Treatment of PD-L1-negative melanoma using anti-PD-1 antibodies and anti-CTLA-4 antibodies

Abstract

This provides a method for treating melanoma. [Solution] The present invention provides a method comprising (i) identifying a patient having PD-L1-negative melanoma, and (ii) administering to the patient a combination of an anti-PD-1 antibody or its antigen-binding moiety and an anti-CTLA-4 antibody or its antigen-binding moiety. The method of the present invention can extend progression-free survival to more than 8 months and / or reduce tumor size by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the tumor size before administration.

Description

[Technical Field]

[0001] Throughout this specification, various publications are cited in parentheses by author name and date or patent or patent publication number. The disclosures of these publications are thereby incorporated herein by reference to provide a more complete description of the state of the art as it was known to those skilled in the art at the time the present invention was described herein and claimed. However, no such references are to be construed as acknowledging that such references constitute prior art of the present invention.

[0002] Field of Invention The present invention relates to a method for treating PD-L1-negative melanoma, comprising administering a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody. [Background technology]

[0003] Background of the Invention Human cancers possess numerous genetic and epigenetic alterations and produce neoantigens that the immune system may recognize (Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system, composed of T and B lymphocytes, has potent anti-cancer activity and possesses broad ability and exquisite specificity to respond to diverse tumor antigens. Furthermore, the immune system exhibits considerable flexibility and memory. The successful utilization of all these properties of the adaptive immune system makes immunotherapy unique among all cancer treatment modalities.

[0004] In recent years, several immune checkpoint pathway inhibitors have begun to offer novel immunotherapeutic approaches for cancer treatment, including the development of ipilimumab (Yervoy®), an antibody (Ab) that binds to and inhibits cytotoxic T lymphocyte antigen-4 (CTLA-4) for the treatment of patients with advanced melanoma, and the development of antibodies such as nivolumab and pembrolizumab (formerly lambrolizumab; USAN Council Statement, (2013) Pembrolizumab: Statement on a nonproprietary name adopted by the USAN Council (ZZ-165), November 27, 2013), which specifically bind to the programmed death-1 (PD-1) receptor and block the inhibitory PD-1 / PD-1 ligand pathway. [Overview of the project] [Problems that the invention aims to solve]

[0005] The future of the emerging field of personalized medicine lies in the fact that advances in pharmacogenomics will increasingly be used to tailor therapeutics to specific subpopulations, and ultimately to individual patients, in order to enhance efficacy and minimize adverse effects. Recent successes include, for example, imatinib mesylate (Gleevec®), a protein tyrosine kinase inhibitor that inhibits bcr-abl tyrosine kinase, for treating Philadelphia chromosome-positive chronic myeloid leukemia (CML); crizotinib (Xalkori®), for treating 5% of patients with late-stage non-small cell lung cancer expressing the mutant anaplastic lymphoma kinase (ALK) gene; and vemurafenib (Zelboraf®), an inhibitor of the mutant B-RAF protein (V600E-BRAF) expressed in about half of melanoma tumors. However, unlike the clinical development of small molecule agents targeting distinct activating mutations found in selected cancer populations, a unique challenge in cancer immunotherapy is the identification of mechanism-based predictive biomarkers to enable patient selection and on-treatment management. [Means for solving the problem]

[0006] Summary of the Invention The present invention provides a method for treating melanoma, comprising (i) identifying a patient having a PD-L1-negative melanoma tumor, and (ii) administering to the patient (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4. The present invention also provides a method for treating melanoma, comprising administering to a patient having a melanoma tumor (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4, wherein the patient has been identified as having a PD-L1-negative melanoma tumor prior to administration.

[0007] The present invention further provides a method for extending progression-free survival beyond eight months in a patient with melanoma tumor, comprising administering to the patient (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4, wherein the patient has been identified as having a PD-L1-negative melanoma tumor prior to administration, and the patient exhibits a progression-free survival beyond eight months. In one embodiment, the patient's progression-free survival is extended beyond approximately 11 months, approximately 12 months, approximately 13 months, approximately 14 months, approximately 15 months, approximately 16 months, approximately 17 months, approximately 18 months, approximately 2 years, approximately 3 years, approximately 4 years, approximately 5 years, approximately 6 years, approximately 7 years, approximately 8 years, approximately 9 years, or approximately 10 years after administration. In one particular embodiment, the patient's progression-free survival is extended beyond 11 months.

[0008] The present invention also provides a method of reducing tumor size by at least 10% in a patient having a melanoma tumor, comprising administering to the patient (a) an anti-PD-1 antibody or antigen-binding portion thereof that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or antigen-binding portion thereof that specifically binds to human CTLA-4, wherein the patient has been identified as having a PD-L1-negative melanoma tumor prior to administration, and the administration reduces tumor size by at least about 10%, about 20%, about 30%, about 40% or about 50% compared to prior to administration.

[0009] The present invention also provides a method of increasing objective response rate to higher than 40% in a patient population each having a melanoma tumor in cancer treatment, comprising administering to the patients (a) an anti-PD-1 antibody or antigen-binding portion thereof that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or antigen-binding portion thereof that specifically binds to human CTLA-4, wherein each patient has been identified as having a PD-L1-negative melanoma tumor prior to administration, and the objective response rate is higher than 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%.

[0010] The present invention also provides a method of selecting a patient suitable for combination therapy with an anti-PD-1 antibody and an anti-CTLA-4 antibody, comprising: (i) identifying a patient having a PD-L1-negative melanoma tumor; and (ii) instructing a healthcare provider to administer to the patient (a) an anti-PD-1 antibody or antigen-binding portion thereof that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or antigen-binding portion thereof that specifically binds to human CTLA-4.

[0011] In one embodiment, the methods disclosed herein further comprise identifying a patient having a melanoma tumor that does not express PD-L1 prior to administration. In one embodiment, the patient is characterized by (i) a long-term progression-free survival of more than 8 months, (ii) a reduction in tumor size of at least about 10%, about 20%, about 30%, about 40% or about 50% compared to the tumor size prior to administration, or (iii) both.

[0012] In one embodiment, the method disclosed herein further includes measuring PD-L1 expression in melanoma tumors. In one embodiment, the measurement further includes evaluating the proportion of cells expressing PD-L1 on the cell surface in a test tissue sample. In one particular embodiment, the presence of PD-L1 is determined using an automated IHC assay.

[0013] The present invention further provides a kit for the treatment of patients with melanoma tumors, comprising: (a) a constant dose of an anti-PD-1 antibody or its antigen-binding moiety in the range of 0.1 to 10 mg / kg body weight; (b) a constant dose of an anti-CTLA-4 antibody or its antigen-binding moiety in the range of 0.1 to 10 mg / kg body weight; and (c) instructions for using the anti-PD-1 antibody or its antigen-binding moiety and the anti-CTLA-4 antibody or its antigen-binding moiety in any of the methods disclosed herein. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows a patient flowchart for a randomized, double-blind, multicenter phase 3 clinical trial.

[0015] [Figure 2A] Figures 2A-C show progression-free survival data for patients in the treatment-intended population (Figure 2A) with positive PD-L1 status (Figure 2B) and negative PD-L1 status (Figure 2C). Each graph shows progression-free survival in months for patients treated with nivolumab monotherapy (black line), ipilimumab monotherapy (dashed line), or a combination of nivolumab and ipilimumab (dotted line) (Figures 2A-C). The number of individuals facing risk for nivolumab, nivolumab + ipilimumab, and ipilimumab, respectively, is shown below each x-axis in months (Figures 2A-C). PD-L1 expression status is based on validated PD-L1 assay data (Figures 2B-C). [Figure 2B]Figures 2A-C show progression-free survival data for patients in the treatment-intended population (Figure 2A) with positive PD-L1 status (Figure 2B) and negative PD-L1 status (Figure 2C). Each graph shows progression-free survival in months for patients treated with nivolumab monotherapy (black line), ipilimumab monotherapy (dashed line), or a combination of nivolumab and ipilimumab (dotted line) (Figures 2A-C). The number of individuals facing risk for nivolumab, nivolumab + ipilimumab, and ipilimumab, respectively, is shown below each x-axis in months (Figures 2A-C). PD-L1 expression status is based on validated PD-L1 assay data (Figures 2B-C). [Figure 2C] Figures 2A-C show progression-free survival data for patients in the treatment-intended population (Figure 2A) with positive PD-L1 status (Figure 2B) and negative PD-L1 status (Figure 2C). Each graph shows progression-free survival in months for patients treated with nivolumab monotherapy (black line), ipilimumab monotherapy (dashed line), or a combination of nivolumab and ipilimumab (dotted line) (Figures 2A-C). The number of individuals facing risk for nivolumab, nivolumab + ipilimumab, and ipilimumab, respectively, is shown below each x-axis in months (Figures 2A-C). PD-L1 expression status is based on validated PD-L1 assay data (Figures 2B-C).

[0016] [Figure 3] Figures 3A and 3B show subgroup analyses of progression-free survival in patients treated with nivolumab monotherapy compared to ipilimumab monotherapy (Figure 3A) and in patients treated with nivolumab plus ipilimumab compared to ipilimumab monotherapy (Figure 3B).

[0017] [Figure 4] Figures 4A–C show the changes in tumor volume of target lesions in patients treated with nivolumab monotherapy (Figure 4A), nivolumab plus ipilimumab (Figure 4B), and ipilimumab monotherapy (Figure 4C). In each graph, the y-axis represents the best-case percentage reduction in target lesion volume from baseline, and the x-axis represents each patient (Figures 4A–C). [Modes for carrying out the invention]

[0018] Detailed description of the invention This invention relates to the identification of an optimal strategy for treating patients with PD-L1-negative melanoma. The invention demonstrates that in patients with PD-L1-negative melanoma tumors, combination therapy with anti-PD-1 and anti-CTLA-4 antibodies provides a better response (e.g., progression-free survival) than monotherapy with either anti-PD-1 or anti-CTLA-4 antibody. Furthermore, combination therapy can increase the objective response rate compared to monotherapy when administered to a given patient population.

[0019] definition term To make this disclosure easier to understand, some terms are defined first. To the extent used herein, unless otherwise expressly provided, each of the following terms has the meaning set forth below. Further definitions are provided throughout the specification.

[0020] The term “and / or” as used herein should be interpreted as a specific disclosure of each of two identified characteristics or components, whether or not they are accompanied by the other. Therefore, the term “and / or” as used herein in examples such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone) and “B” (alone). Similarly, the term “and / or” as used in examples such as “A, B and / or C” is intended to include each of the following aspects of A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone) and C (alone).

[0021] Where a certain aspect is described herein using the term “contains,” it is understood that similar aspects are also provided, described using the terms “consist of” and / or “essentially consisting of.”

[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press explain the common meanings of many of the terms used herein.

[0023] Units, prefixes, and symbols are used in the form accepted by the Systeme International de Unites (SI). Numerical ranges include the numerical values ​​that define those ranges. Titles given herein are not intended to limit the content of this disclosure and are best understood by referring to the specification as a whole. Accordingly, the terms defined below are best explained by referring to the specification as a whole.

[0024] "Administration" means introducing a composition containing a therapeutic agent into a subject's body using any of the various methods and delivery systems known to those skilled in the art. The routes of administration of the formulations disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other non-enteral administration routes, such as by injection or infusion. As used herein, "non-enteral administration" means administration methods other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered via a route that is not non-enteral, in some embodiments, by oral administration. Other non-enteral routes include topical, epithelial, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or topical. The administration may also be carried out, for example, once, multiple times, and / or over one or more periods.

[0025] As used herein, “adverse events” (AEs) are any undesirable and generally unintended or unwanted signs (including abnormal laboratory findings), symptoms, or illnesses related to the application of a medical procedure. For example, an adverse event may be related to the activation of the immune system or the proliferation of immune system cells (e.g., T cells) in response to a procedure. A medical procedure may be associated with one or more AEs, each AE may have the same or different levels of severity. A method that can “modify adverse events” means a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0026] An “antibody” (Ab) is a glycoprotein immunoglobulin or its antigen-binding moiety that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain has a heavy chain variable region (here, V H It includes the heavy chain constant region (abbreviated as C). The heavy chain constant region consists of three constant domains, C H1 , C H2 and C H3 It includes. Each light chain has a light chain variable region (here VL , abbreviated herein) and a light chain constant region. The light chain constant region comprises one constant domain, C L . V H and V L regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each V H and V L comprises 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 region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0027] Immunoglobulins can be derived from any commonly known isotype, 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 antibody class or subclass encoded by the heavy chain constant region gene (e.g., IgM or IgG1). The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless otherwise specified and the context indicates otherwise, the term "antibody" also includes any antigen-binding fragment or antigen-binding portion of said immunoglobulin, including monovalent and bivalent fragments or portions, and single chain antibodies.

[0028] The term “monoclonal antibody” (“mAb”) refers to an antibody molecule with a single molecular composition, i.e., a preparation of an antibody molecule that does not exist in nature, having essentially identical primary sequences and exhibiting single-binding specificity and affinity for a particular epitope. mAbs are an example of isolated antibodies. mAbs can be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0029] A “human” antibody (HuMAb) refers to an antibody having a variable region in which both the framework and CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody includes a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of this invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random in vitro or site-directed mutagenesis or somatic mutation in vivo). However, the term “human antibody” as used herein is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted into a human framework sequence. The terms “human” antibody and “fully human” antibody are used synonymously.

[0030] A “humanized antibody” is an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulin. In some embodiments of the humanized form of an antibody, some, most, or all of the amino acids outside the CDR domain are replaced with amino acids from human immunoglobulin, but some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not eliminate the antibody's ability to bind to a particular antigen. “Humanized” antibodies retain antigen specificity similar to that of the original antibody.

[0031] A "chimeric antibody" refers to an antibody in which the variable region originates from one species and the constant region originates from another species, such as an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody.

[0032] An "anti-antigen" antibody is an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, and an anti-CTLA-4 antibody specifically binds to CTLA-4.

[0033] The "antigen-binding portion" (also called the "antigen-binding fragment") of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to the antigen to which the entire antibody is bound.

[0034] "Cancer" is a broad group of diverse diseases characterized by the uncontrolled proliferation of abnormal cells in the body. This uncontrolled proliferation through cell division leads to the formation of malignant tumors that can invade neighboring tissues and metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors.

[0035] Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an immunosuppressive receptor belonging to the CD28 family. CTLA-4 is exclusively expressed on T cells in vivo and binds to two ligands, CD80 and CD86 (also known as B7-1 and B7-2, respectively). The term "CTLA-4" as used herein includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs that share at least one common epitope with hCTLA-4. The complete hCTLA-4 sequence can be found under GenBank Accession No. AAB59385.

[0036] The term "progression-free survival" used here, sometimes abbreviated as PFS, refers to the length of time a patient survives without progression of the disease during and after treatment for a solid tumor (i.e., melanoma).

[0037] The term "dosing interval" as used herein refers to the time elapsed between multiple administrations of the formulation disclosed herein. Therefore, the dosing interval can be expressed as a range.

[0038] The term "administration frequency" as used herein refers to the frequency with which multiple doses of the disclosed formulation are administered within a given time period. Administration frequency can also be expressed as the number of doses per period, for example, once a week or once every two weeks.

[0039] The use of the term “fixed dose” in relation to the compositions of this invention means that two or more different antibodies are present in a single composition in a specific (fixed) ratio to one another. In some embodiments, the fixed dose is based on the weight of the antibody (e.g., mg). In some embodiments, the fixed dose is based on the concentration of the antibody (e.g., mg / ml). In some embodiments, the ratio is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, about 1:20 The ratios are approximately 0, 200:1, 180:1, 160:1, 140:1, 120:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1 mg of primary antibody versus secondary antibody. For example, a 3:1 ratio of primary and secondary antibodies means that the vial may contain approximately 240 mg of primary antibody and 80 mg of secondary antibody, or approximately 3 mg / ml of primary antibody and 1 mg / ml of secondary antibody.

[0040] With respect to the compositions of the present invention, the use of the term “uniform dose” means a dose administered to a patient regardless of the patient’s body weight or body surface area (BSA). Therefore, the uniform dose is provided as an absolute amount of the drug (e.g., anti-CTLA-4 antibody and / or anti-PD-1 antibody) rather than as mg / kg. For example, a 60kg person and a 100kg person would receive the same dose of the composition (e.g., 240mg of anti-PD-1 antibody and 80mg of anti-CTLA-4 antibody in a single fixed-dose formulation vial containing both 240mg of anti-PD-1 antibody and 80mg of anti-CTLA-4 antibody (or two fixed-dose formulation vials containing 120mg of anti-PD-1 antibody and 40mg of anti-CTLA-4 antibody).

[0041] The term "body weight-based dosage" here means that the dose administered to a patient is calculated based on the patient's body weight. For example, if a patient weighing 60 kg requires 3 mg / kg of anti-PD-1 antibody and 1 mg / kg of anti-CTLA-4 antibody, the appropriate amounts of anti-PD-1 antibody (i.e., 180 mg) and anti-CTLA-4 antibody (i.e., 60 mg) can be drawn at once from a 3:1 fixed-ratio dosage formulation of anti-PD-1 antibody and anti-CTLA-4 antibody.

[0042] The term “anti-PD-1 antibody monotherapy” as used herein includes treatment with an anti-PD-1 antibody without accompanying anti-CTLA-4 antibody therapy. Anti-PD-1 antibody monotherapy includes, or consists of, the administration of one or more doses of an anti-PD-1 antibody to a patient requiring treatment, but does not include the administration of an anti-CTLA-4 antibody. In one embodiment, anti-PD-1 antibody monotherapy includes the administration of one or more doses of an anti-PD-1 antibody to a patient requiring treatment, but does not include the administration of an anti-CTLA-4 antibody. In another embodiment, anti-PD-1 antibody monotherapy includes the administration of one or more doses of an anti-PD-1 antibody to a patient requiring treatment, but does not include the administration of an antibody that specifically targets proteins other than PD-1. In yet another embodiment, anti-PD-1 antibody monotherapy includes the administration of one or more doses of an anti-PD-1 antibody to a patient requiring treatment, but does not include the administration of other anticancer agents.

[0043] "Immune response" refers to the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, of normal human cells or tissues.

[0044] Regarding cell surface PD-L1 expression, "PD-L1 negative" or "PD-L1 expression negative" refers to the absence of a detectable amount of cell surface PD-L1. For example, in an IHC cell surface expression assay using mAb 28-8, a PD-L1 negative tumor or PD-L1 expression negative tumor means that less than 0.01% of cells express a detectable level of PD-L1. In one embodiment, a PD-L1 negative tumor or PD-L1 expression negative tumor means that there are 0 cells expressing a detectable level of PD-L1. In one embodiment, a PD-L1 negative or PD-L1 expression negative tumor is any tumor other than a PD-L1 positive or PD-L1 expression positive tumor.

[0045] The term “PD-L1 positive” or “PD-L1 expression positive” in relation to cell surface PD-L1 expression refers to the percentage of cells in a test tissue sample, including tumor cells and tumor-infiltrating inflammatory cells, that are scored as expressing cell surface PD-L1. For example, in an immunohistochemistry (IHC) cell surface expression assay using mAb 28-8, a PD-L1 positive tumor or PD-L1 expression positive tumor means that at least approximately 0.01%, at least approximately 0.5%, at least approximately 1%, at least approximately 2%, at least approximately 3%, at least approximately 4%, at least approximately 5%, at least approximately 6%, at least approximately 7%, at least approximately 8%, at least approximately 9%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, or at least approximately 30% of the total number of cells express PD-L1. In other embodiments, a PD-L1-positive tumor or PD-L1-expression-positive tumor, which may also be referred to here as a PD-L1-expressing tumor, means that at least about 0.1% to at least about 20% of the total number of cells express PD-L1. In one embodiment, a PD-L1-positive tumor or PD-L1-expression-positive tumor means that at least about 0.1% to at least about 10% of the total number of cells express PD-L1. In one embodiment, a PD-L1-positive or PD-L1-expression-positive tumor means that at least about 1% of the total number of cells express PD-L1 on the cell surface. In another embodiment, a PD-L1-positive or PD-L1-expression-positive tumor means that at least about 5% of the total number of cells express PD-L1 on the cell surface. In one particular embodiment, a PD-L1-positive or PD-L1-expression-positive tumor means that at least about 1% or in the range of 1-5% of the total number of cells express PD-L1 on the cell surface.

[0046] "Programmed death-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is predominantly expressed in pre-activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank Accession No. U64863.

[0047] "Programmed death ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1" used here includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank Accession No. Q9NZQ7.

[0048] The term "patient" as used herein includes all patients with cancer (e.g., melanoma). The terms "subject" and "patient" are used interchangeably here.

[0049] The “therapeutic effective dose” of a drug or therapeutic agent is the amount of the drug that, when used alone or in combination with other therapeutic agents, protects a subject from the onset of the disease or promotes disease relief, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of functional or physical impairment due to disease onset. The ability of a therapeutic agent to promote disease relief can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in predictive animal models of efficacy in humans, or by assays of drug activity in in vitro assays.

[0050] The “treatment” or “therapy” of the subject means any form of intervention or process or administration of an activator applied to the subject with the aim of restoring, reducing, relieving, suppressing, slowing or preventing the manifestation, progression, advancement, severity or relapse of any biochemical signs associated with a symptom, complication, condition or disease.

[0051] "Tumor-infiltrating inflammatory cells" are all types of cells that generally participate in the inflammatory response in a subject and infiltrate tumor tissue. Such cells include tumor-infiltrating lymphocytes (TILs), macrophages, monocytes, eosinophils, histiocytes, and dendritic cells.

[0052] The use of options (e.g., "or") should be interpreted as meaning one of the options, both, or any combination thereof. The singular expression used here should be interpreted as meaning "one or more" of the listed or enumerated components.

[0053] The terms “about” or “essentially include” mean a value or composition that falls within the acceptable margin of error of a particular value or composition as determined by those skilled in the art, which in part depends on how the value or composition was measured or determined, i.e., the limits of the measuring system. For example, “about” or “essentially include” may mean a standard deviation of 1 or more, according to the conventions of the art. Alternatively, “about” or “essentially include” may mean a range of up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg may include any number between 2.7 mg and 3.3 mg (about 10%) or between 2.4 mg and 3.6 mg (about 20%). Furthermore, particularly with respect to biological systems or processes, the term may mean up to one order of magnitude or up to five times the value. When a particular value or composition is described in the specification and claims, unless otherwise specified, the meaning of “about” or “essentially include” should be assumed to be within the acceptable margin of error of that particular value or composition.

[0054] The terms “approximately once a week,” “approximately once a week,” “approximately once every two weeks,” or any other similar terms used herein for dosing intervals are approximate. “Approximately once a week” or “approximately once a week” may include every 7 ± 2 days, i.e., every 5 to 9 days. A “once a week” dosing frequency could therefore be every 5 days, every 6 days, every 7 days, every 8 days, or every 9 days. “Approximately once every two weeks” may include every 14 ± 3 days, i.e., every 11 to 17 days. Similar approximate expressions apply, for example, to every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, and every 12 weeks. In one embodiment, an interval of approximately once every six weeks or approximately once every twelve weeks means that the first dose may be administered on any day of the first week, and the next dose on any day of the sixth or twelfth week, respectively. In another embodiment, an interval of approximately once every six weeks or approximately once every twelve weeks means that the first dose may be administered on a specific day of the week in the first week (e.g., Monday), and the next dose may be administered on the same day of the week in the sixth or twelfth week, respectively (i.e., Monday).

[0055] As stated herein, any concentration range, percentage range, ratio range, or integer range should be interpreted as including all integers within the range and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of the integer), unless otherwise specified.

[0056] Various aspects of the present invention are described in more detail in the following subsections.

[0057] Method of the present invention The present invention relates to a method for treating PD-L1-negative melanoma in subjects requiring treatment. The present invention demonstrates that combination therapy of anti-PD-1 antibody and anti-CTLA-4 antibody is more suitable for treating PD-L1-negative tumors than monotherapy with either anti-PD-1 antibody or anti-CTLA-4 antibody.

[0058] Without being bound by any theory, the present invention demonstrates that in patients with PD-L1-negative tumors who are progression-free, progression-free survival and overall response rates are higher after combination therapy with anti-PD-1 and anti-CTLA-4 antibodies than after treatment with either anti-PD-1 or anti-CTLA-4 antibody alone. Therefore, to enhance the response in patients with PD-L1-negative tumors, the present invention leads to the identification of patients suitable for combination therapy with anti-PD-1 and anti-CTLA-4 antibodies.

[0059] In one embodiment, the present invention provides a method for treating PD-L1-negative melanoma, comprising administering to a patient (a) an anti-PD-1 antibody or its antigen-binding portion that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding portion that specifically binds to human CTLA-4, wherein the patient has been identified as having a PD-L1-negative melanoma tumor prior to administration. In another embodiment, the present invention provides a method for treating melanoma in a patient requiring treatment, comprising (i) identifying a patient having a PD-L1-negative melanoma tumor, and (ii) administering to the patient (a) an anti-PD-1 antibody or its antigen-binding portion that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding portion that specifically binds to human CTLA-4.

[0060] In one embodiment, the present invention provides a method for extending progression-free survival beyond nine months in a patient having a PD-L1-negative melanoma tumor, comprising administering to the patient (a) an anti-PD-1 antibody or its antigen-binding portion that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding portion that specifically binds to human CTLA-4, wherein the patient has been identified as having a PD-L1-negative melanoma tumor prior to administration, and the patient exhibits a progression-free survival beyond nine months. In another embodiment, the present invention provides a method for extending progression-free survival beyond nine months in a patient having a melanoma tumor, comprising (i) identifying a patient having a PD-L1-negative melanoma tumor, and (ii) administering to the patient (a) an anti-PD-1 antibody or its antigen-binding portion that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding portion that specifically binds to human CTLA-4, wherein the patient exhibits a progression-free survival beyond nine months. The present invention can extend a patient's progression-free survival by more than about 10 months, more than about 11 months, more than about 12 months, more than about 13 months, more than about 14 months, more than 15 months, more than 16 months, more than 17 months, more than 18 months, more than 2 years, more than 3 years, more than 4 years, more than 5 years, more than 6 years, more than 7 years, more than 8 years, more than 9 years, or more than 10 years after administration. In certain embodiments, the patient's progression-free survival is extended by more than 10 months.

[0061] In yet another embodiment, the present invention relates to a method for reducing tumor size by at least 10% in a patient having a PD-L1-negative melanoma tumor, comprising administering to the patient (a) an anti-PD-1 antibody or its antigen-binding portion that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding portion that specifically binds to human CTLA-4, wherein the patient has been identified as having a PD-L1-negative melanoma tumor prior to administration, and the administration reduces the tumor size by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to before administration. In another embodiment, the method comprises (i) identifying a patient having a PD-L1-negative melanoma tumor, and (ii) administering to the patient (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4, wherein the administration reduces the tumor size by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to before administration. The tumor size may be reduced by at least about 60%, 70%, 80%, 90%, or 100% after administration. After administration, the tumor may be completely removed from the patient's body.

[0062] The present invention also comprises administering a method for preventing recurrence and / or inducing remission in a patient having a PD-L1-negative melanoma tumor, the method comprising administering to the patient (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4, wherein the patient has been identified as having a PD-L1-negative melanoma tumor prior to administration. In one embodiment, the method of the present invention comprises (i) identifying a patient having a PD-L1-negative melanoma tumor, and (ii) administering to the patient (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4.

[0063] In one embodiment, the present invention provides a method for increasing the objective response rate to more than 40% in a patient population, each having a melanoma tumor, comprising administering to the patient (a) an anti-PD-1 antibody or its antigen-binding portion that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding portion that specifically binds to human CTLA-4, wherein each patient has been identified as having a PD-L1-negative melanoma tumor prior to administration, and the objective response rate is higher than 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. The method further comprises identifying each patient as having a PD-L1-negative melanoma tumor prior to administration. In other embodiments, each patient in the method may further be characterized by (i) a long-term progression-free survival of more than 11 months, (ii) a reduction of at least about 10%, about 20%, about 30%, about 40%, or about 50% of the tumor size compared to the tumor size before administration, or (iii) both. In one embodiment, the patient population may be at least 100 patients with PD-L1-negative melanoma tumors. In another embodiment, the patient population may be at least 200, 300, 400, 500, 600, 700, 800, 900, or 1000 patients with PD-L1-negative melanoma tumors.

[0064] In a further embodiment, the present invention provides a method for selecting an appropriate cancer treatment course in a patient having a PD-L1-negative melanoma tumor, comprising (i) identifying a patient having a PD-L1-negative melanoma tumor, and (ii) instructing a healthcare provider to administer to the patient (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1 and (b) an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4. The method further comprises administering the antibodies to the patient.

[0065] The methods of the present invention, as a result of combination therapy, can treat melanoma tumors, reduce tumor size, inhibit tumor growth, eliminate tumors from patients, prevent tumor recurrence, induce remission in patients, or a combination thereof. In some embodiments, the combination therapy induces a complete response. In other embodiments, the combination therapy induces a partial response.

[0066] melanoma Melanoma (MEL) is a malignant tumor of melanocytes, which are melanin-producing cells found primarily in the skin. Although not more frequent than other skin cancers, it is the most dangerous type of skin cancer if not diagnosed early, and it accounts for the majority (75%) of skin cancer deaths. The incidence of MEL is increasing globally, particularly in Caucasian populations where people with less skin pigmentation are exposed to excessive ultraviolet radiation from sunlight. In Europe, the incidence is <10-20 cases per 100,000 people, in the United States it is 20-30 cases per 100,000 people, and in Australia, where the highest incidence is observed, it is 50-60 cases per 100,000 people (Garbe et al., Eur. J. Cancer. 48(15):2375-90 (2012)). MEL accounts for approximately 5% of all new cancer cases in the United States, and its incidence continues to increase by about 3% per year. This is estimated to be 76,690 new cases and 9,480 related deaths in 2013 (Siegel et al., CA Cancer J. Clin. 63(1):11-30 (2013)).

[0067] For melanoma in situ (stage 0) or early MEL (stages I-II), surgical resection is the primary treatment. Generally, patients with localized disease and tumor thickness of 1.0 mm or less have a good prognosis, with a 5-year survival rate exceeding 90% (NCCN GUIDELINES®, 2013 - Melanoma). For melanoma in situ where surgical resection is not feasible due to comorbidities or tumor location requiring cosmetic considerations, topical imiquimod (INN) and radiotherapy have emerged as treatments, particularly for lentigo malignant melanoma. Chemotherapy agents for treating MEL include dacarbazine, temozolomide and imatinib for melanoma with c-KIT mutations, and paclitaxel in combination with high-dose interleukin-2 and carboplatin. However, these treatments have only moderate success in first-line (1L) and second-line (2L) situations, with response rates of less than 20%.

[0068] For patients with localized melanoma exceeding 1.0 mm in thickness, the survival rate ranges from 50% to 90%. The likelihood of local lymph node metastasis increases with tumor thickness. For stage III MEL (clinically positive nodular and / or migrating disease), the 5-year survival rate is 20% to 70%. By far the most fatal is stage IV MEL, with long-term survival in patients with distant metastatic melanoma being less than 10% (NCCN GUIDELINES®, 2013 - Melanoma).

[0069] The types of melanoma that can be treated by the present invention include, but are not limited to, lentigo malignant, lentigo malignant melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, fibroplastic melanoma, achromatic melanoma, soft tissue melanoma, melanoma with small nerve-like cells, melanoma with Spitz nevus characteristics, or uveal melanoma. The stages of melanoma that can be treated by this method include, (i) Stage I / II (invasive melanoma): primary tumor thickness 1.0 mm, no ulceration, and mitosis <1 / mm 2 Characterized by T1a; primary tumor thickness 1.0 mm, ulceration present or mitosis ≥ 1 / mm 2T1b is characterized by a primary tumor thickness of 1.01-2.0 mm and the absence of ulceration; (ii) Stage II (high-risk melanoma): T2b is characterized by a primary tumor thickness of 1.01-2.0 mm and the presence of ulceration; T3a is characterized by a primary tumor thickness of 2.01-4.0 mm and the absence of ulceration; T3b is characterized by a primary tumor thickness of 2.01-4.0 mm and the presence of ulceration; T4a is characterized by a primary tumor thickness of 4.0 mm in length and the absence of ulceration; or T4b is characterized by a primary tumor thickness of 4.0 mm in length and the presence of ulceration; ( iii) Stage III (local metastasis): N1 characterized by a single positive lymph node; N2 characterized by 2-3 positive lymph nodes or local skin / migrating metastasis; or N3 characterized by 4 positive lymph nodes or 1 lymph node and local skin / migrating metastasis; and (iv) Stage IV (distant metastasis): M1a characterized by distant skin metastasis and normal LDH; M1b characterized by lung metastasis and normal LDH; or M1c characterized by other distant metastasis or any distant metastasis and high LDH. PD-L1-negative tumors treatable by the methods of the present invention may lack PD-L1 expression on the surface of tumor cells and / or tumor-infiltrating inflammatory cells.

[0070] Measurement of PD-L1 expression In one embodiment, the identification of patients suitable for combination therapy using the present invention includes measuring or evaluating PD-L1 expression on the surface of melanoma tumor cells or tumor-infiltrating inflammatory cells. The terms “PD-L1 expressing tumor,” “PD-L1 expressing tumor,” “PD-L1 positive tumor,” and “PD-L1 expression positive tumor” are used interchangeably here. The meanings of these terms are provided elsewhere in this specification. Methods for measuring or evaluating PD-L1 expression can be achieved by any applicable method.

[0071] In one embodiment, for the evaluation of PD-L1 expression, a test tissue sample is obtained from a patient in need of treatment. In another embodiment, the evaluation of PD-L1 expression can be achieved without obtaining a test tissue sample. In one embodiment, the selection of a suitable patient includes (i) providing a test tissue sample obtained from a patient who optionally has cancerous tissue, the test tissue sample containing tumor cells and / or tumor-infiltrating inflammatory cells, and (ii) evaluating the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample, based on the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample being lower than a predetermined threshold. A test tissue sample may be considered PD-L1 negative if the percentage of cells in the test tissue expressing PD-L1 on the cell surface is less than approximately 5%. In other embodiments, a test tissue sample may be considered PD-L1 negative if the percentage of cells in the test tissue expressing PD-L1 on the cell surface is less than approximately 4%, less than approximately 3%, less than approximately 2%, less than approximately 1%, less than approximately 0.5%, less than approximately 0.1%, less than approximately 0.01%, or 0%. In a particular example, a test tissue sample may be considered PD-L1 negative if the percentage of cells in the test tissue expressing PD-L1 on the cell surface is less than approximately 5%.

[0072] It should be understood that in any method involving the measurement of PD-L1 expression in a test tissue sample, the provision of a test tissue sample obtained from a patient is optional. That is, in some embodiments, the method includes this step, while in other embodiments, this step is not included. It should also be understood that in some embodiments, the “measurement” or “evaluation” step for identifying or determining the number or proportion of cells in a test tissue sample expressing PD-L1 on the cell surface may be carried out by a modification method for the PD-L1 expression assay, for example, by performing a reverse transcriptase-polymerase chain reaction (RT-PCR) assay or an IHC assay. In some embodiments, no modification step is included, and PD-L1 expression is evaluated, for example, by reviewing a test result report obtained in a laboratory. In some embodiments, the steps of the method up to and including the evaluation of PD-L1 expression provide an intermediate result that may be provided to a physician or other healthcare provider for use in selecting a suitable candidate for combination therapy of anti-PD-1 and anti-CTLA-4 antibodies. In some embodiments, the step of providing the intermediate result is carried out by a physician or a person working under the direction of a physician. In other embodiments, these steps are performed in an independent laboratory or by an independent technician, such as a laboratory technician.

[0073] In any embodiment of the present invention, 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 another embodiment, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 polypeptide. In a further embodiment, the presence of PD-L1 polypeptide is determined by immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), in vivo contrast imaging, or flow cytometry. In one embodiment, PD-L1 expression is assayed by IHC. In all other embodiments of these methods, cell surface expression of PD-L1 is assayed, for example, using IHC or in vivo contrast imaging.

[0074] Contrast imaging techniques provide essential tools for cancer research and treatment. Recent advances in molecular contrast systems, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), fluorescence reflectance (FRI), fluorescence-mediated tomography (FMT), bioluminescence (BLI), laser scanning confocal microscopy (LSCM), and multiphoton microscopy (MPM), are likely to lead to even greater use of these techniques in cancer research. Some of these molecular contrast systems not only allow physicians to see where tumors reside in the body, but also enable visualization of the expression and activity of specific molecules, cells, and biological processes that influence tumor behavior and / or drug responses (Condeelis and Weissleder, Cold Spring Harb. Perspect. Biol. 2(12):a003848 (2010)). The antibody specificity, combined with PET sensitivity and resolution, makes immunoPET imaging particularly attractive for monitoring and assaying antigen expression in tissue samples (McCabe and Wu, Cancer Biother. Radiopharm. 25(3):253-61 (2010); Olafsen et al., Protein Eng. Des. Sel. 23(4):243-9 (2010)). In any embodiment of the present invention, PD-L1 expression is assayed by immunoPET imaging. In any embodiment of the present invention, the percentage of cells expressing PD-L1 in the test tissue sample is assessed by performing an assay to determine the presence of PD-L1 polypeptide on the cell surface in the test tissue sample. In one embodiment, the test tissue sample is an FFPE tissue sample. In another 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 method. In one embodiment, the IHC assay is performed using an anti-PD-L1 mAb that, if present, binds to the PD-L1 polypeptide.

[0075] Automated IHC assay for cell surface PD-L1 expression In one embodiment of the present invention, an automated IHC method is used to assay the expression of PD-L1 on the cell surface of an FFPE tissue sample. The present invention provides a method for detecting the presence of human PD-L1 antigen in a test tissue sample or for quantifying the level of human PD-L1 antigen or the proportion of cells expressing the antigen in a sample, the method comprising contacting a test sample and a negative control sample with an mAb that specifically binds to human PD-L1 under conditions that allow for the formation of a complex of an antibody or a portion thereof with human PD-L1. In one embodiment, the test and control tissue samples are FFPE samples. Complex formation can then be detected, where the difference in complex formation between the test sample and the negative control sample is an indicator of the presence of human PD-L1 antigen in the sample. Various methods are used to quantify PD-L1 expression.

[0076] In certain embodiments, the automated IHC method includes (a) deparaffinizing and rehydrating the embedded tissue section in an autostaner, (b) recovering the antigen using a decloaking chamber and pH 6 buffer and heating at 110°C for 10 minutes, (c) setting up the reagents in the autostaner, and (d) driving the autostaner to include an endogenous peroxidase neutralization step in the tissue sample, blocking nonspecific protein binding sites on the slide, incubating the slide with primary Ab, incubating with primary postblocker, incubating with NovoLink polymer, adding a chromogenic substrate, developing, and counterstaining with hematoxylin.

[0077] To express PD-L1 in tumor tissue samples, pathologists examine the membrane PD-L1 in each microscopic field. +The number of tumor cells is examined, the percentage of positive cells is estimated by hand calculation, and then averaged to obtain the final percentage. Various staining intensities are defined as 0 / negative, 1+ / weak, 2+ / medium, and 3+ / strong. Generally, the percentage values ​​are first assigned to the 0 and 3+ buckets, and then the intermediate 1+ and 2+ intensities are considered. For highly heterogeneous tissues, the sample is divided into zones, each zone is scored individually, and then incorporated into a set of percentage values. The percentages of negative and positive cells of various staining intensities are determined in each region, and the median values ​​are assigned to each zone. Final percentage values: negative, 1+, 2+, and 3+ are assigned to tissues in each staining intensity category. The sum of all staining intensities must be 100%.

[0078] Staining is also evaluated for tumor-infiltrating inflammatory cells such as macrophages and lymphocytes. In most cases, macrophages serve as an internal positive control, as staining is observed in the majority of macrophages. While staining with 3+ intensity is not required, failure to stain macrophages should be considered to rule out some technical defect. Macrophages and lymphocytes are evaluated for plasma membrane staining, and for all samples, they are recorded only as positive or negative for each cell category. Staining is also characterized according to external / internal tumor immune cell designation. "Internal" means that immune cells are located at the boundary within tumor tissue and / or tumor region without being physically inserted within and / or between tumor cells. "External" means that there is no physical connection to the tumor, and immune cells are found peripherally associated with connective tissue or any adjacent tissue.

[0079] In one embodiment of these scoring methods, samples are scored by two independently working pathologists, and the scores are then tallied. In one embodiment, the distinction between positive and negative cells is scored using appropriate software.

[0080] The organizational score is used as a more quantitative measure of IHC data. The histological score is calculated as follows: Tissue score = [(% tumor × 1 (low intensity)) + (% tumor × 2 (medium intensity)) + (% tumor × 3 (high intensity)]

[0081] To determine the tissue score, the pathologist estimates the percentage of stained cells in each intensity category within the specimen. Because the expression of most biomarkers is heterogeneous, the tissue score more accurately reflects overall expression. The final tissue score ranges from 0 (no expression) to 300 (maximum expression). A test sample may be classified as PD-L1 negative even if a certain level of PD-L1 expression is detected. For example, PD-L1 negative designation may be assigned to specimens with final tissue scores of approximately 15 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, or 0.

[0082] Another method for quantifying PD-L1 expression in IHC of test tissue samples is the adjusted inflammation score (AIS), which is defined as the inflammation density multiplied by the percentage of PD-L1 expression by tumor-infiltrating inflammatory cells (Taube et al., Sci. Transl. Med. 4(127):127ra37 (2012)).

[0083] Anti-PD-1 antibody PD-1 is an important immune checkpoint receptor expressed by activated T and B cells that mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands of PD-1, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), have been identified. These are expressed in antigen-presenting cells and many human cancers, and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1. Inhibition of the PD-1 / PD-L1 interaction has been shown to mediate potent antitumor activity in preclinical models.

[0084] HuMAbs that bind specifically and with high affinity to PD-1 are disclosed in U.S. Patents 8,008,449 and 8,779,105. Other anti-PD-1 mAbs are described, for example, in U.S. Patents 6,808,710, 7,488,802, 8,168,757 and 8,354,509 and PCT Publication No. WO2012 / 145493. Each of the anti-PD-1 HuMAbs disclosed in U.S. Patent 8,008,449 has been shown to exhibit one or more of the following characteristics: (a) Determined by surface plasmon resonance using a Biacore biosensor system, it binds to human PD-1 by 1 × 10⁻¹⁶. -7 (b) binds at M or less; (c) substantially does not bind to human CD28, CTLA-4, or ICOS; (d) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (e) increases interferon-γ production in an MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates an antigen-specific memory response; (i) stimulates an antibody response; and (j) inhibits tumor cell proliferation in vivo. Anti-PD-1 antibodies useful in the present invention include mAbs that specifically bind to human PD-1 and exhibit at least one, at least two, at least three, at least four, or at least five of the above features.

[0085] In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab ("Opdivo") (登録商標)Also known as 5C4, BMS-936558, MDX-1106, or ONO-4538, this fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of antitumor T cell function (US Patent No. 8,008,449; Wang et al., Cancer Immunol Res. 2(9):846-56 (2014)). In other embodiments, the anti-PD-1 antibody or its fragments cross-compete with nivolumab. In other embodiments, the anti-PD-1 antibody or its fragments bind to the same epitope as nivolumab. In some embodiments, the anti-PD-1 antibody has the same CDR as nivolumab.

[0086] In other embodiments, the anti-PD-1 antibody or its fragment cross-competes with pembrolizumab. In some embodiments, the anti-PD-1 antibody or its fragment binds to the same epitope as pembrolizumab. In some embodiments, the anti-PD-1 antibody has the same CDR as pembrolizumab. In other embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab ("Keytruda") (登録商標) Pembrolizumab (also known as lambrolizumab and MK-3475) is a humanized monoclonal IgG4 antibody against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described in U.S. Patents, e.g., 8,354,509 and 8,900,587, and is also referred to at http: / / www.cancer.gov / drugdictionary?cdrid=695789 (last accessed December 14, 2014). Pembrolizumab is approved by the FDA for the treatment of relapsed or refractory melanoma.

[0087] In other embodiments, the anti-PD-1 antibody or its fragment cross-competes with MEDI0608. In yet another embodiment, the anti-PD-1 antibody or its fragment binds to the same epitope as MEDI0608. In one embodiment, the anti-PD-1 antibody has the same CDR as MEDI0608. In another embodiment, the anti-PD-1 antibody is the monoclonal antibody MEDI0608 (formerly AMP-514). MEDI0608 is described, for example, in U.S. Patent No. 8,609,089B2 or at http: / / www.cancer.gov / drugdictionary?cdrid=756047 (last accessed December 14, 2014).

[0088] In one embodiment, the primary antibody is an anti-PD-1 antagonist. An example of an anti-PD-1 antagonist is AMP-224, a B7-DC Fc fusion protein. AMP-224 is described in U.S. Publication 2013 / 0017199 or at http: / / www.cancer.gov / publications / dictionaries / cancer-drug?cdrid=700595 (last accessed July 8, 2015).

[0089] In other embodiments, the anti-PD-1 antibody or its fragment cross-competes with BGB-A317. In one embodiment, the anti-PD-1 antibody or its fragment binds to the same epitope as BGB-A317. In one embodiment, the anti-PD-1 antibody is BGB-A317 having the same CDR as BGB-A317. In one embodiment, the anti-PD-1 antibody is BGB-A317, which is a humanized monoclonal antibody. BGB-A317 is described in U.S. Publication No. 2015 / 0079109.

[0090] In one embodiment, the antibody is pidilizumab (CT-011), which was previously reported to bind to PD-1 but is thought to bind to a different target. Pidilizumab is described in U.S. Patent No. 8,686,119B2 or WO2013 / 014668A1.

[0091] Anti-PD-1 antibodies are also useful in the present invention's compositions, which include isolated antibodies that specifically bind to human PD-1 and cross-compete with nivolumab for binding to human PD-1 (see, for example, U.S. Patents 8,008,449 and 8,779,105; see WO2013 / 173223). The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically inhibit the binding of the other cross-competitive antibody to that particular epitope region. These cross-competitive antibodies are expected to have functional properties very similar to nivolumab for binding to the same epitope region of PD-1. Cross-competitive antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays such as Biacore analysis, ELISA assay, or flow cytometry (see, for example, WO2013 / 173223).

[0092] In one embodiment, an antibody that cross-competes with nivolumab for binding to human PD-1 or that binds to the same epitope region of human PD-1 is an mAb. For administration to human subjects, these cross-competitive antibodies may be chimeric antibodies or humanized or human antibodies. Such chimeric, humanized, or human mAbs can be manufactured and isolated by methods well known in the art.

[0093] The anti-PD-1 antibody useful in the composition of the present invention also includes the antigen-binding portion of the antibody. It has been well proven that the antigen-binding function of an antibody can be realized by a fragment of a full-length antibody. An example of a binding fragment encompassed by the term “antigen-binding portion” of an antibody is (i)V L , V H , C L and C H1 (ii) A monovalent fragment consisting of domains, the Fab fragment; (ii) A bivalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region, the F(ab')2 fragment; (iii) V H and C H1 Fd fragments consisting of domains; and (iv) alternating single-arm V Land V H Includes an Fv fragment consisting of domains.

[0094] The anti-PD-1 antibody suitable for use in the compositions of the present invention is an antibody that binds to PD-1 with high specificity and affinity, blocks the binding of PD-L1 and / or PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, the anti-PD-1 "antibody" comprises an antigen-binding moiety or fragment that binds to the PD-1 receptor, inhibits ligand binding, and exhibits functional properties similar to those of the whole antibody that upregulate the immune system. In some embodiments, the anti-PD-1 antibody or its antigen-binding moiety cross-competes with nivolumab for binding to human PD-1. In other embodiments, the anti-PD-1 antibody or its antigen-binding moiety is a chimeric, humanized, or human monoclonal antibody or a part thereof. In some embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is a human antibody. Antibodies of the IgG1, IgG2, IgG3, or IgG4 isotype can be used.

[0095] In one embodiment, the anti-PD-1 antibody or its antigen-binding moiety comprises a heavy chain constant region of a human IgG1 or IgG4 isotype. In one embodiment, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or its antigen-binding moiety contains the S228P mutation, which replaces a serine residue in the hinge region with a proline residue normally found at the corresponding position in IgG1 isotype antibodies. This mutation present in nivolumab inhibits Fab arm exchange with endogenous IgG4 antibody while maintaining low affinity for Fc receptor activation related to wild-type IgG4 antibody (Wang et al., Cancer Immunol Res. 2(9):846-56 (2014)). In another embodiment, the antibody comprises a light chain constant region which is a human kappa or lambda constant region. In yet another embodiment, the anti-PD-1 antibody or its antigen-binding moiety (mAb) or its antigen-binding moiety. In any embodiment of the therapeutic methods described herein, which involve the administration of an anti-PD-1 antibody, the anti-PD-1 antibody is nivolumab. In other embodiments, the anti-PD-1 antibody is pembrolizumab. In other embodiments, the anti-PD-1 antibody is selected from the human antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in U.S. Patent No. 8,008,449. In yet another embodiment, the anti-PD-1 antibody is MEDI0608 (formerly AMP-514), AMP-224, or BGB-A317.

[0096] Since anti-PD-1 and anti-PD-L1 target the same signaling pathway and have been shown in clinical trials to exhibit similar levels of efficacy in a variety of cancers, including RCC (see Brahmer et al. (2012) N Engl J Med 366:2455-65; Topalian et al. (2012a) N Engl J Med 366:2443-54; WO2013 / 173223), anti-PD-L1 antibodies may be a substitute for anti-PD-1 antibodies in any of the therapeutic methods disclosed herein. In one embodiment, the anti-PD-L1 antibody is BMS-936559 (formerly 12A4 or MDX-1105) (see, for example, U.S. Patent No. 7,943,743; WO2013 / 173223). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446) (see Herbst et al. (2013) J Clin Oncol 31(suppl):3000. Abstract; U.S. Patent No. 8,217,149) or MEDI4736 (Khleif (2013) In: Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands. Abstract 802). In some embodiments, the antibody that cross-competes with the above PD-L1 antibody for binding to human PD-L1 or that binds to the same epitope region of human PD-L1 is an mAb. For administration to human subjects, these cross-competing antibodies may be chimeric antibodies or humanized or human antibodies. Such chimeric, humanized or human mAbs can be manufactured and isolated by methods well known in the art.

[0097] Anti-PD-L1 antibody In one embodiment, the present application encompasses the use of an anti-PD-L1 antibody instead of an anti-PD-1 antibody. In one embodiment, the anti-PD-L1 antibody inhibits the binding of the PD-L1 receptor, i.e., PD-1 to its ligand PD-L1.

[0098] The anti-PD-L1 antibody useful in the present invention is disclosed herein V H and / or V L The invention includes antibodies modified starting from antibodies having one or more sequences, and these modified antibodies may have altered properties from the starting antibody. Anti-PD-L1 antibodies can be modified by the various modifications described above for the purpose of modifying the modified anti-PD-1 antibodies of the present invention.

[0099] The anti-PD-L1 antibodies of the present invention include isolated antibodies selected based on their ability to bind to PD-L1 in formalin-fixed, paraffin-embedded (FFPE) tissue specimens. The use of FFPE samples is essential for long-term follow-up analysis of the correlation between tumor PD-L1 expression and disease prognosis or progression. The use of various antibodies for PD-L1 staining in frozen versus FFPE tissues, and the ability of certain antibodies to distinguish the membranous and / or cytoplasmic morphology of PD-L1, may explain some of the inconsistent data reports in the literature regarding the correlation between PD-L1 expression and disease prognosis (Hamanishi et al., Proc. Natl. Acad. Sci. USA 104(9):3360-3365 (2007); Gadiot et al., Cancer 117(10):2192-2201 (2011)). This disclosure provides several rabbit mAbs that bind with high affinity and specifically to membrane-bound human PD-L1 in FFPE tissue samples, including tumor cells and tumor-infiltrating inflammatory cells.

[0100] In one embodiment, an anti-PD-L1 antibody useful in the present invention method comprises mAb 28-8, shown in SEQ ID NOs. 1 and 2, respectively. The sequences of the heavy and light chain CDR domains of MAb 28-8 are shown in SEQ ID NOs. 3-8, depicted using the Kabat system. In another embodiment, an anti-PD-L1 antibody useful in the present invention comprises mAb 28-1, 28-12, 29-8, and 20-12 or their antigen-binding moieties, for example, Fab, F(ab')2, Fd, Fv, and scFv, di-scFv or bi-scFv, and scFv-Fc fragments, bispecific antibodies, triabodies, tetrabodies, and isolated CDRs.

[0101] Anti-CTLA-4 antibody The anti-CTLA-4 antibody used in this invention binds to human CTLA-4 in such a way as to interfere with the interaction between CTLA-4 and the human B7 receptor. Since the interaction between CTLA-4 and B7 transmits signals that result in the inactivation of T cells having the CTLA-4 receptor, interference with this interaction efficiently induces, enhances, or prolongs the activation of such T cells, thereby inducing, enhancing, or prolonging an immune response.

[0102] High-affinity HuMAbs that specifically bind to CTLA-4 are disclosed in U.S. Patents 6,984,720 and 7,605,238. Other anti-CTLA-4 mAbs are disclosed, for example, in U.S. Patents 5,977,318, 6,051,227, 6,682,736 and 7,034,121. The anti-CTLA-4 HuMAbs disclosed in U.S. Patents 6,984,720 and 7,605,238 have been shown to exhibit one or more of the following characteristics: (a) at least about 10 as determined by Biacore analysis 7 M -1 or about 10 9 M -1 or about 10 10 M -1 ~10 11 M -1 or a higher equilibrium coupling constant (K a (b) Binds specifically to human CTLA-4 with binding affinity reflected in (b) at least about 10 3 , about 10 4 or about 10 5 m -1 s -1 The dynamic coupling constant (k a (c) at least about 10 3 , about 10 4 or about 10 5 m -1 s -1 The dynamic dissociation constant (k d(d) inhibits the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies useful in the present invention include mAbs that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the above characteristics.

[0103] An example of an anti-CTLA-4 antibody used clinically is human mAb 10D1 (now known as ipilimumab, Yervoy) disclosed in U.S. Patent No. 6,984,720. (登録商標) It is marketed as [product name]. Ipilimumab is a useful anti-CTLA-4 antibody in the method disclosed herein. Ipilimumab is a fully human, IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma.

[0104] Another anti-CTLA-4 antibody useful in the method of the present invention is tremelimumab (also known as CP-675,206). Tremelimumab is a human IgG2 monoclonal anti-CTLA-4 antibody. Tremelimumab is described in WO / 2012 / 122444, U.S. Publication 2012 / 263677, or WO Publication 2007 / 113648A2.

[0105] The anti-CTLA-4 antibodies useful in the compositions of the present invention also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with ipilimumab or tremelimumab for binding to human CTLA-4, or that bind to the same epitope region of human CTLA-4 as ipilimumab or tremelimumab. In one embodiment, the antibody that cross-competes with ipilimumab or tremelimumab for binding to human CTLA-4, or that binds to the same epitope region of human CTLA-4, is an antibody containing a heavy chain of a human IgG1 isotype. For administration to human subjects, these cross-competing antibodies are chimeric antibodies or humanized or human antibodies. The useful anti-CTLA-4 antibodies also include antigen-binding moieties of the above antibodies, such as Fab, F(ab')2, Fd, or Fv fragments.

[0106] dose A combination of anti-PD-1 antibody and anti-CTLA-4 antibody can be administered to appropriate patients in therapeutically effective doses. For example, each antibody can be administered in doses ranging from at least about 0.1 to at least about 20.0 mg / kg body weight. In one embodiment, each of the anti-PD-1 and anti-CTLA-4 antibodies is administered individually in doses of at least about 0.1, at least about 0.3, at least about 0.5, at least about 1, at least about 3, at least about 5, at least about 10, or at least about 20 mg / kg, for example, at least about 1 to at least about 10 mg / kg, for example, at least about 1 to at least about 3 mg / kg, for example, at least about 3 mg / kg, for example, at least about 1 mg / kg. Each of the anti-PD-1 antibody and the anti-CTLA-4 antibody may be administered at a frequency of at least approximately once a week, at least approximately once every two weeks, at least approximately once every three weeks, at least approximately once every four weeks, or at least approximately once a month, for a maximum of 6 to 72 doses, or until a clinical benefit is observed or uncontrollable toxicity or disease progression occurs. In one embodiment, the anti-PD-1 antibody is administered at a dose of about 1 or about 3 mg / kg. In one embodiment, a serial regimen includes administering the anti-PD-1 antibody to the subject at a frequency of approximately once a week, at approximately once every two weeks, at approximately once every three weeks, at approximately once every four weeks, or once a month, for a maximum of 6 to 72 doses, or until a clinical benefit is observed or uncontrollable toxicity or disease progression occurs. In another embodiment, anti-PD-1 is administered at a dose of about 1 mg / kg at a frequency of approximately once every three weeks for a maximum of 48 doses. In one embodiment, the anti-CTLA-4 antibody is administered at a dose of about 1 or about 3 mg / kg. In one embodiment, a serial regimen involves administering an anti-CTLA-4 antibody to a subject at a frequency of approximately once weekly, at least once every two weeks, at least once every three weeks, at least once every four weeks, or approximately once a month, for a maximum of 6 to 72 doses, or until a clinical benefit is observed or until uncontrollable toxicity or disease progression occurs. In another embodiment, the anti-CTLA-4 antibody is administered at a dose of approximately 3 mg / kg at a frequency of approximately once every three weeks for a maximum of 48 doses.

[0107] In other embodiments, anti-PD-1 and anti-CTLA-4 antibodies are administered in 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 anti-PD-1 antibody and 1 mg / kg anti-CTLA-4 antibody; or (l) 10 mg / kg anti-PD-1 antibody and 1 mg / kg anti-CTLA-4 antibody. In certain embodiments, the method comprises administering 1 mg / kg anti-PD-1 antibody and 3 mg / kg anti-CTLA-4 antibody.

[0108] In one embodiment, the respective doses of anti-PD-1 and anti-CTLA-4 antibodies are kept constant throughout the induction and maintenance administration schedules. In one embodiment, the regimen is an induction dosing schedule comprising (i) at least two, four, six, eight, or ten combination doses of anti-PD-1 and anti-CTLA-4 antibodies at a frequency of at least approximately every two weeks, at least approximately every three weeks, at least approximately every four weeks, or at least approximately every month, followed by at least two, four, six, eight, or twelve doses of anti-PD-1 antibody alone at a frequency of at least every two weeks, three weeks, or four weeks, or at least every month; and then (ii) a maintenance dosing schedule comprising at least four, six, eight, ten, twelve, or sixteen combination doses of anti-PD-1 and anti-CTLA-4 antibodies at a frequency of at least approximately every eight weeks, at least approximately every twelve weeks, at least approximately every sixteen weeks, or at least once every quarter, or until clinical benefit is observed or until uncontrollable toxicity or disease progression occurs. In other embodiments, the administration schedule includes four doses of approximately 3 mg / kg of anti-PD-1 antibody every two weeks or approximately 1 mg / kg of anti-PD-1 antibody every three weeks + approximately 3 mg / kg of anti-CTLA-4 antibody every three weeks, followed by three or more cycles of approximately 3 mg / kg of anti-PD-1 antibody every two weeks or four doses of 3 mg / kg of anti-CTLA-4 antibody every three weeks. In a particular embodiment, the administration schedule includes four doses of approximately 1 mg / kg of anti-PD-1 antibody followed by CTLA-4 antibody (e.g., approximately 3 mg / kg of anti-CTLA-4 antibody) every three weeks on the same day, followed by approximately 3 mg / kg of anti-PD-1 antibody every two weeks.

[0109] In one embodiment of this method, the maintenance dosing schedule includes a combination of up to 4, 6, 8, 10, 12, or 16 doses of anti-PD-1 and anti-CTLA-4 antibodies. In other embodiments, the combination regimen includes an induction dose schedule comprising (i) 2, 4, 6, or 8 doses of the anti-PD-1 and anti-CTLA-4 antibodies in combination at approximately every two weeks, at least every three weeks, at least every four weeks, or approximately once a month, followed by 2, 4, 6, 8, or 12 doses of the anti-PD-1 antibody alone at approximately every two weeks, at least every three weeks, at least every four weeks, or approximately once a month; and (ii) a maintenance dose schedule comprising 4, 6, 8, 10, 12, or 16 doses of the anti-PD-1 and anti-CTLA-4 antibodies in combination at approximately every eight weeks, at approximately every twelve weeks, at approximately every sixteen weeks, or once a quarter, or until clinical benefit is observed or until uncontrollable toxicity or disease progression occurs.

[0110] In a specific embodiment, anti-PD-1 and anti-CTLA-4 antibodies are administered at doses of approximately 1 mg / kg of anti-PD-1 antibody every three weeks and approximately 3 mg / kg of anti-CTLA-4 antibody every three weeks. In another embodiment, the patient is administered approximately 1 mg / kg of anti-PD-1 antibody, followed by anti-CTLA-4 antibody on the same day, four times every three weeks, and then 3 mg / kg of anti-PD-1 antibody every two weeks.

[0111] In one embodiment of the present invention, anti-PD-1 and anti-CTLA-4 antibodies are formulated for non-enteral administration, such as intravenous administration. In one embodiment, when anti-PD-1 and anti-CTLA-4 antibodies are administered in combination, they are administered within about 30 minutes of each other. Either antibody may be administered first; that is, in one embodiment, the anti-PD-1 antibody is administered before the anti-CTLA-4 antibody, while in another embodiment, the anti-CTLA-4 antibody is administered before the anti-PD-1 antibody. Generally, each antibody is administered intravenously over about 60 minutes. In a further embodiment, anti-PD-1 and anti-CTLA-4 antibodies may be administered simultaneously, either as a single composition in a pharmaceutically acceptable formulation for combined administration, or as separate compositions in a pharmaceutically acceptable formulation in which each antibody is present.

[0112] In one embodiment, the anti-PD-1 antibody and the anti-CTLA-4 antibody are formulated in a single composition. The ratio of the amount of anti-PD-1 antibody to the amount of anti-CTLA-4 antibody in the single composition may be 10:1 to 1:10. In other embodiments, the ratio of the amount of anti-PD-1 antibody to the amount of anti-CTLA-4 antibody in the single composition is 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5. In a particular embodiment, the ratio of the amount of anti-PD-1 antibody to the amount of anti-CTLA-4 antibody in the single composition is 1:3.

[0113] In one embodiment, the composition is administered in a uniform dose regardless of the patient's body weight. For example, each of the anti-PD-1 antibody and the anti-CTLA-4 antibody may be administered in uniform doses of 20 mg, 50 mg, 75 mg, 80 mg, 160 mg, 200 mg, 240 mg, 300 mg, 360 mg, 400 mg, 480 mg, 500 mg, 750 mg, or 1500 mg, regardless of the patient's body weight. In another embodiment, the composition is administered in a body-based dose at any dose disclosed herein. In another embodiment, the amount of anti-PD-1 antibody and the amount of anti-CTLA-4 antibody administered to the patient in a single dose are the same.

[0114] In one embodiment of the method of the present invention, the therapeutically effective dose of the anti-PD-1 antibody or its antigen-binding moiety is a uniform dose (not a body weight-based dose) and includes approximately 60 mg, approximately 80 mg, approximately 100 mg, approximately 120 mg, approximately 140 mg, approximately 160 mg, approximately 180 mg, approximately 200 mg, approximately 220 mg, approximately 240 mg, approximately 260 mg, approximately 280 mg, or approximately 300 mg. In other embodiments, the therapeutically effective dose of the anti-PD-1 antibody or its antigen-binding moiety includes approximately 320 mg, 360 mg, 400 mg, 420 mg, 480 mg, 500 mg, 540 mg, 550 mg, 600 mg, 620 mg, 650 mg, 680 mg, 700 mg, 720 mg, 780 mg, 800 mg, 840 mg, or 900 mg. In one embodiment, the dose of anti-PD-1 antibody in the composition is approximately 60 mg to approximately 300 mg, approximately 60 mg to approximately 100 mg, approximately 100 mg to approximately 200 mg, or approximately 200 mg to approximately 300 mg. In one embodiment, the amount of anti-PD-1 antibody in the composition is at least approximately 80 mg, approximately 160 mg, or approximately 240 mg. In one embodiment, the dose of anti-PD-1 antibody in the composition is at least approximately 240 mg or at least approximately 80 mg.

[0115] In other embodiments, the dose is administered weekly, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, or every ten weeks.

[0116] In one embodiment, an anti-PD-1 antibody or its antigen-binding portion is administered at a dose of approximately 240 mg. In another embodiment, an anti-PD-1 antibody or its antigen-binding portion is administered at a dose of approximately 360 mg. In yet another embodiment, an anti-PD-1 antibody or its antigen-binding portion is administered at a dose of approximately 480 mg. In yet another embodiment, 360 mg of anti-PD-1 antibody or antigen-binding fragment is administered once every three weeks. In yet another embodiment, 480 mg of anti-PD-1 antibody or antigen-binding fragment is administered once every four weeks.

[0117] In other embodiments, the therapeutically effective dose of the anti-CTLA-4 antibody or its antigen-binding moiety is a uniform dose (not a body weight-based dose) and includes approximately 60 mg, approximately 80 mg, approximately 100 mg, approximately 120 mg, approximately 140 mg, approximately 160 mg, approximately 180 mg, approximately 200 mg, approximately 220 mg, approximately 240 mg, approximately 260 mg, approximately 280 mg, or approximately 300 mg. In other embodiments, the therapeutically effective dose of the anti-CTLA-4 antibody or its antigen-binding moiety includes approximately 320 mg, 360 mg, 400 mg, 420 mg, 480 mg, 500 mg, 540 mg, 550 mg, 600 mg, 620 mg, 650 mg, 680 mg, 700 mg, 720 mg, 780 mg, 800 mg, 840 mg, or 900 mg. In one embodiment, the dose of anti-CTLA-4 antibody in the composition is approximately 60 mg to approximately 300 mg, approximately 60 mg to approximately 100 mg, approximately 100 mg to approximately 200 mg, or approximately 200 mg to approximately 300 mg. In one embodiment, the amount of anti-CTLA-4 antibody in the composition is at least approximately 80 mg, approximately 160 mg, or approximately 240 mg. In one embodiment, the dose of anti-CTLA-4 antibody in the composition is at least approximately 240 mg or at least approximately 80 mg.

[0118] In one embodiment, the uniform dose of anti-PD-1 antibody is 80 mg, and the uniform dose of anti-CTLA-4 antibody is 240 mg.

[0119] In one embodiment, the anti-PD-1 antibody is administered at a sub-therapeutic dose, i.e., a dose significantly lower than the usual or FDA-approved dose when administered as monotherapy for cancer treatment. The amount of the secondary antibody in the composition is calculated based on a desired ratio.

[0120] In one embodiment, the composition is administered by intravenous infusion approximately once a week, approximately once every two weeks, approximately once every three weeks, or approximately once a month. In another embodiment, the composition is administered once every three weeks. In another embodiment, the infusion is administered for at least approximately 10 minutes, approximately 20 minutes, approximately 30 minutes, approximately 45 minutes, approximately 60 minutes, approximately 90 minutes, approximately 2 hours, approximately 3 hours, approximately 4 hours, or approximately 5 hours.

[0121] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention may be uniform or variable, so as to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and method of administration without causing excessive toxicity to the patient. The selectable dose level depends on a variety of pharmacokinetic factors, including 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 medical history of the patient being treated, and similar factors well known in the medical field. The compositions of the present invention can be administered by one or more routes of administration using one or more diverse methods well known in the art. As will be recognized by those skilled in the art, the route and / or method of administration will vary depending on the desired outcome.

[0122] kit Also included within the scope of the present invention are kits, including a pharmaceutical kit comprising a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody for therapeutic use, and a diagnostic kit comprising an anti-PD-L1 antibody for assaying membrane-bound PD-L1 expression as a biomarker for patient screening for combination therapy or for predicting the efficacy of combination therapy. The kit typically includes instructions indicating the intended use and instructions for use of the kit's contents. The term “instructions” means any written or recorded material on the kit, with the kit, or otherwise attached to the kit. In one embodiment of the pharmaceutical kit, the anti-PD-1 antibody is packaged together with the anti-CTLA-4 antibody in unit dose form. In one embodiment of the diagnostic kit, the anti-PD-L1 antibody is packaged together with the anti-PD-1 antibody and the anti-CTLA-4 antibody for performing an assay for the detection and / or quantification of PD-L1 expression.

[0123] In one embodiment, the drug kit contains the anti-human PD-1 HuMAb, nivolumab. In another embodiment, the drug kit contains the anti-human PD-L1 HuMAb, BMS-936559. In yet another embodiment, the drug kit contains the anti-human CTLA-4 HuMAb, ipilimumab. In one embodiment, the diagnostic kit contains the amino acid sequences shown in SEQ ID NOs: 1 and 2, respectively. H and V L The kit contains rabbit anti-human PD-L1 mAb, 28-8, including the region. In another embodiment, the diagnostic kit contains mouse anti-human PD-L1 mAb, 5H1 (Dong et al., Nature Med. 8(8):793-800 (2002)).

[0124] In one embodiment, the present invention provides a kit for treating a patient having a melanoma tumor, the kit is (a) an anti-PD-1 antibody or its antigen-binding moiety in a dose range of at least about 0.1 to at least about 10 mg / kg body weight; (b) an anti-CTLA-4 antibody or its antigen-binding moiety in a dose range of at least about 0.1 to at least about 10 mg / kg body weight; and (c) Instructions for using the anti-PD-1 antibody or its antigen-binding moiety and the anti-CTLA-4 antibody or its antigen-binding moiety in the methods disclosed herein. Includes.

[0125] The present invention will be further described by the following embodiments, but these should not be construed as limiting. All references made herein expressly incorporate the contents of all references made herein by reference. [Examples]

[0126] Example 1 A randomized, double-blind, multicenter phase 3 trial was conducted to evaluate the safety and efficacy of nivolumab monotherapy or nivolumab-ipilimumab combination therapy compared to ipilimumab monotherapy in patients with previously untreated metastatic melanoma.

[0127] patient Eligible patients had histologically confirmed stage III (unresectable) or stage IV melanoma and had not previously received systemic treatment for unresectable or metastatic melanoma. Other eligibility criteria included being at least 18 years of age, an ECOG (East Coast Cancer Group) Performance Status score of 0 (indicating no symptoms) or 1 (indicating mild symptoms), disease measurable by computed tomography or magnetic resonance imaging according to RECIST v1.1, availability of tissue taken from metastatic or unresectable tumor for PD-L1 status assessment, and known BRAF V600 mutation status (or consent to BRAF V600 mutation testing according to local standards). Key exclusion criteria included the presence of active brain metastases, ocular melanoma, or autoimmune disease, and any prior treatment with anti-PD-1, anti-PD-L1, anti-PD-L2, or anti-CTLA-4 antibodies. Patients requiring systemic corticosteroid treatment or other immunosuppressant therapy within 14 days of investigational drug administration were excluded.

[0128] Clinical trial design and procedures In a double-blind Phase 3 clinical trial, participants were randomly assigned in a 1:1:1 ratio to receive either 3 mg nivolumab / kg body weight every 2 weeks (+ ipilimumab matched placebo) or 1 mg nivolumab / kg every 3 weeks + 3 mg ipilimumab / kg every 3 weeks for 4 cycles (+ nivolumab matched placebo), followed by 3 mg nivolumab / kg every 2 weeks for 3 or more cycles, or 3 mg ipilimumab / kg every 3 weeks for 4 cycles (+ nivolumab matched placebo).

[0129] Both nivolumab and ipilimumab were administered intravenously. Randomization was stratified by tumor PD-L1 status (positive vs. negative or intermediate), BRAF mutation status (V600 mutation positive vs. wild-type), and Joint Committee on Cancer (M0, M1a or M1b vs. M1c) metastatic stage. Treatment continued until disease progression, unacceptable toxicity, or withdrawal of consent as defined by RECIST v1.1. Patients were treated even with progression, provided they received clinical benefit without clinical deterioration and no substantial adverse effects, as assessed by the investigator.

[0130] Progression-free survival and overall survival were co-primary endpoints. Secondary endpoints included objective response rate, tumor PD-L1 expression as a predictive biomarker for progression-free survival and overall survival, and health-related quality of life. Exploratory endpoints included objective response duration and safety / tolerance of investigational drug treatment.

[0131] evaluation Patients were assessed for tumor response according to RECIST v1.1 at 12 weeks after randomization, then every 6 weeks for 49 weeks, and then every 12 weeks until progression or discontinuation of treatment, whichever was later. Progression-free survival was defined as the period from the date of randomization to the first day when progression or progression or death, whichever was earlier, was confirmed. Patients who were treated despite progression were considered to have disease progression at the time of the first progression event, regardless of subsequent tumor response, as assessed by the investigator. PD-L1 expression on the surface of tumor cells was assessed in the central laboratory by immunohistochemistry in formalin-fixed, paraffin-embedded tumor specimens using an analytically validated automated assay developed by Dako (Carpinteria, CA) with rabbit monoclonal anti-human PD-L1 antibody (clone 28-8). PD-L1 positivity was defined as at least 5% of tumor cells showing cell surface PD-L1 staining of any intensity in a section containing at least 100 evaluable tumor cells. The intermediate state was due to samples where tumor cell surface expression could not be identified by melanin content or strong cytoplasmic staining.

[0132] All patients who received at least one dose of the investigational drug from any of the three treatment groups were included in the safety assessment. The severity of adverse events was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0.18. The safety assessment was continued throughout the treatment period and up to 100 days after the last dose of the investigational drug.

[0133] statistical analysis A trial sample size of approximately 915 patients was planned, randomized to three treatment arms in a 1:1:1 ratio. For progression-free survival comparisons, the number of events planned to be observed at at least 9 months of follow-up assumed approximately 83% power to detect a Type I error of 0.005 (two-sided) with a mean hazard ratio of 0.71. Progression-free survival was compared between nivolumab + ipilimumab and ipilimumab monotherapy, and between nivolumab monotherapy and ipilimumab monotherapy, using a two-sided log-rank test stratified according to PD-L1 status, BRAF mutation status, and metastatic stage (as described above). The trial was not designed for formal statistical comparison between the nivolumab monotherapy group and the nivolumab + ipilimumab group. Hazard ratios and corresponding two-sided 99.5% confidence intervals (CIs) were estimated using the Cox proportional hazards model with treatment group as a single covariate, stratified by the factors described above. Progression-free survival rates at 6, 12, and 18 months, with progression-free survival curves, median 95% CI, and 95% CI, were estimated using the Kaplan-Meier method. Overall survival was analyzed when the minimum follow-up for all patients was 22 months.

[0134] result Patient and treatment From July 2013 to March 2014, a total of 1,296 patients were enrolled at 137 sites in Australia, Europe, Israel, New Zealand, and North America. A total of 945 patients were randomized. 316 patients were assigned to the nivolumab group, 314 to the nivolumab + ipilimumab group, and 315 to the ipilimumab group (Figure 1). Baseline characteristics were equalized across the three groups. A total of 58.0% had stage M1c disease, 36.1% had high lactate dehydrogenase levels, 31.5% had BRAF mutations, and 73.5% had negative PD-L1 status (Table 1). [Table 1-1] [Table 1-2]

[0135] All randomized patients had been followed for at least 9 months at data lock (February 17, 2015); 117 out of 313 patients (37.4%) in the nivolumab group, 93 out of 313 patients (29.7%) in the nivolumab + ipilimumab group, and 50 out of 311 patients (16.1%) in the ipilimumab group continued investigational treatment (Table 2). The most frequent reason for discontinuation in the nivolumab and ipilimumab monotherapy groups was disease progression (154 out of 313 patients [49.2%] and 202 out of 311 patients [65.0%], respectively), while in the nivolumab + ipilimumab group it was investigational drug toxicity (120 out of 313 patients [38.3%]). The number of deaths was 85 (27.2%) in the nivolumab group, 86 (27.5%) in the nivolumab + ipilimumab group, and 114 (36.7%) in the ipilimumab group.

[0136] The median number of doses for patients receiving nivolumab monotherapy or ipilimumab monotherapy was 15 (range 1–38) and 4 (range 1–4), respectively. In the combination group, the median number of doses was 4 (range 1–39) for nivolumab and 4 (range 1–4) for ipilimumab; 147 out of 313 patients (47%) received nivolumab monotherapy four or more times after combination therapy.

[0137] Effectiveness The median progression-free survival was 6.5 months (95% confidence interval [CI], 4.3–9.5) in the nivolumab group, 11.5 months (95% CI, 8.9–16.5) in the nivolumab + ipilimumab group, and 2.9 months (95% CI, 2.8–3.4) in the ipilimumab group (Figure 2A). A significant improvement in progression-free survival was observed in the nivolumab + ipilimumab group compared to the ipilimumab group (hazard ratio, 0.42; 95% CI, 0.31–0.57; P<0.0001) (Figure 2A). A significant improvement in progression-free survival was also observed in the nivolumab group compared to the ipilimumab group (hazard ratio, 0.57; 95% CI, 0.43–0.76; P<0.00001) (Figure 2A). The hazard ratio for comparing the nivolumab + ipilimumab group with the nivolumab group was 0.74 (95% CI, 0.60–0.92).

[0138] Analysis of progression-free survival among pre-specified patient subgroups, including subgroups defined by PD-L1 status, BRAF mutation status, and metastatic stage, showed consistent improvement with nivolumab or nivolumab + ipilimumab compared to ipilimumab (Figure 3). In the combination groups, median PFS was 11.7 months (95% CI, 8.0 to not reached) in patients with BRAF mutations and 11.2 months (95% CI, 8.3 to not reached) in patients with wild-type BRAF (Figure 3B). For patients with PD-L1 positive tumor status, median progression-free survival was 14.0 months (95% CI, 9.1 to not reached), 14.0 months (95% CI, 9.7 to not reached), and 3.9 months (95% CI, 2.8 to 4.2) in the nivolumab group, nivolumab + ipilimumab group, and ipilimumab group, respectively. In patients with PD-L1-negative tumor status, the median progression-free survival was 5.3 months (95% CI, 2.8–7.1 months), 11.2 months (95% CI, 8.0 or less, not reached), and 2.8 months (95% CI, 2.8–3.1) in the nivolumab group, nivolumab plus ipilimumab group, and ipilimumab group, respectively (Figure 2C).

[0139] The investigator-assessed objective response rates were 43.7% (95% CI, 38.1–49.3%), 57.6% (95% CI, 52.0–63.2%), and 19.0% (95% CI, 14.9–23.8%) in the nivolumab group, the nivolumab plus ipilimumab group, and the ipilimumab group, respectively (Table 3). The percentage of patients with a complete response was higher in the nivolumab plus ipilimumab group than in the nivolumab group or the ipilimumab monotherapy group (11.5% vs. 8.9% and 2.2%) (Table 3). The time to objective response was similar across the groups (Table 3), and the median time to response was not reached in any group.

[0140] The median reduction in the sum of the longest diameters of tumor target lesions was -34.5% (interquartile range: -75.4 to 15.4), -51.1% (-75.8 to -10.2), and 5.8% (-28.0 to 33.3) in the nivolumab group, nivolumab + ipilimumab group, and ipilimumab group, respectively (Figure 4). In patients with PD-L1-positive tumors, the objective response rates were 57.5% (95% CI, 45.9–68.5), 72.1% (95% CI, 59.9–82.3), and 21.3% (95% CI, 12.7–32.3) in the nivolumab group, nivolumab + ipilimumab group, and ipilimumab group, respectively; in patients with PD-L1-negative tumors, the objective response rates were 41.3% (95% CI, 34.6–48.4), 54.8% (95% CI, 47.8–61.6), and 17.8% (95% CI, 12.8–23.8) (Table 4). [Table 2]

[0141] [Table 3]

[0142] [Table 4]

[0143] Adverse events Treatment-related adverse events of any grade occurred in 82.1%, 95.5%, and 86.2% of patients in the nivolumab group, nivolumab plus ipilimumab group, and ipilimumab group, respectively (Table 5). The most common adverse events in the nivolumab group were fatigue (34.2% of patients), rash (21.7%), and diarrhea (19.2%). In the nivolumab plus ipilimumab group and ipilimumab group, the most common were diarrhea (44.1% and 33.1%, respectively), fatigue (35.1% and 28.0%), and pruritus (33.2% and 35.4%) (Table 5). The incidence of Grade 3 or 4 treatment-related adverse events was also higher in the nivolumab + ipilimumab group than in the nivolumab or ipilimumab group (55.0% vs. 16.3% and 27.3%), with diarrhea being the most common (2.2%, 9.3%, and 6.1% in the nivolumab, nivolumab + ipilimumab, and ipilimumab groups, respectively) (Table 5). Treatment-related adverse events of any grade leading to discontinuation occurred in 7.7%, 36.4%, and 14.8% of patients in the nivolumab, nivolumab + ipilimumab, and ipilimumab groups, respectively, with diarrhea being the most common (1.9%, 8.3%, and 4.5%, respectively) and colitis (0.6%, 8.3%, and 7.7%, respectively) (Table 5). One death due to investigational drug toxicity was reported in the nivolumab group (neutropenia) and one death in the ipilimumab group (cardiac arrest). However, such adverse events were not associated with these drugs in previous trials. No treatment-related deaths were reported in the combination groups.

[0144] The most frequent grade 3 or 4 treatment-related selective adverse events were diarrhea (2.2%, 9.3%, and 6.1% of patients in the nivolumab, nivolumab + ipilimumab, and ipilimumab groups, respectively), colitis (0.6%, 7.7%, and 8.7%, respectively), elevated alanine aminotransferase (1.3%, 8.3%, and 1.6%, respectively), and elevated aspartate aminotransferase (1.0%, 6.1%, and 0.6%, respectively) (Table 6). With the use of immunomodulatory agents, the recovery rates for grade 3 or 4 selective adverse events were generally similar across treatment groups, ranging from 85% to 100% across organ categories in the nivolumab + ipilimumab group. As observed in previous trials, most endocrine events across all treatment groups did not resolve (Table 7). [Table 5]

[0145] [Table 6]

[0146] [Table 7]

[0147] Consideration In this randomized, double-blind, phase 3 trial, both nivolumab monotherapy and the combination of nivolumab and ipilimumab significantly increased progression-free survival and objective response rates compared to ipilimumab monotherapy in patients with previously untreated advanced melanoma. These results were observed independently of PD-L1 tumor status, BRAF mutation status, or metastatic stage. While the baseline characteristics of the trial participants were typical of patients with advanced melanoma, the BRAF mutation rate (31.5%) was lower than the 40–50% commonly reported for advanced disease. Although not the primary endpoint of the trial, the nivolumab-ipilimumab combination resulted in numerically longer progression-free survival and higher response rates compared to nivolumab monotherapy in the overall trial population. While the time to response was similar between groups, the initial tumor assessment was performed at week 12, so the possibility of earlier responses in the combination remains unknown.

[0148] The median progression-free survival (11.7 months in BRAF mutant patients) reported in this trial for the nivolumab and ipilimumab combination is similar to that recently reported for BRAF and MEK inhibitor combinations in BRAF mutant metastatic melanoma (9.9 months for vemurafenib and cobimetinib; 9.3–11.4 months for dabrafenib and trametinib). Such resistance to targeted therapy is almost inevitable when used as monotherapy and often occurs very rapidly. The proportion of patients with confirmed objective responses to the nivolumab and ipilimumab combination (57.6%) is numerically higher than that observed with PD-1 blockade alone in advanced melanoma (nivolumab [40%] in untreated patients with wild-type BRAF or pembrolizumab [37%] in ipilimumab-untreated patients).

[0149] The results of the subgroup analysis suggest that a significant advantage of the nivolumab-ipilimumab combination over nivolumab monotherapy may arise in the context of negative PD-L1 tumor expression. In the PD-L1-positive group, both nivolumab monotherapy and nivolumab + ipilimumab equally extended progression-free survival compared to ipilimumab monotherapy, but the objective response rate was numerically higher in the combination group compared to nivolumab or ipilimumab monotherapy. Therefore, the use of PD-L1 as a biomarker allows physicians to make more informed decisions regarding the risk-benefit ratio of combination therapy versus monotherapy. Nevertheless, the observation of at least additive activity in the ipilimumab-nivolumab combination in the context of negative PD-L1 expression is of interest in melanoma and other tumor types where PD-1 checkpoint inhibitors are being evaluated.

[0150] Overall, the incidence of adverse events in this trial was lowest in the nivolumab group and highest in the combination group. The overall incidence of Grade 3 or 4 drug-related adverse events was higher in the combination group compared to ipilimumab monotherapy (39.6% vs. 18.6%), with most adverse events showing only slightly higher incidences. In particular, with hepatotoxicity, Grade 3 or 4 ALT / AST assessments were 6–8% in the combination group and approximately 1% in ipilimumab monotherapy. One death was reported in each of the nivolumab and ipilimumab groups, but none in the combination group. Overall, the safety profile of the nivolumab-ipilimumab combination was consistent with past experience with nivolumab or ipilimumab monotherapy. No new safety warnings were found, and most adverse events were manageable with established treatment guidelines, as most selected adverse events were resolved with immunomodulatory agents. These data suggest that the nivolumab-ipilimumab combination can be safely used in a wide range of clinical settings.

[0151] In summary, the report shows increased progression-free survival and objective response rates with nivolumab monotherapy and the combination of nivolumab and ipilimumab compared to ipilimumab monotherapy in untreated advanced melanoma. Adverse events with the combination were managed using an established algorithm with no investigational drug-related deaths. The combination of nivolumab and ipilimumab may be a means of improving the outcomes of either monotherapy, particularly in patients with PD-L1-negative tumors. Overall, nivolumab monotherapy and the combination of nivolumab and ipilimumab are promising treatment options for untreated advanced melanoma.

[0152] This application claims priority under U.S. Provisional Application No. 61 / 153,973 filed April 28, 2015, which is incorporated herein by reference in its entirety.

[0153] array 28-8 V H Amino acid sequence (Oryctolagus cuniculus) (SEQ ID NO: 1) Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly Val Gln Cys Leu Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Thr Ile Thr Asn Tyr HisMet Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Thr Ser Ser Gly Ile Gly Ser Ser Ser Thr Thr Tyr Tyr Ala Thr Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asn Leu Arg Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Tyr Phe Thr Asn Thr Tyr Tyr Ala Leu Asp Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 28-8 V L Amino acid sequence (Oryctolagus cuniculus) (SEQ ID NO: 2) Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro Gly Ala Arg Cys Ala Leu Val Met Thr Gln Thr Pro Ser Ser Thr Ser Thr Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Val Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Gln Arg Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Ser Ala Gly Ser 28-8 heavy chain CDR1 sequence (Oryctolagus cuniculus) (SEQ ID NO: 3) Asn Tyr His Met Phe 28-8 heavy chain CDR2 sequence (Oryctolagus cuniculus) (SEQ ID NO: 4) Val Ile Thr Ser Ser Gly Ile Gly Ser Ser Ser Thr Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 28-8 heavy chain CDR3 sequence (Oryctolagus cuniculus) (SEQ ID NO: 5) Asp Tyr Phe Thr Asn Thr Tyr Tyr Ala Leu Asp Ile 28-8 light chain CDR1 sequence (Oryctolagus cuniculus) (SEQ ID NO: 6) Gln Ala Ser Gln Ser Ile Ser Val Tyr Leu Ala 28-8 light chain CDR2 sequence (Oryctolagus cuniculus) (Sequence ID 7) Ser Ala Ser Thr Leu Ala Ser 28-8 light chain CDR3 sequence (Oryctolagus cuniculus) (Sequence ID 8) Leu Gly Ser Ala Gly Ser Asp Asp Ala Ala

Claims

1. A method for treating melanoma, (i) Identify patients with PD-L1-negative melanoma tumors, and (ii) the patient (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1; and (b) An anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4. A method including administering [a substance].

2. A method for treating melanoma, for patients with melanoma tumors. (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1; and (b) The administration of an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4, Herein, the patient is identified as having a PD-L1-negative melanoma tumor prior to administration.

3. A method for extending progression-free survival beyond eight months in patients with melanoma tumors, wherein the patient (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1; and (b) The administration of an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4, Herein, the patient is identified as having a PD-L1-negative melanoma tumor prior to administration, and the patient exhibits a progression-free survival of more than 8 months.

4. The method according to claim 3, wherein the patient's progression-free survival is extended to more than approximately 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years after administration.

5. The method according to claim 4, wherein the patient's progression-free survival is extended to more than 11 months.

6. A method for reducing tumor size by at least 10% in a patient with a melanoma tumor, wherein the patient (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1; and (b) The administration of an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4, Herein, the patient is identified as having a PD-L1-negative melanoma tumor prior to administration, and the administration reduces the tumor size by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to before administration.

7. The method according to any one of claims 2 to 6, further comprising identifying patients with melanoma tumors that do not express PD-L1 before administration.

8. The method according to any one of claims 1 to 7, wherein the patient is characterized by (i) a long progression-free survival period of more than eight months, (ii) a reduction of at least about 10%, about 20%, about 30%, about 40%, or about 50% of the tumor size compared to the tumor size before administration, or (iii) both.

9. A method for increasing the objective response rate to more than 40% in a patient population, each of which has a melanoma tumor, in cancer treatment, (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1; and (b) The administration of an anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4, Here, each patient is identified as having a PD-L1-negative melanoma tumor prior to administration, and the objective response rate is higher than 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.

10. The method according to claim 9, further comprising identifying each patient in the patient population as having a PD-L1-negative melanoma tumor before administration.

11. The method according to claim 9 or 10, wherein each patient in the patient population is further characterized by (i) a long-term progression-free survival of more than eight months, (ii) a reduction of at least about 10%, about 20%, about 30%, about 40%, or about 50% of the tumor size compared to the tumor size before administration, or (iii) both.

12. The method according to any one of claims 9 to 11, wherein the patient population includes at least approximately 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 patients having PD-L1-negative melanoma tumors.

13. A method for selecting patients suitable for combination therapy with anti-PD-1 antibodies and anti-CTLA-4 antibodies, (i) Identify patients with PD-L1-negative melanoma tumors, and (ii) To the healthcare provider the patient (a) an anti-PD-1 antibody or its antigen-binding moiety that specifically binds to human PD-1; and (b) An anti-CTLA-4 antibody or its antigen-binding moiety that specifically binds to human CTLA-4. A method including instructing the administration of a drug.

14. The method according to any one of claims 1, 7, 10, and 13, wherein the identification includes measuring PD-L1 expression in melanoma tumors.

15. The method according to any one of claims 1 to 14, wherein the administration treats a melanoma tumor.

16. The method according to claim 14 or 15, wherein the measurement comprises providing a test tissue sample obtained from a patient, the test tissue sample comprising tumor cells and / or tumor-infiltrating inflammatory cells.

17. The method according to claim 16, wherein the measurement further includes evaluating the proportion of cells expressing PD-L1 on the cell surface in a test tissue sample.

18. The method according to claim 17, wherein the test tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample.

19. The method according to claim 18, wherein the presence of PD-L1 is determined using an automated IHC assay.

20. The method according to claim 19, wherein the IHC assay is performed using an anti-PD-L1 monoclonal antibody that specifically binds to PD-L1, and the anti-PD-L1 monoclonal antibody is selected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1, and any combination thereof.

21. The method according to any one of claims 1 to 20, wherein the PD-L1-negative melanoma tumor is characterized by the fact that approximately 5%, 4%, 3%, 2%, 1%, or 0% of tumor cells show binding to an anti-PD-L1 antibody or its antigen-binding portion.

22. The method according to any one of claims 1 to 21, wherein the anti-PD-1 antibody or its antigen-binding portion cross-competes with nivolumab for binding to human PD-1.

23. The method according to any one of claims 1 to 22, wherein the anti-PD-1 antibody or its antigen-binding portion is a chimeric, humanized, or human monoclonal antibody or a part thereof.

24. The method according to any one of claims 1 to 23, wherein the anti-PD-1 antibody or its antigen-binding portion comprises a heavy chain constant region of a human IgG1 or IgG4 isotype.

25. The method according to any one of claims 1 to 24, wherein the anti-PD-1 antibody is nivolumab.

26. The method according to any one of claims 1 to 24, wherein the anti-PD-1 antibody is pembrolizumab.

27. The method according to any one of claims 1 to 26, wherein an anti-PD-1 antibody or its antigen-binding portion is administered at a dose of 0.1 to 10.0 mg / kg body weight once every two, three, or four weeks.

28. The method according to claim 27, wherein an anti-PD-1 antibody or its antigen-binding portion is administered at a dose of 1 or 3 mg / kg body weight once every three weeks.

29. The method according to any one of claims 1 to 28, wherein the anti-CTLA-4 antibody or its antigen-binding moiety cross-competes with ipilimumab for binding to human CTLA-4.

30. The method according to any one of claims 1 to 29, wherein the anti-CTLA-4 antibody or its antigen-binding portion is a chimeric, humanized, or human monoclonal antibody or a part thereof.

31. The method according to any one of claims 1 to 30, comprising an anti-CTLA-4 antibody or a heavy chain constant region in which the antigen-binding portion is a human IgG1 isotype.

32. The method according to any one of claims 1 to 31, wherein the anti-CTLA-4 antibody is ipilimumab.

33. The method according to any one of claims 1 to 31, wherein the anti-CTLA-4 antibody is tremelimumab.

34. The method according to any one of claims 1 to 33, wherein an anti-CTLA-4 antibody or its antigen-binding portion is administered at a dose of 0.1 to 10.0 mg / kg body weight once every two, three, or four weeks.

35. The method according to claim 34, wherein an anti-CTLA-4 antibody or its antigen-binding moiety is administered at a dose of 1 or 3 mg / kg body weight once every three weeks.

36. The method according to claim 35, wherein an anti-PD-1 antibody or its antigen-binding portion is administered at a dose of 1 mg / kg body weight once every three weeks, and an anti-CTLA-4 antibody or its antigen-binding portion is administered at a dose of 3 mg / kg body weight once every three weeks.

37. The method according to any one of claims 1 to 36, wherein an anti-PD-1 antibody or its antigen-binding portion is administered simultaneously with an anti-CTLA-4 antibody or its antigen-binding portion.

38. The method according to any one of claims 1 to 36, wherein an anti-PD-1 antibody or its antigen-binding portion is administered before or after an anti-CTLA-4 antibody or its antigen-binding portion.

39. The method according to any one of claims 1 to 36, wherein the anti-PD-1 antibody or its antigen-binding portion and the anti-CTLA-4 antibody or its antigen-binding portion are a single pharmaceutical composition.

40. The method according to claim 39, wherein the pharmaceutical composition comprises an anti-PD-1 antibody and an anti-CTLA-4 antibody in a ratio of 50:1 to 1:

50.

41. The method according to claim 39, wherein the pharmaceutical composition comprises an anti-PD-1 antibody and an anti-CTLA-4 antibody in a ratio of 10:1 to 1:

10.

42. The method according to claim 39, wherein the pharmaceutical composition comprises an anti-PD-1 antibody and an anti-CTLA-4 antibody in a ratio of 3:1 to 1:

3.

43. The method according to claim 39, wherein the pharmaceutical composition comprises an anti-PD-1 antibody and an anti-CTLA-4 antibody in a ratio of 1:

3.

44. The method according to claim 39, wherein the pharmaceutical composition comprises 80 mg of anti-PD-1 antibody and 240 mg of anti-CTLA-4 antibody.

45. The method according to any one of claims 1 to 38, wherein anti-PD-1 and anti-CTLA-4 antibodies are administered to each other within 30 minutes of each other.

46. The method according to any one of claims 1 to 45, further comprising the administration of an anticancer agent.

47. A treatment kit for patients with melanoma tumors, (a) A constant dose of anti-PD-1 antibody or its antigen-binding moiety in the range of 0.1 to 10 mg / kg body weight; (b) a constant dose of anti-CTLA-4 antibody or its antigen-binding moiety in the range of 0.1 to 10 mg / kg body weight; and (c) Instructions for the use of the anti-PD-1 antibody or its antigen-binding moiety and the anti-CTLA-4 antibody or its antigen-binding moiety in any of the methods of claims 1 to 46. A kit that includes this.

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