RU2846266C2 - Methods of treating malignant neoplasm using anti-pd-1 antibodies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to oncology. Presented is a method of treating a malignant new growth expressing PD-L1 in a human patient involving administering anti-PD-1 antibody 400 mg every six weeks into the patient.

EFFECT: invention provides the prolonged dosing regimen of the anti-PD-1 antibody which is safe, effective and more convenient for patients.

18 cl, 4 dwg, 9 tbl, 2 ex

Description

The present invention relates to therapeutic agents that can be used to treat a malignant neoplasm. In particular, the invention relates to a method for treating a malignant neoplasm, comprising administering to a patient in need thereof an antibody against PD-1 or an antigen-binding fragment thereof using the dosage regimens defined herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/630,038, filed February 13, 2018, and U.S. Provisional Patent Application No. 62/732,828, filed September 18, 2018, the contents of which are hereby incorporated by reference in their entirety.

LINK TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of this application has been filed electronically via EFS-Web as an ASCII sequence listing with the file name "24567WOPCT-SEQLIST-25JAN2019.TXT", a creation date of January 25, 2019, and a size of 23.7 KB. This sequence listing, filed via EFS-Web, is a part of this specification and is hereby incorporated by reference in its entirety.

LEVEL OF TECHNOLOGY

PD-1 is considered an important component of immune regulation and 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 in regulating autoimmunity and infection. Nature Immunology (2007); 8:239–245).

Two known PD-1 ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), are expressed in human malignancies arising in a variety of tissues. In large series of samples, such as ovarian cancer, renal cell carcinoma, colorectal cancer, pancreatic cancer, liver cancer, and melanoma, PD-L1 expression was shown to be correlated with poor prognosis and reduced overall survival, independent 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)).

Similarly, PD-1 expression on tumor-infiltrating lymphocytes has been found to be an indicator of 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 correlates with poor prognosis in renal cell carcinoma (Thompson et al ., Clinical Cancer Research 15: 1757–1761 (2007)). Thus, PD-L1-expressing tumor cells are suggested to interact with PD-1-expressing T cells to reduce T cell activation and immune evasion, thereby contributing to impaired tumor immune response.

Immune checkpoint therapeutics targeting the PD-1 axis result in significant improvements in clinical response in many human malignancies (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). Immune therapeutics targeting the PD-1 axis include monoclonal antibodies against the PD-1 receptor (KEYTRUDA™ (pembrolizumab), Merck and Co., Inc., Kenilworth, NJ, USA and OPDIVO™ (nivolumab), Bristol-Myers Squibb Company, Princeton, NJ, USA) and antibodies that bind to the PD-L1 ligand (MPDL3280A; TECENTRIQ™ (atezolizumab), Genentech, San Francisco, CA, USA; IMFINZI™ (durvalumab), AstraZeneca Pharmaceuticals LP, Wilmington, DE; BAVENCIO™ (avelumab), Merck KGaA, Darmstadt, Germany). Both therapeutic approaches have demonstrated antitumor effects in a variety of malignancies.

The development of additional dosing regimens that allow for the administration of a safe and effective dose of anti-PD-1 antibody that is more convenient for patients would be useful.

ESSENCE OF THE INVENTION

The present invention relates to alternative, less frequent dosing regimens for treating a patient with a cancer using an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the dosing regimen is expected to provide a safe and effective dose of the anti-PD-1 antibody or antigen-binding fragment thereof. In particular, the invention relates to a method of treating a cancer in a human patient, comprising administering about 400 mg of an anti-PD-1 antibody or antigen-binding fragment thereof to the patient every six weeks, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) light chain complementarity determining regions (CDRs) comprising the amino acid sequence set forth in SEQ ID NOs: 1, 2, and 3, and heavy chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 6, 7, and 8; or (b) light chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 11, 12, and 13 and heavy chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 14, 15, and 16. In preferred embodiments, the antibody or antigen-binding fragment is pembrolizumab.

In embodiments of the invention, the amount of the anti-PD-1 antibody or antigen-binding fragment thereof administered to the patient is from about 350 mg to about 450 mg. In further embodiments, the amount of the antibody or antigen-binding fragment is about 400 mg. In further embodiments, the amount of the antibody or antigen-binding fragment is 400 mg.

In all of the above treatment methods, compositions and uses of the present invention, the anti-PD-1 antibody or antigen-binding fragment inhibits the binding of PD-L1 to PD-1, and also preferably inhibits the binding of PD-L2 to PD-1. In some preferred embodiments of the treatment methods, compositions and uses of the invention, the anti-PD-1 antibody or antigen-binding fragment thereof 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 the heavy and light chains comprise the amino acid sequences shown in Fig. 1 (SEQ ID NO: 5 and SEQ ID NO: 10).

In some embodiments of any of the above methods of treatment, compositions, and uses, the cancer expresses one or both of PD-L1 and PD-L2. In some embodiments, the expression of PD-L1 in the cancer is increased.

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

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

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows the amino acid sequences of the light chain and heavy chain of an exemplary anti-PD-1 monoclonal antibody that can be used in the present invention (SEQ ID NOs: 5 and 10, respectively). The variable regions of the light chain and heavy chain are underlined (SEQ ID NOs: 4 and 9), and the CDRs are highlighted in bold and boxed.

Fig. 2 shows that the steady-state C _max of pembrolizumab for 400 mg Q6W is in the range corresponding to 2 mg/kg Q3W and 200 mg Q3W to 10 mg/kg Q2W.

Figure 3 shows that the extent of pembrolizumab exposure ( _Cmean and _Cmin ) at steady state for 400 mg Q6W is similar to 2 mg/kg Q3W and 200 mg Q3W.

Figures 4A and 4B show the steady-state pharmacokinetic profiles of pembrolizumab for the 400 mg Q6W dosing regimen compared to the fixed-dose 200 mg Q3W dosing regimen (top), the 2 mg/kg body weight Q3W dosing regimen (middle), and the 10 mg/kg body weight Q2W dosing regimen (bottom). Results are presented for concentrations on a log scale (Figure 4A) and concentrations on a linear scale (Figure 4B).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions and Abbreviations

Throughout the present description and claims, the following abbreviations are used:

AE - adverse event

AUC _ss - area under the concentration-time curve at steady state

BICR - Independent Blinded Centralized Review

C _average,ss - time-averaged concentration at equilibrium

CDR - complementarity determining region

CI - confidence interval

C _max,ss - peak concentrations at equilibrium

C _min,ss - residual concentrations in equilibrium state

CPS - Combined Positivity Score

DOR - duration of response

ECG - electrocardiogram

ECOG - Eastern Cooperative Oncology Group of the United States

E-R - impact (concentration)-response

FFPE - formalin-fixed, paraffin-embedded specimens

FR - frame region

GM - geometric mean

HCC - hepatocellular carcinoma

HNSCC - head and neck squamous cell carcinoma

HL - Hodgkin's lymphoma

IgG - immunoglobulin G

IHC - immunohistochemistry or immunohistochemical analysis

IV - intravenous

LPS - lymphoma proportion score

mAb - monoclonal antibody

MCC - Merkel cell carcinoma

MEL - melanoma

MMR - mismatch repair

MPS - Modified Proportion Scale

MRI - magnetic resonance imaging

MSI-H - with high microsatellite instability

NCI CTCAE - National Cancer Institute Common Terminology Criteria for Adverse Events

NSCLC - non-small cell lung cancer

ORR - objective response rate

OS - overall survival

PD is a progressive disease

PD-1 - programmed death protein 1 (also known as programmed cell death protein-1 and programmed death receptor 1)

PD-L1 - programmed cell death protein 1 ligand 1

PD-L2 - programmed cell death protein 2 ligand 1

PFS - progression free survival

PK - pharmacokinetic

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 event

SC - subcutaneous

TPS - tumor proportion scale

V _H - variable region of the immunoglobulin heavy chain

V _L - variable region of the light chain of immunoglobulin.

For a better understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise defined elsewhere in this description, all other technical and scientific terms used in this description have the meaning commonly understood by a person skilled in the art to which the invention pertains.

The term "or" means either or both of the possibilities unless the context clearly indicates one of the stated possibilities. In some cases, the term "and/or" is used to refer to either or both possibilities.

Within the scope of the invention, including the claims, singular terms include reference to corresponding plural terms unless the context clearly indicates otherwise.

The term "about" with respect to an amount (e.g., mg) of a substance or composition, or a value of a parameter characterizing a step of a method, or the like, refers to variations in the numerical value that arise, for example, during typical measuring, handling, and sampling methods used in the preparation, characterization, and/or use of the substance or composition; as a result of the inherent error of these methods; as a result of differences in the manufacturing, source, or purity of ingredients used to prepare or use the compositions or perform the methods; and the like. In some embodiments, the term "about" can mean variations of ±0.1%, ±0.5%, ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%. With respect to the dose of "about 400 mg", the dose may be from 360 mg to 440 mg, from 370 mg to 430 mg, from 380 mg to 420 mg, from 390 mg to 410 mg, from 395 mg to 405 mg, from 400 mg to 440 mg, or from 390 mg to 440 mg. In alternative embodiments, 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. With respect to the amount of time between administrations in a therapeutic regimen (i.e., the amount of time between administrations of an anti-PD-1 antibody or antigen-binding fragment thereof, e.g., "about 6 weeks," which is used interchangeably herein with the term "about every six weeks"), the term "about" refers to the stated time ± variations due to scheduling and patient/clinician availability around the 6-week target date. For example, the term "about 6 weeks" may refer to 6 weeks ± 5 days, 6 weeks ± 4 days, 6 weeks ± 3 days, 6 weeks ± 2 days, or 6 weeks ± 1 day, or may refer to a period from 5 weeks and 2 days to 6 weeks and 5 days.

Pharmacokinetic "steady state" is the period of time during which any accumulation of drug concentrations following administration of multiple doses is maximized and systemic exposure to the drug is considered uniform after each subsequent dose; in the specific case of pembrolizumab, steady state is achieved after ~16 weeks of administration and after ~16 weeks of administration.

AUC _ss , C _mean,ss and C _min,ss are pharmacokinetic measures of systemic exposure to a drug (e.g. pembrolizumab) in humans after administration and are generally considered determinants of drug efficacy. AUC _ss and C _mean,ss represent the average exposure over a dosing interval but differ in units. "C _min,ss " is the minimum or lowest (trough) drug concentration observed at the end of a dosing interval immediately prior to administration of the next dose.

"C _max,ss " is the maximum or highest (peak) concentration of a drug observed shortly after administration. In the specific case of pembrolizumab, which is administered as an intravenous infusion, the peak concentration is reached immediately after completion of the infusion. C _max,ss is a value that is generally considered a determining factor in safety.

The terms "administering" and "treating" with respect to an animal, human, experimental subject, cell, tissue, organ or biological fluid refer to contacting an exogenous pharmaceutical, therapeutic, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid. As used herein, the terms "treating" or "treating" a malignant neoplasm means administering an anti-PD-1 antibody or antigen-binding fragment thereof to an individual having or diagnosed with a malignant neoplasm to achieve at least one beneficial therapeutic effect, such as, for example, reducing the number of malignant cells, reducing the size of a tumor, reducing the rate of infiltration of peripheral organs by malignant cells or reducing the rate of tumor metastasis or tumor growth. The term "treatment" may include one or more of the following: inducing/enhancing an anti-tumor immune response, decreasing one or more tumor markers, stopping or slowing tumor or hematological malignancy growth or disease progression associated with the binding of PD-1 to its ligands PD-L1 and/or PD-L2 ("PD-1-associated disease") such as cancer, stabilizing a PD-1-associated disease, inhibiting tumor cell growth or survival, eliminating or reducing the size of one or more lesions of cancer or tumors, decreasing one or more tumor markers, improving or eliminating the clinical manifestation of a PD-1-associated disease, reducing the severity or duration of clinical symptoms of a PD-1-associated disease such as cancer, prolonging patient survival relative to the expected survival of a similar untreated patient, and inducing complete or partial remission of a cancer neoplasm or other PD-1-associated disease.

Positive therapeutic effects in cancer can be measured in a number of ways (see W. A. Weber, J. Nucl. Med . 50:1S-10S (2009)). For example, with respect to tumor growth inhibition, according to NCI standards, a T/C ≤42% is a minimum level of antitumor activity. A T/C <10% is considered high antitumor activity, where T/C (%) = median tumor volume of treatment/median tumor volume of control x 100. In some embodiments, the treatment effect achieved by a therapeutically effective amount is any of progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). The term "PFS", also referred to as "time to progression", refers to the length of time during and after treatment that the cancer does not grow, and includes the amount of time that patients have a complete response or partial response, as well as the amount of time that patients have stable disease. The term "DFS" refers to the length of time during and after treatment that the patient does not have evidence of disease. The term "OS" refers to the prolongation of survival compared to untreated individuals or patients. Although an embodiment of the methods of treatment, compositions and uses of the present invention may not be effective in achieving a positive therapeutic effect in every patient, it should be so for a statistically significant number of individuals, as determined by any statistical test known in the art, such as the Student t-test, the chi ² test, the Mann-Whitney U-test, the Kruskal-Wallis (H-test), the Jonckheere-Terpstra test and the Wilcoxon test.

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

The term "antibody" refers to any form of antibody that exhibits the desired biological or binding activity. It is thus used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized antibodies, fully human antibodies, and chimeric antibodies. "Parent antibodies" are antibodies produced by exposing the immune system to an antigen prior to modification of the antibody for its intended use, such as humanization of an antibody for use as a human therapeutic.

In general, the basic structural unit of an antibody comprises a tetramer. Each tetramer comprises two identical pairs of polypeptide chains, each pair comprising one "light" (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain comprises a variable region of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxy-terminal portion of a heavy chain may define a constant region primarily responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. In addition, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and they determine the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with the heavy chain also including a "D" region of approximately 10 more amino acids. Generally, see Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

The variable regions of each light/heavy chain pair form the binding site of the antibody. Thus, essentially an intact antibody has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are essentially the same.

Typically, the variable domains of the heavy and light chains contain three hypervariable regions, also called complementarity-determining regions (CDRs), contained within relatively conserved framework regions (FRs). The CDRs are typically aligned within the framework regions, allowing binding to a specific epitope. Generally, from N-terminus to C-terminus, the variable domains of the light and heavy chains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain generally follow the definitions in Sequences of Proteins of Immunological Interest , Kabat, et al .; National Institutes of Health, Bethesda, Md.; 5 ^th 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.

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

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

An antibody that "specifically binds to" a particular target protein is an antibody that exhibits preferential binding to that target over other proteins, but this specificity does not require absolute binding specificity. An antibody is considered "specific" for a given target if its binding is determinative of the presence of the target protein in a sample, for example, without causing undesirable results such as false positives. Antibodies, or binding fragments thereof, that may be used in the present invention will bind to the target protein with an affinity that is at least two-fold, preferably at least ten-fold, more preferably at least 20-fold, and most preferably at least 100-fold greater than the affinity for non-target proteins. Within the framework of the invention, it is indicated that the antibody specifically binds to a polypeptide containing the specified amino acid sequence, for example, the amino acid sequence of the mature molecule of human PD-1 or human PD-L1, if it binds to polypeptides containing this sequence, but does not bind to proteins that lack this sequence.

The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is identical to or homologous with corresponding sequences in an antibody derived from a particular species (e.g., human) or belonging to a particular class or subclass of antibodies, while the remainder of the chains is identical to or homologous with corresponding sequences in an antibody derived from a particular species (e.g., mouse) or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity.

The term "human antibody" refers to an antibody containing only human immunoglobulin protein sequences. A human antibody may contain mouse carbohydrate chains if it is produced by a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, the terms "mouse antibody" or "rat antibody" refer to an antibody containing only mouse or rat immunoglobulin sequences, respectively.

The term "humanized antibody" refers to forms of antibodies that comprise sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies comprise a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to loops of a non-human immunoglobulin and all or substantially all of the FR regions are FR regions of a human immunoglobulin sequence. A humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically from a human immunoglobulin. The prefix "hum", "hu", or "h" is added to the antibody clone designation when necessary to distinguish humanized antibodies from the parental rodent antibodies. Humanized forms of rodent antibodies will generally contain the same CDR sequences of the parent rodent antibodies, although some amino acid substitutions may be included to improve affinity, enhance the stability of the humanized antibody, or for other reasons.

The terms "malignant neoplasm" or "malignant" refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell growth. Non-limiting examples of malignant neoplasms include carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific, non-limiting examples of such malignancies include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer. Additional malignancies that can be treated in accordance with the present invention include malignancies characterized by increased expression of one or both of PD-L1 and PD-L2 in test tissue samples.

The term "biotherapeutic agent" means a biological molecule, such as an antibody or fusion protein, that blocks ligand/receptor signaling in any biological pathway that supports tumor maintenance and/or growth or suppresses the anti-tumor immune response.

The term "CDR" refers to the complementarity determining regions of the immunoglobulin variable region, typically defined using the Kabat numbering scheme.

The term "platinum chemotherapy" (also known as platins) refers to the use of chemotherapeutic agents used to treat cancer that are platinum coordination complexes. Platinum-containing chemotherapeutic agents are alkylating agents that cross-link DNA, leading to ineffective DNA mismatch repair and, typically, apoptosis. Examples of platins include cisplatin, carboplatin, and oxaliplatin.

A "chemotherapeutic agent" is a chemical compound that can be used in the treatment of a malignancy. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, plant alkaloids that are spindle poisons, cytotoxic/antineoplastic antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens and selective estrogen receptor modulators (SERMs), antiprogesterones, estrogen receptor suppressors (ERDs), estrogen receptor antagonists, luteinizing hormone releasing factor agonists, antiandrogens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Chemotherapeutic agents that can be used in the treatment methods of the present invention include cytostatic and/or cytotoxic agents.

The term "Chothia definition" refers to the numbering system of residues in an antibody described in Al-Lazikani et al. , JMB 273 : 927-948 (1997).

The terms "conservatively modified variants" or "conservative substitution" refer to substitutions of amino acids in a protein with other amino acids that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that the changes can often be made without altering biological activity or other desired property of the protein, such as antigen affinity and/or specificity. Those skilled in the art will appreciate that, in general, substitutions of individual amino acids in non-critical regions of a polypeptide will not significantly alter biological activity (see, e.g., Watson et al. (1987) Molecular biology of the Gene , The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to impair biological activity. Examples of conservative substitutions are provided in Table 1.

Table 1. Examples of conservative amino acid substitutions

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

The term "consists essentially of" and variations thereof such as "consist essentially of" or "consisting essentially of," as used throughout the present specification and claims, means the inclusion of the listed elements or groups of elements and the optional inclusion of other elements of a similar or different nature relative to the listed elements, which do not substantially alter the basic or novel properties of a particular dosage regimen, method, or composition. As a non-limiting example, a PD-1 antigen-binding fragment consisting essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, which do not substantially alter the properties of the binding compound.

The term "comprising" or its variations such as "comprise", "comprises" or "consisting of" is used throughout the present description and claims in an inclusive sense, i.e. to specify the presence of the stated features, but not to exclude the presence or addition of additional features that may significantly improve the operation of any embodiments of the invention or their utility, unless the context otherwise requires due to the language of expression or necessary implied implications.

The term "diagnostic monoclonal antibody against PD-L" means a mAb that specifically binds to the mature form of a particular PD-L (PD-L1 or PD-L2) expressed on the surface of certain mammalian cells. Mature PD-L lacks a presecretory leader sequence, also referred to 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 stated or clearly apparent from the context.

As used herein, the term "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:

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVD PVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 17).

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

Table 2. Monoclonal antibodies 20C3 and 22C3

Mature variable region light chain 20C3 DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYDVVTFGAGTKLELK SEQ ID NO:18 Mature variable region heavy chain 20C3 QVQVQQSGAELAEPGASVKMSCKASGYIFTSYWMHWLKQRPGQGLEWIGYINPSSDYNEYSEKFMDKATLTADKASTTAYMQLISLTSEDSAVYYCARSGWLVHGDYYFDYWGQGTTLTVSS SEQ ID NO:19 Mature variable region light chain 22C3 DIVMSQSPSSLAVSAGEKVTMTCKSSQSLLHTSTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYDVVTFGAGTKLELK SEQ ID NO:20 Mature variable region heavy chain 22C3 QVHLQQSGAELAKPGASVKMSCKASGYTFTSYWIHWIKQRPGQGLEWIGYINPSSGYHEYNQKFIDKATLTADRSSSTAYMHLTSLTSEDSAVYYCARSGWLIHGDYYFDFWGQGTTLTVSS SEQ ID NO:21

Another anti-human PD-L1 mAb that has been described to be useful for IHC detection of PD-L1 expression in FFPE tissue sections (Chen, BJ et al ., Clin Cancer Res 19: 3462–3473 (2013)) is rabbit anti-human PD-L1 mAb, available from Sino Biological, Inc. (Beijing, PR China; Cat. No. 10084-R015).

As used herein, the term "framework region" or "FR" refers to the variable regions of an immunoglobulin, excluding the CDR regions.

The term "isolated antibody" and "isolated antibody fragment" refers to the state of purification and, in such context, means that the molecule in question is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. In general, the term "isolated" is not intended to mean the complete absence of such material or the absence of water, buffers, or salts unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the binding compound as described herein.

As used herein, the term "Kabat definition" means the immunoglobulin alignment and numbering system described by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.).

As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. Conversely, conventional (polyclonal) antibody preparations typically include a plurality of different antibodies having different amino acid sequences in their variable domains, particularly in the CDRs, which are often specific for different epitopes. The modifier "monoclonal" is indicative of the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be produced by the hybridoma method first described by Kohleret al. (1975)Nature256: 495, or can be produced by recombinant DNA methods (see, (e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from antibody phage libraries by methods described, for example, in Clacksonet al. (1991)Nature352: 624-628 and Markset al. (1991)J. Mol. Biol. 222: 581-597. See also Presta (2005)J. Allergy Clin. Immunol. 116:731.

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

Expression of "PD-L1" or "PD-L2" means any detectable level of expression of said PD-L protein on the surface of a cell or said PD-L mRNA in a cell or tissue, unless otherwise indicated. Expression of PD-L protein can be determined using a diagnostic antibody against PD-L in an IHC assay of a tumor tissue section or by flow cytometry. Alternatively, expression of PD-L protein by tumor cells can be determined by PET using a binding agent (e.g., an antibody fragment, an affibody, etc.) that specifically binds to a desired PD-L target, such as PD-L1 or PD-L2. Methods for detecting and measuring PD-L mRNA expression include RT-PCR and real-time quantitative RT-PCR.

Several approaches have been described for quantitative analysis of PD-L1 protein expression in IHC assays on tumor tissue sections. 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).

One approach uses a simple binary endpoint of positive or negative PD-L1 expression, with positivity defined in terms of the percentage of tumor cells exhibiting histologic evidence of cell surface membrane staining. A tumor tissue section is considered positive for PD-L1 expression if it is expressed in at least 1%, and preferably 5%, of all tumor cells.

In another approach, PD-L1 expression in a tumor tissue section is quantified in tumor cells as well as infiltrating immune cells, including mainly lymphocytes. The percentage of tumor cells and infiltrating immune cells showing membranous staining is separately quantified as <5%, 5-9%, and then in 10% increments up to 100%. For tumor cells, PD-L1 expression is considered negative if the score is <5% and positive if the score is ≥5%. PD-L1 expression in the immune infiltrate is recorded as a semiquantitative measurement called the adjusted inflammatory score (AIS), determined by multiplying the percentage of cells with membranous staining by the intensity of the infiltrate, classified as absent (0), mild (score 1, rare lymphocytes), moderate (score 2, focal tumor infiltration with lymphohistiocytic aggregates), or severe (score 3, diffuse infiltration). A tumor tissue section is considered positive for PD-L1 expression by immune infiltrates if the AIS is ≥5.

A tumor tissue section stained by IHC using a diagnostic antibody against PD-L1 can also be assessed for PD-L1 protein expression by assessing PD-L1 expression in both tumor cells and infiltrating immune cells in the tissue section using a scoring method. See WO 2014/165422. One method for scoring PD-L1 involves examining each tumor cell nest in a tissue section for staining and assigning a score to the tissue section using one or both of a modified H-score (MHS) and a modified proportion score (MPS). To assign an MHS score, four separate percentages of all viable tumor cells and stained mononuclear inflammatory cells in all tumor cell nests examined are assessed: (a) cells showing no staining (intensity = 0), (b) weak staining (intensity = 1+), (c) moderate staining (intensity = 2+), and (d) strong staining (intensity = 3+). A cell must have at least partial membranous staining to be included in the percentage of weak, moderate, or strong staining. The percentages analyzed, which add up to 100%, are then entered into the formula 1 × (percentage of weakly staining cells) + 2 × (percentage of moderately staining cells) + 3 × (percentage of strongly staining cells), and the result is assigned to the tissue section as an MHS score. MPS is assigned by assessing the percentage of cells having at least partial membranous staining of any intensity across all viable tumor cells and stained mononuclear inflammatory cells in all tumor cell nests examined, and the resulting percentage is assigned to the tissue section as an MPS score. In some embodiments, a tumor is designated as positive for PD-L1 expression if MHS or MPS is positive.

Another way to score/quantify PD-L1 expression in a tumor is the "combined positivity score" or "CPS," which refers to an algorithm for determining a score for PD-L1 expression in a patient's tumor specimen. CPS is useful in selecting patients for treatment with specific regimens, including therapies that include administration of an anti-PD-1 antibody, in which PD-L1 expression is associated with a higher response rate in a particular patient population relative to the same patient population who do not express PD-L1. CPS is determined by determining the number of viable PD-L1-positive tumor cells, the number of viable PD-L1-negative tumor cells, and the number of viable PD-L1-positive mononuclear inflammatory cells (MICs) in the tumor tissue of a patient bearing a tumor and calculating the CPS using the following formula:

In particular embodiments, the method used to score PD-L1 expression is the "lymphoma proportion score." Lymphoma is characterized by a homogeneous population of confluent cells that disrupt the architecture of a lymph node or metastatic site. The "LPS" or "lymphoma proportion score" is the percentage of this population of cells that express PD-L1. The LPS does not attempt to distinguish true neoplastic cells from reactive cells. PD-L1 expression is characterized by partial or complete membranous staining of any intensity.

Another way to score PD-L1 expression is the "TPS" or "tumor proportion score," which is the percentage of tumor cells expressing PD-L1 on the cell membrane. TPS typically includes the percentage of neoplastic cells expressing PD-L1 at any intensity (weak, moderate, or weak), which can be determined using immunohistochemistry using a diagnostic mAb against human PD-L1, such as the 20C3 antibody and 22C3 antibody described above. Cells are considered to express PD-L1 if membranous staining is present, including cells with partial membranous staining.

PD-L mRNA expression levels can be compared with mRNA expression levels of one or more reference genes frequently used in qRT-PCR, such as ubiquitin C.

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

The term "tissue section" refers to a single piece or fragment of a tissue sample, such as a thin slice of tissue cut from normal tissue or a tumor.

The term "tumor," when used in reference to an individual diagnosed with or suspected of having a malignant neoplasm, refers to a malignant or potentially malignant neoplasia or tissue mass of any size and includes primary tumors and secondary neoplasias. A solid tumor is an abnormal growth or mass of tissue, usually without a cyst or fluid area. The different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. Leukemias (hemoblastoses) do not usually form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

As used herein, the terms "variable regions" or "V region" mean a segment of IgG chains that is variable in sequence between different antibodies. It extends up to Kabat residue 109 in the light chain and residue 113 in the heavy chain.

For the purposes of the invention, the term "RECIST 1.1 Response Evaluation Criteria" means the definitions provided in Eisenhauer, EA et al ., Eur. J. Cancer 45:228-247 (2009), for target lesions or non-target lesions, as appropriate, taking into account the context in which the response is measured.

II. Anti-PD-1 antibodies and antigen-binding fragments thereof that can be used in the present invention

Examples of mAbs that bind to human PD-1 that may be used in the treatment methods, compositions, and uses of the invention are described in U.S. Patent Nos. 7,521,051, 8,008,449, and 8,354,509. Specific anti-human PD-1 mAbs that may be used as a PD-1 antagonist in the treatment methods, compositions, and uses of the present invention include: pembrolizumab (formerly known as MK-3475, SCH 900475, and lambrolizumab), a humanized IgG4 mAb with the structure described in WHO Drug Information , Vol. 27, No. 2, pages 161-162 (2013), comprising the heavy and light chain amino acid sequences shown in FIG. 1, and humanized antibodies h409A11, h409A16 and h409A17 described in WO 2008/156712 and in Table 3.

In some embodiments of the methods of treatment, compositions, kits, and uses of the present invention, an anti-PD-1 antibody or antigen-binding fragment thereof comprises: (a) light chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 1, 2, and 3 and heavy chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 6, 7, and 8; or (b) light chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 11, 12, and 13 and heavy chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 14, 15, and 16. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a human antibody. In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a humanized antibody. In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a chimeric antibody. In specific embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a monoclonal antibody.

In other embodiments of the methods of treatment, compositions, kits and uses of the present invention, the anti-PD-1 antibody or antigen-binding fragment thereof specifically binds to human PD-1 and comprises (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof, and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of 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.

A variant heavy chain variable region sequence or a full-length heavy chain sequence is identical to a reference sequence, except that it includes 17 conservative amino acid substitutions in the framework region (i.e., outside the CDRs) and, preferably, includes less than ten, nine, eight, seven, six, or five conservative amino acid substitutions in the framework region. A variant light chain variable region sequence or a full-length light chain sequence is identical to a reference sequence, except that it includes up to five conservative amino acid substitutions in the framework region (i.e., outside the CDRs) and, preferably, includes less than four, three, or two conservative amino acid substitutions in the framework region.

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

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

Table 3 below provides a list of amino acid sequences of exemplary anti-PD-1 mAbs for use in the treatment methods, compositions, kits, and uses of the present invention.

Table 3. Examples of antibodies against human PD-1

A. Contains the hPD-1.09A light and heavy chain CDRs in WO2008/156712 (pembrolizumab light and heavy chain CDRs) CDRL1 SEQ ID NO:1 CDRL2 SEQ ID NO:2 CDRL3 SEQ ID NO:3 CDRH1 SEQ ID NO:6 CDRH2 SEQ ID NO:7 CDRH3 SEQ ID NO:8 B. Contains the hPD-1.08A light and heavy chain CDRs in WO2008/156712 CDRL1 SEQ ID NO: 11 CDRL2 SEQ ID NO:12 CDRL3 SEQ ID NO:13 CDRH1 SEQ ID NO:14 CDRH2 SEQ ID NO:15 CDRH3 SEQ ID NO:16 C. Contains the mature variable heavy chain region (V _H ) h109A and one of the mature variable light chain regions (V _L ) K09A in WO 2008/156712 V _H heavy chain SEQ ID NO: 9 (V _H of pembrolizumab) V _L light chain SEQ ID NO: 4 (V _L of pembrolizumab) or SEQ ID NO: 22 or SEQ ID NO: 23 D. Contains the mature heavy chain 409 and one of the mature light chains K09A in WO 2008/156712 Heavy chain SEQ ID NO: 10 (pembrolizumab heavy chain) Light chain SEQ ID NO: 5 (pembrolizumab light chain) or SEQ ID NO: 24 or SEQ ID NO: 25

III. Methods and application of the invention

The invention relates to a method of treating a cancer in a human patient, comprising administering about 400 mg of an anti-PD-1 antibody or antigen-binding fragment thereof to the patient once about every six weeks, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises: (a) light chain complementarity determining regions (CDRs) comprising the amino acid sequence set forth in SEQ ID NOs: 1, 2, and 3, and heavy chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 6, 7, and 8; or (b) light chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 11, 12, and 13, and heavy chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 14, 15, and 16. In specific embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is pembrolizumab.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the patient about once every six weeks for 12 weeks or more. In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the patient once every six weeks for 18 weeks or more, 24 weeks or more, 30 weeks or more, 36 weeks or more, 42 weeks or more, 48 weeks or more, 54 weeks or more, 60 weeks or more, 66 weeks or more, 72 weeks or more, 78 weeks or more, 84 weeks or more, or 90 weeks or more.

In a first embodiment (Embodiment E1), the present invention includes a method of treating a cancer in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof once about every six weeks.

In a second embodiment (Embodiment E2), the present invention includes a method of treating unresectable or metastatic melanoma in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (eg, pembrolizumab) or antigen-binding fragment thereof once about every six weeks.

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

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

In a further sub-embodiment of embodiment E3 (embodiment E3-B), the patient has a tumor that expresses PD-L1 (TPS ≥1%) and has been previously treated with platinum-based chemotherapy. In particular embodiments of embodiment E3-B, the patient has disease progression during or after platinum-based chemotherapy.

In another sub-embodiment of embodiment E3 (embodiment E3-C), the patient has a tumor that expresses PD-L1 (TPS ≥1%) and has not previously been treated with platinum-based chemotherapy.

In another sub-embodiment of embodiment E3 (embodiment E3-D), the patient's tumor is not tested for PD-L1 expression. In this embodiment, the patient is treated with an anti-PD-1 antibody or antigen-binding fragment thereof regardless of PD-L1 expression. In certain embodiments, the patient has not previously been treated with platinum-based chemotherapy.

In some embodiments of embodiment E3 (including embodiment E3-A, E3-B, and E3-C), the PD-L1 TPS is determined using an FDA-approved assay.

In some embodiments of embodiment E3 (including embodiment E3-A, E3-B, E3-C, and E3-D), the patient's tumor does not have EGFR or ALK genomic aberrations.

In some embodiments of embodiment E3 (including embodiment E3-A, E3-B, E3-C, and E3-D), the patient's tumor has EGFR or ALK genomic aberrations, and disease progression is observed during or after treatment due to EGFR or ALK aberrations prior to administration of the anti-PD-1 antibody or antigen-binding fragment thereof.

In a fourth embodiment (Embodiment E4), the present invention includes a method of treating metastatic non-small cell lung cancer (NSCLC) in a human patient, comprising: (1) administering to the patient 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof once about every six weeks, and (2) administering to the patient pemetrexed and carboplatin. In sub-embodiments of Embodiment E4, the patient has not previously been treated with anti-cancer therapy prior to initiating a combination regimen using an anti-PD-1 antibody or an antigen-binding fragment thereof, pemetrexed, and carboplatin.

In some embodiments of embodiment E3 and E4 (including sub-embodiments), the patient has non-squamous cell non-small cell lung cancer.

In sub-embodiments of embodiment E4, pemetrexed is administered to the patient in an amount of 500 mg/ ^m2 .

In sub-embodiments of embodiment E4, pemetrexed is administered to the patient by intravenous infusion every 21 days. In particular embodiments, the infusion time is about 10 minutes.

In sub-embodiments of embodiment E4 (embodiment E4-A), the present invention further comprises administering to the patient about 400 mcg to about 1000 mcg of folic acid once daily, beginning about 7 days before pemetrexed is administered to the patient and continuing until about 21 days after the last dose of pemetrexed is administered to the patient. In some embodiments, the folic acid is administered orally.

In sub-embodiments of embodiments E4 and E4-A (embodiment E4-B), the present invention further comprises administering to the patient about 1 mg of vitamin B ₁₂ about 1 week prior to the first administration of pemetrexed and about every three cycles of pemetrexed administration (i.e., about every 9 weeks). In some embodiments, vitamin B ₁₂ is administered intramuscularly.

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

In a fifth embodiment (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 to the patient 400 mg of an anti-PD-1 antibody (eg, pembrolizumab) or an antigen-binding fragment thereof once about every six weeks.

In sub-embodiments of embodiment E5, the patient has previously been treated with platinum-based chemotherapy. In some embodiments, the patient has disease progression during or after platinum-based chemotherapy.

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

In a seventh embodiment (Embodiment E7), the present invention includes a method of treating classical Hodgkin lymphoma (cHL) in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof once about every six weeks, wherein the patient has relapsed after (a) one or more lines of cHL therapy, (b) 2 or more lines of cHL therapy, or (c) 3 or more lines of cHL therapy.

In sub-embodiments of embodiments E6 and E7, the patient is an adult patient.

In alternative sub-embodiments of embodiments E6 and E7, the patient is a pediatric patient.

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

In sub-embodiments of embodiment E8, the patient is not suitable for chemotherapy comprising cisplatin.

In sub-embodiments of embodiment E8, the patient has disease progression during or after platinum-based chemotherapy or within 12 months of neoadjuvant or adjuvant treatment using platinum-based chemotherapy.

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

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

In a sub-variant of embodiment E9, the patient has disease progression following prior anti-tumor treatment.

In a tenth embodiment (Embodiment E10), the present invention includes a method of treating unresectable or metastatic colorectal cancer with MSI-H or MMR deficiency in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (eg, pembrolizumab) or an antigen-binding fragment thereof once about every six weeks.

In a sub-embodiment of embodiment E10, the patient has had disease progression following prior treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.

In an eleventh embodiment (Embodiment E11), the present invention includes a method of treating recurrent, locally advanced or metastatic gastric cancer in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (eg, pembrolizumab) or an antigen-binding fragment thereof once about every six weeks.

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

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

In sub-embodiments of embodiments E11 and E12, the patient has had disease progression during or after one or more prior lines of therapy. In specific embodiments, the prior lines of therapy include a fluoropyrimidine and platinum-based chemotherapy.

In sub-embodiments of embodiments E11 and E12, the patient has had disease progression during or after two or more prior lines of therapy including a fluoropyrimidine and platinum-based chemotherapy.

In sub-embodiments of embodiments E11 and E12, the patient has had disease progression during or after one or more prior lines of therapy including HER2/neu-directed therapy.

In sub-embodiments of embodiments E11 and E12, the patient has had disease progression during or after two or more prior lines of therapy including HER2/neu-directed therapy.

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

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

In a fifteenth embodiment (Embodiment E15), the present invention includes a method of treating non-Hodgkin's lymphoma in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (eg, pembrolizumab) or antigen-binding fragment thereof once about every six weeks.

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

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

In a seventeenth embodiment (Embodiment E17), the present invention includes a method of treating Merkel cell carcinoma (MCC) in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof once about every six weeks. In certain subembodiments of embodiment E17, the cancer is recurrent, locally advanced MCC. In certain subembodiments of embodiment E17, the cancer is metastatic MCC.

In sub-embodiments of embodiment E17, the patient is an adult patient. In alternative sub-embodiments of embodiment E17, the patient is a pediatric patient.

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

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

In a twentieth embodiment (Embodiment E20), the present invention includes a method of treating renal cell carcinoma (RCC) in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (eg, pembrolizumab) or an antigen-binding fragment thereof once about every six weeks.

In sub-embodiments of embodiment E20, the malignancy is late-stage clear cell RCC.

In sub-embodiments of embodiment E20, the patient has advanced renal cell carcinoma (RCC) or metastatic renal cell carcinoma (RCC).

In sub-embodiments of embodiment E20 (embodiment E20A), the patient is additionally treated with axitinib. In sub-embodiments of the invention, axitinib is administered orally.

In specific embodiments of embodiment E20A, the patient is administered 5 mg of axitinib approximately every 12 hours or twice daily.

In alternative embodiments of embodiment E20A, the dose of axitinib is 2.5 mg, 3 mg, 7 mg, or 10 mg twice daily.

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

In a sub-embodiment of embodiment E21, the breast cancer is triple-negative breast cancer.

In a sub-embodiment of embodiment E21, the breast cancer is ER ⁺ / ^HER2- breast cancer.

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

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

In a twenty-fourth embodiment (Embodiment E24), the present invention includes a method of treating salivary gland cancer in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (eg, pembrolizumab) or an antigen-binding fragment thereof once about every six weeks.

In a twenty-fifth embodiment (embodiment E25), the present invention includes a method of treating a cancer in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof once about every six weeks, wherein the cancer is selected from the group consisting of: 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.

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

In a sub-embodiment of embodiment E26, the hematological malignancy is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia protein-1 (MCL-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), and small lymphocytic lymphoma (SLL).

In a twenty-seventh embodiment (embodiment E27), the present invention includes a method of treating a cancer in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody (eg, pembrolizumab) or an antigen-binding fragment thereof once about every six weeks, wherein the patient has a tumor with a high mutational burden.

In particular embodiments, the high mutational load is at least about 10 mutations per 1 million nucleotides of the genome being studied, at least about 11 mutations per 1 million nucleotides of the genome being studied, at least about 12 mutations per 1 million nucleotides of the genome being studied, or at least about 13 mutations per 1 million nucleotides of the genome being studied.

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

In sub-embodiments of embodiment E28, the patient has progressed on one prior line of standard therapy prior to administration of the anti-PD-1 antibody or antigen-binding fragment thereof. In a further embodiment, the patient has progressed on one or more lines of standard therapy prior to administration of the anti-PD-1 antibody or antigen-binding fragment thereof. In another embodiment, the patient has progressed on two or more lines of standard therapy prior to administration of the anti-PD-1 antibody or antigen-binding fragment thereof. In particular embodiments, the standard therapy comprises one or more of paclitaxel, docetaxel, or irinotecan.

In sub-embodiments of embodiment E28, the patient has an advanced stage adenocarcinoma or squamous cell carcinoma of the esophagus or metastatic adenocarcinoma or squamous cell carcinoma of the esophagus.

In sub-embodiments of embodiment E28, the patient has advanced stage Sievert type I gastroesophageal junction adenocarcinoma or metastatic Sievert type I gastroesophageal junction adenocarcinoma.

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

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

In a sub-embodiment of embodiment E29, the patient has been previously treated with standard therapy prior to treatment with an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the prior therapy is BCG vaccine therapy. In certain embodiments, the patient has not responded to BCG vaccine therapy. In some embodiments, the patient is not suitable for radical cystectomy or prefers not to undergo radical cystectomy.

In any of the above-described methods of the invention (including embodiments E1 through E29), the anti-PD-1 antibody or antigen-binding fragment is any of the antibodies or antigen-binding fragments described in Section II of the Detailed Description of the Invention, "Anti-PD-1 Antibodies and Antigen-Binding Fragments That Can Be Used in the Present Invention" herein. In some embodiments, the anti-PD-1 antibody is pembrolizumab, or an antigen-binding fragment thereof, 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 comprising light chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 1, 2, and 3, and heavy chain CDRs comprising the amino acid sequence set forth in SEQ ID NOs: 6, 7, and 8.

In any of the above-described methods of the invention (including embodiments E1-E29), the anti-PD-1 antibody or antigen-binding fragment is administered to the patient once about every six weeks. In specific embodiments, the anti-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.

In embodiments of any of the treatment methods described herein, the patient is administered an intravenous (IV) infusion of a medicament comprising any of the anti-PD-1 antibodies or antigen-binding fragments described herein.

In alternative embodiments of any of the treatment methods of the present invention, any of the anti-PD-1 antibodies or antigen-binding fragments thereof are administered to the patient (e.g., by a clinician) subcutaneously.

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

As noted above, in some embodiments of the methods of the invention, the method further comprises administering an additional therapeutic agent. In particular embodiments, the additional therapeutic agent is an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-LAG3 antibody or antigen-binding fragment thereof, an anti-GITR antibody or antigen-binding fragment thereof, an anti-TIGIT antibody or antigen-binding fragment thereof, an anti-CD27 antibody or antigen-binding fragment thereof, an anti-ILT3 antibody or antigen-binding fragment thereof, or an anti-ILT4 antibody or antigen-binding fragment thereof. In one embodiment, the additional therapeutic agent is a pseudoplague virus vector expressing IL-12. In a further embodiment, the additional therapeutic agent is dinaciclib. In another embodiment, the additional therapeutic agent is navarixin. In a further embodiment, the additional therapeutic agent is vicriviroc.

In a further embodiment, the additional therapeutic agent is an oncolytic virus. In one embodiment, the additional therapeutic agent is a Coxsackie virus or CVA21. In one embodiment, the additional therapeutic agent is CAVATAK™.

In 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.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylolomelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carselesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; Duocarmycin (including synthetic analogues KW-2189 and CBI-TMI); Eleutherobin; Pancratistatin; Sarcodictin; Spongistatin; Nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenestrine, prednimustine, trofosfamide, uramustine; Nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 1I and calicheamicin phi I1, see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; Androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; Adrenal suppressants such as aminoglutethimide, mitotane, trilostane; Folate supplement such as frolinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatrexate; Defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamole; nitracrine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2ʺ-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verraccurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included are antihormonal agents that regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (fareston); aromatase inhibitors that inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole and anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above compounds.

In some embodiments comprising the step of administering an additional therapeutic agent (i.e., in addition to the anti-PD-1 antibody (e.g., pembrolizumab) or antigen-binding fragment thereof), the additional therapeutic agent in the combination treatment may be administered using the same dosing regimen (dose, frequency, and duration of treatment) typically used when the agent is used as a monotherapy to treat the same cancer. In other embodiments, a lower total amount of the additional therapeutic agent is administered to the patient in the combination treatment than when the agent is used as a monotherapy, such as lower doses, less frequent administration, and/or a shorter duration of treatment.

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

The combination treatment according to the invention can be used before or after surgery to remove the tumor and can be used before, during or after radiation therapy. The combination treatment according to the invention can also be used if the patient's tumor is inoperable.

In some embodiments, the combination treatment of the invention is administered to a patient who has not previously been treated with a biotherapeutic or chemotherapeutic agent, i.e., previously untreated. In other embodiments, the combination treatment is administered to a patient who has not achieved a sustained response after prior therapy with a biotherapeutic or chemotherapeutic agent, i.e., previously treated.

The combination treatment of the invention can be used to treat a tumor large enough to be detected by palpation or imaging techniques well known in the art, such as MRI, ultrasound, or CAT scanning. In some embodiments, the combination treatment of the invention is used to treat a late-stage tumor having dimensions 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 .

In some embodiments, the combination treatment of the invention is administered to a human patient having a malignancy that expresses PD-L1. In some embodiments, PD-L1 expression is determined using a diagnostic antibody against human PD-L1 or an antigen-binding fragment thereof in an IHC assay on FFPE sections or frozen sections of a tumor sample removed from the patient. The patient's treating physician may order a diagnostic test to determine PD-L1 expression in a tumor tissue sample of the patient prior to initiating treatment with an antibody against PD-1 or an antigen-binding fragment thereof, but it is contemplated that the treating physician may order the first or subsequent diagnostic tests at any time after initiating treatment, such as after completing a treatment cycle.

The choice of dose of the additional therapeutic agent depends on several factors, including the rate of metabolism of the substance in the serum or tissue, the level of symptoms, the immunogenicity of the substance, and the availability of the target cells, tissue, or organ of the individual being treated. The dose of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Thus, the dose and frequency of administration of each additional therapeutic agent (e.g., a biotherapeutic or chemotherapeutic agent) will depend in part on the specific therapeutic agent, the severity of the malignancy being treated, and the characteristics of the patient. Guidance on the selection of appropriate doses of antibodies, cytokines, and small molecules is 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; Milgromet al. (1999)New Engl. J. Med. 341:1966-1973; Slamonet al. (2001)New Engl. J. Med. 344:783-792; Beniaminovitzet al. (2000)New Engl. J. Med. 342:613-619; Ghoshet al. (2003)New Engl. J. Med. 348:24-32; Lipskyet 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). Determination of the appropriate dosage regimen may be made by the clinician, for example, using parameters or factors known or suspected in the art to influence treatment or predicted to influence treatment, and will depend on, for example, the patient's medical history (e.g., previous therapy), the type and stage of the malignancy being treated, and biomarkers of response to one or more of the therapeutic agents in combination therapy.

IV. Compositions and sets

The 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 approximately 400 mg. The anti-PD-1 antibody can be produced, for example, in CHO cells using conventional cell culture and isolation/purification methods.

In embodiments of the invention, the composition further comprises a histidine buffer with a pH of about 5.0 to 6.0. In particular embodiments, the histidine is present at a concentration of about 10 mM.

In embodiments of the invention, the composition further comprises sucrose. In particular embodiments, sucrose is present at a concentration of about 70 mg/mL.

In embodiments of the invention, the composition further comprises polysorbate 80. In particular embodiments, polysorbate 80 is present at a concentration of about 0.2 mg/mL.

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

In embodiments of the invention, the composition is liquid.

In alternative embodiments, the composition is lyophilized.

In the compositions of the invention, the anti-PD-1 antibody or antigen-binding fragment thereof may be any of the antibodies and antigen-binding fragments described herein, i.e., those described in Section II of the Detailed Description of the Invention, "Anti-PD-1 Antibodies and Antigen-Binding Fragments Thereof That Can Be Used in the Present Invention" (e.g., pembrolizumab).

In some embodiments, a composition comprising an anti-PD-1 antibody as a PD-1 antagonist may be provided as a liquid formulation or prepared by reconstituting a lyophilized powder with sterile water for injection prior to use. WO 2012/135408 describes the preparation of liquid and lyophilized medicaments containing pembrolizumab suitable for use in the present invention.

The invention also relates to a kit for treating a patient with a malignant neoplasm, comprising: (a) 400 mg of an anti-PD-1 antibody or antigen-binding fragment thereof, and (b) instructions for using the anti-PD-1 antibody or antigen-binding fragment thereof in any of the methods for treating a malignant neoplasm presented herein.

In any of the kits of the invention, the anti-PD-1 antibody or antigen-binding fragment may be any of the antibodies or antigen-binding fragments described in Section II of the Detailed Description of the Invention, "Anti-PD-1 Antibodies and Antigen-Binding Fragments Thereof That Can Be Used in the Present Invention."

Kits of the invention may comprise anti-PD-1 antibodies or antigen-binding fragments in a container and a package insert. The container comprises at least one dose (i.e., about 400 mg) of a medicament comprising an anti-PD-1 antibody or antigen-binding fragment thereof, and a package insert or label comprising instructions for treating a patient with a cancer using the medicament. The container may be of the same or a different shape (e.g., vials, syringes, and bottles) and/or may be made of the same or a different material (e.g., plastic or glass). The kit may further comprise other materials that can be used to administer the medicaments, such as diluents, filters, intravenous bags and systems, needles, and syringes. In some preferred embodiments of the kit, the instructions indicate that the medicament is intended for use in treating a patient having a tumor, wherein the tumor expresses PD-L1, as determined 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 a TPS of ≥50% PD-L1. The PD-L1 TPS is the number of tumor cells in a sample that express PD-L1. In further embodiments, the tumor has a TPS of ≥5% PD-L1, ≥10% PD-L1, ≥15% PD-L1, ≥20% PD-L1, ≥25% PD-L1, ≥30% PD-L1, ≥35% PD-L1, ≥40% PD-L1, or ≥45% PD-L1. In another embodiment, the patient's tumor expresses PD-L1 with a CPS of ≥10%. In another embodiment, the patient's tumor expresses PD-L1 with a CPS ≥5%. In another embodiment, the patient's tumor expresses PD-L1 with a CPS ≥1%.

These and other aspects of the invention, including the following examples of specific embodiments, will be apparent from the teachings presented in this description.

GENERAL METHODS

Standard techniques in molecular biology are described in Sambrook, Fritsch and Maniatis (2nd edition 1982 and 1989, 3rd edition 2001)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 ^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods are also described in Ausbel,et al. (2001)Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

Methods for protein purification have been described, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization (Coligan, et al . (2000) Current Protocols in Protein Science, Vol. 1 , John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation have been described (see, e.g., Coligan, et al . (2000) Current Protocols in Protein Science, Vol. 2 , John Wiley and Sons, Inc., New York; Ausubel, et al . (2001) Current Protocols in Molecular Biology, Vol. 3 , John Wiley and Sons, Inc., NY, NY, pp. 16,0,5-16,22,17; Sigma-Aldrich, Co. (2001) Products for Life Science Research , St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory , Piscataway, NJ, pp. 384-391). The production, purification, and fragmentation of polyclonal and monoclonal antibodies have been described (Coligan, et al . (2001) Current Protocols in Immunology, Vol. 1 , John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard methods for characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al . (2001) Current Protocols in Immunology, Vol. 4 , John Wiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies , Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering , Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139–243; Carpenter, et al. (2000) J. Immunol . 165:6205; He, et al . (1998) J. Immunol . 160:1029; Tang et al . (1999) J. Biol. Chem. 274:27371–27378; Baca et al . (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. 6329511).

An alternative to humanization is the use of human antibody libraries displayed on phage libraries 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, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual , Academic Press, San Diego, CA; de Bruin et al . (1999) Nature Biotechnol . 17:397-399).

Purification of the antigen is not necessary to obtain antibodies. Animals can be immunized using cells bearing the antigen of interest. Splenocytes can then be isolated from the immunized animals and fused with a myeloma cell line to produce a hybridoma (see, e.g., Meyaardet al. (1997) Immunity7:283-290; Wrightet al. (2000)Immunity 13:233-242; Prestonet al., above; Kaithamanaet al. (1999)J. Immunol. 163:5157-5164).

Antibodies can be conjugated to, for example, small molecule drugs, enzymes, liposomes, polyethylene glycol (PEG). Antibodies can be used for therapy, diagnosis, kit production, or other purposes, and include antibodies coupled to, for example, dyes, radioisotopes, enzymes, or metals such as 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).

Flow cytometric methods are available, including fluorescence-activated cell sorting (FACS) (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, polypeptides, and antibodies, are available for use, for example, as diagnostic reagents (Molecular Probesy (2003) Catalogue , Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue , St. Louis, MO).

Standard techniques for the histological examination of the immune system have been described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology , Springer Verlag, New York, NY; Hiatt, et al . (2000) Color Atlas of Histology , Lippincott, Williams, and Wilkins, Phila, PA; Louis, et al . (2002) Basic Histology: Text and Atlas , McGraw-Hill, New York, NY).

Software packages and databases are available for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments (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).

All publications mentioned in this specification are incorporated herein by reference for their description of methods and materials that can be used in the present invention.

Taking into account the embodiments of the invention presented in the present description with reference to the accompanying drawings, it should be understood that the invention is not limited to the specific embodiments, and that a person skilled in the art can make various changes and modifications without departing from the scope or spirit of the invention as defined in the claims.

EXAMPLE 1

Six-week (Q6W) dosing regimen of pembrolizumab across tumor types assessed using modeling and simulation

Pembrolizumab, a PD-1-targeted checkpoint inhibitor currently approved for use in a variety of malignancies, demonstrates safety and efficacy when administered at 200 mg or 2 mg/kg Q3W. An alternative extended dosing regimen would provide the benefits of convenience and flexibility for both patients and prescribers. Robust characterization of the pharmacokinetics (PK) and exposure-response (E-R) of pembrolizumab with respect to efficacy and safety enables the use of modeling approaches to inform alternative dosing regimens of pembrolizumab.

The dose for the Q6W pembrolizumab regimen was selected by comparing exposure to the approved Q3W regimens (200 mg and 2 mg/kg) after PK steady-state was achieved; efficacy and safety of the different regimens were compared taking into account ER. PK exposure was simulated up to 24 weeks of steady-state dosing in all individuals using a well-established population PK (time-dependent elimination) model of pembrolizumab that reliably describes PK across tumor types. Efficacy was compared using steady-state exposure, AUC _ss or time-averaged concentrations (C _average,ss ) and trough concentrations (C _min,ss ), which were compared between regimens. The safety profile of pembrolizumab with the Q6W regimen was compared, ensuring that predicted peak concentrations at steady state (C _max,ss ) were lower than those with the maximum administered and well-tolerated dose of 10 mg/kg Q2W.

The PK of pembrolizumab following administration of 400 mg Q6W is predicted to follow a profile similar to that achieved with the approved 200 mg Q3W and 2 mg/kg Q3W dosing regimens (see Fig. 4). Exposure measures compared between the regimens are shown in Table 4. The 400 mg Q6W pembrolizumab dosing regimen was selected based on similar predicted exposures (C _mean,ss or AUC _ss , geometric mean (GM) ~1% higher) when compared to that achieved with 200 mg Q3W (see Fig. 3). Less than 1% of individuals are predicted to have a C _min,ss lower than that achieved with 200 mg Q3W and 2 mg/kg Q3W (Fig. 3). The predicted C _max,ss for 400 mg Q6W is much lower (GM ~65% lower) than for 10 mg/kg Q2W, which has been shown to have acceptable safety across tumor types (see Fig. 2). Given the similar exposure profiles and the established, uniform ER relationship for pembrolizumab at the clinically tested doses, clinical outcomes achieved with 400 mg Q6W are expected to be similar to those achieved with 200 mg Q3W across tumor types.

Using the modeling and simulation approach used herein, the 400 mg Q6W pembrolizumab dosing regimen is expected to result in PK exposure similar to that of the approved 200 mg Q3W and 2 mg/kg dosing regimens. PK simulations showed that, in terms of pembrolizumab exposure, the mean concentration over the dosing interval ( _Cmd ) (or area under the curve [AUC]) at 400 mg Q6W is similar to that of the approved 200 mg Q3W dose, thus allowing for comparison of efficacy between the dosing regimens. Trough concentrations ( _Cmin ) at 400 mg Q6W were generally within the range of concentrations achieved with 2 mg/kg or 200 mg Q3W in the majority (>99%) of patients. Peak concentrations (C _max ) at 400 mg Q6W were much lower than the C _max at the highest clinically tested dose of 10 mg/kg Q2W, confirming that the safety profile of 400 mg Q6W should be comparable to the established safety profile of pembrolizumab. The exposure-response (ER) for pembrolizumab has been shown to be consistent across indications, and OS prediction in melanoma and NSCLC has shown that efficacy at 400 mg Q6W can be expected to be similar to that at 200 mg or 2 mg/kg Q3W given similar exposure; thus, the 400 mg Q6W regimen is expected to be effective across indications.

Table 4. Simulation-based PK exposures of pembrolizumab for the 400 mg Q6W dosing regimen

Alternative dosing regimen Q6W 400 mg Average,ss Relative to 200 mg Q3W,
% GM differences at steady state 0.7% C _min,ss Relative to 2 mg/kg Q3W,
% GM differences at steady state -12% % of patients below lower limit of range for 200 mg and 2 mg/kg Q3W at steady state <1% C _max,ss Relative to 10 mg/kg Q2W,
% GM differences at steady state -66%

EXAMPLE 2

A randomized phase 1 clinical trial of pembrolizumab to evaluate the safety and tolerability of 400 mg intravenous infusion of pembrolizumab Q6W in participants with advanced melanoma

This study is designed to evaluate the pharmacokinetics (PK), safety, and tolerability of pembrolizumab administered every 6 weeks (Q6W). A cohort of 100 participants will receive 400 mg pembrolizumab Q6W. PK, efficacy, and safety data will be collected for this cohort. The study will include male/female participants at least 18 years of age with advanced melanoma. This study will not stratify by age, sex, or other characteristics.

Participants will receive an IV infusion of 400 mg pembrolizumab Q6W in cycles 1–18. PK, efficacy, and safety data will be collected for these participants. The results provide preliminary PK, efficacy, and safety data for pembrolizumab administered Q6W. Given the robust understanding of the clinical pharmacology of pembrolizumab and its well-established E-R profiles, this dosing regimen is expected to result in similar efficacy and safety across all treatment settings for which the 200 mg Q3W pembrolizumab regimen is approved (including monotherapy and combinations with other agents). Thus, the 400 mg Q6W regimen would have a similar risk/benefit profile to 200 mg Q3W as a less frequent dosing regimen in the clinical use of pembrolizumab, based on modeling and simulation analyses (see Example 1).

Study design

The study is a randomized, crossover, multicenter, open-label safety study of pembrolizumab in participants with advanced melanoma, conducted in accordance with Good Clinical Practice (GCP) criteria. This phase 1 study is being conducted in participants with unresectable or metastatic melanoma. The treatment period continues every 42 days for up to 18 cycles (approximately 2 years). Treatment will continue as long as participants are benefiting from treatment and do not experience disease progression or meet any study exclusion criteria. Specifically, the study consists of: (1) a screening period of up to 28 days to verify that the participant is eligible for the study, and (2) an intervention period of approximately 104 weeks of treatment with pembrolizumab. Participants are administered pembrolizumab via IV infusion over 30 minutes Q6W for up to 18 cycles, and (3) a follow-up period in which participants are monitored for AEs for 30 days and serious adverse events (SAEs) for 90 days (30 days if the participant is started on a new anticancer therapy). Participants with ongoing AEs at the time of treatment discontinuation are followed until resolution, stabilization, other explanation for the event, or loss to follow-up.

Participants who discontinue participation for reasons other than radiographic disease progression will undergo post-treatment imaging at follow-up to determine disease status before radiographic disease progression is documented per RECIST 1.1 and, if clinically appropriate, confirmed per iRECIST, before initiation of non-study cancer treatment, withdrawal of consent, loss to follow-up, or end of study. All participants will undergo follow-up by telephone interview regarding overall survival during follow-up, survival to death, withdrawal of consent, loss to follow-up, or end of study.

All participants included in this study will have a diagnosis of advanced melanoma. The results of this study will provide insight into the PK characteristics of pembrolizumab when administered on a Q6W dosing schedule. Safety parameters typically used to evaluate investigational systemic cancer therapies are included as safety endpoints, including, but not limited to, the incidence, cause, and outcome of adverse events (AEs)/serious adverse events (SAEs); and changes in vital signs and laboratory values. AEs will be assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events [NCI CTCAE] version 4.0.

The objective of this study is to characterize the PK profile of pembrolizumab following administration as an IV infusion Q6W. PK data are analyzed after all subjects have completed Cycle 5. PK parameters include AUC, C _max , and C _min . Anti-drug antibody (ADA) formation has the potential to interfere with determination of drug exposure at therapeutic doses and may underlie subsequent infusion-related toxicity. Anti-drug antibody response to pembrolizumab is determined at the start of each of Cycles 1, 2, 4, and 5. Any impact of the presence of ADA on pembrolizumab exposure is investigated.

This study uses ORR based on RECIST 1.1 criteria assessed by independent central review (BICR) as the primary endpoint. Objective response rate is an acceptable measure of clinical benefit in a late-stage trial demonstrating a benefit of a new antineoplastic therapy, particularly if the effect size is large and the therapy has an acceptable risk/benefit profile. Images are submitted to the imaging CRO (iCRO) and reviewed by independent central review blinded to intention-to-treat to minimize bias in response assessment.

Overall survival (OS) is a secondary endpoint and is considered the gold standard for demonstrating the benefit of a new antineoplastic therapy in randomized clinical trials. RECIST 1.1 is used by BICR for imaging assessment of efficacy endpoints and by the local center for study eligibility determination. Modified RECIST 1.1 for the Evaluation of Immunologic Therapeutics (iRECIST) was developed and described by the RECIST Working Group with input from leading experts in the field from industry and academic research, as well as the US Food and Drug Administration and the European Medicines Agency. Unidimensional measurement of target lesions, qualitative assessment of non-target lesions, and response categories are identical to RECIST 1.1 until progression is observed per RECIST 1.1. However, if a participant is clinically stable, additional imaging may be performed to provide radiographic confirmation of progression. Investigators use iRECIST to assess tumor response and progression and to guide treatment decisions and for exploratory efficacy analyses when indicated.

Inclusion Criteria

Participants are eligible for inclusion in the study only if all of the following criteria are met:

The participant has a histologically or cytologically confirmed diagnosis of late-stage melanoma.

The participant has inoperable melanoma stage III or stage IV according to the American Joint Cancer Committee (AJCC) staging system that is refractory to local therapy.

The participant has not been treated for advanced or metastatic disease except that BRAF V600-mutant melanoma is eligible for standard-of-care targeted therapy (eg, a BRAF/MEK inhibitor alone or in combination) and the participant is eligible for the study.

Prior adjuvant or neoadjuvant therapy for melanoma is permissible if at least 4 weeks before randomization and all associated AEs have returned to baseline or stabilized (resolution of toxic effects of last prior therapy to grade 1 or less [except alopecia]). If the individual has undergone major surgery or radiation therapy >30 Gy, he or she must have recovered from the toxicity and/or complications of the procedure.

A woman is eligible to participate if she is not pregnant, not breastfeeding, and agrees to follow specific contraceptive guidance during treatment and for at least 120 days or has provided informed consent.

The participant must have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 (fully active, can continue all pre-illness activities without limitation) or 1 (limited in physically strenuous activity but ambulatory and able to perform light or sedentary work, such as light housework, office work) and must have adequate organ function as defined in Table 5. Samples are collected within 72 hours before the start of the study intervention.

Table 5. Laboratory indicators of adequate organ function

System Laboratory indicator Hematological Absolute Neutrophil Count (ANC) ≥1500/µl Platelets ≥100000/µl Hemoglobin ≥9.0 g/dL or ≥5.6 mmol/L ¹ Kidneys Creatinine OR
Measured or calculated ² creatinine clearance (GFR can also be used instead of creatinine or CrCl) ≤1.5×VPN OR
≥30 mL/min for participant with creatinine levels >1.5× institutional LPN Liver Total bilirubin ≤ 1.5×ULN OR direct bilirubin ≤ULN for participants with total bilirubin levels >1.5×ULN AST (SGOT) and ALT (SGPT) ≤2.5×ULN (≤5×ULN for participants with liver metastases) Clotting International Normalized Ratio (INR) OR Prothrombin Time (PT)
activated partial thromboplastin time (APTT) ≤1.5×ULN if participant is not receiving anticoagulant therapy provided PT or PTT is within the therapeutic range for the intended anticoagulant use ¹ Criteria must be met without erythropoietin dependence and without red blood cell (pRBC) transfusions within the last 2 weeks.
² Creatinine clearance (CrCl) should be calculated according to the standard adopted by the institution.
ALT (SGPT)=alanine aminotransferase (serum glutamic pyruvate transaminase); AST (SGOT)=aspartate aminotransferase (serum glutamic oxaloacetate transaminase); GFR=glomerular filtration rate; ULN=upper limit of normal.

Exclusion criteria

Participants are excluded from the study if they meet any of the following criteria:

A participant is a woman of childbearing potential (WOCBP) who has a positive urine pregnancy test within 72 hours before randomization or treatment assignment. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test is required.

The participant had received prior systemic treatment for unresectable or metastatic melanoma (with the exception specified in the inclusion criteria described above).

The participant has received prior therapy with an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent, or an agent directed against another stimulatory or coinhibitory T cell receptor (eg, OX-40 and CD137), or any other antibody or drug specifically targeting checkpoint pathways other than an anti-CTLA-4 agent that is acceptable in the adjuvant setting.

Participants must have received prior radiation therapy within 2 weeks of starting study treatment. Participants must have recovered from all radiation-related toxicities, do not require corticosteroids, and do not have radiation pneumonitis.

The participant was administered a live vaccine within 30 days prior to the first dose of study drug. Non-limiting examples of live vaccines include the following: measles, mumps, rubella, varicella/shingles, yellow fever, rabies, bacille Calmette-Guérin (BCG), and typhoid vaccines. Injectable seasonal influenza vaccines are generally inactivated viral vaccines and are acceptable; however, intranasal influenza vaccines (e.g., FluMist®) are live attenuated vaccines and are not acceptable.

The participant is currently enrolled in a study, or has enrolled in a study with an investigational drug, or has used an investigational device within 4 weeks prior to the first dose of the investigational drug.

The participant has a diagnosis of immunodeficiency or is receiving chronic systemic steroid therapy (at doses exceeding 10 mg/day prednisone equivalent) or any other form of immunosuppressant therapy within 7 days prior to the first dose of study drug.

Participant has a known additional malignancy that is progressive or requiring active treatment within the last 2 years. Note: Participants with basal cell carcinoma of the skin, squamous cell carcinoma of the skin, or carcinoma in situ (eg, breast carcinoma, cervical carcinoma in situ ) undergoing potentially curative therapy are not excluded.

Participant has known active CNS metastases and/or carcinomatous meningitis. Participants with previously treated brain metastases may participate provided they are radiographically stable (i.e., no evidence of progression) for at least 4 weeks on repeat imaging (note that repeat imaging must be performed during study screening), clinically stable, and have not required steroid treatment for at least 14 days prior to the first dose of study drug.

The participant has severe hypersensitivity (grade ≥3) to pembrolizumab and/or any of the excipients.

The participant has ocular melanoma.

• The participant has an active autoimmune disease that has required systemic treatment within the past 2 years (i.e., using disease-modifying agents, corticosteroids, or immunosuppressive drugs). Replacement therapy (e.g., thyroxine, insulin, or saline corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered a form of systemic treatment and is acceptable.

The participant has a history of (non-infectious) pneumonitis requiring steroids or currently suffers from pneumonitis.

The participant has an active infection that requires systemic therapy.

The participant has a history of human immunodeficiency virus (HIV) infection.

The participant has hepatitis B virus infection (defined as hepatitis B surface antigen [HBsAg] positivity) or known active hepatitis C virus infection (defined as HCV RNA detection [qualitative]).

The participant has a history of or is currently receiving any condition, therapy, or laboratory abnormality that may confound the study results, interfere with participation in the study for the duration of the study, or be considered by the treating investigator to be not in the best interests of the participant.

The participant has a known psychiatric disorder or alcohol or drug dependence that would interfere with his or her ability to comply with the requirements of the study.

The participant is pregnant, breastfeeding, or planning to conceive or give birth to a child within the planned study period, beginning at the screening visit and up to 120 days after the last dose of study drug.

Termination of study intervention and exclusion of participants

Discontinuation of the study intervention does not constitute withdrawal from the study. Because some data on clinical events after discontinuation of the study intervention may be important to the study, they must be collected before the participant's last scheduled follow-up visit, even if the participant has discontinued the study intervention. Thus, all participants who discontinue the study intervention before the end of the protocol-defined treatment period will still remain in the study.

Participants may discontinue the study intervention for any reason or may be withdrawn from the study at the discretion of the investigator if any adverse effects occur. In addition, a participant may discontinue the study intervention at the discretion of the investigator if the study intervention is unacceptable, if the study plan is violated, or for administrative and/or other safety reasons.

The participant must discontinue the study intervention but continue to be monitored as part of the study for any of the following reasons:

The participant or the participant's legal representatives request that the study intervention be discontinued.

The participant misses more than 12 consecutive weeks of study medication administration or has a total of 3 missed doses.

The participant has a medical condition or personal circumstances that, in the opinion of the investigator, imposes an undue risk on the participant by continuing administration of the investigational drug.

The participant has a confirmed positive serum pregnancy test result.

The participant has radiographically confirmed disease progression.

The participant has any progression or recurrence of any malignancy, or has developed another malignancy requiring active treatment.

The participant has an unacceptable adverse event.

The participant has an intercurrent illness, other than another malignancy listed above, that prevents further administration of the medicinal product.

The researcher decides to stop treatment.

The participant has grade 2 recurrent pneumonitis.

The participant underwent 35 administrations (approximately 2 years) of pembrolizumab.

A participant is withdrawn from the study if the participant or the participant's legally authorized representative withdraws consent to participate in the study. If a participant is withdrawn from the study, the participant is no longer administered the study drug or does not follow the protocol visit schedule.

Informed consent

The investigator or authorized health care professional obtains documented consent from each potential participant or the participant's legally authorized representative before participation in a clinical trial. If there is a change in the participant's status during the trial (e.g., health requirements or age of majority), the investigator or authorized health care professional verifies that appropriate consent has been given.

Efficiency/Evaluation

Tumor evaluation includes all known or suspected sites of disease. Imaging may include computed tomography (CT) of the chest, abdomen, and pelvis or magnetic resonance imaging (MRI) at baseline and if disease progression or brain metastases are suspected. CT is particularly preferred for tumor imaging. For the chest, abdomen, and pelvis, contrast-enhanced MRI may be used if iodinated CT is contraindicated or if local practice dictates. MRI is particularly preferred for the brain.

The same imaging modality (ideally using the same scanner and consistent use of contrast) is used for the participant throughout the study. Consistent use of imaging modality helps optimize the reproducibility of assessment of existing and new tumor burden and to improve the accuracy of assessment of response or progression. All scheduled imaging for all study participants is reviewed by the investigator for disease progression. In addition, images (including those obtained by other modalities) obtained at unscheduled times to determine disease progression (as well as images obtained for other reasons but that radiographically document disease progression based on investigator assessment) are also recorded at the study center.

BICR confirmation of measurable disease per RECIST 1.1 at screening will be used to determine participant eligibility. Before participant allocation, BICR confirmation that the participant has at least 1 lesion on imaging that is eligible for selection as a target lesion per RECIST 1.1 is required.

Initial tumor imaging

Baseline tumor imaging at screening will be performed within 28 days prior to the first dose date. Any images obtained after day 1 of treatment cycle 1 are not included in the screening assessment. Study site personnel will evaluate screening images to confirm that the participant has measurable disease in accordance with RECIST 1.1. If brain imaging is performed to document the stability of existing metastases, MRI will be used when available. If there are medical contraindications to MRI, CT with contrast is an acceptable alternative.

Visualization of the tumor during the examination

The first imaging during the study will be performed 12 weeks (84 days ± 7 days) from the date of the first dose. Subsequent tumor imaging will be performed every 9 weeks (63 days ± 7 days) or more frequently if clinically indicated. After 52 weeks (365 days ± 7 days), participants continuing treatment will undergo imaging every 12 weeks (84 days ± 7 days).

Objective response is confirmed by repeat imaging. Tumor imaging to confirm PR or CR will be performed at least 4 weeks after the first sign of response is observed. Participants will then return for regularly scheduled imaging beginning on the next scheduled imaging date. Participants who underwent additional confirmatory imaging may not undergo the next scheduled tumor imaging if it is scheduled in less than 4 weeks; tumor imaging may resume on the subsequent scheduled imaging date.

According to modified iRECIST criteria, disease progression is confirmed at 4-8 weeks after the first radiographic evidence of progressive disease (PD) in clinically stable participants. Participants with unconfirmed disease progression may continue treatment at the investigator's discretion until progression is confirmed by the study center. Participants who underwent confirmatory imaging may not undergo the next scheduled tumor imaging if it is scheduled in less than 4 weeks; tumor imaging may resume at the subsequent scheduled imaging date if the participant is clinically stable. Participants with confirmed disease progression according to iRECIST, as assessed by the study center, will discontinue study treatment.

Tumor imaging at 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 performed within 4 weeks of the discontinuation date, imaging at treatment discontinuation is optional. For participants who discontinue the study intervention due to documented disease progression, this is the final tumor imaging required if the investigator decides not to use iRECIST.

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

Disease assessment according to RECIST 1.1

RECIST 1.1 is used as the primary endpoint for tumor response, date of disease progression, and as the basis for all protocol guidelines related to disease status (e.g., discontinuation of study intervention). Although RECIST 1.1 specifies a maximum of 5 target lesions overall and 2 lesions per organ, this protocol allows for evaluation of a maximum of 10 target lesions overall and 5 lesions per organ if clinically important to allow for broader tumor burden analysis.

Disease assessment by iRECIST

iRECIST is based on RECIST 1.1 but is adapted to account for the unique tumor response seen with immunotherapy drugs. The investigator will use iRECIST to assess tumor response and progression and make treatment decisions. If clinically stable, participants will not be discontinued until progression is confirmed by the investigator working with the local radiography service. In this case, the possibility of continuing treatment despite initial radiographic PD takes into account the observation that some participants may have a transient worsening of clinical tumor manifestations in the first few months after starting immunotherapy and then experience a subsequent response.

Any participant considered clinically unstable is discontinued from the study treatment at the time the first radiographic evidence of PD is detected by the study center and repeat tumor imaging is not required to confirm the iRECIST PD. If the investigator decides to continue treatment, the participant may continue to receive the study treatment and tumor assessment should be repeated in 4-8 weeks to confirm the investigator-assessed iRECIST PD. If repeat imaging does not confirm the investigator-assessed iRECIST PD and the participant continues to be clinically stable, then the study treatment is continued and the regular imaging schedule is followed. If PD is confirmed, participants are discontinued from the study treatment.

If a participant has radiographic progression (iCPD), the study treatment is stopped; however, if the participant achieves clinically significant benefit, continuation of the study treatment is considered as an exception. In this case, if the study treatment is continued, tumor imaging is continued. The set of imaging and treatment requirements after the first radiographic evidence of progression is summarized in Table 6.

Table 6. Imaging and treatment after the first radiographic signs of progressive disease

Clinically stable Clinically unstable Visualization Treatment Visualization Treatment First radiographic evidence of PD according to RECIST 1.1 as assessed by the investigator Repeat imaging in 4-8 weeks to confirm PD Continuation of study treatment may be determined by the investigator and after participant consent. Repeat imaging in 4-8 weeks to confirm PD only at investigator's discretion Termination of treatment First radiographic signs of PD according to RECIST 1.1 Repeat imaging in 4-8 weeks to confirm PD The study intervention may be continued at the discretion of the investigator while awaiting confirmatory tumor imaging at the iRECIST study site Repeat imaging in 4-8 weeks to confirm PD only at investigator's discretion Termination of treatment Repeat tumor imaging confirms iRECIST PD (iCPD) as assessed by investigator No additional visualization required Termination of treatment. No additional visualization required They don't use it On tumor repeat imaging, iRECIST iUPD is observed as assessed by the investigator. Repeat imaging in 4-8 weeks to confirm PD. May be performed at next scheduled imaging visit Continue the study intervention at the discretion of the investigator Repeat imaging in 4-8 weeks to confirm PD only at investigator's discretion Termination of treatment On repeat tumor imaging, iSD, iPR, or iCR according to iRECIST is observed as assessed by the investigator Continue regularly scheduled imaging Continue the study intervention at the discretion of the investigator. Continue regularly scheduled imaging The study intervention may be restarted if the patient's condition has improved and/or is clinically stable, at the discretion of the investigator. The next imaging of the tumor should be performed according to the imaging schedule. Abbreviations: iCPD=iRECIST-confirmed progressive disease; iCR=iRECIST complete response; iPR=iRECIST-confirmed partial response; iRECIST=modified Response Evaluation Criteria in Solid Tumors 1.1 for Immunotherapeutic Agents; iSD=iRECIST stable disease; iUPD=iRECIST-unconfirmed progressive disease; PD=progressive disease; RECIST 1.1=Response Evaluation Criteria in Solid Tumors 1.1; VOP=verification of progression.

Safety assessment

Safety assessments include collection of AE and SAE data, vital sign monitoring and laboratory tests (including pregnancy testing), electrocardiography (ECG) and physical examination, and review of concomitant medications.

Adverse events

The investigator or a certified authorized staff member will assess each individual for potential new or worsening AEs and more frequently if clinically indicated. AE assessment includes, but is not limited to, type, frequency, severity (as classified using the National Cancer Institute Common Terminology Criteria for Adverse Events [NCI CTCAE] version 4.0), timing, seriousness, and relationship to study drug. Adverse events occurring during the study, including baseline signs and symptoms, are recorded.

Complete physical examination

The investigator or a certified authorized staff member performs a complete physical examination during the screening period. Clinically significant abnormalities are recorded as medical history. After the first dose of study drug, new clinically significant abnormalities are recorded as AEs.

Physical examination based on symptoms

For cycles not requiring a full physical examination, the investigator or a certified authorized staff member will perform a symptom-based physical examination as clinically indicated prior to administration of the study drug. New clinically significant abnormalities will be recorded as AEs.

Vital signs

Vital signs are measured in a semi-recumbent position after 5 minutes of rest and include temperature, systolic and diastolic pressure, respiratory rate, pulse, and weight. Height is recorded only during screening.

Electrocardiography

A standard 12-lead ECG is obtained using locally standardized methods. Clinically significant abnormalities at screening are recorded as part of the medical history. Additional ECGs are obtained during the study when clinically necessary. Clinically significant abnormalities observed on the ECG during follow-up are recorded as AEs.

Laboratory analyses of clinical safety

The tests detailed in Table 7 are performed in a local laboratory. Additional tests may be performed at any time during the study if deemed necessary by the investigator.

Table 7. Laboratory safety tests required by the protocol

Laboratory tests Parameters Complete blood count Platelet count Red blood cell counts:
MCV
MCH
% reticulocytes Differential leukocyte formula:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils Red blood cell count Hemoglobin Hematocrit Biochemical blood test Blood Urea Nitrogen (BUN) Potassium Aspartate aminotransferase (AST)/serum glutamic oxaloacetate transaminase (SGOT) Total bilirubin (and direct bilirubin if total bilirubin exceeds the upper limit of normal) Albumen Bicarbonate Chloride Phosphorus Creatinine Sodium Alanine aminotransferase (ALT)/serum glutamic pyruvate transaminase (SGPT) Total protein Glucose Calcium Alkaline phosphatase TSH
Total T3 (or free T3)
Total T4 (or free T4) ^a Routine urine analysis Specific gravity
pH, glucose, protein, blood, ketones, [bilirubin, urobilinogen, nitrite, leukocyte esterase] using test strip
Microscopic examination (if blood or protein is abnormal) Other screening tests Follicle-stimulating hormone and estradiol (if needed only in women of infertile age)
[Serum or urine] [screen for alcohol and drugs (including at least: amphetamines, barbiturates, cocaine, opiates, cannabinoids and benzodiazepines), if possible]
[Serum or urine] β-human chorionic gonadotropin (β-hCG) pregnancy test (if needed in case of WOCBP)
[Serology [(anti-HIV antibodies, hepatitis B surface antigen [HBsAg], and anti-HCV antibodies)] [or some other tests] [if needed] NOTES:
^a T3 and T4 are preferred; if these are not available, free T3 and free T4 may be assayed.
Abbreviations: β-hCG=β-human chorionic gonadotropin; ALT=alanine transaminase; AST=aspartate transaminase; BUN=blood urea nitrogen; HBsAg=hepatitis B surface antigen; HIV=human immunodeficiency virus; MCH=mean corpuscular hemoglobin; MCV=mean corpuscular volume; RBC=red blood cell; SGOT=serum glutamic oxaloacetate transaminase; SGPT=serum glutamic pyruvate transaminase; TSH=thyroid stimulating hormone; WBC=white blood cell; WOCBP=women of childbearing potential.

Period and frequency of collection of AE, SAE and other safety event log information

All AEs, SAEs, and other reportable safety events occurring after the informed consent form is signed but before treatment assignment/randomization must be recorded by the investigator if the participant is in the placebo or other treatment run-in period, if the event causes the participant to withdraw from the study, or is the result of a protocol-defined intervention, including, but not limited to, washout or discontinuation of usual therapy, nutrition, or procedure. All AEs from the time of treatment assignment/randomization through 30 days after discontinuation of the study intervention must be recorded by the investigator.

All AEs that meet criteria for serious AEs from the time of treatment assignment/randomization until 90 days after discontinuation of the study intervention, or 30 days after discontinuation of the study intervention if the participant starts a new anticancer therapy, whichever occurs first, must be recorded by the investigator. In addition, any SAE that comes to the investigator's attention at any time outside the time period defined above is recorded promptly if the event is considered related to the drug.

Statistical methods of efficiency analysis

Objective response rate (ORR) - ORR is calculated as the number of participants recorded as having achieved a confirmed CR or PR as verified by the BICR divided by the number of participants included in the APaT sample. Participants in the APaT sample without an ORR estimate will be considered non-responders. The true ORR is calculated using the 95% exact binomial CI (Clopper-Pearson, 1934).

Progression-free survival (PFS) - The nonparametric Kaplan-Meier method will be used to estimate the distribution of PFS. 95% CIs will be calculated for the median PFS and the point estimate of PFS at various time points during the follow-up period from day 1 of study treatment. Because disease progression is assessed periodically, a PD may occur at any time during the interval between the last assessment when a PD was not documented and the assessment when a PD was documented. The true date of PD will be estimated from the date of the first assessment at which a PD was objectively documented by BICR per RECIST 1.1. Death is always considered a PFS event. Participants who do not experience a PFS event will be censored at the last disease assessment. For PFS analyses, if events (PD or death) occur immediately after multiple missed disease assessments, data are censored at the last disease assessment before the missed visits. In addition, data obtained after the start of a new anticancer therapy are censored at the last disease assessment before the start of the new anticancer therapy. If a participant meets multiple censoring criteria, the censoring criterion that occurs earliest will be used.

Overall survival (OS) - The nonparametric Kaplan-Meier method is used to estimate the distribution of OS. Calculate the 95% CI of the median OS and the point estimate of OS at various time points during the follow-up period from the first day of study treatment.

Duration of response (DOR) - DOR is summarized descriptively using the nonparametric Kaplan-Meier method. Only the subset of participants who achieve a CR or PR are included in the analysis.

Strategy for analyzing key efficacy endpoints

Table 8 shows the approach for the primary analysis of key efficacy endpoints.

Table 8. Strategy for analysis of key efficacy endpoints

End point Statistical method Sample analyzed An approach to missing data analysis Primary endpoints ORR according to RECIST 1.1 according to BICR assessment Exact method based on binomial distribution (Clopper-Pearson method) APaT Participants without an assessment are considered non-responders and are included in the denominator with caution. Key secondary endpoint PFS according to RECIST 1.1 as assessed by BICR Summary statistics using the Kaplan-Meier method APaT Primary rule of censorship OS Summary statistics using the Kaplan-Meier method APaT Censoring by last known date when the participant was alive DOR according to RECIST 1.1 as assessed by BICR Summary statistics using the Kaplan-Meier method APaT Non-responders are excluded from the analysis.
Respondents are censored according to the censorship rules. ^a The statistical models are described in more detail in the text.
Abbreviations: APaT=All subjects intended to treat; BICR=blinded independent central review; DOR=duration of response; ORR=objective response rate; OS=overall survival; PFS=progression-free survival; RECIST=Response Evaluation Criteria in Solid Tumors.

Statistical methods of safety analysis

Safety and tolerability are assessed by clinical assessment of all important parameters, including adverse events and laboratory parameters. Broad AE categories consisting of the percentage of participants with any AE, drug-related AEs, serious AEs, AEs that are both drug-related and serious, and participants who discontinued treatment due to AEs are presented using point estimates with 95% CIs (Table 9).

Table 9. Security Parameter Analysis Strategy

Endpoint Security 95% CI in group Descriptive statistics Any AE X X Any serious AE X X Any AE associated with the drug X X Any serious or drug-related AE X X Discontinuation of treatment due to AE X X Specific AE, SOC or PDLC X Changes from baseline results (laboratory values, vital signs) X Note: 95% CIs will be calculated using the Clopper-Pearson method.
X=results are presented.
Abbreviations: SOC=system organ class; PDLC=predefined change limit.

An AE is any unfavorable medical event in a clinical trial participant that is temporally associated with the use of an investigational intervention, whether or not it is considered related to the investigational intervention. Thus, an AE can be any unfavorable and unexpected sign (including laboratory abnormalities), symptom, or condition (new or worsened) that is temporally associated with the use of a medicinal product. The following are included as AEs:

Any abnormal laboratory test results (complete blood count, blood chemistry, or urinalysis) or other safety assessments (e.g., ECG, radiographic studies, vital sign measurements), including those that worsened from baseline, or that are considered clinically significant by the investigator in the medical and scientific judgment.

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

New conditions identified or diagnosed after administration of the study drug, even if they may have been present before the start of the study.

Signs, symptoms or clinical complications of suspected drug interactions.

Signs, symptoms or clinical complications of suspected overdose of the study drug or concomitant medicinal product.

Worsening of signs and symptoms of malignancy during the study are recorded as AEs. Disease progression assessed by measuring lesions radiographically or by other means is not recorded as an AE unless it results in hospitalization or death.

The following events do not meet the definition of AE in this study:

Medical or surgical procedure (eg, endoscopy, appendectomy): The condition leading to the procedure is an AE.

Situations in which an adverse medical event did not occur (hospitalization for social and/or convenience reasons).

Expected daily fluctuations in pre-existing diseases or conditions present or identified at baseline that have not worsened.

Surgery planned before informed consent was given to treat a pre-existing condition that has not worsened.

If an event is not an AE according to the definition above, it cannot be an SAE, even if the severity conditions are met. An SAE is defined as any adverse medical event that, at any dose:

Leads to death

Life-threatening. The term "life-threatening" in the definition of "severity" refers to an event in which the participant has a risk of death during the event. It does not refer to an event that could hypothetically result in death if it were more severe.

Requires hospitalization or prolongation of an existing hospitalization. Hospitalization is defined as admission to a hospital, regardless of the length of stay, even if the hospitalization is precautionary to continue observation. Hospitalization for an elective procedure to treat a pre-existing condition that has not worsened is not an SAE. A pre-existing condition is a clinical condition diagnosed prior to use of the MSD product and documented in the participant's medical review.

Resulting in persistent or significant disability/incapacity. The term "disability" means significant impairment of an individual's ability to perform normal daily activities. This definition is not intended to include events of relatively minor medical significance, such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, and accidental injury (e.g., ankle sprain), which may interfere with daily activities but do not cause significant impairment.

Causes a congenital abnormality/malformation in the offspring of the participant who was administered the product, regardless of the time of diagnosis.

Medical or scientific judgment is used in deciding whether to report an SAE in other situations, such as important medical events that may not be life-threatening at the time or result in death or hospitalization, but may place the participant at risk or may require medical or surgical intervention to prevent one of the other outcomes listed in the definition above. These events are generally considered serious. Examples of such events include invasive or malignant neoplasms, intensive treatment in the emergency department or at home for allergic bronchospasm, blood dyscrasias, or seizures that do not result in hospitalization, or the development of drug dependence or abuse.

Demographic and baseline characteristics

The number and percentage of individuals screened, individuals assigned to treatment, major reasons for screening exclusion, and major reasons for discontinuation are reported. Demographic variables (eg, age, sex), baseline characteristics, primary and concomitant diagnoses, and prior and concomitant treatments are summarized using descriptive statistics or categorical tables for all included individuals.

Subgroup analysis

To determine whether the response rates were consistent across subgroups, the response rates (with nominal 95% CI) for the primary endpoint were estimated in each category of the following classification variables:

Age category (<65 vs ≥65 years)

Gender (female vs male)

Race (Caucasian vs. non-Caucasian)

Disease stage (III vs IVM1a vs IVM1b vs IVM1c)

Brain Metastases (Yes vs. No)

ECOG status (0 vs 1)

PD-L1 status (positive vs. negative)

BRAF wild-type and mutant BRAF (untreated) versus mutant BRAF (treated).

A forest plot is constructed from which point estimates and CIs for the treatment effect in the subgroup categories specified above are obtained. Any defined subgroups with fewer than 10 participants are excluded from the analysis.

All references cited in this specification are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank or GeneID entries), patent application, or patent were specifically indicated to be incorporated by reference. This statement is made by applicants pursuant to 37 C.F.R. §1.57(b)(1) with respect to each individual publication, database entry (e.g., Genbank or GeneID entries), patent application, or patent, each of which is expressly identified pursuant to 37 C.F.R. §1.57(b)(2), even if such citation is not adjacent to the stated incorporation by reference statement. Citation of references in this specification is not intended as an admission that the reference is prior art or that it constitutes any statement about the content or date of those publications or documents.

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SEQUENCE LIST

<110> MERCK SHARP & DOHME CORP.

Lala, Mallika

Jain, Lokesh

Li, Mengyao

<120> METHODS OF TREATMENT OF MALIGNANT NEOPLASMS

USING ANTIBODIES AGAINST PD-1

<130> 24567-WO-PCT

<150> 62/630,038

<151> 2018-02-13

<150> 62/732,828

<151> 2018-09-18

<160> 25

<170> PatentIn version 3.5

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Arg Ala Ser Lys Gly Val Ser Thr Ser Gly Tyr Ser Tyr Leu His

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Leu Ala Ser Tyr Leu Glu Ser

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Gln His Ser Arg Asp Leu Pro Leu Thr

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<223> Pembrolizumab light chain variable region

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Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

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Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

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Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

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Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

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Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

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Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

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Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

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Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

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Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

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Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

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Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

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Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

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Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

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Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

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Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

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Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

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Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

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Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

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Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

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His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

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Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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Asn Tyr Tyr Met Tyr

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Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys

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Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr

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Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

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Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

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Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

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Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

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Gly Thr Thr Val Thr Val Ser Ser

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<223> Pembrolizumab heavy chain

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Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

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<223> hPD light chain CDR1-1.08A

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Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Phe Ser Tyr Leu His

1 5 10 15

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Leu Ala Ser Asn Leu Glu Ser

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<210> 13

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<223> hPD-1.08A light chain CDR3

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Gln His Ser Trp Glu Leu Pro Leu Thr

1 5

<210> 14

<211> 5

<212> PROTEIN

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Ser Tyr Tyr Leu Tyr

1 5

<210> 15

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<223> hPD heavy chain CDR2-1.08A

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Gly Val Asn Pro Ser Asn Gly Gly Thr Asn Phe Ser Glu Lys Phe Lys

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Ser

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<211> 11

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<223> hPD heavy chain CDR3-1.08A

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Arg Asp Ser Asn Tyr Asp Gly Gly Phe Asp Tyr

1 5 10

<210> 17

<211> 290

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Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

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Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr

245 250 255

Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys

260 265 270

Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu

275 280 285

GluThr

290

<210> 18

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mature variable region of light chain 20C3

<400> 18

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Ser Tyr Asp Val Val Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 19

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mature variable region of heavy chain 20C3

<400> 19

Gln Val Gln Val Gln Gln Ser Gly Ala Glu Leu Ala Glu Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Asp Tyr Asn Glu Tyr Ser Glu Lys Phe

50 55 60

Met Asp Lys Ala Thr Leu Thr Ala Asp Lys Ala Ser Thr Thr Ala Tyr

65 70 75 80

Met Gln Leu Ile Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Trp Leu Val His Gly Asp Tyr Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 20

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mature variable region of light chain 22C3

<400> 20

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Lys Ser Ser Gln Ser Leu Leu His Thr

20 25 30

Ser Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Val Val Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 21

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mature variable region heavy chain 22C3

<400> 21

Gln Val His Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr His Glu Tyr Asn Gln Lys Phe

50 55 60

Ile Asp Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met His Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Trp Leu Ile His Gly Asp Tyr Tyr Phe Asp Phe Trp

100 105 110

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 22

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain variable region K09A-L-16

<400> 22

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 23

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain variable region K09A-L-17

<400> 23

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 24

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Full Size Light Chain K09A-L-16

<400> 24

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 25

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Full Size Light Chain K09A-L-17

<400> 25

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<---

Claims (20)

1. A method of treating a malignancy in a human patient, comprising administering to the patient 400 mg of an anti-PD-1 antibody every six weeks, wherein the anti-PD-1 antibody is a monoclonal antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 5, and where the indicated malignancy expresses PD-L1. 2. The method according to claim 1, wherein the malignant neoplasm is selected from the group consisting of: melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, malignant neoplasm of the gastrointestinal tract, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, bile duct cancer, colorectal cancer, cervical cancer, thyroid cancer, endometrial cancer, squamous cell carcinoma, or salivary gland cancer. 3. The method according to any one of claims 1, 2, wherein the patient has a tumor with a high mutational load. 4. The method of claim 1, wherein the patient has a solid tumor with high microsatellite instability (MSI-H) or mismatch repair deficiency. 5. The method according to claim 1, wherein the malignant neoplasm is an inoperable or metastatic melanoma. 6. The method of claim 1, wherein the malignant neoplasm is metastatic non-small cell lung cancer (NSCLC). 7. The method of claim 1, wherein the malignancy is recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). 8. The method of claim 1, wherein the malignancy is (1) refractory classical Hodgkin lymphoma (cHL) or (2) cHL, and the patient has relapsed after 3 or more lines of cHL therapy. 9. The method according to claim 1, wherein the malignant neoplasm is locally advanced or metastatic urothelial carcinoma. 10. The method according to claim 1, wherein the malignant neoplasm is locally advanced or metastatic gastric cancer or adenocarcinoma of the gastroesophageal junction. 11. The method according to claim 1, wherein the malignant neoplasm is cervical cancer. 12. The method of claim 1, wherein the malignant neoplasm is primary mediastinal large B-cell lymphoma (PMBCL). 13. The method according to claim 1, wherein the malignant neoplasm is a resected high-risk stage III melanoma. 14. The method according to claim 1, wherein the malignant neoplasm is hepatocellular carcinoma. 15. The method of claim 1, wherein the malignant neoplasm is renal cell carcinoma (RCC). 16. The method of claim 1, wherein the malignant neoplasm is recurrent, locally advanced or metastatic Merkel cell carcinoma (MCC). 17. The method according to claim 1, wherein the malignant neoplasm is endometrial cancer. 18. The method according to any one of claims 1 to 17, wherein the anti-PD-1 antibody is pembrolizumab.