnosed with progressive / recurrent or metastatic disease. Metastasis from lung cancer to the adrenal gland is common, with approximately 33% of patients having such metastasis. NSCLC treatments have been gradually improving OS, but the benefit has reached a plateau (median OS for advanced stage patients is just 1 year). Progression after 1L treatment occurs in almost all of these subjects, with the 5-year survival rate being only 3.6% in the refractory setting. From 2005 to 2009, the overall 5-year relative survival rate for lung cancer in the United States was 15.9% (NCCN GUIDELINES®, 2013-Non-Small Cell Lung Cancer).

[0243] Surgery, RT and chemotherapy are the three modalities commonly used to treat patients with NSCLC. As a class, NSCLC is relatively poorly responsive to chemotherapy and RT compared to small cell carcinoma. Generally, in patients with stage I or II disease, surgical resection offers the best chance for care, with chemotherapy increasingly used pre- and post-surgery. RT may also be used as adjuvant therapy for patients with resectable NSCLC, as the first local treatment, or as palliative therapy for patients with incurable NSCLC.

[0244] Patients with stage IV disease who have good performance status (PS) benefit from chemotherapy. Numerous agents are available for stage IV NSCLC, including platinum agents (e.g., cisplatin, carboplatin), taxanes (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, and gemcitabine). Combinations using many of these drugs produce 1-year survival rates of 30% to 40%, superior to single agents. Specific targeted therapies are also being developed for the treatment of advanced lung cancer. For example, bevacizumab (AVASTIN®) is a mAb that blocks vascular endothelial growth factor A (VEGF-A). Erlotinib (Tarceva®) is a small molecule TKI of the epidermal growth factor receptor (EGFR). Crizotinib (XALKORI®) is a small molecule TKI that targets ALK and MET and is used to treat NSCLC in patients with a mutated ALK fusion gene. Cetuximab (Erbitux®) is a mAb that targets EGFR.

[0245] Due to the scarcity of treatment options after 1L therapy, there is a particular unmet need for patients with squamous NSCLC (representing up to 25% of all NSCLCs). Single-agent chemotherapy is the standard of care after progression with platinum-based doublet chemotherapy (Pt doublet), resulting in a median OS of approximately 7 months. Docetaxel remains the benchmark treatment in this line of therapy, although erlotinib may also be used less frequently. Pemetrexed has also been shown to produce clinically equivalent efficacy outcomes with significantly fewer side effects compared to docetaxel in the 2L treatment of patients with advanced NSCLC (Hanna et al., 2004). There are currently no treatments approved for use in lung cancer beyond the 3L setting. Pemetrexed and bevacizumab are not approved in squamous NSCLC, and targeted therapies have limited application. Unmet needs in advanced lung cancer are exacerbated by the recent failure of Oncothyreon and Merck KgaA's STIMUVAX® to improve OS in Phase 3 trials, the inability of ArQule and Daiichi Sankyo's c-Met kinase inhibitor tivantinib to meet survival endpoints, the failure of Eli Lilly's ALIMTA® in combination with Roche's AVASTIN® to improve OS in late-stage trials, and the failure of Amgen and Takeda Pharmaceuticals to meet clinical endpoints with their small molecule VEGF-R antagonist motesanib in late-stage trials.

[0246] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references mentioned throughout this application are hereby incorporated by reference.

[0247] Example 1 Cross-competition between anti-PD-1 HuMAbs for binding to CHO cells expressing human PD-1 Chinese hamster ovary (CHO) cells transfected to express human PD-1 (CHO / PD-1 cells) were incubated with 10 μg / ml of anti-PD-1 HuMAb 5C4 or Fab fragment of human IgG1 (hIgG1) isotype control Ab for 30 min at 4° C., and anti-PD-1 HuMAbs 2D3, 7D3, or 4H1 were added at a concentration of 0.2 μg / ml. Binding of 4H1, 2D3, or 7D3 to CHO / PD-1 cells was detected with fluorescein isothiocyanate (FITC)-conjugated goat anti-hIgG, Fc-gamma specific Ab. In the case of cross-competition assays with 5C4 and 17D8, CHO / PD-1 cells were incubated with the whole molecule of 5C4, and FITC-labeled 17D8 was added. Binding of 2D3, 7D3, 4H1, or 17D8 to CHO / PD-1 cells was measured by flow cytometric analysis using a FACScalibur flow cytometer (Becton Dickinson, San Jose, Calif.).

[0248] The results are depicted in Figure 1. As measured by mean fluorescent intensity (MFI) of staining, the data show that the 5C4 Fab fragment substantially blocked binding of mAb 5C4 itself, as well as 2D3, 7D3 (Figure 1A) and 4H1 (Figure 1B), and that the 5C4 whole mAb substantially blocked binding of 17D8 (Figure 1C) to CHO / PD-1 cells.

[0249] Example 2 Cross-competition between anti-PD-L1 HuMAbs for binding to CHO cells expressing human PD-L1 CHO cells transfected to express hPD-L1 (CHO / PD-L1 cells) were incubated with 10μg / ml each of the 10 unconjugated human anti-PD-L1 mAbs (5F8, 7H1, 10H10, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7, and 13G4) or human IgG1 (hIgG1) isotype control Ab for 20 minutes at 4℃. FITC-conjugated 10H10 (A), 3G10 (B), 10A5 (C), 11E6 (D), 12A4 (E) or 13G4 (F) were added to the cells to a final concentration of 0.09 μg / ml (B, D), 0.27 μg / ml (A, C), 0.91 μg / ml (F), or 2.73 μg / ml (E) without washout of unbound, unconjugated Ab for an additional 20 min at 4° C. Different amounts of different FITC-conjugated HuMAbs were used due to differences in binding efficiency after labeling, and the optimal amounts of these FITC-conjugated HuMAbs were previously determined by dose titration analysis of binding to CHO / PD-L1 cells. Binding of FITC-conjugated 10H10, 3G10, 10A5, 11E6, 12A4 or 13G4 to CHO / PD-L1 cells was measured by flow cytometry.

[0250] The results are depicted in Figure 2. Binding of labeled 10H10 was partially blocked by 10A5, 11E6, and 13G4, but substantially blocked by itself alone (Figure 2A). Conversely, 10H10 substantially blocked binding of itself alone to CHO / PD-L1 cells. Each of the anti-PD-L1 HuMAbs 5F8, 7H1, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7, and 13G4 substantially blocked binding of labeled mAbs 3G10 (Figure 2B), 10A5 (Figure 2C), 11E6 (Figure 2D), 12A4 (Figure 2E), and 13G4 (Figure 2F) to CHO / PD-L1 cells as measured by MFI, with mAbs 5F8 and 13G4 generally blocking binding of the labeled mAbs to a lesser extent.

[0251] Example 3 Cross-competition between anti-PD-L1 mAbs for binding to ovarian carcinoma cells expressing human PD-L1 Anti-PD-L1 HuMAbs 5F8, 12B7, 3G10, 1B12, 13G4, 10H10, 10A5 and 12A4, and a human IgG1 (huIgG1) isotype control Ab were serially diluted from 10μg / ml and incubated with hPD-L1 expressing ES-2 ovarian carcinoma cells for 20 minutes at 4℃. Without washing, biotinylated-12A4 Ab was added to a final concentration of 0.4μg / ml for an additional 20 minutes at 4℃. After washing, bound biotin-12A4 was detected using a fluorescent streptavidin-PE secondary reagent and measured by flow cytometry. Figure 3 shows the fluorescence of bound biotin-12A4 plotted against the concentration of unlabelled hPD-L1 HuMAb. Binding of biotin-12A4 to ES-2 cells was substantially blocked by 12A4 itself as well as 1B12 and 12B7, and moderately significantly blocked by mAbs 5F8, 10A5, 13G4 and 3G10, but not by mAb 10H10.

[0252] Example 4 Design of Phase 1 clinical trials of anti-PD-1 Ab A phase 1 study was conducted to evaluate the safety, antitumor activity and pharmacokinetics of anti-PD-1 in patients with selected advanced solid tumors. BMS-936558 (also referred to herein as nivolumab and 5C4 in U.S. Pat. No. 8,008,449), a human anti-PD-1 mAb, was administered as an intravenous infusion every 2 weeks for each 8-week treatment cycle. Tumor status was reassessed after each cycle. Patients continued treatment for up to 2 years (12 cycles) until they experienced complete remission, unacceptable toxicity, disease progression, or withdrew consent. In otherwise clinically stable patients, study treatment was continued beyond the apparent first disease progression until further progression occurred, as recommended by the proposed immune response criteria (Wolchok et al., 2009). Patients with stable disease (SD) or ongoing objective response (OR: complete response [CR] or partial response [PR]) at the end of treatment were followed for 1 year and offered retreatment for an additional year in case of progression.

[0253] Dose escalation Patients with advanced melanoma (MEL), non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), castration-resistant prostate cancer (CRPC), and colorectal cancer (CRC) were eligible to enroll. Cohorts of 3-6 patients per dose level were enrolled consecutively at 1.0, 3.0, and 10.0 mg / kg. Dose escalation was performed when a minimum of 3 patients completed the safety evaluation period (56 days) at a given dose level, with dose-limiting toxicity in less than one-third of patients. Intrapatient dose escalation was not permitted.

[0254] Cohort Expansion The maximum tolerated dose (MTD) was not reached. Initially, five expansion cohorts of approximately 16 patients each were enrolled at 10 mg / kg for MEL, NSCLC, RCC, CRPC, and CRC. Based on the first signal of activity and a 6.5-month interruption following protocol amendment, further expansion cohorts of approximately 16 patients each were enrolled for MEL (cohorts at 1.0 and 3.0 mg / kg, then randomized to 0.1, 0.3, or 1.0 mg / kg), NSCLC (squamous or non-squamous histology cohorts randomized to 1, 3, or 10 mg / kg), and RCC (at 1.0 mg / kg). Intrapatient dose escalation to 1 mg / kg was allowed for MEL patients with progressive disease after receiving 0.1 or 0.3 mg / kg.

[0255] patient Eligible patients were those with advanced solid tumors; age >18 years; life expectancy >12 weeks; Eastern Cooperative Oncology Group performance status of ≤2; measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST), v1.0 with modifications (see Topalian et al., 2012b); demonstrated adequate hematological, hepatic and renal function; and had received 1-5 prior systemic treatment regimens. Patients with stable treated brain metastases were enrolled. Exclusion criteria included a history of chronic autoimmune disease, previous treatment with T cell-modulating Abs (e.g., anti-CTLA-4, anti-PD-1, anti-PD-L1), conditions requiring immunosuppressant therapy, and chronic infections (e.g., HIV, hepatitis B or C).

[0256] A total of 296 patients with advanced solid tumors, including MEL (n=104), NSCLC (n=122), RCC (n=34), CRPC (n=17), and CRC (n=19), were treated with BMS-936558 for 40 months up to February 2012. By March 2013, 306 patients, including patients with non-small cell lung cancer (n=129), melanoma (n=107), RCC (n=34), CRPC (n=17), and CRC (n=19), were treated with BMS-936558 from October 2008 to January 2012 and all were observed for a minimum of 1 year. Two patients did not receive a complete cycle of treatment and were not considered evaluable for response. Median age was 63 years (range 29-85 years). ECOG behavioral scores were 0 or 1 in 98% of patients. The majority of patients were heavily pretreated, with 47% receiving at least 3 prior regimens. Notable prior treatments included immunotherapy (64%) and B-RAF inhibitors (8%) in MEL patients; platinum-based chemotherapy (94%) and tyrosine kinase inhibitors (TKI, 34%) in NSCLC patients; and nephrectomy (94%), immunotherapy (59%), and antiangiogenic therapy (74%) in RCC patients. Baseline characteristics of the all-treated population (N=306) were similar to those of the efficacy population (response-evaluable patients, N=270). Details of patient pretreatment are provided in Topalian et al. (2012b).

[0257] statistical analysis Baseline characteristics and adverse events for all treated patients (N=306), and efficacy results for 270 patients with lung, melanoma, and kidney cancer in March 2013 are reported. The pharmacokinetic and molecular marker population consisted of treated patients with available data from the exploratory analysis in February 2012. The efficacy population consisted of response-evaluable patients who began treatment at least 8 months prior to the analysis date. Tumor measurements were collected after each treatment cycle (4 times) by the investigator. Individual best objective response based on tumor measurements was assessed by the sponsor by modified RECIST v1.0. Objective responses were confirmed by at least one consecutive tumor assessment. Objective responses and stable disease rates were estimated with confidence intervals using the Clopper-Pearson method. Time-to-event endpoints, including PFS, OS, survival, and duration of response, were estimated using the Kaplan-Meier method. AEs were coded using the Medical Dictionary for Clinical Trials International (MedDRA), version 15.1. Select adverse events with a possible immunological etiology, also referred to as "immune-related adverse events" of "AEs of special interest" (AEOSI), defined as adverse events requiring frequent monitoring and / or unique interventions, were identified using a predefined list of MeDRA terms. Individual best ORs were derived from investigator-reported data by modified RECIST v1.0. ORs were confirmed by at least one consecutive tumor assessment, and OR rates (ORR = {[CR + PR] ÷ n} × 100) were calculated.

[0258] Example 5 Safety evaluation of patients treated with anti-PD-1 antibodies Safety evaluations, including clinical and laboratory assessments, were performed in all treated patients at baseline and at regular intervals for up to 100 days after the last dose of drug. The severity of AEs was graded according to the NCI Common Terminology Criteria for Adverse Events (NCI CTCAE), v3.0. Computed tomography (CT) or magnetic resonance imaging was performed for tumor evaluation at baseline and after each treatment cycle.

[0259] The MTD was not defined beyond the dose of BMS-936558 tested for this study, up to the highest planned dose of 10 mg / kg. Relative BMS-936558 dose intensity of 90% or greater was achieved in 87% of patients (see Topalian et al., 2012b; Topalian et al., 2013 for details). AEs were coded using the Medical Dictionary for Reproductive Medicines (MedDRA), version 14.1. AEOSIs were identified using a predefined list of MeDRA terms. Fifteen of 296 (5%) patients discontinued treatment due to BMS-936558-related AEs. In a preliminary analysis in February 2012, 62 patients (21%) had died, and by March 2013, 195 patients (64%) had died from disease progression, the most common cause of death (Topalian et al., 2012b; Topalian et al., 2013).

[0260] The most common adverse events, regardless of causality, included fatigue, decreased appetite, diarrhea, vomiting, cough, dyspnea, constipation, vomiting, rash, fever, and pruritus (Topalian et al., 2012b; Topalian et al., 2013). Common BMS-936558-related AEs included fatigue, rash, diarrhea, decreased appetite, and vomiting. The majority of events were low grade, with grade 3-4 drug-related AEs observed in 41 of 296 (14%) patients. Treatment-related AEs (any grade) were observed in 230 of 306 (75%) patients, the most common being fatigue, rash, diarrhea, and pruritus (Topalian et al., 2013). The extent, frequency, and severity of BMS-936558-related AEs were generally similar across dose levels treated, with no apparent relationship to treatment duration. Fifty-two of 306 patients (17%) experienced grade 3-4 treatment-related adverse events, with fatigue (2%), pneumonia, lymphopenia, diarrhea, abdominal pain, and hypophosphatemia (1% each) being the most common. Treatment-related serious adverse events occurred in 42 of 306 patients (14%) (Topalian et al., 2013).

[0261] Drug-related AEOSIs with possible immune-related etiology were classified by organ involvement to provide a more accurate estimate of frequency, specifically including pneumonia, vitiligo, colitis, hepatitis, hypophysitis, and thyroiditis. Treatment-related selected adverse events of any grade were observed in 140 of 306 patients (46%), the most common being rash (15%), diarrhea (13%), and pruritus (11%) (Table 2). Grade 3-4 treatment-related selected events were seen in 19 patients (6%).

[0262] Fifty-two patients (23%) out of 230 with drug-related adverse events required management with systemic glucocorticoids and / or other immunosuppressants. Hepatic or gastrointestinal AEOSIs were managed with treatment interruptions, together with administration of corticosteroids when necessary. Among patients treated to date, these AEs were reversible in all cases. Endocrine AEOSIs were managed with replacement therapy. Thirty-two patients (11%) out of 306 discontinued treatment due to treatment-related adverse events. At the discretion of the treating physician, patients were successfully restarted on treatment with BMS-936558. Twenty-one (40%) were able to resume after toxicity had resolved.

[0263] Drug-related pneumonitis (any grade) occurred in 12 of 306 (4%) patients (Topalian et al., 2013). Clinical presentation ranged from radiological abnormalities in asymptomatic patients to progressive, diffuse pulmonary infiltrates and associated symptoms (cough, fever, dyspnea). Grade 3-4 pneumonitis developed in 4 patients (1%), 3 of which were fatal (2 NSCLC patients, 1 CRC). No clear association was noted between the occurrence of pneumonitis and tumor type, dose level, or number of doses given. In 9 of 12 patients, pneumonitis was reversible with treatment discontinuation and / or immunosuppression (glucocorticoids, infliximab, mycophenolate). After the most recent pneumonitis-related death in November 2011, 79 patients continued to receive nivolumab (median 29 weeks, range 2-69 weeks) without further deaths from this or other treatment-related causes.

[0264] Example 6 Pharmacokinetic / pharmacodynamic analysis of anti-PD-1 antibodies For pharmacokinetic (PK) analysis, serial blood samples were collected and serum concentrations of BMS-936558 were quantified using ELISA. For pharmacodynamic (PD) analysis, peripheral blood mononuclear cells were isolated from patients at baseline and after cycle 1, and PD-1 receptor occupancy (RO) by BMS-936558 was estimated on circulating CD3+ T-cells by flow cytometry (Brahmer et al., 2010).

[0265] The maximum concentration of BMS-936558 was measured at median T max The PK of BMS-936558 was observed in the dose range of 0.1-10 mg / kg (n=35), C max and AUC (0-14d) BMS-936558 PD was assessed by PD-1 RO on circulating T-cells. PBMCs from 65 MEL patients treated with one cycle of BMS-936558 showed a range of 64%-70% increase in circulating CD3+ T cells with BMS-936558 at 0.1-10 mg / kg biweekly. + demonstrated central occupancy of PD-1 molecules on T-cells (for details see Topalian et al., 2012b).

[0266] Example 7 Antitumor efficacy demonstrated by anti-PD-1 antibodies Data analyzed in February 2012 Clinical antitumor activity was observed at all BMS-936558 doses tested. ORs (confirmed CR or PR) were observed in a substantial proportion of patients with NSCLC, MEL, and RCC (Tables 1 and 2; Figure 4), and in various sites of metastatic disease, including liver, lung, lymph nodes, and bone (Figures 5-7 and not shown). Tumor regression was followed by a prolonged decrease in tumor burden in the presence of new lesions, as well as in conventional and "immune-related" patterns of response. Individual best overall responses were obtained from investigator-reported data by modified RECIST v1.0. ORs were confirmed by at least one consecutive tumor assessment. At the time of data analysis, two patients with NSCLC treated at 10 mg / kg had unconfirmed responses, and eight additional patients (with MEL, NSCLC, or RCC) had sustained declines in baseline target lesions in the presence of new lesions (i.e., "immune-related" response patterns). These patients were not classified as responders for the purposes of calculating OR rates. Antitumor responses and / or long-term disease stabilization were observed in patients regardless of the prior treatment they had received (see summary of progression-free time for patients with OR and SD in Supplementary Appendix 4 of Topalian et al., 2012b).

[0267] In NSCLC patients, 14 ORs were observed at BMS-936558 doses of 1, 3, or 10 mg / kg, respectively, with response rates of 6%, 32%, and 18%. ORs were observed across NSCLC histologies: 6 responders (33%) of 18 squamous, 7 responders (13%) of 56 non-squamous, and 1 of 2 unknown. All 14 patients with ORs began treatment ≥24 weeks prior to data analysis, and 8 of these had response durations ≥24 weeks (Table 1). Stable disease (SD) lasting ≥24 weeks was observed in 5 (7%) of all NSCLC patients with non-squamous histology. In MEL patients, 26 ORs were observed at doses ranging from 0.1-10 mg / kg, with response rates ranging from 19%-41% depending on dose level. At the 3 mg / kg dose level, OR was noted in 7 of 17 (41%) patients. Seventeen of the 26 MEL patients who achieved OR had begun treatment ≥1 year prior to data analysis, and of these, 13 patients had an OR duration ≥1 year (yr). The remaining eight patients with OR were on study <1 year, and six had responses ranging from 1.9-5.6 months. SD lasting ≥24 weeks was observed in six (6%) patients. In RCC patients, OR occurred in four of 17 (24%) patients treated with a BMS-936558 dose of 1 mg / kg and five of 16 (31%) patients treated with 10 mg / kg. Of the eight RCC patients with OR who began treatment ≥1 year prior to data analysis, five (63%) had an OR duration ≥1 yr. SD lasting ≥24 weeks was observed in an additional nine (27%) patients.

[0268] Table 1. Efficacy population for data evaluated up to February 2012 * (N=236) † Clinical Activity of BMS-936558 in [Table 1]

[0269] *The efficacy population consisted of response-evaluable patients in whom treatment was initiated at least 8 months prior to data analysis in February 2012 and who had measurable disease at baseline and one of the following: at least one treatment scan or clinical evidence of disease progression or death. † CR indicates complete response, MEL indicates melanoma, NSCLC indicates non-small cell lung cancer, ORR indicates objective response rate, PFSR indicates progression-free survival, PR indicates partial response, RCC indicates renal cell carcinoma, SD indicates stable disease, and n indicates the number of patients. ‡ Objective response rates ({[CR+PR]÷n}×100) were calculated based on confirmed responses with confidence intervals calculated using the Clopper-Pearson method. Individual patient responses were adjudicated by RECIST v1.0 with modifications (see Topalian et al., 2012b). § Progression-free survival was the proportion of patients surviving at 24 weeks calculated by Kaplan-Meier methodology with confidence intervals using the Greenwood method. ¶ 1 CR. **One NSCLC patient treated at the 3 mg / kg dose level had an initial assessment of progressive disease and then a PR and was classified as a responder.

[0270] Table 2. BMS-936558 evaluated up to February 2012 * Duration of objective response to [Table 2]

[0271] *MEL means melanoma, NA means not applicable, NSCLC means non-small cell lung cancer, and RCC means renal cell carcinoma. † Time from first response to confirmed progression, death, or, for censored data, to last tumor assessment. ‡One patient was treated beyond the initial assessment of progressive disease and then had a PR; the patient was classified as a responder for purposes of calculating response rate by RECIST v1.0 but was not eligible for calculation of duration of response.

[0272] Data analyzed March 2013 Objective responses were observed in patients with NSCLC (17%), MEL (31%) and RCC (29%), but not in patients with CRC or CRPC. Responses were observed across all nivolumab doses tested; response rates in NSCLC patients receiving 1 mg / kg vs. 3 or 10 mg / kg appeared to be reduced (3%, vs. 24% and 20%, respectively) (Tables 3 and 4). Thirteen patients (4.8%) of 270 with responding histology had unconventional response patterns that did not meet RECIST criteria (e.g., sustained decline in target lesions in the presence of new lesions or regression after initial progression) (Wolchok et al., 2009). Additional patients showed SD at 24 weeks or longer (10% NSCLC, 7% MEL, 27% RCC). Sustained survival, reflected by 1- and 2-year landmark OS rates, was noted in each of the responding populations as follows: NSCLC, 42% and 14%; MEL, 62% and 43%; and RCC, 70% and 50% (Table 3). A median OS of 9.6 months for lung cancer (9.2 and 10.1 months for squamous and non-squamous NSCLC tissues, respectively), 16.8 months for MEL, and over 22 months for RCC was observed; median PFS was 2.3 months for NSCLC, 3.7 months for MEL, and 7.3 months for RCC; and median response duration was 74, 104, and 56 weeks, respectively (Table 4). Of the 16 responders who discontinued treatment for reasons other than disease progression and were followed for at least 24 weeks, 13 (81%) were in response at the time of analysis (Figure 8).

[0273] Table 3. Efficacy population assessed in March 2013 (N=306) * Clinical activity of nivolumab in [Table 3]

[0274] *No objective responses were seen in 19 patients with colorectal cancer or 17 patients with castration-resistant prostate cancer. † Objective response rates ({[CR+PR]÷n}×100) were calculated based on confirmed responses with confidence intervals calculated using the Clopper-Pearson method. Individual patient responses were determined by RECIST v1.0 with modifications (see Methods S1 and Study Protocol, NEJM.org). ‡ Time from first response to confirmed progression, death, or, for censored data (indicated by "+"), time to last tumor assessment. **Of 129 patients with non-small cell lung cancer, one had unknown histology and had no objective response. Other patients had squamous or non-squamous histology as indicated. § NR, not reached; the point at which responders have a 50% or lower chance of progressing was not reached due to insufficient number of events and / or follow-up. ¶ Insufficient duration of follow-up. [ka] One CR was indicated for melanoma and one CR was indicated for renal cancer. # Median overall survival was not reached at 22 months, the longest time to death for any patient with kidney cancer in this trial. ^NE, cannot be rated.

[0275] Table 4. Clinical activity of nivolumab by dose level evaluated in March 2013 [Table 4-1] [Table 4-2]

[0276] *Objective response rates ({[CR+PR]÷n}×100) were calculated based on confirmed responses with confidence intervals calculated using the Clopper-Pearson method. Individual patient responses were adjudicated by RECIST v1.0 with modifications (see Methods S1 and Study Protocol, NEJM.org). † Time from first response to confirmed progression, death, or, for censored data (indicated by "+"), time to last tumor assessment. ‡ NR, not reached; the point at which responders have a 50% or lower chance of progressing was not reached due to insufficient number of events and / or follow-up. § One CR was indicated for melanoma and one CR was indicated for renal cancer. ¶ Five patients with tumor progression had their dose escalated from 0.1 to 1.0 mg / kg, and six had their dose escalated from 0.3 to 1.0 mg / kg. These patients did not respond to treatment. # Median overall survival was not reached at 22 months, the longest time to death for any patient with kidney cancer in this trial. ^NE, cannot be rated.

[0277] Example 8 Correlation between membrane PD-L1 expression and anti-PD-1 responses IHC staining of PD-L1 was performed in pretreated formalin-fixed paraffin-embedded (FFPE) tumor specimens using mouse anti-human PD-L1 mAb 5H1 (Dong et al., 2002) in a standard IHC protocol (Taube et al., 2012; Supp. Materials). Briefly, 5 μm-FFPE sections mounted on glass slides were deparaffinized in xylene and antigen retrieval was performed using Tris-EDTA buffer at pH 9.0 at 120°C for 10 min in an antigen retrieval apparatus (Biocare Medical). Endogenous peroxidase, biotin and proteins were blocked (CAS system K1500, Dako; Avidin / biotin Blocking Kit, SP-2001, Vector Laboratories; Serotec Block ACE), and primary 5H1 Ab was added at a concentration of 2 μg / ml and incubated at 4°C for 20 h. Secondary Ab (biotinylated anti-mouse IgG1, 553441 BD) was applied at a concentration of 1 μg / ml for 30 min at room temperature (RT). Signals were then developed by amplification according to the manufacturer's protocol (CAS system K1500, Dako). Sections were counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and coverslipped.

[0278] The percentage of tumor cells showing cell surface staining for PD-L1 was scored by two independent pathologists blinded to treatment outcome. PD-L1 positivity was defined on a specimen-by-specimen basis by a 5% expression threshold (Taube et al., 2012; Thompson et al., 2006) and, in the case of multiple specimens, when specimens met this criterion. Fisher's exact test was applied to assess the association between PD-L1 expression and OR. Note, however, that this analysis was based, in part, on random biopsies from nonrandom subsets of the population and testing of statistical hypotheses was not prespecified.

[0279] Sixty-one pretreatment tumor specimens from 42 patients (18 MEL, 10 NSCLC, 7 CRC, 5 RCC, and 2 CRPC) were analyzed for tumor cell surface PD-L1 expression (Figure 8). Biopsy specimens from 25 to 42 patients were positive for PD-L1 expression by IHC. Fisher's exact test was applied to assess the association between PD-L1 expression and OR in post-hoc analysis. Surface-PD-L1 in 42 patients + Of the patients, 9 (36%) achieved OR but had PD-L1 - Seventeen patients with tumors did not achieve an OR (Figure 8A). Thus, in a subset of patients, cell surface expression of PD-L1 on tumor cells in pretreatment biopsies is associated with increased OR rates in patients treated with BMS-936558, whereas patients with documented PD-L1-negative tumors did not experience an OR. These data indicate that tumor PD-L1 expression is a molecular marker that can enable patient selection for anti-PD-1 immunotherapy.

[0280] Example 9 Isolation of rabbit mAb directed against membrane hPD-L1 antigen in FFPE tissues Rabbit Abs against human PD-L1 polypeptide were prepared by Epitomics, Inc. (Burlingame, CA) by immunization of rabbits with recombinant human PD-L1 fusion protein. Antiserum titers were assessed using a standard direct ELISA with hPD-L1 antigen and using a cellular ELISA using transfected cells overexpressing hPD-L1. These Abs were also screened for their ability to bind to PD-L1 by IHC assay on FFPE tissue sections. Rabbits with the highest Ab titers were selected for splenectomy. Lymphocytes isolated from the spleen were fused to myeloma cells in 40x96-well plates and screened by ELISA against the immune PD-L1 antigen and by cellular ELISA against cells overexpressing hPD-L1. Positive clones were expanded to 24-well plates and confirmatory screening was performed by direct and cellular ELISA. Supernatants (sups) of clones that were specific for the screening antigen were rescreened by IHC.

[0281] A set of mouse anti-hPD-L1 mAbs were also produced by immunization of mice, using a similar protocol as described above for the rabbit mAbs.

[0282] Of a total of 185 multiclones from both rabbit and mouse immunizations screened, only 10 rabbit multiclone Abs specifically detected the membrane form of hPD-L1. Purified mouse subclones were not found to specifically detect cell surface hPD-L1. 60 subclones (designated numbers 13, 20, 28, 29 and 49, each containing 12 subclones) from the top 5 rabbit multiclones were first screened by IHC on FFPE low-density tissue microarrays (TMA), then confirmed and specificity verified in a limited number of 25 subclones. Rabbit IgG was used as a negative isotype control, and mAb 5H1 (Dong et al., 2002) was used as a positive control. Specificity was further verified by antigen preabsorption assay. By two rounds of IHC, the following 15 purified subclones were selected as highly promising Abs with respect to staining specificity and intensity: 13-1, 13-3, 13-7, 13-8; 20-5, 20-7, 20-12, 20-6; 28-1, 28-8, 28-12; 29-8; 49-5, 49-7 and 49-9. The immune response data for these selected Abs are summarized in Table 5.

[0283] Table 5. Immunoreactivity of rabbit anti-hPD-L1 mAb [Table 5]

[0284] *CHO cells stably transfected with PD-L1 vs. CHO-S control; † PD-L1 positive tissues included placenta and one non-small cell lung carcinoma; detection of up to "very high" expression suggests better sensitivity in detecting membrane PD-L1.

[0285] Additional assays were performed to further characterize the purified Ab clones, including determining binding affinity and cross-competition among the Abs (to identify overlapping versus distinct epitope regions) by surface plasmon resonance. All Abs showed high binding affinity (K D <10 -9 These 15 purified clones were also rescreened by IHC on standard sections or FFPE low-density TMAs on various cell and tissue types known to be positive or negative for cell surface expression of PD-L1. Rabbit IgG was used as an isotype control and mAb 5H1 was used as a positive control. At high concentrations (10 μg / ml), clones 28-x and 49-x showed low to moderate levels of background staining in tissues, whereas clone 13-x showed no background staining, suggesting that the 13-x clone has a wide dynamic range. The 20-x clone showed various degrees of background staining that was mainly cytoplasmic and diffuse. A clone with very strong specific detection of membrane PD-L1, rabbit clone 28-8 (K as determined by SPR) was identified. D = 100 pM) and were selected as the primary Abs for subsequent IHC assays. MAbs 28-1, 28-12, 20-12, and 29-8 had K values ​​of 130 pM, 94 pM, 160 pM, and 1200 pM, respectively. D The heavy chain variable (V H ) and light (kappa) chain variable (V κThe sequences of the 28-8 Ab are set forth in SEQ ID NOs: 35 and 36, respectively. Based on SPR analysis, 28-8 Ab was shown to recognize a different epitope from mouse mAb 5H1. Clones 28-1, 28-12, 29-8 and 20-12 were the next best Abs for robust detection of membrane PD-L1 in FFPE tissues. mAb13-1 had the best specificity for detection of membrane PD-L1, but the maximum detection level was lower than that of the other leading Abs. Western blotting was also performed with plus / minus antigen competition to verify the specificity of the top selected Abs for PD-L1.

[0286] Binding of mAbs 5H1 and 28-8 to membrane PD-L1 was compared in FFPE test tissue samples containing tumor cells and tumor-infiltrating inflammatory cells from different tumor types. Membrane PD-L1 expression was assessed using the histoscore method performed by two independent pathologists. Four NSCLC, two MEL, and two RCC tumors were stained with 28-8 at 2 mg / ml and 5H1 at 5 mg / ml. Data are tabulated in Table 6 and shown graphically in Figure 9. Rabbit mAb 28-8 showed better detection (higher histoscore) for 7 out of 10 samples using 2.5-fold less Ab, and in only one sample had a slightly higher histoscore for 5H1 than mAb 28-8.

[0287] Table 6. Comparison of mAb 28-8 and 5H1 by Histoscore analysis [Table 6]

[0288] Taube et al. (2012) demonstrated cell surface binding of mAb 5H1 by flow cytometry in cultured cells and confirmed the specificity of binding to PD-L1 using a PD-L1 fusion protein to competitively block binding of 5H1 mAb to tissue sections. These authors also compared 5H1, previously described by Gadiot et al. (2011), with a rabbit polyclonal anti-hPD-L1 Ab, 4059, and found that 5H1 displayed a cell surface staining pattern in FFPE samples, whereas pAb 4059 demonstrated widespread cytoplasmic staining. Furthermore, when 5H1 was compared to pAb 4059 by Western blot analysis, in contrast to 5H1, which specifically detected a 50 kDa band of glycosylated PD-L1, Ab 4059 bound to multiple proteins in melanoma cell lysates in addition to the 50 kDa protein corresponding to the expected mass of glycosylated PD-L1 (Taube et al., 2012). Contrary to the results of Taube et al. (2012) and those described herein (summarized in Table 7), Gadiot et al. (2011) reported that mAb 5H1 produced high levels of background staining in FFPE tissue samples and found that pAb 4059 produced sufficient specific staining of PD-L1 in FFPE samples. None of the 13 other Abs tested by Gadiot et al. (2011) stained the FFPE tissues giving high background staining or were blocked by PD-L1 fusion protein competition, highlighting the difficulty of obtaining anti-PD-L1 Abs that specifically bind to PD-L1 in FFPE tissues.

[0289] In this study, an automated IHC assay (see Example 10) was used to evaluate the binding of several commercially available anti-PD-L1 Abs and 5H1 (Dong et al., 2002) to FFPE tissue samples containing a variety of cells expressing PD-L1. The results, summarized in Table 7, show that the commercially available Abs tested did not specifically recognize membrane PD-L1 expression in human tissues known to express PD-L1 or clearly distinguish between CHO cells expressing PD-L1 versus untransfected parental CHO cells not expressing PD-L1. The inability of polyclonal Ab (pAb) 4059 to bind specifically recognized membrane PD-L1 is consistent with the findings of Taube et al. (2012). Binding of 28-8 is similar to that of 5H1 in this assay, but histoscore analysis suggests that 28-8 works better than 5H1.

[0290] Table 7. Binding of mAbs to FFPE samples containing PD-L1-expressing cells [Table 7]

[0291] *CHO cells stably transfected with PD-L1 vs. parental CHO-S negative control; † PD-L1 positive tissues included tonsil and / or thymus; mAb, mouse monoclonal Ab; pAb, rabbit polyclonal Ab.

[0292] Example 10 Development of an automated IHC protocol to assess PD-L1 expression An automated IHC protocol was developed to assay PD-L1 expression in FFPE specimens. Tissue sections (4 μm) were mounted on slides and deparaffinized in an autostainer (Leica) by immersing in xylene twice for 5 min, and rehydrated by immersing in 100% EtOH twice, 95% (v / v) EtOH twice, 70% (v / v) EtOH once, and deionized water (dH2O) once for 2 min each. Antigen retrieval was performed using an antigen retrieval device (Biocare Medical Decloaking Chamber Plus) and Dako pH6 buffer, heated to 110°C (P1) for 10 min, and then transferred to the next step (P2 FAN ON at 98°C; FAN OFF at 90°C). Slides were cooled at room temperature (RT) for 15 min and rinsed with water for approximately 1 min.

[0293] The reagents were placed into an automated stainer (BioGenex i6000) and tissue areas were defined using a pap pen. The IHC assay, run using an autostainer in research mode, included the following steps: neutralize endogenous peroxidase using Peroxidase Block (Leica) for 10 min, then rinse 3 times with IHC Wash Buffer (Dako); apply Protein Block (Leica) to slides and incubate at RT for 10 min, then wash 3 times with Wash Buffer; apply Primary Ab (2 μg / ml) to slides and incubate at RT for 1 h, then wash 6 times with Wash Buffer; add Post Primary Block (NovoLink Kit) to slides and incubate for 30 min, then wash 6 times with Wash Buffer; add NovoLink Polymer (NovoLink Kit) to slides and incubate for 30 min, then wash 6 times with Wash Buffer; add DAB chromogenic substrate (NovoLink Kit), develop for 3 min, then rinse 5 times with dH2O at RT; counterstain with Hematoxylin (NovoLink Kit) for 1 min at RT, then wash 3 times with dH2O for 5time at RT. Primary Abs were selected from rabbit anti-PD-L1 Abs shown in Table 4; mAb28-8 was the preferred Ab. Rabbit IgG (Dako) was used as a negative control. Tissue sections were dehydrated using a Leica autostainer by washing once in 70% EtOH for 2 min, twice in 95% EtOH for 2 min, and three times in 70% EtOH for 2 min, and cleared by washing three times in xylene for 5 min. Sections were permanently mounted on slides with permount, covered with coverslips, and transferred to a chemical hood to dry.

[0294] Example 11 Design of Phase 1 clinical trials of anti-PD-L1 antibodies Test Design A Phase 1 study was conducted to evaluate the safety and tolerability of BMS-936559 (also referred to herein and in U.S. Patent No. 7,943,743) in patients with selected advanced solid tumors. Secondary objectives included initial evaluation of the antitumor activity of BMS-936559 and pharmacokinetic evaluation. Pharmacokinetic measurements included exploratory objectives. Patients were treated in 6-week cycles of BMS-936559 administered as a 60-minute intravenous infusion every 2 weeks on days 1, 15, and 29 of each cycle. Treatment continued for up to 16 cycles until patients experienced unacceptable toxicity, disease progression, or withdrew consent. In some patients who were clinically stable, treatment beyond the first disease progression was permitted until further progression was confirmed.

[0295] Dose escalation Patients with advanced NSCLC, MEL, CRC, RCC, ovarian (OV), gastric (GC), breast (BC) and pancreatic (PC) carcinoma were eligible to enroll. Safety was evaluated at doses of 0.3, 1, 3 and 10 mg / kg using an accelerated titration design. One patient was enrolled in each successive cohort who had a grade 2 drug-related AE during cycle 1. Two additional patients were then enrolled at that dose level, and the study transitioned to a standard 3+3 design. No intrapatient dose escalation or de-escalation was allowed. If less than 1 / 3 of patients had dose-limiting toxicity, the maximum tolerated dose (MTD) was defined as the highest dose.

[0296] Cohort Expansion Initially, five expansion cohorts (n=16 / cohort) were enrolled at 10 mg / kg for patients with NSCLC, MEL, RCC, OV and CRC. Based on initial signals of activity, further expansion cohorts (up to n=16 / cohort) were enrolled at 10 mg / kg for MEL (at 1.0 and 3.0 mg / kg), NSCLC (1, 3 or 10 mg / kg randomized squamous or non-squamous tissue cohorts), and PC, BC and GC.

[0297] patient Patients were required to have documented progressive NSCLC, MEL, RCC, OV, CRC, PC, GC, or BC and had failed at least one prior tumor-appropriate therapy for progressive / metastatic disease (with the exception of PC or GC patients who could be treatment naive). Other inclusion criteria included age ≥ 18 years, life expectancy ≥ 12 weeks, Eastern Cooperative Oncology Group performance status of ≤ 2, measurable disease as defined by RECIST v1.0, and adequate hematological, hepatic, and renal function. Patients with treated brain metastases were allowed if stable for at least 8 weeks. Major exclusion criteria included a history of autoimmune disease or other disease requiring steroid or immunosuppressant therapy, previous treatment with T-cell modulating Abs (including anti-PD-1, anti-PD-L1, and anti-CTLA-4), HIV, or a history of active hepatitis B or C.

[0298] In this extension study, 207 patients with NSCLC (n=75), MEL (n=55), CRC (n=18), RCC (n=17), OV (n=17), PC (n=14), GC (n=7) or BC (n=4) were treated with BMS-936559 for 34 months. This includes safety data. Efficacy was characterized in 160 response-evaluable patients. Baseline demographic characteristics of the total and response-evaluable patient populations were very similar (Brahmer et al., 2012). Of treated patients, 86% had received prior chemotherapy and 28% had received immunological or biological therapy. Prior treatment by tumor type included immunotherapy (56%) and B-RAF inhibitors (9%) in patients with MEL; platinum-based chemotherapy (95%) and tyrosine kinase inhibitors (TKIs; 41%) in patients with NSCLC; and nephrectomy (94%), anti-angiogenic therapy (82%) and immunotherapy (41%) in patients with RCC (for details of patient pretreatment, see Brahmer et al., 2012).

[0299] statistical analysis All 207 patients initiating treatment on the analysis date were used for summary of baseline characteristics and AEs. The efficacy population consisted of 160 response-evaluable patients who initiated treatment at least 7 months prior to the analysis date. AEs were coded using MedDRA v14.1. Individual best overall response was derived from radiological scan measurements by modified RECIST v1.0. ORs were c...

Claims

1. A method of treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion thereof that disrupts the interaction between programmed death-1 (PD-1) and programmed death-ligand-1 (PD-L1), wherein the antibody or antigen-binding portion thereof specifically binds to PD-1 or PD-L1.

2. 2. The method of claim 1, wherein the cancer is selected from the group consisting of melanoma, renal cell carcinoma, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, squamous cell carcinoma of the head and neck, carcinoma of the esophagus, gastrointestinal tract and breast, and hematological malignancies.

3. 10. The method of claim 1, wherein the therapeutically effective amount of the antibody or antigen-binding portion thereof comprises a dose ranging from 0.1 to 10.0 mg / kg body weight administered on a weekly, biweekly, or monthly dosing schedule.

4. (a)(i) providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample contains tumor cells and tumor-infiltrating inflammatory cells; (ii) assessing the proportion of cells in the test tissue sample that express PD-L1 on their cell surface; and (iii) selecting the subject as a suitable candidate based on assessing that the proportion of cells in the test tissue sample that express PD-L1 on their cell surface exceeds a predetermined threshold level. selecting a subject who is an appropriate candidate for immunotherapy comprising: (b) administering to the selected subject a composition comprising a therapeutically effective amount of an anti-PD-1 antibody.

1. A method for immunotherapy of a subject suffering from cancer, comprising:

5. 5. The method of claim 4, wherein the proportion of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 polypeptide on the surface of cells in the test tissue sample.

6. 6. The method of claim 5, wherein the test tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample.

7. 7. The method of claim 6, wherein the presence of PD-L1 polypeptide is determined using an automated IHC assay.

8. 13. The method of claim 12, wherein the IHC assay is performed using an anti-PD-L1 monoclonal antibody that binds to a PD-L1 polypeptide, and the anti-PD-L1 monoclonal antibody is selected from 28-8, 28-1, 28-12, 29-8, and 5H1.

9. 5. The method of claim 4, wherein the predetermined threshold level is 1% of tumor cells or a single tumor-infiltrating inflammatory cell expressing cell surface PD-L1 as determined by automated IHC using mAb 28-8.

10. (a)(i) providing a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample contains tumor cells and tumor-infiltrating inflammatory cells; (ii) assessing the proportion of cells in the test tissue sample that express PD-L1 on their cell surface; and (iii) selecting a subject as not suitable for anti-PD-1 antibody immunotherapy based on an assessment that the proportion of cells in the test tissue sample that express PD-L1 on their cell surface is below a predetermined threshold level. selecting subjects who are not suitable for anti-PD-1 antibody immunotherapy, including: (b) administering a standard-of-care therapeutic agent other than an anti-PD-1 antibody to the selected subject; 10. A method for treating a subject suffering from cancer, comprising:

11. A monoclonal antibody or antigen-binding portion thereof that specifically binds to a PD-L1 polypeptide expressed on the cell surface in a formalin-fixed, paraffin-embedded (FFPE) tissue sample.

12. 17. The monoclonal antibody or antigen-binding portion thereof of claim 16, wherein the antibody or portion thereof comprises CDR1, CDR2 and CDR3 regions in the heavy chain variable region comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO: 35, and CDR1, CDR2 and CDR3 regions in the light chain variable region comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:

36.

13. 17. The monoclonal antibody or antigen-binding portion thereof of claim 16, wherein the antibody or portion thereof comprises a heavy chain variable region comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO: 35, and a light chain variable region comprising consecutively linked amino acids having the sequence set forth in SEQ ID NO:

36.

14. (a) an antibody or antigen-binding portion thereof that specifically binds to and inhibits programmed death-1 (PD-1); and (b) an antibody or antigen-binding portion thereof that specifically binds to and inhibits cytotoxic T-lymphocyte antigen-4 (CTLA-4); 1. A method of treating a subject suffering from cancer, comprising administering to the subject Each antibody is administered at a dose ranging from 0.1 to 20.0 mg / kg body weight in a concurrent regimen comprising: (i) an induction dosing schedule comprising at least 2, 4, 6, 8, or 10 doses of a combination of anti-PD-1 and anti-CTLA-4 antibodies at a dosing frequency of at least once every 2, 3, or 4 weeks, or at least once a month, followed by at least 2, 4, 6, 8, or 12 doses of anti-PD-1 alone at a dosing frequency of at least once every 2, 3, or 4 weeks, or at least once a month; and (ii) a maintenance dosing schedule comprising at least 4, 6, 8, 10, 12, or 16 doses of a combination of anti-PD-1 and anti-CTLA-4 antibodies administered at a dosing frequency of at least once every 8, 12, or 16 weeks, or at least once quarterly; A method comprising:

15. Anti-PD-1 and anti-CTLA-4 antibodies at the following doses: (a) 0.1 mg / kg anti-PD-1 antibody and 3 mg / kg anti-CTLA-4 antibody; (b) 0.3 mg / kg anti-PD-1 antibody and 3 mg / kg anti-CTLA-4 antibody; (c) 1 mg / kg anti-PD-1 antibody and 3 mg / kg anti-CTLA-4 antibody; (d) 3 mg / kg anti-PD-1 antibody and 3 mg / kg anti-CTLA-4 antibody; (e) 5 mg / kg anti-PD-1 antibody and 3 mg / kg anti-CTLA-4 antibody; (f) 10 mg / kg anti-PD-1 antibody and 3 mg / kg anti-CTLA-4 antibody; (g) 0.1 mg / kg anti-PD-1 antibody and 1 mg / kg anti-CTLA-4 antibody; (h) 0.3 mg / kg anti-PD-1 antibody and 1 mg / kg anti-CTLA-4 antibody; (i) 1 mg / kg anti-PD-1 antibody and 1 mg / kg anti-CTLA-4 antibody; (j) 3 mg / kg anti-PD-1 antibody and 1 mg / kg anti-CTLA-4 antibody; (k) 5 mg / kg of an anti-PD-1 antibody and 1 mg / kg of an anti-CTLA-4 antibody; or (l) 10 mg / kg anti-PD-1 antibody and 1 mg / kg anti-CTLA-4 antibody 15. The method of claim 14, wherein the

16. 1. A method of treating a subject suffering from cancer who has previously been treated with an anti-CTLA-4 antibody, comprising administering to the subject an antibody or antigen-binding portion thereof that specifically binds and inhibits PD-1 at a dose ranging from 0.1 to 20.0 mg / kg body weight with a dosing frequency of at least once a week, at least once every 2, 3, or 4 weeks, or at least once a month in a regimen of up to 6 to up to 72 consecutive doses.

17. The method of claim 15 or 16, wherein the anti-PD-1 antibody is nivolumab.

18. The method of claim 15 or 16, wherein the anti-CTLA-4 antibody is ipilimumab.

19. 1. An anti-PD-1 antibody, or antigen-binding portion thereof, for use in treating a subject suffering from cancer in a concomitant regimen comprising combined administration with an anti-CTLA-4 antibody, or antigen-binding portion thereof, wherein the anti-PD-1 and anti-CTLA-4 antibodies are each administered at a dose ranging from 0.1 to 20.0 mg / kg body weight, and the concomitant regimen comprises: (i) an induction dosing schedule comprising at least 2, 4, 6, 8, or 12 doses of a combination of anti-PD-1 and anti-CTLA-4 antibodies at a dosing frequency of at least once every 2, 3, or 4 weeks, or at least once a month, followed by at least 2, 4, 6, 8, or 10 doses of anti-PD-1 alone at a dosing frequency of at least once every 2, 3, or 4 weeks, or at least once a month; and (ii) a maintenance dosing schedule comprising up to 4, 6, 8, 10, 12, or 16 doses of a combination of anti-PD-1 and anti-CTLA-4 antibodies administered at a dosing frequency of at least once every 8, 12, or 16 weeks, or at least once quarterly; An anti-PD-1 antibody or antigen-binding portion thereof, comprising:

20. (a) an antibody or antigen-binding portion thereof that specifically binds to and inhibits PD-1, at a dose ranging from 0.1 to 20.0 mg / kg body weight; (b) an antibody or antigen-binding portion thereof that specifically binds to and inhibits CTLA-4 at a dose ranging from 0.1 to 20.0 mg / kg body weight; and (c) Instructions for using anti-PD-1 and anti-CTLA-4 antibodies in the simultaneous regimen method of claim 14.

10. A kit for treating a subject suffering from cancer, comprising:

21. (a) an antibody or antigen-binding portion thereof that specifically binds to and inhibits PD-1, at a dose ranging from 0.1 to 20.0 mg / kg body weight; and (b) Instructions for using an anti-PD-1 antibody in the sequential regimen method of claim 16.

10. A kit for treating a subject suffering from cancer, comprising:

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