Stable formulation of programmed death receptor 1(PD-1) antibody, and method of using the same

Abstract

To provide a stable formulation of an antibody to a human programmed death receptor PD-1 or an antigen-binding fragment thereof.SOLUTION: An antihuman PD-1 antibody preparation contains: a) approximately 5 mg / mL to approximately 200 mg / mL of an antihuman PD-1 antibody or an antigen-binding fragment thereof; b) buffer; c) stabilizer selected from the group consisting of i) sucrose, trehalose or (2-hydroxypropyl)-β-cyclodextrin, ii) mannitol, sorbitol, L-arginine, a pharmaceutically acceptable salt of L-arginine, L-proline or a pharmaceutically acceptable salt of L-proline; and iii) glycine or a pharmaceutically acceptable salt thereof; d) nonionic surfactant; and e) antioxidant. Also, provided are a method for treating cancer in a human patient who needs treatment of cancer or chronic infection and a method for treating chronic infection which include administering an effective dose of the antihuman PD-1 antibody preparation of the present invention to a patient.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to an antibody or an antigen binding site thereof that binds to human programmed death receptor 1 (PD-1). The present invention also relates to a stable formulation containing the active fragment. Also provided are methods for treating chronic infections, such as erythropoietin, erythroblastosis, and erythroblastosis.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of USSN 62 / 500,238 filed May 2, 2017 (which This application claims the benefit of the above-referenced US Pat. No. 6,399,443, which is incorporated herein by reference in its entirety.

[0003] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The sequence listing of the present application is set forth under file name "24439WOPCT-SEQLIST-26 AP RIL2018.TXT", created on April 26, 2018 and 33.3Kb in size The sequence listing will be submitted electronically via EFS-Web in ASCII format having the following format: This sequence listing submitted via FS-Web is incorporated herein by reference in its entirety. No. 6,399,433, filed on Oct. 23, 2003, and is hereby incorporated by reference in its entirety. [Background technology]

[0004] There are several immune checkpoint therapies that target the programmed death receptor-1 (PD-1) system. This has resulted in dramatic improvements in clinical responses in human cancers (Brahmer et al., NE ngl 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 2015,37 2:2521-32;Robert et al., N Engl J Med 2015,372: 320-30; Topalian et al., N Engl J Med 2012, 366:2 443-54;Topalian et al., J Clin Oncol 2014,32:10 20-30;Wolchok et al., N Engl J Med 2013,369:122 -33) The PD-1 receptor on T cells and its ligands on tumor and immune-infiltrating cells (i.e. Interaction with PD-L1 and PD-L2 regulates T cell-mediated immune responses and These results suggest that the tumours may play a role in immune evasion (Pardol l DM. Nat Rev Cancer 2012,12:252-64). PD-1 Binding of PD to any of its ligands results in the delivery of an inhibitory stimulus to T cells. Immunotherapies targeting the PD-1 system include monoclonal antibodies against the PD-1 receptor [KEY TRUDA™ (pembrolizumab), Merck and Co., Inc. Kenilworth, NJ and OPDIVO (trademark) Nivolumab, Bristol-Myers Squibb, P rinceton, NJ], and also those that bind to the PD-L1 ligand [MPDL 3280A;TECENTRIQ(TM) (atezolizumab) )), Genentech, San Francisco, CA]. Therapeutic approaches have also demonstrated antitumor efficacy in multiple cancer types.

[0005] Antibodies to be used in human subjects must be stored prior to use and transported to the point of administration. Achieving desired levels of an antibody drug in a subject in a reproducible manner is one of the most important steps in the administration of the drug. It is necessary for drugs to be stored in formulations that maintain their biological activity. For example, there is a need for stable formulations of anti-human PD-1 antibodies for the treatment of various cancers and infectious diseases. Preferably, such formulations exhibit a long shelf life and are stable when stored and shipped. It is stable in high concentrations for use, e.g., in subcutaneous administration, and in low concentrations for use, e.g., in intravenous administration. Therefore, it is suitable for administration at low concentrations. Summary of the Invention

[0006] Overview of the Invention The present invention relates to a) an anti-human PD-1 antibody or (i) about 6% to about 20 mM of an antigen-binding fragment; (b) about 5 mM to about 20 mM of a buffer; Approximately 8% weight / volume (w / v) of sucrose, trehalose or (2-hydroxypropanediol (ii) about 3% to about 5% w / v mannitol, so L-arginine, pharma- ceutically acceptable salts of L-arginine, L-proline or or a pharma- ceutically acceptable salt of L-proline; and (iii) about 1.8 to about 2.2% w / v glycine or a pharma- ceutically acceptable salt thereof; d) and e) about 0.01% to about 0.10% of a nonionic surfactant; and f) about 1 mM to about 20 mM The present invention provides an anti-human PD-1 antibody formulation comprising an antioxidant comprising M.

[0007] In one embodiment of the invention, the buffer provides a pH of 5.0 to 6.0.

[0008] In certain embodiments, the stabilizer in the anti-human PD-1 antibody formulation is (i) about 6% to about 8 % w / v of sucrose, trehalose or (2-hydroxypropyl)-β-cyclo (ii) about 3% to about 5% mannitol, sorbitol, L-proline or or a pharma- ceutically acceptable salt of L-proline; and (iii) about 1.8 to about 2.2% w / v glycine or a pharma- ceutically acceptable salt thereof.

[0009] In one embodiment, the anti-human PD-1 antibody formulation further comprises about 1% to about 3% w / v L-arginine or a pharma- ceutically acceptable salt thereof.

[0010] The present invention also relates to a) an anti-human PD-1 antibody having a concentration of about 25 mg / mL to about 200 mg / mL, or b) about 5 mM to about 20 mM histidine buffer; ) about 6% to about 8% w / v sucrose; d) about 0.01% to about 0.04% w / v polysaccharide; Resorbate 80; and e) about 1 mM to about 20 mM L-methionine or a pharmaceutical The present invention provides an anti-human PD-1 antibody formulation comprising an acceptable salt thereof.

[0011] In certain embodiments, the anti-human PD-1 antibody formulation further comprises about 1% to about 3% w / v or a pharma- ceutically acceptable salt thereof.

[0012] The present invention further relates to a method for producing an anti-human PD-1 antibody or an antigen thereof, comprising administering to said subject a subject a) ... binding fragment; b) about 8 mM to about 12 mM histidine buffer; c) about 5 mM ~ about 10 mM methionine; d) about 6% to about 8% w / v sucrose; and e) 0. The present invention provides an anti-human PD-1 antibody formulation containing 0.01% to about 0.04% w / v polysorbate 80. Provide.

[0013] The present invention also relates to a method for producing an anti-human PD-1 antibody or an anti-human PD-1 antibody comprising administering to said patient a concentration of about 125 to about 200 mg / mL of said ... b) about 10 mM histidine buffer; c) about 10 mM L- methionine or a pharma- ceutically acceptable salt thereof; d) about 7% w / v sucrose; and e) providing an anti-human PD-1 antibody formulation comprising about 0.02% w / v polysorbate 80; do.

[0014] In some embodiments, the anti-human PD-1 antibody formulation further comprises about 1.25% to about 2. 5% w / v L-arginine or a pharma- ceutically acceptable salt thereof. In one embodiment, the arginine is L-arginine. The compound is L-arginine HCL.

[0015] The present invention also relates to a) an anti-human PD-1 antibody or its derivative having a concentration of about 5 mg / mL to about 75 mg / mL. b) about 8 mM to about 12 mM histidine buffer; c) ( i) about 6% to about 8% w / v of sucrose, trehalose or (2-hydroxypropyl) (ii) about 3% to about 5% w / v mannitol, sol bitol, L-proline or a pharma- ceutically acceptable salt of L-proline; and (iii) From the group consisting of about 1.8 to about 2.2% w / v glycine or a pharma- ceutically acceptable salt thereof d) about 0.01% to about 0.04% polysorbate 80; and e) an anti-human P antibody containing about 5 mM to about 10 mM methionine or a pharma- ceutically acceptable salt thereof; A D-1 antibody formulation is provided.

[0016] In some embodiments, the anti-human PD-1 antibody formulation further comprises a metal chelator. In certain embodiments, the metal chelator is DTPA. DTPA is present at a concentration of about 10 μM to about 30 μM.

[0017] The present invention also provides a liquid anti-human PD-1 antibody formulation reconstituted (reconstituted) from a lyophilized formulation. The reconstituted solution contains: a) about 125 mg / mL to about 175 mg / mL of anti-human P D-1 antibody or an antigen-binding fragment thereof; b) about 8 mM to about 12 mM histidine Buffer; c) (i) about 3% to about 8% w / v sucrose; (ii) about 2% to about 5% (iii) about L-arginine or a pharma- ceutically acceptable salt of L-arginine; 3% to about 5% mannitol and about 1% to about 2% sucrose; and (iv) i) and ii) in combination with; and d) from about 0.01% to about 0. Contains 0.4% Polysorbate 80.

[0018] In certain embodiments of the invention, the anti-PD-1 antibody is pembrolizumab. olizumab) or an antigen-binding fragment of pembrolizumab.

[0019] The present invention also includes administering to a patient an effective amount of an anti-human PD-1 antibody formulation of the present invention. Methods for treating cancer and chronic infections in human patients in need of such treatment Methods of treatment are provided.

[0020] Detailed Description of the Invention The present invention relates to an anti-PD-1 antibody or an antigen-binding fragment thereof that binds to human PD-1. The present invention provides a stable formulation comprising: The present invention is useful in methods of treatment that involve intravenous or subcutaneous administration to a patient in need thereof. In certain embodiments of the present invention, the anti-PD-1 antibody is pembrolizumab or The formulation of the present invention is an antibody that contains a high concentration of an anti-PD1 antibody. The present invention further addresses the problems of high viscosity and increased aggregation associated with the formulation. Methionine oxidation, including reduction in oxidation of methionine 105 located in CDR3 of the heavy chain of izumab The present invention provides a formulation comprising pembrolizumab or an antigen-binding fragment thereof with reduced .

[0021] The formulations of the present invention are useful for subcutaneous delivery to patients in need thereof. To achieve this, formulations for subcutaneous (SC) delivery require high antibody concentrations (75–200 mg / m l) should be included in the SC injection. Based on the expected dose range, higher concentrations of API are often required in SC formulations. However, high concentrations of antibodies or antigen-binding fragments thereof can lead to other properties of the product that are undesirable, such as: For example, poor injectability and aggregation due to increased viscosity and higher than physiological osmolality. Therefore, antibody products intended for SC administration have the greatest therapeutic benefit. It is preferable to balance the effect of concentration while maintaining drug levels that are optimal for the patient. The product has high protein concentration, low viscosity, osmolality similar to physiological conditions, and typical The increased viscosity at high protein concentrations is due to the low level of aggregation under normal storage conditions. Not only can this make it difficult to withdraw the product from its container with a syringe, but it can also make it difficult to obtain the required dose with a syringe. Advantageously, some of the present inventions can also make it difficult to inject into a patient from the This embodiment provides a method for administering a high concentration of an antibody or antigen-binding fragment thereof to a subject, the subject being able to administer the antibody or antigen-binding fragment thereof to a subject that is acceptable for subcutaneous delivery. The formulations of the present invention are also provided in the examples. As shown in further detail, it does not result in high levels of aggregation.

[0022] Previous forced degradation studies have been conducted to investigate the degradation pathways of products and to isolate impurities. These studies were performed on pembrolizumab drug substance (DS) to characterize In this study, pembrolizumab DS was exposed to various stress conditions. Analysis of the samples demonstrated that under the stress conditions used, pembrolizumab DS was able to inhibit light, peroxides and The main decomposition pathway of pembrolizumab was shown to be peroxide. Oxidation and photolysis of methionine 105 (Met105) in the heavy chain CDR during stress. These included oxidation of Met105 and Fc methionine residues upon exposure to . Lizumab was bioactive under most stress conditions tested relative to degradation levels. However, surface plasmon resonance (SPR) revealed that the peroxide-stressed samples A decrease in affinity for PD-1 was observed for exposed methionine residues or antigen-binding domains. Oxidation of methionine residues in CDRs may affect the biological activity of antibodies. The formulation of the present invention can reduce the oxidation of Met105 in the heavy chain CDR of pembrolizumab. is presented herein.

[0023] I. Definitions and Abbreviations The following abbreviations are used throughout the specification and appended claims:

[0024] API: Active Pharmaceutical Ingredient CDR: Complementarity determining region within an immunoglobulin variable region CE-SDS: Capillary electrophoresis-sodium dodecyl sulfate CHO: Chinese hamster ovary CI: Confidence interval DS: API EC50: Concentration that results in 50% efficacy or binding ELISA: Enzyme-linked immunosorbent assay FFPE: formalin-fixed paraffin-embedded FR: Framework region HC: Heavy chain HNSCC: head and neck squamous cell carcinoma HPBC: 2-hydroxypropyl)-β-cyclodextrin HP-HIC: High-performance hydrophobic interaction chromatography HP-IEX: High performance ion exchange chromatography HP-SEC: High-performance size-exclusion chromatography IC50: Concentration that produces 50% inhibition IgG: Immunoglobulin G IHC: immunohistochemistry or immunohistochemical mAb: Monoclonal antibody MES: 2-(N-morpholino)ethanesulfonic acid NCBI: National Center for Biotechnology Information NSCLC: non-small cell lung cancer PCR: polymerase chain reaction PD-1: Programmed Death 1 (also known as Programmed Cell Death-1 and Programmed Death Receptor 1) ) PD-L1: Programmed cell death 1 ligand 1 PD-L2: Programmed cell death 1 ligand 2 PS80 or PS-80: Polysorbate 80 SBEC: (Sulfobutyl ether)-β-cyclodextrin SWFI: Sterile water for injection TNBC: Type 3 negative breast cancer V H : Immunoglobulin heavy chain variable region VK: immunoglobulin kappa light chain variable region V L : Immunoglobulin light chain variable region VP-DSC: Valerian-Plotnikov Differential scanning calorimetry v / v: volume / volume WFI: Water for injection w / v: weight / volume So that the present invention may be more readily understood, certain scientific and technical terms are specifically defined below. All other terms used herein are intended to be used interchangeably unless specifically defined elsewhere in this specification. All technical and scientific terms have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] As used throughout this specification and the appended claims, the singular forms "a," "an," and "the" are used unless the context clearly dictates otherwise. Unless otherwise stated, it includes its corresponding plural objects.

[0026] The word "or" is used unless the context clearly dictates either one of the stated possibilities. Indicates one or both of the possibilities shown. In some cases, one or both "And / or" is used to emphasize the possibility.

[0027] As used herein, "treatment" of cancer or "treating" cancer refers to the treatment of an immune condition or a cancerous condition. Subjects with a chronic condition, or subjects with cancer or a pathogenic infection (e.g., viral, bacterial, fungal) The formulation of the present invention can be administered to a subject diagnosed with cancer, for example to reduce the number of cancer cells, reduce tumor size, etc. such as reducing the rate of cancer cell invasion into peripheral organs or reducing the rate of tumor metastasis or tumor growth. "Treatment" means obtaining at least one positive therapeutic effect, such as one or more of the following: Induction / enhancement of anti-tumor immune responses, against pathogens, toxins and / or self-antigens. stimulation of an immune response to a viral infection; stimulation of an immune response to a tumor marker greater than or equal to 1 Decreasing the level of PD-1, stopping or slowing the growth of tumors or blood cancers, or inhibiting the activity of PD-1 and its ligand P diseases associated with binding to PD-L1 and / or PD-L2 ("PD-1-associated diseases"); For example, stopping or slowing the progression of cancer, stabilizing PD-1-associated disease, inhibiting the growth or survival of tumor cells, inhibition of the presence of, elimination or reduction in size of, one or more cancerous lesions or tumors, Reduces the levels of PD-1 receptors, improves clinical symptoms of PD-1-related diseases, suppresses PD-1-related diseases, For example, a reduction in the severity or duration of clinical symptoms of cancer, or the expected survival of a similar untreated patient. Extending patient survival compared to placebo, complete or partial recovery from cancer or other PD-1-related disease Induction of partial or complete remission.

[0028] "Immune pathology" or "immune disorder" includes, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders. "Immune pathology" also includes infection (infection), persistent infection, and proliferation. Also includes conditions such as cancer, tumors and angiogenesis, e.g., infections that resist eradication by the immune system. "Cancerous condition" also includes, for example, cancer, cancer cells, tumors, angiogenesis, and and precancerous conditions, such as dysplasia.

[0029] Positive therapeutic effects in cancer can be measured in several ways (WA Weber, JN ucl.Med.50:1S-10S (2009)). For example, tumor growth Regarding inhibition, according to NCI standards, T / C≦42% is the minimal level of antitumor activity. A T / C < 10% was considered a high level of antitumor activity, where T / C (%) = treated Median tumor volume / median tumor volume of control x 100. The treatment achieved by the formulation of the present invention improves progression-free survival (PFS), disease-free survival (DFS) or PFS is also called "time to tumor progression" and refers to the time it takes for cancer to progress. The length of time during or after treatment that the patient does not grow (proliferate) and indicates a complete or partial response The length of time that a patient experienced a satisfactory response and the length of time that a patient experienced stable disease were included. DFS refers to the length of time during or after treatment that a patient remains disease-free. OS refers to the extension of life span compared to untreated or untreated individuals or patients. The formulations, methods of treatment and uses of the present invention achieve a positive therapeutic effect in all subjects. Any statistical test known in the art, including, but not limited to, any method that may be useful for determining the degree of the variance may be used. For example, Student's t-test, chi-square test, Mann and Whitney U test, class Kull-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test It should be effective in a statistically significant number of subjects, as determined by standard experimental design.

[0030] A "patient" (also referred to herein as a "subject" or "individual") is a Mammals (e.g., rats, mice, dogs, cats, rabbits) that may be treated with the formulations of the present invention and most preferably a human. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient. Patients who may benefit from treatment with the formulation, e.g., patients suffering from cancer or an immune condition, Included.

[0031] The term "antibody" refers to any form of antibody that exhibits the desired biological activity. It is used in the broadest sense, particularly to refer to monoclonal antibodies (full-length monoclonal antibodies). including human antibodies), polyclonal antibodies, humanized antibodies, fully human antibodies and chimeric antibodies (including (including but not limited to)

[0032] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer is made up of two polypeptide chains. Each pair contains one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately The amino-terminal portion of each chain has a molecular mass of approximately 10 kDa, which is primarily responsible for antigen recognition. The variable region of each light / heavy chain pair contains 0 to 110 or more amino acids. They form the antibody binding site. Thus, generally, an intact antibody has two binding sites. The carboxy-terminal portions of the chains may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically Generally, they are classified as Mu, Delta, Gamma, Alpha or Epsilon, respectively. Defines the antibody isotype as IgM, IgD, IgG, IgA, or IgE. and in the heavy chain, the variable and constant regions are "J" regions of about 12 or more amino acids. and the heavy chain also includes a "D" region of about 10 or more amino acids. teeth, Fundamental Immunology Ch.7 (Paul, W. ed., 2 nd ed. Raven Press, NY (1989).

[0033] Typically, the variable domains of both the heavy and light chains share a relatively conserved framework. Three hypervariable regions, also called complementarity determining regions (CDRs), are located within the complementarity determining region (FR). The CDRs typically comprise a set of amino acids arranged in a framework that enables binding to a particular epitope. Generally, from the N-terminus to the C-terminus, the light and heavy chain variable domains are aligned by Both main sequences contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is generally as follows: Sequences of Prot eins of Immunological Interest ,Kabat et al.;Na tional 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) Based on the definition in ature 342:878-883.

[0034] An antibody or antigen-binding fragment that "specifically binds" to a particular target protein is An antibody that exhibits preferential binding to its target compared to other proteins, but this specificity is not absolute. Antibodies do not require specific binding to their intended target. The binding can be performed in a sample without causing undesired results, such as false positives. In some cases, the antibody is used to determine the presence of a target protein in a sample. or a binding fragment thereof, which has an affinity for a non-target protein less than that for the non-target protein. at least twice as large, preferably at least 10 times as large, more preferably at least 20 times as large and most preferably at least 100-fold greater affinity to the target protein. As used herein, antibodies may be used that bind to a given amino acid sequence, e.g., mature human PD -1 or a polypeptide containing the amino acid sequence of the human PD-L1 molecule. What is said is that it binds to polypeptides containing that sequence but not to polypeptides lacking that sequence. This is the case when it does not bind to proteins.

[0035] A "chimeric antibody" is an antibody in which a portion of the heavy and / or light chain is Derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass While the remainder of the chain is identical or homologous to a corresponding sequence in an antibody of another species (e.g. mouse) or a corresponding sequence in an antibody belonging to another antibody class or subclass. It is meant antibodies identical or homologous to the sequence, as well as fragments of such antibodies.

[0036] The term "pharmacologically effective amount" or "effective amount" refers to a sufficient amount of a therapeutic composition or formulation to be administered to a patient. This level can vary depending on patient characteristics, e.g. age, It will be appreciated by those skilled in the art that this may vary depending on age, weight, and the like.

[0037] The term "about" refers to the amount of a substance or composition (e.g., mM or M), a formulation component, percentage (v / v or w / v), pH of the solution / formulation, or a step in the method In the case where the value of a parameter to be added is modified, for example, , characterization and / or use of the typical measurement, handling and sampling operations involved. the variations in quantities that can occur due to random errors in these operations; Variations in the amount; This includes variations in quantity that may result from differences in manufacture, origin, or purity of the ingredients used. In certain embodiments, "about" means ±0.1%, 0.5%, 1%, 2%, 3%, This could mean a variation of 4%, 5% or 10%.

[0038] The terms "cancer," "cancerous," or "malignant" refer to a disease typically characterized by uncontrolled cell growth. Cancer refers to or refers to the physiological condition in mammals in which the cancer is caused by a variety of factors, including, but not limited to, cancers. Examples of cancer include carcinomas, lymphomas, and the like. These include, but are not limited to, leukemia, blastoma and sarcoma. More specific examples of cancer include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, hodgkin's lymphoma, and the like. Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal (duct) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic Myeloid leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, Melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain These include cancer of the stomach, bladder, hepatocellular carcinoma, breast, colon and head and neck cancer.

[0039] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. The anti-PD-1 antibody may be any one or more It may be used in conjunction with an appropriate chemotherapeutic agent. Examples of such chemotherapeutic agents include: Alkylating agents, such as thiotepa and cyclosphosphamide (cyclosphosphamide); alkylsulfonates, e.g. busulfan (busulfan), improsulfan and piposulfan aziridines, such as benzodop a) Carboquone, Meturedopa and and uredopa; ethyleneimine and methylaminamine hylamelamine, e.g. altretamine, trimethoprim, Ethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide (t riethiylenethiophosphoramide) and trimethylol mer amine (trimethylolomelamime); acetogenin in) [especially bullatacin and bullatacinone] tacinone)]; camptothecin [synthetic analog topo topotecan]; bryostatin; Callistatin; CC-1065 [adoz elesin, carzelesin and bizelesin esin (including synthetic analogues); cryptophycin (especially cryptophycin 1 and cryptophycin 8; dolastatin (dolastatin ); duocarmycin (synthetic analogues KW-2189 and CBI-TMI included); eleutherobin; Pancrati Statins (pancratistatin); sarcodicty in; spongistatin; nitrogen mustard, e.g., chloramphenicol; Chlorambucil, Chlornaphazine ine), chlorophosphamide, estramusti Estramustine, ifosfamide, meclo Mechlorethamine, mechlorethamine oxide hydrochloride, me Melphalan, Novembichin, Fene Phenesterine, prednimustin e), trofosfamide, uracil mustard cil mustard; nitrosoureas, e.g. carmustine e), chlorozotocin, fotemustine stine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics. [For example, calicheamicin, particularly calicheamicin gamma 1I and calicheamicin phi I1 (e.g., Agnew, Chem. In tl. Ed. Engl., 33:183-186 (1994); dynemicins, such as dynemicin A; bisphosphonates, such as Clodronate; Esperamicin ); and neocarzinostatin chromophore and Related chromoprotein enediyne antibiotic chromophores], aclacinomycin (aclacin omysin), actinomycin, ausramycin uthramycin, azaserine, bleomycin omycin, cactinomycin, carabicin abicin, caminomycin, cardinophilin (ca rzinophilin, chromomycin, dactinomycin Dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin (morpholino-doxorubicin, cyanomorph Folinodoxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin including epirubicin, esorubicin n), idarubicin, marcellomycin ycin, mitomycin, e.g. mitomycin C, Micofu Mycophenolic acid, nogalamycin ycin, olivomycin, peplomycin ycin), potfiromycin, puromycin uromycin, quelamycin, rodorubicin orubicin, streptonigrin, strepton Streptozocin, tubercidin, ubeni Ubenimex, zinostatin, zorubicin ( zorubicin; antimetabolites, e.g. methotrexate te) and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin (d enopterin, methotrexate, pteropterin pteropterin, trimetrexate; Phosphorus analogues, such as fludarabine, 6-mercaptopurine, Thiamiprine, thioguanine; Pyrimidine analogues, such as ancitabine, azacytidine acitidine, 6-azauridine, carmofur, cytara Cytarabine, dideoxyuridine , doxifluridine, enocitabine ine), floxuridine; androgens, e.g. Calusterone, dromostanolone Propionate, epitiostanol, mepitiostan ( mepitiostane, testolactone; anti-adrenergic nals, e.g. aminoglutethimide, mitotane mitotane, trilostane; folic acid supplements, e.g. Florinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; Bess Trabucil (bestrabucil); Bisantrene (bisantrene); Eda edatraxate; defofamine; de Mecolcine (demecolcine); diaziquone; El Formithine; Elliptinium acetate um); epothilone; etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine idamine); maytansinoids, e.g. maytansin maytansine and ansamitocin; Azone (mitoguazone); Mitoxantrone; Pidamol; Nitracrine; Pentos pentostatin; phenamet; pirarubicin (pirarubicin); losoxantrone; podof Podophyllinic acid; 2-ethylhydrazide; Procal Procarbazine; Razoxane; Rhizoxin rhizoxin; sizofuran; spirogermanium rogermanium; tenuazonic acid; tria Diquone (triaziquone); 2,2',2''-trichlorotriethylamine Trichothecenes (especially T-2 toxin, verlaculin rraculin A, roridin A and anguidin dine); urethane; vindesine; dacarbazine rbazine); mannomustine; mitobronitol mitobronitol; mitolactol; pipobroman (pipobroman); gacytosine; arabinoside (ar abinoside ("Ara-C"); cyclophosphamide (cyclophosp hamide; thiotepa; taxoids, such as paclitaxel ( paclitaxel and doxetaxel; chlorambucil (chlorambucil); gemcitabine; 6-thioglucose anine; mercaptopurine; methotrexate thotrexate; platinum analogues, e.g., cisplatin and and carboplatin; vinblastin e); platinum; etoposide (VP-16); ifosfamide (i fosfamide); mitoxantrone; vincristine (vincristine); vinorelbine; novantrone (novantrone); teniposide; edatrexate ( edatrexate; daunomycin; aminopterin aminopterin; xeloda; ibandronate dronate);CPT-11;Topoisomerase inhibitor RFS 2000;Di Fluoromethylornithine (DM FO); retinoids, such as retinoic acid; capecitabine and pharma- ceutically acceptable salts, acids or derivatives of any of the foregoing. Antihormonal drugs that act to regulate or block hormone action on tumors, such as: Also included are antiestrogens and selective estrogen receptor modulators (SEs). RM), such as tamoxifen, raloxifene fene, droloxifene, 4-hydroxytamoxifene phene, trioxifene, keoxifen e), LY117018, onapristone and toremife Toremifene (Fareston); regulates estrogen production in the adrenal glands. Aromatase inhibitors, such as 4(5)-imidazolidinone, which inhibit the aromatase enzyme that blocks zole, aminoglutethimide, megestrol acetate - Megestrol, Exemestane, Formestane (formestan), fadrozole, vorozole ozole, letrozole and anastrozole trozole); and antiandrogens, such as flutamide , nilutamide, bicalutamide, Leuprolide and goserelin; and pharma- ceutically acceptable salts, acids or derivatives of any of the foregoing.

[0040] "Chothia" is a (1997).

[0041] As used herein, "Kabat" refers to the methodology used by Elvin A. Kabat ((1991) S. sequences of Proteins of Immunological In terest,5th Ed.Public Health Service,Nati National Institutes of Health, Bethesda, Md. It refers to the original immunoglobulin alignment and numbering system.

[0042] As used herein, a "proliferative inhibitor" refers to an in vitro or in vivo Cells that overexpress any of the genes identified herein, particularly It means a compound or composition that inhibits the proliferation of cancer cells. Therefore, the proliferation inhibitor is , which significantly reduces the proportion of cells overexpressing such genes in S phase. Examples of growth inhibitors include substances that block cell cycle progression (at a place other than S phase), e.g. These include agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include: Vinca (vincristine and vinblastine) astine), taxanes and topoisomerase II inhibitors , e.g. doxorubicin, epirubicin in), daunorubicin and etoposide Agents that arrest G1, such as DNA alkylating agents, e.g., decalcification agents, Dacarbazine, mechlorethamine ) and cisplatin also affect S-phase arrest. The information is from The Molecular Basis of Cancer, Mendel Sohn and Israel, Chapter 1, entitled "Cell cycle regulation,oncogenes,and antiplastic Murakami et al. drugs(WB Saunders:Philadelph ia, 1995).

[0043] "PD-1 binding fragment," "antigen-binding fragment thereof," "binding fragment thereof" The term "fragment" or "fragment thereof" refers to a fragment or derivative of an antibody. It binds to an antigen (human PD-1) and inhibits its activity (e.g., PDL1 and and blocking the binding of PD-1 to PDL2) while still substantially retaining its biological activity. Thus, the term "antibody fragment" or PD-1 binding fragment includes means a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of fragments include Fab, Fab', F(ab')2 and Fv fragments. Typically, the binding fragment or derivative retains at least one of its PD-1 inhibitory activity. In some embodiments, the amount of the glycerol that is sufficient to exert a desired biological effect is 0%. Any binding fragment having a suitable affinity is useful, but the binding fragment may be The compound or derivative has at least 25%, 50%, 60%, 70%, or 80% of its PD-1 inhibitory activity. Hold 80%, 90%, 95%, 99% or 100% (or more). In this embodiment, the antigen-binding fragment has an affinity for an unrelated antigen. At least 2 times greater, preferably at least 10 times greater, more preferably less and most preferably at least 100-fold greater affinity for the antigen. In one embodiment, the antibody binds to, e.g., by Scatchard analysis. When measured, approximately 10 9 It has an affinity greater than liter / mole. Muns en et al. (1980) Analyt. Biochem. 107:220-239. PD-1 A binding fragment may have conservative amino acid substitutions that do not substantially alter its biological activity. It is intended that the term may include variants such as

[0044] A "humanized antibody" is an antibody that contains sequences from a non-human (e.g., murine) antibody and a human antibody. Such antibodies are intended to refer to a form of antibody that contains minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody contains at least one, and typically two, variable domains. and substantially all, wherein all or substantially all of the hypervariable loops are non-human immunoglobulins. and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody may also optionally contain an immunoglobulin constant region (Fc), typically The antibody may comprise at least a portion of a human immunoglobulin. To increase the affinity of the antibody, to enhance the stability of the humanized antibody, or for other reasons. Although humanized forms of rodent antibodies may contain amino acid substitutions that are similar to those of the parent rodent antibody, humanized forms of rodent antibodies generally have the same structure as the parent rodent antibody. The sequences contain identical CDR sequences to each other.

[0045] The antibodies of the invention may be modified (or blocked) to provide altered effector function. The present invention also includes antibodies having an Fc region modified by a modified Fc domain. 2003 / 086310;WO2005 / 120571;WO2006 / 0057702 ;Presta(2006)Adv.Drug Delivery Rev.58:64 Such modifications have potential beneficial effects in diagnosis and therapy. The Fc region can be used to enhance or suppress various reactions of the immune system, with various effects. Modifications include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation and the addition of multiple Fcs. Changes to the Fc can also be made to the antibody in a therapeutic antibody. It is possible to modify the half-life of the drug, with a longer half-life allowing for less frequent administration. This allows for increased convenience and reduced substance use. 05)J.Allergy Clin.Immunol.116:731,p.734- Please refer to 35.

[0046] By "fully human antibody" is meant an antibody that contains only human immunoglobulin protein sequences. Fully human antibodies are produced in mice, in mouse cells, or derived from mouse cells. When produced in a hybridoma, it may contain murine carbohydrate chains. "Mouse antibody" means an antibody that contains only mouse immunoglobulin sequences. Fully human antibody In humans, transgenic animals with human immunoglobulin germline sequences In the present invention, the vector can be produced by phage display or other molecular biology methods.

[0047] The term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen-binding. The variable regions are referred to as "complementarity determining regions" or "CDRs" [e.g., the Kabat numbering system (Ka bat et al. (1991) Sequences of Proteins of Immun ological interest,5th Ed.Public Health S ervice,National Institutes of Health,Bet Residues 24-3 in the light chain variable domain, as determined by the method of 4 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) and Residues 31-35 (CDRH1), 50-65 (CDRH2) and and 95–102 (CDRH3)], and / or the “hypervariable loop " [i.e., residues 26-32 (L1), 50-52 (L2) in the light chain variable domain and 91–96 (L3) and 26–32 (H1) in the heavy chain variable domain, 53 ~55 (H2) and 96~101 (H3) (Chothia and Lesk (1987 ) J. Mol. Biol. 196:901-917). As used herein, the term "framework" or "FR" residues refers to residues that are defined herein as CDR residues. Variable domain residues other than the hypervariable region residues as defined herein. Sequence definitions for the FR and FR residues follow the standard Kabat sequence definition (Kabat et al. (1987) Sequence ces of Proteins of Immunological Interes National Institutes of Health, Bethesd The provisions of this Agreement shall be determined in accordance with the provisions of the preceding paragraph (a) of the same Act.

[0048] "Conservatively modified variants" or "conservative substitutions" refer to those that are Even if the modification is carried out in a manner known to those skilled in the art, it can be performed generally without altering the biological activity of the resulting molecule. Such exemplary substitutions are preferably those listed in Table 1 below. This can be done in accordance with what is stated therein. [Table 1]

[0049] Also, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially abolish biological activity. Those skilled in the art recognize that the use of fluorescein as a marker for the synthesis of fluorescein is not necessarily a marker for the synthesis of fluorescein. lecular Biology of the Gene,The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.).

[0050] As used throughout this specification and the claims, "consisting essentially of" and and variations thereof, such as "consisting essentially of" refer to the entirety of a listed element or group of elements. The invention relates to a method for the treatment of a disease comprising administering to a subject a subject a subject in need thereof, comprising administering to a subject a subject a subject in need thereof, and Optional substitution of other elements of similar or different nature to the listed elements without qualitatively changing them Inclusive (i.e., may or may not be included). The binding compounds essentially consisting of the recited amino acid sequences have the following characteristics: one or more amino acid substitutions that do not substantially affect the activity of the It may also include.

[0051] The terms "include" and variations such as "includes," "including," "including" and "contains" are used to expressly indicate Unless otherwise stated in the context, either by express language or by necessary implication, the present specification and patents The claims are used in a comprehensive sense throughout, i.e., to refer to any of the embodiments of the invention. excludes the presence or addition of further features which would materially enhance the practice or usefulness of any is used to identify the presence of a stated feature, without

[0052] "Isolated antibody" and "isolated antibody fragment" refer to a purified form, In such contexts, the molecule shown may be any other biological molecule, e.g., a nucleic acid, a protein, It is substantially free of lipids, lipids, carbohydrates, or other substances such as cell debris and growth medium. Generally, the term "isolated" refers to the complete absence of such material. The presence of water, buffers, or salts is not intended, nor is the absence of water, buffers, or salts intended. However, they are not intended to be used in conjunction with experimental or therapeutic studies of the binding compounds described herein. It should not be present in an amount that would substantially interfere with its use.

[0053] As used herein, "monoclonal antibody" or "mAb" or "Mab" refers to a It refers to a population of antibodies that is generally homogeneous, i.e., the antibody molecules that make up the population are present in minute amounts. They are identical in amino acid sequence except for possible natural mutations that may occur. In contrast, conventional (polyclonal) antibody preparations typically are directed against different epitopes. have different amino acid sequences within the variable domains, especially the CDRs, which are often specific to The modifier "monoclonal" refers to a population of antibodies that is substantially homogeneous. and requiring the production of said antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be those Hybrid first described by ler et al. (1975) Nature 256, 495 They can be produced by recombinant DNA techniques (see, e.g., U.S. Pat. No. 4,816, ,567). In addition, "monoclonal antibodies" can be produced by See, e.g., Clackson et al. (1991) Nature 352:624-628 and As described in Marks et al. (1991) J. Mol. Biol 222:581-597 Antibody fragments can be isolated from phage antibody libraries using techniques currently available in the art. 5) See also J. Allergy Clin. Immunol. 116:731.

[0054] "Tumor" as applied to a subject diagnosed with or suspected of having cancer. means a malignant or potentially malignant neoplasm or mass of tissue of any size, including a primary tumor Solid tumors usually contain cystic or liquid areas. Solid tumors are abnormal growths or masses of tissue that do not grow in the body. Various types of solid tumors are caused by the Solid tumors are named for the type of cells they contain. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (blood cancers) generally do not form solid tumors (Nati onal Cancer Institute,Dictionary of Cancer er Terms).

[0055] The term "tumor size" refers to the total size of a tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, for example, by measuring the tumor size. Upon removal from the subject, e.g., using calipers, or by imaging techniques, e.g. Measure the size of the tumor inside the body using bone scans, ultrasound, CT or MRI scans. This can be determined by determining

[0056] As used herein, a "variable region" or "V region" refers to a region that varies in sequence between different antibodies. This refers to the segment of the IgG chain that is denatured. In the light chain, it is Kabat residue 1 09 in the heavy chain and 113 in the heavy chain.

[0057] The term "buffer" includes substances that maintain the solution pH of the formulation of the present invention within an acceptable range; Alternatively, for the freeze-dried formulation of the present invention, the solution may be adjusted to an acceptable pH before freeze-drying. Glass.

[0058] The terms "lyophilization," "freeze-dried," and "lyophilized" refer to a material that is dried. is first frozen, and then the ice or frozen solvent is removed by sublimation in a vacuum environment. In order to enhance the stability of the lyophilized product during storage, It is possible to include excipients.

[0059] The term "pharmaceutical formulation" refers to a preparation in a form that allows the active ingredient to be effective. By this we mean a formulation that does not contain additional components that are toxic to the subject to which it is administered. The terms "formulation" and "pharmaceutical formulation" are used interchangeably throughout this specification. .

[0060] "Pharmaceutically acceptable" means a substance that is reasonably acceptable to a subject for providing an effective amount of the active ingredient being used. can be administered to humans and is generally regarded as safe when administered to humans ", e.g., physiologically tolerated and typically does not cause an allergic or similar undesirable reaction (e.g., Regarding excipients (vehicles, additives) and compositions that do not cause adverse reactions (e.g., stomach upsets, etc.) In another embodiment, the term refers to use in animals, more particularly For more information, see the Federal or State regulatory agencies approved or approved for use in humans. are molecules and compositions listed in the United States Pharmacopeia or other generally recognized pharmacopeias. It is about things.

[0061] A "reconstituted" formulation is a formulation in which the lyophilized protein formulation is dissolved in a diluent to form the protein. The reconstituted formulation is prepared by dispersing a protein in the reconstituted formulation. The composition may be suitable for administration, for example parenterally and in some cases subcutaneously.

[0062] "Reconstitution time" refers to the time it takes to rehydrate the lyophilized formulation with solution to produce a particle-free clarified solution. is the time required.

[0063] A "stable" formulation is one in which the protein contained therein maintains its physical stability and formulations that essentially retain their chemical stability and / or biological activity. Various analytical techniques are available in the art for measuring protein stability. ptide and protein drug delivery,247-301, Edited by Vincent Lee, Marcel Dekker, Inc., New York , NY, Pubs. (1991) and Jones, A. Adv. Drug Del. Stability is a function of selection. The temperature may be measured at a selected temperature for a selected period of time.

[0064] A "stable" formulation is one in which the protein contained therein maintains its physical stability and formulations that essentially retain their chemical stability and / or biological activity. Various analytical techniques are available in the art for measuring protein stability. ptide and protein drug delivery,247-301, Edited by Vincent Lee, Marcel Dekker, Inc., New York , NY, Pubs. (1991) and Jones, A. Adv. Drug Del. Stability is a function of selection. The temperature may be measured at a selected temperature for a selected period of time. For example, in one embodiment For stable formulations, no significant changes were observed at refrigerated temperatures (2-8°C) for at least 12 months. In another embodiment, the stable formulation is stable at refrigerated temperatures (2-8° C.). In another embodiment, the formulation is one in which no significant change is observed for at least 18 months. In this study, stable formulations were tested for the ability to withstand at least three months at room temperature (23-27°C) without any significant changes. In another embodiment, the stable formulation is a formulation that is stable at room temperature (23 to 27°C). ) for at least six months. In this study, a stable formulation is one that will remain stable for at least 12 months at room temperature (23-27°C) without any significant changes being observed. In another embodiment, the stable formulation is a formulation that is stable at room temperature (23 to 27 ℃) for at least 18 months. The criteria for are as follows: Typically, as measured by SEC-HPLC Typically, no more than 10%, preferably no more than 5%, of the antibody monomer is degraded. By visual analysis, it is colorless or transparent to slightly opalescent. Potency, pH and osmolality vary by no more than + / - 10%. , within the range of 60-140%, preferably 80-120%, of the control or reference. Generally, antibody clipping, i.e., the percentage of low molecular weight species, can be measured, e.g. For example, in the measurement by HP-SEC, 10% or less, preferably 5% or less, is observed. Typically, antibody aggregation, i.e., high molecular weight species (%), is measured, for example, by HP-SEC. , and is observed in less than 10%, preferably less than 5%.

[0065] An antibody can be said to "retain its physical stability" in a pharmaceutical formulation if it retains its color and and / or by visual inspection for clarity, UV light scattering, size exclusion chromatography No evidence of aggregation, precipitation and / or denaturation as measured by SEC and dynamic light scattering. The conformational changes of the protein are the result of the protein Fluorescence spectroscopy to determine the tertiary structure of proteins, and FTIR spectroscopy to determine the secondary structure of proteins. It can be evaluated by law.

[0066] An antibody can be said to "retain its chemical stability" in a pharmaceutical formulation if it has significant Chemical stability refers to the ability of a protein to undergo chemical modification without undergoing any chemical changes. These can be assessed by detecting and quantifying the protein's chemical structure. The degradation processes involved include hydrolysis or clipping (size exclusion chromatography and and SDS-PAGE), oxidation (assessed by mass spectrometry or MALDI (evaluated by methods such as peptide mapping combined with TOF / MS), Amidation (ion exchange chromatography, capillary isoelectric focusing, peptide mapping isomerization (evaluated by methods such as isoaspartic acid measurement) and isomerization (isoaspartic acid measurement). (assessed by measuring paragic acid content, peptide mapping, etc.) .

[0067] In a pharmaceutical formulation, an antibody is said to "retain its biological activity" if it The biological activity of the antibody is within the range of biological activity shown at the time of preparation of the pharmaceutical formulation. The biological activity of the antibody can be determined, for example, by an antigen binding assay. When reconstituted or in liquid form, biologically active antibodies and their fragments are Includes

[0068] The term "isotonic" refers to the formulation of interest having essentially the same osmotic pressure as human blood. Isotonic preparations generally have an osmotic pressure of about 270 to 328 mOsm. The tonic pressure is 250-269, and the slightly hypertonic pressure is 328-350 mOsm. Osmolality can be measured, for example, using a vapor pressure or ice osmometer.

[0069] "Non-reducing sugars" are sugars that do not contain or are designed to contain free aldehyde or ketone groups. These are sugars that cannot act as reducing agents because they cannot be converted to , disaccharides, such as, but not limited to, sucrose and trehalose isn't it.

[0070] "Pembrolizumab" (formerly MK-3475, SC H 900475 and lambrolizumab) is referred to herein as Also called "Pembro" and WHO Drug Information, Vol. 27, No. o.2, p.161-162(2013) and has the structure described in Table 2 It is a humanized IgG4 mAb containing the heavy and light chain amino acid sequences and CDRs described in Prescribing information for KEYTRUDA (Merck & Co., Inc., W Hitehouse Station, NJ USA; First US approval, revised September 2017 As stated in the revised 2017 update, pembrolizumab is approved for use in the treatment of unresectable or metastatic melanoma. For the treatment of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) ), classical Hodgkin lymphoma (cHL), urothelial carcinoma, gastric cancer, microsatellite instability US FDA Approval for the Treatment of Certain Patients with Multiple Sclerosis Induced Inflammation (MSI-H) and Non-Small Cell Lung Cancer is approved by.

[0071] II. Formulations of the Invention The formulations of the present invention minimize the formation of antibody aggregates and particulates, high and low molecular weight species. This minimizes oxidation of methionine residues (especially Met105 in pembrolizumab) and ensures that the antibody It ensures long-term retention of biological activity.

[0072] The present invention relates to PD-1 antibodies or antigen-binding For example, the present invention includes (i) an anti-human PD-1 antibody or (ii) a buffer (e.g., histidine or acetone); (iii) stabilizers [e.g., non-reducing sugars, e.g., sucrose or trehalose, Or sorbitol, mannitol, (2-hydroxypropyl)-β-cyclodextrin phosphorus, arginine, proline or glycine], (iv) non-ionic surfactants (e.g. , polysorbate 80), and (v) formulations containing antioxidants (e.g., methionine). In more particular embodiments, the formulations of the invention contain a viscosity-lowering agent (e.g., arginine or a pharma- ceutically acceptable salt thereof) and / or a metal chelator (e.g., DTPA). Includes.

[0073] Anti-PD-1 antibodies and antigen-binding fragments thereof The present invention provides an active pharmaceutical ingredient (API), which is human PD-1 (e.g., human or human A vaccine comprising an antibody or an antigen-binding fragment thereof that specifically binds to a PD-1 antibody (a modified anti-PD-1 antibody) The present invention provides certain biological preparations, as well as methods of using the preparations of the invention. or antigen-binding fragments thereof may be used in the formulations and methods of the invention. In certain embodiments, the API is an anti-PD-1 antibody, which is an anti-PD-1 antibody, such as pembrolizumab and In certain embodiments, the anti-PD-1 antibody is selected from pembrolizumab and nivolumab. In another embodiment, the anti-PD-1 antibody is nivolumab. The amino acid sequences of pembrolizumab and nivolumab, which are typical anti-human PD-1 antibodies, are Alternative PD-1 antibodies and antigen-binding fragments useful in the formulations and methods of the invention are shown. The segments are shown in Table 3.

[0074] In some embodiments, the anti-human PD-1 antibodies or or its antigen-binding fragments, each of which has three light receptors, CDRL1, CDRL2, and CDRL3. and / or the three heavy chain CDRs CDRH1, CDRH2 and CDRH3. include.

[0075] In one embodiment of the invention, CDRL1 is SEQ ID NO:1 or a variant of SEQ ID NO:1. CDRL2 is SEQ ID NO:2 or a variant of SEQ ID NO:2, and CDRL3 is SEQ ID NO: No. 3 or a variant of SEQ ID NO:3.

[0076] In one embodiment, CDRH1 is SEQ ID NO:6 or a variant of SEQ ID NO:6. CDRH2 is SEQ ID NO: 7 or a variant of SEQ ID NO: 7, and CDRH3 is SEQ ID NO: 8 or a variant of SEQ ID NO: 8. or a variant of SEQ ID NO:8.

[0077] In one embodiment, the three light chain CDRs are set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. No. 3, and the three heavy chain CDRs are SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0078] In another embodiment of the invention, CDRL1 is SEQ ID NO:11 or SEQ ID NO:1 1, CDRL2 is SEQ ID NO: 12 or a variant of SEQ ID NO: 12, RL3 is SEQ ID NO:13 or a variant of SEQ ID NO:13.

[0079] In one embodiment, CDRH1 is SEQ ID NO:16 or a variant of SEQ ID NO:16. wherein CDRH2 is SEQ ID NO:17 or a variant of SEQ ID NO:17, and CDRH3 is SEQ ID NO: No. 18 or a variant of SEQ ID NO:18.

[0080] In one embodiment, the three light chain CDRs are set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. No. 3, and the three heavy chain CDRs are SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0081] In another embodiment, the three light chain CDRs are SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13. and SEQ ID NO:13, and the three heavy chain CDRs are SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18. It is 18.

[0082] In another embodiment of the invention, CDRL1 is SEQ ID NO:21 or SEQ ID NO:2 1, CDRL2 is SEQ ID NO:22 or a variant of SEQ ID NO:22, RL3 is SEQ ID NO:23 or a variant of SEQ ID NO:23.

[0083] In yet another embodiment, CDRH1 is SEQ ID NO:24 or the sequence of SEQ ID NO:24. and CDRH2 is SEQ ID NO:25 or a variant of SEQ ID NO:25, and CDRH 3 is SEQ ID NO:26 or a variant of SEQ ID NO:26.

[0084] In another embodiment, the three light chain CDRs are SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. and SEQ ID NO:23, and the three heavy chain CDRs are SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26. It is 26.

[0085] Some of the antibodies and antigen-binding fragments of the formulations of the invention contain a light chain variable region and a heavy chain variable region. In some embodiments, the light chain variable region comprises SEQ ID NO: 4 or SEQ ID NO: The heavy chain variable region comprises SEQ ID NO: 9 or a variant of SEQ ID NO: 9. In embodiments, the light chain variable region comprises SEQ ID NO: 14 or a variant of SEQ ID NO: 14, The heavy chain variable region comprises SEQ ID NO: 19 or a variant of SEQ ID NO: 19. In the present invention, the heavy chain variable region comprises SEQ ID NO:27 or a variant of SEQ ID NO:27, and the light chain variable region comprises SEQ ID NO:27. SEQ ID NO: 28 or a variant of SEQ ID NO: 28, SEQ ID NO: 29 or a variant of SEQ ID NO: 29, or or SEQ ID NO: 30 or a variant of SEQ ID NO: 30. In such an embodiment, In such embodiments, the variant light or heavy chain variable region sequence may be 1, 2, 3, Identical to the reference sequence except for having 4 or 5 amino acid substitutions. In some embodiments, the substitutions are in the framework regions (ie, outside the CDRs). In some embodiments, 1, 2, 3, 4, or 5 of the amino acid substitutions are conservative substitutions. be.

[0086] In one embodiment of the formulation of the invention, the antibody or antigen-binding fragment is A light chain variable region comprising or consisting of SEQ ID NO: 4 and a heavy chain variable region comprising or consisting of SEQ ID NO: 9. In another embodiment, the antibody or antigen-binding fragment comprises a The present invention relates to a light chain variable region comprising or consisting of SEQ ID NO: 14 and a light chain variable region comprising or consisting of SEQ ID NO: 19. In one embodiment of the formulation of the present invention, the antibody or The antigen-binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO:28 and a sequence and a heavy chain variable region comprising or consisting of sequence number 27. The antibody or antigen-binding fragment may have a light chain that comprises or consists of SEQ ID NO:29. and a heavy chain variable region comprising or consisting of SEQ ID NO: 27. In one embodiment, the antibody or antigen-binding fragment comprises or comprises SEQ ID NO:30. and a heavy chain variable region comprising or consisting of SEQ ID NO:27.

[0087] In another embodiment, the formulation of the present invention comprises L Domain or V H Domestic At least 95%, 90%, 85%, 80%, 75% or 50% of one of the V with sequence homology L Domain and / or V H PD-1 specific domain In another embodiment, the antibody or antigen binding protein exhibits a specific binding activity. The antibody or antigen-binding protein of the formulation of the invention may be up to 1, 2, 3, 4, or 5 or more V with amino acid substitutions of L and V H domain and exhibits specific binding to PD-1 .

[0088] In any of the above embodiments, the API is a full-length polypeptide that specifically binds human PD-1. It may be an anti-PD-1 antibody or an antigen-binding fragment thereof. The API is any of the immunoglobulins including IgM, IgG, IgD, IgA, and IgE. Preferably, the antibody is an IgG antibody. Any IgG isotype, including IgG1, IgG2, IgG3 and IgG4 can be used. L and V H Different constant domains in the region For example, the specific intended use of the antibody (or fragment) of the present invention may include If modified effector functions are required, heavy chain constant domains other than IgG1 may be used. IgG1 antibodies have long half-lives and effector functions, such as complement activation and antibody and such activity is not desirable for all uses of antibodies. In such cases, for example, IgG4 constant domains may be used.

[0089] In some embodiments of the invention, the API is an amino acid sequence set forth in SEQ ID NO:5. a light chain comprising or consisting of a sequence of amino acid residues set forth in SEQ ID NO: 10; and a heavy chain comprising or consisting of the sequence of In this case, the API comprises or consists of the sequence of amino acid residues set forth in SEQ ID NO:15. and a light chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 20. In another embodiment, the API is an anti-PD-1 antibody comprising a heavy chain selected from the group consisting of SEQ ID NO:3. 2 and a light chain comprising or consisting of the amino acid residue sequence set forth in SEQ ID NO: 31. and a heavy chain comprising or consisting of the sequence of amino acid residues described. In additional embodiments, the API is an amino acid sequence set forth in SEQ ID NO:33. a light chain comprising or consisting of a sequence of amino acid residues set forth in SEQ ID NO:31; and a heavy chain comprising or consisting of the sequence of the base. In one embodiment, the API comprises the sequence of amino acid residues set forth in SEQ ID NO:34 or and a light chain consisting of the same and comprising or comprising the sequence of amino acid residues set forth in SEQ ID NO:31. and a heavy chain consisting of the above. I is pembrolizumab or a biosimilar of pembrolizumab. In this formulation, the API is nivolumab or a biosimilar of nivolumab.

[0090] Typically, amino acid sequence variants of the anti-PD-1 antibodies and antigen-binding fragments of the invention include , a reference antibody or antigen-binding fragment (e.g., heavy chain, light chain, V H , V L or human At least 75%, more preferably at least 80%, of the amino acid sequence of the More preferably at least 85%, more preferably at least 90%, and most preferably at least They have amino acid sequences with at least 95, 98 or 99% amino acid sequence identity. Identity or homology with respect to sequences is measured herein by aligning the sequences. and gapping, if necessary, without considering any conservative substitutions as part of the sequence identity. Candidate sequences that are identical to anti-PD-1 residues after introducing the loop to obtain the highest percentage of sequence identity The percentage of amino acid residues in the antibody sequence that are N-terminal, C-terminal or internal Any extensions, deletions or insertions of a sequence will not be understood to affect sequence identity or homology. It is not something to be interpreted.

[0091] Sequence identity is the degree of similarity between two polypeptides when the two sequences are optimally aligned. The degree of sequence identity is determined by the BLAST algorithm. The parameters of the algorithm can be determined using the following formula: The sequences are chosen to give the maximum match between each of the sequences over the entire length of the reference sequence. The following references concern the BLAST algorithm, which is often used for sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., ( 1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (19 93)Nature Genet.3:266-272;Madden,TL et al.,( 1996)Meth.Enzymol.266:131-141;Altschul,S .F. et al., (1997) Nucleic Acids Res. 25: 3389-340 2;Zhang, J. et al., (1997) Genome Res. 7:649-656;W ootton, JC et al., (1993) Comput. Chem. 17:149-16 3;Hancock, JM et al. (1994) Comput.Appl.Biosci .10:67-70;ALIGNMENT SCORING SYSTEMS:Dayh off, MO et al., “A model of evolutionary change e in proteins”.Atlas of Protein Sequence and Structure,(1978)vol.5,suppl.3.MOD ayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Fou nd., Washington, DC; Schwartz, RM et al., “Matric es for detecting distant relationships.” Atlas of Protein Sequence and Structure, (1978)vol.5,suppl.3.“MODayhoff(ed.),pp.3 53-358,Natl.Biomed.Res.Found.,Washington ,DC;Altschul,SF,(1991)J.Mol.Biol.219:5 55-565; States, DJ et al., (1991) Methods 3:66-7 0;Henikoff, S. et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, S. F. et al. (1993) J.Mol.Evol.36:290-300;ALIGNMENT STATISTI CS: Karlin, S. et al. (1990) Proc. Natl. Acad. Sci. U SA 87:2264-2268;Karlin, S. et al. (1993) Proc.Na tl.Acad.Sci.USA 90:5873-5877;Dembo, A. et al. 1994) Ann. Prob. 22:2022-2039; and Altschul, S. .F.“Evaluating the statistical significa nce of multiple distinct local alignments s”.Theoretical and Computational Methods in Genome Research (edited by S. Suhai), (1997)pp.1 -14, Plenum, New York.

[0092] Similarly, light chains of either class may be used in the compositions and methods herein. In particular, kappa, lambda or variants thereof are useful in the present compositions and methods. . [Table 2] TIFF2024102215000004.tif143161 [Table 3]

[0093] In some embodiments of the formulations of the invention, the API (i.e., an anti-PD-1 antibody or The antigen-binding fragment thereof is present at a concentration of about 25 mg / mL to about 200 mg / mL. In additional embodiments, the API is at a concentration of about 5 mg / mL to about 25 mg / mL. In some embodiments of the formulations of the present invention, the API (i.e., the anti-PD- 1) The antibody or antigen-binding fragment thereof is at a concentration of about 5 mg / mL to about 200 mg / mL. In another embodiment, the API is present at about 5 mg / mL, about 10 mg / mL, Approx. 25mg / mL, approx. 50mg / mL, approx. 75mg / mL, approx. 100mg / mL, approx. 12 5mg / mL, approx. 130mg / mL, approx. 150mg / mL, approx. 165mg / mL, approx. 16 It is present in concentrations of about 7 mg / mL, about 175 mg / mL, and about 200 mg / mL.

[0094] In one embodiment, the API is present in a concentration of about 165 to about 170 mg / mL. .

[0095] In one embodiment, the API is present in a concentration of about 167 mg / mL.

[0096] In one embodiment, the API is present in a concentration of about 130 mg / mL.

[0097] In additional embodiments, the API is at about 5 mg / mL to about 75 mg / mL, about 50 mg / mL g / mL ~ approx. 200 mg / mL, approx. 75 mg / mL ~ approx. 200 mg / mL, approx. 100 mg / mL ~ approx. 200mg / mL, approx. 25mg / mL ~ approx. 175mg / mL, approx. 50mg / m L ~ approx. 175 mg / mL, approx. 75 mg / mL ~ approx. 175 mg / mL, approx. 100 mg / mL ~175mg / mL, approx. 25mg / mL~150mg / mL, approx. 50mg / mL~approx. 150mg / mL, about 75mg / mL to about 150mg / mL, about 100mg / mL to about 1 50mg / mL, about 25mg / mL to about 125mg / mL, about 50mg / mL to about 125 mg / mL, about 75 mg / mL to about 125 mg / mL, about 25 mg / mL to about 100 mg / mL, about 125mg / mL to about 175mg / mL, about 125mg / mL to about 200mg / mL, or at a concentration of about 5 mg / mL to 200 mg / mL.

[0098] Pharmaceutical Excipients The formulations of the present invention include at least one excipient that stabilizes the formulation. In the present invention, the formulation comprises multiple stabilizers.

[0099] In some embodiments of the formulations of the present invention, the stabilizer is a non-reducing sugar. In some embodiments, the non-reducing sugar is glucose. The base sugar is sucrose. In additional embodiments, the non-reducing sugar is trehalose. In yet other embodiments, the non-reducing sugar is lactose. The non-reducing sugar is raffinose.

[0100] In some embodiments, the anti-human PD-1 antibody formulation of the present invention has a weight percent of about 6% to about 8%. Amount / volume (w / v) of sucrose, trehalose or (2-hydroxypropyl)-β -Cyclodextrin, about 3% to about 5% w / v of mannitol, sorbitol, L-alanine Pharmacologically acceptable salts of L-arginine or L-proline or L - a pharma- ceutically acceptable salt of proline and about 1.8 to about 2.2% w / v glycine or or a pharma- ceutically acceptable salt thereof.

[0101] In some embodiments, the stabilizer is about 6% to about 8% w / v sucrose.

[0102] In some embodiments, the stabilizer is about 6% to about 8% w / v trehalose. .

[0103] In some embodiments, the stabilizer is about 6% to about 8% w / v (2-hydroxypropyl acrylate). propyl)-β-cyclodextrin.

[0104] In some embodiments, the stabilizer is sucrose, trehalose or (2-hydroxybutyrate). (hydroxypropyl)-β-cyclodextrin, which is present in an amount of about 6% to about 8% w / v. In other embodiments, sucrose, trehalose or (2-hydroxybutyrate) is present. Dipropyl-β-cyclodextrin is present in an amount of about 6.5% to about 7.5% w / v. In yet another embodiment, sucrose, trehalose or (2-hydroxypropyl) 6.25% w / v, and about 6.5% w / v. % w / v, approx. 6.75% w / v, approx. 7% w / v, approx. 7.25% w / v, approx. 7.5 % w / v, about 7.75% w / v or about 8% w / v.

[0105] In some embodiments, the stabilizer is about 3% to about 5% w / v mannitol. .

[0106] In some embodiments, the stabilizer is about 3% to about 5% w / v sorbitol. .

[0107] In some embodiments, the stabilizer is about 3% to about 5% w / v L-arginine or is a pharma-ceutically acceptable salt thereof.

[0108] In certain embodiments, the formulations of the invention contain arginine, e.g., L-arginine or and pharma- ceutically acceptable salts thereof. In additional embodiments, the formulations of the present invention comprise In another embodiment, the formulation comprises arginine hydrochloride (i.e., arginine HCl). In another embodiment, the arginine is L-arginine. do.

[0109] In some embodiments, the stabilizer is about 3% to about 5% w / v proline, e.g., L -proline, or a pharma- ceutically acceptable salt thereof. The formulations of the invention include proline hydrochloride (i.e., proline HCl). For this purpose, the formulation contains L-proline.

[0110] In some embodiments, the stabilizer is mannitol, sorbitol, L-arginine. , a pharma- ceutically acceptable salt of L-arginine, L-proline or a pharma- ceutically acceptable salt of L-proline. An acceptable salt is present in an amount of about 3% to about 5% w / v. For these, mannitol, sorbitol, L-arginine, and pharma- ceutical tolerance of L-arginine are The salts thereof, L-proline or pharma- ceutically acceptable salts of L-proline, are about 3.5% to about 4 In yet another embodiment, mannitol, sorbitol, L-arginine, a pharma- ceutically acceptable salt of L-arginine, L-proline or L - A pharma- ceutically acceptable salt of proline is about 3% w / v, about 3.25% w / v, about 3.5 % w / v, approx. 3.75% w / v, approx. 4% w / v, approx. 4.25% w / v, approx. 4.5 % w / v, about 4.75% w / v or about 5% w / v.

[0111] In some embodiments, the stabilizer is about 1.8 to about 2.2% w / v glycine or is a pharma-ceutically acceptable salt thereof.

[0112] In certain embodiments, the formulations of the invention contain glycine or a pharma- ceutically acceptable salt thereof. In additional embodiments, the formulations of the invention include sodium glycinate.

[0113] In certain embodiments, the stabilizer is glycine, which is present at about 150 mM to about 200 mM. mM, or about 150 mM, about 160 mM, about 170 mM, about 175 mM, about 180 mM , about 190 mM or about 200 mM.

[0114] In one embodiment, the stabilizer is glycine, which is about 1.8 to about 2.2% w / v, about 1.5 to about 2.5%, or about 1.8 to about 2.5%, or about 1.5 to about 2.2% In certain embodiments, glycine is present in an amount of about 1.8%, about 2.0%, about 2. It is present in an amount of .2% or about 2.5%.

[0115] In some embodiments, the anti-human PD-1 antibody formulation of the present invention comprises: (1) about 6% to about 8% w / v sucrose, trehalose or (2-hydroxypropyl)-β-cyclohexyl (2) about 3% to about 5% mannitol, sorbitol, L-proline or or a pharma- ceutically acceptable salt of L-proline; and (3) about 1.8 to about 2.2% w / v of glycine or a pharma- ceutically acceptable salt thereof.

[0116] The formulations of the present invention optionally contain arginine, e.g., L-arginine, or a pharmaceutical form thereof. The glycerol may contain a suitably acceptable salt, which provides additional stability to the formulation and also controls viscosity, This allows for the formulation of high API concentrations. Arginine or a pharma- ceutical acceptable salt is present in the formulation in an amount of 0.25% to about 3% weight / volume. In additional embodiments, L-arginine or a pharma- ceutically acceptable salt is Approx. 0.25% w / v, approx. 0.50% w / v, approx. 0.75% w / v, approx. 1.0% w / v, approx. 1.25% w / v, approx. 1.5% w / v, approx. 1.75% w / v, approx. 2.0% w / v, about 2.25% w / v, about 2.5% w / v, about 2.75% w / v, or In another embodiment, L-arginine or A physiologically acceptable salt is about 0 to about 2.75% w / v, 0 to about 2.5% w / v, 0 to about 2 .25% w / v, 0~about 2% w / v, 0~about 1.75% w / v, 0~about 1.5% w / v, 0 to approx. 1.25% w / v, 0 to approx. 1.0% w / v, approx. 0.5% to approx. 3.0% w / v, about 0.5% to about 2.75% w / v, about 0.5% to about 2.5% w / v, about 0 .5%~about 2.25% w / v, about 0.5%~about 2% w / v, about 0.5%~about 1.75 % w / v, approx. 0.5% to approx. 1.5% w / v, approx. 0.5% to approx. 1.25% w / v, approx. 0.5%~about 1.0% w / v, about 1.0%~about 3.0% w / v, about 1.0%~about 2 .75% w / v, about 1.0% to about 2.5% w / v, about 1.0% to about 2.25% w / v, about 1.0% to about 2% w / v, about 1.0% to about 1.75% w / v, about 1.0% to about 1.5% w / v, about 1.5% to about 3.0% w / v, about 1.5% to about 2.75% w / v, about 1.5% to about 2.5% w / v, about 1.5% to about 2.25% w / v, about 1.5% present in an amount of about 2% w / v, or about 2% to about 3% w / v.

[0117] In some embodiments of the invention, the stabilizer is about 6% to about 8% w / v sucrose. , trehalose or (2-hydroxypropyl)-β-cyclodextrin; about 3% About 5% w / v of mannitol, sorbitol or proline or a pharma- ceutical acceptable salt thereof and about 1.8 to about 2.2% w / v glycine or a pharma- ceutically acceptable salt thereof. and the formulation further comprises L-arginine or a pharma- ceutically acceptable salt thereof. This includes salts that are used in pharmaceutical formulations, especially when the API is in high concentration (e.g., 75 mg / mL to 200 mg / m L), may be added in any of the amounts listed above to reduce the viscosity of the formulation. In such an embodiment, L-arginine or a pharma- ceutically acceptable salt thereof is L-arginine or a pharma- ceutically acceptable salt may be added to reduce viscosity. It may also function to stabilize the formulation, and L-arginine or pharmacologic It is understood that acceptable salts may provide additional stability to formulations that do not contain such salts.

[0118] As noted above, in certain embodiments, the formulations of the present invention are administered at high concentrations (e.g., about 75 ml The API concentration ranges from about 200 mg / mL to about 200 mg / mL. In an embodiment, the formulation of the invention comprises arginine, e.g., L-arginine, or a combination thereof. and a physiologically acceptable salt, such as L-arginine in an amount of about 0.25% to about 3.0% w / v. It may also include.

[0119] an anti-PD-1 antibody or antigen-binding fragment thereof and a stabilizer (in the amounts specified above); In addition to the above-mentioned components / concentrations, the formulations of the present invention also include a buffer. For example, the buffer is present in an amount of about 5 mM to about 20 mM, which is in the range of about 4.5 to 6.4. This results in a pH of 0.01.

[0120] In some embodiments of the invention, the buffer is added to the formulation in the range of about 4.5 to about 6.5 In another embodiment, the buffer provides a pH in the range of about 5.0 to about 6.0. In yet another embodiment, the pH is about 5.3 to about 5.8. In an embodiment, the pH is from about 6.0 to about 6.4.

[0121] In certain embodiments, the buffer is about 5.0, about 5.1, about 5.2, about 5.3, Approx. 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.2 or about 6.4. Examples of buffers that control the pH in this range include succinic acid, phosphate (sodium or potassium), histidine, sodium acetate, phosphate (sodium Tris(tris(hydroxymethyl)aminomethane), diethanolamine These include nolamine, citrate (sodium) and other organic acid buffers.

[0122] In certain embodiments of the invention, the buffer has a pH of about 5.0 to about 6.0. In some embodiments, the buffer is acetic acid or acetate. In some preferred embodiments, the buffer is an L-histidine buffer. In embodiments in which the formulation is lyophilized, the buffer is acetate. It is preferable that the buffer is not acetate. is not suitable for the freeze-drying process.

[0123] If a pH range is indicated as, for example, "pH 5.5 to 6.0," that range is , are intended to include the values ​​indicated. For lyophilized formulations, unless otherwise indicated In this case, the pH refers to the pH after reconstitution of the lyophilized formulation of the present invention. The pH is typically pH is measured at 25° C. using a standard glass bulb pH meter. A solution containing "histidine buffer at pH X" is a solution containing histidine buffer at pH X solution, i.e. pH is intended to mean the pH of the solution.

[0124] Anti-PD-1 antibody or antigen-binding fragment thereof, stabilizer and buffer (as described above) In addition to the specified amounts / concentrations, the formulations of the present invention also contain an antioxidant. In one embodiment, the antioxidant is methionine. is L-methionine or a pharma- ceutically acceptable salt thereof. The thionine is L-methionine. In another embodiment, the antioxidant is L-methionine. HCl. In another embodiment, the antioxidant is histidine.

[0125] In some embodiments, an antioxidant (e.g., L-methionine) is added to the formulation of the invention. In another embodiment, the antioxidant is present in an amount of about 5 mM to about 20 mM. In particular, the amount of about 5 mM to about 20 mM, about 5 mM to about 15 mM, or about 5 mM to about 10 mM. In certain embodiments, the antioxidant is at about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM , about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM Or is present in an amount of about 20 mM.

[0126] In embodiments where the antioxidant is histidine, the histidine is present in an amount of up to 100 mM. In such an embodiment, histidine may be present in the formulations of the invention. It can function as a buffer and as an antioxidant.

[0127] Anti-PD-1 antibody or antigen-binding fragment thereof, stabilizer, buffer and antioxidant In addition to the agent (in the amounts / concentrations specified above), the formulations of the present invention also include a surfactant. Surfactants are typically used to provide stability and reduce and / or prevent aggregation. or protein during processing conditions such as purification, filtration, lyophilization, shipping, storage and transportation. In some embodiments of the present invention, In this case, the surfactant is used to effectively inhibit the active ingredient, i.e., the anti-PD-1 antibody or its antigen-binding fragment. This is useful for providing additional stability to the segment.

[0128] Surfactants that may be useful in the formulations of the present invention include, but are not limited to, the following: Non-limiting examples of surfactants include: non-ionic surfactants, such as polyoxyethylene sorbitan Fatty acid ester (trade name: Tween (registered trademark) (Unique American Polysorbate (Po) sold under the tradename Polysorbate (Po) of 1999 and 2000 by Pharmacy, LLC, Wilmington, DE. lysorbate)], e.g. polysorbate 20 (polyoxyethylene sorbitan mono nolaurate), Polysorbate 40 (Polyoxyethylene sorbitan monopalmitate) ), Polysorbate 60 (Polyoxyethylene sorbitan monostearate) and Poly Sorbate 80 (polyoxyethylene sorbitan monooleate); Polyoxyethylene Alkyl ethers, such as Brij® 58 (Uniquema America) as LLC, Wilmington, DE) and Brij® 35; Poloxamer (e.g., poloxamer 188); Triton® X-100 (Uni on Carbide Corp., Houston, TX) and Triton (Registered Trademark) X-114; NP40; Span 20, Span 40, Span 60, Span Span 65, Span 80 and Span 85; copolymers of ethylene and propylene glycol ( For example, non-ionic surfactants of the pluronic® series, e.g. For example, pluronic F68, pluronic 10R5, luronic® F108, pluronic® F127, plu ronic(registered trademark) F38, pluronic(registered trademark) L44, pluroni cL62 (BASF Corp., Ludwigshafen, Germany) ny); and sodium dodecyl sulfate (SDS).

[0129] The amount of surfactant included in the formulation of the present invention is an amount sufficient to perform the desired function, i.e. That is, the active pharmaceutical ingredient (i.e., anti-PD-1 antibody or its antigen-binding fragment) in the formulation The minimum amount required to stabilize the surfactant is typically about 0.008%. In some embodiments of this aspect of the invention, In this embodiment, the surfactant is contained in the formulation at about 0.01% to about 0.04%, about 0.01% to about 0. .03%, about 0.01% to about 0.02%, about 0.015% to about 0.04%; about 0.015 % to about 0.03%, about 0.015% to about 0.02%, about 0.02% to about 0.04%, about 0 Present in an amount of 0.02% to about 0.035%, or from about 0.02% to about 0.03%. In an embodiment, the surfactant is present in an amount of about 0.02%. The surfactants were approximately 0.01%, approximately 0.015%, approximately 0.025%, approximately 0.03%, and approximately 0 It is present in an amount of .035% or about 0.04%.

[0130] In an exemplary embodiment of the invention, the surfactant is polysorbate 20, polysorb A non-ionic surfactant selected from the group consisting of Bate 80 and F127. In a preferred embodiment, the surfactant is polysorbate 80.

[0131] In certain embodiments, the PD-1 formulations of the invention contain about 0.01% to about 0.04% PD-1. In other embodiments, the formulations of the present invention contain about 0.008%, about 0.01% , about 0.015%, about 0.02%, about 0.025%, about 0.03%, about 0.035%, about In certain embodiments, the present invention comprises a method for the preparation of a medicament ... The formulation of the invention contains about 0.02% PS80.

[0132] The present invention also provides a method for administering a pharmaceutical composition to a patient in need thereof, wherein the formulation is contained within a glass vial or an injection device (e.g., a syringe). The present invention provides an anti-human PD-1 antibody formulation as described herein, which is

[0133] In some embodiments of the formulations of the invention, the anti-human PD-1 antibody formulations are stored at 2-8° C. After 12 months of storage, it has one or more of the following attributes: a) The percentage of heavy and light chains is 90.0% or more as measured by reduced CE-SDS. , b) The percentage of intact IgG is 90.0% or more as measured by non-reduced CE-SDS; and c) The monomer percentage is 95% or more as measured by HP-SEC.

[0134] In another embodiment, the present invention provides a method for producing reduced CE-S after storage at 2-8° C. for 12 months. The heavy and light chain ratio is 96% or more as measured by DS, The present invention provides an anti-human PD-1 antibody formulation.

[0135] In another embodiment, the present invention provides a method for the preparation of a non-reduced CE- The percentage of intact IgG in the formulation is 97% or greater as measured by SDS. The present invention provides an anti-human PD-1 antibody formulation.

[0136] In another embodiment, the present invention relates to a method for producing a HP-SEC gel, comprising the steps of: 5. The anti-human P antibody described herein has a monomer percentage of 98.5 or more as measured by A D-1 antibody formulation is provided.

[0137] In an additional embodiment, the present invention provides a method for the preparation of HP-S after storage at 2-8° C. for 12 months. The percentage of high molecular weight species is 1.5% or less as determined by EC, as described herein. The present invention provides an anti-human PD-1 antibody formulation.

[0138] In another embodiment, the present invention provides a method for producing a cellulose acetate ester copolymer that is compatible with HP-SEC after storage at 25° C. for 12 months. The monomeric percentage of the anti-human P antibody described herein is 98.0% or more as measured by the method of the present invention. A D-1 antibody formulation is provided.

[0139] In an additional embodiment, the present invention provides a method for the preparation of a medicament ... 2% or less of high molecular weight species as measured by ELISA. A D-1 antibody formulation is provided.

[0140] In another embodiment, the present invention provides a method for the production of glycerol by HP-SEC after storage at 40° C. for 3 months. The monomer percentage is 94.0% or more, 94.5% or more, or 95.0% or more. The present invention provides anti-human PD-1 antibody formulations as described herein.

[0141] In an additional embodiment, the present invention provides a method for the production of HP-SEC after storage at 40° C. for 3 months. The percentage of high molecular weight species is 5.5% or less, 5.0% or less, or 4.4% or less, as measured by The present invention provides an anti-human PD-1 antibody formulation as described herein,

[0142] Specific Aspects and Embodiments of the Invention In one aspect (A1), the present invention provides a method for treating a pulmonary circulation comprising: (a) administering to a patient a dose of from about 5 mg / mL to about 200 mg / m L of an anti-human PD-1 antibody or an antigen-binding fragment thereof; (b) about 5 mM to about 20 mM (c) (i) about 6% to about 8% weight / volume (w / v) sucrose, (ii) about 3 % to about 5% w / v of mannitol, sorbitol, L-arginine, L-arginine a pharma- ceutically acceptable salt thereof, L-proline or a pharma- ceutically acceptable salt thereof; and and (iii) about 1.8 to about 2.2% w / v glycine or a pharma- ceutically acceptable salt thereof. (d) about 0.01% to about 0.10% of a non-ionic surfactant and e) about 1 mM to about 20 mM of an antioxidant. provide.

[0143] In one embodiment of aspect (A1), the formulation has a pH of 4.5 to 6.4.

[0144] In one embodiment of aspect (A1), the formulation has a pH of 5.0 to 6.0.

[0145] In one embodiment of aspect (A1), the formulation has a pH of 5.3 to 5.8.

[0146] In one embodiment of aspect (A1), the formulation has a pH of about 5.5.

[0147] In one embodiment of aspect (A1), the buffer is histidine or acetate. It is.

[0148] In one embodiment of aspect (A1), the buffer is about 10 mM histidine. do.

[0149] In one embodiment of aspect (A1), the buffer contains about 10 mM L-histidine. It is.

[0150] In one embodiment of aspect (A1), the buffer is about 10 mM acetate. do.

[0151] In one embodiment of aspect (A1), the stabilizer is about 6% to about 8% w / v sucrose. It is a.

[0152] In one embodiment of aspect (A1), the stabilizer is about 6% to about 8% w / v trehalose. It's loin.

[0153] In one embodiment of aspect (A1), the stabilizer is about 6% to about 8% w / v of (2- Hydroxypropyl)-β-cyclodextrin.

[0154] In one embodiment of aspect (A1), the stabilizer is about 3% to about 5% w / v mannitol. It's Thor.

[0155] In one embodiment of aspect (A1), the stabilizer is about 3% to about 5% w / v sorbitol. It's Thor.

[0156] In one embodiment of aspect (A1), the stabilizer is about 3% to about 5% w / v L-alanine. arginine or a pharma- ceutically acceptable salt thereof.

[0157] In one embodiment of aspect (A1), the stabilizer is about 3% to about 5% w / v L-alanine. and arginine or a pharma- ceutical acceptable salt thereof, and the pH of the formulation is about 6.0 to about 6.4. do.

[0158] In one embodiment of aspect (A1), the stabilizer is about 3% to about 5% w / v L-alanine. It is arginine.

[0159] In one embodiment of aspect (A1), the stabilizer is about 3% to about 5% w / v arginine. Nin-HCl.

[0160] In one embodiment of aspect (A1), the stabilizer is about 3% to about 5% w / v L-proline. lorin or a pharma- ceutically acceptable salt thereof.

[0161] In one embodiment of aspect (A1), the stabilizer is about 3% to about 5% w / v L-proline. It's Lorin.

[0162] In one embodiment of aspect (A1), the stabilizer is about 3% to about 5% w / v L-proline. Lorin HCl.

[0163] In one embodiment of aspect (A1), the stabilizer is present in an amount of about 160 mM to about 200 mM. Lysine or a pharma- ceutically acceptable salt thereof.

[0164] In one embodiment of aspect (A1), the stabilizer is present in an amount of about 1.8 to about 2.2% w / v. Glycine or a pharma- ceutically acceptable salt thereof.

[0165] In one embodiment of aspect (A1), the nonionic surfactant is present in an amount of from about 0.01% to about It is 0.04% polysorbate 80.

[0166] In one embodiment of aspect (A1), the nonionic surfactant is about 0.02% poly Resolvate 80.

[0167] In one embodiment of aspect (A1), the antioxidant is about 1 mM to about 20 mM L-methyl. Thionine or a pharma- ceutically acceptable salt thereof.

[0168] In one embodiment of aspect (A1), the antioxidant is about 5 mM to about 15 mM L-methyl. Thionine or a pharma- ceutically acceptable salt thereof.

[0169] In one embodiment of aspect (A1), the antioxidant is about 10 mM L-methionine or or a pharma- ceutically acceptable salt thereof.

[0170] In one embodiment of aspect (A1), the antioxidant is L-methionine.

[0171] In one aspect (A2), the present invention provides a method for treating a chronic pulmonary disease comprising: (a) administering to a patient a dose of from about 5 mg / mL to about 200 mg / m L of an anti-human PD-1 antibody or an antigen-binding fragment thereof; (b) about 5 mM to about 20 mM (c) (i) about 6% to about 8% weight / volume (w / v) sucrose, (ii) about 3 % to about 5% w / v of mannitol, sorbitol, L-proline or L-proline a pharma- ceutically acceptable salt; and (iii) about 1.8 to about 2.2% w / v glycine or or a pharma- ceutically acceptable salt thereof; (d) from about 0.01% to about 0.10% nonionic surfactant; and (e) about 1 mM to about 20 mM antioxidant. The present invention provides an anti-human PD-1 antibody formulation comprising the

[0172] In one embodiment of aspect (A2), the formulation contains about 1% to about 3% w / v L-alanine. The compound further comprises ginine or a pharma- ceutically acceptable salt thereof.

[0173] In one embodiment (A3), the present invention provides a method for treating a chronic pulmonary disease comprising: (a) administering to a patient a dose of about 25 mg / mL to about 200 mg / mL of a pulmonary circulation; mL of an anti-human PD-1 antibody or antigen-binding fragment thereof; (b) about 5 mM to about 20 mL of an anti-human PD-1 antibody or antigen-binding fragment thereof; (c) about 6% to about 8% w / v sucrose; (d) about 0 (e) about 1 mM to about 20 mM polysorbate 80; The present invention provides an anti-human PD-1 antibody formulation containing L-methionine or a pharma- ceutical acceptable salt thereof. do.

[0174] In one embodiment of aspect (A3), the formulation contains about 1% to about 3% w / v L-alanine. The compound further comprises ginine or a pharma- ceutically acceptable salt thereof.

[0175] In one embodiment of aspect (A3), the formulation comprises about 1.25% to about 2.5% w / v or a pharma- ceutically acceptable salt thereof.

[0176] In one embodiment of aspect (A3), the histidine buffer is from about 8 mM to about 12 mM. It is present in a concentration of mM.

[0177] In one embodiment of aspect (A3), the histidine buffer is L-histidine. be.

[0178] In one embodiment of aspect (A3), L-methionine or a pharma- ceutically acceptable The salt is present at a concentration of about 5 mM to about 15 mM.

[0179] In one embodiment of aspect (A3), the polysorbate 80 is about 0.02% w / are present in a weight ratio of v.

[0180] In one embodiment of aspect (A3), sucrose is present in a weight ratio of about 7% w / v. There is.

[0181] In one embodiment (A4), the present invention provides an antibody against rheumatoid arthritis, comprising: (a) about 75 to about 200 mg / mL of an anti-rheumatoid arthritis vaccine; (b) an anti-PD-1 antibody or an antigen-binding fragment thereof; (c) about 5 mM to about 10 mM L-methionine or a pharma- ceutical acceptable salt thereof; (d) about 6% to about 8% w / v sucrose; and (e) 0.01% to about An anti-human PD-1 antibody formulation is provided that contains 0.04% w / v polysorbate 80.

[0182] In one embodiment (A5), the present invention provides a method for treating a chronic inflammatory bowel disease comprising administering to a patient a therapeutically effective amount of an anti-inflammatory drug, comprising administering to the ... and (b) a human PD-1 antibody or an antigen-binding fragment thereof; (c) about 10 mM L-methionine or a pharma- ceutically acceptable salt thereof; (d) (e) about 0.02% w / v polysorbate 80 The present invention provides an anti-human PD-1 antibody formulation comprising the compound.

[0183] In one embodiment of aspect (A5), the formulation comprises about 1.25% to about 2.5% w / v or a pharma- ceutically acceptable salt thereof.

[0184] In one embodiment of aspect (A5), the formulation comprises about 1.25% to about 2.5% w / v It further contains L-arginine.

[0185] In one embodiment of aspect (A5), the formulation comprises about 1.25% to about 2.5% w / v L-arginine-HCl.

[0186] In one embodiment of aspect (A5), the formulation has a pH of 5.0 to 6.0.

[0187] In one embodiment of aspect (A5), the formulation has a pH of 5.3 to 5.8.

[0188] In one embodiment (A6), the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising the steps of: (a) about 5 mg / mL to about 75 mg / mL (b) an anti-human PD-1 antibody or an antigen-binding fragment thereof; (c) (i) about 6% to about 8% weight / volume (w / v) sucrose buffer; tetrose, trehalose or (2-hydroxypropyl)-β-cyclodextrin; i) About 3% to about 5% w / v of mannitol, sorbitol, L-arginine, L-arginine a pharma- ceutically acceptable salt of ginine, L-proline or a pharma- ceutically acceptable salt of L-proline and (iii) about 1.8 to about 2.2% w / v glycine or a pharma- ceutically acceptable salt thereof. (d) about 0.01% to about 0.04% of a polyisoprene salt; (e) about 5 mM to about 10 mM L-methionine or a pharmaceutical The present invention provides an anti-human PD-1 antibody formulation comprising an acceptable salt thereof.

[0189] In one embodiment of aspect (A6), an anti-human PD-1 antibody or its antigen-binding activity The fragments are present in a concentration of about 5 mg / mL to about 25 mg / mL.

[0190] In one embodiment (A7), the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising the steps of: (a) about 5 mg / mL to about 75 mg / mL (b) an anti-human PD-1 antibody or an antigen-binding fragment thereof; histidine buffer; (c) about 6% to about 8% weight / volume w / v sucrose; (d) about 0.01% to about 0.04% polysorbate 80; and (e) about 5 mM to about 10 1. An anti-human PD-1 antibody formulation comprising 1 mM L-methionine or a pharma- ceutical acceptable salt thereof. provide.

[0191] In one embodiment of aspect (A7), an anti-human PD-1 antibody or its antigen-binding activity The fragments are present in a concentration of about 5 mg / mL to about 25 mg / mL.

[0192] In one embodiment of aspect (A7), L-methionine or a pharma- ceutically acceptable derivative thereof is The salt used is L-methionine-HCl.

[0193] In one embodiment (A8), the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising the steps of: (a) about 5 mg / mL to about 75 mg / mL (b) an anti-human PD-1 antibody or an antigen-binding fragment thereof; histidine buffer; (c) about 6% to about 8% w / v trehalose; (d) about 0. (e) about 5 mM to about 10 mM methyl The present invention provides an anti-human PD-1 antibody formulation comprising onin or a pharma- ceutical acceptable salt thereof.

[0194] In one embodiment of aspect (A8), an anti-human PD-1 antibody or its antigen-binding activity The fragments are present in a concentration of about 5 mg / mL to about 25 mg / mL.

[0195] In one embodiment (A9), the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising the steps of: (a) about 5 mg / mL to about 75 mg / mL (b) an anti-human PD-1 antibody or an antigen-binding fragment thereof; histidine buffer; (c) about 6% to about 8% w / v (2-hydroxypropyl) -β-cyclodextrin; (d) about 0.01% to about 0.04% polysorbate 80; and (e) about 5 mM to about 10 mM L-methionine or a pharma- ceutically acceptable salt thereof. The present invention provides an anti-human PD-1 antibody formulation comprising the

[0196] In one embodiment of aspect (A9), an anti-human PD-1 antibody or its antigen-binding activity The fragments are present in a concentration of about 5 mg / mL to about 25 mg / mL.

[0197] In one embodiment (A10), the present invention provides a method for treating a chronic pulmonary disease comprising: (a) administering to a patient a dose of from about 5 mg / mL to about 75 mg / m L of an anti-human PD-1 antibody or antigen-binding fragment; (b) about 8 mM to about 12 mM histidine buffer; (c) about 3% to about 5% w / v mannitol; (d) about 0.0 (e) about 5 mM to about 10 mM L-methionine; The present invention provides an anti-human PD-1 antibody formulation comprising thionine or a pharma- ceutically acceptable salt thereof.

[0198] In one embodiment of aspect (A10), an anti-human PD-1 antibody or antigen-binding The reactive fragments are present at a concentration of about 5 mg / mL to about 25 mg / mL.

[0199] In one embodiment (A11), the present invention provides a method for treating a chronic pulmonary disease comprising: (a) administering to a patient a dose of from about 5 mg / mL to about 75 mg / m L of an anti-human PD-1 antibody or an antigen-binding fragment thereof; (b) about 8 mM to about 12 mM (c) about 3% to about 5% w / v sorbitol; (d) about 0 (e) about 5 mM to about 10 mM L -Providing an anti-human PD-1 antibody formulation containing methionine or a pharma- ceutical acceptable salt thereof .

[0200] In one embodiment of aspect (A11), an anti-human PD-1 antibody or antigen-binding The reactive fragments are present at a concentration of about 5 mg / mL to about 25 mg / mL.

[0201] In one embodiment (A12), the present invention provides a method for treating a chronic pulmonary disease comprising: (a) administering to a patient a dose of from about 5 mg / mL to about 75 mg / m L of an anti-human PD-1 antibody or an antigen-binding fragment thereof; (b) about 8 mM to about 12 mM (c) about 3% to about 5% w / v L-proline or an agent thereof (d) about 0.01% to about 0.04% polysorbate 80; and (e) an antibody comprising about 5 mM to about 10 mM L-methionine or a pharma- ceutical acceptable salt thereof; A human PD-1 antibody formulation is provided.

[0202] In one embodiment of aspect (A12), an anti-human PD-1 antibody or antigen-binding The reactive fragments are present at a concentration of about 5 mg / mL to about 25 mg / mL.

[0203] In one embodiment of aspect (A12), L-proline or a pharma- ceutically acceptable derivative thereof is The salt used is L-proline.

[0204] In one embodiment of aspect (A12), L-proline or a pharma- ceutically acceptable The salt is L-proline HCl.

[0205] In one embodiment (A13), the present invention provides a method for treating a chronic pulmonary disease comprising: (a) administering to a patient a dose of from about 5 mg / mL to about 75 mg / m L of an anti-human PD-1 antibody or an antigen-binding fragment thereof; (b) about 8 mM to about 12 mM (c) about 3% to about 5% w / v L-arginine or (d) about 0.01% to about 0.04% polysorbate 80; and and (e) about 5 mM to about 10 mM L-methionine or a pharma- ceutically acceptable salt thereof. The present invention provides an anti-human PD-1 antibody formulation.

[0206] In one embodiment of aspect (A13), an anti-human PD-1 antibody or antigen-binding The reactive fragments are present at a concentration of about 5 mg / mL to about 25 mg / mL.

[0207] In one embodiment of aspect (A13), the pH of the formulation is from about 6.0 to about 6.4. .

[0208] In one embodiment of aspect (A13), L-arginine or a pharma- ceutically acceptable salt thereof is The salt used is L-arginine.

[0209] In one embodiment of aspect (A13), L-arginine or a pharma- ceutically acceptable salt thereof is The salt used is L-arginine HCl.

[0210] In one embodiment (A14), the present invention provides a method for treating a chronic pulmonary disease comprising: (a) administering to a patient a dose of from about 5 mg / mL to about 75 mg / m L of an anti-human PD-1 antibody or an antigen-binding fragment thereof; (b) about 8 mM to about 12 mM (c) about 160 mM to about 200 mM glycine or an agent thereof; (d) about 0.01% to about 0.04% polysorbate 80; and (e) an antibody comprising about 5 mM to about 10 mM L-methionine or a pharma- ceutical acceptable salt thereof; A human PD-1 antibody formulation is provided.

[0211] In one embodiment of aspect (A14), an anti-human PD-1 antibody or antigen-binding The reactive fragments are present at a concentration of about 5 mg / mL to about 25 mg / mL.

[0212] In one embodiment of aspect (A14), glycine or a pharma- ceutically acceptable derivative thereof is The salt is glycine.

[0213] In one embodiment of aspect (A14), glycine or a pharma- ceutically acceptable derivative thereof is The salt is glycine HCl.

[0214] In one embodiment of aspect (A14), glycine or a pharma- ceutically acceptable derivative thereof is The salt is glycine succinate.

[0215] In one embodiment of any of aspects (A6) to (A14), the formulation comprises a metal chelate. The composition further comprises a stimulant.

[0216] In one embodiment of any one of aspects (A6) to (A14), the formulation further comprises DTPA. The compound includes a hydroxypropyl amine, which is present at a concentration of about 10 μM to about 30 μM.

[0217] In some embodiments of any of aspects (A1)-(A14), the formulation is a liquid. do.

[0218] In some embodiments of any of aspects (A1)-(A14), the formulation is lyophilized. Reconstituted solution from the formulation.

[0219] In any of the specific aspects and embodiments described herein, any anti- PD-1 antibodies or antigen-binding fragments thereof (i.e., antibodies that specifically bind to human PD-1) The antibody or antigen-binding fragment thereof, e.g., pembrolizumab or its antigen-binding In certain embodiments, the nucleic acid fragments described herein may be used. and one of the anti-PD-1 antibodies or antigen-binding fragments thereof, e.g., Antibodies and antigen-binding fragments thereof" section are used.

[0220] In some embodiments of the invention, any of the formulations described herein may be water-soluble. In another embodiment, the present invention provides a method for the preparation of a liquid suspension comprising the steps of: and freeze-drying the aqueous formulation to obtain the reconstituted formulation of the present invention. The agent is provided.

[0221] Lyophilized pharmaceutical composition Lyophilized formulations of therapeutic proteins have several advantages. Lyophilized formulations generally: Offers better chemical stability and therefore longer half-life than solution formulations. The dry formulation is available in different concentrations depending on clinical factors such as route of administration or dose. For example, lyophilized formulations can be reconstituted at high concentrations (i.e. It can be reconstituted in small volumes (small doses) or at lower concentrations for intravenous administration. When high doses are required for individual subjects, especially in the case of subcutaneous administration, where the injection volume must be minimized, In some cases, higher concentrations may be required. One such lyophilized antibody formulation is described in U.S. Pat. No. 267,958, the entirety of which is incorporated herein by reference. Another lyophilized formulation of a therapeutic protein is disclosed in U.S. Pat. No. 7,247,707. No. 6,399,413, which is incorporated herein by reference in its entirety.

[0222] Typically, the drug product (DP, in an exemplary embodiment, a humanized anti-PD-1 antibody) is In anticipation of reconstitution at high concentrations of pembrolizumab or its antigen-binding fragment, That is, the lyophilized formulation is prepared in anticipation of reconstitution in a small amount of water. Subsequent dilutions with water or isotonic buffers are easily performed to dilute the DP to lower concentrations. Typically, the formulation is approximately isotonic when reconstituted with a high DP concentration, for example for subcutaneous administration. The excipients are incorporated into the lyophilized formulations of the present invention at levels that provide a formulation with a lower DP concentration. Reconstitution with a larger volume of water to obtain a solution of 100% necessarily reduces the tonicity of the reconstituted solution. However, such a reduction is of little consequence in non-subcutaneous, e.g., intravenous, administration. If isotonicity at lower DP concentrations is desired, lyophilized powders may be the target. Reconstitute with a relatively small volume of water and then dilute with an isotonic diluent, e.g., 0.9% sodium chloride. Further dilution is possible.

[0223] In one embodiment of the invention, a humanized anti-PD-1 antibody (or an antigen-binding fragment thereof) is The fragments are formulated as lyophilized powders for reconstitution and use for intravenous administration. In one embodiment, the antibody (or antigen-binding fragment thereof) has a It is provided in 50 mg / vial and is reconstituted with sterile water for injection prior to use. The constituent antibodies were aseptically diluted in 0.9% Sodium Chloride Injection, USP in a sterile IV container. In some embodiments, the target pH of the reconstituted formulation is 5.5±0.5. In some embodiments, the lyophilized formulations of the invention contain, for example, about 20, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 25 5, 30, 40, 50, 60, 75, 100, 125, 150, 175 mg / mL or more This allows the protein to be reconstituted to such a high concentration.

[0224] A lyophilized formulation is, by definition, essentially dry and therefore requires careful consideration to describe it. The concept of dosage is not useful. Lyophilized formulations are described in terms of the weight of the components in a unit dose vial. It is more useful to do this, but it varies by dose or vial size, so In describing the lyophilized formulation of the present invention, It is useful to express the amount of an ingredient as a ratio of the weight of the ingredient to the weight of the drug substance (DS) in the This ratio may be expressed as a percentage. Such ratios may vary depending on vial size, dosage amount and Regardless of the reconstitution method, this reflects the inherent properties of the lyophilized formulation of the present invention.

[0225] In another embodiment, a lyophilized preparation of the anti-human PD-1 antibody or antigen-binding fragment is provided. The agent may be a pre-lyophilization solution used to prepare the lyophilized formulation, e.g., a pre-lyophilization formulation. In one embodiment, the pre-lyophilized solution is about 10 mg / mL, The antibody or antigen-binding fragment thereof at a concentration of about 25 mg / mL or about 50 mg / mL. Such pre-lyophilization solutions have a pH of 4.4 to 5.2 (approximately 4.4, 4.5, 4.6). , 4.7, 4.8, 4.9, 5.0, 5.1 and 5.2), for example preferably p The pH may be about H 4.8 or about pH 5.5.

[0226] In yet another embodiment, the anti-human PD-1 antibody or antigen-binding fragment is A lyophilized formulation is defined with respect to the reconstituted solution obtained from the lyophilized formulation.

[0227] The reconstituted solution is approximately 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90 or 100 mg / mL, or higher concentrations, e.g., 150 mg / mL, 167 mg / mL, 200 mg / mL or up to about 250 mg / mL of antibody or its antigen binding Such a reconstitution solution may contain a pH of about 5.5, or a pH of about 5.0. can range from about 6.0.

[0228] The lyophilized formulations of the present invention are formed by lyophilization (freeze-drying) of a pre-lyophilization solution. Freeze drying involves freezing the formulation and then sublimating the water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic or collapse temperature of the formulation. For primary drying, shelf temperatures are typically set at appropriate pressures ranging from about 50 to 250 mTorr. The temperature range is approximately -30 to 25°C (however, the product will remain frozen during primary drying). The formulation, size and type of container (e.g., glass vial) in which the sample will be placed; The volume of liquid determines the time it takes to dry, which can range from a few hours to a few days (e.g., 40-60 minutes). The time period can range from 10 minutes to 30 minutes (hours) depending mainly on the type and size of the vessel and the protein used. Depending on the type of product, the secondary drying step may be carried out at temperatures between about 0 and 40°C. This depends on the desired residual moisture level and typically takes at least about 5 hours. Typically, the moisture content of the lyophilized formulation is less than about 5%, preferably less than about 3%. The pressure can be the same as that used during the primary drying step. The lyophilization conditions are may vary depending on the vial size.

[0229] In some cases, protein reconstitution is performed to avoid the transfer step. It may be desirable to lyophilize the protein formulation in a container, in which case the container may be , for example, 3, 5, 10, 20, 50 or 100 cc vials.

[0230] The lyophilized formulations of the present invention are reconstituted prior to administration. 25, 30, 40, 50, 60, 75, 80, 90 or 100 mg / mL or more Higher concentrations, e.g., 150mg / mL, 200mg / mL, 250mg / mL or 300 The reconstituted formulation may be reconstituted at a concentration of about 500 mg / mL to about 500 mg / mL. Subcutaneous delivery of the reconstituted formulation is intended. High protein concentrations are particularly useful when the drug is administered intravenously. For this route of administration, a lower protein concentration (e.g., about 5-50 mg / mL) is desirable. Maybe.

[0231] Reconstitution is generally carried out at a temperature of about 25° C. to ensure complete hydration, but may be carried out at any temperature desired. Other temperatures may also be used. The time required for reconstitution may vary depending on, for example, the type of diluent, excipients, etc. Typical diluents include sterile water, bacteriostatic water for injection (BW FI), pH buffer solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution or Dextran Contains kistrose solution.

[0232] The present invention provides a liquid anti-human PD-1 antibody formulation that is reconstituted from a lyophilized formulation, The composition of the solution is: a) about 125 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or its b) about 8 mM to about 12 mM histidine buffer; c) ( (i) about 3% to about 8% weight / volume (w / v) sucrose; (ii) about 2% to about 5% w / v / v L-arginine or a pharma- ceutically acceptable salt thereof; (iii) about 3% to about 5% (iv) a combination of i) and ii); and d) from about 0.01% to about 0.04% of a polyisoprene. Includes Rubet 80.

[0233] In some embodiments of the present invention, the stabilizer is present in an amount of about 3% to about 8% weight / volume (w / v Contains sucrose.

[0234] In some embodiments of the present invention, the stabilizer is about 2% to about 5% w / v L-arginine. The present invention includes nin or a pharma- ceutically acceptable salt thereof.

[0235] In some embodiments of the invention, the stabilizer is about 3% to about 5% mannitol and Contains about 1% to about 2% sucrose.

[0236] In some embodiments of the present invention, the stabilizer is about 4% to about 4.5% mannitol and and about 1% to about 2% sucrose.

[0237] In some embodiments of the present invention, the stabilizer is present in an amount of about 3% to about 8% weight / volume (w / v of sucrose and about 2% to about 5% w / v L-arginine or a pharma- ceutically acceptable In certain embodiments, the stabilizer is sucrose and L-arginine. In another embodiment, the stabilizer comprises sucrose and L-arginine-HCl. include.

[0238] In certain embodiments, the stabilizer is 2-4% w / v L-arginine or an equivalent thereof. Other embodiments include a combination of a physiologically acceptable salt and 3.5-6% w / v sucrose. In one embodiment, the stabilizer comprises about 3% L-arginine or a pharma- ceutically acceptable salt thereof and about 5% In another embodiment, the stabilizer is about 2% L- It contains a combination of arginine or a pharma- ceutically acceptable salt thereof and about 5% sucrose. In this embodiment, the stabilizer is about 2% L-arginine or a pharma- ceutically acceptable salt thereof. It contains a combination of salt and about 3.7% sucrose.

[0239] Liquid pharmaceutical composition Liquid antibody formulations are prepared by dissolving the drug substance (e.g., anti-humanized PD-1) in a liquid form (e.g., aqueous pharmaceutical formulation). As the final step in the purification process, pembrolizumab (in the desired buffer) is taken and mixed with In this embodiment, the solution may be prepared by buffer exchange into a freeze-dried solution. There is no step. The drug substance in the final buffer is concentrated to the desired concentration. Sucrose, Excipients such as thionin and polysorbate 80 are added to the drug substance to provide a suitable balance. The final formulated drug substance is diluted to the final protein concentration using a buffer. The mixture is then filtered, for example using a 0.22 μm filter, into the final container (e.g., glass vial or Such liquid formulations are filled into a 10 mM histidine (pH 5.0 or 6.0) solution. .5), 7% sucrose, 0.02% polysorbate 80, 25-200mg / mL Pembrolizumab and 1.5-2.5% arginine or its pharma- ceutical acceptable salts This is exemplified by the final liquid formulation containing a salt.

[0240] III.How to use The present invention also relates to any of the formulations of the present invention, i.e., any of the formulations described herein. The formulation (described in the section "Specific Aspects and Embodiments of the Invention" herein as aspect (A1 ) to (A14) (hereinafter referred to as "Aspects (A1) to (A14)"). A method for treating cancer in a subject, comprising administering to the subject an effective amount of In some embodiments of the method, the formulation is administered to the subject by intravenous administration. In other embodiments, the formulation is administered to the subject by subcutaneous administration.

[0241] In any of the methods of the present invention, the cancer is melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, Cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary cancer, prostate cancer (e.g., hormone deficiency) refractory prostate adenocarcinoma), pancreatic cancer, colon cancer, esophageal cancer, liver cancer, thyroid cancer, glioblastoma, glioma, and and other neoplastic malignancies.

[0242] In some embodiments, the lung cancer is non-small cell lung cancer.

[0243] In another embodiment, the lung cancer is small cell lung cancer.

[0244] In some embodiments, the lymphoma is Hodgkin's lymphoma.

[0245] In other embodiments, the lymphoma is non-Hodgkin's lymphoma. In some embodiments, the lymphoma is mediastinal large B-cell lymphoma. The lymphoma is diffuse large B-cell lymphoma (DLBCL).

[0246] In some embodiments, the breast cancer is triple negative breast cancer.

[0247] In other embodiments, the breast cancer is ER+ / HER2- breast cancer.

[0248] In some embodiments, the bladder cancer is urothelial carcinoma.

[0249] In some embodiments, the head and neck cancer is nasopharyngeal carcinoma. In some embodiments, the cancer is thyroid cancer. In other embodiments, the cancer is salivary gland cancer. In, the cancer is squamous cell carcinoma of the head and neck.

[0250] In some embodiments, the cancer is characterized by high levels of microsatellite instability (MSI). -H) metastatic colorectal cancer.

[0251] In some embodiments, the cancer is characterized by high levels of microsatellite instability (MSI). -H).

[0252] In some embodiments, the cancer is a solid tumor with a high mutational burden.

[0253] In some embodiments, the cancer is melanoma, non-small cell lung cancer, recurrent or refractory Classic Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial cancer, esophageal cancer, gastric cancer, DLBCL and and hepatocellular carcinoma.

[0254] In other embodiments of the methods of treatment, the cancer is a heme malignancy. In terms of hemolytic malignancies, hemolytic malignancies include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and ), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), 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-1 protein (Mcl-1), myelodysplastic syndrome ( Myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma (NHL) or small lymphocytic lymphoma (SLL) .

[0255] Disease-free survival and overall survival associated with the presence of tumor-infiltrating lymphocytes in biopsy or surgical material Malignant diseases showing improved survival rates, such as melanoma, colorectal cancer, liver cancer, kidney cancer, gastric / esophageal cancer, etc. Cancers of the esophagus, breast, pancreas and ovary are included in the methods and treatments described herein. It is known that such cancer subtypes are susceptible to immune regulation by T lymphocytes. refractory or recurrent malignancies whose growth may be inhibited using the antibodies described herein; This also includes diseases.

[0256] In some embodiments, the formulations of the invention (e.g., embodiments (A1)-(A14)) include , ovarian cancer, kidney cancer, colorectal cancer, pancreatic cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer and melanoma for elevated expression of PD-L1 and / or PD-L2 in a test tissue sample, The anti-PD-1 antibody, e.g., a humanized anti-PD-1 antibody, is administered to a subject having a cancer that is to be further characterized. Additional cancers that may benefit from treatment with the -1 antibody pembrolizumab include, for example, Kaposi's For sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervical cancer, vulvar cancer, anal cancer, penile cancer and oral cancer Viruses known to be causally related, e.g., human immunodeficiency virus, hepatitis virus Persistent infection with classes A, B and C, Epstein-Barr virus, and human papillomavirus This includes cancers associated with

[0257] In one embodiment, the present invention provides a method for the preparation of any of the formulations of the present invention (e.g., embodiment (A1 The present invention also includes a method for treating cancer in a human patient, comprising administering to the patient one of the compounds listed in claim 1 to (A14).

[0258] In one embodiment, the present invention provides a method for the preparation of any of the formulations of the present invention (e.g., embodiment (A1 ) to (A14)) in a human patient. Methods for treating melanoma are included.

[0259] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4)) to the patient. In certain embodiments, the patient is treated with a tumor-specific inhibitor of high PD-L1 expression [tumor ratio Tumor Proportion Score (TPS) ≥ 50%) In another embodiment, the patient has a tumor that has not been previously treated with platinum-containing chemotherapy. For this study, patients were required to have tumors with PD-L1 expression (TPS ≥ 1%) and to undergo platinum-containing chemotherapy. In yet another embodiment, the patient has been previously treated with PD-L1 expression (T Patients had tumors with PS ≥ 1%) and had not been previously treated with platinum-containing chemotherapy. In certain embodiments, the patient exhibits disease progression during or after platinum-containing chemotherapy. did.

[0260] In one embodiment, PD-L1 TPS is determined by an FDA approved test.

[0261] In certain embodiments, the patient's tumor does not have an EGFR or ALK genomic aberration.

[0262] In one embodiment, the patient's tumor has an EGFR or ALK genomic abnormality and is treated with anti-P Prior to administration of the D-1 antibody or its antigen-binding fragment, showed disease progression during or after receiving treatment with refractory leukemia.

[0263] In one embodiment, the present invention provides: (1) a formulation of the present invention (e.g., embodiment (A1) to embodiment (A2) (A14)) to a patient, and (2) pemetrexed ed) and carboplatin to the patient, Methods for treating metastatic non-small cell lung cancer (NSCLC) in human patients are included. In the present invention, patients are treated with a combination of the formulation of the present invention, pemetrexed and carboplatin. They had not been previously treated with anticancer drugs before starting the regimen.

[0264] In certain embodiments, the patient has non-squamous non-small cell lung cancer.

[0265] In one embodiment, pemetrexed is administered at 500 mg / m 2 is administered to the patient in an amount of In a subembodiment, pemetrexed is administered to the patient by intravenous infusion every 21 days. In certain embodiments, the infusion time is about 10 minutes.

[0266] In an embodiment of the invention where a patient is treated with a formulation of the invention in combination with pemetrexed The present invention provides a method for administering pemetrexed to a patient, comprising administering about 400 μg of pemetrexed once a day for about 7 days prior to administration of pemetrexed to the patient. Approximately 1000 μg of folic acid is administered to the patient and approximately 10 minutes after the administration of the final dose of pemetrexed to the patient. In one embodiment, the method further comprises administering folic acid orally to the subject for 21 days after the first administration of the folic acid. In some embodiments, the present invention provides a method for administering pemetrexed to a patient about one week prior to the first administration of pemetrexed. and approximately 1 mg every 3 cycles of pemetrexed (i.e., approximately every 9 weeks). Vitamin B 12 In one embodiment, the method further comprises administering to the patient vitamin B 12 In one embodiment, the present invention provides a method for administering pemetrexed intramuscularly on the day before administration of pemetrexed. and administering to the patient about 4 mg of dexamethasone twice daily on the same day and the following day. In certain embodiments, dexamethasone is administered orally.

[0267] In one embodiment, the present invention provides a method for the preparation of any of the formulations of the present invention (e.g., embodiment (A1 ) to (A14)) in a human patient. In one embodiment, the patient is receiving platinum-based chemotherapy for cervical squamous cell carcinoma (HNSCC). In certain embodiments, the patient has been previously treated with platinum-containing chemotherapy. showed disease progression at or after treatment.

[0268] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4)) to a patient. In one embodiment, the method includes the treatment of cHL. In certain embodiments, the patient is an adult patient. In an embodiment, the patient is a pediatric patient.

[0269] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4)) administering to the patient a method for treating locally advanced or metastatic urothelial cancer in a human patient. In one embodiment, the patient is not a candidate for cisplatin-containing chemotherapy. In certain embodiments, the patient is treated with platinum-containing chemotherapy during or after chemotherapy, or Disease within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy Shows progress.

[0270] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4) administering to the patient a Methods for treating light instability-high (MSI-H) or mismatch repair deficient solid tumors In certain embodiments, the patient has demonstrated disease progression following prior anti-cancer treatment.

[0271] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4) administering to the patient a Methods for treating light instability-high (MSI-H) or mismatch repair deficient colorectal cancer. In certain embodiments, the patient is receiving fluoropyrimidine, oxaliplatin and isoformic acid. The patient showed disease progression after prior treatment with rinotecan.

[0272] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4)) for treating recurrent locally advanced or metastatic gastric cancer in a human patient, comprising administering to the patient This includes methods for treating cancer.

[0273] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4)) for treating recurrent locally advanced or metastatic gastric cancer in a human patient, comprising administering to the patient In certain embodiments, the patient's tumor is PD-L1 [Combined Positive Score (C PS) > 1]. In certain embodiments, the patient is receiving fluoropyrimidine and platinum-containing Showing disease progression during or after a series of two or more prior treatments, including chemotherapy. In this setting, patients are treated with a series of two or more prior therapies that include HER2 / neu targeted therapy. These may indicate disease progression at or after the procedure.

[0274] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4)) to the patient, wherein the patient is Lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, lymphoma , kidney cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer and salivary gland cancer.

[0275] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4) ...

[0276] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4)) a method for treating non-Hodgkin's lymphoma in a human patient, comprising administering to the patient In certain embodiments, the non-Hodgkin's lymphoma is mediastinal large B-cell lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma. It is a tumor.

[0277] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4) ... In some embodiments, the breast cancer is triple negative breast cancer. In this case, the breast cancer is ER+ / HER2- breast cancer.

[0278] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4))) to the patient.

[0279] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4) ...

[0280] In one embodiment, the present invention relates to a formulation of the present invention (e.g., aspect (A1) to (A1 4) ...

[0281] Antagonist anti-PD-1 antibodies or antibody fragments may be used to prevent or treat infections and infections. Therefore, the present invention relates to a method for the treatment of a pulmonary artery disease, comprising administering an effective amount of the formulation of the present invention to a patient. The present invention provides a method for treating a chronic infection in a mammalian subject, comprising administering to an elephant a In some particular embodiments of the method, the formulation is administered to the subject by intravenous administration. In an embodiment, the formulation is administered to the subject by subcutaneous administration.

[0282] These substances act alone to stimulate immune responses against pathogens, toxins and self-antigens. It can be used alone or in combination with a vaccine. Human immunodeficiency virus, hepatitis viruses class A, B and C, Epstein-Barr virus Viruses, human cytomegalovirus, human papillomavirus and herpesviruses ( Immunity to viruses infectious to humans, including but not limited to Antagonist anti-PD-1 antibodies or antibody flagella can be used to stimulate an immune response. ment stimulates the immune response against infections by bacterial or fungal parasites and other pathogens Viral infections with hepatitis B, hepatitis C and HIV can be used to treat chronic It is one of those considered to be viral infections.

[0283] The formulations of the invention may be administered to a patient in combination with one or more "additional therapeutic agents." Therapeutic agents of interest are biotherapeutics (VEGF, EGFR, Her2 / neu, VEGF receptor , other growth factor receptors, CD20, CD40, CD-40L, OX-40, 4-IBB and and antibodies against ICOS), antiproliferative agents , immunogenic substances [e.g., attenuated cancer cells, tumor antigens, tumor-derived antigens, or nucleic acids] Antigen-presenting cells such as dendritic cells, immune-stimulating cytokines (e.g., IL-2, IFN-γ, Nα2, GM-CSF), and immune stimulating cytokines, e.g., GM-CSF ( (including but not limited to) a cell transfected with a gene encoding do.

[0284] As noted above, in some embodiments of the methods of the invention, the methods include administering an additional treatment In certain embodiments, the additional therapeutic agent is an anti-inflammatory agent. LAG3 antibody or antigen-binding fragment thereof, anti-GITR antibody or antigen-binding fragment thereof Antibody or antigen-binding fragment thereof, anti-TIGIT antibody or antigen-binding fragment thereof, anti-CD27 antibody or or an antigen-binding fragment thereof. In one embodiment, the additional therapeutic agent The target substance is a Newcastle disease virus vector expressing IL-12. In one embodiment, the additional therapeutic agent is dinaciclib. In yet other embodiments, the additional therapeutic agent is a STING agonist.

[0285] Suitable routes of administration include, for example, parenteral delivery, e.g., intramuscular, subcutaneous, and intrathecal, direct to the brain. Drugs can be administered in a variety of conventional ways, including intravenously, intravenously, or intraperitoneally. It can be administered by oral, intra-arterial or intravenous injection. Methods of administration (eg, subcutaneous administration) require lyophilized formulations that allow for reconstitution at high concentrations.

[0286] The choice of dosage of the additional therapeutic agent will depend on the serum or tissue turnover rate of the agent, the severity of the symptoms, and the severity of the disease. the degree of immunogenicity of the substance, and the engraftment of target cells, tissues or organs in the individual being treated. The dosage of additional therapeutic agent depends on several factors, including the availability of the agent at acceptable levels. Therefore, each additional therapeutic agent (e.g., The dose and frequency of administration of a therapeutic agent (pharmacotherapy or chemotherapeutic agent) will depend, in part, on the particular therapeutic agent, therapeutic The type of treatment depends on the severity of the cancer being treated and the characteristics of the patient. Antibodies, cytokines and small molecules Guidance is available for selecting appropriate doses of :Wawrzynczak(1996)Antibody Therapy,Bios Scientific Pub.Ltd,Oxfordshire,UK;Kresin a (ed.) (1991) Monoclonal Antibodies, Cytokine s and Arthritis,Marcel Dekker,New York,N Y;Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003)Ne w Engl. J. Med. 348:601-608; Milgrom et al. (1999) N ew Engl. J. Med. 341:1966-1973; Slamon et al. (2001 )New Engl.J.Med.344:783-792;Beniaminovit z et al. (2000) New Engl.J.Med.342:613-619;Ghosh (2003) New Engl. J. Med. 348:24-32; Lipsky et al. ( 2000)New Engl.J.Med.343:1594-1602;Physic ians'Desk Reference 2003(Physicians'Desk Reference,57th Ed);Medical Economics Co mpany;ISBN:1563634457;57th Edition (November 2002). Determining the dosing regimen may be based, for example, on any known factors in the art that may affect treatment. using parameters or factors that are suspected or expected to affect treatment This can be done by a clinician, for example by taking into account the patient's clinical history (e.g., past medical the type and stage of cancer being treated, and one of the therapeutic agents in the combination therapy. It will depend on biomarkers of response to these.

[0287] Facilitates selection of pharma- ceutically acceptable carriers or excipients for additional therapeutic substances Various references are available for this purpose. See, for example, Remi ngton's Pharmaceutical Sciences and USPh armacopeia:National Formulary,Mack Publi shing Company, Easton, PA (1984); Hardman et al. (2 001)Goodman and Gilman's The Pharmacolog ical Basis of Therapeutics,McGraw-Hill,N ew York, NY; Gennaro (2000) Remington: The Sc. ience and Practice of Pharmacy,Lippincott t, Williams and Wilkins, New York, NY; Avis et al. (eds. )(1993)Pharmaceutical Dosage Forms:Paren teral Medications,Marcel Dekker,NY;Liebe (eds.) (1990) Pharmaceutical Dosage Form s:Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990)Pharmaceutical Dosage Forms:Disper se Systems, Marcel Dekker, NY; Weiner and Kot koskie(2000)Excipient Toxicity and Safet y, Marcel Dekker, Inc., New York, NY.

[0288] The pharmaceutical antibody formulations can be administered by continuous infusion or, for example, at daily intervals, 1 to 7 times a week, at weekly intervals, or 2 Administration may be at weekly intervals, three-weekly intervals, monthly intervals, bimonthly intervals, etc. Protocols include the maximum dose or frequency of administration that avoids significant undesirable side effects. The total weekly dose is generally at least 0.05 μg / kg, 0.2 μg / kg, 0.5 μg / kg, 1μg / kg, 10μg / kg, 100μg / kg, 0.2mg / kg, 1 .0mg / kg, 2.0mg / kg, 10mg / kg, 25mg / kg, 50mg / kg For example, Yang et al. (2003) New Engl. J. Med. 34 9:427-434; Herold et al. (2002) New Engl. J. Med. 34 6:1692-1698;Liu et al. (1999) J. Neurol. Neurosurg .Psych.67:451-456;Portielji et al. (20003) Cance r Immunol. Immunother. 52:133-144. The desired dose of a molecular therapeutic, e.g., a peptidomimetic, natural product, or organic compound, is The dose is approximately the same as for antibodies or polypeptides on a per kg basis.

[0289] In certain embodiments, administration is by administration of a pharmaceutical formulation, i.e., pembro, over the course of treatment. Subjects will be administered escalating doses of 1.0, 3.0 and 10 mg / kg of a formulation containing lizumab. The formulation containing pembrolizumab may be a reconstituted liquid formulation. The time course can be variable and can be as desired. This may be continued as long as efficacy is achieved. In one embodiment, the dose escalation is about 10 mg / In one embodiment, the subject is treated with melanoma or other tumor markers. Subjects have a histologic or cytologic diagnosis of a solid tumor of any type, and in some cases, subjects have a cytologic diagnosis of a solid tumor of any type, including idiopathic pulmonary disease. In certain embodiments, the subject may have a disease that is treatable with other chemotherapy agents. However, in other embodiments, the subject is treatment naive.

[0290] In yet additional embodiments, the dosing regimen is the same as described herein over the entire course of treatment. Any one of the pharmaceutical preparations (i.e., preparations containing pembrolizumab) described in the literature Such a fixed administration regimen includes administering a dose of 10 mg / kg, 3 mg / kg or 10 mg / kg. For dimen, the dosing interval is about 14 days (± 2 days). is approximately 21 days (± 2 days).

[0291] In certain embodiments, the dosing regimen is about 0.005 with intrapatient dose escalation. In one embodiment, the method comprises administering a dose of from about 10 mg / kg to about 10 mg / kg. Doses of 5 mg / kg or 10 mg / kg are administered at intervals of 3 weeks or 2 weeks. In yet additional embodiments, patients with melanoma or other solid tumors are In these embodiments, patients are administered a dose of 3 mg / kg every 3 weeks. Patients should have a disabling disease, but may have had previous surgery.

[0292] In certain embodiments, a 30 minute period of any of the pharmaceutical formulations described herein is An IV infusion is administered to the subject. In some embodiments of the escalating dose, the administration interval is 10 min after the first administration. There is about 28 days (± 1 day) between the second administration. In some embodiments, the second administration and the third administration are The interval between doses is about 14 days (± 2 days). In one embodiment, the interval between doses is about 14 days (± 2 days). For doses after the second dose, it is approximately 14 days (± 2 days).

[0293] In one embodiment, WO2012 / 018538 or WO2008 / 1567 The use of cell surface markers and / or cytokine markers as described in 12 Bioassays for monitoring, diagnosis, patient selection and / or treatment regimens ( These include blocking the PD-1 pathway.

[0294] Subcutaneous administration may be by using a syringe or other injection device (e.g., Inject-e ase® device), injector pen; or needleless device (e.g., MediJ This can be done using the BioJector and BioJector®.

[0295] Embodiments of the invention also include (a) therapeutic (e.g., treatment of the human body); (b) pharmaceutical; (c) anti-inflammatory; (d) induction or enhancement of a tumor immune response; (e) reduction in the number of one or more tumor markers in the patient; (e) preventing or slowing the growth of tumors or blood cancers; (f) preventing the progression of PD-1-associated diseases. (g) preventing or delaying progression of cancer; (h) stabilizing PD-1-associated disease; (i) (j) inhibiting the growth or survival of tumor cells; (j) eliminating or reducing the size of one or more cancerous lesions or tumors. (k) reducing the progression, onset or severity of PD-1-associated diseases, such as cancer; (m) reducing the severity or duration of clinical symptoms of PD-1-associated disease; and (m) prognosis in similar untreated patients. n) the prolongation of patient survival compared to expected survival, n) the prolongation of patient survival compared to expected survival due to a cancerous condition or other PD-1-related (i) induction of complete or partial remission of a related disease, or (o) treatment of cancer, (ii) for use as a medicament or composition for (iii) The present invention includes one or more of the biological agents for use in the manufacture of a medicament for.

[0296] General method Standard methods in molecular biology include those described by Sambrook, Fritsch and Man iatis (1982 & 1989 2 nd Edition, 2001 3 rd E dition)Molecular Cloning,A Laboratory Ma nual,Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY; Sambrook and Russell l(2001)Molecular Cloning, 3 rd ed, Cold Spring ng Harbor Laboratory Press,Cold Spring H arbor,NY;Wu(1993)Recombinant DNA,Vol.217 ,Academic Press,San Diego,CA. Standards A typical method is described in Ausbel et al. (2001) Current Protocols in Molecular Biology,VoIs.1-4,John Wiley an and also described by MD Sons, Inc., New York, NY, which is a method for producing bacterial cells. Cloning and DNA mutagenesis in mammalian cells and yeast (Vol. 1) (Vol. 2), glycoconjugates and protein expression (Vol. 3), as well as bioinformatics (Vol. 4).

[0297] Protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization Methods for preparation have been described (Coligan et al. (2000) Current Press, otocols in Protein Science,Vol.1,John Wi ley and Sons, Inc., New York). Chemical analysis, chemical modification, translation Post-modification, preparation of fusion proteins, glycosylation of proteins have been described (e.g., Coligan et al. (2000) Current Protocols in Prote in Science,Vol.2,John Wiley and Sons,Inc. .,New York;Ausubel et al. (2001)Current Protoco ls 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;A mersham Pharmacia Biotech(2001)BioDirect (See, for example, P. Ory, Piscataway, NJ, pp. 384-391.) The production, purification and fragmentation of reclonal and monoclonal antibodies are described. (Coligan et al. (2001) Current Protocols in Immunology unology,Vol.1,John Wiley and Sons,Inc.,N ew York;Harlow and Lane (1999)Using Antibod ies,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques are available for characterizing ligand / receptor interactions (e.g., Coligan et al. (2001) Current Protocols in Immuno logy, Vol. 4, John Wiley, Inc., New York. sea ​​bream).

[0298] Monoclonal, polyclonal and humanized antibodies can be produced (e.g. , Sheperd and Dean (eds.) (2000) Monoclonal Antibodies odies,Oxford Univ.Press,New York,NY;Kont Ermann and Dubel (eds.) (2001) Antibody Engineer ing, Springer-Verlag, New York;Harlow and La ne(1988)Antibodies A Laboratory Manual,C old Spring Harbor Laboratory Press,Cold Spring Harbor, NY, pp. 139-243; Carpenter et al. (2 000)J.Immunol.165:6205;He et al. (1998)J.Immuno l.160:1029;Tang et al. (1999) J.Biol.Chem.274:27 371-27378;Baca et al. (1997) J.Biol.Chem.272:106 78-10684; Chothia et al. (1989) Nature 342:877-88 3;Foote and Winter (1992) J. Mol. Biol. 224:487 -499; see U.S. Patent No. 6,329,511).

[0299] An alternative to humanization is the use of human antibody libraries displayed on phage, or trans. The first step is to use a human antibody library in a transgenic mouse (Vaughan et al., 2013). (1996) Nature Biotechnol. 14:309-314; Bar bas(1995)Nature Medicine 1:837-839;Mende z et al. (1997) Nature Genetics 15:146-156;Hooge nboom and Chames (2000) Immunol.Today 21:371 -377;Barbas et al. (2001) Phage Display: A Labora tory Manual,Cold Spring Harbor Laborator y Press, Cold Spring Harbor, New York; Kay et al. (1996)Phage Display of Peptides and Prot eins:A Laboratory Manual,Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biote chnol.17:397-399).

[0300] Purification of the antigen is not necessary for the production of antibodies. Immunizing an animal with cells containing the antigen of interest Spleen cells are then isolated from the immunized animal and the spleen cells are transformed with myeloma cells. It is possible to fuse the endothelial cells with a cellular line to obtain hybridomas (see, for example, Meyaard et al. 1997)Immunity 7:283-290;Wright et al. (2000)Immunity unity 13:233-242; Preston et al., supra; Kaithana et al. (1999) J. Immunol. 163:5157-5164).

[0301] Antibodies can be coupled to, for example, small drug molecules, enzymes, liposomes, and polyethylene glycol (PEG). The antibodies may be useful for therapeutic, diagnostic, kit or other purposes, e.g. Examples include antibodies bound to dyes, radioisotopes, enzymes or metals, such as colloidal gold ( See, e.g., Le Doussal et al. (1991) J. Immunol. 146:169-1 75;Gibellini et al. (1998) J.Immunol.160:3891-38 98;Hsing and Bishop (1999) J. Immunol. 162:280 4-2811;Everts et al. (2002) J.Immunol.168:883-88 9).

[0302] Methods for flow cytometry including fluorescence activated cell sorting and detection system (FACS) are available. It is possible (see, for example, Owens et al. (1994) Flow Cytometry Pri ciples for Clinical Laboratory Practice ,John Wiley and Sons,Hoboken,NJ;Givan(20 01) Flow Cytometry, 2 nd ed.;Wiley-Liss,Hob oken,NJ;Shapiro(2003)Practical Flow Cyto See Metry, John Wiley and Sons, Hoboken, NJ. (e.g., for use as diagnostic reagents), including nucleic acid primers and probes. Suitable fluorescent reagents for modifying nucleic acids, polypeptides and antibodies are available (Mol ecular Probesy(2003)Catalogue,Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003 )Catalogue,St. Louis,MO).

[0303] Standard histological methods for the immune system have been described (e.g., Muller-Harme et al. link (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 At (See, e.g., McGraw-Hill, New York, NY).

[0304] For example, antigen fragments, leader sequences, protein folding, functional domains A software package for determining glycosylation sites and sequence alignments and databases are available (e.g., GenBank, Vector NTI (R) Suite (Informax, Inc., Bethesda, MD); GC G Wisconsin Package (Accelrys, Inc., San Di ego, CA); DeCypher (registered trademark) (TimeLogic Corp., C Menne et al. (2000)Bioinform atics 16:741-742;Menne et al. (2000)Bioinformat ics Applications Note 16:741-742;Wren et al.(2 002)Comput.Methods Programs Biomed.68:17 7-181;von Heijne(1983)Eur.J.Biochem.133: 17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0305] Analysis method Suitable analytical methods for evaluating product stability include size exclusion chromatography (SEC), Dynamic light scattering (DLS), differential scanning calorimetry (DSC), iso-asp quantification , potency, UV at 340 nm, UV spectroscopy and FTIR. SEC (J. Pha rm.Scien.,83:1645-1650,(1994);Pharm.Res. ,11:485(1994);J.Pharm.Bio.Anal.,15:1928( 1997);J.Pharm.Bio.Anal.,14:1133-1140(198 6)) measures the percentage of monomer in the product and provides information on the amount of soluble aggregates. Pharm.Res.,15:200(1998);Pharm.Res.,9:109 (1982)) provides information on protein denaturation temperature and glass transition temperature. American Lab, November (1991) measured the average diffusion coefficient, Provides information on the amount of soluble and insoluble aggregates. UV at 340 nm is 340 nm It measures the scattered light intensity of the sample and provides information about the amount of soluble and insoluble aggregates. The spectrophotometric method measures the absorbance at 278 nm and provides information on protein concentration. rJPharm.Biopharm.,45:231(1998);Pharm.R es.,12:1250(1995);J.Pharm.Scien.,85:1290 (1996); J. Pharm. Science, 87:1069(1998)) is an amide The IR spectrum of a single region is measured and provides information on the secondary structure of the protein.

[0306] The iso-asp content in the samples was determined using the Isoquant isoaspartate detection system (I soquant Isoaspartate Detection System)(P The kit is used to measure the activity of the enzyme protein isoaspartyl. Isoasparagine methyltransferase (PIMT) is used to detect isoasparagine residues in target proteins. PIMT specifically detects the presence of S-carboxyl residues at the alpha-carboxyl position. Catalyzes the transfer of a methyl group from adenosyl-L-methionine to isoaspartic acid, This process produces S-adenosyl-L-homocysteine ​​(SAH), which is a relatively small It is a complex molecule and is typically analyzed by reversed-phase HPLC using the SAH HPLC standard provided in the kit. C.

[0307] The potency or bioidentity of an antibody can be measured by its ability to bind to its antigen. Specific binding of the antibody to its antigen can be determined by any method known to those skilled in the art, for example, ELISA (enzyme-linked immunosorbent assay). The amount of IgG1 may be quantified by immunoassays such as combined immunosorbent assay (MIA).

[0308] All publications cited herein are incorporated by reference in their entirety for purposes of illustration only and are not intended to be limiting unless otherwise specified. No. 6,393,945, and the materials therein are incorporated by reference for purposes of describing and disclosing such materials.

[0309] Various embodiments of the present invention are described herein with reference to the accompanying drawings, in which: It is not intended to be limited to the precise embodiments, but various changes and modifications may be made in accordance with the appended claims. Any changes or modifications that may be made within the scope of the present invention will be apparent to those skilled in the art without departing from the scope or spirit of the present invention as defined in the following claims. It should be understood that the present invention may be administered by any person skilled in the art. [Brief description of the drawings]

[0310] [Figure 1] 1A-1C show the results of an HP-HIC study measuring the oxidation of Met-105 for high concentration pembrolizumab formulations over a 12 week period. Results are shown for test formulations (see Example 2) stored at 5° C. (FIG. 1A), 25° C. (FIG. 1B), and 40° C. (FIG. 1C). [Diagram 2] 2A-2C show the results of the HP-HIC study of the formulations in Example 3. Results are shown for test formulations stored for 9 months at 5° C. (FIG. 2A), 25° C. (FIG. 2B), and 40° C. (FIG. 2C). [Diagram 3]Figures 3A-3C show the results of HP-SEC analysis (measured as % mAb) of the formulations described in Example 4. Results are shown for formulations stored at 5°C (Figure 3A), 25°C (Figure 3B), and 40°C (Figure 3C). [Figure 4] Figures 4A-4C show the results of HP-HIC analysis of the formulations described in Example 4. The results shown show the percentage of Pre-Peak 1+2 (oxidized species) formulations stored for 12 weeks at 5°C (Figure 3A), 25°C (Figure 3B) and 40°C (Figure 3C). The dashed lines in Figures 4B and 4C show the results from Formulation 1, Example 3, as a comparative example. [Diagram 5] FIG. 5 shows the amount of aggregation for each formulation in Example 5 over an 8 week storage period at 40° C. as measured by HP-SEC. [Figure 6] FIG. 6 shows the oxidation of Met-105 in the formulations of Example 6, Study 2 over 8 weeks at 40° C. as measured by HP-HIC. [Figure 7] FIG. 7 shows the oxidation of Met-105 in the formulations of Example 6, Study 3 over 8 weeks at 40° C. as measured by HP-HIC.

[0311] Example 1 material and method CE-SDS The samples were analyzed by CE-SDS technique. In this case, the proteins were The capsid was denatured with sodium dodecyl sulfate (SDS) under reducing and non-reducing conditions. Electrophoresis (CE) [Beckman-Coulter ProteomeLab PA800 CE System and IgG Purity / Homogeneity Assay Kit (Purity / H The method was performed using the Protein Isolation Assay Kit. Proteins are separated based on the apparent molecular weight of the protein. Under non-reducing conditions, the main IgG peak All species other than IgG were classified as impurities. Under reducing conditions, IgG was degraded into heavy and light chains. All other species were classified as impurities. In both cases, the results were The area fractions were reported as the corrected area fractions of each peak calculated from the area fractions of the peaks.

[0312] HP-IEX : High performance ion exchange chromatography (HP-IEX) was used to determine the charge The profile was evaluated using a Dionex ProPac WCX-10 An ion-exchange HPLC method was performed using a sample column and a UV detector at 280 nm. The sample was diluted with purified water and 80 μg was injected for analysis. The mobile phase used for the IEX analysis was The mobile phase gradient was as follows (Mobile phase A: 24 mM MES, pH 6, 4% acetonite). mobile phase B: 20 ​​mM phosphate, 95 mM NaCl, pH 8, 4% a Acetonitrile (v / v). The main peak is the main component of the chromatogram, which is acidic and is used as a control for characterization of the basic variant. The acidic variant is the major peak The main cause of the formation of the acidic variants is the deacetylation of Asn in the main peak. This is due to midation and the presence of sialic acid compared to the main peak. The variants eluted later than the main peak, and the main cause of the formation of the basic variants was the C This is due to incomplete removal of the terminal Lys. Other causes include the loss of the light or heavy chains or both. Incomplete cyclization of N-terminal glutamine (Gln) to pyroGlu, and also in the main peak This is due to the isomerization of Asp in to isoAsp.

[0313] HP-SEC : Sample purity assessed by size exclusion chromatography (SEC) In this case, the proportion of monomer and the proportion of high molecular weight species (HMW) and late eluting peaks (L The proportion of HMW species was determined. The presence of HMW species indicates protein aggregates, while the presence of LMW species indicates protein aggregates. indicates protein fragments. High performance-size exclusion chromatography (HP-SEC) The sample was diluted to 1.0 mg / mL with water. ) on a HP column equipped with a YMC-Pak-Diol 200 column and a UV detector. The sample was injected into the LC. Proteins in the sample were separated by size and analyzed by UV absorption at 280 nm. It was detected by.

[0314] HP-SEC Arg : Check sample purity with size exclusion chromatography (SEC) In this case, the proportion of monomer and the proportion of high molecular weight species (HMW) and late eluting peaks were evaluated. The proportion of LMW species was determined by high performance-size exclusion chromatography (HP-S EC) was performed by mixing the sample with a mobile phase (50 mM sodium phosphate, 450 mM arginine monohydrate, The column temperature was adjusted to 100° C. by diluting the solution to 5.0 mg / mL in 100 mL of 100% ethanol (HCl, pH 7.0). The temperature was set at 25°C and the flow rate was maintained at 0.5 mL / min using isocratic elution. The diluted sample (30 μL) was transferred to a YMC-PACK Diol 200 column. The samples were then injected into an HPLC system equipped with a column and UV detector. The proteins were separated and detected by UV absorption at 280 nm.

[0315] A350 : As an index of turbidity, a 96-well plate Spectramax reader was used. The UV absorbance at 350 nm was measured using a 100% PBS. The absorbance readings were compared against blank plate readings. Ranked and normalized with respect to sample pathlength.

[0316] HP-HIC : High-performance hydrophobic interaction chromatography (HP-HIC) was used to separate non-oxidized molecules. The oxidation products were evaluated. Preliminary analytical characterization revealed heavy chain Met105 oxidation on one heavy chain. The proportion of pre-peaks determined to be oxidized species, including The percentage of the peak was determined by diluting the sample to 5.0 mg / mL in purified water. The HP-HIC method was then carried out. The sample was then transferred to a Tosoh Phenyl-5PW 10 μL was injected into an HPLC equipped with a column and a UV detector at 280 nm. The analysis consisted of a mobile phase containing a gradient of the following components: Mobile phase A: 5 mM Sodium phosphate, pH 7.0; Mobile phase B: 400 mM sulfuric acid in 2% acetonitrile The buffer used was 5 mM ammonium phosphate, 5 mM sodium phosphate, pH 6.9.

[0317] VP-DSC :Valerian-Plotnikov Differential scanning calorimetry (VP-DSC) was used to determine the thermal stability and conformational stability of monoclonal antibodies. DSC is capable of determining the conformational stability of proteins. The time series for the heat capacity of the placebo solution with respect to the incremental temperature that causes the temperature transition during the injection Heat capacity of protein solution (C p In the case of monoclonal antibodies, multiple amphoteric The folding transitions (Tonset, Tm1, Tm2) are the unfolding transitions of the individual domains. This is typically seen in DSC thermograms corresponding to the dings.

[0318] Example 2 Evaluation of the stability of high-concentration pembrolizumab formulations To evaluate the stability of formulations containing high concentrations (160 mg / ml) of pembrolizumab Initial studies were conducted to evaluate the effect of various formulation excipients.

[0319] Pembrolizumab drug substance stock solution was diluted to 10 mM acetate by tangential flow filtration. (pH 5.0) (189 mg / mL) and 10 mM histidine (pH 5.5) (18 The protein stock solution was prepared in 0.7 mg / mL. By adding each buffer, the pembrolizumab formulation was diluted to a target concentration of 160 mg / mL. The test formulations containing high concentrations of pembrolizumab (see Table 4) were The excipients were added to the protein solution in a volume of 3 mL to 4 mL in a 6R glass vial. Add to achieve the target levels of each excipient and use acetate or histidine buffer. Formulation 2 was prepared at 125 mg / mL (surfactant-free) and mixed to the final volume. Millipore centricon device (10,000 The solution was concentrated to a concentration of >160 mg / mL using a 50% MWCO. A formulation matrix containing all ingredients except Ab and surfactants) with a concentration of 160 mM After adjusting the concentration to 100 µg / mL, PS80 was added. After preparing the sample, place it in a vial. Store at 2-8°C until filling. [Table 4]

[0320] The pembrolizumab formulation solution was placed in a Millex GV 0.22μm PVDF 33 Filter using a mm filter and place into a glass low volume HPLC vial (Waters #1 86000384c, 12 x 32mm Glass Screw Neck Vials, Per Vial The vials were capped and wrapped in parafilm to prevent evaporation. Samples were placed in environmental stability chambers at 2-8°C, 25°C, and 40°C. Each sample box was then placed in a stable They were double bagged before being placed in the qualitative chamber.

[0321] The stability of the formulation was evaluated by HP-SEC (to assess purity) and HP-IEX (to assess charge profile). The results were evaluated over a 12-week period using a 5°C storage (The term "5°C" is used throughout this specification and the examples.) The term "2 to 8°C" is used interchangeably with "2 to 8°C" to indicate 5°C + / - 3°C (standard deviation) over a 12 week period. Therefore, all formulations were considered stable at 5°C storage. Over the course of the study, HP-SEC (a small percentage of mAb) was measured for each formulation. Changes were observed with HP-IEX (slight decrease in the proportion of major substances) and HP-IEX (slight decrease in the proportion of major substances). Using either of these two techniques, the results for the five formulations at 25°C were: No differences were observed (data not shown). For all formulations, more significant changes were observed compared to the same formulations stored at 25°C. The smallest decrease in the percentage of mAb by HP-SEC after 12 weeks was observed in formulation 4 at 40°C. This was observed in comparison with other formulations stored at 40°C for 12 weeks. These results suggest that this formulation may have improved stability (data not shown). At any of these temperatures, there was no difference in the charge profiles among the five formulations. No differences were observed (i.e., data for each 4-week interval were similar among the five formulations). (data not shown).

[0322] In addition, the oxidation of methionine-105 (Met-105) was measured for each of the test preparations. The change in Met-105 oxidation was monitored by HP-HIC for 12 weeks. At 25° C., no changes were observed in any of the formulations (FIG. 1A). In all five formulations, the proportion of total prepeak 1+2 (one oxidized Met-10 per molecule) 5 or approximately 50% oxidized Met-105) was observed (Figure 1B ), which was more pronounced at 40°C (Figure 1C). The lowest amount of β-amyloides was observed in formulation 4 over 12 weeks, indicating that this formulation may have improved stability. This suggests that this may be the case.

[0323] Example 3 Evaluation of the stability of high-concentration pembrolizumab formulations containing arginine as a viscosity-reducing agent Further studies are required to evaluate the stability of formulations containing higher concentrations of pembrolizumab. (The amount used in Example 2 was 160 mg / mL, but in this example, 200 mg / mL was used.) In this study, arginine (3%, 2%) was used alone or or in combination with sucrose to evaluate their effect on the storage stability of pembrolizumab Ta.

[0324] For this study, pembrolizumab was concentrated by concentration and diafiltration. The concentrated drug substance was prepared at 234 mg / mL in 10 mM histidine (pH 5.4). Add excipient stock solutions and 10 mM histidine buffer (see Table 5) to the protein stock solution. By adding 200 ml of pembrolizumab formulations (#1 to #3), Prepared in a 1 L volumetric flask to a target concentration of 200 mg / mL. Each of the following was then filtered using a Stericup 250mL apparatus with a 0.22μm PVDF membrane. Filter and load into a 1 mL syringe (Hypak S with BD Hypak SCF stopper). CF 1mL) and 2mL vials (Nuova Ompi 0612090.56 57) with a drug fill volume of 1 mL. Samples were staged and protected from light. 12 months in an environmental stability chamber at 2-8°C and 9 months in a stability chamber at 25°C The test formulations for this study were placed in a stability chamber at 40°C for 1 month and 40°C for 5 months. It did not contain any antioxidants. [Table 5]

[0325] Visual observation, A350, HP-SEC (purity), HP-IEX (charge profile), H The formulations were evaluated by P-HIC (Met-105 oxidation) and CE-SDS. When stored for 12 months, whether stored in a vial or in a syringe, Visual observation, A350, HP-SEC, HP-IEX, HP-HIC and CE-SDS( No changes were observed between the different formulations depending on the NR. The agent was considered stable at a storage condition of 5°C.

[0326] Each of the test formulations was visually inspected for precipitate formation or color change (data not shown). All formulations showed some yellow coloration at 25°C by visual assessment. The formulations containing sucrose in the absence of arginine (formulations 1A and 1B) showed some improvement after 3 months. By 9 months, all samples showed some precipitation. Formulations containing 2% sucrose and 2% arginine were bioavailable. In some vials, a gel formed in the gel (formulation 3A) as early as one month. This was evident in the 3B formulation but not in the syringe (3B formulation). Rapid changes were observed in all formulations at 40°C over the entire time period. In the case of the syringe, the yellow color was observed after one month in the sucrose-only formulations (1A and 1B). Coloration was observed.

[0327] The A350 value was measured as an index of turbidity (data not shown). The samples containing sucrose (formulations 1A and 1B) and the vials were treated with sucrose and arginine. For the sample containing the combination (Formulation 3A), the trend in turbidity increase over the 6-month study period was The increase in A350 value of the sucrose samples at 6 months was due to the yellow coloration. The A350 of the sucrose-arginine sample correlated with the observations in the syringe. At 40°C, all the samples were found to have significantly higher oxidative stress than the samples containing the same combination. A more pronounced trend of increasing turbidity over time was observed for the sucrose-containing samples. The pulls (formulations 1A and 1B) turned amber in color, and therefore the A350 at 6 months was not measured. In addition, the sucrose-arginine formulation in the vial (Formulation 3A) Due to gel formation, the A350 was not measured at 6 months. Arginine-containing formulations (without sucrose, formulations 2A and 2B) in syringes and The A350 results for the intravenous sucrose-arginine formulation (formulation 3B) were comparable.

[0328] Purity of the formulation was determined by HP-SEC over a 9 month period (data not shown). In all five formulations, there was a slight decrease in the percentage of mAb (% mAb) and a corresponding A trend towards increasing proportion of HMW in the vials and syringes was observed over time. The sucrose-containing formulations (formulations 1A and 1B) showed the greatest change compared to the other formulations. The LMW percentage data showed significant variation at intermediate time points (3M, 6M) and were not conclusive. At 40°C, all formulations showed a decrease in the percentage of mAb and the corresponding A trend towards increased HMW and LMW percentages was observed over the nine-month period. However, no clear differences were observed between the different formulations.

[0329] The charge profile was determined by HP-IEX (data not shown). At 25°C, five In all formulations, there was a slight decrease in the proportion of major and total basic substances and a corresponding decrease in the proportion of acidic substances. A trend towards an increased proportion of patients with arginine-containing (formulations 2A and 2B) and sucrose + arginine-containing formulations (formulations 3A and 3B). The greatest change was observed in the sucrose-containing formulations in the al or syringe (formulations 1A and 1B). At 40°C, a decrease in the percentage of major and total bases was observed in all five formulations. A more pronounced trend for the increase in the proportion of acidic substances was observed after 3 months. No clear differences were observed between the formulations. No obvious differences in stability profiles were observed for the formulations.

[0330] Met-105 oxidation was measured by HP-HIC. At 5°C, no significant changes were observed for any of the formulations. Even with the addition of 100 mg / kg of Met-105, minimal changes in Met-105 oxidation were observed over the 9-month period (Figure 2A). At 25°C, there was a slight increase in the proportion of total prepeak 1+2 in all formulations in the vial. A tendency for the drug to increase in volume was observed, which was not observed in the corresponding formulation in a syringe (Figure 1). 2B). In the vial containing sucrose (without arginine), the arginine-containing formulation Over a 9-month period, the effect of sucrose on oxidation was significantly greater than that of sucrose (with or without sucrose). At 40°C, a higher increase was observed in the arginine-containing formulations (vials and syringes). ) and sucrose preparations in syringes showed a rapid increase in oxidation rate after 3 months. The other formulations showed no improvement after 3 months due to gel formation or amber coloring (Figure 2C). Not tested (see above).

[0331] Example 4 Evaluation of formulations containing high concentrations of pembrolizumab with alternative viscosity-reducing agents Examining the utility of alternative excipients as viscosity-reducing agents in high-concentration pembrolizumab formulations To confirm this, additional studies were performed using a microrecovery HPLC buffer with a loading volume of 400 μL. A series of formulations were prepared in vials (Table 6). The formulations in this study did not contain antioxidants. Samples were stored in stability chambers at 5°C, 25°C and 40°C for 12 weeks. The test formulations in this study did not contain antioxidants. [Table 6]

[0332] For formulations stored at 5°C for 12 weeks, HP-SEC analysis (proportion of mAb No change in purity was observed when the samples were stored at 25°C (measured as the total amount of the sample) (Figure 3A). Regarding the sample, a slight trend toward a decrease in the proportion of mAb was observed in all five formulations. It was observed that formulation 4 (150 mM camphorsulfonic acid) showed the greatest reduction ( The percentage of mAb also decreased for the formulations stored at 40°C (Figure 3B). A trend of 0.01 was observed, with no clear differences observed among the five formulations tested (Figure 3). C).

[0333] Similar to the results obtained by HP-SEC, at 5°C, as assessed by HP-IEX, No differences in charge profiles were observed among the five formulations over 12 weeks. Again, all five formulations showed a reduction in the percentage of the primary endpoint over 12 weeks. A trend was observed at 25°C and formulations 3 and 4 (150 mM camphorsulfonic acid and 1 The most significant decrease was measured at 40°C for all the tested compounds (50 mM guanidine HCL). For each of the five formulations, HP-IEX resulted in a more pronounced reduction in the proportion of major components. was observed over time, and no clear differences were observed among the five formulations (data not shown). .

[0334] After 12 weeks at 5°C, all five formulations showed no significant changes in solubility as measured by HP-HIC. Regarding the ratio of Met-105 oxidation (prepeak 1 + 2), no difference was observed. However, for formulation 4, the pre-peak (except for pre-peak 1 + 2) An increase in the proportion of pre-peaks was observed (data not shown). Pre-peaks other than 1+2 were both This includes the contribution of molecules in which the methionine residues are oxidized. A slight increase was observed for formulations 3 (2% histidine, 2% sucrose) and 4 (150 ml) at 25°C. M camphorsulfonic acid) (Figure 4B), and the peaks other than 1+2 were An increase in the proportion of repeaks was observed (data not shown). For formulations 1, 2 and 5, Minimal changes in the proportion of total prepeak 1+2 were detectable (Figure 4B). and formulation 2 showed a significant increase in the proportion of pre-peaks other than 1+2 (data not shown).

[0335] The percentage of total pre-peak 1+2 for all five formulations over 12 weeks at 40°C storage conditions A more significant increase was observed in the total pre-peak 1+2, again with formulations 3 and 4 showing (Fig. 4C) and in the proportion of prepeaks other than 1+2 (data not shown). Formulations 1, 2, and 5 showed minimal changes in pre-peak 1+2, but (Fig. 4C), a significant change in the proportion of prepeaks other than 1+2 was observed, which was consistent with both ) and oxidation of Met-105 residue (data not shown).

[0336] Example 5 Analysis of formulations containing arginine in combination with glutamine Previous studies have shown that an equimolar mixture of arginine and glutamine is a model for the development of biologics. These results suggest that the temperature-induced aggregation of monoclonal antibodies can be suppressed (Kheddo et al., Int.J.Pharmaceutics 473:126-33(2014)). Fu Kuda et al. (Pharm. Res. 31:992-1001 (2014)) reported that Arg- HCl can inhibit antibody aggregation at near-neutral pH, but does not inhibit aggregation at acidic pH or at high temperatures. In that study, the effect of Arg-HCl at high temperatures was shown to be The buffer was then added to the buffer to obtain an equimolar mixture of arginine and aspartic acid or glutamic acid. It is possible to synthesize these compounds, which resulted in inhibition of aggregation (Fukuda et al.).

[0337] Investigating the effect of an equimolar mixture of glutamine and arginine on pembrolizumab formulations To this end, a series of compositions were formulated and tested as described below. The lolizumab drug substance was concentrated and diafiltered to 10 mM histidine (p Stock solutions of the following excipients were prepared in Penbu Formulation C1 was prepared by adding lolizumab drug substance to obtain the target composition listed in Table 7. ~C6 was prepared: L-arginine hydrochloride (475 mM stock), L-glutamic acid (170 mM stock), polysorbate-80 (2% w / w stock) and Clause (40% w / v stock). The formulated drug substance batches were then packed into a 0.22 μm PVDF The solution was filtered using a syringe filter and filled into a 2 mL glass vial (filling amount: 0 0.5mL). Use a 13mm serum stopper to The vial was capped and sealed using a 13 mm flip-off seal. The cells were incubated at 40°C in a walk-in incubator with 75% relative humidity (RH). Vials were tested at T0, 1 week, 2 weeks, 4 weeks and 8 weeks.

[0338] The thermal degradation was tested by DSC (Table 7) and the viscosity of each formulation was measured (Table 7). It was shown that there was no improvement in the thermal denaturation behavior in the presence of an equimolar mixture of Arg and Glu. Compared with C1, which contains neither arginine nor glutamine, A decrease in viscosity was observed for all formulations containing 1:1 Glu (Table 7). The amount of aggregation in each of these formulations after storage at 12 weeks at ℃ was also measured by HP-SEC. The results showed that there was no reduction in aggregation in formulations containing an equimolar mixture of Arg:Glu. (Figure 5). [Table 7]

[0339] Example 6 Effect of antioxidants and chelating agents on stability in the presence of metal ions. Study 1: Metal ions are commonly used, for example, in the processing of monoclonal antibody products and associated buffers. The presence of metal ions can be introduced during the production of biological products from the steel tanks used. A series of tests were conducted to determine the effect of antioxidants and chelating agents on formulation stability under The experimental formulations were evaluated by HP-SEC (Table 8). Diafiltration yielded 252.3 ml in 10 mM histidine (pH 5.5). Stock solutions of the following excipients were added to the pembrolizumab drug substance to obtain Formulations D1-D5 were prepared by obtaining the target compositions listed in Table 8: Methionine (100 mM stock), ferrous chloride (0.0227% w / v stock), poly Sorbate-80 (2% w / w stock) and sucrose (40% w / v stock) ck).

[0340] The formulated drug substance batches were filtered using 0.22 μm PVDF syringe filters; Filled into a 2 mL glass vial (filling volume: 0.5 mL). 13 mm cell stopper Cap the vial using a cap and seal the vial using a 13 mm flip-off seal. The vials were then sealed. The vials were then incubated in a walk-in incubator at 40°C / 75% RH. Vials were tested at T0, 1 week, 2 weeks, 4 weeks and 8 weeks.

[0341] The base formulation tested in this study (Formulation 1) contained sucrose and Contains high concentration of pembrolizumab with PS80 (see Table 8, no. D1) On the other hand, formulation D3 contains a basic formulation plus metal ions together with an antioxidant (methionine), and formulation D5 contained a base formulation plus metal ions and a chelating agent (DTPA). [Table 8]

[0342] The results showed that methionine did not prevent aggregation in the presence of metal ions (Table 8, Preparation D3). Base formulation in the presence of metal ions without chelating agents / antioxidants (D4) ) or a formulation containing methionine in the presence of metal ions (formulation D3). The formulation containing DTPA in the presence of ions (formulation D5) showed lower agglutination after 8 weeks. The percentage of aggregates observed.

[0343] The formulations were also evaluated by HP-HIC at 40°C for 8 weeks to determine the presence of metal ions. The effects of methionine and DTPA on oxidation in the presence of DMSO were determined (data not shown). Compared to Fe 2+ In the presence of DTPA and methionine in the formulations , a slight decrease in oxidation was observed.

[0344] Study 2: Stability in the presence of metal ions at 40°C and additional antioxidants and antioxidants against oxidation The effect of rate agents (formulations E1 to E9, Table 9) was evaluated by HP-SEC and HP-HIC. did. [Table 9]

[0345] HP-SEC results (see Table 9) show that 5 mM and 10 mM methionine The formulations containing EDTA (formulations E1 and E2) showed a low increase in aggregates in the absence of metal ions. The results show that the formulations containing methionine and DTPA (formulations E3 and E7) ) showed a smaller increase in aggregation levels in the presence of metal ions compared to EDTA (formulation E6). The greatest increase in aggregates was observed for the formulation containing glutathione in the presence of metal ions (formulation E9).

[0346] The oxidation of the formulations at 40° C. was also examined by HP-HIC over an 8 week period (see FIG. 6). In the formulations containing 5 mM and 10 mM methionine (formulations E1 and E2), Regarding HP-100, the reduction in oxidation levels over the 8-week period was greater than that of the other formulations tested. This was observed by HIC analysis. Formulations containing TPA (formulation E6) showed low levels of cysteine ​​in the presence of metal ions over an 8-week period. The results also showed that DTPA was more effective than EDTA in preventing oxidation over time. Sodium citrate and glutathione were shown to be effective in preventing the formation of methionine, DTPA, and Alternatively, it appeared to be less effective than EDTA at inhibiting oxidation in the presence of metal ions.

[0347] Study 3: To further optimize excipient levels, the effects of antioxidants and chelating agents were examined separately or in combination. Additional studies were conducted to evaluate combinations (with or without metal ions) After formulation, samples were stored at 40°C for 8 weeks and then incubated at T=0, 4 weeks, and 8 weeks for HP-S Evaluated by EC (see Table 10). [Table 10]

[0348] In this study, the presence of arginine and glutamic acid (formulations H10 and H11 ) appeared to affect the aggregation rate.

[0349] The oxidation of the samples at 40°C was also measured over a period of 8 weeks by HP-HIC. Results showed that the presence of 10 mM methionine significantly reduced aggregation over an 8-week period compared to other formulations. This shows that the result was a decrease in speed (see FIG. 7).

[0350] Overall, studies 1-3 demonstrated that the presence of L-methionine in pembrolizumab formulations significantly increased the rate of oxidation. The results showed that methionine, DTPA, and EDTA could reduce the oxidative stress in a concentration-dependent manner. No additional benefit was observed.

[0351] Example 7 Viscosity of high-concentration anti-PD-1 antibody formulations Study 1 - Viscosity as a function of protein concentration Study 1A: In one experiment, unformulated pembroke lysate was measured as a function of protein concentration. The viscosity of pembrolizumab (pembrolizumab in 10 mM histidine buffer) was measured. Ultrafiltration / dialysis of pembrolizumab in 0 mM histidine buffer (pH 5.4) Samples were prepared by filtration. Concentration values ​​were measured by SoloVPE. The viscosity was measured using an MVROC device and the values ​​are shown in Table 11 below. [Table 11]

[0352] Study 1B: In another experiment, 10 mM histidine in the presence of 3% (w / v) arginine was The effect of pembrolizumab on the solubility of pembrolizumab formulated in ethanol (pH 5.5) as a function of pembrolizumab concentration. The viscosity was also measured using 10 mM histidine in the presence of 3% (w / v) arginine (pH 5.4) Ultrafiltration of pembrolizumab in buffer - Diafiltration was used to obtain the samples for this experiment. Samples were collected at various stages of the UV / DF process to determine the concentration and viscosity. The concentration values ​​were measured by SoloVPE and the viscosity values ​​were measured using the MVROC device. The values ​​are shown in Table 12. [Table 12]

[0353] Study 1C: In another experiment, the viscosity of formulated pembrolizumab samples was measured. 7% (w / v) sucrose, 0.02% in mM histidine buffer (pH 5.5) (w / v) Polysorbate 80, 10 mM methionine, 100-200 mg / ml Pembrolizumab formulations were prepared at various concentrations in the range of mL. The solutions were prepared by mixing: (1) 10 mM histidine (pH 5.5) buffer; (2) 236 mg / mL pembrolizumab drug substance in water; (3) 49% (w / w) sucrose , 0.14% (w / w) polysorbate 80, 10 mM histidine (pH 5.4) 85 mM methionine; and (3) 10 mM histidine (pH 5.5). Viscosity measurements were taken using a viscometer (see Table 13). [Table 13]

[0354] Study 2: Effect of different excipients on the viscosity of high-concentration pembrolizumab solutions In this study, various excipients were prepared in 10 mM histidine (pH 5.5) buffer. A stock solution of the agent was prepared. Lizumab drug substance samples were spiked with these excipient stocks to achieve the target excipient concentrations. Pembrolizumab concentration was measured by oloVPE and viscosity was measured using the MVROC device. (Table 14). Sample pH was also measured. [Table 14]

[0355] Study 3: Effects of arginine, histidine, and methionine on the viscosity of pembrolizumab solutions Effect of concentration of In this study, the effect of arginine, histidine, and erythritol on the viscosity of pembrolizumab solutions was The effects of sodium acetate and methionine concentrations were investigated. 10 mM histidine (pH 5. 4) Pembrolizumab drug substance (>200 mg / mL) in Mix with various amounts of stock solutions of methionine to obtain 200 mg / mL or 167 mg / mL Various concentrations of excipients in solutions containing the target concentration of pembrolizumab in mL were obtained. Results are summarized in Table 15. [Table 15]

[0356] Data from the study were obtained using penmb12 in 10 mM histidine buffer (pH 5.5). We show that the viscosity of lolizumab increases exponentially with increasing protein concentration. (Table 11). The data show that the presence of 3% (w / v) arginine significantly reduces the solubility of 10 mM histidine (p H5.5) It has also been shown to reduce the viscosity of pembrolizumab in solution. An exponential increase in viscosity with increasing mbrolizumab concentration is still observed (Table 12). 7% (w / v) sucrose at protein concentrations ranging from 100 to 200 mg / mL; 0.02% polysorbate 80, 10 mM methionine with 10 mM histidine ( The viscosity values ​​of pembrolizumab formulated in 10 mM (pH 5.5) were The viscosity of the formulated pembrolizumab was similar to the corresponding value measured in buffer alone. The intensity of the drug significantly increases at concentrations of approximately 150 mg / mL or higher (Table 13).

[0357] Different excipients affected the viscosity of high-concentration pembrolizumab solutions to different degrees (Table 14) The excipients that had the greatest effect on pembrolizumab viscosity were L-arginine, L -histidine, guanidine hydrochloride, DMSO and camphorsulfonic acid (Table 1 4) Table 15 also shows that L-arginine and L-hysitidine have an effect on the viscosity of pembrolizumab solution. The addition of up to 40 mM L-methionine reduced the pentaerythrocyte concentration in a concentration-dependent manner. This indicates that it does not reduce the viscosity of brolizumab.

[0358] Example 8 Long-term stability of high-concentration anti-PD-1 antibody formulation Determine the long-term stability of high-concentration pembrolizumab formulations in the presence and absence of methionine This study examined the effect of acid resistance on the stability of pembrolizumab. The effect of the concentration and presence of activating agents (e.g., methionine) was investigated. Stock solutions of excipients were The formulations were prepared and added to the pembrolizumab drug substance to obtain the final composition shown in Table 16. 5℃+ / -3℃, 25℃+ / -3℃ / 60%±5% relative humidity and 40℃+ / -2℃ / Formulations K2 and K3 were staged in an environmental stability chamber at 75% ± 5% relative humidity. 3 was staged with limited stability compared to formulations K1 and K4. The planning period is 36 months, with 25°C ending in 6 months and 40°C ending in 3 months. Results over a period of up to 12 months are shown below. [Table 16]

[0359] Visual inspection of all test formulations revealed that they exhibited no visible changes over a 12-month period at 5°C. The formulations were found to be substantially free of viscous particles. It was also evaluated by HP-IEX, with the results shown in Tables 17-19 and summarized below.

[0360] pH: All formulations had the same initial pH value of 5.7. No change in pH was observed for any of the formulations.

[0361] Potency by binding ELISA: Potency by binding ELISA was observed for all formulations, regardless of storage time and conditions. No changes in efficacy were observed. All potency values ​​were within the acceptance criteria of 60-140% of the reference value. (For samples stored at 5°C, values ​​ranged from 85 to 10 over a 12-month storage period.) 6; data not shown).

[0362] Reduced and non-reduced CE-SDS: Purity by CE-SDS was measured under reducing and non-reducing conditions. Purity (%) or percent intact IgG (%) as a function of time at 5°C for up to 12 months At 5°C, no measurable changes were observed in the reduced CE-SDS (heavy and Purity (%) based on the nucleotide and light chain (LC) of formulations K1 and K4 exceeded 96.5% over 12 months. For formulations K2 and K3, the percentage was 97.0% or higher over the same period. At 40°C, a slight decrease in purity was observed for all formulations. After 1 month, the expected decrease in purity was observed. ) at 5°C for formulations K1 and K2 over a 12-month period at the time of testing. For K2, it is 98.2 or more, for formulation K2, it is 97.1 or more, and for formulation K4, it is 97.1 or more. All results were consistent with those of both reduced and non-reduced CE-SDS. Regarding the above, the clinical acceptability criteria were met in more than 90.0% of cases.

[0363] Purity by HP-SEC: Under the recommended storage conditions of 5°C, initial levels of serotonin were reduced by 12 months for both formulations. No measurable change in the proportion of HMW from the cellulose was observed. Over the course of the experiment, an increase in the proportion of HMW and a corresponding decrease in the proportion of monomeric monomers were observed. No LMW species were detected in any of the formulations or storage conditions. Over a 3-month period, the proportion of HMW increased for the various formulations, with a corresponding increase in the proportion of monomeric For formulations K1 to K3, an increase in HMW was observed with increasing concentration. At 40°C, LMW species were observed at 3 months for all formulations.

[0364] Oxidation by HP-HIC: By monitoring pre-peak 1+2 by HP-HIC, the methionine 105 The oxidation of formulations K1 to K4 did not change over 12 months at 5°C. At 25°C, the levels of prepeak 1+2 increased slightly over 6 months, whereas At 25°C and 40°C, the increase was more evident over the course of three months. The oxidative changes were more pronounced for agent K4, which does not contain L-methionine. As the only formulation, these results suggest that the inclusion of L-methionine significantly reduced the rate of oxidation. This shows that it has brought about

[0365] Charge non-uniformity by HP-IEX: Charge heterogeneity was observed by monitoring the main peak along with various acidic and basic species. At 5°C, the individual peaks including the main peak were not observed for all formulations up to 12 months. No measurable changes were found in any of the peaks at 25°C over 6 months. The main peak decreased over the course of the acidic species (acidic variant, acidic 1 and pre-main peak). and basic mutant peaks were all increased. At 40°C, the main peak increased more rapidly over the course of 3 months. As in the case of 25°C, the acidic mutant, acidic 1, premature and basic mutants showed a significant decrease. An increase was observed for the isomeric peak, while a decrease was observed for the basic 1 and basic 2 peaks. It was recognized.

[0366] Turbidity: Turbidity of pharmaceutical batches with respect to stability is assessed by measuring the optical density at 350 nm. No measurable change in turbidity was observed at 5°C for any of the formulations. The samples stored at 25°C showed a slight increase, while the samples stored at 40°C showed a turbidity increase. The increase in turbidity at 25°C and 40°C was a strong indicator of stability. This is consistent with the increase in high molecular weight species observed with

[0367] Overall, based on the 12-month stability data, formulations K1-K4 are stable for up to 12 months. Stable at 5°C for 30 min with no measurable changes in product quality attributes Some decomposition was observed during monitoring of properties for 6 months at 25°C and 3 months at 40°C. The stability of the product at 5°C will be further evaluated over a 36 month period. [Table 17] TIFF2024102215000020.tif36145 [Table 18] [Table 19]

[0368] Examples 9 to 13: Examples (9-13) show that the antibody and excipients are present in amounts less than intended in the final formulation. However, these preparations are not intended to be The pembrolizumab / stabilizer and pembrolizumab / surfactant molar ratios of the formulation were maintained. Antioxidants, buffers and metal chelators concentrations were tested at final target concentrations. Pembrolizumab (5 mg / mL), PS80 (0.0016%), and stabilizers (e.g. For example, the concentration of sucrose was reduced by a factor of five to produce a more concentrated final target formulation. For example, formulation 1A in Table 20 is a 25 mg / mL penicillin solution. The same molar ratios were used as in the formulation containing brolizumab, 7% sucrose, and 0.02% PS80. The formulations disclosed in Examples 9-12 are representative of contemplated final formulations, The results disclosed suggest how the final, higher concentration formulations may behave. Some of the parameters considered (e.g., aggregation) are expected to be related to intermolecular interactions. It is understood that the reduced potential for action may be affected by the reduced concentration. To complement this, more aggressive stabilization regimens ( The study was continued at 50°C for 10 days.

[0369] Antioxidants (e.g., methionine), buffers (e.g., histidine) and metal ions The concentrations of doping agents (e.g., DTPA and EDTA) are not reduced as much as the other excipients. Methionine was converted to Met- by removing oxidizing agents (e.g., dissolved oxygen). 105 and thus is a functional excipient used to reduce the oxidation of pembryoly maintains the chemical stability of izumab. Similarly, DTPA prevents undesired proteolysis. It is a functional excipient used to complex metal ion impurities that may cause It is more difficult to maintain chemical stability at low pembrolizumab concentrations, so methionine The concentrations of nin and DTPA were kept constant at 10 mM and 20 μM, respectively. If methionine is effective in low-concentration formulations, it will most likely be effective in higher-concentration formulations. It is expected that the addition of 10 mM L-histidine and / or Alternatively, L-histidine hydrochloride is expected to maintain buffering capacity at the intended pH of the test formulation. can be.

[0370] Example 9 Evaluation of the stability of low-concentration pembrolizumab formulations combined with methionine To evaluate the stability of formulations containing low concentrations (5 mg / ml) of pembrolizumab, Initial formulation studies were conducted to evaluate the effect of various formulation excipients. Examples 9-12 In all studies, the arginine used was L-arginine or L-arginine HCl. where the glycine used was glycine and the proline used was L-proline.

[0371] Pembrolizumab bulk in 10 mM histidine (pH 5.5) (41.2 mg / mL) The drug was mixed with a polysorbate 80 (PS80) solution (approximately 0.36 mg / mL) and then Protein in 10 mM histidine, pH 5.5, adjusted to final volume with buffer. A stock solution of protein (20 mg / mL) / PS80 (0.16 mg / mL) was obtained. Add excipient stock solutions and their respective buffers to the PS80 stock solution. The pembrolizumab formulation was prepared to a target concentration of 5 mg / mL. All stock solutions were diluted with MillaporeExpress® PLUS® prior to use. Filtered through a Stericup® 0.22 μm PES filter. The test formulations containing pembrolizumab were prepared in a volume of 1 mL in a 96-well plate. The excipients were added to the protein / PS80 stock solution to achieve the target levels of each excipient. The solution was brought to the final volume with cysteine ​​buffer (pH 5.5) (see Table 20). (2-hydroxypropyl)-β-cyclodextrin [CAVITRON (trademark)] and (sold under the name sulfobutyl ether)-β-cyclodextrin [C APTISOL®] are HPBC and S, respectively. The well plate is sealed with a 96-well silicone sealing mat. It was covered and then vacuum sealed (twice) in a moisture barrier bag to minimize the possibility of evaporation. The samples were incubated at 2-8°C (the term "5°C" as used throughout this specification and the examples means 5°C ± 3°C). (used interchangeably with "2-8°C" to indicate the standard deviation) and 50°C environmental stability chart The patients were classified into stages according to the member. [Table 20]

[0372] Each of the test formulations was visually inspected for precipitate formation or color change (data not shown). After a 10-day stability period, the turbidity (A350) and UP-SEC (purity) were evaluated. (to determine charge profile) and HP-IEX (to evaluate the stability of the formulation) Formulations 9A and 9B containing SBEC were turbid upon removal from the stability chamber. Conversely, formulations 6A and 6B containing HPBC showed no visible signs of turbidity. Formulations 9A and 9B showed no symptoms, only turbidity, as shown in Table 21. and was not further tested by UP-SEC and HP-IEX. P-SEC results showed that for formulations stored at 5°C for 10 days, arginine No changes were observed except for the formulations containing methionine (with or without methionine). In the above-mentioned arginine-containing preparation, UP-SEC was used to A slight decrease (<0.5%) in the percentage of Ab was observed. All other formulations were stored at 5°C. At 50°C, the UP-SEC (reduction in the percentage of mAb) and HP-SEC were considered stable. IEX (a decrease in the proportion of primary products and a significant increase in the proportion of pre-primary products; data not shown) More significant changes were observed over the study period for each of the formulations. Remarkably, formulations 7A and 7B at 50°C showed no significant changes after 10 days of storage at 50°C. The greatest reduction in the percentage of mAb (relative to the percentage of aggregates) after 10 days was observed compared to other formulations. A corresponding increase in the stability of these formulations was observed by UP-SEC. Turbidity for formulations 7A and 7B after 10 days at 50°C The (A350) results support the UP-SEC results and indicate that the stability of formulations 7A and 7B This shows a decline in

[0373] Cyclodextrins maximize both conformational and colloidal stability. It has been shown in the literature that the use of cyclohexane stabilizes protein formulations. Cyclodextrins have been studied primarily with IgG1 proteins. However, the mechanism by which this stabilization occurs has been the subject of much debate. As shown, the turbidity and UP-SEC measurements of formulations 6A and 6B (containing HPBC) It showed comparable stability to other stabilizers tested at 50°C for 10 days. Unlike other cyclodextrin formulations tested (9A and 9B; including SBEC), , showed high turbidity after 10 days at 50°C.

[0374] During the 10-day test period, at 50°C, no changes were observed in any of the formulations (not tested). No obvious differences in charge profiles were observed. (Data not shown). The addition of methionine (10 mM) to the test formulation did not affect the overall had little effect on preventing aggregation across the body. [Table 21]

[0375] Example 10 Analysis of low-concentration pembrolizumab formulations containing methionine in combination with a metal chelator Effects of metal chelators (DTPA and EDTA) on low-concentration pembrolizumab formulations To investigate the effect of acetaminophen on the efficacy and safety of acetaminophen in treating rheumatoid arthritis, a series of compositions were formulated and tested, as described below. Pembrolizumab (20 mg / mL) / PS8 in 10 mM histidine (pH 5.5) 0 (0.16 mg / mL) stock solution was prepared as described in Example 9 above. The following excipients were added: sucrose (5% w / v), mannitol (5% w / v), trehalose (5% w / v), sorbitol (5% w / v), glycine (5 % w / v), HPBC (5% w / v), arginine (5% w / v), proline (5 % w / v), methionine (2% w / v), DTPA (0.01% w / v) and E A stock solution of DTA (0.01% w / v) was added to the pembrolizumab / PS80 stock solution. Add to the solution to achieve the target composition (histidine (pH 5.5) buffer) listed in Table 22. Preparations 1 to 8 (C to F) were mixed in a 96-well plate. All stock solutions used in the formulations were diluted in MillaporeExplorer prior to use. ss®PLUS Stericup® 0.22μm PES Filter The well plate was covered with a 96-well silicone sealing mat and then sealed with a The samples were vacuum sealed (twice) in wet bags to minimize the possibility of evaporation. The cultures were staged in an environmental stability chamber at 37 °C for 10 days. [Table 22]

[0376] Each of the test formulations was evaluated for precipitation or color change over a 10-day test period. The formulations containing SBEC were visually inspected (data not shown). Similar to the results of Example 9, Upon removal from the stability chamber after 10 days at 50°C, turbidity was visible. and turbidity measurements (data not shown) confirmed the stability of these formulations compared to other formulations tested. As a result, formulations 9C-9F were not subjected to further testing. For the formulations, the results of turbidity and UP-SEC measurements correspond to the results listed in Table 21. The results were very similar to those of the preparations containing arginine (7C-7F). The addition of metal chelating agents has little or no effect on stability over 10 days at 50°C This suggests that the arginine-containing formulations (7C to 7F) ) represents the maximum change in percentage of mAb (monomer) and the highest turbidity value over the stability period. Formulations 7C–7F showed very clear differences in the proportion of mAb. However, the UP-SEC results for 7D and 7F showed that methionine was used to inhibit aggregation. These results show the additional benefit of incorporating metal chelators in combination with methionine and EDTA. The combination of methionine and DTPA (7F) had a slightly higher mAb activity than the combination of methionine and DTPA (7D). During the 10-day test period at 50°C, the No obvious differences in the charge profiles were observed for all three samples (except for 7C–7F, which were not tested). (data not shown). [Table 23] TIFF2024102215000027.tif88148

[0377] Example 11 Evaluation of the effect of pH on the stability of low-concentration pembrolizumab formulations combined with methionine Price Further studies will be conducted to evaluate the stability of the formulation in histidine buffers at various pH values. In this study, histidine buffers with pH values ​​of 4.5, 6.0, and 6.4 were used. We evaluated pharmacokinetics and its effect on pembrolizumab stability.

[0378] For this study, pembrolizumab drug formulation was plated at 5 mg / mL in a 96-well plate. The pH was adjusted to the target pH with dilute HCl or NaOH. Embrolizumab / PS80 stock solution (pH 5.5) to 4.5, 6.0 and 6.4 Pembrolizumab (20 mg / mL) / PS80 ( 0.16 mg / mL) stock solutions were prepared. Excipient stock solutions were similarly prepared to the target p H and MillaporeExpress® PLUS Ste The mixture was filtered through a 0.22 μm PES filter. The solution was mixed with pembrolizumab (20 mg / mL) / PS80 (0.16 mg / mL) stock solution. The following sucrose (5% w / v) was added to the sucrose solution to achieve the target concentrations listed in Table 24: v), mannitol (5% w / v), trehalose (5% w / v), sorbitol ( 5% w / v), glycine (5% w / v), HPBC (5% w / v), arginine ( 5% w / v), proline (5% w / v) and methionine (2% w / v). The plate was covered with a 96-well silicone sealing mat and then vacuum sealed (2 The samples were then stored in 5°C and 50°C environmental stability chambers. The stages were classified over a 10-day period. [Table 24]

[0379] The formulations were visually observed, and analyzed for turbidity (A350), UP-SEC (purity) and HP-IEX (charge After a test period of 10 days at 50°C, the pH was 4.5 (7G and At 7H and 8H, the formulations containing arginine were visibly cloudy. All other formulations showed no visible signs of aggregation. Turbidity (A350) values ​​were measured after 10 days. The results are shown in Table 25. The measured turbidity value of the pH 6.0 formulation was higher than that of the pH 4.5 and 6. 4, which indicates improved stability at pH 6.0. A tendency for increased turbidity was observed in all formulations at pH 6.4. The purity of the formulations was tested by UP-SEC after 10 days. All formulations at pH 4.5 had mA b) and a corresponding increase in the proportion of aggregates (HMW). For all formulations tested at various pH values, the addition of methionine to the formulations was There was a reduction in aggregation after 10 days, with the greatest change observed in all formulations at pH 4.5. Surprisingly, formulations 5G and 5H were significantly superior to the baseline formulation and showed stable The minimum rate of change in the percentage of mAb (and the subsequent minimum increase in the percentage of aggregates) over the lifespan This indicates that these formulations are superior to other formulations at pH 4.5. This suggests a relatively enhanced stability of

[0380] The charge profile of the formulation after 10 days at 50°C was determined by HP-IEX (data not shown). All formulations at each pH showed similar charge profiles for each stabilizer tested. The pH 4.5 formulation showed the lowest acidic peak percentage and peak profile after the stability period. The proportion of the major peak was significantly increased (>30%) by the proportion of the corresponding basic variant. The charge profile of the formulation at pH 6.0 was similar to that of Example 9 (histidine pH 5.5). The pH 6.4 formulation had an opposite charge profile to the pH 4.5 formulation. where a significant change (>30%) in the proportion of acidic variants was observed in the proportion of the pre-major peak. The percentage of the main peak in all formulations was observed with only a slight increase in pH. Regardless of the type, the effects were very similar in all formulations (41-43%). Addition of 100 mM NaCl did not result in any significant change in the charge profile. [Table 25] TIFF2024102215000030.tif184154

[0381] Example 12 Evaluation of pembrolizumab formulations containing different concentrations of methionine and DTPA Previous studies have shown that the addition of methionine (an antioxidant) and metal chelators reduces aggregation. (See Example 10.) To investigate the effect of antioxidant and metal chelator concentrations on inhibition, A series of compositions were formulated and tested as described in. Histidine (pH 5.5) buffer. Pembrolizumab (20 mg / mL) / PS80 (0.16 mg / mL) stock solution in The sucrose solution was prepared as previously described in Example 9. , mannitol, trehalose, sorbitol, glycine, HPBC, arginine and A stock solution of proline (20 mg / mL) was prepared using pembro / PS80 strain. Methionine was used as an antioxidant, and DTPA was used as a metal chelator. As mentioned above, all stock solutions used in the formulations were diluted in a Mill before use. aporeExpress® PLUS Stericup® 0.2 The solution was filtered through a 2 μm PES filter. Methionine (20 mg / mL) and DTPA ( A stock solution of 0.1 mg / mL of pembro / PS80 / Add stabilizer solution and then add histidine (pH 5.5) buffer to a final volume of 1 mL. The final formulations with the compositions listed in Table 26 were prepared by adding the following: Cover with a 96-well silicone sealing mat, then vacuum seal (twice) in a moisture-proof bag. The possibility of evaporation was minimized. Samples were incubated for 10 min in 5°C and 50°C environmental stability chambers. The stages were classified over the 3-day test period. [Table 26] TIFF2024102215000032.tif19168

[0382] The formulation was visually observed, and turbidity (A350), UP-SEC (purity), and HP-IEX (charge profile) were analyzed. After a 10-day test period at 50°C, all formulations showed No visible turbidity or discoloration was observed in any of the formulations (Arginine formulation 7N The turbidity values ​​for the various formulations tested (except for 100% ethanol) were very similar among the various formulations tested and are shown in Example 9. The turbidity data showed that the methionine concentration at various concentrations was very similar to that reported in the literature (data not shown). The effect of adding dimethylformamide alone or in combination with various concentrations of DPTA was unclear. Similar to the results shown in Examples 9 and 10, the formulations containing arginine showed the greatest increase in the proportion of HMW species over the stability study period (Table 27), whereas formulation 7 Q and 7T (15 mM methionine) showed a slightly lower proportion of HMW species. These formulations may have greater stability than other formulations with lower methionine concentrations. A similar trend was observed for all other formulations, in this case at 15 mM The lowest proportion of HMW species was observed in formulations containing methionine and at least 10 μM DTPA. Although some agreement was observed, the differences were very small.

[0383] Over a 10-day test period at 50°C, no significant changes were observed in any of the formulations (not tested). No obvious differences in charge profiles were observed for all the 1000-1500 carbon nanotubes (except for 7C–7F). , the percentage of the main peak for all formulations compared to the formulations stored at 5°C for 10 days. A significant decrease (from about 53% to about 41%) and the proportion of acidic variants and pre-major peaks An increase in the proportion of [Table 27] TIFF2024102215000034.tif227149

[0384] The effect of methionine and DTPA concentrations on stability and oxidation after 10 days at 50°C. To evaluate the efficacy and safety of selected formulations, HP-HIC analysis was performed on selected formulations shown in Table 27. Results for formulations 1N, 1Q, and 1T showed that the presence of 15 mM methionine improved the stability period. Overall, the results shown in Table 28 indicate that the results of the study resulted in minimal changes in oxidation over the course of the study. The results showed that the addition of methionine to the formulation reduced the oxidation rate in a concentration-dependent manner. The addition of DTPA as a chelating agent (formulations 1X to 1Z) In this case, even when combined with the addition of methionine (formulations 1N-1T), additional No benefit was observed. [Table 28]

[0385] Example 13 Evaluation of the effect of pH on the stability of low-concentration pembrolizumab formulations combined with methionine Price To evaluate the effect of histidine buffer on the stability of pembrolizumab formulations To do so, the formulation shown in Example 9 was diluted with 10 mM It was prepared in water for injection (no pH adjustment) rather than histidine (pH 5.5). Pembrolizumab drug substance (approximately 45 mg / m Penbuxin (1.0 L) was dialyzed against water for injection (WFI) to a final concentration of 15.5 mg / mL. PS80 was added to the lolizumab solution (15.5 mg / mL), and then the SteriFlip Pembrolizumab (registered trademark) was purified by filtration through a 0.22 μm PVE filter unit. Prepare lizumab (15.5 mg / mL) / PS80 (0.16 mg / mL) stock solution Excipient stock solutions (prepared in WFI and filtered through a 0.22 μm PVE filter) The pembrolizumab / PS80 stock solution was added to obtain the 100% 150% ... The test formulations were prepared by obtaining the following concentrations: Additionally, 10 mM methionine was added to assess its ability to reduce aggregation over the stability study period. The formulations were supplemented with either 20 μM DTPA or 50 μM EDTA. The drugs were prepared in 6-well plates, where the final drug volume was 1 mL. The package was sealed with a silicone seal mat and then vacuum sealed in a moisture-proof bag (twice). The formulations were staged at 5°C and 50°C for 10 days. [Table 29]

[0386] The prepared formulations were analyzed by appearance, differential scanning fluorescence analysis (thermal denaturation), turbidity, UP-SEC (purity) and Both formulations were tested by HP-IEX (charge profile). The measured turbidity values ​​were 0.01 to 0.01 mm, and the samples were not discolored or turbid when removed from the container. The results were very similar across all formulations tested. Regarding arginine formulations (7A to 7F, Examples 1 and 2) prepared in a buffer In contrast to the turbidity measurements, the formulations prepared in WFI (7A'-7F') showed good stability. After the test period, significantly lower turbidity values ​​were observed (data not shown).

[0387] The formulations prepared in WFI were compared to those prepared in Examples 9 and 10. Similar preparations showed modest improvement in thermal denaturation behavior upon shifting from Tm1 to higher temperatures ( Table 30. Exceptions to this statement are mannitol formulations 2E' (EDTA) and 2F' (Met + EDTA), which are similar to the formulations prepared in Examples 9 and 10. The Tm1 values ​​were shown (data not shown). Formulations 6A' to 6F' did not show any temperature transition (thermal transition). This is probably due to the fact that the SYPRO™ dyes preferentially access the cyclodextrin pocket. This may be due to prior binding of metal chelators to the cytoplasm. The presence of EDTA (50 μM) alone as a phosphatase inhibitor did not result in Met, Met+DTPA, DTPA or resulted in lower Tm1 values ​​than the corresponding formulation containing Met+EDTA.

[0388] To evaluate the self-buffering capacity of pembrolizumab formulations, we stably monitored the pH change of unbuffered formulations. The efficacy was measured over the study period (see Table 30). DTA) showed minimal change in pH over 10 days at 50°C. Regarding HPBC formulations (6A'-6F'), formulation 6B using methionine In the arginine formulations (7A'-7F'), minimal changes in pH were observed. The smallest change in pH observed with methionine was observed with formulation 7A' (containing neither methionine nor a metal chelator). This was observed in [Table 30] TIFF2024102215000038.tif179163

[0389] The amount of aggregation for each formulation over a 10-day period at 50°C was also measured by UP-SEC. The percentage of mAb and aggregates (HMW species) for all formulations were compared for formulations 7A'-7F'. They are very similar except for the arginine formulation, which shows an increased proportion of HMW species (approximately 4 %. UP-SEC data showed that methionine (10 mM), DTPA (20 μM), The addition of 50 μM EDTA or a combination of these significantly reduced aggregation. No clear evidence was found.

[0390] The charge profile of the formulation after 10 days at 50°C was determined by HP-IEX (data not shown). All formulations showed similar charge profiles with respect to each stabilizer tested. Compared with the formulations stored at 5°C for 10 days, the percentage of the main peak was significantly higher for all formulations. A significant decrease (from about 53% to about 41%) and the proportion of acidic variants and the pre-major peak An increase in the percentage of glycerol was observed (data not shown). After 1 day, the percentage of pre-major peak and base were comparable to the similar formulations in Examples 9 and 10. The proportion of the acidic peaks was higher (about 28%) along with the proportion of the methyl variants. Addition of onion, DTPA, EDTA or their combinations did not significantly alter the charge profile. It did not bring about any transformation.

[0391] Example 14 Development of a highly concentrated lyophilized pharmaceutical formulation containing pembrolizumab The experimental design and a summary of the results from all formulation batches are shown in Table 32. All formulations were Histidine buffer (pH 5.5) and polysorbate 80 (PS 80) were used. Various concentrations of pembrolizumab and excipients were used together with various cryoprotectants. Various test formulations were manufactured using the following pembrolizumab drug substance batches: The test formulations for the research study were formulated using: 204mg / mL pembro in 10mM histidine buffer (pH 5.5) Lizumab; 2 in 3% arginine HCl, 10 mM histidine buffer (pH 5.5) 88 mg / mL pembrolizumab.

[0392] The freeze-drying process used in the study was the model LYOSTAR 3, described in Table 31. (SP Scientific).

[0393] Batch 0021 : 103 mg / mL pembrolizumab formulated in sucrose Batch 0021 was produced from the above. 2.30 mL volumes were placed in 2R vials, 6R vials and The samples were filled into 10R vials. A 55 hour freeze-dry cycle was used ("lyo"). (See "Cycle A"; Table 31). After lyophilization, residual moisture, injectable volume and swelling were Reconstitution volumes were measured. Generally, reconstitution times ranging from 17 to 28 minutes were observed.

[0394] Batch 0022 : Sucrose, mannitol and arginine hydrochloride (Arg.HCl ) from 48 to 103 mg / mL pembrolizumab formulated in various combinations. Batch 0022 was manufactured. Volumes of 2.30 to 4.97 mL were placed in 2R vials and 6R vials. The primary antibody was filled in 10R vials, 10R vials and 10R vials. Therefore, the freeze-drying cycle used was similar to freeze-drying cycle A. The freeze cycle was extended to 60 hours ("Freeze Cycle B"; see Table 31). Reconstitution times ranged from 4 to 23 min. Residual moisture was 0.24-0.26%.

[0395] Batch 0024 : Various combinations of sucrose and arginine hydrochloride (Arg.HCl) Batch 0024 from 104-200 mg / mL pembrolizumab formulated in The drug substance used was 288 mg of 3% Arg, 10 mM His (pH 5.5). The volume was 1.24 to 2.30 mL in 2R vials. One shelf was filled with 7% sucrose vials for the freeze-drying process. The secondary drying time was reduced from 8 hours to 6 hours. The freeze-drying cycle had a secondary drying time that was 2 hours shorter compared to freeze-drying cycle A ("Freeze-drying cycle B"). (Cycle C; see Table 31). Generally, reconstitution times range from 37 to 42 minutes. Dissolving the freeze-dried cake involved some difficulties, which were improved by the introduction of a centrifugation step. Residual moisture was measured at 0.27%.

[0396] Batch 0025 : 25 mg / mL pembrolizumab formulated in sucrose was used. Batch 0025 was formulated using 10.66 mL of ... The usual form of freeze-drying cycle A was employed (approximately 136 hours, ("freeze cycle D") (See Table 31.) Among other modifications, a longer primary drying step was used. In general, reconstitution times ranged from 2 minutes 20 seconds to 4 minutes 40 seconds. Residual moisture was 0.4 to 1.0 g / L. The rate was 0.5%.

[0397] Batch 0027 : 25mg / mL and 35mg / mL Pembroliz in sucrose Batch 0027 was formulated using mab. Volumes of 6.8-9.5 mL were dispensed into 15R vials. The normal form of freeze-drying cycle A was adopted (approximately 136 hours, "freeze cycle"). "ClE"; see Table 31), while the longer primary drying step is a two-step drying process (- The vials were reconstituted with a smaller amount of water (1.0 mL). In general, reconstitution times ranged from 4 minutes 20 seconds to 6 minutes 15 seconds. Residual moisture was 0.1 The syringe discharge rate from the reconstituted body was very slow, and the 27G 1 This was difficult to achieve using a 3mL syringe fitted with a 2'' needle.

[0398] Batch 0028 (1) 25 mg / mL or 35 mg / mL penbub in sucrose Lorisumab (6.78-9.6mL volume filled in 15R or 20R vials) or (2) 35 mg / mL pembrolizumab in arginine (filled in a 15R vial) Batch 0028 was formulated using a packed volume of 6.78 mL. The vials were used without modification (approximately 55 hours). In general, reconstitution times ranged from 5 minutes 10 seconds to 6 minutes 15 seconds. The concentration ranged from 0.60 to 0.85%. Filling and emptying the arginine-containing reconstituted solution was easier than with the sucrose formulation. This was believed to be a result of viscosity.

[0399] This example shows the protein concentration of 48mg / mL to 200mg / mL. Lyophilized formulations prepared using starting solutions containing mbrolizumab were evaluated. All formulations The cake was reconstituted with SWFI and reconstituted. The reconstituted solutions were evaluated for tonicity, viscosity and reconstitution time. Reconstitute with less than full volume of water to give approximately 167 mg / mL (i.e., approximately 200 mg / vial). The pre-freezing solution was prepared using a high concentration (>100 mg / mL) of pre-freezing solution. The cakes were generally smaller in nature.

[0400] Reconstitution times for the various formulations ranged from 2 to 42 minutes (see Table 32). The reconstitution time increased with increasing protein concentration of the pre-frozen solution. The results showed that arginine hydrochloride had a similar effect on shortening reconstitution time to the excipients tested. This indicates that it may help reduce the viscosity of the reconstituted solution. Other excipients (e.g., sucrose, mannitol) and freeze-drying process parameters The effect of the parameters was found to be negligible. [Table 31] TIFF2024102215000040.tif35164 [Table 32] TIFF2024102215000042.tif24981

Claims

1. a) 5 mg / mL to 100 mg / mL of an anti-human Programmed Death Receptor 1 (PD-1) antibody or antigen-binding fragment thereof; b) 5 mM to 20 mM histidine buffer; c) i) 6% to 8% weight / volume (w / v) of trehalose or (2-hydroxypropyl)-β-cyclodextrin; ii) 3% to 5% w / v of mannitol, sorbitol, L-arginine, a pharma- ceutically acceptable salt of L-arginine, L-proline, or a pharma- ceutically acceptable salt of L-proline; and iii) 1.8 to 2.2% w / v glycine or a pharma- ceutically acceptable salt thereof A stabilizer selected from the group consisting of: d) 0.01% to 0.10% polysorbate 80; An anti-human PD-1 antibody formulation comprising: The anti-human PD-1 antibody formulation, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises three light chain CDRs comprising CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2, and CDRL3 of SEQ ID NO:3, and three heavy chain CDRs comprising CDRH1 of SEQ ID NO:6, CDRH2 of SEQ ID NO:7, and CDRH3 of SEQ ID NO:

8.

2. The anti-human PD-1 antibody formulation of claim 1, wherein the formulation has a pH of 4.5 to 6.

4.

3. The anti-human PD-1 antibody formulation of claim 1, wherein the formulation has a pH of 5.0 to 6.

0.

4. The anti-human PD-1 antibody formulation of claim 1, wherein the formulation has a pH of 6.0 to 6.

4.

5. The anti-human PD-1 antibody formulation of claim 1, comprising 1 mM to 20 mM L-methionine or a pharma- ceutically acceptable salt thereof.

6. The stabilizer, i) 6% to 8% weight / volume (w / v) trehalose or (2-hydroxypropyl)-β-cyclodextrin; ii) 3% to 5% mannitol, sorbitol, or L-proline or a pharma- ceutically acceptable salt of L-proline; and iii) 1.8 to 2.2% w / v glycine or a pharma- ceutically acceptable salt thereof The anti-human PD-1 antibody formulation according to any one of claims 1 to 4, selected from the group consisting of:

7. The anti-human PD-1 antibody formulation of claim 5, further comprising 1% to 3% w / v L-arginine or a pharma- ceutically acceptable salt thereof.

8. The anti-human PD-1 antibody formulation of claim 1, 2, or 4, wherein the stabilizer is 3% to 5% w / v L-arginine or L-arginine HCl, and the pH of the formulation is 6.0 to 6.

4.

9. The anti-human PD-1 antibody formulation of claim 8, further comprising 1% to 3% w / v L-arginine or a pharma- ceutically acceptable salt thereof.

10. The anti-human PD-1 antibody formulation of claim 9, wherein L-arginine or a pharma- ceutically acceptable salt thereof is present in a weight ratio of 1.25% to 2.5%.

11. The anti-human PD-1 antibody formulation of any one of claims 1 to 10, wherein the histidine buffer is present at a concentration of 8 mM to 12 mM.

12. The anti-human PD-1 antibody formulation according to claims 1 to 11, wherein L-methionine or a pharma- ceutically acceptable salt thereof is present at a concentration of 5 mM to 15 mM.

13. The anti-human PD-1 antibody formulation according to any one of claims 1 to 12, wherein polysorbate 80 is present at a weight ratio of 0.02% w / v.

14. The anti-human PD-1 antibody formulation according to any one of claims 1 to 13, wherein the concentration of the anti-human PD-1 antibody or antigen-binding fragment thereof is 5 mg / mL to 100 mg / mL.

15. The anti-human PD-1 antibody formulation of any one of claims 1 to 6 and 8 to 10, wherein the stabilizer is trehalose present at a weight ratio of 7% w / v.

16. The stabilizer is trehalose and the formulation is a) 5 to 100 mg / mL of an anti-human PD-1 antibody or antigen-binding fragment thereof; b) 8 mM to 12 mM histidine buffer; c) 6 mM to 8 mM trehalose; d) 0.01% to 0.04% w / v Polysorbate 80 The anti-human PD-1 antibody formulation according to claim 1,

17. a) 5 to 100 mg / mL of an anti-human PD-1 antibody or antigen-binding fragment thereof; b) 10 mM histidine buffer; c) 7% w / v trehalose; and d) 0.02% w / v Polysorbate 80 The anti-human PD-1 antibody formulation of claim 16,

18. The anti-human PD-1 antibody formulation of claim 17, wherein the pharma- ceutically acceptable salt of L-methionine is L-methionine HCl.

19. The anti-human PD-1 antibody formulation according to any one of claims 16 to 18, further comprising 1.25% to 2.5% w / v L-arginine or a pharma- ceutically acceptable salt thereof.

20. The anti-human PD-1 antibody formulation of claim 19, wherein the L-arginine or a pharma- ceutically acceptable salt thereof is L-arginine HCl.

21. The anti-human PD-1 antibody formulation of any one of claims 1 to 6 and 8 to 20, wherein the formulation has a pH of 5.3 to 5.

8.

22. The anti-human PD-1 antibody formulation of any one of claims 1 to 21, wherein the formulation further comprises a metal chelator.

23. The anti-human PD-1 antibody formulation of claim 22, wherein the metal chelator is DTPA present at a concentration of 10 μM to 30 μM.

24. The anti-human PD-1 antibody formulation according to any one of claims 1 to 23, which is liquid.

25. The anti-human PD-1 antibody formulation according to any one of claims 1 to 24, which is a reconstituted solution from a lyophilized formulation.

26. A liquid anti-human PD-1 antibody formulation reconstituted from a lyophilized formulation, the reconstituted solution comprising: a) 5 mg / mL to 100 mg / mL of an anti-human PD-1 antibody or antigen-binding fragment thereof; b) 8 mM to 12 mM histidine buffer; c) i) 3% to 8% weight / volume (w / v) trehalose; ii) 2% to 5% w / v L-arginine or a pharma- ceutically acceptable salt thereof; iii) 3% to 5% mannitol and 1% to 2% trehalose; and iv) A combination of i) and ii) a stabilizer selected from the group consisting of: d) 0.01% to 0.04% Polysorbate 80 A liquid anti-human PD-1 antibody formulation comprising: The anti-human PD-1 antibody formulation, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises three light chain CDRs comprising CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2, and CDRL3 of SEQ ID NO:3, and three heavy chain CDRs comprising CDRH1 of SEQ ID NO:6, CDRH2 of SEQ ID NO:7, and CDRH3 of SEQ ID NO:

8.

27. The anti-human PD-1 antibody formulation of any one of claims 1 to 26, wherein the formulation is contained in a glass vial or an injection device.

28. The formulation exhibited the following attributes after storage for 12 months at 2-8°C: a) the percentage of heavy and light chains is 90.0% or more as measured by reduced CE-SDS; b) The percentage of intact IgG is 90.0% or more as measured by non-reducing CE-SDS; And c) the percentage of monomer is 95% or more as measured by HP-SEC; The anti-human PD-1 antibody formulation according to any one of claims 1 to 27, comprising one or more of the following:

29. The anti-human PD-1 antibody formulation of any one of claims 1 to 28, wherein the ratio of heavy chains to light chains is greater than 96% as measured by reduced CE-SDS after storage at 2-8°C for 12 months.

30. The anti-human PD-1 antibody formulation of any one of claims 1 to 29, wherein the percentage of intact IgG in the formulation is greater than 97% as measured by non-reducing CE-SDS after storage at 2 to 8°C for 12 months.

31. The anti-human PD-1 antibody formulation of any one of claims 1 to 30, which has a monomer percentage of greater than 98.5% as measured by HP-SEC after storage at 2 to 8°C for 12 months.

32. The anti-human PD-1 antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:

4. Ｌ region, and a V comprising the amino acid sequence set forth in SEQ ID NO:

9. Ｈ The anti-human PD-1 antibody formulation according to any one of claims 1 to 31, comprising a region.

33. The anti-human PD-1 antibody formulation of any one of claims 1 to 32, wherein the formulation comprises a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5, and a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

10.

34. The anti-human PD-1 antibody formulation of any one of claims 1 to 33, wherein the formulation comprises an anti-human PD-1 antibody that is pembrolizumab.

35. 35. The anti-human PD-1 antibody formulation according to any one of claims 1 to 34, for use in a method for treating a chronic infection in a human patient in need thereof, comprising administering to the patient an effective amount of the anti-human PD-1 antibody formulation.

36. 35. The anti-human PD-1 antibody formulation of any one of claims 1 to 34, for use in a method for treating cancer in a human patient in need of such treatment, comprising administering to the patient an effective amount of the anti-human PD-1 antibody formulation.

37. The anti-human PD-1 antibody formulation according to claim 36, wherein the cancer is melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, prostate cancer, or glioblastoma.

38. The anti-human PD-1 antibody formulation according to claim 37, wherein the cancer is triple-negative breast cancer or ER+ / HER2- breast cancer.

39. The anti-human PD-1 antibody formulation according to claim 37, wherein the cancer is a non-Hodgkin's lymphoma that is primary mediastinal B-cell lymphoma or diffuse large B-cell lymphoma.

40. The anti-human PD-1 antibody formulation of claim 36, wherein the cancer is a microsatellite instability-high (MSI-H) or mismatch repair deficient solid tumor.

41. The anti-human PD-1 antibody formulation according to claim 36, wherein the cancer is non-small cell lung cancer, melanoma, Hodgkin's lymphoma, urothelial carcinoma, head and neck cancer, gastric cancer, or MSI-H cancer.

42. The anti-human PD-1 antibody formulation according to claim 37, wherein the cancer is metastatic non-small cell lung cancer (NSCLC).

43. The anti-human PD-1 antibody formulation of claim 42, wherein the patient has a tumor with high PD-L1 expression (Tumor Proportion Score (TPS) > 50%) and has not been previously treated with platinum-containing chemotherapy.

44. The anti-human PD-1 antibody formulation of claim 42, wherein the patient has a tumor with PD-L1 expression (TPS > 1%) and has been previously treated with platinum-containing chemotherapy.

45. The anti-human PD-1 antibody formulation of claim 42, wherein the patient has a tumor with PD-L1 expression (TPS > 1%) and has not been previously treated with platinum-containing chemotherapy.

46. The anti-human PD-1 antibody formulation of any one of claims 42 to 45, wherein the patient's tumor does not have EGFR or ALK genomic abnormalities.

47. The anti-human PD-1 antibody formulation of any one of claims 42 to 46, wherein the method further comprises administering to the patient pemetrexed and carboplatin.

48. The anti-human PD-1 antibody formulation of any one of claims 35 to 47, wherein the effective amount comprises a dose of anti-human PD-1 antibody selected from the group consisting of 1.0, 3.0 and 10 mg / kg patient body weight.

49. The anti-human PD-1 antibody formulation of any one of claims 35 to 47, wherein the effective amount of the anti-human PD-1 antibody formulation comprises a dose of 200 mg of anti-human PD-1 antibody.

50. The anti-human PD-1 antibody formulation according to any one of claims 35 to 47, wherein the anti-human PD-1 antibody formulation is administered subcutaneously in the treatment method.

51. The anti-human PD-1 antibody formulation according to any one of claims 35 to 47, wherein the anti-human PD-1 antibody formulation is administered intravenously in the treatment method.

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