Methods of treating cancer with anti-PD-1 antibodies

Abstract

The present invention relates to methods of treating cancer with anti-PD-1 antibodies. The method comprises administering to the patient a specific amount of a PD-1 antagonist, such as an anti-PD-1 antibody or an antigen-binding fragment thereof (e.g., pemmumab), about every six weeks. In some embodiments, the amount of the anti-PD-1 antibody or antigen-binding fragment thereof is about 400 mg. In certain embodiments, the PD-1 antagonist is pembromab or an antigen binding fragment thereof. Also provided are compositions and kits comprising a dose of an anti-PD-1 antibody or antigen-binding fragment thereof, and uses thereof for the treatment of cancer.

Description

[0001] This application is a divisional application of Chinese patent application 201980013286.8, "Method for treating cancer with anti-PD-1 antibody", filed on February 8, 2019. Technical Field

[0002] This invention relates to therapies that can be used to treat cancer. Specifically, this invention relates to methods for treating cancer, comprising administering an anti-PD-1 antibody or an antigen-binding fragment thereof to a patient in need using a dosage regimen specified herein.

[0003] Cross-references to related applications This application claims the benefits of U.S. Provisional Application No. 62 / 630,038, filed February 13, 2018, and U.S. Provisional Application No. 62 / 732,828, filed September 18, 2018, the contents of which are incorporated herein by reference in their entirety.

[0004] Reference to the electronically submitted sequence list The sequence list of this application was submitted electronically via EFS-Web as a sequence list in ASCII format, with the filename "24567WOPCT-SEQLIST-25JAN2019.TXT", a creation date of January 25, 2019, and a size of 23.7 kb. This sequence list submitted via EFS-Web is a part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0005] PD-1 is considered to play an important role in immune regulation and the maintenance of peripheral tolerance. 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).

[0006] Two known ligands of PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), are expressed in human cancers arising in various tissues. In large ensembles of cancers such as ovarian cancer, renal cell carcinoma, 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., 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)).

[0007] Similarly, PD-1 expression on tumor-infiltrating lymphocytes has been 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 associated with poor prognosis of renal cell carcinoma (Thompson et al., Clinical Cancer Research 15: 1757-1761 (2007)). Therefore, it has been proposed that PD-L1-expressing tumor cells interact with PD-1-expressing T cells to reduce T cell activation and evade immune surveillance, thereby impairing the immune response against the tumor.

[0008] Immune checkpoint therapy targeting the PD-1 axis has led to groundbreaking 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). Immunotherapy targeting the PD-1 axis includes monoclonal antibodies against the PD-1 receptor (KEYTRUDA™ (pembrolizumab), Merckand Co., Inc., Kenilworth, NJ, USA; and OPDIVO™ (nivorumab), Bristol-Myers Squibb Company, Princeton, NJ, USA) as well as those binding to PD-L1 ligands (MPDL3280A; TECENTRIQ™ (atezumab), Genentech, San Francisco, CA, USA; IMFINZI™ (dulvacizumab), AstraZeneca Pharmaceuticals LP, Wilmington, DE; BAVENCIO™ (avelumab), MerckKGaA, Darmstadt, Germany). Both treatments have demonstrated antitumor activity in many cancer types.

[0009] Developing additional dosing schedules would be beneficial, allowing for the administration of safer and more effective doses of anti-PD-1 antibodies that are more convenient for patients. Summary of the Invention

[0010] This 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 anticipated dosing schedule provides a safe and effective dose of the anti-PD-1 antibody or an antigen-binding fragment thereof. Specifically, this invention provides a method of treating cancer in a human patient comprising administering approximately 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof to the patient every six weeks, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises: (a) a light chain complementarity-determining region (CDR) comprising the amino acid sequences shown in SEQ ID NO: 1, 2, and 3 and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NO: 6, 7, and 8; or (b) a light chain CDR comprising the amino acid sequences shown in SEQ ID NO: 11, 12, and 13 and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NO: 14, 15, and 16. In a preferred embodiment of the invention, the antibody or antigen-binding fragment is pembrolizumab.

[0011] In an embodiment of the invention, the amount of the anti-PD-1 antibody or its antigen-binding fragment administered to the patient is from about 350 mg to about 450 mg. In a further embodiment, the amount of the antibody or antigen-binding fragment is about 400 mg. In a further embodiment, the amount of the antibody or antigen-binding fragment is 400 mg.

[0012] In all the treatment methods, compositions, and uses described 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 one specific embodiment, the anti-PD-1 antibody comprises a heavy chain and a light chain, and wherein the heavy chain and light chain comprise Figure 1 The amino acid sequences shown are (SEQ ID NO:5 and SEQ ID NO:10).

[0013] In some embodiments of any of the above-described treatment methods, 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.

[0014] In some embodiments of any of the methods described herein, the anti-PD-1 antibody or antigen-binding fragment is administered subcutaneously to the patient.

[0015] In an alternative implementation of any of the methods described herein, the anti-PD-1 antibody or antigen-binding fragment is administered intravenously to the patient.

[0016] This invention specifically relates to the following technical solutions: 1. A method of treating cancer in a human patient, comprising administering approximately 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof to the patient every approximately six weeks, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises: (a) A light chain complementarity-determining region (CDR) comprising the amino acid sequences shown in SEQ ID NO:1, 2 and 3 and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NO:6, 7 and 8; or (b) A light chain CDR containing the amino acid sequences shown in SEQ ID NO:11, 12 and 13 and a heavy chain CDR containing the amino acid sequences shown in SEQ ID NO:14, 15 and 16.

[0017] The method of Scheme 1, wherein the anti-PD-1 antibody or its antigen-binding fragment 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 as shown in SEQ ID NO:4 or a variant of SEQ ID NO:4, (ii) The amino acid sequence as shown in SEQ ID NO:22 or a variant of SEQ ID NO:22, or (iii) The amino acid sequence as shown in SEQ ID NO:23 or a variant of SEQ ID NO:23.

[0018] The method of Scheme 1 or 2, wherein the anti-PD-1 antibody or its antigen-binding fragment comprises a variable heavy chain region and a light chain variable region, the variable heavy chain region comprising an amino acid sequence as shown in SEQ ID NO:9, and the light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:4.

[0019] In method 1 or 2, wherein the anti-PD-1 antibody or its antigen-binding fragment is a monoclonal antibody comprising: (a) A heavy chain containing the amino acid sequence shown in SEQ ID NO:10 or a variant of SEQ ID NO:10, and (b) A light chain containing 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.

[0020] The method of any one of Schemes 1-4, wherein the anti-PD-1 antibody or its antigen-binding fragment is a monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:10, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:5.

[0021] The method of any one of schemes 1-5, 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, or salivary gland cancer.

[0022] The method of any one of schemes 1-6, wherein the patient has a tumor with a high mutation burden.

[0023] The method of any one of schemes 1-5, wherein the patient has a solid tumor with microsatellite instability-high (MSI-H) or mismatch repair deficiency.

[0024] The method of any one of schemes 1-5, wherein the cancer is unresectable or metastatic melanoma.

[0025] The method of any one of schemes 1-5, wherein the cancer is metastatic non-small cell lung cancer (NSCLC).

[0026] The method of Protocol 10, wherein the patient has a tumor with high PD-L1 expression [(tumor proportion score (TPS) ≥ 50%)] and has not previously been treated with platinum-based chemotherapy.

[0027] The method of Protocol 10, wherein the patient has a tumor with PD-L1 expression (TPS ≥1%) and has been previously treated with platinum-based chemotherapy.

[0028] The method of any one of schemes 11-12, wherein the patient’s tumor does not have EGFR or ALK genomic aberrations.

[0029] The method of any one of schemes 10-13, wherein the method further comprises administering pemetrexed and carboplatin to the patient.

[0030] The method of scheme 14, wherein the patient has non-squamous non-small cell lung cancer, and the pemetrexed is administered at 500 mg / m² approximately every 21 days. 2 The appropriate amount was administered to the patient.

[0031] The method of scheme 14 or scheme 15 further includes administering folic acid to the patient once daily, starting about 7 days before administering pemetrexed and continuing until about 21 days after administering the last dose of pemetrexed.

[0032] The method of any of protocols 14-16 further includes administering approximately 1 mg of vitamin B to the patient approximately one week prior to the first administration of pemetrexed and approximately every three cycles of pemetrexed administration. 12 .

[0033] The method of any one of schemes 14-17 further includes administering dexamethasone to the patient twice a day, one day before, one day after, and one day after pemetrexed administration.

[0034] The method of scheme 10, wherein the NSCLC is squamous and the patient is also treated with carboplatin-paclitaxel or nab-paclitaxel.

[0035] The method of any one of schemes 1-5, wherein the cancer is recurrent or metastatic head and neck squamous cell carcinoma (HNSCC).

[0036] The method of any one of schemes 1-5, wherein the cancer is (1) refractory classical Hodgkin lymphoma (cHL) or (2) cHL, and the patient has relapsed after 3 or more lines of therapy against cHL.

[0037] The method of any one of schemes 1-5, wherein the cancer is locally advanced or metastatic urothelial carcinoma.

[0038] The method of scheme 22, wherein the patient’s tumor expresses PD-L1 [combined positive score >10].

[0039] The method of scheme 22, wherein the patient is not suitable for platinum-based chemotherapy or has disease progression during or after platinum-based chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-based chemotherapy.

[0040] The method of any one of schemes 1-5, wherein the cancer is locally advanced or metastatic gastric cancer or gastroesophageal junction adenocarcinoma.

[0041] The method of any one of schemes 1-5, wherein the cancer is cervical cancer.

[0042] The method of scheme 26, wherein the cervical cancer is recurrent or metastatic cervical cancer, and the patient has disease progression during or after chemotherapy.

[0043] Methods 25, 26, or 27, wherein the patient’s tumor expresses PD-L1 [combined positive score (CPS) ≥1].

[0044] The method of any one of schemes 1-5, wherein the cancer is primary mediastinal large B-cell lymphoma (PMBCL).

[0045] The method of scheme 29, wherein the patient has refractory PMBCL or has relapsed after two or more lines of prior therapy.

[0046] The method of any one of schemes 1-5, wherein the cancer is a resected high-risk stage III melanoma.

[0047] The method of any one of schemes 1-5, wherein the cancer is hepatocellular carcinoma.

[0048] The method of any one of schemes 1-5, wherein the cancer is renal cell carcinoma (RCC).

[0049] The method of scheme 32, wherein the cancer is advanced clear cell RCC.

[0050] The method of any one of schemes 1-5, wherein the cancer is recurrent, locally advanced or metastatic Merkel cell carcinoma (MCC).

[0051] The method of any one of schemes 1-35, wherein the anti-PD-1 antibody or its antigen-binding fragment is administered to the patient via intravenous or subcutaneous administration.

[0052] The method of any one of schemes 1-36, wherein the anti-PD-1 antibody or its antigen-binding fragment is pembrolizumab.

[0053] The composition comprises approximately 400 mg of pembrolizumab and a pharmaceutically acceptable carrier.

[0054] The composition of Scheme 38 further comprises 10 mM histidine, pH 5.5, 7% sucrose, and 0.02% polysorbate 80.

[0055] A kit for treating patients with cancer, the kit comprising: (a) Approximately 400 mg of anti-PD-1 antibody or its antigen-binding fragment, and (b) Instructions for use of the anti-PD-1 antibody or its antigen-binding fragment in any of the methods in schemes 1-36.

[0056] The kit for Scheme 40, wherein the anti-PD-1 antibody is pembrolizumab.

[0057] The use of the composition of any one of Schemes 38-39 or the kit of any one of Schemes 40-41 for the treatment of an individual suffering from cancer.

[0058] The use of scheme 42, wherein the cancer is 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, or salivary gland cancer. Attached Figure Description

[0059] Figure 1 The amino acid sequences of the light and heavy chains of exemplary anti-PD-1 monoclonal antibodies that can be used in this invention are shown (SEQ ID NO: 5 and 10, respectively). The variable regions of the light and heavy chains are underlined (SEQ ID NO: 4 and 9), and CDRs are in bold and framed.

[0060] Figure 2 The results showed that the steady-state Cmax of pembrolizumab at 400 mg Q6W was in the range of 2 mg / kg Q3W and 200 mg Q3W to 10 mg / kg Q2W.

[0061] Figure 3 The results showed that steady-state pembrolizumab exposure (Cavg and Cmin) was similar relative to 2 mg / kg Q3W and 200 mg Q3W.

[0062] Figure 4A and 4B The steady-state pharmacokinetic profile of the 400 mg Q6W dosing regimen is shown compared to the 200 mg, Q3W flat dosing regimen (top), the Q3W, 2 mg / kg weight-based dosing regimen (middle), and the Q2W, 10 mg / kg weight-based dosing regimen (bottom). Logarithmic scale concentrations are provided. Figure 4A ) and linear scale concentration ( Figure 4B The result of ). Detailed Implementation

[0063] I. Definitions and abbreviations As used throughout this specification and the appended claims, the following abbreviations apply: AE adverse events AUCss: Area under the concentration-time curve at steady state BICR's uninformed independent central review Cavg,ss: Time-averaged concentration at steady state CDR Complementary Determinant Region CI confidence interval Cmax,ss Peak concentration at steady state Cmin,ss Valley Concentration at Steady State CPS combined positive score Duration of DOR response ECG (electrocardiogram) ECOG Eastern Collaborative Oncology Group ER exposure (concentration) - response FFPE formalin-fixed paraffin-embedded FR Frame Area GM geometric mean HCC (hepatocellular carcinoma) HNSCC (Head and Neck Squamous Cell Carcinoma) HL Hodgkin lymphoma IgG Immunoglobulin G IHC immunohistochemistry or immunohistochemical IV. Intravenous LPS lymphoma proportion score mAb monoclonal antibody MCC Merkel cell carcinoma MEL melanoma MMR mismatch repair MPS Modified Ratio Scoring MRI (Magnetic Resonance Imaging) MSI-H Microsatellite Instability - High NCI, CTCAE, National Cancer Institute – General Terminology Standard for Adverse Events NSCLC (Non-small Cell Lung Cancer) Objective response rate (ORR) Overall OS Survival PD disease progression PD-1 (also known as programmed cell death-1 and programmed death receptor 1) PD-L1 programmed cell death ligand 1 PD-L2 programmed cell death 1 ligand 2 PFS No Progression Survival PK Pharmacokinetics Q2W, one dose every two weeks. Q3W, one dose every three weeks. Q6W, one dose every six weeks. RCC renal cell carcinoma SAE Serious Adverse Events SC subcutaneous TPS Tumor Proportion Score V H Immunoglobulin heavy chain variable region V L immunoglobulin light chain variable region To facilitate a better understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0064] A reference to "or" indicates one or both possibilities unless the context clearly indicates one of the possibilities. In some cases, "and / or" is used to emphasize one or both possibilities.

[0065] As used herein (including the appended claims), unless the context clearly indicates otherwise, the singular forms of words (such as “a”, “an”, and “the”) include their respective multiple indicators.

[0066] When referring to the amount (e.g., mg) of a modified substance or composition, or the value of a parameter in a step of a characterization method, the term "about" refers to possible variations in the value, for example, through typical measurement, processing, and sampling procedures involved in the preparation, characterization, and / or use of the substance or composition; through unintentional errors in these procedures; through differences in the manufacture, origin, or purity of the ingredients used in making or using the composition or carrying out the procedure; etc. In some embodiments, "about" may mean 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 may 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 implementations, the dose 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 regimen (i.e., the amount of time between administrations of the anti-PD-1 antibody or its antigen-binding fragment, e.g., “about 6 weeks,” which is used interchangeably herein with “approximately every six weeks”), “about” means a specified time ± possible variation due to patient / clinician scheduling and availability around the target date of 6 weeks. For example, “about 6 weeks” could mean 6 weeks ± 5 days, 6 weeks ± 4 days, 6 weeks ± 3 days, 6 weeks ± 2 days, or 6 weeks ± 1 day, or could mean 5 weeks 2 days to 6 weeks 5 days.

[0067] The pharmacokinetic "steady state" is the period during which the drug concentration has been maximized by multiple doses and systemic drug exposure is considered uniform after each subsequent dose administered; in the specific case of pembrolizumab, steady state is reached at ~16 weeks after administration and thereafter.

[0068] AUCss, Cavg,ss, and Cmin,ss are pharmacokinetic measures of systemic exposure to a drug (e.g., pembrolizumab) in humans after administration and are generally considered to be drivers of drug efficacy. AUCss and Cavg,ss represent the average exposure over the dosing interval, but differ in 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.

[0069] "Cmax,ss" is the maximum or highest (peak) drug concentration observed shortly after administration. In the specific case of pembrolizumab (which is administered via intravenous infusion), the peak concentration occurs immediately after the infusion ends. Cmax,ss is generally considered a measure of driving force for driver safety.

[0070] "Administration" and "treatment," when applied 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 the animal, human, subject, cell, tissue, organ, or biological fluid. As used herein, "treat" or "treating" cancer means administering an anti-PD-1 antibody or antigen-binding fragment to a subject with cancer or diagnosed with cancer to achieve at least one positive therapeutic effect, such as, for example, reducing the number of cancer cells, reducing tumor size, reducing the rate of cancer cell invasion into peripheral organs, or reducing the rate of tumor metastasis or tumor growth. "Treatment" may include one or more of the following: inducing / increasing an anti-tumor immune response, reducing the number of one or more tumor markers, halting or delaying the growth of a tumor or hematologic malignancy or the progression of a disease ("PD-1-related disease") (such as cancer) associated with the binding of PD-1 to its ligands PD-L1 and / or PD-L2, stabilizing PD-1-related disease, 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 abolishing the clinical presentation of PD-1-related disease, reducing the severity or duration of clinical symptoms of PD-1-related disease (such as cancer), prolonging the survival of a patient relative to the expected survival of an untreated similar patient, and inducing complete or partial remission of cancerous conditions or other PD-1-related diseases.

[0071] There are many ways to measure the positive effects of cancer treatment (see WA Weber, J. Nucl. Med.50:1S-10S (2009)). For example, regarding tumor growth inhibition, according to NCI criteria, a T / C of ≤42% is the minimum level of antitumor activity. A T / C of <10% is considered a high level of antitumor activity, where T / C (%) = median tumor volume treated / median tumor volume in the control group × 100. In some implementations, treatment achieved by an effective dose is any of progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also known as “time to tumor progression,” indicates the length of time during and after treatment when cancer does not grow, and includes the amount of time a patient has experienced a complete or partial response, as well as the amount of time a patient has experienced stable disease. DFS refers to the length of time a patient remains disease-free during and after treatment. OS refers to the extension of life expectancy compared to a naive or untreated individual or patient. Although one embodiment of the treatment methods, compositions, and uses of the present invention may not effectively achieve a positive therapeutic effect in every patient, it should be achieved in a statistically significant number of subjects, as determined by any statistical test known in the art (such as Student's t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test).

[0072] The term "patient" (which may be alternatively referred to herein as "subject" or "individual") means a mammal (e.g., rat, mouse, dog, cat, rabbit), preferably a human, capable of being 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.

[0073] The term "antibody" refers to any form of antibody that exhibits the desired biological or binding activity. Therefore, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized antibodies, fully human antibodies, and chimeric antibodies. "Parental antibody" is an antibody obtained by exposing the immune system to an antigen prior to the modification of an antibody for its intended use, such as the humanization of antibodies used as human therapeutic agents.

[0074] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain defines a constant region primarily responsible for effector function. Human light chains are typically classified as κ and λ light chains. Furthermore, human heavy chains are generally classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, the variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, with the heavy chain also including "D" regions of approximately 10 or more amino acids. See also... Fundamental Immunology Ch. 7 (Paul, W., editor, 2nd edition. RavenPress, NY (1989).

[0075] The variable region of each light / heavy chain pair forms the antibody binding site. Therefore, typically, a complete antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are usually the same.

[0076] Typically, both heavy and light chain variable domains contain three hypervariable regions, also known as complementarity-determining regions (CDRs), located within relatively conserved framework regions (FRs). CDRs are usually aligned with the framework regions to enable the binding of specific epitopes. Typically, from the N-terminus to the C-terminus, both light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Amino acid assignment to each domain is generally based on 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.

[0077] The term "hypervariant region" refers to the amino acid residues of the antibody responsible for antigen-binding. The hypervariant region contains amino acid residues from the "complementarity-determining region" or "CDR" (i.e., CDRL1, CDRL2, and CDRL3 in the light chain variable domain, and CDRH1, CDRH2, and CDRH3 in the heavy chain variable domain). See, Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (The CDR region of the antibody is defined by the sequence); see also Chothia and Lesk (1987). J. Mol. Biol. 196: 901-917 (The CDR region of the antibody is defined by its structure). The terms "frame" or "FR" residues refer to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0078] Unless otherwise specified, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, that is, an antibody fragment that retains the ability to specifically bind to an antigen bound by a full-length antibody, such as a fragment retaining one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments.

[0079] An antibody that "specifically binds" to a specific target protein is one that exhibits preferential binding to that target compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered "specific" to its intended target if antibody binding confirms the presence of the target protein in a sample, for example, without producing undesirable results such as false positives. Antibodies or their binding fragments used in this invention will bind to the target protein with an affinity at least twice, preferably at least 10 times, more preferably at least 20 times, and most preferably at least 100 times, that of binding to non-target proteins. As used herein, an antibody is said to specifically bind to a polypeptide containing a given amino acid sequence, such as the amino acid sequence of a mature human PD-1 or human PD-L1 molecule, if it binds to a polypeptide containing that sequence but not to a protein lacking that sequence.

[0080] "Chimeric antibody" refers to an antibody in which portions of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species (e.g., human) or belonging to a particular class or subclass of antibodies, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species (e.g., mouse) or belonging to another class or subclass of antibodies; and fragments of such antibodies, provided they exhibit the desired biological activity.

[0081] "Human antibody" refers to an antibody that contains only the sequence of human immunoglobulin proteins. Human antibodies may contain mouse carbohydrate chains, if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, "mouse antibody" or "rat antibody" refers to an antibody that contains only the sequence of mouse or rat immunoglobulins, respectively.

[0082] "Humanized antibody" refers to an antibody form containing sequences derived from non-human (e.g., mouse) antibodies as well as human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Typically, humanized antibodies will substantially contain all of at least one, usually two, variable domains, where all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin sequences. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. When it is necessary to distinguish humanized antibodies from parental rodent antibodies, the prefix "hum," "hu," or "h" is added to the antibody clone name. Humanized forms of rodent antibodies will typically contain the same CDR sequence as the parental rodent antibody, although certain amino acid substitutions may be included to increase affinity, increase the stability of the humanized antibody, or for other reasons.

[0083] The terms “cancer,” “cancerous,” or “malignant” refer to or describe a physiological condition in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, germ cell tumors, and sarcomas. 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 (intestinal) cancers, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver tumor, breast cancer, colon cancer, and head and neck cancer. Additional cancers treatable according to the invention include those characterized by elevated expression of one or both of PD-L1 and PD-L2 in the test tissue sample.

[0084] "Biotherapeutic agents" refer to a biomolecule, such as an antibody or fusion protein, that blocks ligand / receptor signaling in any biological pathway that supports tumor maintenance and / or growth or inhibits antitumor immune responses.

[0085] "CDR" or "CDRs" refers to the complementarity-determining region in the variable region of immunoglobulins, which is usually defined using the Kabat numbering system.

[0086] "Platinum-based chemotherapy" (also known as platinum) refers to the use of chemotherapeutic agents as coordination complexes of platinum for the treatment of cancer. Platinum-based chemotherapeutic agents are alkylating agents that cross-link DNA, disable DNA mismatch repair, and often induce apoptosis. Examples of platinum-based chemotherapeutic agents include cisplatin, carboplatin, and oxaliplatin.

[0087] "Chemotherapy agents" are chemical compounds that can be used to treat cancer. Categories of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle toxins, plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, anti-estrogens and selective estrogen receptor modulators (SERMs), antiprogesterone agents, estrogen receptor downregulators (ERDs), estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists, anti-androgens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, and antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Chemotherapy agents that can be used in the treatment methods of this invention include cell growth inhibitors and / or cytotoxic agents.

[0088] “Chothia” refers to Al-Lazikani and others. JMB The antibody numbering system described in 273:927-948 (1997).

[0089] "Conservative modification variants" or "conservative substitutions" refer to the substitution of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and stiffness), such that the changes can be made frequently without altering the protein's biological activity or other desired properties, such as antigen affinity and / or specificity. Those skilled in the art will recognize that, generally, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987)). Molecular Biology of the Gene (See The Benjamin / Cummings Pub. Co., p. 224 (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to impair biological activity. Exemplary conserved substitutions are illustrated in Table 1.

[0090] Table 1. Exemplary Conserved Amino Acid Substitutions

[0091] As used throughout the specification and claims, “consists essentially of” and its variations (such as “consist essentially of” or “consisting essentially of”) indicate the inclusion of any of the stated elements or groups of elements, and optionally include other elements having properties similar to or different from those of the stated elements that do not significantly alter the fundamental or novel properties of the specified dosing regimen, method, or composition. As a non-limiting example, the PD-1 antigen-binding fragment consisting essentially of the stated amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues that do not materially affect the properties of the binding compound.

[0092] The word “comprising” or variations such as “comprise”, “comprises”, or “composed of” are used throughout the specification and claims in an inclusive sense, that is, specifying the presence of the stated feature, but not excluding the presence or addition of further features that may substantially enhance the operation or utility of any embodiment of the invention, unless the context requires otherwise due to explicit language or necessary meaning.

[0093] "Diagnostic anti-PD-L monoclonal antibody" refers to a mAb that specifically binds to the mature form of a designated 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 should be understood to refer to the same molecule unless otherwise indicated or readily apparent from the context.

[0094] 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 amino acids 19-290 of the following sequence: .

[0095] Specific examples of diagnostic anti-human PD-L1 mAbs that can be used for immunohistochemical (IHC) detection of PD-L1 expression in formalin-fixed, paraffin-embedded (FFPE) tumor tissue sections are antibodies 20C3 and 22C3, described in WO 2014 / 100079. These antibodies contain the amino acid sequences of the light and heavy chain variable regions as shown in Table 2 below:

[0096] Another anti-human PD-L1 mAb has been reported for use in 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 disclosed in Sino Biological, Inc. (Beijing, PR China; catalog number 10084-R015).

[0097] As used in this article, "frame region" or "FR" refers to the immunoglobulin variable region excluding the CDR region.

[0098] "Isolated antibody" and "isolated antibody fragment" refer to a purified state and, in this context, mean that the named molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, carbohydrates, or other substances such as cell debris and growth media. Generally, the term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the experimental or therapeutic application of the binding compound as described herein.

[0099] As used in this article, "Kabat" refers to the immunoglobulin matching 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.).

[0100] As used herein, "monoclonal antibody" or "mAb" or "Mab" refers to a substantially homogeneous group of antibodies, meaning that the antibody molecules constituting the group are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically comprise many different antibodies with different amino acid sequences in their variable domains, particularly their CDRs (which are often specific to different epitopes). The modifier "monoclonal" indicates the characteristics of antibodies obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the production of the antibody by any specific method. For example, monoclonal antibodies to be used according to the invention can be produced by means of Kohler et al. (1975). NatureThe monoclonal antibody was first prepared using the hybridoma method described in 256:495, or it can be prepared using recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). The "monoclonal antibody" can also be used, for example, by Clackson et al. (1991). Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol The technique described in 222:581-597 is used to isolate from a phage antibody library. See also Presta (2005). J. Allergy Clin. Immunol. 116:731.

[0101] "Anti-PD-1 antibody" used in any treatment method, composition, and use of the present invention comprises a monoclonal antibody (mAb) that specifically binds to human PD-1 or an antigen-binding fragment thereof. Alternative names or synonyms for PD-1 and its ligands include: for PD-1; PDCD1, PD1, CD279, and SLEB2; for PD-L1, PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H; and for PD-L2, PDCD1L2, PDL2, B7-DC, Btdc, and CD273. In any treatment method, composition, and use of the present invention in which a human individual is being treated, the PD-1 antibody or its antigen-binding fragment 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 amino acid sequence of human PD-1 can be found at NCBI locus number NP_005009. The amino acid sequences of human PD-L1 and PD-L2 can be found in NCBI loci 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 may include a human constant region. In some embodiments, the constant region is selected from the IgG1, IgG2, IgG3, and IgG4 constant regions, and in a preferred embodiment, the human constant region is the 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.

[0102] “PD-L1” or “PD-L2” expression refers to any detectable expression level of PD-L protein on the cell surface or PD-L mRNA within cells or tissues, unless otherwise defined. PD-L protein expression can be detected in IHC assays of tumor tissue sections using diagnostic PD-L antibodies or by flow cytometry. Alternatively, PD-L protein expression in tumor cells can be detected by PET imaging using a binding agent (e.g., antibody fragment, affinity, etc.) that specifically binds to the desired PD-L target (e.g., PD-L1 or PD-L2). Techniques used to detect and measure PD-L mRNA expression include RT-PCR and real-time quantitative RT-PCR.

[0103] Several methods for quantifying PD-L1 protein expression in IHC assays 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).

[0104] One approach employs a simple binary endpoint for PD-L1 expression positivity or negativity, where a positive result is defined in terms of the percentage of tumor cells exhibiting histological evidence of cell-surface membrane staining. Tumor tissue sections are counted as positive for PD-L1 expression at least 1%, preferably 5%, of total tumor cells.

[0105] In another method, PD-L1 expression in tumor tissue sections was quantified in tumor cells and in infiltrating immune cells that predominantly consist of lymphocytes. The percentages of membrane-stained tumor cells and infiltrating immune cells were individually quantified as <5%, 5% to 9%, and then incremented by 10% to a maximum of 100%. For tumor cells, PD-L1 expression was counted as negative if the score was <5%, and as positive if the score was ≥5%. PD-L1 expression in the immune infiltrate was reported as a semi-quantitative measure known as the modulated inflammation score (AIS), which was determined by multiplying the percentage of membrane-stained cells by the infiltrate intensity, graded as none (0), mild (score of 1, sparse lymphocytes), moderate (score of 2, focal infiltration of tumors via lymphocyte histiocytic aggregates), or severe (score of 3, diffuse infiltration). If the AIS was ≥5, the tumor tissue section was counted as positive for PD-L1 expression via the immune infiltrate.

[0106] PD-L1 expression in both tumor cells and infiltrating immune cells in tissue sections can also be scored for PD-L1 protein expression using a scoring method. This involves scoring tissue sections from tumors that have been stained with diagnostic PD-L1 antibodies via IHC. See WO 2014 / 165422. One PD-L1 scoring method includes examining staining of each tumor nest in a tissue section and assigning one or both of a modified H score (MHS) and a modified proportional score (MPS) to the tissue section. To assign an MHS, four separate percentages are estimated for all viable tumor cells and stained mononuclear inflammatory cells in all examined tumor nests: (a) unstained cells (intensity = 0), (b) weakly stained (intensity = 1+), (c) moderately stained (intensity = 2+), and (d) strongly stained (intensity = 3+). Cells must be at least partially membrane stained to be included in the weak, moderate, or strong staining percentages. The estimated percentages (summing up to 100%) are then entered into the following formula: 1 x (percentage of weakly stained cells) + 2 x (percentage of moderately stained cells) + 3 x (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 all intensities among all viable tumor cells and stained mononuclear inflammatory cells in all examined tumor nests, 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 PD-L1 expressing positive.

[0107] Another method for scoring / quantifying PD-L1 expression in tumors is the "Combined Positive Score" or "CPS," which refers to an algorithm for determining PD-L1 expression scores from a patient's tumor sample. CPS can be used to select patients for treatment with a specific regimen that includes the administration of an anti-PD-1 antibody, where PD-L1 expression in the specific patient population is associated with a higher response rate compared to the same patient population that does not express PD-L1. CPS is calculated by determining the number of surviving PD-L1-positive tumor cells, the number of surviving PD-L1-negative tumor cells, and the number of surviving PD-L1-positive mononuclear inflammatory cells (MICs) in tumor tissue from a patient with a tumor, and then calculating CPS using the following formula: (# PD-L1 positive tumor cells) + (# PD-L1 positive MIC) x 100% (PD-L1 positive tumor cells) + (PD-L1 negative tumor cells).

[0108] In the specific implementation plan, the PD-L1 expression scoring method used is the "Lymphoma Proportion Score" (LPS). Lymphoma is characterized by a homogeneous population of confluenced cells that erases the structure of lymph nodes or metastatic sites. The LPS, or "Lymphoma Proportion Score," is the percentage of this cell population expressing PD-L1. When determining the LPS, no attempt is made to distinguish between true neoplastic cells and reactive cells. PD-L1 expression is characterized by partial or complete membrane staining at any intensity.

[0109] Another scoring method for PD-L1 expression is the "TPS" or "tumor proportion score," which is the percentage of tumor cells expressing PD-L1 on their cell membrane. TPS typically includes the percentage of proliferative cells expressing PD-L1 at any intensity (weak, moderate, or strong), which can be determined using immunohistochemical assays with diagnostic anti-human PD-L1 mAbs (e.g., antibody 20C3 and antibody 22C3) as described above. Cells are considered to express PD-L1 if membrane staining is present (including cells with partial membrane staining).

[0110] The expression level of PD-L mRNA can be compared with the expression level of one or more reference genes (such as ubiquitin C) that are frequently used in quantitative RT-PCR.

[0111] In some embodiments, the level of PD-L1 expression (protein and / or mRNA) in malignant cells and / or tumor-infiltrating immune cells is determined as “overexpression” or “elevated” based on a comparison with the PD-L1 expression (protein and / or mRNA) levels of an appropriate control. For example, the control PD-L1 protein or mRNA expression level may be the level quantified in the same type of non-malignant cells or in sections from matched normal tissue. In some preferred embodiments, elevated PD-L1 expression in a tumor sample is determined if the PD-L1 protein (and / or PD-L1 mRNA) in the tumor sample is at least 10%, 20%, or 30% higher than that in a control.

[0112] A "tissue slice" refers to a single portion or piece of tissue sample, such as a thin slice of tissue cut from a sample of normal tissue or a tumor.

[0113] When applied to a subject diagnosed with or suspected of having cancer, the term "tumor" refers to a malignant or potentially malignant growth or mass of tissue of any size, including primary tumors and secondary growths. A solid tumor is an abnormal growth or mass of tissue that typically does not contain cysts or fluid-filled areas. Different types of solid tumors are named according to the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (blood cancers) typically does not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

[0114] As used herein, "variable region" or "V region" refers to a segment of the IgG chain that is variable in the sequence between different antibodies. It extends to Kabat residue 109 in the light chain and Kabat residue 113 in the heavy chain.

[0115] As used in this article, "RECIST 1.1 response criteria" refers to Eisenhauer, EA et al. Eur. J. Cancer The definition of appropriate target or non-target lesions as described in 45:228-247 (2009) based on the context of measurement response.

[0116] II. PD-1 antibody and antigen-binding fragments that can be used in this invention Examples of mAbs that bind to human PD-1 and can be used in the therapeutic methods, compositions, and uses of the present invention are described in US 7,521,051, US 8,008,449, and US 8,354,509. Specific anti-human PD-1 mAbs that can be used as PD-1 antagonists in the therapeutic methods, compositions, and uses of the present invention include: pembrolizumab (formerly known as MK-3475, SCH 900475, and lambolizumab), humanized IgG4 mAbs (which have... WHO Drug Information The structure described in Volume 27, Issue 2, pp. 161-162 (2013), and which contains Figure 1 The heavy and light chain amino acid sequences shown are shown in the figure, and the humanized antibodies h409A11, h409A16 and h409A17 (described in WO 2008 / 156712 and Table 3).

[0117] In some embodiments of the treatment methods, compositions, kits, and uses of the present invention, the anti-PD-1 antibody or its antigen-binding fragment comprises: (a) a light chain CDR comprising the amino acid sequences shown in SEQ ID NO: 1, 2, and 3 and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NO: 6, 7, and 8; or (b) a light chain CDR comprising the amino acid sequences shown in SEQ ID NO: 11, 12, and 13 and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NO: 14, 15, and 16. In some embodiments of the present invention, the anti-PD-1 antibody or its antigen-binding fragment is a human antibody. In other embodiments, the anti-PD-1 antibody or its antigen-binding fragment is a humanized antibody. In other embodiments, the anti-PD-1 antibody or its antigen-binding fragment is a chimeric antibody. In a specific embodiment, the anti-PD-1 antibody or its antigen-binding fragment is a monoclonal antibody.

[0118] In other embodiments of the treatment methods, compositions, kits, and uses of the present invention, the PD-1 antibody or its antigen-binding fragment specifically binds to human PD-1 and comprises: (a) a heavy chain variable region comprising an amino acid sequence as shown in 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.

[0119] Variants of the heavy chain variable region sequence or full-length heavy chain sequence are identical to the reference sequence, except that they have up to 17 conserved amino acid substitutions in the frame region (i.e., outside the CDR), and preferably fewer than 10, 9, 8, 7, 6, or 5 conserved amino acid substitutions in the frame region. Variants of the light chain variable region sequence or full-length light chain sequence are identical to the reference sequence, except that they have up to 5 conserved amino acid substitutions in the frame region (i.e., outside the CDR), and preferably fewer than 4, 3, or 2 conserved amino acid substitutions in the frame region.

[0120] In another embodiment of the treatment methods, compositions, kits, and uses of the present invention, the PD-1 antibody or its antigen-binding fragment is a monoclonal antibody that specifically binds to human PD-1 and comprises: (a) a heavy chain comprising an amino acid sequence as shown in SEQ ID NO:10 or a variant thereof; and (b) a light chain comprising an amino acid sequence as shown in 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.

[0121] In yet another embodiment of the treatment methods, compositions, and uses of the present invention, the PD-1 antibody or its antigen-binding fragment is a monoclonal antibody that specifically binds to human PD-1 and comprises: (a) a heavy chain comprising an amino acid sequence as shown in SEQ ID NO:10 or thereof; and (b) a light chain comprising an amino acid sequence as shown in SEQ ID NO:5 or thereof.

[0122] Table 3 below provides a list of exemplary anti-PD-1 mAbs in the treatment methods, compositions, kits, and uses of the present invention.

[0123] III. Methods and uses of the present invention This invention provides a method for treating cancer in a human patient, comprising administering approximately 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof to the patient every approximately six weeks, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises: (a) a light chain complementarity-determining region (CDR) comprising the amino acid sequences shown in SEQ ID NO:1, 2, and 3 and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NO:6, 7, and 8; or (b) a light chain CDR comprising the amino acid sequences shown in SEQ ID NO:11, 12, and 13 and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NO:14, 15, and 16. In a specific embodiment of the invention, the anti-PD-1 antibody or antigen-binding fragment thereof is pembrolizumab.

[0124] In some embodiments of the invention, the anti-PD-1 antibody or its antigen-binding fragment is administered to the patient approximately every six weeks for 12 weeks or longer. In other embodiments, the anti-PD-1 antibody or its antigen-binding fragment is administered to the patient every six weeks for 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.

[0125] In a first embodiment (E1), the present invention includes a method of treating 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 every approximately six weeks.

[0126] In a second embodiment (E2), the invention includes a method of treating unresectable or metastatic melanoma 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 every approximately six weeks.

[0127] In a third embodiment (E3), the invention includes a method of treating metastatic non-small cell lung cancer (NSCLC) 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 every approximately six weeks.

[0128] In a sub-implementation of implementation scheme E3 (implementation scheme E3-A), the patient has a tumor with high PD-L1 expression [(tumor proportion score (TPS) ≥50%)] and has not previously been treated with platinum-based chemotherapy.

[0129] In a further sub-implementation of implementation scheme E3 (implementation scheme E3-B), the patient has a tumor with PD-L1 expression (TPS ≥1%) and has previously been treated with platinum-based chemotherapy. In a specific implementation scheme E3-B, the patient has disease progression during or after receiving platinum-based chemotherapy.

[0130] In another sub-implementation of implementation scheme E3 (implementation scheme E3-C), the patient has a tumor with PD-L1 expression (TPS ≥1%) and has not previously been treated with platinum-based chemotherapy.

[0131] In yet another sub-implementation of implementation scheme E3 (implementation scheme E3-D), the patient's tumor is not tested against PD-L1 expression. In this implementation scheme, the patient is treated with an anti-PD-1 antibody or its antigen-binding fragment, regardless of PD-L1 expression. In this specific implementation scheme, the patient has not previously been treated with platinum-based chemotherapy.

[0132] In certain implementation schemes of scheme E3 (including schemes E3-A, E3-B, and E3-C), PD-L1 TPS are determined by FDA-approved testing.

[0133] In certain implementation schemes of scheme E3 (including schemes E3-A, E3-B, E3-C, and E3-D), the patient's tumor does not have EGFR or ALK genomic aberrations.

[0134] In certain embodiments of implementation scheme E3 (including implementation schemes E3-A, E3-B, E3-C, and E3-D), the patient's tumor has EGFR or ALK genomic aberrations and has disease progression during or after treatment targeting the EGFR or ALK aberrations, and before receiving the anti-PD-1 antibody or its antigen-binding fragment.

[0135] In a fourth embodiment (E4), the present invention includes a method of treating metastatic non-small cell lung cancer (NSCLC) in a human patient, comprising: (1) administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or its antigen-binding fragment to the patient every approximately six weeks, and (2) administering pemetrexed and carboplatin to the patient. In a sub-implementation of E4, the patient has not previously been treated with anticancer agents before initiating a combination therapy regimen of the anti-PD-1 antibody or its antigen-binding fragment, pemetrexed, and carboplatin.

[0136] In some embodiments of implementation schemes E3 and E4 (including their sub-implementations), the patient has non-squamous non-small cell lung cancer.

[0137] In the sub-implementation of implementation plan E4, pemetrexed is administered at 500 mg / m². 2 The appropriate amount was administered to the patient.

[0138] In a sub-implementation of implementation plan E4, pemetrexed is administered to the patient every 21 days via intravenous infusion. In a specific implementation plan, the infusion time is approximately 10 minutes.

[0139] In a sub-implementation of implementation scheme E4 (implementation scheme E4-A), the invention further includes administering approximately 400 μg to approximately 1000 μg of folic acid once daily to the patient, starting approximately 7 days before administering pemetrexed and continuing until approximately 21 days after administering the last dose of pemetrexed. In some embodiments, the folic acid is administered orally.

[0140] In sub-implementations of implementation schemes E4 and E4-A (implementation scheme E4-B), the invention further includes administering approximately 1 mg of vitamin B to the patient approximately one week prior to the first administration of pemetrexed and approximately every three cycles of pemetrexed administration (i.e., approximately every nine weeks). 12 In some implementations, vitamin B... 12 Intramuscular application.

[0141] In sub-implementations of implementation schemes E4, E4-A, and E4-B (implementation scheme E4-C), the invention further includes administering approximately 4 mg of dexamethasone twice daily to the patient one day before, on the day of, and one day after pemetrexed administration. In some embodiments, dexamethasone is administered orally.

[0142] In a fifth embodiment (E5), the present invention includes a method of treating recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) 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 every approximately six weeks.

[0143] In a sub-implementation of implementation scheme E5, the patient has previously been treated with platinum-based chemotherapy. In some implementations, the patient experienced disease progression during or after platinum-based chemotherapy.

[0144] In a sixth embodiment (E6), the present invention includes 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 every approximately six weeks.

[0145] In a seventh embodiment (E7), the invention includes a method of treating classical Hodgkin lymphoma (cHL) in a human patient, comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or its antigen-binding fragment to the patient every approximately six weeks, wherein the patient has relapsed following (a) first-line or multiple-line therapy against cHL, (b) second-line or more-line therapy against cHL, or (c) third-line or more-line therapy against cHL.

[0146] In the sub-implementations of implementation schemes E6 and E7, the patient is an adult patient.

[0147] In alternative sub-implementations of implementation schemes E6 and E7, the patient is a pediatric patient.

[0148] In an eighth embodiment (E8), the present invention includes 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 every approximately six weeks.

[0149] In a sub-implementation of implementation plan E8, the patient is not suitable for cisplatin-containing chemotherapy.

[0150] In a sub-implementation of implementation scheme E8, the patient experiences disease progression during or after platinum-based chemotherapy or within 12 months of receiving neoadjuvant or adjuvant therapy with platinum-based chemotherapy.

[0151] In a sub-implementation of implementation scheme E8, the patient's tumor expresses PD-L1 (CPS). > 10).

[0152] In a ninth embodiment (E9), the invention includes a method of treating unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair (MMR) deficient solid tumors in human patients, comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to said patient every approximately six weeks.

[0153] In one sub-implementation of implementation scheme E9, the patient has disease progression following prior anticancer treatment.

[0154] In a tenth embodiment (E10), the present invention includes a method of treating unresectable or metastatic, MSI-H or MMR-deficient colorectal 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 every approximately six weeks.

[0155] In one sub-implementation of implementation scheme E10, the patient has disease progression following prior treatment with fluoropyrimidine, oxaliplatin, and irinotecan.

[0156] In the eleventh embodiment (E11), the present invention includes a method of 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 every approximately six weeks.

[0157] In the twelfth embodiment (E12), the present invention includes a method of treating recurrent locally advanced or metastatic gastroesophageal junction adenocarcinoma 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 every approximately six weeks.

[0158] In the sub-implementations of implementation schemes E11 and E12, the patient's tumor expresses PD-L1 [combined positive score (CPS) ≥1].

[0159] In sub-implementations of implementation schemes E11 and E12, the patient experiences disease progression during or after first-line or multiple-line prior therapy. In specific implementations, the first-line prior therapy includes fluoropyrimidine-based chemotherapy and platinum-based chemotherapy.

[0160] In the sub-implementations of implementation schemes E11 and E12, the patient has disease progression during or after two or more lines of prior therapy (including fluoropyrimidine-based chemotherapy and platinum-based chemotherapy).

[0161] In the sub-implementations of implementation schemes E11 and E12, the patient has disease progression during or after first-line or multiple lines of prior therapy (including HER2 / neu-targeted therapy).

[0162] In the sub-implementations of implementation schemes E11 and E12, the patient has disease progression during or after two or more lines of prior therapy (including HER2 / neu-targeted therapy).

[0163] In a thirteenth embodiment (E13), the present invention includes a method of treating 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 every approximately six weeks, wherein the patient has cancer selected from the following: melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, and salivary gland cancer.

[0164] In the fourteenth embodiment (E14), the present invention includes a method of treating 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 every approximately six weeks, wherein the patient has small cell lung cancer.

[0165] In the fifteenth embodiment (E15), the present invention includes a method of treating non-Hodgkin's lymphoma 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 every approximately six weeks.

[0166] In one sub-implementation of implementation scheme E15, the non-Hodgkin lymphoma is primary mediastinal large B-cell lymphoma (PMBCL). In some embodiments where the patient has PMBCL, the patient has refractory PMBCL. In some embodiments, the patient has relapsed after one or more lines of prior therapy. In some embodiments, the patient has relapsed after two or more lines of prior therapy. In some embodiments, the patient has not previously been treated with another line of therapy.

[0167] In the sixteenth embodiment (E16), the present invention includes 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 every approximately six weeks, and (2) administering (i) carboplatin and paclitaxel, or (ii) carboplatin and nab-paclitaxel to the patient.

[0168] In a seventeenth embodiment (E17), the present invention includes a method of treating Merkel cell carcinoma (MCC) 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 every six weeks. In a specific sub-implementation of E17, the cancer is recurrent locally advanced MCC. In a specific sub-implementation of E17, the cancer is metastatic MCC.

[0169] In a sub-implementation of implementation scheme E17, the patient is an adult patient. In an alternative sub-implementation of implementation scheme E17, the patient is a pediatric patient.

[0170] In the eighteenth embodiment (E18), the invention includes a method for adjuvant treatment of melanoma in a human patient, comprising administering 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof to a patient every approximately six weeks, wherein the patient has previously undergone resection of one or more melanoma lesions. In a sub-implementation of E18, the method includes treating resected high-risk stage III melanoma.

[0171] In a nineteenth embodiment (E19), the invention includes a method of treating hepatocellular carcinoma (HCC) 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 every six weeks. In some embodiments of E19, the patient has previously been treated with sorafenib.

[0172] In the twentieth embodiment (E20), the present invention includes a method of treating renal cell carcinoma (RCC) 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 every approximately six weeks.

[0173] In a sub-implementation of implementation scheme E20, the cancer is advanced clear cell RCC.

[0174] In a sub-implementation of implementation plan E20, the patient has advanced or metastatic renal cell carcinoma (RCC).

[0175] In a sub-implementation of implementation scheme E20 (implementation scheme E20A), the patient is further treated with axitinib. In a sub-implementation of the invention, axitinib is administered orally.

[0176] In the specific implementation plan of E20A, the patient takes approximately 5 mg of axitinib every 12 hours or twice a day.

[0177] In an alternative implementation scheme to scheme E20A, axitinib is administered at a dose of 2.5 mg, 3 mg, 7 mg, or 10 mg twice daily.

[0178] In the twenty-first embodiment (E21), the present invention includes a method of treating breast 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 every approximately six weeks.

[0179] In one sub-implementation of implementation scheme E21, the breast cancer is triple-negative breast cancer.

[0180] In one sub-implementation of implementation scheme E21, the breast cancer is ER+ / HER2- breast cancer.

[0181] In the twenty-second embodiment (E22), the present invention includes a method of treating nasopharyngeal 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 every approximately six weeks.

[0182] In the twenty-third embodiment (E23), the present invention includes a method of treating thyroid 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 every approximately six weeks.

[0183] In the twenty-fourth embodiment (E24), the present invention includes a method of treating salivary gland 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 every approximately six weeks.

[0184] In the twenty-fifth embodiment (E25), the present invention includes a method of treating 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 every approximately 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.

[0185] In the twenty-sixth embodiment (E26), the present invention includes a method of treating 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 every approximately six weeks, wherein the cancer is a hematologic malignancy.

[0186] In a sub-implementation of implementation scheme E26, the hematologic malignancy is selected from: 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 / histocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid leukemia-1 protein (MCL-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), and small lymphocytic lymphoma (SLL).

[0187] In the twenty-seventh embodiment (E27), the present invention includes a method of treating 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 every approximately six weeks, wherein the patient has a tumor with a high mutational burden.

[0188] In a specific implementation, a high mutation load is defined as at least approximately 10 mutations per megabase examined, at least approximately 11 mutations per megabase examined, at least approximately 12 mutations per megabase examined, or at least approximately 13 mutations per megabase examined.

[0189] In the twenty-eighth embodiment (E28), the present invention includes a method of treating esophageal 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 every approximately six weeks.

[0190] In a sub-implementation of implementation scheme E28, the patient received first-line prior standard therapy prior to receiving the anti-PD-1 antibody or its antigen-binding fragment. In a further implementation, the patient received first-line or multiple-line prior standard therapy prior to receiving the anti-PD-1 antibody or its antigen-binding fragment. In another implementation, the patient received two or more lines of prior standard therapy prior to receiving the anti-PD-1 antibody or its antigen-binding fragment. In a specific implementation, the standard therapy comprises one or more of the following: paclitaxel, docetaxel, or irinotecan.

[0191] In a sub-implementation of implementation scheme E28, the patient has advanced or metastatic adenocarcinoma or squamous cell carcinoma of the esophagus.

[0192] In a sub-implementation of implementation scheme E28, the patient has advanced or metastatic Siewert type I adenocarcinoma of the esophagogastric junction.

[0193] In a sub-implementation of implementation scheme E28, the patient's tumor expresses PD-L1 (combined positive score (CPS) ≥10).

[0194] In a twenty-ninth embodiment (E29), the present invention includes a method of treating high-risk non-muscle-invasive bladder cancer (NMIBC) 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 every six weeks. In some embodiments, the patient has NMIBC with carcinoma in situ (CIS) or CIS plus papillary lesions.

[0195] In one sub-implementation of implementation scheme E29, the patient was previously treated with standard therapy prior to treatment with the anti-PD-1 antibody or its antigen-binding fragment. In some implementations, the prior therapy is BCG therapy. In a specific implementation, the patient did not respond to BCG therapy. In some implementations, the patient is not suitable for radical cystectomy or has chosen not to undergo radical cystectomy.

[0196] In any of the methods of the present invention described above (including embodiments E1-E29), the PD-1 antibody or antigen-binding fragment is any antibody or antigen-binding fragment described in Part II, “PD-1 Antibodies and Antigen-Binding Fragments Usable in the Invention,” of the detailed description of the invention herein. In some embodiments, the anti-PD-1 antibody is pembrolizumab or its antigen-binding fragment, or an antibody that cross-competes with pembrolizumab for binding to human PD-1. In some embodiments, the anti-PD-1 antibody is a variant of pembrolizumab; i.e., an antibody or antigen-binding fragment having a light chain CDR comprising the amino acid sequences shown in SEQ ID NO: 1, 2, and 3 and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NO: 6, 7, and 8.

[0197] In any of the methods of the present invention described above (including embodiments E1-E29), the PD-1 antibody or antigen-binding fragment is administered to the patient approximately every six weeks. In specific embodiments, the PD-1 antibody or antigen-binding fragment is administered to the patient every six weeks, every six weeks ± 5 days, ± 4 days, ± 3 days, ± 2 days, or ± 1 day.

[0198] In any implementation of any treatment method described herein, an intravenous infusion of a drug comprising any of the anti-PD-1 antibodies or antigen-binding fragments described herein is administered to the patient.

[0199] In any alternative implementation of the treatment described herein, any anti-PD-1 antibody or antigen-binding fragment may be administered subcutaneously to the patient (e.g., by a clinician) or subcutaneously to the patient.

[0200] In any of the methods described herein (including embodiments E1-E29 and their sub-implementations), the method may further include one or more “additional therapeutic agents” (as used herein, “additional therapeutic agent” means an additional agent relative to the anti-PD-1 antibody or its antigen-binding fragment). The additional therapeutic agent may be, for example, a chemotherapeutic agent other than the anti-PD-1 antibody, a biological therapeutic agent (including, but not limited to, antibodies against CTLA4, VEGF, EGFR, Her2 / neu, VEGF receptor, other growth factor receptors, CD20, CD40, CD-40L, OX-40, 4-1BB, and ICOS), an immunogenic agent (e.g., weakened 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 (e.g., but not limited to GM-CSF).

[0201] As shown above, in some embodiments of the method of the present invention, the method further includes administering an additional therapeutic agent. In specific embodiments, the additional therapeutic agent is an anti-CTLA4 antibody or its antigen-binding fragment, an anti-LAG3 antibody or its antigen-binding fragment, an anti-GITR antibody or its antigen-binding fragment, an anti-TIGIT antibody or its antigen-binding fragment, an anti-CD27 antibody or its antigen-binding fragment, an anti-ILT3 antibody or its antigen-binding fragment, or an anti-ILT4 antibody or its antigen-binding fragment. 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 denaciline. In another embodiment, the additional therapeutic agent is navarixin. In a further embodiment, the additional therapeutic agent is vicriviroc.

[0202] In a further embodiment, the additional therapeutic agent is an oncolytic virus. In one embodiment, the additional therapeutic agent is Coxsackievirus or CVA21. In one embodiment, the additional therapeutic agent is CAVATAK™.

[0203] In yet another embodiment, the additional therapeutic agent is a STING agonist. In a further embodiment, the additional therapeutic agent is an IL-27 antagonist. In one embodiment, the additional therapeutic agent is a PARP inhibitor. In one embodiment, the additional therapeutic agent is a multi-kinase inhibitor. In one embodiment, the additional therapeutic agent is a MEK inhibitor. In one embodiment, the additional therapeutic agent is a 4-1BB agonist.

[0204] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piperazine; azacyclopropane derivatives such as benzodopa, carboquinone, meturedopa, and uredopa; ethylene imines and methylmelamines including altretamine, triethlenemelamine, triethenylphosphamide, triethenylthiophosphamide, and trimethylolomelamine; polyacetogenins (especially bulbatacin and bulbatacinone); camptothecin (including its synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (Including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlornaphazine, cholophosphamide, estradiol, ifosfamide, dichloromethyldiethylamine, dichloromethyldiethylamine hydrochloride, melphalan, etc. Novembichin, phenesterine, prednisone, trofenoxam, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, formustine, lomustine, nimustine, ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin phiI1, see, for example, Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;And new carcinogen chromophores and related chromopyrins (ene diyne antibiotic chromophores), aclacinomysin, actinomycin D, authramycin, azaserine, bleomycin, actinomycin C, carabicin, caminomycin, carzinophilin, chromomycinis, danomycin, detorubicin, 6-diaza-5-oxo-L-leucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-) Doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, isorubicin, idarubicin, ephedrine, mitomycins such as mitomycin C, mycophenolic acid, nogamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozotocin, tuberculin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5- FU; folic acid analogs, such as folate, methotrexate, pteropterin, trimethyltroxa; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, 6-azacitidine, 6-azouridine, carmoflu, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluxuridine; androgen derivatives, such as dimethyltestosterone, drotalbutone propionate, epitiostanol, mepitios... tane), testosterone; antiadrenergic drugs, such as aminoglucopyranoside, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldehyde phosphoramide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; eletine;Epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazine; procarbazine; razoxane; rhizomycin; sizofuran; spirogermanium; tenuazonic acid acid); triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; piperobroman; gacytosine; cytarabine (“Ara-C”); cyclophosphamide; thiotepa; taxane (t axoids, such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vincristine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; mitoxantrone dihydrochloride injection (novantrone); teniposide; edaraxacin; danomycin; aminopterin; capecitabine (xeloda); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine;And pharmaceutically acceptable salts, acids, and derivatives of any of the aforementioned drugs. This also includes anti-hormonal agents used to regulate or inhibit hormones acting on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifen, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene. (Fareston); aromatase inhibitors that inhibit aromatase, which regulate the production of estrogen in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglucopyranoside, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing drugs.

[0205] In some embodiments that include the step of administering an additional therapeutic agent (i.e., in addition to a PD-1 antibody (e.g., pembrolizumab) or its antigen-binding fragment), the agent is administered using the same dosing regimen (dosage, frequency, and duration of treatment) commonly used when the additional therapeutic agent in the combination therapy is used as a monotherapy for the same cancer. In other embodiments, the patient receives a lower total amount of the agent, e.g., a smaller dose, a lower frequency of administration, and / or a shorter duration of treatment, compared to when the additional therapeutic agent in the combination therapy is used as a monotherapy.

[0206] Additional therapeutic agents in combination therapy can be administered orally, intratumorally, or parenterally, including via intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, local, and percutaneous routes. For example, the combination therapy may comprise an anti-PD-1 antibody or its antigen-binding fragment and an anti-CTLA antibody or its antigen-binding fragment (both of which can be administered intravenously or subcutaneously), and a chemotherapy agent (which can be administered orally).

[0207] The combination therapy of the present invention can be used before or after surgical removal of the 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.

[0208] In some embodiments, the combination therapy of the present invention is administered to patients who have not previously been treated with biological or chemotherapeutic agents, i.e., those who are untreated. In other embodiments, the combination therapy is administered to patients who have not achieved a durable response after prior therapy with biological or chemotherapeutic agents, i.e., those who have undergone treatment.

[0209] The combination therapy of the present invention can be used to treat tumors that are large enough to be detected by palpation or by imaging techniques well known in the art, such as MRI, ultrasound, or CAT scan. In some embodiments, the combination therapy of the present invention is used to treat tumors with a diameter of at least about 200 mm. 3 300 mm 3 400 mm 3 500 mm 3 750 mm 3 or up to 1000 mm 3 Late-stage tumors of that size.

[0210] In some embodiments, the combination therapy of the present invention is administered to human patients with cancer expressing PD-L1. In some embodiments, PD-L1 expression is detected in an IHC assay using a diagnostic anti-human PD-L1 antibody or its antigen-binding fragment on an FFPE or frozen tissue section taken from the patient's tumor sample. Prior to the initiation of treatment with the anti-PD-1 antibody or its antigen-binding fragment, the patient's physician may schedule a diagnostic test to determine PD-L1 expression in a tumor tissue sample taken from the patient; however, it is envisioned that the physician may schedule the first or subsequent diagnostic test at any time after the start of treatment (e.g., after the completion of a treatment cycle).

[0211] The choice of dosage for additional therapeutic agents depends on several factors, including the serum or tissue conversion rate of the entity, the level of symptoms, the immunogenicity of the entity, and the availability of target cells, tissues, or organs in the individual being treated. The dosage of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Therefore, the dosage and frequency of administration for each additional therapeutic agent (e.g., a biologic or chemotherapeutic agent) will depend in part on the specific agent, the severity of the cancer being treated, and the patient characteristics. Guidelines for selecting appropriate doses of antibodies, cytokines, and small molecules are available. 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. J. Med. 341:1966-1973; Slamon et al. (2001) New 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 edition); Medical Economics Company; ISBN:1563634457; 57th edition (November 2002). The determination of an appropriate dosing regimen can be made by a clinician, for example, using parameters or factors known or suspected in the art to affect or predict their effect on treatment, and will depend on, for example, the patient's clinical history (e.g., prior therapy), the type and stage of the cancer to be treated, and biomarkers of response to one or more therapeutic agents in combination therapy.

[0212] IV. Compositions and Kits The present invention also relates to compositions 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, for example, in CHO cells using conventional cell culture and recovery / purification techniques.

[0213] In embodiments of the invention, the composition further comprises a histidine buffer at a pH of about 5.0 to 6.0. In a specific embodiment, histidine is present at a concentration of about 10 mM.

[0214] In embodiments of the invention, the composition further comprises sucrose. In a specific embodiment, sucrose is present at a concentration of about 70 mg / mL.

[0215] In embodiments 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.

[0216] In some embodiments, the composition comprises 10 mM histidine (pH 5.5), 7% sucrose, 0.02% polysorbate 80, and 400 mg of anti-PD-1 antibody or its antigen-binding fragment.

[0217] In an embodiment of the present invention, the composition is a liquid.

[0218] In an alternative embodiment, the composition is lyophilized.

[0219] In the compositions of the present invention, the anti-PD-1 antibody or its antigen-binding fragment may be any antibody or antigen-binding fragment described in Part II of the detailed description of the invention, “PD-1 antibodies and antigen-binding fragments that may be used in the present invention” (e.g., pembrolizumab).

[0220] In some embodiments, compositions comprising anti-PD-1 antibodies as PD-1 antagonists may be provided as liquid formulations or prepared by reconstituted lyophilized powders with sterile water for injection prior to use. WO 2012 / 135408 describes the preparation of liquid and lyophilized pharmaceutical preparations containing pembrolizumab suitable for use in this invention.

[0221] The present invention also relates to a kit for treating patients with cancer, the kit comprising: (a) 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof, and (b) instructions for use of the anti-PD-1 antibody or an antigen-binding fragment thereof in any of the methods described herein for treating cancer.

[0222] In any kit of the present invention, the PD-1 antibody or antigen-binding fragment may be any antibody or antigen-binding fragment described in Part II of the detailed description of the invention, “PD-1 Antibodies and Antigen-Binding Fragments Usable in the Invention”.

[0223] The kit of the present invention may provide an anti-PD-1 antibody or an antigen-binding fragment thereof in a container and packaging insert. The container contains at least one dose (i.e., about 400 mg) of the drug comprising an anti-PD-1 antibody or an antigen-binding fragment thereof, and a packaging insert or label containing instructions for using the drug to treat a patient with cancer. The container may be constructed of the same or different shapes (e.g., vials, syringes, and bottles) and / or materials (e.g., plastic or glass). The kit may further include other materials that may be useful in drug administration, such as diluents, filters, IV bags and tubing, needles, and syringes. In some preferred embodiments of the kit, the instructions indicate that the drug is intended for the treatment of a patient with a tumor, wherein the tumor expresses PD-L1 by, for example, an IHC assay. In some embodiments, the tumor has… >A tumor proportion score (TPS) of 1% PD-L1. In another embodiment, the tumor has > 50% PD-L1 TPS. PD-L1 TPS is the number of tumor cells expressing PD-L1 in the sample. In a further embodiment, the tumor has... > 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% TPS of PD-L1. In another embodiment, the patient's tumor expresses PD-L1, wherein CPS is > 10%. In another embodiment, the patient's tumor expresses PD-L1, wherein CPS is > 5%. In another embodiment, the patient's tumor expresses PD-L1, wherein CPS is > 1%.

[0224] These and other aspects of the invention (including the exemplary embodiments listed below) will be apparent from the teachings contained herein.

[0225] General methods The standard methods in molecular biology are described in Sambrook, Fritsch, and Maniatis (2nd edition, 1982 & 1989; 3rd edition, 2001). Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd Edition , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA (Volume 217, Academic Press, San Diego, CA). The standard method also appeared in Ausbel et al. (2001). Current Protocols in Molecular Biology, Volumes 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Volume 1), cloning in mammalian cells and yeast (Volume 2), glycoconjugates and protein expression (Volume 3), and bioinformatics (Volume 4).

[0226] Methods for protein purification are described, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization (Coligan et al. (2000)). Current Protocols in Protein Science, Volume 1, John Wiley and Sons, Inc., New York. It describes chemical analysis, chemical modification, post-translational modification, the generation of fusion proteins, and protein glycosylation (see, for example, Coligan et al. (2000)). Current Protocols in ProteinScience Volume 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology Volume 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) BioDirectory Piscataway, NJ, pp. 384-391. Describes the generation, purification, and fragmentation of polyclonal and monoclonal antibodies (Coligan et al. (2001)). Current Protocols in Immunology Volume 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, ibid.). Standard techniques for characterizing ligand / receptor interactions are available (see, for example, Coligan et al. (2001)). Current Protocols in Immunology Volume 4, John Wiley, Inc., New York.

[0227] Monoclonal, polyclonal, and humanized antibodies can be prepared (see, for example, Sheperd and Dean (ed.) (2000)). Monoclonal Antibodies Oxford University Press, New York, NY; edited by Kontermann and Dubel (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. (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 Patent No. 6,329,511).

[0228] An alternative to humanization is to use human antibody libraries displayed on bacteriophages or human antibody libraries 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, ColdSpring 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).

[0229] Antigen purification is not necessary for antibody production. Animals can be immunized with cells carrying the target antigen. Spleen cells can then be isolated from the immunized animal, and these spleen cells can be fused with myeloma cell lines to generate hybridomas (see, for example, Meyaard et al. (1997)). Immunity 7: 283-290; Wright et al. (2000) Immunity 13: 233-242; Preston et al., ibid.; Kaithamana et al. (1999) J. Immunol. 163: 5157-5164).

[0230] Antibodies can be conjugated to, for example, small drug molecules, enzymes, liposomes, and polyethylene glycol (PEG). Antibodies can be used for therapeutic, diagnostic, kit, or other purposes, and include antibodies conjugated to, for example, dyes, radioisotopes, enzymes, or metals (e.g., colloidal gold) (see, for example, 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).

[0231] Methods for flow cytometry, including fluorescence-activated cell sorting (FACS), are available (see, for example, Owens et al. (1994)). Flow Cytometry Principles for Clinical Laboratory Practice ,John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd 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, peptides and antibodies) for use as, for example, diagnostic reagents are available (Molecular Probes (2003)). Catalogue , Molecular Probesy,Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue St. Louis, MO).

[0232] The standard approach to the histology of the immune system is described (see, for example, 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, Phila, PA; Louis et al. (2002) Basic Histology: Text and Atlas , McGraw-Hill, New York, NY).

[0233] Software packages and databases for determining, for example, antigen fragments, leader sequences, protein folds, functional domains, glycosylation sites, and sequence alignment are available (see, for example, GenBank, Vector NTI® Suite (Informax, Inc., Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (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).

[0234] For the purpose of describing and disclosing methods and materials that may be used in conjunction with the present invention, all publications mentioned herein are incorporated by reference.

[0235] Various embodiments of the invention have been described herein with reference to the accompanying drawings. It should be understood that the invention is not limited to those precise embodiments, and that various changes and modifications can be made therein without departing from the scope or spirit of the invention as defined in the appended claims.

[0236] Example 1 Based on evaluations using modeling and simulation, a six-week (Q6W) dosing schedule for pembrolizumab across multiple tumor types was determined. Pembrolizumab (currently approved as an anti-PD-1 checkpoint inhibitor for multiple cancer indications) has been demonstrated to be safe and effective when administered at a dose of 200 mg or 2 mg / kg every 3 weeks. Alternative extended dosing regimens would offer convenience and flexibility to both patients and prescribers. Robust characterization of pembrolizumab pharmacokinetic (PK) and exposure (concentration)-response (ER) relationships for both efficacy and safety allows for the use of model-based approaches to support alternative dosing regimens for pembrolizumab.

[0237] After achieving PK steady state, the dose of pembrolizumab in the Q6W schedule was selected by matching the exposure to the approved Q3W (200 mg and 2 mg / kg) regimens; based on knowledge of ER, a bridge was established between the regimens for potency and safety. PK exposure for up to 24 weeks of dosing was simulated using an established population PK model of pembrolizumab (with time-dependent elimination) (which adequately describes PK across multiple tumor types) to ensure steady state in all subjects. Potency was bridged using steady-state exposure measures, AUCss or time-averaged concentrations (Cavg, ss), and trough concentrations (Cmin, ss) (which were compared between regimens). The safety profile of pembrolizumab in the Q6W schedule was bridged by ensuring that the predicted peak concentration (Cmax, ss) in steady state was below 10 mg / kg for those at the maximum clinically administered and well-tolerated doses in Q2W.

[0238] The expected pharmacokinetic (PK) profile of pembrolizumab after administration of 400 mg Q6W is similar to that of the approved 200 mg Q3W and 2 mg / kg Q3W dosing regimens (see Figure 4). Exposure metrics compared between the regimens are summarized in Table 4. Based on similar predicted exposures (Cavg, ss, or AUCss, geometric mean (GM) ~1% higher) compared to those achieved at 200 mg Q3W, the 400 mg Q6W dosing regimen of pembrolizumab was selected (see Figure 4). Figure 3 Less than 1% of subjects were predicted to have lower Cmin,ss compared to those in the 200 mg Q3W and 2 mg / kg Q3W regimens. Figure 3 The predicted Cmax,ss of 400 mg Q6W was significantly lower (GM lower than ~65%) than that achieved with 10 mg / kg Q2W (which has been shown to have acceptable safety across multiple tumor types) (see [link to relevant documentation]). Figure 2 Given the similar exposure profile and established flat ER relationship of pembrolizumab at the doses tested in clinical trials, clinical outcomes across tumor types are expected to be similar to those achieved with 400 mg Q6W as with those achieved with 200 mg Q3W.

[0239] Based on the modeling and simulation methods used in this paper, the 400 mg Q6W dosing regimen of pembrolizumab was expected to result in PK exposure similar to the approved 200 mg Q3W and 2 mg / kg dosing regimens. PK simulations showed that, in terms of pembrolizumab exposure – the mean concentration (Cavg) (or area under the curve [AUC]) over the 400 mg Q6W dosing interval was similar to the mean concentration (Cavg) (or area under the curve [AUC]) at the approved 200 mg Q3W dose, thus bridging the efficacy between dosing regimens. In most (>99%) patients, the trough concentration (Cmin) at 400 mg Q6W was generally within the range achieved with those at 2 mg / kg or 200 mg Q3W. The peak concentration (Cmax) at 400 mg Q6W was significantly lower than the Cmax at 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 results indicate that the exposure-response (ER) of pembrolizumab is flat across indications, and the OS projections in melanoma and NSCLC suggest that, given similar exposures, the potency at 400 mg Q6W is expected to be similar to that at 200 mg or 2 mg / kg Q3W; therefore, the 400 mg Q6W cross-indication efficacy is expected.

[0240] Table 4. Overview of pembrolizumab PK exposure metrics based on a simulated 400 mg Q6W dosing regimen

[0241] Example 2 A phase 1 randomized clinical trial evaluating the safety and tolerability of intravenous infusion of 400 mg pembrolizumab every 6 weeks in participants with advanced melanoma. This study was designed to evaluate the pharmacokinetics (PK), safety, and tolerability of pembrolizumab when administered every 6 weeks (Q6W). A cohort of 100 participants was given 400 mg of pembrolizumab every 6 weeks. PK, potency, and safety data were collected from this participant cohort. Male / female participants with advanced melanoma were enrolled in this study. Stratification based on age, sex, or other characteristics was not used in this study.

[0242] From cycle 1 to cycle 18, participants received an IV infusion of 400 mg pembrolizumab Q6W. 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 robust understanding of the clinical pharmacology of pembrolizumab and its well-established ER profile, this dosing schedule is expected to produce similar efficacy and safety in all treatment settings where 200 mg Q3W pembrolizumab is approved (including as monotherapy and in combination with other agents). Therefore, the 400 mg Q6W regimen would have a similar benefit-risk profile to the 200 mg Q3W regimen (a less frequent dosing regimen in clinical use of pembrolizumab based on modeling and simulation analysis) (see Example 1).

[0243] Research Design This study was conducted in accordance with Good Clinical Practice (GCP) and was a randomized, crossover, multicenter, open-label safety study of pembrolizumab in participants with advanced melanoma. The Phase 1 study was conducted in participants with unresectable or metastatic melanoma. Treatment continued for up to 18 cycles (approximately 2 years) every 42 days. Treatment continued as long as participants were benefiting from treatment and had no disease progression or did not meet any criteria for study withdrawal. More specifically, the study consisted of: (1) a screening period of up to 28 days to ensure participants were eligible for the study; and (2) an intervention period of approximately 104 weeks with pembrolizumab. Participants received pembrolizumab via IV infusion every 6 weeks over 30 minutes for up to 18 cycles; and (3) a follow-up period during which participants were monitored for adverse events (AEs) for 30 days and for serious adverse events (SAEs) for 90 days (or 30 days if a participant started a new anticancer therapy). Follow up with participants who have progressive adverse events at the time of treatment discontinuation until the event is resolved, stabilized, otherwise explained, or the participant loses follow-up.

[0244] Participants whose disease status was discontinued due to reasons other than radiographic disease progression underwent post-treatment follow-up imaging until disease progression was recorded according to RECIST 1.1 radiographic documentation, and when clinically appropriate, as confirmed on-site by iRECIST, non-study cancer treatment was initiated, consent was withdrawn, and they became out of follow-up or the study ended. During the survival follow-up period, all participants were followed up by telephone regarding overall survival until death, when a participant withdrew consent, became out of follow-up, or the study ended.

[0245] All participants enrolled in this study had a diagnosis of advanced melanoma. The results of this study will contribute to understanding the PK characteristics of pembrolizumab when administered in a Q6W dosing regimen. Safety endpoints will include safety parameters commonly used to assess investigational systemic anticancer therapy, including but not limited to the incidence, causality, and outcomes of adverse events (AEs) / serious adverse events (SAEs); and changes in vital signs and laboratory values. AEs will be evaluated as defined by the National Cancer Institute Common Terminology Criteria for Adverse Events [NCI CTCAE] version 4.0.

[0246] The objective of this trial was to characterize the pharmacokinetic profile of pembrolizumab following intravenous infusion at Q6W. PK data were analyzed after all participants completed cycle 5. PK parameters included AUC, Cmax, and Cmin. Antidrug antibody (ADA) formation can confound drug exposure at therapeutic doses and trigger subsequent infusion-related toxicities. Antidrug antibody responses to pembrolizumab were determined at the start of cycles 1, 2, 4, and 5. Any effect of the presence of ADAs on pembrolizumab exposure was investigated.

[0247] This study used ORR based on RECIST 1.1 criteria, as assessed by an uninformed independent central review (BICR), as the primary endpoint. Objective response rate is an acceptable measure of clinical benefit in late-stage studies demonstrating the superiority of novel anti-tumor therapies, especially if the magnitude of this effect is large and the therapy has an acceptable risk / benefit profile. Images were submitted to an imaging CRO (iCRO) and read by an uninformed independent central reviewer to minimize bias in response assessment.

[0248] Overall survival (OS) is a secondary endpoint and is widely recognized as the gold standard for demonstrating the superiority of novel anticancer therapies in randomized clinical trials. RECIST 1.1 is used by BICR for evaluating efficacy on imaging and by local sites for eligibility determination. The RECIST Working Group has developed and published a modified RECIST 1.1 (iRECIST) evaluation for immunotherapy-based agents, with input from leading experts in industry and academia, and participation from the US Food and Drug Administration and the European Medicines Agency. One-dimensional measurements of the target lesion, qualitative evaluation of non-target lesions, and response categories are the same as in RECIST 1.1 until progression is seen through RECIST 1.1. However, if the participant is clinically stable, additional imaging may be performed to confirm radiographic progression. Investigators use iRECIST to evaluate tumor response and progression, and to make treatment decisions and for exploratory power analyses (where specified).

[0249] Inclusion criteria Participants are eligible for inclusion in the study only if all of the following criteria are met: • Participants have a histologically or cytologically confirmed diagnosis of advanced melanoma. • According to the American Joint Committee on Cancer (AJCC) staging system, the participants had unresectable stage III or IV melanoma and were not suitable for local therapy. • Participants were not receiving treatment for advanced or metastatic disease, except in the following circumstances: BRAF V600 mutant melanoma may have already received standard-of-care targeted therapy (e.g., BRAF / MEK inhibitors, alone or in combination) and met the study criteria. • Previous adjuvant or neoadjuvant melanoma therapy is permitted if it was completed at least 4 weeks prior to randomization and all relevant adverse events (AEs) have returned to baseline or stabilized (toxicities of the most recent prior therapy resolved to grade 1 or lower [[except for alopecia]]). If the subject received a major surgical or radiation therapy >30 Gy, they must have recovered from toxicities and / or complications caused by the intervention.

[0250] A female participant is eligible to participate if she is not pregnant, is not breastfeeding, and agrees to follow specific contraceptive guidelines or provide informed consent for at least 120 days during the treatment period.

[0251] Participants should have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 (fully active, able to perform all pre-disease manifestations without restriction) or 1 (limited to strenuous physical activity but able to walk and perform light or sedentary work, such as lighthouse work or office work), and should have adequate organ function as defined in Table 5. Samples should be collected within 72 hours prior to the start of the study intervention.

[0252] Table 5. Sufficient Laboratory Values ​​for Organ Function

[0253] Exclusion criteria Participants were excluded from the study if any of the following criteria applied: • Participants must be women of childbearing potential (WOCBP) who have a positive urine pregnancy test within 72 hours prior to randomization or treatment allocation. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test is required.

[0254] • Participants had received prior systemic therapy for unresectable or metastatic melanoma (in addition to the inclusion criteria above).

[0255] • Participants have received agents targeting anti-PD-1, anti-PD-L1, or anti-PD-L2, or another stimulatory or co-inhibitory T-cell receptor (e.g., OX-40 and CD137), or any other antibody or drug that specifically targets checkpoint pathways other than anti-CTLA-4, permitted in an assistive setting.

[0256] • Participants must have received prior radiation therapy within 2 weeks of the start of the study treatment. Participants must have recovered from all radiation-related toxicities, not require corticosteroids, and not have radiation pneumonitis.

[0257] • Participants had received a live vaccine within 30 days prior to their first dose of the study drug. Examples of live vaccines include, but are not limited to, the following: measles, mumps, rubella, varicella / shingles (fowlpox), yellow fever, rabies, BCG, and typhoid vaccines. Seasonal influenza vaccines administered by injection are typically kill-the-virus vaccines and are permitted; however, intranasal influenza vaccines (e.g., FluMist®) are live attenuated vaccines and are not permitted.

[0258] • Participants are currently participating in or have already participated in a study of the investigational drug, or have used the investigational device within 4 weeks prior to the first dose of the study intervention.

[0259] • Participants were diagnosed with immunodeficiency or were receiving chronic systemic steroid therapy (more than 10 mg of prednisone equivalent per day) or any other form of immunosuppressive therapy within 7 days prior to the first dose of the study drug.

[0260] • Participants have a known additional malignancy that is progressing or already requires active treatment within the past 2 years. Note: Participants with basal cell carcinoma, squamous cell carcinoma, or carcinoma in situ (e.g., breast cancer, cervical cancer in situ) who have already undergone potentially curative therapies are not excluded.

[0261] • Participants have known active CNS metastases and / or carcinomatous meningitis. Participants with previously treated brain metastases may participate if they are radiographically stable (i.e., no evidence of progression) for at least 4 weeks via repeat imaging (note that repeat imaging should be performed during study screening), are clinically stable, and do not require steroid treatment for at least 14 days prior to the first dose of treatment intervention.

[0262] • Participants had severe hypersensitivity reactions (≥ grade 3) to pembrolizumab and / or any of its excipients.

[0263] • Participants had ocular melanoma.

[0264] • Participants have an active autoimmune disease that has required systemic treatment (i.e., use of disease modifiers, corticosteroids, or immunosuppressants) within the past 2 years. Replacement therapies (e.g., thyroid hormone, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency) are not considered a form of systemic treatment and are permitted.

[0265] • Participants had a history of (non-infectious) pneumonia, required steroids, or had current pneumonia.

[0266] • The participant had an active infection that required systemic treatment.

[0267] • Participants had a known history of human immunodeficiency virus (HIV) infection.

[0268] • Participants had a known history of hepatitis B (defined as hepatitis B surface antigen [HBsAg] reactivity) or a known active hepatitis C virus infection (defined as detection of HCV RNA [qualitative]).

[0269] • Participants have a history or current evidence of any medical condition, treatment, or laboratory abnormality (which may confound the study results, interfere with participation throughout the study duration), or, according to the investigator, are not in the best interest of the participant to participate.

[0270] • Participants have a known mental illness or substance abuse disorder that may interfere with their cooperation with the research requirements.

[0271] • From the start of screening and follow-up to 120 days after the last dose of the study intervention, the subject is expected to become pregnant, breastfeed, or anticipate pregnancy or give birth within the expected study duration.

[0272] Termination of research intervention and participant withdrawal Discontinuation of a study intervention does not equate to withdrawal from the study. Because certain data regarding clinical events following intervention discontinuation may be important to the study, they must be collected through the participant's last scheduled follow-up, even after the participant has discontinued the intervention. Therefore, all participants who discontinue their study intervention before the completion of the protocol-defined treatment period will continue to participate in the study.

[0273] Participants may withdraw from the research intervention at any time for any reason, or at the researcher's discretion if any adverse effects occur. Furthermore, researchers may require participants to withdraw from the research intervention if it is inappropriate, violates the research plan, or for administrative and / or other safety reasons.

[0274] Participants must discontinue the study intervention for any of the following reasons, but continue to be monitored during the study: • A participant or a legitimate, acceptable representative of the participant requests the termination of the research intervention.

[0275] • Participants who interrupted the study intervention for more than 12 consecutive weeks or missed administration 3 times cumulatively.

[0276] • The researchers believed that the participants' medical conditions or personal circumstances would put them at unnecessary risk due to the continued administration of the research intervention.

[0277] • Participants had a confirmed positive serum pregnancy test.

[0278] • Participants had confirmed progression of radiographic disease. • Participants must have any progression or recurrence of any malignant tumor, or any occurrence of another malignant tumor requiring active treatment. • Participants have unacceptable negative experiences.

[0279] • Participants had comorbidities in addition to the aforementioned malignancy, which prevented further treatment administration.

[0280] • The researchers decided to discontinue the treatment.

[0281] • Participant had recurrent grade 2 pneumonia • Participants had completed 35 treatments with pembrolizumab (approximately 2 years). If a participant or their legally authorized representative withdraws their consent from the study, that participant is removed from the study. If a participant withdraws from the study, they will no longer receive study treatment or be followed up in the scheduled protocol follow-up.

[0282] Informed consent Researchers or designated medically qualified personnel obtain written consent from each potential participant or each participant's legally acceptable representative before participating in a clinical study. If a participant's condition changes during the study (e.g., most participants become healthier or of a different age), the researcher or designated medically qualified personnel ensure that appropriate consent is obtained.

[0283] Efficacy / Evaluation Tumor evaluation includes all known or suspected sites of disease. Imaging may include computed tomography (CT) or magnetic resonance imaging (MRI) of the chest, abdomen, and pelvis, at baseline and when disease progression or brain metastasis is suspected. CT imaging of the tumor is strongly preferred. For the chest, abdomen, and pelvis, contrast-enhanced MRI may be used when CT with iodine-containing contrast agents is contraindicated or when required by local practice. For the brain, MRI is the strongly preferred imaging modality.

[0284] Throughout the study, the same imaging techniques (ideally, the same scanner and consistent use of contrast agents) were used among participants. Consistent use of imaging techniques helps optimize the reproducibility of assessments of existing and new tumor burden and improves the accuracy of assessments of response or progression. Images from all scheduled events for all study participants were reviewed by the investigator for disease progression. In addition, images obtained at non-scheduled time points to determine disease progression (including those obtained by other means) (and images obtained for other reasons but based on investigator-assessed capture radiographic progression) were also submitted at the study site.

[0285] During screening, confirmation of measurable disease by BICR based on RECIST 1.1 will be used to determine participant eligibility. Prior to participant allocation, BICR confirmation is required that the participant's images show at least one lesion suitable for selection as a target lesion according to RECIST 1.1.

[0286] Initial tumor imaging Initial tumor imaging at screening was performed within 28 days prior to the day of the first dose. Any images obtained after day 1 of cycle 1 of treatment were not included in the screening evaluation. The field study team reviewed the screening images to confirm that the participant had measurable disease according to RECIST 1.1. If brain imaging was performed to document the stability of existing metastases, MRI was used whenever possible. If MRI was medically contraindicated, contrast-treated CT was an acceptable alternative.

[0287] Tumor imaging during the study Imaging was evaluated at the time of the first study, 12 weeks (84 days ± 7 days) from the day of the first dose. Tumor imaging was then performed every 9 weeks (63 days ± 7 days) or more frequently (if clinically applicable). Participants still undergoing treatment after 52 weeks (365 days ± 7 days) will be imaged every 12 weeks (84 days ± 7 days).

[0288] Objective response is confirmed through repeated imaging. Tumor imaging is performed at least 4 weeks after the first indicator of response is observed to confirm PR or CR. Participants are then returned to their scheduled imaging sessions, starting at the next scheduled imaging time point. If this is less than 4 weeks late, participants receiving additional imaging for confirmation do not need to undergo the next scheduled tumor imaging session; tumor imaging can continue at a subsequently scheduled imaging time point.

[0289] According to the modified iRECIST, in clinically stable participants, disease progression is confirmed on-site 4 to 8 weeks after the first radiological evidence of disease progression (PD). Participants with unconfirmed disease progression may continue treatment at the investigator's discretion until on-site confirmation of progression. If it is less than 4 weeks late, participants receiving confirmatory imaging do not need to undergo a subsequent scheduled tumor imaging session; if clinically stable, tumor imaging may continue at a later scheduled imaging time point. Participants whose disease progression is confirmed on-site by iRECIST will discontinue study treatment.

[0290] Tumor imaging at the end of treatment and follow-up For participants who discontinue the study intervention, tumor imaging is performed at the time of treatment discontinuation (±4-week window). If prior imaging was obtained within 4 weeks prior to the discontinuation date, imaging is not required at the time of treatment discontinuation. For participants who discontinue the study intervention due to documented disease progression, this is the final tumor imaging requirement if the investigator chooses not to perform iRECIST.

[0291] For participants whose disease progression was not recorded after discontinuing the study intervention, every effort should be made to continue monitoring disease status using tumor imaging on the same imaging schedule used concurrently with treatment every 12 weeks (±7 days) until the start of new anticancer therapy, disease progression, pregnancy, death, withdrawal of consent, or study termination, whichever comes first.

[0292] RECIST 1.1 evaluation of the disease RECIST 1.1 is used as the primary measure for evaluating tumor response and the date of disease progression, and serves as the basis for all protocol guidelines related to disease status (e.g., discontinuation of study interventions). Although RECIST 1.1 mentions a maximum of 5 target lesions in total, 2 per organ, the protocol allows for a maximum of 10 target lesions in total, 5 per organ, if clinically relevant, to achieve a broader sampling of tumor burden.

[0293] iRECIST evaluation of the disease iRECIST is based on RECIST 1.1, but is applicable to cases considering unique tumor responses seen with immunotherapy. Investigators will use iRECIST to evaluate tumor response and progression and make treatment decisions. When clinically stable, participants are not discontinued until progression is confirmed by an investigator in collaboration with their local radiology department. This permission to continue treatment, despite initial radiation-induced disease progression (PD), takes into account the observation that some participants may have experienced transient tumor flare-ups in the first few months after starting immunotherapy and then undergo subsequent disease response.

[0294] When initial radiographic evidence of disease (PD) is evaluated on-site, any participant deemed clinically unstable is discontinued from the study intervention, and repeat tumor imaging is not required to confirm PD via iRECIST. If the investigator decides to continue treatment, the participant may continue the study intervention and should undergo repeat tumor evaluation 4 to 8 weeks later, as assessed by the investigator, to confirm PD via iRECIST. If repeat imaging does not confirm PD via iRECIST, as assessed by the investigator, and the participant remains clinically stable, the study intervention continues and follows the regular imaging schedule. If PD is confirmed, the participant is discontinued from the study intervention.

[0295] If a participant has confirmed radiographic progression (iCPD), the study intervention is discontinued; however, if the participant achieves a clinically meaningful benefit, an exception to continue the study intervention is considered. In such cases, if the study intervention continues, tumor imaging will continue. An overview of imaging and treatment requirements following the first radiographic evidence of progression is provided in Table 6.

[0296] Table 6 Imaging and treatment following initial radiological evidence of disease progression Safety assessment Safety evaluation includes collecting adverse events (AEs) and severe adverse events (SAEs), monitoring vital signs and laboratory evaluations (including pregnancy tests), electrocardiogram (ECG) performance and physical examination, and validation of concurrent medication.

[0297] Adverse events Each subject is evaluated by an investigator or a qualified designated person to assess for potential new or worsening adverse events (AEs), and more frequently if clinically appropriate. AE evaluation includes, but is not limited to, type, incidence, severity (classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events [NCI CTCAE] version 4.0), timing, severity, and relevance to the investigational drug. Adverse events occurring during the study period are documented, including baseline signs and symptoms.

[0298] Complete physical examination Researchers or qualified designated personnel conduct a full physical examination during the screening period. Clinically significant abnormalities are documented as part of the medical history. New clinically significant abnormalities following the first dose of the study intervention are documented as adverse events (AEs).

[0299] Targeted physical examination For cycles that do not require a full physical examination, the investigator or a qualified designated person shall conduct a clinically appropriate physical examination prior to administration of the study intervention. New, clinically significant abnormalities are documented as adverse events (AEs).

[0300] vital signs Vital signs were measured in a semi-supine position after 5 minutes of rest and included temperature, systolic and diastolic blood pressure, respiratory rate, pulse rate, and weight. Height was collected only at screening.

[0301] electrocardiogram A standard 12-lead ECG was performed using local standard procedures. Clinically significant abnormalities observed during screening were documented in the patient's medical history. One or more additional ECGs were performed during the study if clinically necessary. Clinically significant findings observed on follow-up ECGs were documented as adverse events (AEs).

[0302] Clinical safety laboratory evaluation The tests detailed in Table 7 were conducted by the local laboratory. Additional tests may be performed at any time during the study if determined by the investigator.

[0303] Table 7. Laboratory evaluation of the safety requirements of the program

[0304] The time period and frequency for collecting information on AE, SAE, and other reportable safety incidents. If a participant is receiving placebo run-in or other run-in treatment, the investigator must report any adverse events (AEs), special adverse events (SAEs), or other reportable safety events that occur after the consent form is signed but before treatment allocation / randomization, resulting in the exclusion of the participant from the study, or as a result of interventions specified in the protocol (including but not limited to washout or discontinuation of routine therapies, diets, or procedures). The investigator must report all AEs from the time of treatment allocation / randomization to 30 days after the termination of the study intervention.

[0305] If a participant begins a new cancer therapy, all adverse events (AEs) meeting the stringent criteria from the time of treatment allocation / randomization to 90 days or 30 days after the cessation of the study intervention (whichever is earlier) must be reported by the investigator. Furthermore, any SAE that comes to the investigator's attention at any time outside the aforementioned specified timeframes and is deemed drug-related must be reported immediately.

[0306] Statistical methods for effectiveness analysis Objective Response Rate (ORR) – ORR is calculated as the ratio of the number of participants who reported a confirmed CR or PR that had been validated by the BICR to the number of participants included in the APaT population. Participants in the APaT analysis population who did not undergo ORR evaluation are counted as non-responders. For the true ORR, a 95% exact binomial CI is calculated (based on the method of Clopper and Pearson, 1934).

[0307] Progression-free survival (PFS) – A nonparametric Kaplan-Meier method was used to estimate the PFS distribution. The median PFS and the 95% CI of the estimated PFS point were calculated at different follow-up times from day 1 of study treatment. Because disease progression is assessed periodically, PD can occur at any time within the time interval between the last assessment where PD was not recorded and the assessment where PD was recorded. The true date of PD will be approximated to the date of the first assessment of PD objectively recorded by BICR based on RECIST 1.1. Death is always considered a PFS event. Participants who did not experience a PFS event will be examined at their last disease assessment. For PFS analysis, if an event (PD or death) occurs immediately after more than one missed disease assessment, the data are examined at the last disease assessment before the missed follow-up. Similarly, data after a new cancer therapy are examined at the last disease assessment before starting a new anticancer therapy. If a participant meets multiple examination criteria, the earliest examination criterion will be applied.

[0308] Overall survival (OS) - A nonparametric Kaplan-Meier method was used to estimate the OS distribution. Median OS and the 95% CI of the OS point estimates were calculated at each follow-up time starting from day 1 of the study treatment.

[0309] Duration of Response (DOR) - DOR is descriptively summarized using a nonparametric Kaplan-Meier method. Only a subset of participants who exhibited CR or PR are included in this analysis.

[0310] Analysis strategy for key efficacy endpoints Table 8 summarizes the main analytical methods used for key efficacy endpoints.

[0311] Table 8. Analytical Strategies for Key Efficacy Endpoints

[0312] Statistical methods for security analysis Safety and tolerability were evaluated through clinical examination of all relevant parameters, including adverse events and laboratory parameters. A broad AE category was summarized using point estimates and 95% CI, comprising: any AE, drug-related AE, serious AE, drug-related and serious AE, and the percentage of participants who discontinued due to AE (Table 9).

[0313] Table 9. Analysis Strategies for Security Parameters

[0314] An AE is any adverse medical event that occurs in a clinical research participant and is temporally related to the use of the study intervention, whether or not it is considered related to the study intervention. Therefore, an AE can be any adverse and unexpected sign (including abnormal laboratory findings), symptom, or illness (new or worsening) that is temporally related to the use of a medication. The following are included as AEs: • Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or other safety assessments (e.g., ECG, radiological scans, vital sign measurements), including those that deteriorate from baseline or those deemed clinically significant in the investigator’s medical and scientific judgment.

[0315] • Exacerbation of a pre-existing chronic or intermittent condition, including an increase in the frequency and / or intensity of the condition.

[0316] • New conditions detected or diagnosed after the study intervention is administered, even if they may have existed before the study began.

[0317] • Signs, symptoms, or clinical sequelae of suspected drug-drug interactions.

[0318] • Investigate signs, symptoms, or clinical sequelae of suspected overdose during intervention or concomitant medication.

[0319] • The worsening of signs and symptoms of malignancy during the study period is reported as an AE. Disease progression assessed by radiography or other methods of measuring malignancy is not reported as an AE unless the event results in hospitalization or death.

[0320] For the purposes of this study, the following events do not meet the definition of AE: • Medical or surgical procedures (e.g., endoscopy, appendectomy): The condition that leads to the procedure is an AE.

[0321] • No adverse medical events occurred (social and / or convenient admission).

[0322] • Expected daily fluctuations of a pre-existing disease or condition that is present or detected at the start of the study and has not worsened.

[0323] • Plan surgery to treat a pre-existing condition that has not yet worsened before obtaining informed consent.

[0324] If an event is not an AE according to the above definition, it is not a SAE, even if it meets the criteria for a serious condition. An SAE is defined as any adverse medical event occurring at any dose where the following occurs: • Caused death • Life-threatening. The term "life-threatening" in the definition of "serious" refers to an event in which participants are at risk of death at the time of the event. It does not refer to an event that is assumed to cause death (if it is more serious).

[0325] • Hospitalization is required for patients who require hospitalization or extension of existing hospitalization. Hospitalization is defined as hospitalization of a patient regardless of the length of stay, even if hospitalization is a precautionary measure for continued observation. Hospitalization for selective procedures for pre-existing conditions that have not yet worsened is not a SAE. Pre-existing conditions are clinical conditions diagnosed and documented in the participant's medical history prior to the use of the MSD product.

[0326] • Leading to persistent or significant disability / incapacity. The term disability means a substantial impairment of a person’s ability to perform normal life functions. This definition is not intended to include experiences of relatively minor medical significance, such as simple headaches, nausea, vomiting, diarrhea, flu, and accidental injuries (e.g., ankle sprains) that may interfere with or prevent daily life functions but do not constitute a substantial impairment.

[0327] • It refers to congenital abnormalities / birth defects in the offspring of participants who took the product, regardless of the time of diagnosis.

[0328] In determining whether an SAE report is appropriate for other circumstances (such as significant medical events that may not immediately threaten life or lead to death or hospitalization, but could endanger the participant or may require medical or surgical intervention to prevent one of the other outcomes listed in the above definition), a medical or scientific judgment is made. These events are generally considered serious. Examples of such events include aggressive or malignant cancer, intensive treatment of allergic bronchospasm in the emergency room or at home, blood-fluid imbalances or seizures that do not lead to hospitalization, or the development of drug dependence or substance abuse.

[0329] Demographic and baseline characteristics The number and percentage of subjects screened and assigned are displayed, along with the main reasons for screening failure and discontinuation. For all enrolled subjects, demographic variables (e.g., age, sex), baseline characteristics, primary and secondary diagnoses, and prior and concomitant therapies are summarized using descriptive statistics or classification tables.

[0330] Subgroup analysis To determine whether response rates were consistent across subgroups, estimates of the primary endpoint response rate (within the nominal 95% CI) were made within each category of the following categorical variables: • Age category (<65 years old vs. ≥65 years old) • Gender (female vs. male) • Race (White vs. Non-white) • Disease stages (III vs. IVM1a vs. IVM1b vs. IVM1c) • Brain metastasis (Yes vs. No) • ECOG status (0 vs. 1) • PD-L1 status (positive vs. negative) • BRAF wild-type vs. BRAF mutant (untreated) vs. BRAF mutant (treated) A forest plot was generated, providing point estimates and CIs for the treatment effects across the subgroup categories listed above. Any specified subgroups with fewer than 10 participants were excluded from the analysis.

[0331] All references cited herein are incorporated by reference to the same extent that each individual publication, database entry (e.g., Genbank or GeneID entry), patent application, or patent specifically indicates incorporation by reference. The applicant, in accordance with 37 CFR §1.57(b)(1), intends this statement to apply to each and every individual publication, database entry (e.g., Genbank or GeneID entry), patent application, or patent, which is expressly identified in accordance with 37 CFR §1.57(b)(2), even if such citation does not directly approximate the specific expression incorporated by reference. The citation of references herein is not intended as an admission that the reference is relevant prior art, nor is it intended to constitute any admission of the content or date of such references or documents.

Claims

1. A method of treating cancer in a human patient, comprising administering approximately 400 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof to the patient every approximately six weeks, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises: (a) A light chain complementarity-determining region (CDR) comprising the amino acid sequences shown in SEQ ID NO:1, 2 and 3 and a heavy chain CDR comprising the amino acid sequences shown in SEQ ID NO:6, 7 and 8; or (b) A light chain CDR containing the amino acid sequences shown in SEQ ID NO:11, 12 and 13 and a heavy chain CDR containing the amino acid sequences shown in SEQ ID NO:14, 15 and 16.

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

4.

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

5.

6. The method of any one of claims 1-5, 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, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary gland cancer.

7. The method of any one of claims 1-6, wherein the patient has a tumor with a high mutation burden.

8. The method of any one of claims 1-5, wherein the patient has a solid tumor with high microsatellite instability (MSI-H) or mismatch repair deficiency.

9. The method of any one of claims 1-5, wherein the cancer is an unresectable or metastatic melanoma.

10. The method of any one of claims 1-5, wherein the cancer is metastatic non-small cell lung cancer (NSCLC).

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