Methods of treating cancer with anti-PD-1 and anti-CTLA-4 antibodies

Abstract

The present invention relates to a method for treating cancer in a patient comprising administering to the patient a specific amount of an anti-PD-1 antibody or antigen-binding fragment thereof per about six weeks, in combination with administering to the patient an anti-CTLA4 antibody per about six weeks. In certain embodiments, the PD-1 antagonist is pembromab or an antigen binding fragment thereof. Also provided are compositions comprising a dose of an anti-PD-1 antibody or antigen-binding fragment thereof and a dose of an anti-CTLA4 antibody or antigen-binding fragment thereof, and uses thereof for the treatment of cancer.

Description

[0001] This application is a divisional application of a patent application with an application date of February 8, 2019, application number 201980013384.1, and name “Method for treating cancer with anti-PD-1 antibodies and anti-CTLA-4 antibodies”. Technical Field

[0002] The present invention relates to therapies useful for treating cancer. In particular, the present invention relates to methods for treating cancer comprising administering a combination of an anti-PD-1 antibody or antigen-binding fragment thereof and an anti-CTLA4 antibody or antigen-binding fragment thereof to a patient in need thereof using a dosage regimen as specified herein.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 62 / 630,038, filed on February 13, 2018, U.S. Provisional Application No. 62 / 732,838, filed on September 18, 2018, and U.S. Provisional Application No. 62 / 740,741, filed on October 3, 2018, the contents of each of which are incorporated herein by reference in their entirety.

[0005] References to electronically submitted sequence listings

[0006] The sequence listing of this application is electronically submitted via EFS-Web as a sequence listing in ASCII format, with a file name of "24695WOPCT-SEQLIST-06FEB2019.TXT", a creation date of February 6, 2019, and a size of 56.0 kb. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background of the Invention

[0008] PD-1 is considered an important player in immune regulation and peripheral tolerance maintenance. PD-1 is moderately expressed on naive T, B and NKT cells and is upregulated by T / B cell receptor signaling on lymphocytes, monocytes and myeloid cells (Sharpe et al., The function of programmed cell death 1 and its ligands inregulating autoimmunity and infection. Nature Immunology (2007); 8: 239-245).

[0009] Two known ligands of PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), are expressed in human cancers arising from a wide variety of tissues. In large sample sets such as ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, and melanoma, PD-L1 expression has been shown to be associated with poor prognosis and reduced overall survival, regardless of subsequent treatment (Dong et al., Nat Med. 8(8):793-800 (2002); Yang et al. Invest Ophthalmol Vis Sci. 49:2518-2525 (2008); Ghebeh et al. Neoplasia 8:190-198 (2006); Hamanishi et al., Proc. Natl. Acad. Sci. USA 104:3360-3365 (2007); Thompson et al., Cancer 5:206-211 (2006); Nomi et al., Clin. Cancer Research 13:2151-2157 (2007); Ohigashi et al., Proc. Natl. Acad. Sci. USA 104:3360-3365 (2007); Thompson et al., Cancer 5:206-211 (2006); Nomi et al., Clin. Cancer Research 13:2151-2157 (2007); Ohigashi et al., Proc. Natl. Acad. Sci. USA 104:3360-3365 (2007); et al., Clin. Cancer Research 11: 2947-2953 (2005); Inman et al., Cancer 109: 1499-1505 (2007); Shimauchi et al. Int. J. Cancer 121: 2585-2590 (2007); Gao et al. Clin. Cancer Research 15:971-979 (2009); Nakanishi J. Cancer Immunol Immunother. 56:1173-1182 (2007); and Hino et al., Cancer 00:1-9 (2010)).

[0010] Similarly, PD-1 expression on tumor-infiltrating lymphocytes was found to mark dysfunctional T cells in breast cancer and melanoma (Ghebeh et al, BMC Cancer. 2008 8: 5714-15 (2008); Ahmadzadeh et al., Blood 114: 1537-1544 (2009)), and was associated with poor prognosis in renal cancer (Thompson et al., Clinical Cancer Research 15: 1757-1761 (2007)). Therefore, it has been proposed that tumor cells expressing PD-L1 interact with T cells expressing PD-1 to attenuate T cell activation and escape immune surveillance, thereby contributing to an impaired immune response against the tumor.

[0011] Immune checkpoint therapy targeting the PD-1 axis has led to breakthrough improvements in clinical responses in a variety of human cancers (Brahmer et al., N Engl J Med 2012, 366:2455-65; Garon et al. N Engl J Med 2015, 372:2018-28; Hamid et al., N Engl J Med 2013, 369:134-44; Robert et al., Lancet 2014, 384:1109-17; Robert et al., N Engl J Med 2015, 372:2521-32; Robert et al., N Engl J Med 2015, 372:320-30; Topalian et al., N Engl J Med 2012, 366:2443-54; Topalian et al., J Clin Oncol ... 2014, 32: 1020-30; Wolchok et al., N Engl J Med 2013, 369: 122-33). Immunotherapies targeting the PD-1 axis include monoclonal antibodies directed to the PD-1 receptor (KEYTRUDA TM (pembrolizumab), Merck and Co., Inc., Kenilworth, NJ, USA, and OPDIVO TM (nivolumab), Bristol-Myers Squibb Company, Princeton, NJ, USA), and those that bind to PD-L1 ligands (MPDL3280A; TECENTRIQ TM (atezolizumab), Genentech, San Francisco, CA, USA; IMFINZI TM (durvalumab), AstraZeneca Pharmaceuticals LP, Wilmington, DE; BAVENCIO TM (avelumab, Merck KGaA, Darmstadt, Germany). Both treatments have demonstrated antitumor effects in a wide range of cancer types.

[0012] It has been proposed that the efficacy of such antibodies may be enhanced if administered in combination with other approved or experimental cancer therapies, such as radiation, surgery, chemotherapeutic agents, targeted therapies, agents that inhibit other signaling pathways that are dysregulated in tumors, and other immune enhancers. One such agent that has been tested in combination with an antagonist of PD-1 is an antagonist of cytotoxic T lymphocyte-associated antigen 4 (abbreviated CTLA4).

[0013] CTLA4 has a very close relationship with CD28 molecules in terms of gene structure, chromosomal location, sequence homology and gene expression. Both are receptors for the co-stimulatory molecule B7 and are mainly expressed on the surface of activated T cells. After binding to B7, CTLA4 can inhibit the activation of mouse and human T cells and play a negative regulatory role in the activation of T cells.

[0014] CTLA4 mAb or CTLA4 ligand can prevent CTLA4 from binding to its natural ligand, thereby blocking CTLA4 transduction of T cell negative regulatory signals and enhancing the responsiveness of T cells to various antigens. In this regard, the results of in vivo and in vitro studies are basically the same. At present, some CTLA4 mAbs are being tested in clinical trials for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso et al., CTLA-4blockade in tumor models: an overview of preclinical and translational research. Cancer Immun. 13: 5 (2013)).

[0015] As an important factor affecting the function of T cells, CTLA4 and CTLA4 mAb can produce specific therapeutic effects on diseases by interfering with the immune microenvironment in the body. They have high efficacy and make up for the shortcomings of traditional drugs, opening up new avenues for gene therapy. CTLA4 and CTLA4 mAb are being tested in experiments and at various stages of clinical trials. For example, in autoimmune diseases, they have been shown to effectively inhibit airway hyperresponsiveness in asthma animal models, prevent the development of rheumatic diseases, mediate immune tolerance to allogeneic transplants in vivo, and so on. On the other hand, although biological gene therapy has not shown any adverse reactions in short-term clinical trials, attention should be paid to its potential effects after long-term application. For example, excessive blocking of CTLA4-B7 signaling by CTLA4 mAb can lead to the development of autoimmune diseases. Since antibodies can specifically bind to their antigens and induce lysis of target cells or block the progression of pathology, the development and utilization of drugs based on antibodies, especially humanized antibodies, are of great significance in the clinical treatment of human malignancies and other immune diseases.

[0016] It would be beneficial to develop additional dosing regimens that are more convenient for patients and allow safe and effective doses of anti-PD-1 antibodies to be administered alone or in combination with anti-CTLA4 antibodies. It would be beneficial to develop methods for treating PD-1 / PD-L1 refractory cancers by administering anti-PD-1 antibodies and anti-CTLA4 antibodies using the dosing regimens provided herein. Summary of the invention

[0017] The present invention provides an alternative, less frequent dosing regimen for treating cancer patients with an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the dosing regimen is expected to provide a safe and effective dose of the anti-PD-1 antibody or an antigen-binding fragment thereof. It also provides an alternative, less frequent dosing regimen for treating cancer patients with an anti-PD-1 antibody or an antigen-binding fragment thereof in combination with an anti-CTLA4 antibody or an antigen-binding fragment thereof. Specifically, the present invention provides a method for treating cancer in a human patient, comprising administering to the patient about 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof every six weeks, wherein the anti-PD-1 antibody or an antigen-binding fragment thereof comprises: (a) a light chain complementary determining region (CDR) comprising the amino acid sequence shown in SEQ ID NOs: 1, 2, and 3; and a heavy chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 6, 7, and 8; or (b) a light chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 11, 12, and 13; and a heavy chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 14, 15, and 16. In a preferred embodiment of the present invention, the antibody or antigen-binding fragment is pembrolizumab.

[0018] Also provided is a method of treating cancer in a human patient, comprising administering to the patient about 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof and about 25 mg, 50 mg, 75 mg or 100 mg of an anti-CTLA4 antibody or an antigen-binding fragment thereof, wherein the anti-PD-1 antibody and the anti-CTLA4 antibody or an antigen-binding fragment thereof are each administered to the patient every six weeks, wherein the anti-PD-1 antibody or an antigen-binding fragment thereof comprises: (a) a light chain complementary determining region (CDR) comprising the amino acid sequence of SEQ ID NOs: 1, 2 and 3 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 6, 7 and 8; (b) a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 11, 12 and 13; and a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 14, 15 and 16; and wherein the anti-CTLA4 antibody or an antigen-binding fragment thereof comprises a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 39, 40 and 41 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: The heavy chain CDR of the amino acid sequence shown in NO:36, 37 and 38.

[0019] In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment comprises a light chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 1, 2 and 3, and a heavy chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 6, 7 and 8; and the anti-CTLA4 antibody or its antigen-binding fragment comprises a light chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 39, 40 and 41, and a heavy chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 36, 37 and 38.

[0020] In another embodiment, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:4; and the anti-CTLA4 antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:50 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:49.

[0021] In all of the above embodiments, the amount of the anti-PD-1 antibody or antigen-binding fragment thereof administered to the patient is about 350 mg to about 450 mg. In further embodiments, the amount of the anti-PD-1 antibody or antigen-binding fragment thereof is about 400 mg. In further embodiments, the amount of the anti-PD-1 antibody or antigen-binding fragment thereof is 400 mg.

[0022] In all the above-mentioned treatment methods, compositions and uses herein, the PD-1 antibody or antigen-binding fragment inhibits the binding of PD-L1 to PD-1, and preferably also inhibits the binding of PD-L2 to PD-1. In some preferred embodiments of the treatment methods, compositions and uses of the present invention, the PD-1 antibody or antigen-binding fragment is a monoclonal antibody that specifically binds to PD-1 and blocks the binding of PD-L1 to PD-1. In a specific embodiment, the anti-PD-1 antibody comprises a heavy chain and a light chain, and wherein the heavy chain and the light chain comprise Figure 1 In another embodiment, the anti-PD-1 antibody comprises a heavy chain and a light chain, and wherein the heavy chain and the light chain comprise Figure 1 The anti-CTLA4 antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain comprise the amino acid sequences shown in SEQ ID NOs: 57 and 58.

[0023] In some preferred embodiments of the methods of treatment, compositions and uses of the present invention, the anti-CTLA4 antibody or antigen-binding fragment is a monoclonal antibody that specifically binds to CTLA4. In one embodiment, the anti-CTLA4 antibody or antigen-binding fragment thereof comprises a light chain CDR comprising the amino acid sequences shown in SEQ ID NOs: 39, 40 and 41; and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NOs: 36, 37 and 38. In a specific embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain and light chain variable regions comprise the amino acid sequences shown in SEQ ID NOs: 50 and 49, respectively. In another embodiment, the anti-CTLA antibody is a monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 58.

[0024] In some embodiments of any of the above methods of treatment, compositions, and uses, the cancer expresses one or both of PD-L1 and PD-L2. In some embodiments, PD-L1 expression is elevated in the cancer. In further embodiments, the cancer is PD-1 / PD-L1 refractory (e.g., a cancer that has not responded to prior treatment with an anti-PD-1 or anti-PD-L1 agent). In further embodiments, the cancer is PD-1 / PD-L1 refractory melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The amino acid sequences of the light and heavy chains of exemplary anti-PD-1 monoclonal antibodies useful in the present invention are shown (SEQ ID NOs: 5 and 10, respectively). The light and heavy chain variable regions are underlined (SEQ ID NOs: 4 and 9), while the CDRs are bold and boxed.

[0026] Figure 2 The pembrolizumab Cmax at steady state for 400 mg Q6W was shown to be within the range of 2 mg / kg Q3W and 200 mg Q3W to 10 mg / kg Q2W.

[0027] Figure 3 It was shown that pembrolizumab exposure (Cavg and Cmin) at steady-state was similar for 400 mg Q6W relative to 2 mg / kg Q3W and 200 mg Q3W.

[0028] Figure 4A and 4BThe pharmacokinetic profiles of pembrolizumab at steady state are shown for the 400 mg Q6W dosing regimen compared to the 200 mg flat dosing regimen (top) and the Q3W, 2 mg / kg body weight based dosing regimen (bottom). The results are provided as logarithmic scale concentration ( Figure 4A ) and linear scale concentration ( Figure 4B ). DETAILED DESCRIPTION

[0029] I. Definitions and Abbreviations

[0030] As used throughout the specification and appended claims, the following abbreviations apply:

[0031] AE adverse event

[0032] AUCss Area under the concentration-time curve at steady state

[0033] BICR Blindness Covering Independent Center Review

[0034] Cavg,ss Time-averaged concentration at steady state

[0035] CDR complementarity determining region

[0036] CI confidence interval

[0037] Cmax,ss Peak concentration at steady state

[0038] Cmin,ss Trough concentration at steady state

[0039] CPS Composite Positive Score

[0040] CTLA4 Cytotoxic T lymphocyte-associated antigen 4

[0041] DOR Response Duration

[0042] ECG Electrocardiogram

[0043] ECOG Eastern Cooperative Oncology Group

[0044] ER exposure (concentration) response

[0045] FFPE Formalin fixed paraffin embedded

[0046] FR Framework Region

[0047] GM Geometric mean

[0048] HCC Hepatocellular carcinoma

[0049] HNSCC Head and neck squamous cell carcinoma

[0050] HL Hodgkin's lymphoma

[0051] IgG Immunoglobulin G

[0052] IHC Immunohistochemistry or Immunohistochemistry

[0053] IV

[0054] LPS lymphoma proportion score

[0055] mAb monoclonal antibody

[0056] MCC Merkel cell carcinoma

[0057] MEL Melanoma

[0058] MMR mismatch repair

[0059] MPS Modified Ratio Score

[0060] MRI Magnetic Resonance Imaging

[0061] MSI-H microsatellite instability-high

[0062] NCI CTCAE National Cancer Institute – Common Terminology Criteria for Adverse Events

[0063] NSCLC Non-small cell lung cancer

[0064] ORR objective response rate

[0065] OS overall survival

[0066] PD progressive disease

[0067] PD-1 Programmed death 1 (also known as programmed cell death 1 and programmed death receptor 1)

[0068] PD-L1 Programmed cell death 1 ligand 1

[0069] PD-L2 Programmed cell death 1 ligand 2

[0070] PFS Progression-free survival

[0071] PK Pharmacokinetics

[0072] Q2W every two weeks

[0073] Q3W every three weeks

[0074] Q6W every six weeks

[0075] RCC Renal Cell Carcinoma

[0076] SAE Serious Adverse Event

[0077] SC Subcutaneous

[0078] TPS tumor proportion score

[0079] V H Immunoglobulin heavy chain variable region

[0080] V L Immunoglobulin light chain variable region

[0081] Certain technical and scientific terms are specifically defined below to make it easier to understand the present invention. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meaning commonly understood by ordinary technicians in the field to which the present invention belongs.

[0082] Unless the context clearly indicates one of the indicated possibilities, references to "or" mean either or both possibilities. In some cases, "and / or" is used to highlight either or both possibilities.

[0083] As used herein, including the appended claims, singular forms of words, such as "a," "an," and "the," include their corresponding plural references unless the context clearly dictates otherwise.

[0084] The term "about" when modifying the amount (e.g., mg) of a substance or composition, or the value of a parameter of a step in a characterization method, etc., refers to a variation in the numerical amount, which may occur, for example, by typical measurement, handling and sampling procedures associated with the preparation, characterization and / or use of the substance or composition, by inadvertent errors in these procedures, by differences in the manufacture, source or purity of ingredients used to make or use the composition or perform the procedure, etc. In certain embodiments, "about" can represent a variation of ±0.1%, ±0.5%, ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9% or ±10%. When referring to a dose of "about 400 mg", the dose can be 360 ​​mg to 440 mg, 370 mg to 430 mg, 380 mg to 420 mg, 390 mg to 410 mg, 395 mg to 405 mg, 400 mg to 440 mg, or 390 mg to 440 mg. In alternative embodiments, the dosage may be 360 ​​mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, or 440 mg. When referring to the amount of time between administrations in a therapeutic treatment regimen (i.e., the amount of time between administrations of an anti-PD-1 antibody or antigen-binding fragment thereof and an anti-CTLA4 antibody or antigen-binding fragment thereof, e.g., "about 6 weeks," which is used interchangeably herein with "every about six weeks"), "about" refers to a specified time ± variation, which may occur due to patient / clinician schedules and availability around a 6-week target date. For example, "about 6 weeks" may refer to 6 weeks ± 5 days, 6 weeks ± 4 days, 6 weeks ± 3 days, 6 weeks ± 2 days, or 6 weeks ± 1 day, or may refer to 5 weeks 2 days to 6 weeks 5 days.

[0085] Pharmacokinetic "steady state" is the period of time during which any accumulation of drug concentrations attributable to multiple doses has been maximized and systemic drug exposure is considered uniform after administration of each subsequent dose; in the specific case of pembrolizumab, steady state is achieved at and after approximately 16 weeks of administration.

[0086] AUCss, Cavg,ss and Cmin,ss are pharmacokinetic measures of systemic exposure to a drug (e.g., pembrolizumab) in humans following drug administration and are generally considered drivers of drug efficacy. AUCss and Cavg,ss represent the average exposure over the dosing interval, but in different units. "Cmin,ss" represents the minimum or lowest (trough) drug concentration observed at the end of the dosing interval, just before the next dose is administered.

[0087] "Cmax,ss" is the maximum or highest (peak) drug concentration observed shortly after drug administration. In the specific case of pembrolizumab, which is administered as an intravenous infusion, peak concentrations occur immediately after the infusion is completed. Cmax,ss is a metric that is generally considered to be the driving force behind safety.

[0088] "Administering" and "treating" as they apply to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Treatment" or "treatment" of cancer, as used herein, refers to the administration of an anti-PD-1 antibody or antigen-binding fragment thereof, alone or in combination with an anti-CTLA4 antibody or antigen-binding fragment thereof, to a subject having cancer or diagnosed with cancer to achieve at least one positive therapeutic effect, e.g., a decrease in the number of cancer cells, a decrease in tumor size, a decrease in the rate of tumor cell infiltration into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth. "Treatment" may include one or more of the following: inducing / increasing anti-tumor immune responses, reducing the amount of one or more tumor markers, halting or delaying the growth of tumors or blood cancers or diseases associated with PD-1 binding to its ligands PD-L1 and / or PD-L2 ("PD-1-associated diseases") such as cancer progression, stabilization of PD-1-associated diseases, inhibiting the growth or survival of tumor cells, eliminating or reducing the size of one or more cancerous lesions or tumors, reducing the level of one or more tumor markers, improving or eliminating clinical manifestations of PD-1-associated diseases, reducing the severity or duration of clinical symptoms of PD-1-associated diseases such as cancer, prolonging patient survival relative to the expected survival of similar untreated patients, and inducing complete or partial remission of cancer conditions or other PD-1-associated diseases.

[0089] The positive treatment effect of cancer can be measured in a variety of ways (see WA Weber, J. Nucl. Med. 50: 1S-10S (2009)). For example, with respect to tumor growth inhibition, according to the NCI standard, T / C ≤ 42% is the lowest level of anti-tumor activity. T / C < 10% is considered a high level of anti-tumor activity, T / C (%) = median tumor volume treated / median tumor volume of the control × 100. In some embodiments, the treatment achieved by a therapeutically effective amount is any one of progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS). PFS, also known as "Time to Tumor Progression", refers to the length of time that cancer does not grow during and after treatment, including the amount of time a patient experiences a complete response or partial response, and the amount of time a patient experiences stable disease. DFS refers to the length of time a patient remains disease-free during and after treatment. OS refers to an expected extension of life expectancy compared to an untreated or untreated individual or patient. Although embodiments of the methods of treatment, compositions and uses of the invention may not be effective in achieving a positive therapeutic effect in every patient, this should be determined by any statistical test known in the art (e.g., Student's t-test, chi-square test, 2 This was done in a statistically significant number of subjects as determined by the PCR test, the U test according to Mann and Whitney, the Kruskal-Wallis test (H test), the Jonckheere-Terpstra test, and the Wilcoxon test).

[0090] The term "patient" (also referred to herein as "subject" or "individual") refers to a mammal (e.g., rat, mouse, dog, cat, rabbit), most preferably a human, that can be treated with the methods and compositions of the present invention. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient.

[0091] The term "antibody" refers to any form of an antibody that exhibits the desired biological or binding activity. It is therefore used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized, fully human antibodies, and chimeric antibodies. "Parent antibody" refers to an antibody obtained by exposing the immune system to an antigen before modifying the antibody for its intended use, such as humanization of an antibody for human therapy.

[0092] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of the heavy chain can define a constant region that is primarily responsible for effector function. Typically, human light chains are divided into kappa light chains and lambda light chains. In addition, human heavy chains are typically divided into mu, delta, gamma, alpha or epsilon, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. In the light chain and the heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See generally Fundamental Immunology Ch.7 (Paul, W., ed., 2nded. Raven Press, NY (1989).

[0093] The variable regions of each light / heavy chain pair form an antibody combining site. Thus, in general, a complete antibody has two combining sites. Except for bifunctional or bispecific antibodies, these two combining sites are usually the same.

[0094] Typically, the variable regions of both the heavy and light chains contain three hypervariable regions, also known as complementarity determining regions, located within relatively conserved framework regions. The CDRs are typically aligned through the framework regions to enable binding to specific epitopes. In general, from N-terminus to C-terminus, both the light and heavy chain variable domains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain generally conforms to the following definitions: Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252: 6609-6616; Chothia, et al., (1987) J Mol. Biol. 196: 901-917 or Chothia, et al., (1989) Nature 342: 878-883.

[0095] The term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs" (i.e., L1, CDRL2, and CDRL3 in the light chain variable region and CDRH1, CDRH2, and CDRH3 in the heavy chain variable region). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of antibodies by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (defining the CDR regions of antibodies by structure). The term "framework" or "FR" residues refers to variable region residues other than the hypervariable region residues defined herein as CDR residues.

[0096] Unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by a full-length antibody, such as a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments.

[0097] An antibody that "specifically binds" to a particular target protein is an antibody that exhibits preferential binding to the target compared to other proteins, but the specificity does not require absolute binding specificity. If the binding of the antibody determines the presence of the target protein in the sample, for example without producing undesirable results such as false positives, the antibody is considered to be "specific" for its intended target. The antibodies or binding fragments thereof used in the present invention will bind to the target protein with an affinity that is at least twice as high as that with non-target proteins, preferably at least ten times as high, more preferably at least 20 times as high, and most preferably at least 100 times as high. As used herein, if an antibody binds to a polypeptide comprising a given amino acid sequence, such as the amino acid sequence of a mature human PD-1 or human PD-L1 molecule, but does not bind to a protein lacking the sequence, the antibody is said to specifically bind to a polypeptide comprising the sequence.

[0098] "Chimeric antibodies" refer to antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species (e.g., humans) or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity.

[0099] "Human antibody" refers to an antibody that contains only human immunoglobulin sequences. If produced in mice, mouse cells, or hybridomas derived from mouse cells, human antibodies may contain mouse sugar chains. Similarly, "mouse antibody" or "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively.

[0100] "Humanized antibody" refers to a form of an antibody comprising sequences from non-human (e.g., mouse) antibodies and human antibodies. Such an antibody comprises a minimal sequence derived from a non-human immunoglobulin. Generally speaking, a humanized antibody will comprise substantially all of at least one (usually two) variable region, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies may also selectively comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. If necessary, a prefix "hum", "hu" or "h" is added to the antibody clone name to distinguish between a humanized antibody and a parent rodent antibody. The humanized form of a rodent antibody typically comprises the same CDR sequence as the parent rodent antibody, although certain amino acid substitutions may be included to improve affinity, improve the stability of the humanized antibody, or for other reasons.

[0101] The terms "cancer", "cancerous" or "malignant" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondroma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer. Additional cancers that may be treated according to the invention include those characterized by elevated expression of one or both of PD-L1 and PD-L2 in a test tissue sample.

[0102] "Biotherapeutic agent" refers to a biological molecule, such as an antibody or fusion protein, that blocks ligand / receptor signaling in any biological pathway that supports tumor maintenance and / or growth or suppresses anti-tumor immune responses.

[0103] "CDR" or "CDR" refers to the complementarity determining region in an immunoglobulin variable region, generally defined using the Kabat numbering system.

[0104] "Platinum chemotherapy" (also called platins) refers to the treatment of cancer with chemotherapeutic agents that are coordination compounds of platinum. Platinum-containing chemotherapeutic agents are alkylating agents that cross-link DNA, leading to ineffective DNA mismatch repair and often causing apoptosis. Examples of platinum include cisplatin, carboplatin, and oxaliplatin.

[0105] "Chemotherapeutic agents" are compounds used to treat cancer. The types of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, kinase inhibitors, spunatoxin plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens and selective estrogen receptor modulators (SERMs), antiprogestins, estrogen receptor downregulators (ERDs), estrogen receptor antagonists, luteinizing hormone releasing hormone agonists, antiandrogens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, antisense oligonucleotides that inhibit abnormal cell proliferation or tumor growth-related gene expression. Chemotherapeutic agents that can be used in the treatment methods of the present invention include cytostatics and / or cytotoxic agents.

[0106] "Chothia" refers to the antibody numbering system described in Al-Lazikani et al., JMB 273:927-948 (1997).

[0107] "Conservatively modified variants" or "conservative substitutions" refer to amino acids in a protein that are replaced by other amino acids with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity or other desired properties of the protein (e.g., antigen affinity and / or specificity). Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not significantly change biological activity (see Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of amino acids with similar structure or function are less likely to destroy biological activity. Exemplary conservative substitutions are listed in Table 1.

[0108] Table 1. Exemplary conservative amino acid substitutions

[0109] Original residue Conservative substitution Ala(A) Gly; Ser Arg(R) Lys; His Asn(N) Gln; His Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr;Met;Leu Pro(P) Ala Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

[0110] As used throughout the specification and claims, "consisting essentially of" and variations such as "consisting essentially of" or "consisting essentially of" indicate the inclusion of any of the recited elements or groups of elements, and optionally other elements of similar or different properties to the recited elements, which do not substantially change the basic or novel properties of the particular dosing regimen, method, or composition. As a non-limiting example, a PD-1 antigen-binding fragment consisting essentially of the recited amino acid sequence may also include one or more amino acids that do not substantially affect the properties of the binding compound, including substitutions of one or more amino acid residues.

[0111] “Comprise” or variations such as “comprises,” “includes,” or “comprising” are used throughout the specification and claims in an inclusive sense, i.e., specifying the presence of stated features but not excluding the presence or addition of further features that may materially affect the operation or use of any embodiment of the invention, unless the context requires otherwise by explicit language or necessary meaning.

[0112] As used herein, "co-formulation" or "co-formulation" or "co-formulation" or "co-formulation" refers to at least two different antibodies or antigen-binding fragments thereof that are formulated together and stored in a single bottle or container (e.g., injection device) as a combination product, rather than being formulated and stored separately and then mixed before administration or administered separately. In one embodiment, the co-formulation comprises an anti-PD-1 antibody and an anti-CTLA4 antibody.

[0113] "Diagnostic anti-PD-L monoclonal antibody" refers to a mAb that specifically binds to the mature form of a specified PD-L (PD-L1 or PD-L2) expressed on the surface of certain mammalian cells. Mature PD-L lacks a pre-secretory leader sequence, also known as a leader peptide. The terms "PD-L" and "mature PD-L" are used interchangeably herein and are understood to refer to the same molecule unless otherwise indicated or apparent from the context.

[0114] As used herein, diagnostic anti-human PD-L1 mAb or anti-hPD-L1 mAb refers to a monoclonal antibody that specifically binds to mature human PD-L1. The mature human PD-L1 molecule consists of 19-290 amino acids of the following sequence:

[0115] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNI

[0116] IQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITV

[0117] KVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFN

[0118] VTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 17).

[0119] Specific examples of diagnostic anti-human PD-L1 mAbs that can be used for immunohistochemistry (IHC) detection of PD-L1 expression in formalin-fixed, paraffin-embedded (FFPE) tumor tissue sections are antibodies 20C3 and 22C3, which are described in WO2014 / 100079. These antibodies comprise the light chain and heavy chain variable region amino acid sequences shown in Table 2 below:

[0120]

[0121]

[0122] Another anti-human PD-L1 mAb reportedly useful for IHC detection of PD-L1 expression in FFPE tissue sections (Chen, BJ et al., Clin Cancer Res 19:3462-3473 (2013)) is a rabbit anti-human PD-L1 mAb publicly available from Sino Biological, Inc. (Beijing, China; Catalog No. 10084-R015).

[0123] As used herein, "framework region" or "FR" refers to an immunoglobulin variable region excluding the CDR regions.

[0124] "Isolated antibodies" and "isolated antibody fragments" refer to a purified state, which in this context means that the referenced molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. In general, the term "isolated" is not intended to refer to the complete absence of such materials or the absence of water, buffers, or salts unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the binding compounds described herein.

[0125] As used herein, "Kabat" refers to the immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.).

[0126] "Monoclonal antibody" or "mAb" or "Mab", as used herein, refers to a substantially homogeneous antibody population, i.e., the antibody molecules comprising the population are identical in amino acid sequence, except for possible small amounts of naturally occurring mutations. In contrast, conventional (polyclonal) antibody preparations typically include a plurality of different antibodies whose variable regions have different amino acid sequences, particularly their CDRs, which are typically specific for different epitopes. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous antibody population, and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be prepared by recombinant DNA methods (see, e.g., US Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described, for example, by Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0127] "Anti-CTLA4 antibodies" that can be used in any of the treatment methods, compositions, kits, and uses of the present invention include monoclonal antibodies (mAbs) or antigen-binding fragments thereof that specifically bind to human CTLA4 and block the interaction of CTLA4 with its ligands CD80 (B7.1) and CD 86 (B7.2). The anti-CTLA4 antibody can be a human antibody, a humanized antibody, or a chimeric antibody, and can include a human constant region. In some embodiments, the human constant region is selected from IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen-binding fragment is selected from Fab, Fab'-SH, F(ab')2, scFv, and Fv fragments.

[0128] "Anti-PD-1 antibodies" useful in any of the methods of treatment, compositions, kits and uses of the present invention include monoclonal antibodies (mAbs) or antigen-binding fragments thereof that specifically bind to human PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. In any of the methods of treatment, compositions and uses of the present invention for treating human individuals, the PD-1 antibody or antigen-binding fragment thereof is a PD-1 antagonist that blocks the binding of human PD-L1 to human PD-1, or blocks the binding of both human PD-L1 and PD-L2 to human PD-1. The human PD-1 amino acid sequence can be found in NCBI seat number: NP_005009. Human PD-L1 and PD-L2 amino acid sequences can be found in NCBI locus numbers: NP_054862 and NP_079515, respectively. The anti-PD-1 antibody can be a human antibody, a humanized antibody, or a chimeric antibody, and can include a human constant region. In some embodiments, the human constant region is selected from IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen binding fragment is selected from Fab, Fab'-SH, F(ab')2, scFv, and Fv fragments.

[0129] "PD-L1" or "PD-L2" expression refers to any detectable level of expression of a specified PD-L protein on the surface of a cell or a specified PD-L mRNA within a cell or tissue, unless otherwise defined. PD-L protein expression can be detected in IHC analysis of tumor tissue sections with a diagnostic PD-L antibody or by flow cytometry. Alternatively, PD-L protein expression of tumor cells can be detected by PET imaging, using a binding agent (e.g., antibody fragment, affibody, etc.) that specifically binds to the desired PD-L target (e.g., PD-L1 or PD-L2). Techniques for detecting and measuring PD-L mRNA expression include RT-PCR and real-time quantitative RT-PCR.

[0130] Several methods for quantifying PD-L1 protein expression in IHC analysis of tumor tissue sections have been described. See, for example, Thompson et al., PNAS 101 (49): 17174-17179 (2004); Thompson et al., Cancer Res. 66: 3381-3385 ​​(2006); Gadiot et al., Cancer 117: 2192-2201 (2011); Taube et al., Sci Transl Med 4, 127ra37 (2012); and Toplian et al., New Eng. J Med. 366 (26): 2443-2454 (2012).

[0131] One approach uses a simple binary endpoint of PD-L1 expression as positive or negative, with a positive result determined by the percentage of tumor cells showing histological evidence of cell surface membrane staining. Tumor tissue sections are considered positive for PD-L1 expression if at least 1%, and preferably 5%, of the total number of tumor cells are present.

[0132] In another approach, PD-L1 expression in tumor tissue sections is quantified in tumor cells and infiltrating immune cells, which mainly contain lymphocytes. The percentages of tumor cells and infiltrating immune cells showing membrane staining are quantified as <5%, 5% to 9%, and then incremented by 10% to 100%, respectively. For tumor cells, PD-L1 expression is scored as negative if the score is <5% and as positive if the score is ≥5%. PD-L1 expression in immune infiltrates is reported as a semiquantitative measurement called the adjusted inflammatory score (AIS), which is determined by multiplying the percentage of membrane-stained cells by the intensity of infiltration, which is divided into none (0), mild (1 point, rare lymphocytes), moderate (2 points, local infiltration of the tumor by lymphohistiocytic aggregates) or severe (3 points, diffuse infiltration). If the AIS is ≥5, the tumor tissue section is counted as positive for PD-L1 expression of the immune infiltrate.

[0133] Tumor tissue sections that have been stained by IHC with a diagnostic PD-L1 antibody can also be scored for PD-L1 protein expression by using a scoring method to assess the expression of PD-L1 in both tumor cells and infiltrating immune cells in tissue sections. See WO 2014 / 165422. A PD-L1 scoring process includes examining each tumor nest in a tissue section for staining and assigning one or two modified H scores (MHS) and modified proportion scores (MPS) to the tissue section. To assign the MHS, four percentages of all live tumor cells and stained mononuclear inflammatory cells in all examined tumor nests were evaluated: (a) unstained cells (intensity = 0), (b) weakly stained cells (intensity = 1+), (c) moderately stained cells (intensity = 2+), (d) strongly stained cells (intensity = 3+). Cells must have at least partial membrane staining to be included in the weak, moderate, or strong staining percentages. The estimated percentages (summing to 100%) are then entered into the formula 1x (percentage of weakly stained cells) + 2x (percentage of moderately stained cells) + 3x (percentage of strongly stained cells), and the result is assigned to the tissue section as the MHS. The MPS is assigned by estimating the percentage of cells with at least partial membrane staining of any intensity among all viable tumor cells and stained mononuclear inflammatory cells in all tumor nests examined, and the resulting percentage is assigned to the tissue section as the MPS. In some embodiments, if the MHS or MPS is positive, the tumor is designated as positive for PD-L1 expression.

[0134] Another method of scoring / quantifying PD-L1 expression in a tumor is the "combined positive score" or "CPS," which refers to an algorithm that determines a PD-L1 expression score from a patient's tumor sample. The CPS can be used to select patients for treatment with a specific treatment regimen (including treatments in which an anti-PD-1 antibody is administered) where the expression of PD-L1 is associated with a higher response rate in a specific patient population relative to the same patient population that does not express PD-L1. The CPS is determined by determining the number of viable PD-L1-positive tumor cells, the number of viable PD-L1-negative tumor cells, and the number of viable PD-L1-positive mononuclear inflammatory cells (MICs) in tumor tissue of a patient having a tumor and calculating the CPS using the following formula:

[0135]

[0136] In a specific embodiment, the PD-L1 expression scoring method used is the "lymphoma proportion score". Lymphomas are characterized by a homogeneous, fused population of cells that obliterate the architecture of the lymph nodes or the architecture of the metastatic site. "LPS" or "lymphoma proportion score" refers to the percentage of a population of cells that express PD-L1. In determining the LPS, no attempt is made to distinguish between true tumor cells and reactive cells. PD-L1 expression is characterized by partial or complete membrane staining intensity.

[0137] Another scoring method for PD-L1 expression is the "TPS" or "tumor proportion score," which is the percentage of tumor cells that express PD-L1 on the cell membrane. The TPS generally includes the percentage of tumor cells that express PD-L1 at any intensity (weak, moderate, or strong), which can be determined by immunohistochemical analysis using a diagnostic anti-human PD-L1 mAb (e.g., antibody 20C3 and antibody 22C3 described above). If membrane staining is present, including cells with partial membrane staining, the cell is considered to express PD-L1.

[0138] The PD-L mRNA expression level can be compared with the mRNA expression level of one or more reference genes commonly used in quantitative RT-PCR (such as ubiquitin c).

[0139] In some embodiments, the PD-L1 expression level (protein and / or mRNA) of malignant cells and / or tumor-infiltrating immune cells is determined to be "overexpressed" or "elevated" based on comparison with the PD-L1 expression level (protein and / or mRNA) of an appropriate control. For example, the control PD-L1 protein or mRNA expression level can be the level quantified in non-malignant cells of the same type or in a matched normal tissue section. In some preferred embodiments, if the PD-L1 protein (and / or PD-L1 mRNA) in the tumor sample is at least 10%, 20% or 30% higher than the PD L1 protein (and / or PD-L1 mRNA) in the control sample, it is determined that the PD-L1 expression in the tumor sample is elevated.

[0140] "Tissue section" refers to a single portion or fragment of a tissue sample, such as a thin slice of tissue cut from a normal tissue or tumor sample.

[0141] "Tumor" applies to subjects diagnosed with or suspected of having cancer and refers to any malignant or potentially malignant tumor or mass of tissue of any size, including primary tumors and secondary tumors. A solid tumor is an abnormal growth or mass of tissue that does not usually contain cysts or areas of fluid. Different types of solid tumors are named after the type of cells that form the solid tumor. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (blood cancers) do not usually form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

[0142] As used herein, "variable region" or "V region" refers to the segment of the IgG chain whose sequence varies between different antibodies. It extends to Kabat residues 109 in the light chain and 113 in the heavy chain.

[0143] As used herein, "RECIST 1.1 response criteria" refers to the definitions described by Eisenhauer, EA et al., Eur. J. Cancer 45:228-247 (2009) for target lesions or non-target lesions, as appropriate, based on the context in which the response is measured.

[0144] II. PD-1 Antibodies and Antigen-Binding Fragments That Can Be Used in the Present Invention

[0145] Examples of mAbs that bind to human PD-1 that can be used in the methods of treatment, compositions, and uses of the present invention are described in U.S. Patents 7,521,051, 8,008,449, and 8,354,509. Specific anti-human PD-1 mAbs used as PD-1 antagonists in the methods of treatment, compositions, and uses of the present invention include: pembrolizumab (formerly known as MK-3475, SCH 900475, and lambrolizumab), which is a humanized IgG4 mAb having a structure described in WHO Drug Information, Vol. 27, No. 2, pp. 161-162 (2013), comprising Figure 1 The heavy and light chain amino acid sequences are shown, as well as the humanized antibodies h409A11, h409A16 and h409A17, which are described in WO2008 / 156712 and Table 3.

[0146] In some embodiments of the methods of treatment, compositions, kits and uses of the present invention, the anti-PD-1 antibody or antigen-binding fragment thereof comprises: (a) a light chain CDR comprising the amino acid sequence of SEQ ID NO: 1, 2 and 3 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NO: 6, 7 and 8; or (b) a light chain CDR comprising the amino acid sequence of SEQ ID NO: 11, 12 and 13 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NO: 14, 15 and 16. In some embodiments of the present invention, the anti-PD-1 antibody or antigen-binding fragment thereof is a human antibody. In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a humanized antibody. In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a chimeric antibody. In a specific embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0147] In other embodiments of the methods of treatment, compositions, kits and uses of the present invention, the PD-1 antibody or antigen-binding fragment thereof specifically binds to human PD-1 and comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof, and (b) a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 22 or a variant thereof; and SEQ ID NO: 23 or a variant thereof.

[0148] The variant of the heavy chain variable region sequence or the full-length heavy chain sequence is identical to the reference sequence, except that it has a maximum of 17 conservative amino acid substitutions in the framework region (i.e., outside the CDR), and preferably has less than 10, 9, 8, 7, 6 or 5 conservative amino acid substitutions in the framework region. The variant of the light chain variable region sequence or the full-length light chain sequence is identical to the reference sequence, except that it has a maximum of five conservative amino acid substitutions in the framework region (i.e., outside the CDR), and preferably has less than four, three or two conservative amino acid substitutions in the framework region.

[0149] In another embodiment of the methods of treatment, compositions, kits and uses of the present invention, the PD-1 antibody or antigen-binding fragment thereof is a monoclonal antibody that specifically binds to human PD-1 and comprises (a) a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 10 or a variant thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 5 or a variant thereof; SEQ ID NO: 24 or a variant thereof; or SEQ ID NO: 25 or a variant thereof.

[0150] In another embodiment of the methods of treatment, compositions and uses of the present invention, the PD-1 antibody or antigen-binding fragment thereof is a monoclonal antibody that specifically binds to human PD-1 and comprises (a) a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 10 and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 5.

[0151] Table 3 below lists the amino acid sequences of exemplary anti-PD-1 mAbs for use in the treatment methods, compositions, kits, and uses of the present invention.

[0152]

[0153]

[0154] III. Anti-CTLA4 Antibodies and Antigen-Binding Fragments Useful in the Present Invention

[0155] In one embodiment of the methods of treatment, compositions, kits and uses of the invention, the anti-CTLA-4 antibody is the human monoclonal antibody 10D1, currently known as ipilimumab and as YervoyTM Marketed, disclosed in US Patent No. 6,984,720 and WHO Drug Information 19(4):61(2005). In another embodiment, the anti-CTLA-4 antibody is tremelimumab, also known as CP-675,206, which is an IgG2 monoclonal antibody described in US Patent Application Publication No. 2012 / 263677 or PCT International Application Publication No. WO 2012 / 122444 or WO 2007 / 113648A2.

[0156] In further embodiments of the treatment methods, compositions, kits and uses of the present invention, the anti-CTLA4 antibody or antigen-binding fragment thereof comprises: a light chain CDR comprising the amino acid sequences described in SEQ ID NOs: 26, 27 and 28 and a heavy chain CDR comprising the amino acid sequences described in SEQ ID NOs: 29, 30 and 31.

[0157] In other embodiments of the treatment methods, compositions, kits and uses of the present invention, the anti-CTLA4 antibody is a monoclonal antibody or an antigen-binding fragment thereof, which binds to human CTLA4 and comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:32, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:33.

[0158]

[0159]

[0160] In one embodiment of the methods of treatment, compositions, kits and uses of the present invention, the anti-CTLA-4 antibody is a monoclonal antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CTLA4 antibody is an antigen-binding fragment of SEQ ID NO: 34 and / or SEQ ID NO: 35, wherein the antigen-binding fragment specifically binds to CTLA4.

[0161] In one embodiment of the treatment methods, compositions, kits and uses of the present invention, the anti-CTLA-4 antibody is any anti-CTLA-4 antibody or antigen-binding fragment thereof disclosed in International Application Publication No. WO 2016 / 015675 A1. In one embodiment, the anti-CTLA4 antibody is a monoclonal antibody comprising the following CDRs:

[0162] CDRH1, comprising the amino acid sequence GFTFSDNW (SEQ ID NO: 36);

[0163] CDRH2, comprising the amino acid sequence IRNKPYNYET (SEQ ID NO: 37);

[0164] CDRH3 comprising the amino acid sequence TAQFAY (SEQ ID NO: 38)

[0165] and / or

[0166] CDRL1, comprising the amino acid sequence ENIYGG (SEQ ID NO: 39);

[0167] CDRL2, comprising the amino acid sequence GAT (SEQ ID NO:40); and

[0168] CDRL3 comprising an amino acid sequence selected from the group consisting of: QNVLRSPFT (SEQ ID NO:41); QNVLSRHPG (SEQ ID NO:42); or QNVLSSRPG (SEQ ID NO:43).

[0169] In one embodiment of the treatment methods, compositions, kits and uses of the present invention, the anti-CTLA4 antibody is 8D2 / 8D2(RE) or a variant thereof, 8D2H1L1 or a variant thereof, 8D2H2L2 or a variant thereof, 8D3H3L3 or a variant thereof, 8D2H2L15 or a variant thereof, or 8D2H2l17 or a variant thereof.

[0170]

[0171]

[0172] In another embodiment of the methods of treatment, compositions, kits and uses of the present invention, the anti-CTLA4 antibody is a variant of 8D2 / 8D2(RE), a variant of 8D2H1L1, a variant of 8D2H2L2, a variant of 8D2H2L15 or a variant of 8D2H2117, wherein the methionine (Met) at position 18 of the amino acid sequence of the VH chain is independently substituted with an amino acid selected from leucine (Leu), valine (Val), isoleucine (Ile) or alanine (Ala). In an embodiment of the present invention, the anti-CTLA4 antibody comprises the sequence of 8D2H2L2 variant 1 listed in the above table.

[0173] In another embodiment of the treatment methods, compositions, kits and uses of the present invention, the anti-CTLA4 antibody is 8D2H2L2 variant 1, having the full heavy chain amino acid sequence shown in SEQ ID NO:57 and the full light chain sequence shown in SEQ ID NO:58.

[0174]

[0175] In one embodiment of the treatment methods, compositions, kits and uses of the present invention, the anti-CTLA4 antibody is any anti-CTLA4 antibody or antigen-binding fragment thereof disclosed in International Application Publication No. WO2018 / 035710A1 published on March 1, 2018.

[0176] IV. Methods and uses of the present invention

[0177] The present invention provides a method for treating cancer in a human patient, comprising administering to the patient about 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof about once every six weeks, wherein the anti-PD-1 antibody or the antigen-binding fragment thereof comprises: (a) a light chain complementary determining region (CDR) comprising the amino acid sequence of SEQ ID NOs: 1, 2, and 3 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 6, 7, and 8; or (b) a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 11, 12, and 13 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 14, 15, and 16. In a specific embodiment, the anti-PD-1 antibody or the antigen-binding fragment thereof is pembrolizumab.

[0178] The present invention also provides a method for treating cancer in a human patient, comprising administering to the patient about 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof once every six weeks, wherein the anti-PD-1 antibody or the antigen-binding fragment thereof comprises: (a) a light chain complementary determining region (CDR) comprising the amino acid sequence of SEQ ID NOs: 1, 2 and 3 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 6, 7 and 8; or (b) a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 11, 12 and 13 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 14, 15 and 16; and wherein the anti-CTLA4 antibody or the antigen-binding fragment thereof comprises: (c) a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 39, 40 and 41 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: 36, 37 and 38; (d) a light chain CDR comprising the amino acid sequence of SEQ ID NOs: 39, 40 and 42 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NOs: or (e) a light chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 39, 40 and 43 and a heavy chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 36, 37 and 38.

[0179] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment comprises a light chain CDR and a heavy chain CDR, wherein the light chain CDR comprises the amino acid sequence shown in SEQ ID NOs: 1, 2 and 3, and the heavy chain CDR comprises the amino acid sequence shown in SEQ ID NOs: 6, 7 and 8; and the anti-CTLA4 antibody or its antigen-binding fragment comprises a light chain CDR and a heavy chain CDR, wherein the light chain CDR comprises the amino acid sequence shown in SEQ ID NOs: 39, 40 and 43, and the heavy chain CDR comprises the amino acid sequence shown in SEQ ID NOs: 36, 37 and 38.

[0180] In some embodiments of the present invention, at 12 weeks or longer, anti-PD-1 antibodies or antigen-binding fragments thereof and anti-CTLA4 antibodies or antigen-binding fragments thereof are administered to patients once every about six weeks. In other embodiments, at 18 weeks or longer, 24 weeks or longer, 30 weeks or longer, 36 weeks or longer, 42 weeks or longer, 48 weeks or longer, 54 weeks or longer, 60 weeks or longer, 66 weeks or longer, 72 weeks or longer, 78 weeks or longer, 84 weeks or longer, or 90 weeks or longer, anti-PD-1 antibodies or antigen-binding fragments thereof and anti-CTLA4 antibodies or antigen-binding fragments thereof are administered to patients once every six weeks. In one embodiment, administration occurs on the same day. In a sub-embodiment, anti-PD-1 antibodies or antigen-binding fragments thereof and anti-CTLA4 antibodies or antigen-binding fragments thereof are administered simultaneously on the same day (e.g., in a single formulation or as separate formulations at the same time). In another embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof and the anti-CTLA4 antibody or antigen-binding fragment thereof are administered sequentially in either order on the same day (e.g., as separate formulations). In one embodiment of sequential administration on the same day, the anti-PD-1 antibody or antigen-binding fragment thereof is administered first. In another embodiment of sequential administration on the same day, the anti-CTLA4 antibody or antigen-binding fragment thereof is administered first.

[0181] In a first embodiment (embodiment E1), the present invention comprises a method of treating cancer in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient once about six weeks. In another embodiment of (embodiment 1), the present invention further comprises administering 25 mg, 50 mg, 75 mg, or 100 mg of an anti-CTLA4 antibody or an antigen-binding fragment thereof to the patient once about six weeks. In one embodiment, 25 mg of anti-CTLA4 antibody is administered once about six weeks. In one embodiment, 50 mg of anti-CTLA4 antibody is administered once about every six weeks. In another embodiment, 75 mg of anti-CTLA4 antibody is administered once about every six weeks. In another embodiment, 100 mg of anti-CTLA4 antibody is administered once about every six weeks. In a further embodiment, the cancer is PD-1 / PD-L1 refractory (e.g., it is a cancer that has not responded to previous treatment with an anti-PD-1 or anti-PD-L1 agent). In a further embodiment, the cancer is PD-1 / PD-L1 refractory melanoma. In another embodiment, the cancer is melanoma, non-small cell lung cancer, head and neck cancer, urinary tract cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, kidney cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, pancreatic cancer, brain tumor, glioblastoma, sarcoma, bone tumor or Merkel cell carcinoma.

[0182] In a second embodiment (Embodiment E2), the invention comprises a method of treating unresectable or metastatic melanoma in a human patient, the method comprising administering to the patient 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof approximately once every six weeks.

[0183] In a third embodiment (Embodiment E3), the invention comprises a method of treating metastatic non-small cell lung cancer (NSCLC) in a human patient, the method comprising administering to the patient 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof approximately once every six weeks.

[0184] In a sub-embodiment of embodiment E3 (embodiment E3-A), the patient has a tumor with high PD-L1 expression [(tumor proportion score (TPS) ≥ 50%)] and has not previously received platinum-containing chemotherapy.

[0185] In another sub-embodiment of embodiment E3 (embodiment E3-B), the patient has a tumor that expresses PD-L1 (TPS ≥ 1%) and has previously received platinum-containing chemotherapy. In a specific embodiment of embodiment E3-B, the patient has disease progression during or after receiving platinum-containing chemotherapy.

[0186] In another sub-embodiment of Embodiment E3 (Embodiment E3-C), the patient has a tumor that expresses PD-L1 (TPS ≥ 1%) and has not previously received platinum-containing chemotherapy.

[0187] In another sub-embodiment of embodiment E3 (embodiment E3-D), the patient's tumor is not tested for PD-L1 expression. In this embodiment, the patient is treated with an anti-PD-1 antibody or antigen-binding fragment thereof, regardless of PD-L1 expression. In a specific embodiment, the patient has not previously received platinum-containing chemotherapy.

[0188] In certain embodiments of embodiment E3, including embodiments E3-A, E3-B, and E3-C, the PD-L1 TPS is determined by an FDA-approved test.

[0189] In certain embodiments of embodiment E3, including embodiments E3-A, E3-B, E3-C, and E3-D, the patient's tumor does not have an EGFR or ALK genomic abnormality.

[0190] In certain embodiments of embodiment E3, including embodiments E3-A, E3-B, E3-C, and E3-D, the patient's tumor has an EGFR or ALK genomic abnormality and has disease progression while or after receiving treatment for the EGFR or ALK abnormality prior to receiving the anti-PD-1 antibody or antigen-binding fragment thereof.

[0191] In a fourth embodiment (embodiment E4), the invention includes a method of treating metastatic non-small cell lung cancer (NSCLC) in a human patient, the method comprising: (1) administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks, and (2) administering pemetrexed and carboplatin to the patient. In a subembodiment of embodiment E4, the patient has not received anti-cancer treatment prior to initiating the combination treatment regimen of the anti-PD-1 antibody or antigen-binding fragment thereof, pemetrexed, and carboplatin.

[0192] In certain embodiments of embodiments E3 and E4, including subembodiments thereof, the patient has non-squamous non-small cell lung cancer.

[0193] In a sub-embodiment of embodiment E4, pemetrexed is administered at 500 mg / m 2 The amount is administered to the patient.

[0194] In a sub-embodiment of embodiment E4, pemetrexed is administered to the patient by intravenous infusion every 21 days. In a specific embodiment, the infusion time is about 10 minutes.

[0195] In a sub-embodiment of embodiment E4 (embodiment E4-A), the invention further comprises administering to the patient about 400 μg to about 1000 μg of folic acid once a day, starting from about 7 days before the administration of pemetrexed to the patient and continuing until about 21 days after the last dose of pemetrexed is administered to the patient. In certain embodiments, the folic acid is administered orally.

[0196] In a sub-embodiment of embodiments E4 and E4-A (embodiment E4-B), the invention further comprises administering to the patient about 1 mg of vitamin B1 about 1 week prior to the first administration of pemetrexed and every 3 cycles of pemetrexed administration (i.e., about every 9 weeks). 12 In certain embodiments, vitamin B 12 Intramuscular administration.

[0197] In a sub-embodiment of embodiments E4, E4-A, and E4-B (embodiment E4-C), the invention further comprises administering to the patient about 4 mg of dexamethasone twice daily on the day before, on the day after, and on the day after administration of pemetrexed. In certain embodiments, dexamethasone is administered orally.

[0198] In a fifth embodiment (Embodiment E5), the present invention comprises a method of treating recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0199] In a sub-embodiment of embodiment E5, the patient was previously treated with platinum-containing chemotherapy. In certain embodiments, the patient has disease progression on or after platinum-containing chemotherapy.

[0200] In a sixth embodiment (Embodiment E6), the present invention comprises a method of treating refractory classical Hodgkin lymphoma (cHL) in a human patient, comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0201] In a seventh embodiment (embodiment E7), the invention includes a method of treating classical Hodgkin lymphoma (cHL) in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks, wherein the patient has relapsed after (a) first or more lines of treatment for cHL, (b) second or more lines of treatment for cHL, or (c) third or more lines of treatment for cHL.

[0202] In subembodiments of embodiments E6 and E7, the patient is an adult patient.

[0203] In alternative subembodiments of embodiments E6 and E7, the patient is a pediatric patient.

[0204] In an eighth embodiment (Embodiment E8), the present invention comprises a method of treating locally advanced or metastatic urothelial carcinoma in a human patient, comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0205] In a subembodiment of embodiment E8, the patient is ineligible for cisplatin-containing chemotherapy.

[0206] In a subembodiment of embodiment E8, the patient has disease progression during or after platinum-containing chemotherapy or within 12 months after neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

[0207] In a sub-embodiment of embodiment E8, the patient's tumor expresses PD-L1 (CPS ≥ 10).

[0208] In a ninth embodiment (embodiment E9), the present invention comprises a method for treating unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair (MMR)-deficient solid tumors in a human patient, comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0209] In a subembodiment of embodiment E9, the patient has disease progression following prior anti-cancer therapy.

[0210] In a tenth embodiment (embodiment E10), the present invention comprises a method for treating unresectable or metastatic, MSI-H or MMR-deficient colorectal cancer in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0211] In a subembodiment of embodiment E10, the patient has disease progression following prior treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.

[0212] In an eleventh embodiment (embodiment E11), the present invention comprises a method for treating recurrent locally advanced or metastatic gastric cancer in a human patient, comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0213] In a twelfth embodiment (embodiment E12), the present invention comprises a method for treating recurrent locally advanced or metastatic gastroesophageal junction adenocarcinoma in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0214] In subembodiments of embodiments E11 and E12, the patient's tumor expresses PD-L1 [combined positive score (CPS) ≥ 1].

[0215] In subembodiments of embodiments E11 and E12, the patient has disease progression on or after one or more lines of therapy. In a specific embodiment, the prior line of treatment comprises a fluoropyrimidine- and platinum-containing chemotherapy.

[0216] In subembodiments of embodiments E11 and E12, the patient has disease progression on or after two or more lines of prior treatment comprising a fluoropyrimidine- and platinum-containing chemotherapy.

[0217] In subembodiments of embodiments E11 and E12, the patient has disease progression on or after one or more lines of prior therapy including a HER2 / neu targeted therapy.

[0218] In subembodiments of embodiments E11 and E12, the patient has disease progression on or after two or more lines of prior therapy including a HER2 / neu targeted therapy.

[0219] In a thirteenth embodiment (embodiment E13), the present invention comprises a method of treating cancer in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks, wherein the patient has a cancer selected from the group consisting of melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, lymphoma, kidney cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, and salivary gland cancer.

[0220] In a fourteenth embodiment (embodiment E14), the present invention comprises a method of treating cancer in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks, wherein the patient has small cell lung cancer.

[0221] In a fifteenth embodiment (Embodiment E15), the present invention comprises a method of treating non-Hodgkin lymphoma in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0222] In a sub-embodiment of embodiment E15, non-Hodgkin lymphoma is primary mediastinal large B-cell lymphoma (PMBCL). In certain embodiments, when the patient suffers from PMBCL, the patient suffers from refractory PMBCL. In some embodiments, the patient relapses after the previous treatment of one or more lines. In some embodiments, the patient relapses after the previous treatment of two or more lines. In some embodiments, the patient was not previously treated with the treatment of another line.

[0223] In a sixteenth embodiment (Embodiment E16), the present invention comprises a method of treating metastatic squamous NSCLC in a human patient, comprising: (1) administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks, and (2) administering to the patient (i) carboplatin and paclitaxel or (ii) carboplatin and nab-paclitaxel.

[0224] In a seventeenth embodiment (embodiment E17), the invention comprises a method of treating Merkel cell carcinoma (MCC) in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks. In a specific subembodiment of embodiment E17, the cancer is recurrent locally advanced MCC. In a specific subembodiment of embodiment E17, the cancer is metastatic MCC.

[0225] In a subembodiment of embodiment E17, the patient is an adult patient. In an alternative subembodiment of embodiment E17, the patient is a pediatric patient.

[0226] In an eighteenth embodiment (embodiment E18), the invention comprises a method of adjuvant treatment of melanoma in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks, wherein the patient has previously had one or more melanoma lesions resected. In a subembodiment of embodiment E18, the method comprises treating resected high-risk stage III melanoma.

[0227] In a nineteenth embodiment (embodiment E19), the invention comprises a method of treating hepatocellular carcinoma (HCC) in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks. In some embodiments of embodiment E19, the patient was previously treated with sorafenib.

[0228] In a twentieth embodiment (embodiment E20), the present invention comprises a method of treating renal cell carcinoma (RCC) in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0229] In a subembodiment of embodiment E20, the cancer is advanced clear cell RCC.

[0230] In a subembodiment of embodiment E20, the patient has advanced or metastatic renal cell carcinoma (RCC).

[0231] In a subembodiment of embodiment E20 (embodiment E20A), the patient is further treated with axitinib. In a subembodiment of the invention, axitinib is administered orally.

[0232] In a specific embodiment of embodiment E20A, the patient takes 5 mg of axitinib approximately every 12 hours or twice a day.

[0233] In an alternative embodiment of embodiment E20A, the dose of axitinib is 2.5 mg, 3 mg, 7 mg or 10 mg twice daily.

[0234] In a twenty-first embodiment (embodiment E21), the present invention comprises a method of treating breast cancer in a human patient, the method comprising administering to the patient 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof approximately once every six weeks.

[0235] In a subembodiment of embodiment E21, the breast cancer is triple-negative breast cancer.

[0236] In a subembodiment of embodiment E21, the breast cancer is ER+ / HER2- breast cancer.

[0237] In a twenty-second embodiment (embodiment E22), the present invention comprises a method of treating nasopharyngeal carcinoma in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0238] In a twenty-third embodiment (Embodiment E23), the present invention comprises a method of treating thyroid cancer in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0239] In a twenty-fourth embodiment (embodiment E24), the present invention comprises a method of treating salivary gland cancer in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0240] In the twenty-fifth embodiment (embodiment E25), the present invention comprises a method of treating cancer in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks, wherein the cancer is selected from melanoma, non-small cell lung cancer, relapsed or refractory classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal cancer, gastric cancer, cervical cancer, PMBCL, MSI-H cancer, hepatocellular carcinoma, and Merkel cell carcinoma.

[0241] In a twenty-sixth embodiment (embodiment E26), the present invention comprises a method of treating cancer in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks, wherein the cancer is a heme malignancy.

[0242] In a sub-embodiment of embodiment E26, the heme malignancy is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid leukemia-1 protein (MCL-1), myelodysplastic syndrome, non-Hodgkin lymphoma (NHL), and small lymphocytic lymphoma (SLL).

[0243] In a twenty-seventh embodiment (embodiment E27), the present invention comprises a method of treating cancer in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks, wherein the patient has a tumor with a high mutational load.

[0244] In certain embodiments, a high mutation load is at least about 10 mutations per megabase of the genome examined, at least about 11 mutations per megabase of the genome examined, at least about 12 mutations per megabase of the genome examined, or at least about 13 mutations per megabase of the genome examined.

[0245] In a twenty-eighth embodiment (embodiment E28), the present invention comprises a method of treating esophageal cancer in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks.

[0246] In a sub-embodiment of embodiment E28, the patient received a first line of prior standard therapy prior to receiving the anti-PD-1 antibody or antigen-binding fragment thereof. In another embodiment, the patient received one or more lines of standard therapy prior to receiving the anti-PD-1 antibody or antigen-binding fragment thereof. In another embodiment, the patient received two or more lines of standard therapy prior to receiving the anti-PD-1 antibody or antigen-binding fragment thereof. In a specific embodiment, the standard therapy comprises one or more of the following: paclitaxel, docetaxel, or irinotecan.

[0247] In a subembodiment of embodiment E28, the patient has advanced or metastatic adenocarcinoma or squamous cell carcinoma of the esophagus.

[0248] In a subembodiment of embodiment E28, the patient has advanced or metastatic Siewert type I adenocarcinoma of the esophagogastric junction.

[0249] In a sub-embodiment of embodiment E28, the patient's tumor expresses PD-L1 (combined positive score [CPS] ≥ 10).

[0250] In a twenty-ninth embodiment (Embodiment E29), the present invention comprises a method of treating a high-risk non-muscle invasive bladder cancer (NMIBC) in a human patient, the method comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to the patient approximately once every six weeks. In some embodiments, the patient has NMIBC with carcinoma in situ (CIS) or CIS plus papillary disease.

[0251] In a sub-embodiment of embodiment E29, the patient has been previously treated with standard therapy prior to treatment with the anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the previous treatment is Bacillus Calmette-Guérin (BCG) therapy. In specific embodiments, the patient is unresponsive to BCG therapy. In some embodiments, the patient is not eligible for radical cystectomy or chooses not to undergo radical cystectomy.

[0252] In any of the above methods of the present invention (including embodiments E1-E29), the PD-1 antibody or antigen-binding fragment is any antibody or antigen-binding fragment listed in Section 2 of the detailed description of the present invention, "PD-1 antibodies and antigen-binding fragments that can be used in the present invention".

[0253] Embodiments of any of the methods of the invention described above (including embodiments E1-E29) may further include administering about 25 mg, 50 mg, 75 mg, or 100 mg of an anti-CTLA4 antibody or an antigen-binding fragment thereof once every about six weeks. In one embodiment, 25 mg of an anti-CTLA4 antibody is administered once every about six weeks. In one embodiment, 50 mg of an anti-CTLA4 antibody is administered once every about six weeks. In another embodiment, 75 mg of an anti-CTLA4 antibody is administered once every about six weeks. In another embodiment, 100 mg of an anti-CTLA4 antibody is administered once every about six weeks. In one embodiment, 25 mg of an anti-CTLA4 antibody is administered once every six weeks. In one embodiment, 50 mg of an anti-CTLA4 antibody is administered once every six weeks. In another embodiment, 75 mg of an anti-CTLA4 antibody is administered once every six weeks. In another embodiment, 100 mg of an anti-CTLA4 antibody is administered once every six weeks. In some embodiments of any of the above methods, an anti-CTLA4 antibody and an anti-PD-1 antibody are administered together. In other embodiments, an anti-CTLA4 antibody and an anti-PD-1 antibody are co-formulated together. In any of the above methods of the present invention, the anti-CTLA4 antibody or antigen-binding fragment is any antibody or antigen-binding fragment listed in Section 3 of the detailed description of the present invention, "Anti-CTLA4 antibodies and antigen-binding fragments useful in the present invention".

[0254] In some embodiments, the anti-PD-1 antibody is pembrolizumab or an antigen-binding fragment thereof, or an antibody that cross-competes with pembrolizumab. In some embodiments, the anti-PD-1 antibody is a variant of pembrolizumab; that is, an antibody or antigen-binding fragment having a light chain CDR and a heavy chain CDR, wherein the light chain CDR comprises the amino acid sequence shown in SEQ ID NO: 1, 2 and 3, and the heavy chain CDR comprises the amino acid sequence shown in SEQ ID NO: 6, 7 and 8.

[0255] In any of the above methods, the anti-PD-1 antibody or antigen-binding fragment thereof and the anti-CTLA4 antibody or antigen-binding fragment thereof are administered to the patient once every about six weeks. In a specific embodiment, the anti-PD1 antibody or antigen-binding fragment thereof and the anti-CTLA4 antibody or antigen-binding fragment thereof are administered to the patient every six weeks, every six weeks ±5 days, ±4 days, ±3 days, ±2 days or ±1 day.

[0256] In an embodiment of any method of the invention, a medicament comprising any anti-PD-1 antibody or antigen-binding fragment thereof or any anti-CTLA4 antibody or antigen-binding fragment thereof, each as described herein, is infused intravenously (IV) to a patient. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof and the anti-CTLA4 antibody or antigen-binding fragment thereof are administered simultaneously by IV infusion (e.g., in a single formulation or in separate formulations simultaneously).

[0257] In another embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof and the anti-CTLA4 antibody or antigen-binding fragment thereof are administered sequentially in any order by intravenous infusion on the same day (e.g., as separate formulations). In a sub-embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is administered first. In another embodiment, the anti-CTLA4 antibody or antigen-binding fragment thereof is administered first.

[0258] In alternative embodiments, any anti-PD-1 antibody or antigen-binding fragment thereof, or any anti-CTLA4 antibody or antigen-binding fragment thereof, each described herein, is administered subcutaneously to the patient (e.g., by a clinician).

[0259] In any of the methods described herein, including embodiments E1-E29 and sub-embodiments thereof, the method may further include one or more "additional therapeutic agents" (as used herein, "additional therapeutic agent" refers to an additional therapeutic agent relative to a PD-1 antagonist and an anti-CTLA4 antibody or antigen-binding fragment thereof). The additional therapeutic agent can be, for example, a chemotherapeutic agent other than an anti-PD-1 antibody or an anti-CTLA4 antibody, a biotherapeutic agent (including but not limited to antibodies to vascular endothelial growth factor, EGFR, Her2 / neu, vascular endothelial growth factor receptor, other growth factor receptors, CD20, CD40, CD-40L, OX-40, 4-1BB, and ICOS), an immunogenic agent (e.g., attenuated cancer cells, tumor antigens, antigen presenting cells, such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF), and cells transfected with genes encoding immunostimulatory cytokines such as but not limited to GM-CSF).

[0260] As described above, in some embodiments of the method of the present invention, the method further comprises administering an additional therapeutic agent. In a specific embodiment, the additional therapeutic agent is an anti-LAG3 antibody or an antigen-binding fragment thereof, an anti-GITR antibody or an antigen-binding fragment thereof, an anti-TIGIT antibody or an antigen-binding fragment thereof, an anti-CD27 antibody or an antigen-binding fragment thereof. In one embodiment, the additional therapeutic agent is a Newcastle disease virus vector expressing IL-12. In a further embodiment, the additional therapeutic agent is denacil.

[0261] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneamines and methylamylamines, including octrimecide, triethyleneamine, triethylenephosphamide, triethylenethiophosphamide, and trishydroxymethylmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozel esin, carzelesin and bizelesin synthetic analogs); scutellarin (particularly scutellarin 1 and scutellarin 8); dostatin; duocarmycins (including synthetic analogs, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamineoxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, clozotocin, fortemustine, lomustine, nimustine, laromustine; antibiotics, such as diacetylene antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin phi1I, see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carzinophilin, chromophores, dactinomycin, daunorubicin, desoxorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolidonedoxorubicin, and antimetabolites such as methotrexate and 5-fluorouracil (5-FU);folic acid analogs, such as norpterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs, such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calcena, propoxyphenanolone, epithianol, mepistatin, butyrolactone; antiadreners, such as aminoglutamine, mitotane, tristanolactone; folic acid supplements, such as folic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilones; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazine; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; podophyllinic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulanol; piperobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; mitoxantrone; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine;and pharmaceutically acceptable salts, acids or derivatives of any of the above substances. Also included are anti-hormonal drugs used to regulate or inhibit tumor hormone effects, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, proxifen, keoxifene, LY117018, onapristone and toremifene (Fareston); aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as 4 (5) -imidazole, aminoglutamine, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole and anastrozole; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above substances. ;

[0262] In some embodiments comprising the step of administering an additional therapeutic agent (i.e., in addition to an anti-PD-1 antibody (e.g., pembrolizumab) or antigen-binding fragment thereof and an anti-CTLA4 antibody or antigen-binding fragment thereof), the additional therapeutic agent in the combination therapy can be administered using the same dosing regimen (dose, frequency, and duration of treatment) that is typically used when the therapeutic agent is used as a monotherapy to treat the same cancer. In other embodiments, the total amount of additional therapeutic agent received by the patient in the combination therapy is less than the total amount when treated alone, e.g., a smaller dose, less frequent dose, and / or a shorter duration of treatment.

[0263] The additional therapeutic agent in the combination therapy can be administered orally, intratumorally or parenterally, including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical and transdermal administration routes. For example, the combination therapy can include an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-CTLA4 antibody or antigen-binding fragment thereof, both of which can be administered intravenously or subcutaneously, and a chemotherapeutic agent that can be administered orally.

[0264] The combination therapy of the present invention can be used before or after surgery to remove a tumor, and can be used before, during or after radiation therapy. The combination therapy of the present invention can also be used when the patient's tumor is unresectable.

[0265] In some embodiments, the combination therapy of the invention is administered to patients who have not been previously treated with a biologic or chemotherapeutic agent, i.e., patients who have failed to respond to treatment. In other embodiments, the combination therapy is administered to patients who have failed to achieve a sustained response after previous treatment with a biologic or chemotherapeutic agent, i.e., patients who have experience with treatment.

[0266] The combination therapy of the present invention can be used to treat tumors that are large enough to be detected by palpation or imaging techniques well known in the art (e.g., magnetic resonance imaging, ultrasound, or computed tomography). In some embodiments, the combination therapy of the present invention is used to treat tumors that are at least about 200 mm in size. 3 、300mm 3 , 400mm 3 , 500mm 3 , 750mm 3 or up to 1000mm 3 of advanced tumors.

[0267] In some embodiments, the combination therapy of the present invention is administered to a human patient with a cancer that expresses PD-L1. In some embodiments, PD-L1 expression is detected using a diagnostic anti-human PD-L1 antibody or antigen-binding fragment thereof in an IHC assay performed on FFPE or frozen tissue sections of a tumor sample removed from the patient. The patient's physician may order a diagnostic test to determine the expression of PD-L1 in a tumor tissue sample removed from the patient before initiating treatment with an anti-PD-1 antibody or antigen-binding fragment thereof, but it is contemplated that the physician may order a first or subsequent diagnostic test at any time after initiating treatment, such as after a treatment cycle has ended.

[0268] The dose of the additional therapeutic agent selected depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissues, or organs in the treated individual. The dose of the additional therapeutic agent should achieve an acceptable level of side effects. Therefore, the dose and frequency of administration of each additional therapeutic agent (e.g., a biotherapeutic or chemotherapeutic agent) will depend in part on the specific therapeutic agent, the severity of the cancer being treated, and the patient's characteristics. We provide guidance for selecting appropriate doses of antibodies, cytokines, and small molecules. See, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348: 601-608; Milgrom et al. (1999) New Engl. Engl.J.Med.344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342: 613-619; Ghosh et al. (2003) New Engl. J. Med. 348: 24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). The clinician can determine the appropriate dosing regimen, for example, using parameters or factors known or suspected in the art to affect treatment or expected to affect treatment, and will depend on, for example, the patient's clinical history (e.g., previous treatments), the type and stage of cancer to be treated, and biomarkers of response to one or more therapeutic agents in the combination therapy.

[0269] V. Compositions and kits

[0270] The present invention also relates to a composition comprising a dose of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier or excipient, wherein the dose is about 400 mg. The anti-PD-1 antibody can be produced in, for example, CHO cells using conventional cell culture and recovery / purification techniques.

[0271] In an embodiment of the invention, the composition further comprises a histidine buffer at about pH 5.0 to pH 6.0. In a specific embodiment, the concentration of histidine is about 10 mM.

[0272] In an embodiment of the invention, the composition further comprises sucrose. In a specific embodiment, the sucrose is present at a concentration of about 70 mg / mL.

[0273] In an embodiment of the invention, the composition further comprises polysorbate 80. In a specific embodiment, polysorbate 80 is present at a concentration of about 0.2 mg / mL.

[0274] In some embodiments, the composition comprises 10 mM histidine, pH 5.5, 7% sucrose, 0.02% polysorbate 80, and 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0275] In an embodiment of the invention, the composition is a liquid.

[0276] In an alternative embodiment, the composition is freeze-dried.

[0277] In the composition of the present invention, the anti-PD-1 antibody or its antigen-binding fragment can be any antibody and antigen-binding fragment described herein, i.e., any antibody and antigen-binding fragment shown in Section 2 of the detailed description of the present invention, "PD-1 antibodies and antigen-binding fragments that can be used in the present invention" (e.g., pembrolizumab).

[0278] In some embodiments, the composition comprising an anti-PD-1 antibody as a PD-1 antagonist can be provided as a liquid formulation, or prepared by reconstituting a freeze-dried powder with sterile water for injection before use. WO 2012 / 135408 describes the preparation of liquid and freeze-dried drugs comprising pembrolizumab suitable for use in the present invention.

[0279] In some embodiments, the anti-CTLA4 antibody is formulated as described in WO 2018 / 204343 (PCT / us2018 / 030420). In some embodiments, the anti-CTLA4 antibody and the anti-PD-1 antibody are co-formulated as described in WO 2018 / 204343.

[0280] The present invention also relates to a kit for treating a cancer patient, the kit comprising: (a) 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof, and (b) instructions for using the anti-PD-1 antibody or an antigen-binding fragment thereof in any of the cancer treatment methods described herein.

[0281] The present invention also relates to a kit for treating a cancer patient, the kit comprising: (a) about 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof, (b) about 25 mg, 50 mg, 75 mg, or 100 mg of an anti-CTLA4 antibody or an antigen-binding fragment thereof, and (c) instructions for using the anti-PD-1 antibody or its antigen-binding fragment and the anti-CTLA antibody or its antigen-binding fragment in any of the cancer treatment methods described herein. In one embodiment, the kit comprises 25 mg of an anti-CTLA4 antibody. In one embodiment, the kit comprises 50 mg of an anti-CTLA4 antibody. In another embodiment, the kit comprises 75 mg of an anti-CTLA4 antibody. In a further embodiment, the kit comprises 100 mg of an anti-CTLA4 antibody.

[0282] In any kit of the present invention, the PD-1 antibody or antigen-binding fragment may be any antibody or antigen-binding fragment set forth in Section 2 of the Detailed Description of the Present Invention, "PD-1 Antibodies and Antigen-binding Fragments Useful in the Present Invention". In addition, in any kit of the present invention, the CTLA4 antibody or antigen-binding fragment may be any antibody or antigen-binding fragment described in Section 3 of the Detailed Description of the Present Invention, entitled "Anti-CTLA4 Antibodies and Antigen-binding Fragments Useful in the Present Invention".

[0283] The kit of the present invention may provide anti-PD-1 antibodies or antigen-binding fragments thereof and anti-CTLA4 or antigen-binding fragments thereof and package inserts in separate containers. The kit of the present invention may provide anti-PD-1 antibodies or antigen-binding fragments thereof and anti-CTLA4 antibodies or antigen-binding fragments thereof together in the same formulation (e.g., as a co-formulation). The container of the kit contains at least one dose (i.e., about 400 mg) of a drug containing an anti-PD-1 antibody or antigen-binding fragment thereof and at least one dose (e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg) of a drug containing an anti-CTLA4 antibody or antigen-binding fragment thereof and a package insert or label, the package insert or label containing instructions for using the drugs contained therein to treat cancer patients. The container may be composed of the same or different shapes (e.g., bottles, syringes, and bottles) and / or materials (e.g., plastic or glass). The kit may also include other materials that can be used for administration, such as diluents, filters, intravenous bags and lines, needles, and syringes. In some preferred embodiments of the kit, the instructions state that the drug is intended for use in treating patients with tumors, wherein the tumor is determined to express PD-L1 by, for example, an IHC test. In some embodiments, the tumor has a tumor proportion score (TPS) of ≥1% PD-L1. In another embodiment, the tumor has a TPS of ≥50% PD-L1. PD-L1 TPS refers to the number of tumor cells in a sample expressing PD-L1. In a further embodiment, the tumor has a TPS of ≥5% PD-L1, ≥10% PD-L1, ≥15% PD-L1, ≥20% PD-L1, ≥25% PD-L1, ≥30% PD-L1, ≥35% PD-L1, ≥40% PD-L1, or ≥45% PD-L1. In another embodiment, the patient's tumor expresses PD-L1, CPS ≥10%. In another embodiment, the patient's tumor expresses PD-L1, CPS ≥5%. In another embodiment, the patient's tumor expresses PD-L1, CPS ≥1%.

[0284] These and other aspects of the invention, including the exemplary embodiments set forth below, will become apparent from the teachings contained herein.

[0285] General approach

[0286] Standard methods of molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 1989 2 nd Edition,2001 3 rdEdition) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods are also found in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutation generation in bacterial cells (Vol. 1), cloning in mammals and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0287] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described in (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described in (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) Bio Directory, Piscataway, NJ, pp. 384-391). The production, purification and fragmentation of polyclonal and monoclonal antibodies are described in (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0288] Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1999) J. Biol. Chem. 275:27371-27378). al. (1997) J. Biol. Chem. 272: 10678-10684; Chothia et al. (1989) Nature 342: 877-883; Foote and Winter (1992) J. Mol. Biol. 224: 487-499; US Pat. No. 6, 329, 511).

[0289] An alternative to humanization is to use human antibody libraries displayed on phage or in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

[0290] Purification of the antigen is not necessary for the production of antibodies. Animals can be immunized with cells carrying the antigen of interest. Splenocytes can then be isolated from the immunized animal and fused with myeloma cell lines to produce hybridomas (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana et al. (1999) J. Immunol. 163:5157-5164).

[0291] The antibody can be conjugated, for example, to a small drug molecule, an enzyme, a liposome, polyethylene glycol (PEG). The antibody can be used for therapeutic, diagnostic, kit or other purposes, and includes antibodies coupled, for example, to dyes, radioisotopes, enzymes or metals such as colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol. 146: 169-175; Gibellini et al. (1998) J. Immunol. 160: 3891-3898; Hsing and Bishop (1999) J. Immunol. 162: 2804-2811; Everts et al. (2002) J. Immunol. 168: 883-889).

[0292] Methods for flow cytometry, including fluorescence activated cell sorting (FACS), are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2 nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies for use as, for example, diagnostic reagents are available (Molecular Probesy (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).

[0293] Standard methods for the histology of the immune system are described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Philadelphia, PA; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY). Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folds, functional domains, glycosylation sites, and sequence alignments are available (see, e.g., GenBank, Vector Library, and GenBank). Suite (Informax, Inc., Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al. (2000) Bioinformatics 16:741-742; Menne, et al. (2000) Bioinformatics Applications Note 16: 741-742; Wren, et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0294] All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing methodologies and materials that might be used in the present invention.

[0295] While different embodiments of the present invention have been described herein with reference to the accompanying drawings, it should be understood that the invention is not limited to these precise embodiments and that various changes and modifications may be made thereto by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0296] Example 1

[0297] Six-week (Q6W) dosing schedule of pembrolizumab in multiple tumor types based on evaluation using modeling and simulation

[0298] Pembrolizumab is an anti-PD-1 checkpoint inhibitor currently approved for multiple cancer indications that has demonstrated safety and efficacy when administered at doses of 200 mg or 2 mg / kg Q3W. Alternative extended dosing schedules would provide benefits of convenience and flexibility to both patients and prescribers. The robust nature of pembrolizumab pharmacokinetics (PK) and exposure (concentration)-response (ER) relationships for efficacy and safety allow the use of model-based approaches to support alternative dosing schedules for pembrolizumab.

[0299] After reaching PK steady-state, the dose of the Q6W regimen of pembrolizumab was selected by matching exposure to the approved Q3W (200 mg and 2 mg / kg) regimen; based on the understanding of ER, the efficacy and safety between the two treatment regimens were bridged. PK exposures for up to 24 weeks of dosing were simulated using an established population PK model for pembrolizumab (with time-dependent elimination) to ensure that all subjects were at steady state, which adequately described PK in multiple tumor types. Efficacy was bridged using exposure metrics AUCss or time-averaged concentrations (Cavg, ss) and trough concentrations (Cmin, ss) at steady state, which were compared between regimens. A bridge to the safety profile of pembrolizumab in the Q6W schedule was established by ensuring that the predicted peak concentrations at steady state (Cmax, ss) were lower than those of the maximum clinically administered dose of 10 mg / kg Q2W and well-tolerated doses.

[0300] The PK of pembrolizumab following dosing at 400 mg Q6W is predicted to follow a similar profile to that of the approved 200 mg Q3W and 2 mg / kg Q3W dosing regimens (see Figure 4). Table 4 summarizes the exposure metrics compared between the regimens. The 400 mg Q6W dosing regimen of pembrolizumab is based on the same regimen as the 200 mg Q3W (see Figure 4). Figure 3 ) were selected compared to similar predicted exposures (Cavg,ss or AUCss, geometric mean (GM) approximately 1% higher). Less than 1% of the subjects were predicted to have Cmin,ss below 200 mg Q3W and 2 mg / kg Q3W ( Figure 3 The predicted Cmax,ss of 400 mg Q6W is much lower than that achieved with 10 mg / kg Q2W (GM is approximately 65% ​​lower), which has been shown to have an acceptable safety profile in multiple tumor types (see Figure 2 Given the similar exposure profiles and established ER relationships of pembrolizumab at the clinically tested doses, clinical results achieved with 400 mg Q6W are expected to be similar to those with 200 mg Q3W across tumor types.

[0301] Based on the modeling and simulation methods used herein, it is expected that the 400 mg Q6W dosing regimen of pembrolizumab will result in PK exposures similar to the approved 200 mg Q3W and 2 mg / kg dosing regimens. PK simulations demonstrated that the average concentrations over the dosing interval (Cavg) (or area under the curve [AUC]) at 400 mg Q6W were similar to those at the approved 200 mg Q3W dose in terms of pembrolizumab exposure, thus bridging the efficacy between dosing regimens. Trough concentrations (Cmin) at 400 mg Q6W were generally within the range of those achieved at 2 mg / kg or 200 mg Q3W in most patients (greater than 99%). Peak concentrations (Cmax) at 400 mg Q6W were much lower than the Cmax of the highest clinically tested dose of 10 mg / kg Q2W, supporting that the safety profile of 400 mg Q6W should be comparable to the established safety profile of pembrolizumab. The exposure-response (ER) of pembrolizumab was demonstrated to be stable across the indications, and OS predictions in melanoma and NSCLC demonstrated that the effects of 400 mg Q6W were expected to be similar to those of 200 mg or 2 mg / kg Q3W given similar exposures; therefore, 400 mg Q6W is expected to be effective in the indications.

[0302] Table 4. Summary of PK exposure parameters for pembrolizumab based on simulated 400 mg Q6W dosing regimen

[0303]

[0304]

[0305] Example 2

[0306] A Phase I Randomized Clinical Study of Pembrolizumab to Evaluate the Safety and Tolerability of 400 mg Pembrolizumab Infused Q6W in Participants with Advanced Melanoma

[0307] This study was designed to evaluate the pharmacokinetics (PK), safety, and tolerability of pembrolizumab when administered every 6 weeks (Q6W). A group of 100 subjects received 400 mg pembrolizumab Q6W. PK, efficacy, and safety data were collected from this group of participants. Male / female participants with advanced melanoma who were at least 18 years old were included in the study. No stratification based on age, sex, or other characteristics was used in this study.

[0308] Participants received IV injections of 400 mg pembrolizumab Q6W in cycles 1 to 18. PK, efficacy, and safety data were collected from these participants. The results provide preliminary PK, efficacy, and safety data for pembrolizumab when administered Q6W. Based on a deep understanding of the clinical pharmacology of pembrolizumab and its improved ER profile, it is expected that such a change in dosing regimen will produce similar efficacy and safety in all approved treatment settings for 200 mg Q3W pembrolizumab, including monotherapy and in combination with other agents. Therefore, based on modeling and simulation analysis, the 400 mg Q6W regimen has a similar benefit-risk profile to 200 mg Q3W, which is a less frequent dosing regimen in clinical use of pembrolizumab (see Example 1).

[0309] Study Design

[0310] This study was a randomized, crossover, multicenter, open-label safety study of pembrolizumab in participants with advanced melanoma, conducted in accordance with Good Clinical Practice (GCP). This Phase 1 study was conducted in patients with unresectable or metastatic melanoma. The treatment period lasted for 18 cycles (approximately 2 years) every 42 days. Treatment continued as long as the participant benefited from treatment and did not have disease progression or meet any study withdrawal criteria. In more detail, the study consisted of: (1) a screening period of up to 28 days to ensure that participants were eligible for the study; (2) an intervention period of approximately 104 weeks of pembrolizumab treatment. Participants received pembrolizumab by intravenous infusion, 30 minutes Q6W, for up to 18 cycles, and (3) a follow-up period during which participants were monitored for adverse events for 30 days and serious adverse events (SAEs) for 90 days (30 days if the participant started new anticancer treatment). Participants with persistent adverse events at the time of discontinuation of treatment were followed until the problem was resolved, stabilized, the event was otherwise explained, or the participant could no longer be followed.

[0311] Participants who discontinued for reasons other than radiographic disease progression had post-treatment follow-up imaging of disease status until radiographic documentation of disease progression according to RECIST 1.1 and confirmed by sites according to iRECIST when clinically appropriate, initiation of non-study cancer treatment, withdrawal of consent, loss to follow-up, or end of study. All participants were followed by telephone during the survival follow-up period for overall survival until death, participant withdrawal of consent, loss to follow-up, or end of study.

[0312] All participants enrolled in this study will be diagnosed with advanced melanoma. The results of this study will help understand the PK characteristics of pembrolizumab when administered at a Q6W dosing schedule. Safety parameters commonly used to evaluate investigational systemic anticancer therapies are included as safety endpoints, including but not limited to the incidence, causality, and outcome of adverse events (AEs) / serious adverse events (SAEs); and changes in vital signs and laboratory values. Adverse events will be evaluated according to the definitions of the National Cancer Institute Common Terminology Criteria for Adverse Events [NCI CTCAE] version 4.0.

[0313] One objective of this trial was to characterize the PK profile of pembrolizumab following intravenous Q6W dosing. PK data were analyzed after all participants completed cycle 5. PK parameters included AUC, Cmax, and Cmin. The formation of anti-drug antibodies (ADA) can potentially confound drug exposure at therapeutic doses and induce subsequent infusion-related toxicity. Anti-drug antibody responses to pembrolizumab were determined at the start of each of the first, second, fourth, and fifth cycles. Any effect of the presence of ADA on pembrolizumab exposure was explored.

[0314] This study used ORR assessed by blinded independent central review (BICR) based on RECIST 1.1 criteria as the primary endpoint. Objective response rate is an accepted measure of clinical benefit for late-stage studies demonstrating superiority of a new anti-tumor therapy, especially when the magnitude of effect is large and the treatment has an acceptable risk / benefit profile. Images were submitted to an imaging CRO (iCRO) and read by an independent central reviewer who was blinded to treatment assignment to minimize bias in response assessment.

[0315] Overall survival (OS) is a secondary endpoint that has been recognized as the gold standard for demonstrating the benefit of a new anti-cancer therapy in a randomized clinical study. RECIST 1.1 is used by BICR when evaluating effectiveness measures on images and by local sites when determining eligibility. The RECIST Working Group, with the participation of leading experts from industry and academia, and with the participation of the U.S. Food and Drug Administration and the European Medicines Agency, developed and published modified RECIST 1.1 for the evaluation of immune-based therapeutics (iRECIST). The unidimensional measurement of target lesions, qualitative assessment of non-target lesions, and response categories are the same as RECIST 1.1 until progression is seen by RECIST 1.1. However, if the participant is clinically stable, additional imaging may be performed to confirm radiographic progression. Investigators use iRECIST to assess tumor response and progression and make treatment decisions, as well as to conduct exploratory efficacy analyses when specified.

[0316] Inclusion criteria

[0317] Participants were eligible for inclusion in the study only if all of the following criteria applied:

[0318] ●Participants had a histologically or cytologically confirmed diagnosis of advanced melanoma.

[0319] ●Participants had unresectable stage III or IV melanoma according to the American Joint Committee on Cancer (AJCC) staging system that was not amenable to local therapy.

[0320] ● Participants were not receiving treatment for advanced or metastatic disease, except for the following:

[0321] Patients with BRAF V600 mutant melanoma may have received standard targeted therapy (e.g., BRAF / MEK inhibitors alone or in combination) and be eligible for this study.

[0322] ● Prior adjuvant or neoadjuvant melanoma therapy was allowed if completed at least 4 weeks before randomization and all relevant adverse events had resolved to baseline or were stable (resolution of toxic effects of the most recent prior therapy to grade 1 or less [except alopecia]). If participants had received major surgery or radiation therapy greater than 30 Gy, they must have recovered from toxicity and / or complications of the intervention.

[0323] Female participants were eligible to participate if they were not pregnant, not breastfeeding, and agreed to follow specific contraceptive instructions during treatment and for at least 120 days or provided informed consent.

[0324] Participants should have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 (fully active, able to perform all pre-disease manifestations without limitation) or 1 (limited to vigorous exercise but ambulatory and able to perform work of a light or sedentary nature, such as light indoor work, office work) and should have adequate organ function as defined in Table 5. Samples were collected within 72 hours before the start of the study intervention.

[0325] Table 5. Adequate organ function laboratory values

[0326]

[0327]

[0328] Exclusion criteria

[0329] Participants were excluded from the study if they met any of the following criteria:

[0330] ●Participants are women of childbearing potential (WOCBP) with a positive urine pregnancy test within 72 hours prior to randomization or treatment assignment. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test is required.

[0331] ●Participants have previously received systemic treatment for unresectable or metastatic melanoma (unless noted in the inclusion criteria above).

[0332] ●Participants have previously received anti-PD-1, anti-PD-L1, or anti-PD-L2 therapy, or have received treatment with an agent targeting another stimulatory or co-inhibitory T-cell receptor (e.g., OX-40 and CD137), or have received any other antibody or drug specifically targeting a checkpoint pathway, except for anti-CTLA-4, which is allowed in the adjuvant setting.

[0333] ● Participants had received radiation therapy within 2 weeks of starting study treatment. Participants must have recovered from all radiation-related toxicities, not required corticosteroids, and not developed radiation pneumonitis.

[0334] ● Participants received a live vaccine within 30 days before the first dose of study drug. Examples of live vaccines include, but are not limited to: measles, mumps, rubella, varicella / zoster (chickenpox), yellow fever, rabies, Bacillus Calmette-Guérin (BCG), and typhoid vaccines. Injectable seasonal influenza vaccines, which are usually inactivated virus vaccines, are permitted; however, intranasal influenza vaccines (e.g., ) is a live attenuated vaccine and is not allowed.

[0335] ● Participant is currently participating in or has participated in a study of an investigational drug or has used an investigational device within 4 weeks prior to the first dose of the study intervention.

[0336] ●Participants were diagnosed with immunodeficiency or were receiving long-term systemic steroid therapy (doses exceeding 10 mg prednisone equivalents per day) or any other form of immunosuppressive therapy within 7 days before the first dose of study drug.

[0337] ● Participants have known other malignancies that are developing or require active treatment in the past 2 years. Note: Participants with basal cell carcinoma of the skin, squamous cell carcinoma of the skin, or carcinoma in situ (such as breast cancer, carcinoma in situ) who have received potential treatment are not excluded.

[0338] ● Participants with known CNS metastases and / or carcinomatous meningitis. Participants with previously treated brain metastases may participate if they are radiologically stable (i.e., without signs of progression) for at least 4 weeks as determined by repeat imaging (note that repeat imaging should be performed during the study screening period), clinically stable, and steroid-free for at least 14 days prior to the first dose of study intervention.

[0339] ● Participants have severe allergy (≥Grade 3) to pembrolizumab and / or any of its excipients. ● Participants have ocular melanoma.

[0340] ●Participants had an active autoimmune disease that required systemic treatment (ie, use of disease-modifying agents, corticosteroids, or immunosuppressive medications) in the past 2 years. Replacement therapy (eg, thyroxine, insulin, or physiologic corticosteroid replacement for adrenal or pituitary insufficiency) was not considered a form of systemic therapy and was permitted.

[0341] ● Participants have a history of (non-infectious) pneumonia requiring steroids or current pneumonia. ● Participants have active infection requiring systemic treatment.

[0342] ●Participants have a known history of human immunodeficiency virus (HIV) infection.

[0343] ●Participants had a known history of hepatitis B (defined as hepatitis B surface antigen [HBsAg] reactivity) or known active hepatitis C virus (defined as detectable HCV RNA (qualitative)) infection.

[0344] ● History or current evidence of any illness, treatment, or laboratory abnormality in the participant that could confound the study results, interfere with the participant's participation throughout the study, or be treated in a manner that the investigator determines is not in the best interest of the participant's participation.

[0345] ● The participant has a known mental illness or substance abuse disorder that would interfere with cooperation with the research requirements.

[0346] ● Participants are pregnant or breastfeeding, or anticipate pregnancy or childbearing, during the expected duration of the study (starting from the screening visit to 120 days after the last dose of the study intervention).

[0347] Discontinuation of study intervention and participant withdrawal

[0348] Discontinuation of the study intervention does not constitute withdrawal from the study. Because certain data on clinical events after discontinuation of the study intervention may be important to the study, these data must be collected by the participant's last scheduled follow-up visit, even if the participant has discontinued the study intervention. Therefore, all participants who discontinue the study intervention before the end of the protocol-specified treatment period will continue to participate in the study.

[0349] Participants may discontinue the study intervention at any time for any reason or withdraw from the study intervention at the investigator's discretion if any adverse reaction occurs. In addition, the investigator may discontinue a participant's study intervention if the study intervention is inappropriate, violates the study protocol, or for administrative and / or other safety reasons.

[0350] Participants had to discontinue the study intervention but continued to be monitored in the study for any of the following reasons:

[0351] ● The participant or his / her legally acceptable representative requests that the study intervention be discontinued.

[0352] ●Participants discontinued study intervention dosing for more than 12 consecutive weeks or had three cumulative missed doses.

[0353] ● The investigator believes that the participant's medical condition or personal circumstances place him or her at unnecessary risk from continued study intervention.

[0354] ●Participants had a confirmed positive serum pregnancy test.

[0355] ●Participant has confirmed radiographic disease progression.

[0356] ● Participants experience progression or recurrence of any malignancy, or develop other malignancies requiring active treatment.

[0357] ●Participants had unacceptable adverse experiences.

[0358] ● The participant has a concurrent disease other than another malignancy listed above that precludes further treatment.

[0359] ●The investigator decided to stop treatment.

[0360] ●Participant has recurrent secondary pneumonia.

[0361] ●Participants had completed 35 treatments with pembrolizumab (approximately 2 years).

[0362] If a participant or their legally acceptable representative withdraws consent for the study, the participant is withdrawn from the study. If a participant withdraws from the study, they will no longer receive study treatment or be followed at scheduled protocol visits.

[0363] Informed consent

[0364] Prior to participation in a clinical research study, the researcher or medically qualified designee shall obtain written consent from each potential participant or each participant’s legally acceptable representative. If a participant’s status changes during the research (e.g., health or age of majority requirement), the researcher or medically qualified designee shall ensure that appropriate consent is obtained.

[0365] Effectiveness / Evaluation

[0366] Tumor evaluation includes all known or suspected sites of disease. Imaging may include computed tomography (CT) or magnetic resonance imaging of the chest, abdomen, and pelvis at baseline and when disease progression or brain metastases are suspected. Obtaining tumor images by CT is strongly preferred. For the chest, abdomen, and pelvis, contrast-enhanced MRI may be used when CT with iodinated contrast is contraindicated or when local practice dictates. For the brain, MRI is the strongly preferred imaging modality.

[0367] Participants should use the same imaging modality technology (ideally using the same scanner, and consistent use of contrast) throughout the study. Consistent use of imaging technology will help optimize the reproducibility of assessments of existing and new tumor burden and improve the accuracy of assessments of response or progression. All scheduled images for all study participants should be reviewed by the investigator for disease progression. In addition, images obtained at unscheduled time points for determination of disease progression (including images obtained by other modalities) (as well as images obtained for other reasons but based on the investigator's assessment to capture radiologic progression) should also be archived at the study site.

[0368] Confirmation of measurable disease based on RECIST 1.1 at screening by the BICR will be used to determine participant eligibility. Prior to assignment of participants, BICR confirmation that the participant's imaging shows at least one lesion suitable for selection as a target lesion based on RECIST 1.1 is required.

[0369] Initial tumor imaging

[0370] Initial tumor imaging at screening was performed within 28 days prior to the first dose. Any imaging obtained after treatment on Day 1 of Cycle 1 was not included in the screening assessment. Screening images were reviewed by the site study team to confirm the presence of measurable disease in the participant according to RECIST 1.1. If brain imaging was performed to document the stability of existing metastases, MRI was used when possible. If MRI was medically contraindicated, CT with contrast was an acceptable alternative.

[0371] Tumor imaging during the study

[0372] Imaging assessments in the first study were performed 12 weeks (84 ± 7 days) after the first dose. Subsequent tumor imaging was performed every 9 weeks (63 ± 7 days) or more frequently as clinically indicated. After 52 weeks (365 ± 7 days), participants who continued to receive treatment would be imaged every 12 weeks (84 ± 7 days).

[0373] Objective response is confirmed by repeat imaging assessment. Tumor imaging will be performed at least 4 weeks after the first sign of response is observed to confirm PR or CR. Participants will then return to regularly scheduled imaging starting at the next scheduled imaging time point. Participants who undergo additional imaging for confirmation, if it is less than 4 weeks later, do not need to undergo the next scheduled tumor imaging; tumor imaging can be resumed at the subsequent scheduled imaging time point.

[0374] Disease progression was confirmed on-site 4 to 8 weeks after the first radiographic evidence of progressive disease (PD) in clinically stable participants according to modified iRECIST. Participants without confirmed disease progression could continue treatment at the investigator's discretion until progression was confirmed on-site. Participants who underwent confirmatory imaging were not required to undergo the next scheduled tumor imaging if it was less than 4 weeks later; tumor imaging could resume at a later scheduled imaging time point if clinically stable. Participants with confirmed disease progression by iRECIST, based on on-site assessment, would discontinue study treatment.

[0375] End of Treatment and Follow-up Tumor Imaging

[0376] For participants who discontinued the study intervention, tumor imaging was performed at the time of treatment discontinuation (±4-week window). Imaging at treatment discontinuation was not mandatory if the prior imaging was obtained within 4 weeks prior to the discontinuation date. For participants who discontinued the study intervention due to documented disease progression, this was the final required tumor imaging if the investigator chose not to perform iRECIST.

[0377] For participants who discontinue study intervention without documented disease progression, every effort should be made to continue monitoring disease status by tumor imaging using the same imaging schedule used while on treatment every 12 weeks (±7 days) until initiation of new anticancer treatment, disease progression, pregnancy, death, withdrawal of consent, or end of study, whichever occurs first.

[0378] RECIST 1.1 Disease Assessment

[0379] RECIST 1.1 was used as the primary endpoint for assessing tumor response, date of disease progression, and as the basis for all protocol guidelines related to disease status (e.g., discontinuation of study interventions). Although RECIST 1.1 mentions a total of up to 5 target lesions, 2 per organ, the protocol allows a total of up to 10 target lesions, 5 per organ, if clinically relevant, enabling a more extensive sampling of tumor burden.

[0380] iRECIST Disease Assessment

[0381] iRECIST is based on RECIST 1.1 but is adapted to account for unique tumor responses seen from immunotherapy drugs. Investigators will use iRECIST to assess tumor response and progression and make treatment decisions. When clinically stable, participants will not be discontinued until the investigator, in collaboration with the local radiology department, confirms progression. This permission to continue treatment despite initial radiological PD takes into account the observation that some participants can experience transient tumor flares in the first few months after starting immunotherapy, followed by subsequent disease responses.

[0382] Any participant deemed clinically unstable discontinues study intervention at the first radiographic evidence of PD as assessed on site, and tumor imaging is not repeated to confirm PD by iRECIST. If the investigator decides to continue treatment, the participant may continue to receive study intervention, and tumor assessment should be repeated 4 to 8 weeks later to confirm PD by iRECIST as assessed by the investigator. If repeat imaging does not confirm PD by iRECIST as assessed by the investigator, and the participant continues to be clinically stable, study intervention will continue and follow the regular imaging schedule. If PD is confirmed, the participant discontinues study intervention.

[0383] If a participant had confirmed radiographic progression (iCPD), study intervention was discontinued; however, an exception was considered to continue study intervention if the participant achieved a clinically meaningful benefit. In this case, tumor imaging continued if study intervention was continued. Table 6 summarizes imaging and treatment requirements after first radiographic evidence of progression.

[0384] Table 6. Imaging and treatment after first radiographic evidence of progressive disease

[0385]

[0386]

[0387] Safety Assessment

[0388] Safety assessments included the collection of AEs and SAEs, monitoring of vital signs and laboratory assessments (including pregnancy testing), performance of electrocardiograms (ECGs) and physical examinations, and verification of concomitant medications.

[0389] Adverse Events

[0390] The investigator or qualified designee will assess each participant for the presence of potentially new or worsening AEs, more frequently if clinically indicated. AEs will be assessed including, but not limited to, type, incidence, severity (graded by the National Cancer Institute Common Terminology Criteria for Adverse Events [NCI CTCAE] version 4.0), timing, severity, and relatedness to study drug. Adverse events occurring during the study will be recorded, including baseline signs and symptoms.

[0391] Comprehensive physical examination

[0392] The investigator or qualified designee performed a complete physical examination during the screening period. Clinically significant abnormal findings were recorded as medical history. New clinically significant abnormal findings after the first dose of study intervention were recorded as AEs.

[0393] Targeted physical examination

[0394] For cycles where a full physical examination was not required, the Investigator or qualified designee performed a clinically indicated direct physical examination prior to administration of the study intervention. New clinically significant abnormal findings were recorded as AEs.

[0395] Signs of life

[0396] After resting for 5 minutes, vital signs were measured in the semi-supine position, including temperature, systolic and diastolic blood pressure, respiratory rate, pulse rate, and weight. Height was collected only at screening.

[0397] Electrocardiogram

[0398] A standard 12-lead ECG was performed using local standard procedures. Clinically significant abnormal findings at screening were recorded as medical history. Additional ECGs were performed during the study when clinically indicated. Clinically significant findings found on follow-up ECGs were recorded as AEs.

[0399] Clinical safety laboratory evaluation

[0400] The tests detailed in Table 7 were performed by local laboratories. Additional testing was performed at any time during the study as determined necessary by the investigator.

[0401] Table 7 Safety laboratory evaluation required by the protocol

[0402]

[0403]

[0404] Time period and frequency of collecting information on AEs, SAEs, and other reportable safety events

[0405] All AEs, SAEs, and other reportable safety events occurring after the signing of the informed consent form but before treatment assignment / randomization must be reported by the investigator if the participant is receiving placebo pre-stimulation or other pre-stimulation treatment, if the event leads to the participant's exclusion from the study, or is the result of a protocol-specific intervention (including but not limited to washout or discontinuation of usual care, diet, or procedures). All AEs from the time of treatment assignment / randomization until 30 days after discontinuation of the study intervention must be reported by the investigator.

[0406] All AEs meeting serious criteria must be reported by the investigator from the time of treatment assignment / randomization until 90 days after discontinuation of study intervention or 30 days after discontinuation of study intervention if the participant initiates new anticancer therapy, whichever comes first. In addition, any SAE brought to the attention of the investigator at any time outside of the above specified time periods is reported immediately if the event is considered drug-related.

[0407] Statistical Methods for Power Analysis

[0408] Objective Response Rate (ORR) - ORR was calculated as the ratio of the number of participants who reported having achieved a confirmed CR or PR validated by BICR divided by the number of participants included in the APaT cohort. Participants in the APaT analysis cohort without an ORR assessment were considered non-responders. The 95% exact binomial CI was calculated for the true ORR according to the method of Clopper and Pearson, 1934.

[0409] Progression-free survival (PFS) - The nonparametric Kaplan-Meier method was used to estimate the PFS distribution. The median PFS and 95% CI of the PFS point estimate will be calculated for different follow-up times from the first day of study treatment. Because disease progression is assessed regularly, PD can occur at any time in the interval between the last assessment in which PD is not recorded and the assessment in which PD is recorded. The true date of PD will be approximately the same as the date of the first assessment in which PD is objectively recorded by BICR according to RECIST 1.1. Death is always considered a PFS event. Participants who do not experience a PFS event will be censored at the last disease assessment. For the PFS analysis, if the event (PD or death) is immediately after more than one missed disease assessment, the data will be censored at the last disease assessment before the missed visit. In addition, data after new anticancer treatment will be censored at the last disease assessment before the start of new anticancer treatment. If a participant meets multiple censoring criteria, the earliest censoring criterion will be applied.

[0410] Overall Survival (OS) - OS distribution was estimated using the nonparametric Kaplan-Meier method. Median OS and 95% CI for OS point estimates were calculated for different follow-up times from the first day of study treatment.

[0411] Duration of Response (DOR)-DOR was summarized descriptively using the nonparametric Kaplan-Meier method. Only the subset of participants who showed a CR or PR was included in this analysis.

[0412] Analytical Strategy for Key Efficacy Endpoints

[0413] Table 8 summarizes the primary analysis methods for key efficacy endpoints.

[0414] Table 8. Analysis strategy for key efficacy endpoints

[0415]

[0416]

[0417] Statistical Methods for Safety Analysis

[0418] Safety and tolerability were assessed by clinical review of all relevant parameters, including adverse experiences and laboratory parameters. Broad AE categories, including the percentage of participants with any AE, drug-related AE, serious AE, drug-related and serious AE, and discontinuation due to AE, were summarized by point estimates with 95% CI (Table 9).

[0419] Table 9. Analysis strategy for safety parameters

[0420]

[0421]

[0422] An AE is any adverse medical event in a clinical study participant that is transiently associated with the use of the study intervention, whether or not considered related to the study intervention. Thus, an AE can be any unfavorable and unexpected sign (including abnormal laboratory findings), symptom, or disease (new or worsening) that is transiently associated with drug use. The following are included as AEs:

[0423] Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis)

[0424] or other safety assessments (e.g., ECG, radiological scans, vital sign measurements), including those that are worse from baseline or are considered clinically significant in the medical and scientific judgment of the Investigator.

[0425] ●Aggravation of a chronic or intermittent pre-existing condition, including an increase in the frequency and / or intensity of the condition.

[0426] ●A new condition detected or diagnosed after the study intervention is administered, even though it may have existed before the start of the study.

[0427] ●Signs, symptoms, or clinical sequelae of suspected drug interactions.

[0428] ●Signs, symptoms, or clinical sequelae of suspected overdose of the study intervention or concomitant medications.

[0429] • Worsening of signs and symptoms of malignancy during the study was reported as an AE. Disease progression assessed by radiographic or other methods of measuring malignant lesions was not reported as an AE unless the event resulted in hospitalization or death.

[0430] The following events did not meet the definition of AEs for the purposes of this study:

[0431] ●Medical or surgical procedures (eg, endoscopy, appendectomy): Conditions leading to such procedures are AEs.

[0432] ● Situation in which no adverse medical events occurred (social and / or convenient hospitalization).

[0433] ● Expected day-to-day fluctuations of pre-existing diseases or conditions present or detected at study entry, without exacerbation.

[0434] ●Surgery planned before informed consent to treat a pre-existing condition that has not worsened.

[0435] If an event is not an AE according to the above definition, it cannot be an SAE even if it meets the serious criteria. An SAE is defined as any adverse medical event occurring at any dose that:

[0436] ● Lead to death

[0437] ● Life-threatening. The term “life-threatening” in the definition of “serious” refers to events where the participant was at risk of death at the time of the event. It does not refer to events that, if more serious, could hypothetically result in death.

[0438] ● Requirement for inpatient hospitalization or prolongation of an existing hospitalization. Hospitalization is defined as an inpatient admission, regardless of the length of stay, even if the hospitalization is a precaution for ongoing observation. Hospitalization for an elective procedure to treat a pre-existing condition that has not worsened is not an SAE. A pre-existing condition is a clinical condition that was diagnosed prior to use of an MSD product and documented in the participant’s medical history.

[0439] ● Resulting in persistent or severe disability / incapacity. The term disability refers to a substantial impairment of a person's ability to carry out normal life functions. This definition is not intended to include experiences of relatively minor medical significance, such as simple headaches, nausea, vomiting, diarrhea, influenza, and accidental trauma that may interfere with or prevent daily life functions but does not constitute a substantial impairment (e.g., a sprained ankle).

[0440] ●A congenital anomaly / birth defect in a child of a participant taking this product, regardless of when diagnosed.

[0441] Exercise medical or scientific judgment in deciding whether SAE reporting is appropriate for other situations, such as important medical events that may not be immediately life-threatening or result in death or hospitalization but that could endanger the participant or may require medical or surgical intervention to prevent one of the other outcomes indicated in the above definitions. These events are generally considered serious. Examples of such events include invasive or malignant cancer, intensive treatment in the emergency room or at home for allergic bronchospasm that does not result in hospitalization, blood disorders, or seizures, or the development of drug dependence or drug abuse.

[0442] Demographics and baseline characteristics

[0443] The number and percentage of subjects screened, assigned, the main reason for screening failure, and the main reason for discontinuation are presented. Demographic variables (e.g., age, sex), baseline characteristics, primary and secondary diagnoses, and prior and concomitant treatments are summarized by descriptive statistics or categorical tables for all enrolled subjects.

[0444] Subgroup analysis

[0445] To determine whether the response rate was consistent across subgroups, estimates of the response rate for the primary endpoint (with nominal 95% CI) were estimated in the following categories of categorical variables:

[0446] Age category (<65 years vs ≥65 years)

[0447] Gender (female vs male)

[0448] Race (white vs non-white)

[0449] Disease stage (stage III vs IVM1a vs IVM1b vs IVM1c)

[0450] Brain metastases (yes vs no)

[0451] ECOG status (0 vs 1)

[0452] PD-L1 status (positive vs negative)

[0453] BRAF wild-type vs BRAF mutant (no prior treatment) vs BRAF mutant (prior treatment)

[0454] Forest plots were generated that provide estimated point estimates and CIs for the treatment effects in each of the above subgroups. Any specific subgroup with fewer than 10 participants was excluded from the analysis.

[0455] Example 3

[0456] Patients with PD-1-refractory melanoma were given 400 mg Q6W of pembrolizumab in combination with an anti-CTLA4 antibody.

[0457] Study Groups 1 and 2 will explore the anti-tumor activity of an anti-CTLA4 antibody (e.g., antibody 8D2H2L2 variant 1) with or without pembrolizumab in participants with advanced melanoma that is refractory to anti-PD1 / L1.

[0458] Group I: Participants will receive 25 mg of anti-CTLA4 antibody (e.g., antibody 8D2H2L2 variant 1) in combination with 400 mg of pembrolizumab on Day 1 of Cycle 1 and all subsequent cycles. Both anti-CTLA4 antibody and pembrolizumab will be continuously administered on a Q6W schedule for up to 2 years.

[0459] An interim safety analysis will be performed when the first 6 DLT-evaluable participants complete their DLT evaluations. If the observed DLT rate is higher than 25%, pembrolizumab 200 mg Q3W may be substituted for pembrolizumab 400 mg Q6W in newly enrolled participants.

[0460] Group II (n=up to 40): Participants will receive 25 mg of anti-CTLA4 antibody (eg, antibody 8D2H2L2 variant 1) as monotherapy on a Q6W schedule on Day 1 of Cycle 1 and all subsequent cycles for up to 2 years.

[0461] All references cited herein are incorporated by reference as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application or patent was specifically and individually introduced by reference. This statement of incorporation by reference is intended to be linked by applicant pursuant to 37 CFR § 1.57 (b) (1) to each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application or patent, each of which is specifically identified in accordance with 37 CFR § 1.57 (b) (2), even if such reference is not directly adjacent to a specific statement of incorporation by reference. Citation of a reference herein is not intended to admit that the reference is relevant prior art, nor does it constitute any admission of the contents or date of these publications or documents.

Claims

1. A method of treating cancer in a human patient, comprising administering to the patient about 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof every about six weeks, and administering to the patient about 25 mg, about 50 mg, about 75 mg, or about 100 mg of an anti-CTLA4 antibody or an antigen-binding fragment thereof every six weeks, wherein the anti-PD-1 antibody or the antigen-binding fragment thereof comprises: (a) a light chain complementary determining region (CDR) comprising the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3; and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8; or (b) a light chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 11, 12 and 13; and a heavy chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 14, 15 and 16; And wherein the anti-CTLA4 antibody or antigen-binding fragment thereof comprises: (c) a light chain CDR comprising the amino acid sequences shown in SEQ ID NOs: 39, 40 and 41; and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NOs: 36, 37 and 38; (d) a light chain CDR comprising the amino acid sequences shown in SEQ ID NOs: 39, 40 and 42; and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NOs: 36, 37 and 38; or (e) light chain CDRs comprising the amino acid sequences shown in SEQ ID NOs: 39, 40 and 43; and heavy chain CDRs comprising the amino acid sequences shown in SEQ ID NOs: 36, 37 and 38.

2. The method of claim 1, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9 or a variant of SEQ ID NO: 9, and (b) a light chain variable region comprising: (i) the amino acid sequence shown in SEQ ID NO: 4 or a variant of SEQ ID NO: 4, (ii) the amino acid sequence shown in SEQ ID NO:22 or a variant of SEQ ID NO:22, or (iii) the amino acid sequence shown in SEQ ID NO: 23 or a variant of SEQ ID NO:

23.

3. The method of claim 1 or 2, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

4.

4. The method of claim 1 or 2, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is a monoclonal antibody comprising: (a) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10 or a variant of SEQ ID NO: 10, and (b) a light chain comprising the amino acid sequence shown in SEQ ID NO:5, a variant of SEQ ID NO:5, SEQ ID NO:24, a variant of SEQ ID NO:24: SEQ ID NO:25, or a variant of SEQ ID NO:

25.

5. The method of any one of claims 1 to 4, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is a monoclonal antibody comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

5.

6. The method of any one of claims 1-5, wherein the anti-CTLA4 antibody or antigen-binding fragment thereof is a monoclonal antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:44; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:45; (b) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 46; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:47; (c) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 48; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:49; (d) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 50; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:49; (e) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 51; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:52; (f) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 53; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 54; or (g) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 55; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

56.

7. The method of claim 6, wherein the monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:50 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

49.

8. The method of claim 7, wherein the monoclonal antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:57 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

58.

9. A method for treating cancer in a human patient, comprising administering to each of the patients about 400 mg of an anti-PD-1 antibody and about 25 mg, about 50 mg, about 75 mg, or about 100 mg of an anti-CTLA4 antibody every six weeks, wherein the anti-PD-1 antibody comprises (i) a heavy chain comprising the amino acid sequence of SEQ ID NO:10 and (ii) a light chain comprising the amino acid sequence of SEQ ID NO:5, and wherein the anti-CTLA4 antibody comprises (iii) a heavy chain comprising the amino acid sequence of SEQ ID NO:57 and (iv) a light chain comprising the amino acid sequence of SEQ ID NO:

58.

10. The method of any one of claims 1-9, wherein the cancer is PD-1 / PD-L1 refractory melanoma.

11. The method of any one of claims 1-19, wherein the cancer is selected from melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, kidney cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, pancreatic cancer, brain tumor, glioblastoma, sarcoma, bone tumor or Merkel cell carcinoma.

12. The method of any one of claims 1-11, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the patient by intravenous or subcutaneous administration.

13. The method of any one of claims 1-12, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is pembrolizumab. E 14. The method of any one of E 1-13, wherein the anti-PD-1 antibody and the anti-CTLA4 antibody are co-administered.

15. The method of any one of claims 1-13, wherein the anti-PD-1 antibody and the anti-CTLA4 antibody are co-formulated.

16. The method of any one of claims 1-15, comprising administering 25 mg of the anti-CTLA4 antibody or antigen-binding fragment thereof.

17. The method of any one of claims 1-15, comprising administering 50 mg of the anti-CTLA4 antibody or antigen-binding fragment thereof.

18. The method of any one of claims 1-15, comprising administering 75 mg of the anti-CTLA4 antibody or antigen-binding fragment thereof.

19. The method of any one of claims 1-15, comprising administering 100 mg of the anti-CTLA4 antibody or antigen-binding fragment thereof.

20. A kit for treating a patient having cancer, the kit comprising: (a) about 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof, (b) about 25 mg, 50 mg, 75 mg, or 100 mg of an anti-CTLA4 antibody or antigen-binding fragment thereof; and (c) instructions for using the anti-PD-1 antibody or antigen-binding fragment thereof and the anti-CTLA-4 antibody or antigen-binding fragment thereof in the method of any one of claims 1-19.

21. The kit of claim 20, wherein the anti-PD-1 antibody is pembrolizumab.

22. The kit of claim 20 or 21, wherein the anti-CTLA4 antibody is a monoclonal antibody comprising a light chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 39, 40 and 41 and a heavy chain CDR comprising the amino acid sequence shown in SEQ ID NOs: 36, 37 and 38.

23. The kit of any one of claims 20-22, wherein the anti-CTLA4 antibody is a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:50 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

49.

24. The kit of any one of claims 20-23, wherein the anti-CTLA4 antibody is a monoclonal antibody comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO:57 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

58.

25. The kit of any one of claims 20-24, comprising about 25 mg of the anti-CTLA4 antibody or antigen-binding fragment thereof.

26. The kit of any one of claims 20-25, comprising about 50 mg of the anti-CTLA4 antibody or antigen-binding fragment thereof.

27. The kit of any one of claims 20-25, comprising about 75 mg of the anti-CTLA4 antibody or antigen-binding fragment thereof.

28. The kit of any one of claims 20-25, comprising about 100 mg of the anti-CTLA4 antibody or antigen-binding fragment thereof.

29. Use of the kit of any one of claims 20-28 for treating an individual suffering from cancer.

30. The use of claim 29, wherein the cancer is PD-1 / PD-L1 refractory melanoma.

31. The use of claim 29, wherein the cancer is melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, kidney cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, pancreatic cancer, brain tumor, glioblastoma, sarcoma, bone tumor or Merkel cell carcinoma.

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