Treatment of lung cancer using combination of Anti-PD-1 antibody and Anti-CTLA-4 antibody

Abstract

To provide a composition for treating a subject suffering from lung cancer.SOLUTION: The composition comprises a combination of an antibody or antigen-binding portion thereof that specifically binds to the programmed cell death-1 (PD-1) receptor and inhibits PD-1 activity (an anti-PD-1 antibody or antigen-binding portion thereof) and an antibody or antigen-binding portion thereof that specifically binds to cytotoxic T-lymphocyte antigen-4 (CTLA-4) and inhibits CTLA-4 activity (an anti-CTLA-4 antibody or antigen-binding portion thereof) for use in treating a subject suffering from lung cancer, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose ranging from about 0.1 to 5.0 mg per kg of body weight approximately once every two weeks, and the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose ranging from about 1 to 5.0 mg per kg of body weight approximately once every 6 to 12 weeks.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method of treating lung cancer in a subject comprising administering to the subject a combination of an anti-programmed cell death-1 (PD-1) antibody and an anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibody. [Background technology]

[0002] 2. Background of the Invention Human cancers display many genetic and epigenetic mutations that give rise to potential neoantigens that can be recognized by the immune system (Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system, consisting of T and B lymphocytes, has the potential to be a powerful anticancer agent with a broad ability and excellent specificity to respond to diverse tumor antigens. Furthermore, the immune system displays considerable plasticity and memory components. Successfully harnessing all these properties of the adaptive immune system would make immunotherapy unique among all cancer treatment modalities.

[0003] PD-1 is an important immune checkpoint receptor that is expressed by activated T and B cells and mediates immune suppression. PD-1 is a member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands of PD-1, programmed cell death ligand-1 (PD-L1) and programmed cell death ligand-2 (PD-L2), have been identified and are expressed on antigen-presenting cells as well as in many human cancers, and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1.

[0004] Nivolumab (formerly known as 5C4, BMS-936558, MDX-1106 or ONO-4538) is a fully human IgG4(S228P)PD-1 immune checkpoint inhibitor antibody that selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2), thereby preventing downregulation of antitumor T cell function (U.S. Pat. No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0005] Ipilimumab (YERVOY®) 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 (Hodi et al. (2010) N Engl J Med 363:711-23). ​​Coadministration of nivolumab and ipilimumab in a phase 1 clinical trial resulted in rapid and significant tumor regression in a significant proportion of patients with advanced melanoma and was significantly more effective than either antibody alone (Wolchok et al. (2013) N Engl J Med 369(2):122-33; WO2013 / 173223). However, it was previously unknown whether this combination of immunomodulatory antibodies would be similarly effective in other tumor types.

[0006] Non-small cell lung cancer (NSCLC) is the leading cause of cancer death in the United States and worldwide (NCCN GUIDELINES®, Version 3.2014 - Non-Small Cell Lung Cancer: available at www.nccn.org / professionals / physician_gls / pdf / nscl.pdf, last accessed May 14, 2014). NSCLC is relatively insensitive to chemotherapy, but patients with stage IV disease show good performance status (PS) benefit from treatment with chemotherapy agents including platinum agents (e.g., cisplatin, carboplatin), taxane agents (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, and gemcitabine, as well as various combinations of these agents. Summary of the Invention

[0007] Summary of the Invention The present invention relates to a method of treating a subject suffering from lung cancer, comprising administering to the subject a combination of: (a) an antibody or antigen-binding portion thereof that specifically binds to a programmed cell death-1 (PD-1) receptor and inhibits PD-1 activity, wherein the PD-1 antibody or antigen-binding portion thereof is administered about once every two weeks (Q2W) at a dose ranging from about 0.1 mg / kg to about 5.0 mg / kg of body weight; and (b) an antibody or antigen-binding portion thereof that specifically binds to a cytotoxic T-lymphocyte antigen-4 (CTLA-4) and inhibits CTLA-4 activity, wherein the CTLA-4 antibody or antigen-binding portion thereof is administered about once every six weeks (Q6W) or once every twelve weeks (Q12W) at a dose ranging from about 1 mg / kg to about 5.0 mg / kg of body weight. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In other embodiments, the NSCLC has squamous histology. In yet other embodiments, the NSCLC has non-squamous histology.

[0008] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, cross-competes with nivolumab for binding to human PD-1. In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is a chimeric antibody, a humanized antibody, or a human monoclonal antibody or portion thereof. In other embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, comprises a heavy chain constant region of a human IgG1 or IgG4 isotype. In some embodiments, the anti-PD-1 antibody is nivolumab. In one embodiment, the anti-PD-1 antibody is pembrolizumab (pembrolizumab).

[0009] In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is a chimeric antibody, a humanized antibody, or a human monoclonal antibody or a portion thereof. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof comprises a heavy chain constant region of the human IgG1 isotype. In some embodiments, the anti-CTLA-4 antibody is ipilimumab. In other embodiments, the anti-CTLA-4 antibody is tremelimumab. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof cross-competes with ipilimumab for binding to human CTLA-4.

[0010] In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of about 1 mg / kg body weight or about 3 mg / kg body weight once about every two weeks. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of about 1 mg / kg body weight. In other embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of about 3 mg / kg body weight once about every two weeks, and the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of about 1 mg / kg body weight once about every 12 weeks. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of about 1 mg / kg body weight once about every two weeks, and the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of about 1 mg / kg body weight once about every six weeks.

[0011] In some embodiments, the subject treated by the method of the present invention shows a progression-free survival (PFS) of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years or at least about 5 years. In some embodiments, the subject shows a progression-free survival of at least about 8 months after the first administration.

[0012] In certain embodiments, a subject has a longer progression-free survival when the subject is treated with an anti-PD-1 antibody, or antigen-binding portion thereof, administered at a dose of 3 mg / kg body weight once every two weeks, and an anti-CTLA-4 antibody, or antigen-binding portion thereof, administered at a dose of 1 mg / kg body weight once every 12 weeks ("Regimen A") than when the subject is treated with an anti-PD-1 antibody, or antigen-binding portion thereof, administered at a dose of 1 mg / kg body weight once every two weeks, and an anti-CTLA-4 antibody, or antigen-binding portion thereof, administered at 1 mg / kg body weight once every six weeks ("Regimen B"). In some embodiments, the progression-free survival of subjects treated with Regimen A is at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months longer than the progression-free survival of subjects treated with Regimen B. In some embodiments, the progression free survival of subjects treated with Regimen A is at least about 3 months longer than the progression free survival of subjects treated with Regimen B.

[0013] In certain embodiments, a subject has a longer progression-free survival when the subject is treated with an anti-PD-1 antibody, or antigen-binding portion thereof, administered at a dose of 3 mg / kg body weight once every two weeks, and an anti-CTLA-4 antibody, or antigen-binding portion thereof, administered at a dose of 1 mg / kg body weight once every 12 weeks ("Regimen A") than when the subject is treated with an anti-PD-1 antibody, or antigen-binding portion thereof, administered at a dose of 3 mg / kg body weight once every two weeks ("Regimen C"). In some embodiments, the progression-free survival of subjects treated with Regimen A is at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks longer than the progression-free survival of subjects treated with Regimen C. In some embodiments, the progression free survival of subjects treated with Regimen A is at least about 3 months longer than the progression free survival of subjects treated with Regimen C.

[0014] In some embodiments, the subject has a lung cancer with PD-L1 expression of 1% or more, 5% or more, 10% or more, 25% or more, or 50% or more. In some embodiments, the combination is administered as long as clinical benefit is observed or until disease progression or unmanageable toxicity occurs. In one embodiment, the anti-PD-1 and anti-CTLA-4 antibodies are formulated for intravenous administration. In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are administered sequentially to the subject. In some embodiments, the anti-PD-1 and anti-CTLA-4 antibodies are administered within 30 minutes of each other. In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, is administered before the anti-CTLA-4 antibody, or antigen-binding portion thereof. In another embodiment, the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered before the anti-PD-1 antibody, or antigen-binding portion thereof. In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are administered simultaneously as separate compositions. In certain embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are administered simultaneously as a single composition.

[0015] In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a sub-therapeutic dose. In some embodiments, the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered at a sub-therapeutic dose. In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are each administered at a sub-therapeutic dose.

[0016] The present invention also relates to a kit for treating a subject suffering from lung cancer, comprising: (a) an anti-PD-1 antibody, or antigen-binding portion thereof, in an amount ranging from about 4 mg to about 500 mg; (b) a CTLA-4 antibody, or antigen-binding portion thereof, in an amount ranging from about 40 mg to about 500 mg; and (c) instructions for using the PD-1 antibody, or antigen-binding portion thereof, and the CTLA-4 antibody, or antigen-binding portion thereof, in the methods described herein.

[0017] Other features and advantages of the present invention will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references cited herein, including scientific literature, journal reports, GenBank entries, patents, and patent applications, are expressly incorporated herein by reference. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 shows the administration schedule for nivolumab (nivo) and ipilimumab (ipi).

[0019] [Figure 2-1] Figures 2A-2D show the % change in target lesions from baseline (Figure 2D) for multiple treatment arms: nivolumab 1 mg / kg + ipilimumab 1 mg / kg Q3W (Figure 2A); nivolumab 1 mg / kg Q2W + ipilimumab 1 mg / kg Q6W (Figure 2B); nivolumab 3 mg / kg Q2W + ipilimumab 1 mg / kg Q12W (Figure 2C); and nivolumab 3 mg / kg Q2W + ipilimumab 1 mg / kg Q6W. [Figure 2-2] Same as above

[0020] [Diagram 3] Figure 3 shows the best % change from baseline in target lesion tumor burden by tumor PD-L1 expression.

[0021] [Figure 4] FIG. 4 shows duration of response of nivo+ipi in first line NSCLC.

[0022] [Diagram 5] Figure 5 shows efficacy of nivo+ipi across all tumor PD-L1 expression levels.

[0023] [Figure 6]Figures 6A and 6B show the efficacy of nivo+ipi in first line NSCLC by smoking status (Figure 6A) and EGFR mutation status (Figure 6B).

[0024] [Figure 7] 7A-7D show an example of a pathological CR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description of the Invention The present invention relates to a method for treating a patient with lung cancer comprising administering to said patient a combination of an anti-PD-1 antibody and another anti-cancer agent, hi some embodiments, the other anti-cancer agent is an anti-CTLA-4 antibody.

[0026] term In order that the present description may be more readily understood, certain terms are first defined. Unless otherwise specified herein, as used herein, each of the following terms shall have the meaning set forth below. Further definitions are set forth throughout the present description.

[0027] By "administering" is meant the physical introduction of a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. As used herein, the term "parenteral administration" refers to a method of administration other than enteral administration and topical administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intrathoracic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, intraperitoneal, intraperitoneal, epidural, subcutaneous, subcuticular, intraarticular, subcapsular, intraarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the combination is administered by a non-parenteral route. In some embodiments, it is administered orally. Other parenteral routes include topical, epithelial or mucosal routes of administration, such as intranasal, intravaginal, rectal, sublingual or topical administration. Administration can also be, for example, once, multiple times, and / or one or more times over an extended period of time.

[0028] As used herein, an "adverse event" (AE) is an unfavourable and generally unintended or undesirable sign (including an abnormal laboratory finding), symptom or disease associated with the use of a medical treatment. For example, an adverse event may be associated with activation of the immune system or proliferation of immune system cells (e.g., T cells) in response to the treatment. A medical treatment may have one or more associated AEs, each of which may have the same or different levels of severity. Reference to a method that can "modify an adverse event" refers to a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0029] "Antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (herein referred to as V HThe heavy chain constant region includes three constant domains, C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (referred to herein as V L The light chain constant region comprises one constant domain, C L Includes: V H and V L The regions are further divided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L contains three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0030] Immunoglobulins may be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. By "isotype" is meant the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region. The term "antibody" includes, by way of example, but not limited to, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; totally synthetic antibodies; and single chain antibodies. Non-human antibodies may be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated and unless the context dictates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the above immunoglobulins, including monovalent and bivalent fragments or portions and single chain antibodies.

[0031] By "isolated antibody" is meant an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds PD-1 is substantially free of antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds PD-1 may have cross-reactivity to other antigens, such as PD-1 molecules from different species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0032] The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules essentially identical in primary sequence, which display a single binding specificity and affinity for a particular epitope. A mAb is an example of an isolated antibody. Monoclonal antibodies may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0033] A "human" antibody (HuMAb) refers to an antibody having a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0034] By "humanized antibody" is meant an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with the corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abolish the ability of the antibody to bind to a particular antigen. A "humanized" antibody retains antigen-binding characteristics similar to that of the original antibody.

[0035] By "chimeric antibody" is meant an antibody in which the variable region is derived from one species and the constant region is derived from another species, e.g., the variable region is derived from a murine antibody and the constant region is derived from a human antibody.

[0036] An "anti-antigen" antibody refers to 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.

[0037] An "antigen-binding portion" of an antibody (also called an "antigen-binding fragment") means one or more fragments of an antibody that retain the ability to specifically bind to the antigen bound by the full-length antibody.

[0038] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and proliferation results in the formation of malignant tumors that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system and bloodstream.

[0039] "Cytotoxic T-lymphocyte antigen-4 (CTLA-4)" refers to an immune inhibitory receptor belonging to the CD28 family. CTLA-4 is expressed in vivo only on T cells and binds to two ligands, CD80 and CD86 (also called B7-1 and B7-2, respectively). As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), its variants, isoforms and species homologs, and analogs that share at least one epitope in common with hCTLA-4. The complete hCTLA-4 sequence can be found in GenBank Accession No. AAB59385.

[0040] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of developing or recurring a disease by methods that involve inducing, enhancing, suppressing or otherwise modifying an immune response. "Treatment" or "treatment" of a subject refers to any therapeutic intervention or process performed on a subject, or the administration of an active ingredient to a subject, for the purpose of ameliorating, alleviating, ameliorating, inhibiting, delaying or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical manifestation associated with a disease.

[0041] As used herein, "PD-L1 positive" may be used interchangeably with "at least about 1% PD-L1 expression." In one embodiment, PD-L1 expression may be measured by methods known in the art. In another embodiment, PD-L1 expression is measured by automated IHC. Thus, a PD-L1 positive tumor may contain at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of tumor cells expressing PD-L1 as measured by automated IHC. In one embodiment, "PD-L1 positive" means that there are at least 100 cells expressing PD-L1 on the surface of the cells.

[0042] "Programmed cell death-1 (PD-1)" refers to an immune inhibitory receptor belonging to the CD28 family. PD-1 is expressed primarily on preactivated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms and species homologs of hPD-1, and analogs that share at least one common epitope with hPD-1. The complete hPD-1 sequence can be found in GenBank Accession No. U64863.

[0043] "Programmed cell death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other is PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found in GenBank Accession No. Q9NZQ7.

[0044] A "subject" includes a human or non-human animal. The term "non-human animal" includes, but is not limited to, non-human primates, vertebrates such as sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0045] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is that amount of the agent, when used alone or in combination with another therapeutic agent, that protects a subject from developing a disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of the disease, or prevention of impairment or disability due to the affliction of the disease. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those of skill in the art, such as assaying the activity of the agent in human subjects in clinical trials, animal model systems predictive of efficacy in humans, or in in vitro assays.

[0046] As used herein, a "sub-therapeutic dose" refers to a dose of a therapeutic compound (e.g., an antibody) that is less than the usual or typical dose of the therapeutic compound when administered alone for the treatment of a hyperproliferative disease (e.g., cancer).

[0047] As an example, an "anti-cancer agent" promotes the regression of cancer in a subject or prevents further tumor growth. In some embodiments, a therapeutically effective amount of the agent promotes the regression of cancer to the point of eliminating the cancer. "Promoting the regression of cancer" means that an effective amount of the agent, alone or in combination with an anti-tumor agent, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free periods, or prevention of functional impairment or disability due to disease affliction. In addition, the terms "effective" and "effectiveness" in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the agent to promote the regression of cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (side effects) at the cell, organ and / or organism level resulting from the administration of the drug.

[0048] As an example of tumor treatment, a therapeutically effective amount of an anti-cancer agent can inhibit cell proliferation or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% compared to untreated subjects. In other embodiments of the present invention, tumor regression can be observed, which lasts for at least about 20 days, at least about 40 days, or at least about 60 days. Apart from these ultimate measures of therapeutic efficacy, the evaluation of immunotherapeutic agents must also take into account "immune-related" response patterns.

[0049] By "immune-related" response pattern is meant a clinical response pattern often observed in cancer patients treated with immunotherapeutic agents that induce cancer-specific immune responses or modify natural immune processes to produce antitumor effects. This response pattern is characterized by a beneficial therapeutic effect seen after an initial increase in tumor burden or the appearance of new lesions, which in the evaluation of conventional chemotherapeutic agents would be classified as disease progression and may be synonymous with drug failure. Thus, proper evaluation of immunotherapeutic agents may require longitudinal monitoring of the effects of these agents on the target disease.

[0050] A therapeutically effective amount of an agent includes a "prophylactically effective amount," which is the amount of agent that inhibits the development or recurrence of cancer when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer or at risk of having a recurrence of cancer (e.g., a subject with a pre-malignant condition). In some embodiments, a prophylactically effective amount completely prevents the development or recurrence of cancer. "Inhibiting" the development or recurrence of cancer means reducing the likelihood of the development or recurrence of cancer or completely preventing the development or recurrence of cancer.

[0051] The use of the alternatives (e.g., "or") should be understood to mean either one, both, or a combination thereof of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any described or listed members.

[0052] The term "about" or "essentially comprising" refers to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which may depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" may refer to one or more standard deviations per practice in the art. Alternatively, "about" or "essentially comprising" may refer to 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 (±10%) or between 2.4 mg and 3.6 mg (±20%). Furthermore, particularly with respect to biological systems or processes, these terms may refer to up to an order of magnitude or up to 5 times a value. When a particular value or composition is described in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be considered to be within an acceptable error range of that particular value or composition.

[0053] As used herein, the terms "about once every week," "about once every two weeks," or other similar dosing interval terms refer to approximate numbers. "About once every week" can include every 7 days ± 1 day, i.e., every 6 to 8 days. "About once every two weeks" can include every 14 days ± 3 days, i.e., every 11 to 17 days. Similar approximations apply, for example, to about once every 3 weeks (Q3W), about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, and about once every 7 weeks. In some embodiments, a dosing interval of about once every 6 weeks or about once every 12 weeks means that the first dose can be administered on any day in the first week, and then the next dose can be administered on any day in the sixth or twelfth week, respectively. In other embodiments, a dosing interval of about once every 6 weeks or about once every 12 weeks means that a first dose is administered on a particular day (e.g., a Monday) in the first week, and then the next dose is administered on the same day (i.e., a Monday) in the sixth or twelfth week, respectively.

[0054] As used herein, the term "weight-based dose" means that the dose administered to a patient is calculated based on the patient's weight. For example, if a patient weighing 60 kg requires 3 mg / kg of anti-PD-1 antibody, one skilled in the art can calculate and use the appropriate amount of anti-PD-1 antibody for administration (i.e., 180 mg).

[0055] The use of the term "fixed dose" with respect to the methods of the invention means that two or more different antibodies (e.g., an anti-PD-1 antibody and a second antibody, e.g., an anti-CTLA-4 antibody) in a single composition are present in the composition in a specific (fixed) ratio to each other. In some embodiments, the fixed dose is based on the weight (e.g., mg) of the antibodies. In some embodiments, the fixed dose is based on the concentration (e.g., mg / ml) of the antibodies. 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 1:200, about 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1. For example, a 3:1 ratio of anti-PD-1 antibody and anti-CTLA-4 antibody could mean that a vial contains about 240 mg of anti-PD-1 antibody and 80 mg of anti-CTLA-4 antibody, or about 3 mg / ml of anti-PD-1 antibody and 1 mg / ml of anti-CTLA-4 antibody.

[0056] The term "flat dose" with respect to the methods and dosages of the present invention refers to a dose administered to a patient regardless of the patient's weight or body surface area (BSA). Thus, a flat dose is not provided as a mg dose per kg of body weight, but rather as an absolute amount of agent (e.g., anti-PD-1 antibody). For example, a 60 kg human and a 100 kg human may be administered the same dose of antibody (e.g., 240 mg of anti-PD-1 antibody).

[0057] Any concentration range, percentage range, ratio range, or integer range described herein should be understood to include, unless otherwise stated, all integer values ​​within the recited range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of an integer).

[0058] Various aspects of the invention are described in further detail in the following subsections.

[0059] Methods of the Invention The present invention provides a method of treating a subject suffering from lung cancer, comprising administering to the subject a therapeutically effective amount of a combination of: (a) an anti-cancer agent that is an antibody or antigen-binding portion thereof that specifically binds to the PD-1 receptor and inhibits PD-1 activity; and (b) an anti-CTLA-4 antibody. In some embodiments, the lung cancer is NSCLC, since NSCLC comprises more than 85% of lung cancers. In other embodiments, the subject is a human patient. In some embodiments, the patient is a chemotherapy-naive patient (e.g., a patient who has not previously undergone chemotherapy). In other embodiments, the subject of the combination therapy has undergone another cancer therapy (e.g., chemotherapy), but is resistant or refractory to such another cancer therapy. In certain embodiments, the subject of the combination therapy has cancer cells that express a mutated form of the EGFR or KRAS gene. In some embodiments, the subject has cancer cells that are PD-L1+. In some embodiments, the subject has cancer cells that are PD-L1-. In some embodiments, the subject is a never-smoker. In some embodiments, the subject is a former smoker. In one embodiment, the subject is a current smoker. In some embodiments, the subject has squamous cancer cells. In some embodiments, the subject has non-squamous cancer cells.

[0060] In certain embodiments, a therapy of the invention (e.g., administration of an anti-PD-1 antibody and a CTLA-4 antibody) effectively extends a subject's survival. In some embodiments, a combination therapy of anti-PD-1 antibodies of the invention increases a subject's progression-free survival. In some embodiments, a combination therapy of anti-PD-1 antibodies of the invention increases a subject's progression-free survival compared to a standard therapy. In some embodiments, a combination therapy of anti-PD-1 antibodies of the invention increases a subject's progression-free survival compared to an anti-PD-1 antibody alone. In some embodiments, a combination therapy of anti-PD-1 antibodies of the invention increases a subject's progression-free survival compared to other anti-PD-1 antibody combination therapies. After administration of the anti-PD-1 antibody combination therapy, subjects with lung cancer may exhibit an overall survival of at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after said administration.

[0061] In other embodiments, the survival or overall survival of a subject is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, or at least about 1 year, as compared to another subject treated with standard therapy (e.g., docetaxel) alone, an anti-PD-1 antibody alone, or a combination therapy with a different dosing schedule. For example, the survival or overall survival of a subject treated with a combination therapy of anti-PD-1 antibodies described herein is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or at least about 75%, as compared to another subject treated with standard therapy (e.g., docetaxel) alone, an anti-PD-1 antibody alone, or a combination therapy with a different dosing schedule.

[0062] In some embodiments, the method of the present invention effectively increases the progression-free survival of the subject.In some embodiments, the subject shows a progression-free survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

[0063] In some embodiments, a subject has a longer progression-free survival when an anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose of 3 mg / kg of body weight once every two weeks, and an anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered at a dose of 1 mg / kg of body weight once every 6 to 12 weeks (e.g., every 6 weeks or every 12 weeks) than when the subject is administered an anti-PD-1 antibody, or antigen-binding portion thereof, at a dose of 3 mg / kg of body weight once every two weeks. In some embodiments, the progression free survival of a subject treated with an anti-PD-1 antibody, or antigen-binding portion thereof, administered at a dose of 3 mg / kg body weight once every two weeks, and an anti-CTLA-4 antibody, or antigen-binding portion thereof, administered at a dose of 1 mg / kg body weight once every six or twelve weeks is at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year longer than when a subject is treated with an anti-PD-1 antibody, or antigen-binding portion thereof, administered at a dose of 3 mg / kg body weight once every two weeks. In some embodiments, the progression-free survival of a subject is at least about 3 months longer when the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose of 3 mg / kg body weight once every 2 weeks, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered at a dose of 1 mg / kg body weight once every 6 or 12 weeks than when the subject is administered the anti-PD-1 antibody, or antigen-binding portion thereof, at a dose of 3 mg / kg body weight once every 2 weeks. In some embodiments, the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered every 6 weeks. In other embodiments, the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered every 12 weeks.

[0064] In some embodiments, a subject has a longer progression free survival when the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose of 3 mg / kg body weight once every two weeks, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered at a dose of 1 mg / kg body weight once every six or twelve weeks than when the subject is administered the anti-PD-1 antibody, or antigen-binding portion thereof, at a dose of 1 mg / kg body weight once every two weeks, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, at a dose of 1 mg / kg body weight once every six weeks. In certain embodiments, the progression free survival of a treated subject administered an anti-PD-1 antibody, or antigen-binding portion thereof, at a dose of 3 mg / kg body weight once every two weeks and an anti-CTLA-4 antibody, or antigen-binding portion thereof, at a dose of 1 mg / kg body weight once every six or twelve weeks is at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months longer than when a subject is treated with an anti-PD-1 antibody, or antigen-binding portion thereof, at a dose of 1 mg / kg body weight once every two weeks and an anti-CTLA-4 antibody, or antigen-binding portion thereof, at a dose of 1 mg / kg body weight once every six weeks. In some embodiments, progression-free survival is at least about 3 months longer when an anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of 3 mg / kg body weight once every 2 weeks and an anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 1 mg / kg body weight once every 6 or 12 weeks than when a subject is treated with an anti-PD-1 antibody or antigen-binding portion thereof at a dose of 1 mg / kg body weight once every 2 weeks and an anti-CTLA-4 antibody or antigen-binding portion thereof at a dose of 1 mg / kg body weight once every 6 weeks, or when a subject is treated with standard therapy. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered every 6 weeks. In other embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered every 12 weeks.

[0065] The PD-L1 status of the tumor in the subject can be measured before administering the compositions described herein or before utilizing the methods described herein. In one embodiment, the PD-L1 expression level of the tumor is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In another embodiment, the PD-L1 status of the tumor is at least about 1%. In other embodiments, the PD-L1 status of the subject is at least about 5%. In certain embodiments, the PD-L1 status of the tumor is at least about 10%. In one embodiment, the PD-L1 status of the tumor is at least about 25%. In certain embodiments, the PD-L1 status of the tumor is at least about 50%.

[0066] In some embodiments, the median progression-free survival (mPSF) of subjects having tumors with PD-L1 expression greater than or equal to 1% is at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year longer than the median progression-free survival of subjects having tumors with PD-L1 expression less than 1%. In some embodiments, the progression free survival of subjects having tumors with PD-L1 expression greater than or equal to 1% is at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

[0067] To assess PD-L1 expression, in one embodiment, a test tissue sample can be obtained from a patient in need of treatment. In another embodiment, assessing PD-L1 expression can be accomplished without obtaining a test tissue sample. In some embodiments, selecting a suitable patient includes (i) providing said test tissue sample, optionally comprising tumor cells and / or tumor-infiltrating inflammatory cells, obtained from a patient having cancer of the tissue; and (ii) assessing the proportion of cells in the test tissue sample that express PD-L1 on their surface based on assessing that the proportion of cells in the test tissue sample that express PD-L1 on their surface is higher than a predetermined threshold level.

[0068] However, it should be understood that in any method involving measuring PD-L1 expression in a test tissue sample, the step involving providing a test tissue sample obtained from a patient is an optional step. It should also be understood that in certain embodiments, the "measuring" or "assessing" step to identify or determine the number or percentage of cells in a test tissue sample that express PD-L1 on their cell surface is performed by modified methods of assaying for PD-L1 expression, for example, by performing a reverse transcriptase-polymerase chain reaction (RT-PCR) assay or an IHC assay. In certain other embodiments, no transformation step is involved and PD-L1 expression is assessed, for example, by reviewing a report of experimental test results. In some embodiments, each step of the method up to or including assessing PD-L1 expression provides an intermediate result that can be provided to a physician or other health care provider for use in selecting appropriate candidates for anti-PD-1 antibody or anti-PD-L1 antibody therapy. In some embodiments, the steps of providing the intermediate result are performed by an attending physician or a person acting under the direction of an attending physician. In other embodiments, these steps are performed by an independent laboratory or by an independent person such as a laboratory technician.

[0069] In certain embodiments of any of the methods of the invention, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 RNA. In further embodiments, the presence of PD-L1 RNA is determined by RT-PCR, in situ hybridization, or RNase protection assay. In other embodiments, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of a PD-L1 polypeptide. In further embodiments, the presence of a PD-L1 polypeptide is determined by immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some embodiments, PD-L1 expression is assayed by IHC. In all other embodiments of these methods, cell surface expression of PD-L1 is assayed using, for example, IHC or in vivo imaging.

[0070] Imaging technologies have provided important tools in cancer research and treatment. Recent developments in molecular imaging systems, including positron emission tomography (PET), single photon emission computed tomography (SPECT), fluorescence reflectance imaging (FRI), fluorescence mediated tomography (FMT), bioluminescence imaging (BLI), laser scanning confocal microscopy (LSCM) and multiphoton microscopy (MPM), may foreshadow even greater use of these technologies in cancer research. Some of these molecular imaging systems allow clinicians to not only see where tumors are located in the body, but also visualize the expression and activity of specific molecules, cells and biological processes that affect tumor dynamics and / or responsiveness to therapeutic agents (Condeelis and Weissleder, “In vivo imaging in cancer”, Cold Spring Harb. Perspect. Biol. 2(12):a003848 (2010)). The antibody specificity, combined with the sensitivity and resolution of PET, makes immunoPET imaging particularly useful for monitoring and assaying expression of antigens in tissue samples (McCabe and Wu, "Positive progress in immunoPET-not just a coincidence", Cancer Biother. Radiopharm. 25(3):253-61 (2010); Olafsen et al., "ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies)", Protein Eng. Des. Sel. 23(4):243-9 (2010)). In certain embodiments of any of the methods of the invention, PD-L1 expression is assayed by immunoPET imaging. In certain embodiments of the methods of the invention, the percentage of cells expressing PD-L1 in a test tissue sample is assessed by performing an assay that determines the presence of PD-L1 polypeptide on the surface of cells in the test tissue sample. In an embodiment, the test tissue sample is a FFPE tissue sample.In other embodiments, the presence of a PD-L1 polypeptide is determined by an IHC assay. In further embodiments, the IHC assay is performed using an automated process. In some embodiments, the IHC assay is performed using an anti-PD-L1 mAb that binds to a PD-L1 polypeptide.

[0071] In one embodiment of the method of the present invention, an automated IHC method is used to assay the expression of PD-L1 on the surface of cells in FFPE tissue specimens.The present invention provides a method for detecting the presence of human PD-L1 antigen in a test tissue sample or quantifying the level of human PD-L1 antigen or the proportion of cells expressing the antigen in the sample, the method comprising contacting the test sample and a negative control sample with a mAb that specifically binds to human PD-L1 under conditions that allow the formation of a complex between the antibody or a portion thereof and human PD-L1.In one embodiment, the test sample and the control tissue sample are FFPE samples.Then, the formation of a complex is detected, where a difference in the complex formation between the test sample and the negative control sample indicates the presence of human PD-L1 antigen in the sample.Various methods are used to quantify PD-L1 expression.

[0072] In certain embodiments, the automated IHC method includes the following steps: (a) deparaffinizing and rehydrating tissue sections mounted on an autostainer; (b) retrieving antigens using a decloaking chamber and pH 6 buffer by heating to 110°C for 10 minutes; (c) loading reagents onto the autostainer; and (d) running the autostainer to include the steps of neutralizing endogenous peroxidase in the tissue sample; blocking nonspecific protein binding sites on the slide; incubating the slide with primary antibody; incubating with post-primary blocking agent; incubating with NovoLink Polymer; adding chromogen substrate to develop color; and counterstaining with hematoxylin.

[0073] To assess PD-L1 expression in tumor tissue samples, pathologists performed a microscopic analysis of membrane PD-L1 expression in each field. + Tumor cells are counted, the percentage of cells that are positive is estimated, and then averaged to derive the final percentage. Different staining intensities are defined as 0 / negative, 1+ / weak, 2+ / moderate, and 3+ / strong. Generally, percentage values ​​are first assigned to 0 and 3+ buckets, and then intermediate 1+ and 2+ intensities are considered. For highly heterogeneous tissues, the sample is divided into zones, each zone is scored separately, and then combined into a single set of percentage values. The percentages of negative and positive cells for different staining intensities are determined from each region, and the median value is given to each zone. A final percentage value is given to the tissue for each staining intensity category: negative, 1+, 2+, and 3+. The sum of all staining intensities should be 100%. In one embodiment, the threshold number of cells that need to be PD-L1 positive is at least about 100, at least about 125, at least about 150, at least about 175, or at least about 200 cells. In one embodiment, the threshold number of cells that need to be PD-L1 positive is at least about 100 cells.

[0074] Staining is also evaluated in tumor-infiltrating inflammatory cells such as macrophages and lymphocytes. In most cases, staining is observed in many macrophages, so macrophages are used as an internal positive control. Although it is not necessary to stain with 3+ intensity, the absence of staining of macrophages should be considered to exclude technical failure. Macrophages and lymphocytes are evaluated for cell membrane staining and only scored for all samples as positive or negative for each cell category. Staining is also characterized according to extra / intratumoral immune cell orientation. "Internal" means that the immune cells are within the tumor tissue and / or on the border of the tumor area, without being physically intercalated between tumor cells. "External" means that the immune cells are not physically associated with the tumor, but are found in peripheral tissues associated with connective tissue or related adjacent tissues.

[0075] In certain embodiments of these scoring methods, samples are scored by two pathologists working independently and then the scores are combined. In certain other embodiments, the identification of positive and negative cells is scored using appropriate software.

[0076] A more quantitative measure of IHC data is the histoscore, which is calculated as follows: Histoscore = [(%tumor × 1 (low intensity)) + (%tumor × 2 (moderate intensity)) + (%tumor × 3 (high intensity)].

[0077] To determine the Histoscore, a pathologist estimates the percentage of stained cells in each intensity category within a specimen. Because expression of most biomarkers is heterogeneous, the Histoscore is a more true representation of overall expression. Final Histoscore ranges from 0 (no expression) to 300 (maximal expression).

[0078] Another means to quantify PD-L1 expression in test tissue samples IHC is to determine the adjusted inflammation score (AIS), which is defined as the percentage of PD-L1 expression by tumor-infiltrating inflammatory cells multiplied by the density of inflammation (Taube et al., “Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape,” Sci. Transl. Med. 4(127):127ra37 (2012)).

[0079] The method of the present invention can treat any stage of non-squamous NSCLC. There are at least seven stages used for NSCLC: occult (hidden) stage, stage 0 (in situ carcinoma), stage I, stage II, stage IIIA, stage IIIB, and stage IV. In the occult stage, cancer is not seen by imaging or bronchoscopy. In stage 0, cancer cells are seen in the lining of the airways.

[0080] In one embodiment, the method of the present invention treats stage I non-squamous NSCLC. Stage I NSCLC is divided into stage IA and stage IB. In stage IA, tumor is only in lung and is 3 cm or less. In stage IB, cancer has not spread to lymph nodes, and one or more of the following applies: 1) tumor is larger than 3 cm but smaller than 5 cm; 2) cancer has spread to main bronchus and is at least 2 cm below where trachea connects to main bronchus; 3) cancer has spread to the innermost layer of the membrane that covers the lung; or 4) part of lung collapses or develops pneumonia (lung inflammation) in the area where trachea connects to main bronchus.

[0081] In another embodiment, the method of the present invention treats stage II non-squamous NSCLC. Stage II NSCLC is divided into stage IIA and IIB. In stage IIA, cancer spreads to lymph nodes or not. When cancer spreads to lymph nodes, it can only spread to lymph nodes on the same side of the chest as the tumor, and the lymph nodes with cancer are in the lung or near the main bronchus, and one or more of the following applies: 1) tumor is smaller than 5 centimeters; 2) cancer spreads to the main bronchus and is at least 2 centimeters below where the trachea connects to the main bronchus; 3) cancer spreads to the innermost layer of the membrane that covers the lung; or 4) part of the lung collapses or develops pneumonia (lung inflammation) in the area where the trachea connects to the main bronchus. A tumor is considered to be in stage IIA if the cancer has not spread to the lymph nodes and one or more of the following is true: 1) the tumor is larger than 5 centimeters but smaller than 7 centimeters; 2) the cancer has spread to the main bronchi and is at least 2 centimeters below where the trachea connects to the main bronchi; 3) the cancer has spread to the innermost layer of the membrane that covers the lung; or 4) part of the lung collapses or pneumonia (inflammation of the lung) develops in the area where the trachea connects to the main bronchi. In stage IIB, the cancer may or may not have spread to the lymph nodes. When the cancer has spread to the lymph nodes, it can only spread to lymph nodes on the same side of the chest as the tumor, and the lymph nodes with the cancer are in the lung or near the main bronchus and one or more of the following are true: 1) the tumor is larger than 5 centimeters but smaller than 7 centimeters; 2) the cancer has spread to the main bronchus and is at least 2 centimeters below where the trachea connects to the main bronchus; 3) the cancer has spread to the innermost layer of the membrane that covers the lung; or 4) part of the lung collapses or pneumonia (inflammation of the lung) develops in the area where the trachea connects to the main bronchus.A tumor is also considered to be stage IIB if the cancer has not spread to lymph nodes and one or more of the following are true: 1) the tumor is larger than 7 centimeters; 2) cancer has spread to the main bronchus (at least 2 centimeters below where the trachea connects to the main bronchus), chest wall, diaphragm, or nerves that control the diaphragm; 3) cancer has spread to the membrane around the heart or to the inside wall of the chest wall; 4) the entire lung has collapsed or you have developed pneumonia (inflammation of the lungs); or 5) there are one or more separate tumors in the same lobe of the lung.

[0082] In another embodiment, the method of the present invention treats stage III non-squamous NSCLC. Stage IIIA is classified into three stages. These three stages are based on 1) the size of the tumor; 2) where the tumor was found; and 3) whether the lymph nodes (if any) have cancer. In stage IIIA first stage NSCLC, the cancer has spread to lymph nodes on the same side of the chest as the tumor, and the lymph nodes with cancer are located near the breastbone or where the main bronchus enters the lung. In addition, 1) the tumor can be of any size; 2) a portion of the lung (where the trachea joins the main bronchus) or the entire lung may have collapsed or developed pneumonia (inflammation of the lung); 3) there may be one or more separate tumors in the same lobe of the lung; and 4) the cancer may have spread to any of the following: a) the main bronchus, but not the area where the trachea joins the bronchus, b) the chest wall, c) the diaphragm or the nerves that control it, d) the membrane around the lung or the inner wall of the chest wall, e) the membrane around the heart. In stage IIIA second stage NSCLC, the cancer has spread to lymph nodes on the same side of the chest as the tumor, and the lymph nodes with cancer are inside the lung or near the main bronchus. In addition, 1) the tumor can be of any size; 2) the entire lung may have collapsed or developed pneumonia (inflammation of the lung); 3) there may be one or more separate tumors in either lobe of the lung that has cancer; and 4) the cancer may have spread to any of the following: a) the main bronchus, but not the area where the trachea joins the bronchus, b) the chest wall, c) the diaphragm or the nerves that control it, d) the membranes around the lung or the inner wall of the chest wall, e) the heart or the membranes around it, f) the large blood vessels leading to or from the heart, g) the trachea, h) the esophagus, i) the nerves that control the larynx (vocal cords), j) the sternum (chest bone) or spine, or k) the carina (where the trachea joins the main bronchus).In stage IIIA third stage NSCLC, cancer has not spread to lymph nodes, the tumor can be of any size, and cancer has spread to one of the following: a) the heart, b) the large blood vessels leading to or from the heart, c) the trachea, d) the esophagus, e) the nerves that control the larynx (vocal cords), f) the sternum (chest bone) or spine, or g) the carina (where the trachea joins the main bronchi). Stage IIIB is divided into two stages based on 1) the size of the tumor, 2) where the tumor was found, and 3) whether the lymph nodes have cancer. In stage IIIB first stage NSCLC, cancer has spread to lymph nodes in the chest on the opposite side of the tumor. Additionally, 1) the tumor may be of any size; 2) a portion of the lung (where the trachea joins the main bronchus) or the entire lung may have collapsed or developed pneumonia (inflammation of the lungs); 3) there may be one or more separate tumors in any lobe of the lung that has cancer; and 4) the cancer may have spread to any of the following: a) the main bronchus, b) the chest wall, c) the diaphragm or the nerves that control it, d) the membranes around the lungs or the inner wall of the chest wall, e) the heart or the membranes around it, f) the large blood vessels that lead to or from the heart, g) the trachea, h) the esophagus, i) the nerves that control the larynx (vocal cords), j) the sternum (chest bone) or spine, or k) the carina (where the trachea joins the main bronchus). In stage IIIB second stage NSCLC, cancer has spread to lymph nodes on the same side of the chest as the tumor. Lymph nodes that have cancer are located near the sternum (chest bone) or where the bronchi enter the lungs. Furthermore, 1) the tumor can be of any size; 2) there can be separate tumors in different lobes of the same lung; and 3) the cancer may have spread to any of the following: a) the heart, b) the heart or membranes around it, c) the trachea, d) the esophagus, e) the nerves that control the larynx (vocal cords), f) the sternum (chest bone) or spine, or g) the carina (where the trachea joins the main bronchi).

[0083] In some embodiments, the method of the present invention treats stage IV non-squamous NSCLC.In stage IV NSCLC, tumor can be any size, and cancer can spread to lymph nodes.In stage IV NSCLC, one or more of the following applies: 1) there is one or more tumors in both lungs; 2) cancer is found in the lung or in the fluid around the heart; and 3) cancer spreads to other parts of the body, such as the brain, liver, adrenal gland, kidney or bone.

[0084] The present invention provides a combination therapy for the treatment of lung cancer, in which an anti-PD-1 antibody is combined with another anti-cancer agent that is an antibody or antigen-binding portion thereof that specifically binds to CTLA-4 and inhibits CTLA-4 activity. The combination of the anti-PD-1 antibody, nivolumab, and the anti-CTLA-4 antibody, ipilimumab, has been demonstrated herein to produce early and durable anti-tumor activity in NSCLC patients, particularly with certain dosing schedules (see Example 1). Thus, in some embodiments, the anti-CTLA-4 antibody used in combination with the anti-PD-1 antibody is ipilimumab. In some embodiments, the anti-CTLA-4 antibody is tremelimumab. In other embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is an antibody or portion thereof that cross-competes with ipilimumab for binding to human CTLA-4. In certain other embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is a chimeric, humanized, or human mAb or portion thereof. In yet other embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof comprises a heavy chain constant region of the human IgG1 or IgG4 isotype. In some embodiments, the anti-CTLA-4 antibody comprises a heavy chain constant region of the human IgG1 isotype.

[0085] Due to the durability of clinical benefits previously demonstrated with immunotherapy through immune checkpoint inhibition (see, e.g., WO 2013 / 173223), the combination therapy may, in another embodiment, comprise a finite number of doses, e.g., about 1-10 doses, or may be administered at longer intervals, e.g., about once every 3-6 months or about once every 1-2 years or longer.

[0086] In certain embodiments of the methods of the present invention, the anti-PD-1 antibody is nivolumab. In other embodiments, it is pembrolizumab. In yet other embodiments, the anti-CTLA-4 antibody is ipilimumab. In a further embodiment, the anti-CTLA-4 antibody is tremelimumab. Generally, the anti-PD-1 and anti-CTLA-4 antibodies are formulated for intravenous administration. In some embodiments, when the anti-PD-1 and anti-CTLA-4 antibodies are administered in combination, they are administered within 30 minutes of each other. Either antibody may be administered first, i.e., in some embodiments, the anti-PD-1 antibody is administered before the anti-CTLA-4 antibody, while in other embodiments, the anti-CTLA-4 antibody is administered before the anti-PD-1 antibody. Generally, each antibody is administered by intravenous infusion over a period of 60 minutes. In some embodiments, the anti-PD-1 and anti-CTLA-4 antibodies are administered in combination, either as a single composition in a pharma- ceutically acceptable formulation for administration in combination, or mixed as separate compositions containing each antibody in a pharma- ceutically acceptable formulation.

[0087] In certain embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a sub-therapeutic dose. In certain other embodiments, the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered at a sub-therapeutic dose. In further embodiments, both the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are each administered at a sub-therapeutic dose.

[0088] Anti-PD-1 or anti-PD-L1 antibodies useful in the present invention HuMAbs that specifically bind to PD-1 with high affinity are described in U.S. Patent No. 8,008,449. Other anti-PD-1 monoclonal antibodies are described, for example, in U.S. Patent Nos. 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 described in U.S. Patent No. 8,008,449 has been demonstrated to exhibit one or more of the following properties: (a) a PD-1 specific binding affinity of 1×10 2 or more, as determined by surface plasmon resonance using a Biacore biosensor system; -7 K below M D (b) binds to human PD-1 in a situ hybridization assay; (c) does not substantially 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 a MLR assay; (f) binds to human PD-1 and cynomolgus 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 growth in vivo. Anti-PD-1 antibodies that can be used in the present invention include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in some embodiments, at least five, of the above characteristics. In some embodiments, the anti-PD-1 antibody is nivolumab. In one embodiment, the anti-PD-1 antibody is pembrolizumab.

[0089] In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1. In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as "OPDIVO®"; formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively inhibits interaction with PD-1 ligands (PD-L1 and PD-L2), thus preventing downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). In other embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as nivolumab. In some embodiments, the anti-PD-1 antibody has the same CDRs as nivolumab.

[0090] In another embodiment, the anti-PD-1 antibody or fragment thereof cross-competes with pembrolizumab. In some embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as pembrolizumab. In some embodiments, the anti-PD-1 antibody has the same CDRs as pembrolizumab. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as "KEYTRUDA®", 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, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587; see also www.cancer.gov / drugdictionary-cdrid=695789 (last accessed December 14, 2014). Pembrolizumab is approved by the FDA for the treatment of recurrent or refractory melanoma.

[0091] In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with MEDI0608. In yet other embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as MEDI0608. In some embodiments, the anti-PD-1 antibody has the same CDRs as MEDI0608. In other embodiments, the anti-PD-1 antibody is the monoclonal antibody MEDI0608 (formerly AMP-514). MEDI0608 is described, for example, in U.S. Patent No. 8,609,089 B2.

[0092] In one embodiment, the immune checkpoint inhibitor is AMP-224, a B7-DC Fc fusion protein, which is described in U.S. Patent Application Publication No. 2013 / 0017199 or at www.cancer.gov / publications / dictionaries / cancer-drug-cdrid=700595 (last accessed July 8, 2015).

[0093] In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with BGB-A317. In some embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as BGB-A317. In some embodiments, the anti-PD-1 antibody has the same CDRs as BGB-A317. In some embodiments, the anti-PD-1 antibody is BGB-A317, a humanized monoclonal antibody. BGB-A317 is described in U.S. Patent Application Publication No. 2015 / 0079109.

[0094] Anti-PD-1 antibodies that can be used in the methods described herein also include isolated antibodies that specifically bind to human PD-1 and cross-compete with nivolumab for binding to human PD-1 (see, e.g., U.S. Patent Nos. 8,008,449 and 8,779,105; 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 other anti-chain competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have similar functional properties to those of nivolumab due to their binding to the same epitope of PD-1. Cross-competing antibodies can be easily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO2013 / 173223).

[0095] In some embodiments, the antibody that cross-competes with human PD-1 or binds to the same epitope region as human PD-1 antibody nivolumab is a monoclonal antibody.For administration to human subjects, these cross-competing antibodies are chimeric antibodies, or humanized or human antibodies.Such chimeric, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0096] Anti-PD-1 antibodies that can be used in the methods described herein also include antigen-binding portions of the above antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H1(iv) an Fd fragment consisting of the V domain of a single arm of an antibody; L and V H It includes an Fv fragment consisting of domains.

[0097] Anti-PD-1 antibodies suitable for use in the methods or compositions described herein are those that bind to PD-1 with high specificity and affinity, block binding of PD-L1 and / or PD-L2, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods described herein, an anti-PD-1 "antibody" includes an antigen-binding portion or fragment that exhibits functional properties similar to those of the whole antibody in binding to the PD-1 receptor, inhibiting ligand binding, and upregulating the immune system. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1. In other embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is a chimeric, humanized, or human monoclonal antibody or portion 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.

[0098] In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof comprises a heavy chain constant region of a human IgG1 or IgG4 isotype. In certain other embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or antigen-binding portion thereof comprises a S228P mutation, which replaces a serine residue in the hinge region with a proline residue normally found at the corresponding site in IgG1 isotype antibodies. This mutation, present in nivolumab, prevents Fab arm exchange with endogenous IgG4 antibodies while maintaining a low affinity for activating Fc receptors associated with wild-type IgG4 antibodies (Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). In yet other embodiments, the antibody comprises a light chain constant region that is a human kappa or lambda constant region. In other embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is a mAb or antigen-binding portion thereof. In certain embodiments of any of the therapeutic methods described herein that include 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 human antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in U.S. Patent No. 8,008,449. In yet other embodiments, the anti-PD-1 antibody is MEDI0608 (formerly AMP-514), AMP-224, or BGB-A317.

[0099] Anti-PD-1 antibodies that can be used in the methods described herein also include antigen-binding portions of the above antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H1(iv) an Fd fragment consisting of the V domain of a single arm of an antibody; L and V H It includes an Fv fragment consisting of domains.

[0100] In some embodiments, the anti-PD-1 antibody used in the methods may be replaced with another PD-1 or anti-PD-L1 antagonist. For example, an anti-PD-L1 antibody may be used instead of an anti-PD-1 antibody in the methods described herein, since the anti-PD-L1 antibody blocks the interaction between PD-1 and PD-L1, thereby exerting a similar effect on the PD-1 signaling pathway. Thus, in one embodiment, the present invention relates to a method of treating a subject suffering from NSCLC, comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody and an anti-CTLA-4 antibody.

[0101] In some embodiments, the anti-PD-L1 antibody useful in the methods is BMS-936559 (formerly 12A4 or MDX-1105) (see, e.g., U.S. Patent No. 7,943,743; WO 2013 / 173223).

[0102] In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446) (see, e.g., Herbst et al. (2013) J Clin Oncol 31(suppl):3000. Abstract; U.S. Patent No. 8,217,149).

[0103] In other embodiments, the anti-PD-L1 antibody is MEDI4736 (also referred to as Durvalumab; see Khleif (2013) In: Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands. Abstract 802, U.S. Patent No. 8,779,108 filed May 6, 2014 or US 2014 / 0356353).

[0104] In a further embodiment, the anti-PD-L1 antibody is MSB0010718C (also known as Avelumab; see US2014 / 0341917).

[0105] Because anti-PD-1 and anti-PD-L1 antibodies 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; WO 2013 / 173223), an anti-PD-L1 antibody can be used in place of the anti-PD-1 antibody in any of the treatment methods described herein. In one embodiment, the anti-PD-L1 antibody is BMS-936559 (formerly 12A4 or MDX-1105) (see, e.g., U.S. Patent No. 7,943,743; WO2013 / 173223). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446) (see, e.g., 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 antibodies that cross-compete with the above PD-L1 antibodies for binding to human PD-L1 or bind to the same epitope region of human PD-L1 as the above PD-L1 antibodies are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, or can be humanized or human antibodies. Such chimeric, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0106] In some embodiments, the immune checkpoint inhibitor used in the present invention, e.g., an anti-PD-1 antagonist, is a PD-1 Fc fusion protein.

[0107] The anti-PD-1 antibody or antigen-binding portion thereof, or the anti-PD-L1 antibody or antigen-binding portion thereof of the invention may be administered in an amount of about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 3.0 mg / kg, or about 4.0 mg / kg. g, about 3.5 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg, about 5.5 mg / kg, about 6.5 mg / kg, about 7.5 mg / kg, about 8.0 mg / kg, about 8.5 mg / kg, about 9.0 mg / kg, about 9.5 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 20 mg / kg, or a dose greater than about 20 mg / kg (either the first dose or the second dose). In other embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, or the anti-PD-L1 antibody, or antigen-binding portion thereof, is administered at a dose of about 0.1 to about 20.0 mg / kg, about 0.1 to about 15.0 mg / kg, about 0.1 to about 10.0 mg / kg, about 0.1 to about 9.5 mg / kg, about 0.1 to about 9.0 mg / kg, about 0.1 to about 8.5 mg / kg, about 0.1 to about 8.0 mg / kg, about 0.1 to about 7.5 mg / kg, about 0.1 to about 7.0 mg / kg, about 0.1 to about 6.5 mg / kg, about 0.1 to about 6 ... The subject may be administered a dose selected from the group consisting of: about 0.1 to about 5.5 mg / kg, about 0.1 to about 5.0 mg / kg, about 0.1 to about 4.5 mg / kg, about 0.1 to about 4.0 mg / kg, about 0.1 to about 3.5 mg / kg, about 0.1 to about 3.0 mg / kg, about 0.3 to about 10.0 mg / kg, about 0.3 to about 9.0 mg / kg, about 0.3 to about 6.0 mg / kg, about 0.3 to about 3.0 mg / kg, about 3.0 to about 10.0 mg / kg, about 3.0 to about 9.0 mg / kg, or about 3.0 to about 6.0 mg / kg. In some embodiments, the subject is administered 0.3 mg / kg of an anti-PD-1 antibody, such as nivolumab, or an anti-PD-L1 antibody.In other embodiments, the subject is administered 2.0 mg / kg of an anti-PD-1 antibody, such as nivolumab, or an anti-PD-L1 antibody.In other embodiments, the subject is administered 10 mg / kg of an anti-PD-1 antibody, such as nivolumab, or an anti-PD-L1 antibody.

[0108] Anti-CTLA-4 Antibodies Useful in the Invention The anti-CTLA-4 antibodies of the invention bind to human CTLA-4 in a manner that blocks the interaction of CTLA-4 with the human B7 receptor. Because the interaction of CTLA-4 with B7 transmits a signal that leads to the inactivation of T cells that bear the CTLA-4 receptor, blocking the interaction effectively induces, enhances or prolongs the activation of such T cells and therefore induces, enhances or prolongs an immune response.

[0109] HuMAbs that specifically bind to CTLA-4 with high affinity are described in U.S. Patent Nos. 6,984,720 and 7,605,238. Other anti-CTLA-4 monoclonal antibodies are described, for example, in U.S. Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121. The anti-CTLA-4 HuMAbs described in U.S. Patent Nos. 6,984,720 and 7,605,238 have been demonstrated to exhibit one or more of the following characteristics: (a) a cytoplasmic affinity of at least about 10 as determined by Biacore analysis; 7 M -1 , about 10 9 M -1 or about 10 10 M -1 From 10 11 M -1 or higher equilibrium binding constant (K a (b) specifically binds human CTLA-4 with a binding affinity reflected by at least about 10 3 , about 10 4 , or about 10 5 m -1 s -1 The binding rate constant (k a );(c) at least about 10 3 , about 10 4 , or about 105 m -1 s -1 Dissociation rate constant (k d and (d) inhibit binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies that can be used in the present invention include monoclonal antibodies that specifically bind to human CTLA-4 and exhibit at least one, at least two, or, in one embodiment, at least three of the above properties. One example of a clinical anti-CTLA-4 antibody is human mAb 10D1 (now known as ipilimumab and commercially available as YERVOY®), described in U.S. Pat. No. 6,984,720. Ipilimumab is an anti-CTLA-4 antibody for use in the methods described herein. Another anti-CTLA-4 antibody that can be used in the methods of the present invention is tremelimumab.

[0110] One example of a clinical anti-CTLA-4 antibody is the human mAb 10D1 described in U.S. Patent No. 6,984,720 (now known as ipilimumab and commercially available as YERVOY®). Ipilimumab is an anti-CTLA-4 antibody for use in the methods described herein. Ipilimumab is a fully human IgG1 monoclonal antibody that inhibits the binding of CTLA-4 to its B7 ligand, thereby stimulating the activity of T cells and improving overall survival (OS) of patients with advanced melanoma.

[0111] Another anti-CTLA-4 antibody useful in the methods 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, US Patent Application Publication No. 2012 / 263677, or WO Publication No. 2007 / 113648A2.

[0112] Anti-CTLA-4 antibodies that can be used in the methods described herein 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 bind to the same epitopic region of human CTLA-4 as ipilimumab or tremelimumab. In some embodiments, the antibodies that cross-compete with ipilimumab or tremelimumab for binding to human CTLA-4 or bind to the same epitopic region of human CTLA-4 as ipilimumab or tremelimumab are antibodies that comprise a heavy chain of the human IgG1 isotype. For administration to a human subject, these cross-competing antibodies are chimeric, humanized or human antibodies. Anti-CTLA-4 antibodies that can be used also include antigen-binding portions of the above antibodies, such as Fab, F(ab")2, Fd or Fv fragments.

[0113] Ipilimumab (YERVOY®) is a fully human IgG1 monoclonal antibody that inhibits the binding of CTLA-4 to its B7 ligand, thereby stimulating the activity of T cells and improving overall survival (OS) in patients with advanced melanoma (Hodi et al. (2010) N Engl J Med 363:711-23). ​​Coadministration of nivolumab and ipilimumab in a phase 1 clinical trial resulted in rapid and deep tumor regression in a significant proportion of patients with advanced melanoma and was significantly more effective than treatment with either antibody alone (Wolchok et al. (2013) N Engl J Med 369(2):122-33; WO2013 / 173223). However, it was previously unknown whether this combination of immunomodulatory antibodies would be similarly effective in other tumor types.

[0114] Standard treatment for lung cancer Standard treatments for different types of cancer are well known to those skilled in the art. For example, the National Comprehensive Cancer Network (NCCN), an alliance of 21 major cancer centers in the United States, publishes the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®), which provide up-to-date information on the standard of care for various cancers (see NCCN Guidelines® (2014)). www.nccn.org / professionals / physician_gls / f_guidelines.asp Available at: http: / / www.nature.com / news2014 / 05 / 14 / last-accessed-14-May-2014).

[0115] NSCLC is the leading cause of cancer deaths in the United States and worldwide, including breast, colon, and prostate cancers combined. In the United States, 228,190 new cases of lung and bronchus will be diagnosed, and approximately 159,480 will die from the disease (Siegel et al. (2014) CA Cancer J Clin 64(1):9-29). The majority of patients (approximately 78%) will be diagnosed with progressive disease / recurrence or metastasis. Metastasis from lung cancer to the adrenal glands is common, with approximately 33% of all metastatic patients having such metastasis. NSCLC therapy has shown gradually improved OS, but the benefit has plateaued (the average OS for advanced stage patients is just 1 year). Progression occurs after first-line therapy (1L therapy) in almost all of these subjects, and the 5-year survival rate is only 3.6% in the refractory group. Between 2005 and 2009, the relative 5-year survival rate for lung cancer in the United States was 15.9% (NCCN Guidelines®, Version 3.2014 - Non-Small Cell Lung Cancer, www.nccn.org / professionals / physician_gls / pdf / nscl.pdf Available at: http: / / www.nature.com / news2014 / 05 / 14 / last-accessed-14-May-2014).

[0116] Surgery, radiation therapy (RT) and chemotherapy are three commonly used therapies to treat patients with NSCLC. As a class, NSCLC is relatively insensitive to chemotherapy and RT compared to small cell carcinoma. In general, for patients with stage I or II disease, surgical resection offers the best chance of cure, and there is an increasing use of chemotherapy both pre- and post-operatively. RT can also be used as adjuvant therapy for patients with resectable NSCLC, as a first-line local treatment, or as palliative therapy for patients with refractory NSCLC.

[0117] Patients with stage IV disease who have good performance status (PS) benefit from chemotherapy. Many agents are useful for stage IV NSCLC, including platinum agents (e.g., cisplatin, carboplatin), taxanes (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, and gemcitabine. Combinations using many of these agents result in 1-year survival rates of 30% to 40%, which are superior to single agents. Specific targeted therapies for the treatment of advanced lung cancer have also been developed. For example, bevacizumab (AVASTIN®) is a mAb that inhibits vascular endothelial growth factor A (VEGF-A). Erlotinib (TARCEVA®) is a small molecule TKI of the epidermal growth factor receptor (EGFR). Crizotinib (XALKORI®) is a small molecule TKI that targets ALK and MET and is used to treat NSCLC in patients carrying a mutated ALK fusion gene. Cetuximab (ERBITUX®) is a mAb that targets EGFR.

[0118] There is a particular unmet need for patients with squamous cell NSCLC (which accounts for up to 25% of all NSCLCs) due to the paucity of treatment options after first-line (1L) therapy. After progression on platinum-based combination therapy (Pt-doublet), single-agent chemotherapy is the standard of care, resulting in a median OS of approximately 7 months. Docetaxel remains the benchmark treatment for this therapy, but erlotinib is also available at a low frequency. Pemetrexed has also been shown to produce clinically comparable efficacy results and to have significantly fewer side effects compared to docetaxel in second-line (2L) therapy for patients with advanced NSCLC (Hanna et al. (2004) J Clin Oncol 22:1589-97). Currently, no treatments are approved for use in lung cancer beyond the third-line (3L) therapy. Pemetrexed and bevacizumab are not approved for squamous NSCLC, and targeted therapies have limited application. Oncothyreon and Merck KgaA's STIMUVAX® failed to improve OS in Phase 3 trial; ArQule and Daiichi Sankyo Co., Ltd. failure of Eli Lilly's ALIMTA® and Roche's AVASTIN® combination to improve OS in late-stage trials; and Amgen and Takeda Pharmaceutical Co.'s announcement that they are evaluating a small molecule VEGF-R inhibitor in late-stage trials. Antagonist The recent failure of motesanib, a new marker of efficacy and safety in patients with advanced lung cancer, to meet clinical endpoints creates an unmet need in advanced lung cancer.

[0119] Immunotherapy for Lung Cancer There is a clear need for effective agents for patients who have progressed on multiple lines of targeted therapy, as well as therapies that extend survival for longer periods beyond the current standard of care. Novel approaches involving immunotherapy, particularly the inhibition of immune checkpoints including the CTLA-4, PD-1 and PD-L1 inhibitory pathways, have recently shown promise (Creelan et al. (2014) Cancer Control 21(1):80-89). Thus, ipilimumab in combination with chemotherapy has shown promising results in both small cell and non-small cell lung cancer. In addition, dual checkpoint blockade strategies, such as those combining anti-PD-1 and anti-CTLA-4, have proven highly effective in the treatment of melanoma (Wolchok et al. (2013) N Engl J Med 369(2):122-33; WO 2013 / 173223), and other combinations including anti-PD-L1, anti-LAG-3, or anti-KIR are being tested to increase tumor response rates and durability. Similar to melanoma, NSCLC patients may benefit from either combinations of different immunotherapeutic agents or combinations of such agents with targeted agents or other therapies, including surgery, radiation, standard cancer chemotherapy, or vaccines. The combination of nivolumab and ipilimumab has proven highly effective in treating melanoma with manageable side effects (Wolchok et al. (2013) N Engl J Med 369(2):122-33), but it was not previously known whether this combination is significantly more effective in human subjects than the individual agents for the treatment of NSCLC and other cancers.

[0120] Pharmaceutical Compositions and Dosages The therapeutic agent of the present invention can be made into a composition, for example, a pharmaceutical composition comprising one or more antibodies and a pharma- ceutically acceptable carrier. As used herein, "pharma- ceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In one embodiment, the carrier for the composition comprising the antibody is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., administration by injection or infusion). The pharmaceutical composition of the present invention can include one or more pharma- ceutical acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

[0121] The present invention provides a dosage regimen that can provide a desired response, such as a maximum therapeutic response and / or minimal adverse effects. For administration of an anti-PD-1 antibody, particularly in combination with an anti-CTLA-4 antibody, the dosage can range from about 0.01 to about 20 mg, about 0.1 to about 10 mg, about 0.01 to about 5 mg, about 1 to about 5 mg, about 2 to about 5 mg, about 1 to about 3 mg, about 7.5 to about 12.5 mg, or about 0.1 to about 30 mg per kg of subject body weight. For example, the dosage can be about 0.1, about 0.3, about 1, about 2, about 3, about 4, about 5, or about 10 mg per kg of body weight. The dosing schedule is generally designed to achieve an exposure that results in sustained receptor occupancy (RO) based on the general pharmacokinetic properties of the antibody. An example of a treatment regimen involves administration about once every week, about once every two weeks, about once every three weeks, about once every four weeks, about once every month, about once every three to six months, or longer. In some embodiments, an anti-PD-1 antibody such as nivolumab is administered to a subject about once every two weeks. The anti-PD-1 antibody may be administered in at least two doses, each dose being in an amount of about 0.01 mg / kg to about 5 mg / kg, e.g., 3 mg / kg, with a dosing interval of two weeks between the two doses. In some embodiments, the anti-PD-1 antibody is administered in at least three, four, five, six, or seven doses (i.e., multiple doses), each dose being in an amount of about 0.01 mg / kg to about 5 mg / kg, e.g., 3 mg / kg, with a dosing interval of two weeks between two consecutive given doses. Dosage and dosing schedules may vary during the course of treatment. In one embodiment, the dosage regimen of the anti-PD-1 antibodies of the invention comprises about 0.3-1 mg, about 5 mg, 1-5 mg, or about 1 to about 3 mg per kg of body weight, administered intravenously, with the antibody administered about every 14-21 days in cycles of about 6 weeks or up to about 12 weeks until complete response or confirmed disease progression. In some embodiments, the antibody treatment or any combination therapy described herein is continued for at least about 1 month, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 18 months, at least about 24 months, at least about 3 years at least about 5 years, or at least about 10 years.

[0122] When used in combination with an anti-CTLA-4 antibody, the dosage of the anti-PD-1 antibody can be lower compared to the single-agent dosage. A typical dosage of nivolumab is subtherapeutic, lower than 3 mg / kg, but not less than 0.001 mg / kg. A subtherapeutic dose of the anti-PD-1 antibody used in the methods of the present invention is higher than 0.001 mg / kg and lower than 3 mg / kg. In some embodiments, the subtherapeutic dose is about 0.001 mg to about 1 mg, about 0.01 mg to about 1 mg, about 0.1 mg to about 1 mg, or about 0.001 mg to about 0.1 mg per kg of body weight. In some embodiments, the subtherapeutic dose is at least about 0.001 mg, at least about 0.005 mg, at least about 0.01 mg, at least about 0.05 mg, at least about 0.1 mg, at least about 0.5 mg, or at least about 1.0 mg per kg of body weight. Receptor occupancy data from 15 subjects receiving nivolumab at 0.3 mg / kg to 10 mg / kg indicate that PD-1 occupancy does not appear to be dose-dependent in this dose range. At all doses, the mean occupancy was 85% (range 70% to 97%) with a mean plateau occupancy of 72% (range 59% to 81%) (Brahmer et al. (2010) J Clin Oncol 28:3167-75). Thus, dosing at 0.3 mg / kg allows sufficient exposure to produce maximal biological activity.

[0123] In some embodiments of the invention, the anti-PD-1 antibody is administered at a dose of 3 mg / kg. In other embodiments of the invention, the anti-PD-1 antibody is administered at a dose of 1 mg / kg.

[0124] In some embodiments, the dose of the anti-PD-1 antibody (or anti-PD-L1 antibody) is a fixed dose in the pharmaceutical composition. In other embodiments, the methods of the present invention can be used with a fixed dose (a dose given to the patient regardless of the patient's weight). In some embodiments, the fixed dose of the anti-PD-1 antibody or antigen-binding portion thereof is at least about 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, 200 mg, 220 mg, 240 mg, 260 mg, 280 mg, 300 mg, 360 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, or 600 mg. For example, the fixed dose of nivolumab can be about 240 mg. For example, the fixed dose of pembrolizumab can be about 200 mg. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of about 240 mg. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of about 360 mg. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of about 480 mg. In some embodiments, a constant dose of the anti-PD-1 antibody or antigen-binding portion thereof is administered once every week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks. In one embodiment, 360 mg of the anti-PD-1 antibody or antigen-binding fragment thereof is administered once every three weeks. In another embodiment, 480 mg of the anti-PD-1 antibody or antigen-binding fragment thereof is administered once every four weeks.

[0125] Ipilimumab (YERVOY (登録商標)) is approved for the treatment of melanoma at 3 mg / kg intravenously, once every three weeks for four doses. Thus, while in some embodiments about 3 mg / kg is the maximum dose of ipilimumab used in combination with an anti-PD-1 antibody, in certain embodiments, an anti-CTLA-4 antibody such as ipilimumab, when combined with an anti-PD-1 antibody, can be administered at a dose within the range of about 0.3 to about 10 mg / kg, about 0.5 to about 10 mg / kg, about 0.5 to about 5 mg / kg, about 1 to about 5 mg / kg, about 1 to about 4 mg / kg, or about 1 to about 3 mg / kg of body weight about every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, or 20 weeks. In other embodiments, the anti-CTLA-4 antibody is administered on a different dosing schedule than the anti-PD-1 antibody. In some embodiments, ipilimumab is administered about every week, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, about every 9 weeks, about every 10 weeks, about every 11 weeks, about every 12 weeks, about every 13 weeks, about every 14 weeks, about every 15 weeks, or about every 20 weeks. The dose of ipilimumab is generally less than 3 mg / kg, but not less than 0.001 mg / kg, which is subtherapeutic. The subtherapeutic dose of anti-CTLA-4 antibody used in the method of the present invention is greater than 0.001 mg / kg and less than 3 mg / kg. In some embodiments, a sub-therapeutic dose is about 0.001 mg to about 1 mg, about 0.01 mg to about 1 mg, about 0.1 mg to about 1 mg, or about 0.001 mg to about 0.1 mg per kg of body weight. In some embodiments, a sub-therapeutic dose is at least about 0.001 mg, at least about 0.005 mg, at least about 0.01 mg, at least about 0.05 mg, at least about 0.1 mg, at least about 0.5 mg, or at least about 1.0 mg per kg of body weight.It has been shown that the combined administration of 3 mg / kg nivolumab and 3 mg / kg ipilimumab exceeds the maximum tolerated dose (MTD) in the melanoma population, while the combination of 1 mg / kg nivolumab + 3 mg / kg ipilimumab or 3 mg / kg nivolumab + 1 mg / kg ipilimumab was found to be tolerable in melanoma patients (Wolchok et al. (2013) N Engl J Med 369(2):122-33). Thus, nivolumab is tolerated up to 10 mg / kg administered intravenously every two weeks, but in some embodiments, the dose of the anti-PD-1 antibody does not exceed about 3 mg / kg when combined with an anti-CTLA-4 antibody. In some embodiments, the dose of the anti-CTLA-4 antibody is about 1 mg / kg.

[0126] In some embodiments, based on the risk / benefit assessment and PK-PD assessment, the dosages used include a combination of about 1 mg / kg nivolumab + about 1 mg / kg ipilimumab or about 3 mg / kg nivolumab + about 1 mg / kg ipilimumab, and in some embodiments, nivolumab is administered at a frequency of about once every 2 weeks. In some embodiments, ipilimumab is administered at a frequency of about once every 6 or 12 weeks. In certain other embodiments, nivolumab is administered at a dosage of about 1 mg / kg or about 3 mg / kg in combination with ipilimumab administered at a dosage of about 1 mg / kg, nivolumab is administered every 2 weeks, and ipilimumab is administered every 6 or 12 weeks. In certain other embodiments, nivolumab is administered at a dosage of 1 mg / kg every 2 weeks in combination with ipilimumab administered at a dosage of about 1 mg / kg every 6 weeks. In some embodiments, nivolumab is administered at a dose of 1 mg / kg every 2 weeks in combination with ipilimumab administered at a dose of about 1 mg / kg every 12 weeks. In some embodiments, nivolumab is administered at a dose of 3 mg / kg every 2 weeks in combination with ipilimumab administered at a dose of about 1 mg / kg every 6 weeks. In other embodiments, nivolumab is administered at a dose of 3 mg / kg every 2 weeks in combination with ipilimumab administered at a dose of about 1 mg / kg every 12 weeks.

[0127] In some embodiments, the anti-PD-1 antibody and the anti-CTLA-4 antibody are formulated as a single composition, where the dose of the anti-PD-1 antibody and the dose of the anti-CTLA-4 antibody are combined in a fixed dose at a ratio of 1:50, 1:40, 1:30, 1:20, 1:10, 1:5, 1:3, 1:1, 3:1, 5:1, 10:1, 20:1, 30:1, 40:1, or 50:1. In some embodiments, the dose of the anti-CTLA-4 antibody is a fixed dose given to the patient regardless of body weight. In some embodiments, the fixed dose of the anti-CTLA-4 antibody is at least about 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, or 200 mg. In certain embodiments, the fixed dose of the anti-CTLA-4 antibody is about 80 mg.

[0128] Dosage and frequency vary depending on the half-life of the antibody in the subject. Generally, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. Dosage and frequency vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are generally administered over a long period of time with long intervals. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages are often required with relatively short intervals until disease progression slows or stops, or until the patient shows partial or complete improvement of disease symptoms. The patient can then be administered a prophylactic regimen.

[0129] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied so as to obtain an amount of the active ingredient that is not excessively toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration.The selected dosage level can vary depending on 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 previous medical history of the patient being treated, and similar factors well known in the medical field.The composition of the present invention can be administered by one or more routes of administration using one or more of a variety of methods well known in the art.As can be understood by those skilled in the art, the route and / or mode of administration can vary depending on the desired result.

[0130] kit Also within the scope of the present invention are kits comprising an anti-PD-1 antibody and another anti-cancer agent for therapeutic use. The kit typically includes a label and instructions indicating the intended use of the contents of the kit. The term label includes any written or recorded material that accompanies or is included with the kit or is otherwise contained in the kit. Thus, the present invention provides a kit for treating a subject suffering from lung cancer, comprising: (a) a PD-1 antibody or antigen-binding portion thereof in an amount ranging from about 4 mg to about 500 mg; (b) a CTLA-4 antibody or antigen-binding portion thereof in an amount ranging from about 40 mg to about 500 mg; and (c) instructions for use of the PD-1 antibody or antigen-binding portion thereof and the CTLA-4 antibody or antigen-binding portion thereof in the methods described herein. In some embodiments, the kit comprises the PD-1 antibody or antigen-binding portion thereof and the CTLA-4 antibody or antigen-binding portion thereof as separate compositions. In some embodiments, the kit comprises the PD-1 antibody or antigen-binding portion thereof and the CTLA-4 antibody or antigen-binding portion thereof as a single composition. In some embodiments, the anti-PD-1 antibody and the anti-CTLA-4 antibody can be co-packaged in a unit dosage form. In certain embodiments for treating human patients, the kit comprises an anti-human PD-1 antibody as described herein, such as nivolumab or pembrolizumab. In other embodiments, the kit comprises an anti-human CTLA-4 antibody as described herein, such as ipilimumab or tremelimumab.

[0131] This invention is further illustrated by the following examples which should not be construed as further limiting. All references throughout this specification are expressly incorporated herein by reference. EXAMPLES

[0132] Example 1 Treatment of NSCLC with Nivolumab and Ipilimumab Study design Four different arms were created to test doses and dosing schedules of nivolumab ("nivo") and ipilimumab ("ipi"). These arms were designed to test different dosing schedules than the initial study of the nivo+ipi combination. A summary of prior combination results is shown in Table 1. NR = not reached. [Table 1]

[0133] Nivo 3mg / kg Q3W data is based on database lock March 2015. Nivo 1mg / kg+Ipi 3mg / kg Q3W and Nivo 3mg / kg+Ipi 1mg / kg Q3W data is based on database lock September 2015. mDOR (median duration of response) estimates are the time from first response to documented progression, death within 100 days of last nivolumab dose, or last tumor assessment (if censored + data).

[0134] Four additional groups were tested: 1 mg / kg ipi and 1 mg / kg nivo q3w; 1 mg / kg nivo q2w and 1 mg / kg ipi q6w; 3 mg / kg nivo q2w and 1 mg / kg ipi q12w; 3 mg / kg nivo q2w and 1 mg / kg ipi q6w. Patient characteristics, mutations, and baseline disease are shown in Table 2. [Table 2] Some numbers may not add up to 100% due to rounding.PD-L1 expression rates are based on patients with known PD-L1 symptoms (nivo 1+ipi 1 Q3W, n=25; nivo 1 Q2W+ipi 1 Q6W, n=28; nivo 3 Q2W+ipi 1 Q12W, n=31; nivo 3 Q2W+ipi Q6W, n=30).

[0135] The most common treatment-related adverse events (AEs) are shown in Table 3. At the time of analysis, 47% to 81% of patients (across study arms) had discontinued the study, most commonly due to disease progression. [Table 3] NC = not measured. Treatment-related adverse events were less frequent and less severe with the new dosing schedule compared with the initial nivolumab + ipilimumab cohort (see Table 1). The new dosing schedule was associated with less frequent treatment-related adverse events leading to discontinuation compared with the initial nivolumab + ipilimumab cohort (Table 3). Discontinuation rates were similar to nivolumab monotherapy (see Table 1). There were no treatment-related deaths.

[0136] Selected adverse events related to treatment can be found in Table 4. Selected adverse events are those likely to have an immunological causation requiring frequent monitoring / intervention. The most common categories of treatment-related selected adverse events (all grades > 20%) across groups were dermatological, endocrine, gastrointestinal and hepatic. [Table 4]

[0137] A summary of response rates, progression-free survival and overall survival is shown in Table 5. Confirmed overall response rates (ORR) ranged from 13% to 39% across all groups, with 21% to 42% of patients achieving stable disease. Two patients in the nivolumab 3 mg / kg Q2W + ipilimumab 1 mg / kg Q12W group and one patient in the nivolumab 3 mg / kg Q2W + ipilimumab 1 mg / kg Q6W group demonstrated unconventional immune responses, including a 42%, 47% and 44% maximum reduction in target lesions after disease progression or simultaneous appearance of new lesions. Median DOR (duration of response) was not achieved in any group. Reduction in tumor burden was observed across all groups (Figures 2A-2D). Surprisingly, nivolumab 3mg / kg Q2W + ipilimumab 1mg / kg Q12W and nivolumab 3mg / kg Q2W + ipilimumab 1mg / kg Q6W showed a significant increase in patients with complete responses as well as extended progression-free survival compared with nivolumab 1mg / kg Q2W + ipilimumab 1mg / kg Q6W. [Table 5]

[0138] Duration of response for nivo+ipi in first-line NSCLC can be found in Table 4. Twelve of 15 (80%) responders in the Q6W group and 14 of 18 (77%) responders in the Q12W group had documented responses by the time of first scan at week 11 + / - 5 days. Twelve of 15 (80%) and 12 of 18 (67%) responders in the Q6W and Q12W groups, respectively, had ongoing responses at the time of database lock.

[0139] Efficacy of treatment according to baseline tumor PD-L1 expression can be found in Table 6. Tumor PD-L1 expression was assessed in pretreatment (archived or fresh) tumor samples using an automated Bristol-Myers Squibb / Dako immunohistochemistry assay and evaluated for ORR and PFS. See Phillips, T. et al. Appl. Immunohistochem Mol Morphol 23: 541-549 (2015). All patients had pretreatment tumor samples available, and 76% (113 / 148) had samples evaluable for PD-L1 expression. In addition, clinical activity was observed regardless of tumor PD-L1 expression (Figure 3), although there is preliminary evidence of greater activity in ≥1% of tumors expressing PD-L1. 85% (35 / 41) of confirmed CRs were ongoing at the time of analysis. Median DOR was not achieved in any group, regardless of tumor PD-L1 expression. Further evaluation can be found in Table 7, and the effect of nivo+ipi on PD-L1 expression levels can be found in Figure 5. [Table 6] [Table 7]

[0140] Median PFS and overall response rate (ORR) were higher in current and former smokers. See Table 8 and Figure 6A. Responses were observed in patients with non-squamous NSCLC regardless of EGFR mutation status. See Table 9 and Figure 6B. These data indicate that clinical activity was observed regardless of smoking status or EGFR mutation status. [Table 8] [Table 9] NR is due to a high rate of ongoing response or insufficient number of events and / or follow-up (not reached). The symbol + indicates censored values. These values ​​include patients with non-squamous histology treated with nivo 1mg / kg Q2W + ipi 1mg / kg Q6W, nivo 3mg / kg Q2W + ipi 1mg / kg Q12W or nivo 3mg / kg Q2W + ipi 1mg / kg Q6W. DCR includes patients with confirmed complete response (CR), partial response (PR) and stable disease (SD). The symbol + indicates censored values.

[0141] conclusion Earlier studies with nivolumab at 1 mg / kg and ipilimumab at 3 mg / kg or nivolumab at 3 mg / kg and ipilimumab at 1 mg / kg every 3 weeks showed clinical activity, but the dosing schedule was associated with toxicity. In this study, the new dosing schedule showed unexpected synergistic activity and increased clinical activity, as well as a tolerable safety profile. First-line therapy with nivolumab + ipilimumab shows high levels of clinical activity characterized by deep and durable responses in patients with advanced NSCLC. Furthermore, nivolumab + ipilimumab combination treatment is associated with a favorable safety profile. The frequency of treatment-related grade 3 to 4 adverse events leading to discontinuation was low, and there were no treatment-related deaths.

[0142] Example 2 A case of pathological CR in one patient receiving Nivo 3 Q2W + Ipi 1 Q6W A 54-year-old male (former smoker, smoking index 52 pack-years) with metastatic large cell lung cancer (PD-L1<1%; patient was included as having partial response and unknown PD-L1 expression at the time of analysis at database lock) was treated with Nivo 3 Q2W + Ipi 1 Q6W. The patient had a 53% tumor size reduction by RECIST and radiological residual disease in the lung and mediastinal lymph nodes without distant metastases. See Figures 7A-7D.

[0143] Aspects E1. A method of treating a subject suffering from lung cancer, comprising administering to the subject a combination of: (a) an antibody or antigen-binding portion thereof that specifically binds to the programmed cell death-1 (PD-1) receptor and inhibits PD-1 activity, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose ranging from about 0.1 to about 5.0 mg / kg body weight about once every two weeks; and (b) an antibody or antigen-binding portion thereof that specifically binds to cytotoxic T-lymphocyte antigen-4 (CTLA-4) and inhibits CTLA-4 activity, wherein the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose ranging from about 1 to about 5.0 mg / kg body weight about once every 6 to 12 weeks. method.

[0144] E2. The method of E1, wherein the lung cancer is non-small cell lung cancer (NSCLC).

[0145] E3. The method of E2, wherein the NSCLC has squamous histology.

[0146] E4. The method of E2, wherein the NSCLC has non-squamous histology.

[0147] E5. The method of any of E1-4, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, cross-competes with nivolumab for binding to human PD-1.

[0148] E6. The method of any one of E1-5, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is a chimeric antibody, a humanized antibody, or a human monoclonal antibody, or a portion thereof.

[0149] E7. The method of any one of E1-E6, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, comprises a heavy chain constant region of a human IgG1 or IgG4 isotype.

[0150] E8. The method of any one of E1-E7, wherein the anti-PD-1 antibody is nivolumab.

[0151] E9. The method of any one of E1-E7, wherein the anti-PD-1 antibody is pembrolizumab.

[0152] E10. The method of any one of E1-E9, wherein the anti-CTLA-4 antibody, or antigen-binding portion thereof, is a chimeric antibody, a humanized antibody, or a human monoclonal antibody, or a portion thereof.

[0153] E11. The method of any one of E1-E10, wherein the anti-CTLA-4 antibody, or antigen-binding portion thereof, comprises a heavy chain constant region of the human IgG1 isotype.

[0154] E12. The method of any one of E1-E11, wherein the anti-CTLA-4 antibody is ipilimumab.

[0155] E13. The method of any one of E1-E11, wherein the anti-CTLA-4 antibody is tremelimumab.

[0156] E14. The method of any one of E1-E13, wherein the anti-CTLA-4 antibody, or antigen-binding portion thereof, cross-competes with ipilimumab for binding to human CTLA-4.

[0157] E15. The method of any one of E1-14, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose of about 1 mg / kg or about 3 mg / kg of body weight about once every two weeks.

[0158] E16. The method of any one of E1-E15, wherein the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of about 1 mg per kg of body weight.

[0159] E17. The method of any one of E1-E16, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose of about 3 mg / kg body weight once about every 2 weeks, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered at a dose of about 1 mg / kg body weight once about every 12 weeks.

[0160] E18. The method of any one of E1-E16, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose of about 1 mg / kg of body weight about once every two weeks, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered at a dose of about 1 mg / kg of body weight about once every six weeks.

[0161] E19. The method of any one of E1-E18, wherein the subject exhibits a progression free survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the first administration.

[0162] E20. The method of E19, wherein the subject exhibits a progression free survival of at least about 18 months after the first administration.

[0163] E21. The method of any one of E1-E20, wherein when a subject is treated with an anti-PD-1 antibody, or antigen-binding portion thereof, at a dose of about 3 mg / kg body weight once about every two weeks, and an anti-CTLA-4 antibody, or antigen-binding portion thereof, at a dose of about 1 mg / kg body weight once about every 12 weeks ("Regimen A"), the subject has a longer progression-free survival than when the subject is treated with an anti-PD-1 antibody, or antigen-binding portion thereof, at a dose of 1 mg / kg body weight once every two weeks, and an anti-CTLA-4 antibody, or antigen-binding portion thereof, at a dose of 1 mg / kg body weight once every six weeks ("Regimen B").

[0164] E22. The method of E21, wherein the progression free survival of subjects treated with regimen A is at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months longer than the progression free survival of subjects treated with regimen B.

[0165] The method of E21 or E22, wherein the progression free survival of subjects treated with regimen A is at least about 3 months longer than the progression free survival of subjects treated with no regimen B.

[0166] E24. The method of any one of E1-E22, wherein when a subject is treated with an anti-PD-1 antibody, or antigen-binding portion thereof, at a dose of 3 mg / kg body weight once every two weeks, and an anti-CTLA-4 antibody, or antigen-binding portion thereof, at a dose of 1 mg / kg body weight once every 12 weeks ("Regimen A"), the subject has a longer progression-free survival than when the subject is treated with an anti-PD-1 antibody, or antigen-binding portion thereof, at a dose of 3 mg / kg body weight once every two weeks ("Regimen C").

[0167] E25. The method of E23, wherein the progression free survival of subjects treated with regimen A is at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks longer than the progression free survival of subjects treated with regimen C.

[0168] E26. The method of E24 or E25, wherein the progression free survival of subjects treated with regimen A is at least about 3 months longer than the progression free survival of subjects treated with regimen C.

[0169] E27. The method of any one of E1-E26, wherein the subject has a lung cancer with PD-L1 expression greater than or equal to 1%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 25%, or greater than or equal to 50%.

[0170] E28. The method of any one of E1-E27, wherein said combination composition is administered for as long as clinical benefit is observed or until disease progression or unmanageable toxicity occurs.

[0171] E29. The method of any one of E1-E28, wherein the anti-PD-1 and anti-CTLA-4 antibodies are formulated for intravenous administration.

[0172] E30. The method of any one of E1-29, wherein an anti-PD-1 antibody, or antigen-binding portion thereof, and an anti-CTLA-4 antibody, or antigen-binding portion thereof, are administered sequentially to the subject.

[0173] E31. The method of any one of E1-30, wherein the anti-PD-1 and anti-CTLA-4 antibodies are administered within 30 minutes of each other.

[0174] E32. The method of any one of E1-E31, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered prior to the anti-CTLA-4 antibody, or antigen-binding portion thereof.

[0175] E33. The method of any one of E1-E31, wherein the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered prior to the anti-PD-1 antibody, or antigen-binding portion thereof.

[0176] E34. The method of any one of E1-E29, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are administered simultaneously as separate compositions.

[0177] E35. The method of any one of E1-E29, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are administered simultaneously as a single composition.

[0178] E36. The method of any one of E1-E35, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered in a sub-therapeutic dose.

[0179] E37. The method of any one of E1-36, wherein the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered in a sub-therapeutic dose.

[0180] E38. The method of any one of E1-E37, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are each administered in a sub-therapeutic dose.

[0181] E39. A kit for the treatment of a subject suffering from lung cancer, comprising: (a) an amount of a PD-1 antibody or antigen-binding portion thereof ranging from about 4 mg to about 500 mg; (b) a CTLA-4 antibody or antigen-binding portion thereof in an amount ranging from about 40 mg to about 500 mg; and (c) Instructions for using a PD-1 antibody, or antigen-binding portion thereof, and a CTLA-4 antibody, or antigen-binding portion thereof, in any of the methods of E1-E38. Kit including:

Claims

1. 1. A composition comprising, in combination, an antibody or antigen-binding portion thereof that specifically binds to a programmed cell death-1 (PD-1) receptor and inhibits PD-1 activity (an anti-PD-1 antibody or antigen-binding portion thereof) and an antibody or antigen-binding portion thereof that specifically binds to a cytotoxic T-lymphocyte antigen-4 (CTLA-4) and inhibits CTLA-4 activity (an anti-CTLA-4 antibody or antigen-binding portion thereof) for use in treating a subject suffering from lung cancer, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose ranging from about 0.1 to 5.0 mg / kg of body weight about once every two weeks; and The composition, wherein the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose ranging from about 1 to 5.0 mg per kg of body weight approximately once every 6 to 12 weeks.

2. The composition of claim 1 , wherein the lung cancer is non-small cell lung cancer (NSCLC).

3. The composition of claim 1 or 2, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, cross-competes with nivolumab for binding to human PD-1.

4. The composition of any one of claims 1 to 3, wherein the anti-CTLA-4 antibody, or antigen-binding portion thereof, cross-competes with ipilimumab for binding to human CTLA-4.

5. (i) whether the anti-PD-1 antibody is nivolumab; (ii) the anti-CTLA-4 antibody is ipilimumab; or (iii) The composition of any one of claims 1 to 4, wherein the anti-PD-1 antibody is nivolumab and the anti-CTLA-4 antibody is ipilimumab.

6. 6. The composition of any one of claims 1 to 5, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose of about 1 mg or about 3 mg per kg of body weight about once every two weeks.

7. The composition of any one of claims 1 to 6, wherein the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of about 1 mg per kg of body weight.

8. 8. The composition of any one of claims 1 to 7, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose of about 3 mg / kg body weight about once every two weeks, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered at a dose of about 1 mg / kg body weight about once every 12 weeks.

9. 8. The composition of any one of claims 1 to 7, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered at a dose of about 1 mg / kg body weight about once every two weeks, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered at a dose of about 1 mg / kg body weight about once every six weeks.

10. The composition of any one of claims 1 to 9, wherein the subject has a lung cancer with PD-L1 expression rate of 1% or more, 5% or more, 10% or more, 25% or more, or 50% or more.

11. 11. The composition of any one of claims 1 to 10, administered for as long as clinical benefit is observed or until disease progression or unmanageable toxicity occurs.

12. The composition of any one of claims 1 to 11, wherein the anti-PD-1 antibody and the anti-CTLA-4 antibody are formulated for intravenous administration.

13. The composition of any one of claims 1 to 12, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are administered sequentially to the subject.

14. The composition of any one of claims 1 to 12, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are administered simultaneously as separate compositions or are administered simultaneously as a single composition.

15. (i) the anti-PD-1 antibody, or antigen-binding portion thereof, is administered in a sub-therapeutic dose; (ii) the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered in a sub-therapeutic dose; or (iii) the anti-PD-1 antibody, or antigen-binding portion thereof, and the anti-CTLA-4 antibody, or antigen-binding portion thereof, are each administered at a sub-therapeutic dose; 15. The composition according to any one of claims 1 to 14.

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