Stable high-concentration sodium chloride formulation containing PD-1 antibody and method of use thereof

Abstract

It generally relates to the field of pharmaceutical formulations of antibodies or antigen-binding fragments thereof against the human programmed cell death receptor PD-1. The formulation may further contain a histidine buffer, an inorganic salt, a sugar polyol, and a non-ionic surfactant. The pharmaceutical formulation exhibits low viscosity and a significant degree of antibody stability even after being subjected to thermal and other physical stresses. Methods of making and using such antibody formulations are also provided.

Description

[Technical field]

[0001] Disclosed herein are stable, highly concentrated formulations comprising an antibody or antigen-binding fragment thereof that binds to human programmed death receptor 1 (PD-1). Also disclosed herein are methods of preparing the disclosed formulations and methods of treating cancer using the formulations. [Background technology]

[0002] Antibodies as therapeutic agents are increasingly being used in clinics. However, antibodies generally have similar structures, but different primary amino acid sequences, even for antibodies that bind to the same target protein. The characteristics of the primary amino acid sequence of an antibody are one of the major determinants that determine the solubility and / or stability characteristics of the antibody in various formulations. An antibody formulation that provides solubility and stability for one antibody may not be sufficient for another antibody, resulting in precipitation or fragmentation of the antibody. This is especially true when a subcutaneous antibody formulation is desired.

[0003] Subcutaneous injections have received increasing attention for the delivery of protein therapeutics because of their potential to provide patient self-administration. Rapid, small-volume injections allow patients to administer antibody therapeutics without the need for intravenous infusions, which typically require a hospital visit. However, many antibodies require a fixed dose to be effective, which generally requires the antibody to be concentrated to a small amount. The volume limitations of the subcutaneous route of administration are an important factor to consider for subcutaneous administration, necessitating high antibody concentrations. Challenges then arise regarding protein solubility, physical, and chemical stability, and the difficulty of manufacturing, storing, and delivering subcutaneous antibody formulations. For example, antibodies can lose solubility during processing and / or storage, and form particles in certain formulations, which reduces the efficacy of subcutaneous administration. High viscosity is another problem to overcome, as the concentrated nature of the antibody in the subcutaneous formulation limits the injectability of the product. Also, in the manufacturing process, highly viscous antibody formulations are difficult to process, especially in ultrafiltration and sterile filtration. Finally, subcutaneous antibody formulations must maintain the structure and function of the antibody. Subcutaneous antibody formulations that cause proteolysis or degradation of the antibody structure will be less effective, as will those that impair the ability of the antibody to bind to the target protein. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there is a long felt need in the art for subcutaneous antibody formulations of anti-human PD-1 antibodies for the treatment of various cancers and infectious diseases that would have good antibody solubility, stability, long shelf life, and be amenable to administration at high concentrations. [Means for solving the problem]

[0005] The present disclosure provides a stable, low-viscosity and highly concentrated antibody formulation. about 10 mg / mL to about 200 mg / mL of an anti-programmed death receptor 1 (PD-1) antibody, or an antigen-binding fragment thereof; a formulation buffer providing a pH of about 5.0 to about 7.0; Sugar polyols, Viscosity reducers, and a non-ionic surfactant, A low-viscosity pharmaceutical formulation, wherein the pharmaceutical formulation has a viscosity of 30 cP or less and an osmotic pressure of about 200 mOsmol / kg to about 400 mOsmol / kg.

[0006] A formulation comprising a PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising: (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 1; (b) HCDR2 of SEQ ID NO: 2; (c) HCDR3 of SEQ ID NO: 3; and (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 4; (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

[0007] A formulation, wherein the formulation buffer is selected from the group consisting of histidine, acetate, citrate, succinate, phosphate, a mixture of histidine and acetate, or a mixture of histidine and citrate.

[0008] A formulation wherein the formulation buffer is histidine.

[0009] The formulation, wherein the concentration of the buffer is 15 mM to 25 mM.

[0010] A formulation, wherein the formulation comprises 20 mM histidine buffer.

[0011] The formulation has a pH of 5.5 to 6.0.

[0012] A formulation wherein the sugar polyol is selected from the group consisting of trehalose, sucrose, sorbitol, mannitol, maltose, dextran, or (2-hydroxypropyl)-b-cyclodextrin.

[0013] A formulation wherein the sugar polyol is trehalose.

[0014] A formulation having a trehalose concentration of 70 mM to 240 mM.

[0015] A formulation having a trehalose concentration of 80 mM to 160 mM.

[0016] A formulation having a trehalose concentration of 70 mM to 100 mM.

[0017] A formulation in which the trehalose concentration is 80 mM.

[0018] A formulation wherein the viscosity-lowering agent is an inorganic salt selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, sodium acetate, sodium sulfate, ammonium chloride, or ammonium sulfate.

[0019] A formulation wherein the inorganic salt is sodium chloride at a concentration of 50 mM to 150 mM.

[0020] The formulation, wherein the concentration of sodium chloride is 50 mM to 100 mM.

[0021] A formulation wherein the sodium chloride concentration is 70 mM.

[0022] A formulation wherein the non-ionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, or poloxamer 188.

[0023] A formulation having a concentration of polysorbate 20 of 0.02% to 0.08%.

[0024] A formulation in which the concentration of polysorbate 20 is 0.08%.

[0025] A formulation comprising 20 mM Histidine-Histidine HCl, 100 mM NaCl, 70 mM Trehalose, and 0.08% Polysorbate 20, and having a pH of pH 6.0.

[0026] A formulation comprising 20 mM Histidine-Histidine HCl, 50 mM NaCl, 100 mM Trehalose, and 0.02% Polysorbate 20, and having a pH of pH 6.0.

[0027] A formulation comprising 20 mM Histidine-Histidine HCl, 70 mM NaCl, 80 mM Trehalose, and 0.08% Polysorbate 20, and a pH of pH 6.0.

[0028] The formulation, wherein the concentration of the anti-human PD-1 antibody or antigen-binding fragment thereof is about 10 mg / mL to 200 mg / mL.

[0029] 1. A method for producing an antibody formulation, the method comprising: a. Adding trehalose and sodium chloride to an antibody to obtain an antibody preparation having a trehalose concentration of 50 mM or more and a sodium chloride concentration of 25 mM or more (the antibody comprises a heavy chain variable region comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 1; (b) HCDR2 of SEQ ID NO: 2; (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 4; (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6). b. Concentrating the antibody preparation of (a) to 150-200 mg / mL; and c. adding polysorbate 20 to the antibody formulation of (b) to obtain an antibody formulation having a polysorbate 20 concentration of 0.01 mg / mL or more; The method of claim 1, wherein the antibody formulation of (c) is an aqueous solution and has a viscosity of 30 cP or less at 25° C., and the antibody formulation of (c) is stable to agitation, freezing and thawing, and heat stress.

[0030] 1. A method for treating cancer in a human patient in need of treatment, comprising subcutaneously administering an effective amount of an anti-human PD-1 antibody formulation.

[0031] The method comprises administering the anti-human PD-1 antibody formulation subcutaneously at a dose of about 100 mg to about 1000 mg.

[0032] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 200 mg.

[0033] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 300 mg.

[0034] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 400 mg.

[0035] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 500 mg.

[0036] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously once a week.

[0037] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously once every two weeks.

[0038] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously once every three weeks.

[0039] The method, wherein the cancer is lung cancer (including small cell lung cancer or non-small cell lung cancer), adrenal cancer, liver cancer, gastric cancer, cervical cancer, melanoma, kidney cancer, breast cancer, colon cancer, leukemia, bladder cancer, bone cancer, brain cancer, endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumor, or esophageal cancer.

[0040] The method, wherein the human patient is administered at least one other therapeutic agent which is zanubrutinib, pamiparib, an anti-CTLA4 antibody, an anti-4-1BB antibody, an anti-OX40 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, a CD40 agonist, a TLR agonist, a CAR-T cell, or a chemotherapeutic agent.

[0041] In some embodiments, the antibody formulation comprises an anti-PD-1 antibody or antigen-binding fragment thereof, a formulation buffer, a sugar polyol, a viscosity-lowering agent, and a non-ionic surfactant. In some embodiments, the formulation buffer provides a pH range of 5.0 to 7.0. In some embodiments, the viscosity of the antibody formulation is 30 centipoise (cP) or less. In some embodiments, the osmolality of the antibody formulation is about 200 mOsmol / kg to about 400 mOsmol / kg. In some embodiments, the antibody formulation is stable to agitation, freeze-thawing, and heat stress.

[0042] In some embodiments, the antibody formulation may comprise about 10 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or antigen-binding fragment thereof, a formulation buffer, a sugar polyol, a viscosity-lowering agent, and a non-ionic surfactant, and has a pH of about 6.0±0.5. In some embodiments, the antibody formulation may consist essentially of about 100 mg / mL to about 180 mg / mL of an anti-PD-1 antibody, a formulation buffer, a sugar polyol, a viscosity-lowering agent, and a non-ionic surfactant, and has a pH of 6.0±0.5.

[0043] In some embodiments, the formulation buffer is selected from the group consisting of histidine, acetate, citrate, succinate, phosphate, a mixture of histidine and acetate, and a mixture of histidine and citrate. In some embodiments, the formulation buffer can be a histidine buffer. In some embodiments, the concentration of the histidine buffer is about 10 mM to about 30 mM. In some embodiments, the concentration of the histidine buffer is about 20 mM histidine.

[0044] In some embodiments, the sugar polyol is selected from the group consisting of trehalose, sucrose, sorbitol, mannitol, maltose, dextran, or (2-hydroxypropyl)-b-cyclodextrin. In some embodiments, the sugar polyol can be trehalose. In some embodiments, the trehalose is α,α-trehalose dihydrate. In other embodiments, the sugar polyol can be sucrose. In some embodiments, the concentration of the sugar polyol can be about 25 mM to about 240 mM. In some embodiments, the concentration of the sugar polyol can be about 50 mM to about 150 mM, preferably about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.

[0045] In some embodiments, the viscosity-reducing agent is an inorganic salt. In some embodiments, the viscosity-reducing agent is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, sodium acetate, sodium sulfate, ammonium chloride, or ammonium sulfate. In some embodiments, the viscosity-reducing agent is sodium chloride. In some embodiments, the concentration of sodium chloride can be about 25 mM to about 150 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.

[0046] In some embodiments, the non-ionic surfactant is selected from the group consisting of polysorbate 80 (PS80), polysorbate 20 (PS20), or poloxamer 188. In some embodiments, the concentration of the non-ionic surfactant can be about 0.01 to about 1 mg / mL. In some embodiments, the concentration of the polysorbate is about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, or about 0.8 mg / mL. In some embodiments, the polysorbate is polysorbate 20.

[0047] In some embodiments, the antibody formulation comprises about 100 mg / mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, about 150 mg / mL, about 155 mg / mL, about 160 mg / mL, about 165 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about The antibody formulation comprises 185 mg / mL, about 190 mg / mL, about 195 mg / mL, or about 200 mg / mL of an anti-PD-1 antibody or antigen-binding fragment thereof, about 20 mM histidine buffer, about 70 mM to about 100 mM α,α-trehalose dihydrate or sucrose, about 50 mM to about 100 mM sodium chloride, and about 0.2 mg / mL to about 0.8 mg / mL of polysorbate 20, and the antibody formulation has a pH of 6.0±0.5. In some embodiments, the viscosity of the antibody formulation is 30 cP or less at 25° C.

[0048] In some embodiments of the invention, the anti-PD-1 antibody is tislelizumab (BGB-A317) or an antigen-binding fragment of tislelizumab.

[0049] Also provided herein are methods of making a stable, low-viscosity antibody formulation, the methods comprising: adding trehalose or sucrose and sodium chloride to an antibody to obtain an antibody formulation having a trehalose or sucrose concentration of 50 mM or more and a sodium chloride concentration of 25 mM or more; concentrating the antibody to about 200 mg / mL; and adding polysorbate 20 to the antibody formulation to obtain an antibody formulation comprising a polysorbate 20 concentration of 0.01 mg / mL or more, wherein the antibody formulation is an aqueous solution and has a viscosity of 30 cP or less at 25° C.

[0050] Also provided herein is a method of treating cancer in a human patient suffering from cancer, the method comprising subcutaneously administering to the patient an effective amount of a PD-1 antibody formulation described herein.

[0051] Provided herein are methods of treating cancer in a human patient suffering from a PDL-1-expressing cancer, the methods comprising subcutaneously administering to the patient an effective amount of a PD-1 antibody formulation described herein. [Brief description of the drawings]

[0052] [Figure 1] FIG. 1A shows the results of a SEC-HPLC study (FIG. 1B) and a CZE study (FIG. 1B) for a 10 mg / ml tislelizumab formulation stored at 40° C. for 2 weeks (denoted as "40C2W" on the graph) and exposed to light for 2 weeks (denoted as "pho2W" on the graph). TO refers to the starting point of the sample. [Diagram 2] A-B show the amount of aggregates (FIG. 2A) and monomers (FIG. 2B) (measured by SEC-HPLC) for each formulation after freeze / thaw (labeled "3FT" on the graph), shaking (labeled "SK" on the graph), and heat stress (labeled "40C4W" on the graph). T0 refers to the starting point of the sample. "25C6M" indicates that the formulation was stored at 25° C. for 6 months. "5C6M" indicates that the formulation was stored at 5° C. for 6 months. [Diagram 3] 1 shows the results of a CZE study in which a subcutaneous antibody formulation was stored at 40° C. for 4 weeks (denoted as "40C4W" on the graph). TO refers to the starting time point. "25C6M" indicates that the formulation was stored at 25° C. for 6 months. "5C6M" indicates that the formulation was stored at 5° C. for 6 months. [Figure 4] The purity of the formulation over 4 weeks at 40° C. (labeled "40C4W" on the graph) is shown as measured by CE-SDS under non-reducing conditions. TO refers to the starting time point. "25C6M" indicates that the formulation was stored at 25° C. for 6 months. "5C6M" indicates that the formulation was stored at 5° C. for 6 months. [Diagram 5] The results of SEC-HPLC (FIGS. 5A and 5B), CZE (FIG. 5C), and CE-SDS(NR) (FIG. 5D) studies for formulation F18 stored at 25° C. (denoted as "25C") and 5±3° C. (denoted as "5C") are shown. "0" month refers to the starting time point of the samples. [Figure 6A]1 shows bioavailability data for F18 subcutaneous formulation in a Sprague Dawley rat model. [Figure 6B] 1 shows bioavailability data for F18 subcutaneous formulation in a Sprague Dawley rat model. [Figure 7] 1 shows bioavailability data for F18 subcutaneous formulation in a monkey model. [Figure 8] Photographs showing the absence of injection site reactions in both rats and minipigs upon administration of subcutaneous formulations at various sites on the animals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] definition Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0054] As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include the corresponding plural referents unless the context clearly indicates otherwise.

[0055] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0056] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprises" and variations such as "comprises" and "comprises" will be understood to mean the inclusion of a recited amino acid sequence, DNA sequence, step, or group thereof, but excluding any other amino acid sequences, DNA sequences, steps. As used herein, the term "comprising" can be substituted with the terms "containing," "including," or, where appropriate, "having."

[0057] The terms "administration," "administering," "treating," and "treatment" herein, when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contacting an exogenous pharmaceutical, therapeutic, diagnostic, or antibody formulation with the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell includes contacting a reagent with the cell and contacting a reagent with a fluid (wherein the fluid is in contact with the cell). The terms "administration" and "treatment" also refer to in vitro and ex vivo treatments, e.g., treatment of a cell with a reagent, diagnostic, binding compound, or another cell. The term "subject" herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit), and most preferably a human. In one aspect, treating any disease or disorder refers to alleviating the disease or disorder (i.e., delaying or preventing or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treat", "treating", or "treatment" refers to reducing or improving at least one physical parameter, including one that may not be discernible by the patient. In yet another embodiment, "treat", "treating", or "treatment" refers to modulating a disease or disorder physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both.

[0058] The term "therapeutically effective amount" as used herein refers to an amount of an anti-PD-1 antibody that, when administered to a subject to treat a disease, or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or condition. A "therapeutically effective amount" may vary depending on the agent, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject to be treated, and / or the weight of the subject to be treated. The appropriate amount in any given case will be apparent to those skilled in the art or can be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combined objects for effective treatment of a disease, disorder, or condition. In some embodiments of the present disclosure, the subject is a human.

[0059] A "pharmaceutical formulation" or "formulation" refers to an antibody preparation in a form in which the active ingredient is capable of acting effectively and which does not contain additional components that would be toxic to the subject to which the formulation is administered.

[0060] A "stable" formulation is one in which the antibody is prepared so as to maintain the physical and / or chemical stability and / or biological activity of the antibody over time.Various analytical techniques for measuring protein stability are available in the art and are reviewed in: Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).Stability can be measured at a selected temperature over a selected period of time.

[0061] The term "antibody" herein is used in the broadest sense and specifically encompasses antibodies (including full-length monoclonal antibodies) and antibody fragments, so long as they recognize an antigen (e.g., PD-1). Antibodies are usually monospecific, but may also be referred to as idiospecific, heterospecific, or multispecific. An antibody molecule binds to a specific antigenic determinant or epitope on an antigen through a specific binding site.

[0062] The term "monoclonal antibody" or "mAb" or "Mab" herein refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules in the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations usually contain a large number of different antibodies with different amino acid sequences in the variable domains, particularly in the complementarity determining regions (CDRs), which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by a particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, e.g., Kohler G et al., Nature 1975 256:495-497; US Pat. No. 4,376,110; Ausubel FM et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow E et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan JE et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The mAbs disclosed herein can be of any immunoglobulin class, including IgG, IgM, IgD, IgE, IgA, and any subclass thereof. Hybridomas producing mAbs can be cultured in vitro or in vivo. High titers of mAbs can be obtained by in vivo production, and cells from individual hybridomas are injected intraperitoneally into mice (e.g., initially primed Balb / c mice) to generate ascites fluid containing high concentrations of the desired mAb. MAbs of isotype IgM or IgG can be purified from such ascites fluid or culture supernatants using column chromatography methods well known to those skilled in the art.

[0063] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer comprises a pair of two identical polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that are primarily involved in antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region that is primarily involved in effector function. Human light chains are typically classified as kappa light chains and lambda light chains. Human heavy chains are further typically classified as α, δ, ε, γ, or μ, which define the antibody isotype as IgA, IgD, IgE, IgG, and IgM, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 or more amino acids.

[0064] The variable regions of each light / heavy chain (VL / VH) pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are generally the same.

[0065] Typically, both the heavy and light chain variable domains have three Hypervariable regions, also called "complementarity determining regions (CDRs)", which Located between relatively conserved framework regions (FR). CDRs are usually aligned by the framework regions, allowing binding to a specific epitope. In general, both the light and heavy chain variable domains include, from the N-terminus to the C-terminus, FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4), in that order. The assignment of amino acids to each domain generally follows the definitions set forth in: Sequences of Proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0066] The term "hypervariable region" refers to the amino acid residues of an antibody which are involved in antigen binding. The hypervariable region comprises the amino acid residues of the "CDRs" (i.e., VL-CDR1, VL-CDR2, and VL-CDR3 of the light chain variable domain and VH-CDR1, VH-CDR2, and VH-CDR3 of the heavy chain variable domain). See Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining antibody CDR regions by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (defining antibody CDR regions by structure). The term "framework" or "FR" residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0067] Unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain Fv (ScFv)); nanobodies and multispecific antibodies formed from antibody fragments.

[0068] An antibody that specifically binds to a particular target protein is also described as specifically binding to the particular target protein. This indicates that the antibody preferentially binds to that target compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered to be "specific" for its intended target if its binding determines the presence of the target protein in a sample and does not produce undesirable results, such as false positives. Antibodies or binding fragments thereof useful in the present invention include: It will bind to the target protein with an affinity that is at least 2-fold, preferably at least 10-fold, more preferably at least 20-fold, and most preferably at least 100-fold higher than its affinity to a non-target protein. Antibodies herein are said to specifically bind to a polypeptide comprising a given amino acid sequence.

[0069] The term "human antibody" herein refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycochains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

[0070] The term "humanized antibody" refers to forms of antibodies that contain non-human (e.g., murine) and human antibody sequences. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody will contain substantially all of at least one, and usually two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR regions being those of a human immunoglobulin sequence. Optionally, the humanized antibody will also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The prefixes "hum", "hu", "Hu", or "h" are added to the antibody clone name when necessary to distinguish the humanized antibody from the parent rodent antibody. Humanized forms of rodent antibodies generally contain the same CDR sequences as the parent rodent antibody, but may contain certain amino acid substitutions to increase affinity, to increase the stability of the humanized antibody, or for other reasons.

[0071] The antibodies of the present application have potential therapeutic applications in the treatment of cancer. The term "cancer" or "tumor" herein means or refers to a mammalian physiological condition that is usually characterized by unregulated cell growth. Examples of cancer include, but are not limited to, lung cancer (including small cell lung cancer or non-small cell lung cancer), adrenal cancer, liver cancer, gastric cancer, cervical cancer, melanoma, kidney cancer, breast cancer, colon cancer, leukemia, bladder cancer, bone cancer, brain cancer, endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumor, or esophageal cancer.

[0072] Furthermore, the antibodies of the present application have potential therapeutic uses in the control of viral infections and other human diseases that are mechanistically involved in immune tolerance or "exhaustion." In the context of the present application, the term "exhaustion" refers to a process that leads to the exhaustion of the ability of immune cells to respond to cancer or chronic viral infection.

[0073] Anti-PD-1 antibody The present disclosure provides anti-PD-1 antibodies and subcutaneous formulations thereof. For example, tislelizumab (BGB-A317), disclosed in U.S. Patent No. 8,735,553, is an anti-PD-1 antibody, the sequence of which is provided in Table 1 below. [Table 1-1] [Table 1-2]

[0074] Anti-PD-1 antibodies may include, but are not limited to, tislelizumab, pembrolizumab, or nivolumab. Tislelizumab is disclosed in US8,735,553. Pembrolizumab (formerly known as MK-3475), disclosed in US8,354,509 and US8,900,587 by Merck, is a humanized IgG4-K immunoglobulin that targets the PD-1 receptor and inhibits the binding of the PD-1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is under clinical investigation for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin's lymphoma (cHL). Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Patent No. 8,008,449 and WO 2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin's lymphoma.

[0075] Further framework modifications in the Fc region In yet another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to modify the effector function of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has a modified affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al.

[0076] In another embodiment, one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0077] In yet another embodiment, one or more amino acid residues are altered to alter the ability of the antibody to fix complement. This approach is described, for example, in PCT Publication WO94 / 29351 by Bodmer et al. In certain embodiments, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced with one or more allotypic amino acid residues for the IgG1 subclass and the kappa isotype. Allotypic amino acid residues also include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses and the constant regions of the light chains of the kappa isotype as described in Jefferis et al., MAbs.1:332-338 (2009).

[0078] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for the Fcγ receptor. This approach is described, for example, in PCT Publication WO00 / 42072 by Presta. Additionally, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0079] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (i.e., the antibody lacks or has reduced glycosylation) can be generated. The glycosylation can be altered to, for example, increase the affinity of the antibody for an "antigen". Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such glycosylation can increase the affinity of the antibody for the antigen. Such approaches are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0080] Additionally or alternatively, antibodies can be made with altered types of glycosylation (e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues or increased bisecting GlcNAc structures). Such altered glycosylation patterns have been demonstrated to enhance the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells to express recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation. PCT Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Lecl3 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in reduced fucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases, such as beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNAc structures, resulting in increased ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0081] In another aspect, where reduced ADCC is desired, human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and little CDC effector function (Moore GL, et al., 2010 MAbs, 2:181-189). Meanwhile, native IgG4 has been found to be less stable under stress conditions (e.g., in acidic buffer or during increased temperature) (Angal, S. 1993 Mol Immunol, 30:105-108; Dall'Acqua, W. et al., 1998 Biochemistry, 37:9266-9273; Aalberse et al., 2002 Immunol, 105:9-19). Reduced ADCC can be achieved by operably linking an antibody to an IgG4 engineered with a combination of modifications that reduce or eliminate FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biological drugs, one of the less desirable inherent properties of IgG4 is the dynamic separation of the two heavy chains in solution to form half-antibodies, which results in bispecific antibodies generated in vivo by a process called "Fab arm exchange" (Van der Neut Kolfschoten M, et al., 2007 Science, 317: 1554-157). Mutation of serine to proline at position 228 (EU numbering system) appears to inhibit separation of IgG4 heavy chains (Angal, S. 1993 Mol Immunol, 30: 105-108; Aalberse et al., 2002 Immunol, 105: 9-19).Some of the amino acid residues in the hinge and gamma Fc regions have been reported to affect antibody interaction with Fcγ receptors (Chappel SM, et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, KL et al., 1999 Eur J Immunol, 29:2613-2624; Clynes, RA et al, 2000 Nature Medicine, 6:443-446; Arnold JN, 2007 Annu Rev immunol, 25:21-50). Furthermore, some of the rare IgG4 isoforms occurring in the human population may also result in different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet, 25:349-55; Aalberse et al., 2002 Immunol, 105:9-19). It is possible to engineer the hinge and Fc regions of human IgG4 and introduce a number of modifications to generate PD-1 antibodies with reduced ADCC, CDC, and instability. These engineered IgG4Fc molecules can be found in SEQ ID NOs: 83-88, U.S. Patent No. 8,735,553.

[0082] Treatment The antibodies or antigen-binding fragments of the present disclosure are useful for a variety of applications, including, but not limited to, methods for treating a PD-1-associated disorder or disease. In one embodiment, the PD-1-associated disorder or disease is cancer.

[0083] In one embodiment, the present disclosure provides a method of treating cancer. In certain embodiments, the method comprises administering an effective amount of an anti-PD-1 antibody or antigen-binding fragment to a patient in need thereof. The cancer may include, but is not limited to, lung cancer (including small cell lung cancer or non-small cell lung cancer), adrenal cancer, liver cancer, gastric cancer, cervical cancer, melanoma, kidney cancer, breast cancer, colon cancer, leukemia, bladder cancer, bone cancer, brain cancer, endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumor, or esophageal cancer.

[0084] The antibodies or antigen-binding fragments of the present invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration if localized treatment is desired. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. A variety of administration schedules are contemplated herein, including but not limited to single or multiple administrations at various times, bolus administration, and pulse infusion.

[0085] The antibodies or antigen-binding fragments of the invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to the physician. The antibodies need not be formulated with one or more agents currently used to prevent or treat the disorder in question, but are optionally formulated with them. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and routes of administration as described herein, or about 1-99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0086] For prevention or treatment of disease, the appropriate dosage of the antibody or antigen-binding fragment of the invention will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antibody, and the judgment of the attending physician.

[0087] Antibodies directed against PD-1 have been shown to be safe when administered to human cancer patients at various dose ranges and dosing cycles. The subcutaneous antibody formulations disclosed herein can be administered at 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg. The subcutaneous antibody formulations can be administered twice daily, daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, once every two weeks, once every three weeks, once monthly, once every two months, once every three months, once every four months, once every five months, or once every six months. In some embodiments, the dosing regimen includes administering 100 mg of tislelizumab once every three weeks. In some embodiments, the dosing regimen includes administering 200 mg of tislelizumab once every three weeks. In some embodiments, the dosing regimen includes administering 300 mg of tislelizumab once every three weeks. In some embodiments, the dosing regimen includes administering 400 mg of tislelizumab once every three weeks. In some embodiments, the dosing regimen includes administering 500 mg of tislelizumab once every three weeks. In some embodiments, the dosing regimen includes administering 600 mg of tislelizumab once every three weeks.

[0088] In certain embodiments, tislelizumab can be administered in combination with other therapies, such as zanubrutinib, pamiparib, anti-CTLA4 antibodies, anti-4-1BB antibodies, anti-OX40 antibodies, anti-TIGIT antibodies, anti-TIM-3 antibodies, CD40 agonists, TLR agonists, CAR-T cells, or chemotherapeutic agents.

[0089] Pharmaceutical Compositions and Formulations Compositions, including pharmaceutical formulations, that include anti-PD-1 antibodies or antigen-binding fragments thereof are also provided. In certain embodiments, the compositions include one or more antibodies or antigen-binding fragments that bind to PD-1. These compositions may further include a suitable carrier, such as a pharma- ceutically acceptable excipient, including a buffering agent.

[0090] Pharmaceutical formulations of the anti-PD-1 antibodies or antigen-binding fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antigen-binding fragments having the desired purity with one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers (e.g., phosphate, citric acid, and other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol; alkyl parabens (e.g., methyl or propyl paraben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins (e.g., serum alcohols, glycerides, and the like). Examples of suitable surface active agents include, but are not limited to, liposomes, gelatins, or immunoglobulins; hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., polyethylene glycol (PEG) or polysorbate 20).

[0091] The present disclosure provides various pharmaceutical formulations of the anti-PD-1 antibodies described in detail herein. By way of example, the formulation may include an anti-PD-1 antibody, a buffering agent (e.g., histidine) that provides a particular pH, a sugar polyol (e.g., sucrose or trehalose), a viscosity-reducing agent (e.g., NaCl), and a non-ionic surfactant (e.g., polysorbate 20).

[0092] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0093] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes. EXAMPLES

[0094] The examples and descriptions of specific embodiments are used for illustration purposes, rather than for limiting the invention defined in the claims. As will be readily understood, numerous variations and combinations of the features described above can be utilized without departing from the invention as set forth in the claims. All such variations are intended to be included within the scope of the present invention. All cited references are incorporated herein by reference in their entirety.

[0095] Analysis method The Methods section outlines the methods used in Examples 1-5 below.

[0096] SEC-HPLC The formation of soluble aggregates is analyzed by size exclusion chromatography (SEC) on a Waters HPLC system. Using an isocratic gradient method, proteins are separated based on molecular size on a TSKgel G3000™ SWXL column maintained at 37±5° C. Molecular weight species are eluted and detected by UV absorption at 280 nm. The distribution of aggregates, monomers, and fragments is quantified by peak areas of standards and samples.

[0097] CZE The charge heterogeneity of samples is determined using a PA800 Plus™ (Beckman) by capillary zone electrophoresis (CZE), also called free solution capillary electrophoresis. Samples are separated based on electrophoretic mobility caused by differences in charge and hydrodynamic radius of the analytes in a capillary filled with a buffer containing caproic acid. Samples are analyzed in situ when an external electric field is applied, resulting in specific peak patterns representing different charge variants of the antibodies (acidic, basic, and major charge variants). Samples are injected under pressure and migrated proteins are detected by UV absorbance at 214 nm.

[0098] CE-SDS(NR) The purity of the samples is determined by capillary gel electrophoresis (CE) using a PA800 Plus™ (Beckman). Samples are denatured with sodium dodecyl sulfate (SDS) and separated based on size in a capillary filled with gel that acts as a sieving medium. For non-reduced (NR) samples, the alkylating agent N-ethylmaleimide (NEM) is added to avoid any fragmentation caused by the sample preparation and ensure that the major IgG peak remains intact. Samples are electrokinetically injected and a UV detector is used to detect the migrated proteins by UV absorbance at 200 nm. The reportable value for non-reduced samples is the time-corrected area percent (TCA)% of the major IgG peak.

[0099] Protein concentration Protein concentration is determined by UV 280 nm.

[0100] viscosity The viscosity of the antibody formulation was measured with a chip-based microVISC™ instrument (Rheosense), where the pressure difference correlates with the dynamic viscosity of the solution. Sample sizes were approximately 70-100 μL. Aliquots were loaded into 400 mL microVISC™ disposable pipettes and connected to the tip. Measurements were performed at a shear rate of 500 S. -1 , and perform this in triplicate at a temperature of approximately 25°C.

[0101] thermal stability Stability determinations were performed using Uncle™ (Unchained Labs), which combines three different measurement modes: fluorescence, static light scattering (SLS), and dynamic light scattering (DLS). SLS and intrinsic fluorescence methods were performed to determine the onset of aggregation (Tagg) and melting temperature (Tm) of the formulations, respectively.

[0102] Turbidity As an indicator of turbidity, UV absorbance is measured at 350 nm using a 96-well plate Molecular Devices M2e™ reader. Absorbance readings are controlled against blank well readings and normalized to sample path length.

[0103] Visible particles Visible particles were examined against black and white backgrounds using approximately 2000 lux white fluorescent lighting. The vial under investigation was gently rotated and examined for at least 5 seconds against each background.

[0104] Particles that cannot be seen with the naked eye Micro-flow imaging (MFI, Micro-FlowImaging™ 5200, ProteinSimple) was used to analyze particles that are not visible to the naked eye. Water flushes were performed between each analysis. In addition, water flushes were performed prior to sample analysis to ensure that background counts were adequate for testing. The average cumulative counts per mL were reported.

[0105] Example 1: Effect of pH on the stability of low concentration PD-1 antibody formulations Tislelizumab (Table 1) was prepared and purified to determine the antibody stability within a range of pH. Initially, low concentrations of antibody (10 mg / ml) were prepared by dialyzing tislelizumab into 20 mM disodium hydrogen phosphate-citrate buffers of different pH (5.0, 5.5, 6.0, 6.5, and 7.0) using a 10 kDa MWCO dialysis cassette. The antibody solution was filtered through a Millex™ GP 0.22 μm PES 33 mm filter and filled into 2 mL ready-to-use glass vials (Schott). To evaluate the effect of low concentrations on antibody stability, antibody samples were placed in a 40°C stability chamber for 2 weeks (labeled "40C2W" in Figures 1A-B) and then exposed to light for 2 weeks in a 10°C stability chamber (labeled "pho2W" in Figures 1A-B). As a control, samples were evaluated at the starting time point (labeled as T0 in the graphs). All samples were measured for purity by SEC-HPLC and charge heterogeneity by CZE, and the results are shown graphically in Figures 1A-B.

[0106] A clear trend was observed in the pH-dependent charge profile, with low pH formulations exhibiting a higher main peak% under both heat and light stress. The highest antibody aggregates were observed in the pH 7.0 formulation under heat stress at pH 5.0 and light stress. The SEC-HPLC results (Figure 1A) and CZE results (Figure 1B) taken together showed that low concentration anti-PD-1 antibody formulations at pH 5.5-6.5 were relatively more stable than those at pH values ​​of 5.0 and 7.0. Therefore, it can be concluded that the pH range of pH 5.5-6.5 provides antibody stability.

[0107] Example 2: Effect of pH on the viscosity and stability of high concentration PD-1 antibody formulations This data was used in the high antibody concentration experiments, with the pH range determined for the low antibody concentration. To prepare stock solutions of tislelizumab, tislelizumab was buffer exchanged into the various buffers listed in Table 2 using 10 kDa MWCO dialysis cassettes. To prepare the trehalose-containing (150 mM) high concentration formulation, the high concentration trehalose stock solution was spiked into the tislelizumab stock solution, which was then concentrated to approximately 150 mg / ml tislelizumab using a 30 kDa Amicon Ultra™ centrifugal filter.

[0108] Protein concentration and viscosity were measured. The results are shown in Table 2. The results showed that formulations formulated in histidine-histidine HCl buffer at pH 5.5 and pH 6.0 showed the lowest viscosity. At a concentration of 150 mg / ml of tislelizumab in the same histidine-histidine HCl buffer, the viscosity increased when the pH was above 6.0. As shown in Table 2, tislelizumab formulations F4 and F5 provided good results in terms of good viscosity and low aggregation. Tislelizumab antibody formulation F5 was notable for a viscosity (cP) of 15.53 and an aggregation index of 2.99.

[0109] To determine the freeze-thaw stability of high concentration (approximately 150 mg / mL) formulations of tislelizumab, each of the formulated tislelizumab solutions was filtered through a Millex™ GP 0.22 μm PES 33 mm filter and filled into 2 mL glass vials. Samples were subjected to three cycles of freezing at −40° C. and thawing at room temperature (freeze-thaw (3FT)). In addition, samples were stored at 40° C. for two weeks (40C2W). Aggregate formation was assessed by SEC-HPLC. As a control, samples were analyzed at the start time point and denoted as T0 in the table. The results are summarized in Table 2. The SEC-HPLC results showed that similar amounts of aggregates were formed across the range of formulations tested in the freeze-thaw experiment, with most of the values ​​between the start time point and the end of freeze-thaw being identical. This indicates that these tislelizumab antibody formulations provided adequate stability to the antibody at high concentrations. For tislelizumab antibody formulations stored at 40°C for 2 weeks (40C2W), an increase in aggregates of approximately 0.6% to 1.5% was detected, again indicating that these formulations provided adequate stability for high concentrations of tislelizumab antibody. The lowest amount of aggregates was observed in formulation 4 (F4) stored at 40°C for 2 weeks and formulation 5 (F5) during the freeze-thaw cycle.

[0110] Considering both aggregation and solution viscosity, high-concentration anti-PD-1 antibody formulations containing histidine-histidine HCl (particularly pH 6.0) buffer produced the best results. [Table 2]

[0111] Example 3: Evaluation of structural and colloidal stability of low concentration PD-1 antibody formulations containing NaCl This experiment determined the effect of pH and NaCl on the structural and colloidal stability of anti-PD-1 antibodies. In this study, NaCl and trehalose stock solutions of various concentrations were prepared in 20 mM histidine-histidine HCl buffers at pH 5.5, 6.0, and 6.5. Tislelizumab was then buffer exchanged into 20 mM histidine-histidine HCl buffers (pH 5.5, 6.0, and 6.5) by dialysis to prepare stock solutions of tislelizumab. Trehalose and / or NaCl stock solutions were spiked into the tislelizumab stock solutions to achieve the desired target excipient concentrations (Table 3). The final antibody concentration of each sample was adjusted to 10 mg / ml.

[0112] The structural and colloidal stability of tislelizumab in formulations containing NaCl (Table 3) was evaluated by measuring the denaturation midpoint temperature (Tm) and aggregation onset temperature (Tagg). The Tm and Tagg values ​​showed a clear downward trend with increasing NaCl concentration, but showed a slight upward trend with increasing pH. The addition of 150 mM NaCl significantly decreased the structural and colloidal stability of the tislelizumab formulation. These results indicate that at low antibody concentrations, tislelizumab formulations at pH 5.5-6.0 containing 50-100 mM NaCl showed the best structural and colloidal stability. [Table 3]

[0113] Example 4: Viscosity of high concentration PD-1 antibody formulations This experiment determined the effect of various concentrations of trehalose and NaCl on the viscosity of high-concentration anti-PD-1 antibody formulations. In this study, various concentrations of NaCl and trehalose stock solutions were prepared in 20 mM histidine-histidine HCl buffer (pH 6.0). Tislelizumab was then buffer-exchanged into 20 mM histidine-histidine HCl buffer (pH 6.0) by dialysis to prepare high-concentration tislelizumab stock solutions. Trehalose stock solutions and / or NaCl stock solutions were spiked into the tislelizumab high-concentration antibody stock solutions (Tables 4 and 5). Samples were concentrated to various concentrations by using 30 kDa Amicon Ultra™ centrifugal filters.

[0114] Viscosity analysis, flow rate 500S -1 , and was carried out at a temperature of about 25°C. The results are shown in Table 4. The data show that the viscosity of the anti-PD-1 antibody formulations increased exponentially with increasing antibody concentration. Compared to the base buffer formulation, the addition of 100 mM trehalose and 240 mM trehalose increased the viscosity at both the 100 mM trehalose concentration and especially at the higher concentration of 240 mM trehalose. Subcutaneous formulations have an element of "injectability", which is the ability of a subcutaneous formulation to be administered through the needle of a syringe (e.g., 20-25 gauge). Thus, the addition of a viscosity-reducing agent was necessary.

[0115] In particular, the addition of 50 mM NaCl reduced the viscosity of the high-concentration anti-PD-1 antibody formulation. In contrast, the addition of 100 mM NaCl caused a comparable viscosity reduction, and 140 mM NaCl caused a slightly more significant viscosity reduction effect. As shown in Example 3, tislelizumab antibody formulated with 50-100 mM NaCl at pH 5.5-6.0 showed the best structural and colloidal stability. Considering both the reduction in viscosity and the structural and colloidal stability, the addition of 50-100 mM NaCl to the high-concentration anti-PD-1 antibody formulation was investigated. [Table 4]

[0116] The viscosity and osmolality of anti-PD-1 antibody formulations containing 50-100 mM NaCl in the presence of trehalose were also studied (shown in Table 5). Results showed similar viscosity values ​​for the combination of 50 mM NaCl and 100 mM trehalose (with a viscosity of 13.98 cP at 154.70 mg / ml tislelizumab). The combination of 70 mM NaCl and 80 mM trehalose had a viscosity of 13.43 cP at 152 mg / ml tislelizumab, and 100 mM NaCl and 70 mM trehalose had a viscosity of 11.52 cP at 153.64 mg / ml tislelizumab.

[0117] When the antibody concentration reached approximately 180 mg / ml, the viscosity values ​​were all around 30 cP. In terms of viscosity, all of the formulations tested showed improved injection performance. Specifically, the viscosities generated by the high-concentration tislelizumab formulations showed good compatibility with syringes containing 23- or 25-gauge needles typically used for subcutaneous administration. Furthermore, the viscosity of the combination of 50 mM NaCl and 100 mM trehalose (33.19 cP at an antibody concentration of 184.51 mg / ml) was higher than that of 50 mM NaCl alone (26.92 cP at 182.49 mg / mL), indicating that the addition of up to 100 mM trehalose was considered based on both viscosity and osmolality. [Table 5]

[0118] Example 5: Stability of high concentration PD-1 antibody formulations The stability of high concentration tislelizumab antibody was evaluated in various formulations listed in Table 6. All formulations were prepared in 20 mM histidine-histidine HCl buffer (pH 6.0). Formulations F15 and F16 were prepared with a combination of 100 mM NaCl and 70 mM trehalose, whereas formulation F17 was prepared with a combination of 50 mM NaCl and 100 mM trehalose. - In these formulations, the concentration of polysorbate 20 ranged from 0 to 0.8 mg / mL (equivalent to 0.08%).

[0119] Tislelizumab was buffer exchanged into 20 mM histidine-histidine HCl buffer (pH 6.0) by dialysis to obtain a stock solution of tislelizumab. A stock solution of a combination of NaCl and trehalose was prepared in 20 mM histidine-histidine HCl buffer (pH 6.0) and mixed into the tislelizumab stock solution. The sample was then concentrated to approximately 150 mg / mL using a 30 kDa Amicon Ultra™ centrifugal filter. Formulations of various concentrations of polysorbate 20 (PS20:0, 0.2 mg / ml, and 0.8 mg / ml) were made by adding high concentration PS20 stock solution. Each of the formulated solutions was filtered using a 0.22 μm PES syringe filter and filled into 2 mL ready-to-use glass vials with a drug product fill volume of 0.5 mL.

[0120] Freeze-thaw studies were performed by subjecting the vials to three cycles of freezing at -40°C and thawing at room temperature (denoted as "3FT" on the graph). To study the high temperature and agitation stability of the formulations, samples were either stored in a stability chamber at 40°C for 4 weeks (denoted as "40C4W" on the graph) or mechanically stressed by agitation for 48 hours (denoted as "SK" on the graph). The formulations were evaluated by A350, SEC (purity), CZE (charge profile), and CE-SDS(NR) (purity). As a control, samples were analyzed at the start time point (denoted as T0 on the graph). These results are presented in Table 8 and Figures 2-4.

[0121] The turbidity of the pharmaceutical products was determined by measuring the optical density at 350 nm. Samples stored at 40°C and 25°C showed a slight increase in turbidity, and the A350 of the sample without PS20 was found to be higher than the other samples. Under shaking conditions, a more pronounced increase in A350 was observed for the sample without PS20, while no significant change was observed for the other samples. There was no measurable change in turbidity due to freeze-thaw stress for any of the formulations.

[0122] At 40°C, there was a slight decrease in antibody stability and a corresponding increase in aggregate amount was observed in formulations F15, F16, and F17. Furthermore, there was no significant change in the SEC purity of the formulations due to shaking and freeze-thaw stress. The SEC purity of all formulations was within the clinical acceptance criteria of 95.0% of monomer. Samples stored at 40°C showed a decrease in the main peak % and total basic peak % (data not shown) with a corresponding increase in the acidic peak % (data not shown). Purity by CE-SDS (NR) showed that storage at 40°C for 4 weeks resulted in only a slight reduction in monomer. There was no difference between the different formulations at 40°C.

[0123] In conclusion, these results showed that high concentration tislelizumab formulations containing PS20 (e.g., F16 and F17) were stable upon agitation, freeze-thaw, and heat stress, and storage at 5°C and 25°C for 6 months. Subsequent additional studies were conducted to determine the long-term and accelerated stability of formulation 18 (F18) containing 70 mM NaCl and 80 mM trehalose. Concentrated tislelizumab drug substance was prepared to approximately 200 mg / mL in 20 mM histidine-histidine HCl buffer (pH 6.0) by concentration and diafiltration. Formulation F18 was prepared by blending stock solutions of NaCl, trehalose, and polysorbate 20 with tislelizumab drug substance to obtain the target composition listed in Table 6. Each of the formulated solutions was filtered using a 0.22 μm PES syringe filter and filled into 2 mL ready-to-use glass vials with a drug product fill volume of 2 mL. Samples were staged and placed in stability chambers at 5±3° C. and 25° C. with two vials per time point. The planned duration of the study was 24 months at 5±3° C. and 6 months at 25° C.

[0124] Table 9 summarizes the visible and subvisible particle results for formulation F18 at 152 mg / ml up to 24 months. SEC (purity), CZE (charge profile), and CE-SDS(NR) (purity) results up to 24 months are presented in Figure 5A-D. Formulation F18 at 152 mg / ml was visually inspected and found to be virtually free of visible particles at 24 months at 5 ± 3 °C and 6 months at 25 °C. No significant changes were observed in particles not visible to the naked eye at 5 ± 3 °C, and an increase in particles 10 μm or larger was detected at 25 °C. Additionally, no significant changes were detected in aggregates, SEC monomers, major CZE peaks, and CE-SDS(NR) purity at 5 ± 3 °C. Storage at 25 °C for 6 months resulted in a slight increase in aggregates and a decrease in the major peaks of the sample in CZE and CE-SDS(NR) purity. However, for liquid formulations with intended storage conditions of 5±3°C, an increase in subvisible particles and aggregates and a decrease in the major peaks of CZE and CE-SDS(NR) purity at 25°C are acceptable.

[0125] Therefore, taking all these results together, formulations F16, F17, and F18 are suitable subcutaneous anti-PD-1 antibody formulations suitable for clinical use. In particular, based on several months of stability data, formulation F18 at 152 mg / ml is stable under the recommended storage conditions (up to 24 months at 5°C) with no measurable changes in the quality attributes of the product. [Table 6] [Table 7] [Table 8] [Table 9]

[0126] Example 6: Bioavailability of Subcutaneous Injection Formulation 18 (F18, Table 6) was used to test the bioavailability of the subcutaneous formulation. Tislelizumab was administered intravenously (iv) to mice and compared to F18 administered at 10 mpk (mg / kg) subcutaneously in the back of the animal, 10 mpk subcutaneously in the abdomen of the animal, or 20 mpk subcutaneously in the abdomen of the animal. C57 mice with human PD-1 knock-in were used in this study. The overall bioavailability of the subcutaneous injection was 75.8%. The data is shown below in Table 10. [Table 10]

[0127] Another study used NOD-SCID mice. Tislelizumab was administered intravenously (iv) to mice and compared to 10 mpk (mg / kg) F18 administered subcutaneously to the back of the animals, 20 mpk administered subcutaneously to the back of the animals, 10 mpk administered subcutaneously to the abdomen of the animals, or 20 mpk administered subcutaneously to the abdomen of the animals. Overall bioavailability from the subcutaneous injections was 54.8%. This data is shown below in Table 11. [Table 11]

[0128] The F18 formulation was also tested in larger animals (Sprague Dawley rats). Tislelizumab was administered intravenously at 100mpk (10mg / ml), subcutaneously in the abdominal region of rats at 100mpk (150mg / ml), subcutaneously in the abdominal region at 100mpk (100mg / ml), subcutaneously in the abdominal region at 200mpk (150mg / ml), or subcutaneously in the dorsal region at 100mpk (150mg / ml). The overall bioavailability was 57.2% and the data is shown in Figures 6A-B.

[0129] The F18 tislelizumab formulation was also tested in a minipig model. In this study, 6mpk was administered intravenously to minipigs, either by subcutaneous injection in the leg or by subcutaneous injection behind the ear of the animals. The overall bioavailability of tislelizumab by subcutaneous injection was 79.8%. The data is presented in Table 12. [Table 12]

[0130] Finally, the subcutaneous tislelizumab formulation was tested in a monkey PK study. Three monkeys were dosed with 30 mg / kg of the F18 tislelizumab formulation and blood was collected at 0, 4, 8, 24, 48 hours, 4 days, 7 days, 14 days, and 21 days after dosing. The results are shown in Figure 7, where the subcutaneous results were compared to the AUC of a previously conducted monkey study using the IV formulation and dosing. There were no obvious changes in body weight and no clinical pathology. There were no histopathological changes at the injection site.

[0131] The subcutaneous formulation of tislelizumab was well tolerated with no injection site reactions in either the rat or minipig models regardless of the injection site or concentration used as shown in FIG. 8.

Claims

1. Low viscosity pharmaceutical formulation, Antiprogrammed cell death receptor 1 (PD-1) antibody in concentrations of approximately 10 mg / mL to approximately 200 mg / mL, or its antigen-binding fragment. A formulation buffer that provides a pH of approximately 5.0 to 7.

0. Glycopolyols, Viscosity reducing agent, and Contains a nonionic surfactant, The low-viscosity pharmaceutical preparation having a viscosity of 30 cP or less and an osmotic pressure of about 200 mOsmol / kg to about 400 mOsmol / kg.

2. The formulation according to claim 1, wherein the PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region including (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 1; (b) HCDR2 of SEQ ID NO: 2; (c) HCDR3 of SEQ ID NO: 3; and a light chain variable region including (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 4; (e) LCDR2 of SEQ ID NO: 5; and (f) LCDR3 of SEQ ID NO:

6.

3. The formulation according to claim 2, wherein the formulation buffer is selected from the group consisting of histidine, acetate, citrate, succinate, phosphoric acid, a mixture of histidine and acetic acid, or a mixture of histidine and citric acid, and preferably the formulation buffer is histidine.

4. The formulation according to claim 3, wherein the concentration of the buffer solution is a) 15 mM to 25 mM, or b) 20 mM.

5. The formulation according to claim 4, wherein the pH is 5.5 to 6.

0.

6. The formulation according to claim 1, wherein the sugar polyol is selected from the group consisting of trehalose, sucrose, sorbitol, mannitol, maltose, dextran, or (2-hydroxypropyl)-β-cyclodextrin, and optionally the sugar polyol is trehalose.

7. The concentration of the aforementioned trehalose is a) 70mM to 240mM, b) 80mM to 160mM, c) 70 mM to 100 mM, d) The formulation according to claim 1, wherein the concentration is 80 mM.

8. The formulation according to claim 1, wherein the viscosity-reducing agent is an inorganic salt selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, sodium acetate, sodium sulfate, ammonium chloride, or ammonium sulfate.

9. The inorganic salt is a) 50 mM to 150 mM, b) The formulation according to claim 8, wherein the sodium chloride is 70 mM in concentration.

10. The formulation according to claim 1, wherein the nonionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, or poloxamer 188.

11. The concentration of the polysorbate 20 is a) 0.02% to 0.08%, or b) The formulation according to claim 10, wherein the concentration is 0.08%.

12. a) The formulation comprises 20 mM histidine-histidine HCl, 100 mM NaCl, 70 mM trehalose, and 0.08% polysorbate 20, and has a pH of 6.

0. b) The formulation comprises 20 mM histidine-histidine HCl, 50 mM NaCl, 100 mM trehalose, and 0.02% polysorbate 20, and has a pH of 6.0, or c) The formulation according to claim 1, wherein the formulation comprises 20 mM histidine-histidine HCl, 70 mM NaCl, 80 mM trehalose, and 0.08% polysorbate 20, and has a pH of 6.

0.

13. The formulation according to claim 1, wherein the concentration of the anti-human PD-1 antibody or its antigen-binding fragment is approximately 100 mg / mL to 200 mg / mL.

14. A method for producing an antibody preparation, wherein the method is a) To obtain an antibody preparation having a trehalose concentration of 50 mM or more and a sodium chloride concentration of 25 mM or more by adding trehalose and sodium chloride to an antibody, wherein the antibody comprises a heavy chain variable region including (a) HCDR1 (heavy chain complementarity determination region 1) of SEQ ID NO: 1; (b) HCDR2 of SEQ ID NO: 2; (c) HCDR3 of SEQ ID NO: 3; and a light chain variable region including (d) LCDR1 (light chain complementarity determination region 1) of SEQ ID NO: 4; (e) LCDR2 of SEQ ID NO: 5; and (f) LCDR3 of SEQ ID NO:

6. b) Concentrating the antibody preparation in (a) to 150-200 mg / mL, c) Adding polysorbate 20 to the antibody preparation of (b) to obtain an antibody preparation in which the concentration of polysorbate 20 is 0.01 mg / mL or higher, The method wherein the antibody preparation (c) is an aqueous solution, has a viscosity of 30 cP or less at 25°C, and the antibody preparation (c) is stable against stirring, freeze-thaw cycles, and heat stress.

15. A formulation according to any one of claims 1 to 13 for treating cancer in human patients requiring cancer treatment.

16. The aforementioned formulation, a) Approximately 100 mg to approximately 1000 mg of the anti-human PD-1 antibody, b) Approximately 200 mg of the anti-human PD-1 antibody, c) Approximately 300 mg of the anti-human PD-1 antibody, d) Approximately 400 mg of the anti-human PD-1 antibody, or e) Approximately 500 mg of the anti-human PD-1 antibody The formulation according to claim 15, which is administered subcutaneously in the specified dose.

17. The aforementioned formulation, a) Once a week, b) Once every two weeks, c) The formulation according to claim 16, which is administered subcutaneously once every three weeks.

18. The preparation according to claim 15, wherein the cancer is lung cancer (including small cell lung cancer or non-small cell lung cancer), adrenal cancer, liver cancer, gastric cancer, cervical cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, leukemia, bladder cancer, bone cancer, brain cancer, endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumor, or esophageal cancer.

19. The formulation according to claim 15, wherein the human patient is administered at least one other therapeutic agent.

20. The formulation according to claim 19, wherein the at least one other therapeutic agent is zanubrutinib, pamiparib, anti-CTLA4 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-TIGIT antibody, anti-TIM-3 antibody, second PD-1 antibody, CD40 agonist, TLR agonist, CAR-T cell, or chemotherapeutic agent.

21. The formulation according to claim 1, wherein the formulation comprises 20 mM histidine buffer, has a pH of 5.5 to 6.0, the sugar polyol is trehalose, the concentration of the trehalose is 70 mM to 240 mM, the viscosity reducing agent is an inorganic salt, the inorganic salt is sodium chloride at a concentration of 50 mM to 150 mM, the nonionic surfactant is polysorbate 20, and the concentration of the polysorbate 20 is 0.04%.

22. The formulation according to claim 21, wherein the concentration of sodium chloride is 50 mM to 100 mM, preferably 70 mM.

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