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

Abstract

Pharmaceutical formulations of antibodies or antigen-binding fragments thereof against the human programmed cell death receptor PD-1 are provided. The formulations may further comprise a formulation buffer, a viscosity-lowering agent, and a non-ionic surfactant. The pharmaceutical formulations of the invention exhibit 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 using the formulations to treat cancer. [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 properties 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 invention provides stable, low-viscosity and highly concentrated antibody formulations. 1. A low viscosity pharmaceutical formulation comprising: 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; Viscosity reducers, and a non-ionic surfactant, A low-viscosity pharmaceutical formulation, wherein the formulation has a viscosity of 35 cP or less and an osmotic pressure of about 200 mOsmol / kg to about 400 mOsmol / kg.

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

[0010] The formulation, wherein the concentration of the buffer is 15 mM to 25 mM.

[0011] A formulation comprising a 20 mM histidine buffer or a 20 mM acetate buffer.

[0012] The formulation has a pH of 5.0 to 6.0.

[0013] An anti-human PD-1 antibody formulation, wherein the viscosity-reducing agent is an arginine salt.

[0014] A formulation in which the arginine salt is an equal mixture of 50 mM to 280 mM L-arginine and L-glutamic acid (ArgGlu).

[0015] A formulation, wherein the arginine salt is 50 mM to 280 mM of a complex salt of L-arginine and L-glutamic acid (ArgGlu).

[0016] A formulation in which the arginine salt is an equal mixture of 50 mM to 280 mM L-arginine and L-aspartic acid (ArgAsp).

[0017] A formulation, wherein the arginine salt is 50 mM to 280 mM of a complex salt of L-arginine and L-aspartic acid (ArgAsp).

[0018] The formulation, wherein the arginine salt is 50 mM to 280 mM L-arginine hydrochloride (ArgHCl).

[0019] A formulation wherein the non-ionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, or poloxamer 188.

[0020] A formulation having a concentration of polysorbate 80 of 0.02% to 0.08%.

[0021] A formulation in which the concentration of polysorbate 20 is 0.05%.

[0022] A formulation comprising 20 mM Histidine-Histidine HCl, 140 mM ArgGlu, 0.05% Polysorbate 80, and a pH of pH 5.5.

[0023] A formulation comprising 20 mM acetate, 140 mM ArgAsp, 0.05% polysorbate 80, and a pH of pH 5.5.

[0024] A formulation comprising 20 mM Histidine-Histidine HCl, 140 mM ArgHCl, 0.05% Polysorbate 80, and a pH of pH 5.5.

[0025] The formulation, wherein the concentration of the anti-human PD-1 antibody or antigen-binding fragment thereof is about 100 mg / mL to 200 mg / mL.

[0026] 1. A method for treating cancer in a human patient in need of such treatment, comprising subcutaneously administering an effective amount of an anti-human PD-1 antibody formulation.

[0027] The method, wherein the anti-human PD-1 antibody formulation is administered at a dose of about 100 mg to about 1000 mg.

[0028] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 200 mg.

[0029] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 300 mg.

[0030] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 400 mg.

[0031] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 500 mg.

[0032] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously once every three weeks.

[0033] The method, wherein the anti-human PD-1 antibody formulation is administered once a week.

[0034] The method, wherein the anti-human PD-1 antibody formulation is administered once every two weeks.

[0035] The method, wherein the anti-human PD-1 antibody formulation is administered once every three weeks.

[0036] 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.

[0037] The method wherein the human patient is administered at least one other therapeutic agent.

[0038] The method, wherein the at least one other therapeutic agent 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 second PD-1 antibody, a CD40 agonist, a TLR agonist, a CAR-T cell, or a chemotherapeutic agent.

[0039] In some embodiments, the antibody formulation comprises an anti-PD-1 antibody or antigen-binding fragment thereof, a formulation buffer, 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 50 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 freeze-thaw stress and storage at 5°C and 25°C for 3 months.

[0040] In some embodiments, the antibody formulation may comprise or consist essentially of about 75 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or antigen-binding fragment thereof, a formulation buffer, a viscosity-lowering agent, and a non-ionic surfactant, and has a pH of about 5.5±0.5. In some embodiments, the antibody formulation may consist essentially of about 100 mg / mL to about 200 mg / mL of an anti-PD-1 antibody, a formulation buffer, a viscosity-lowering agent, and a non-ionic surfactant, and has a pH of 5.5±0.5.

[0041] 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.

[0042] In other embodiments, the formulation buffer is an acetate buffer, hi some embodiments, the concentration of the acetate buffer is about 5 mM to about 30 mM, preferably about 20 mM.

[0043] In some embodiments, the viscosity-reducing agent is an amino acid or a derivative thereof. In some embodiments, the amino acid or a derivative thereof is L-arginine, arginine hydrochloride (ArgHCl), arginine acetate, arginine citrate, arginine succinate, arginine phosphate, arginine sulfate, arginine glutamate, arginine aspartate, L-proline, lysine hydrochloride, sodium glutamate (NaGlu), histidine glutamate, or histidine hydrochloride. In some embodiments, the amino acid or a derivative thereof is arginine hydrochloride, arginine acetate, arginine citrate, arginine glutamate, arginine aspartate, lysine hydrochloride, preferably arginine hydrochloride, arginine glutamate, and arginine aspartate.

[0044] In some embodiments, the concentration of the amino acid or derivative thereof is about 25 mM to about 400 mM. In some embodiments, the concentration of the amino acid or derivative thereof is about 50 mM to about 280 mM, preferably about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, and about 140 mM.

[0045] In some embodiments, the concentration of the polysorbate is about 0.01 to about 1.0 mg / mL, preferably about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, or about 0.5 mg / mL. In some embodiments, the polysorbate is polysorbate 80 or polysorbate 20, preferably polysorbate 80.

[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 0.01 to about 1.0 mg / mL, 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 80.

[0047] In some embodiments, the antibody formulation consists of 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 190 mg / mL, or about 200 mg / mL of an anti-PD-1 antibody or antigen-binding fragment thereof.

[0048] In some embodiments, the pharmaceutical composition comprises: a) about 150 mg / mL to about 200 mg / mL of an anti-human PD-1 antibody or antigen-binding fragment thereof; b) about 20 mM histidine buffer or about 20 mM acetate buffer; c) about 100 mM to about 140 mM arginine hydrochloride; and d) about 0.2 to about 0.5 mg / mL polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.5±0.5. In certain embodiments, the arginine hydrochloride is a complex salt of arginine hydrochloride or a mixture of L-arginine and hydrochloric acid.

[0049] In another embodiment, the pharmaceutical composition comprises: a) about 150 mg / mL to about 200 mg / mL of an anti-human PD-1 antibody or antigen-binding fragment thereof; b) about 20 mM histidine buffer or about 20 mM acetate buffer; c) about 100 mM to about 140 mM arginine glutamate; and d) about 0.2 to about 0.5 mg / mL polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.5±0.5. In a specific embodiment, the arginine glutamate is a complex salt of arginine glutamate or a mixture of L-arginine and L-glutamic acid.

[0050] In another embodiment, the pharmaceutical composition comprises: a) about 150 mg / mL to about 200 mg / mL of an anti-human PD-1 antibody or antigen-binding fragment thereof; b) about 20 mM histidine buffer or about 20 mM acetate buffer; c) about 100 mM to about 140 mM arginine aspartate; and d) about 0.2 to about 0.5 mg / mL polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.5±0.5. In a specific embodiment, the arginine aspartate is a complex salt of arginine aspartate or a mixture of L-arginine and L-aspartic acid.

[0051] In some embodiments, the composition may have a viscosity at 25°C of less than about 50 cP, less than about 40 cP, preferably less than about 35 cP.

[0052] In some embodiments of the invention, the PD-1 antibody is tislelizumab (BGB-A317) or an antigen-binding fragment of tislelizumab.

[0053] 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 an antibody formulation described herein.

[0054] Provided herein are methods of treating cancer in a human patient suffering from a PD-L1-expressing cancer, the methods comprising subcutaneously administering to the patient an effective amount of an antibody formulation described herein.

[0055] In some embodiments, the present disclosure provides a method of treating cancer with an anti-PD-1 subcutaneous antibody formulation in combination with another therapeutic agent, such as zanubrutinib, pamiparib, sitravatinib, an anti-CTLA4 antibody, an anti-4-1BB antibody, an anti-OX40 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, a second PD-1 antibody, a CD40 agonist, a TLR agonist, a CAR-T cell, or a chemotherapeutic agent. [Brief description of the drawings]

[0056] [Figure 1] The amount of aggregates (FIG. 1A) and monomers (FIG. 1B) of each of the formulations (measured by SEC-HPLC) is shown. "T0" refers to the starting point of the sample. "40C4W" refers to the sample stored at 40° C. for 4 weeks. "3FT" refers to the sample that was subjected to 3 freeze-thaw cycles. "25C3M" refers to the sample stored at 25° C. for 3 months. "5C3M" refers to the sample stored at 5° C. for 3 months. "25C6M" refers to the sample stored at 25° C. for 6 months. "5C6M" refers to the sample stored at 5° C. for 6 months. [Diagram 2] Figure 1 shows the results of a CZE study of the formulation. "T0" refers to the starting point of the sample. "40C4W" refers to the sample stored at 40°C for 4 weeks. "25C3M" refers to the sample stored at 25°C for 3 months. "5C3M" refers to the sample stored at 5°C for 3 months. "25C6M" refers to the sample stored at 25°C for 6 months. "5C6M" refers to the sample stored at 5°C for 6 months. [Diagram 3] Purity of the formulation as measured by CE-SDS under non-reducing conditions is shown. "T0" refers to the starting point of the sample. "40C4W" refers to the sample stored at 40°C for 4 weeks. "25C3M" refers to the sample stored at 25°C for 3 months. "5C3M" refers to the sample stored at 5°C for 3 months. "25C6M" refers to the sample stored at 25°C for 6 months. "5C6M" refers to the sample stored at 5°C for 6 months. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] definition Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0058] 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.

[0059] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0060] 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."

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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 an intended target if its binding determines the presence of the target protein in a sample (e.g., does not produce undesirable results such as false positives). An antibody or binding fragment thereof useful in the present invention will bind to a target protein with an affinity 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. An antibody herein is said to specifically bind to a polypeptide comprising a given amino acid sequence.

[0073] 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.

[0074] 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.

[0075] The antibody of the present application has potential therapeutic use 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, stomach 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.

[0076] 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.

[0077] 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]

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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).

[0082] 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).

[0083] 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.

[0084] 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).

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] Pharmaceutical Compositions and Formulations Compositions, including pharmaceutical formulations, that comprise an anti-PD-1 antibody or antigen-binding fragment thereof, or a polynucleotide that comprises a sequence encoding an anti-PD-1 antibody or antigen-binding fragment are also provided. In certain embodiments, the compositions comprise one or more antibodies or antigen-binding fragments that bind to PD-1, or one or more polynucleotides that comprise a sequence encoding one or more antibodies or antigen-binding fragments that bind to PD-1. These compositions may further comprise a suitable carrier, such as a pharma- ceutically acceptable excipient, including a buffering agent.

[0094] Pharmaceutical formulations of the anti-PD-1 antibodies or antigen-binding fragments described herein are prepared in the form of a lyophilized formulation or aqueous solution by mixing such antibodies or antigen-binding fragments having the desired purity with any one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic 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., octadecyldimethylbenzyl ammonium 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 albumin, gelatin, etc.). Examples of suitable surfactants include, but are not limited to, 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), polysorbate 20, or polysorbate 80).

[0095] 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.

[0096] 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

[0097] 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.

[0098] Analysis method The Methods section outlines the methods used in Examples 1-5 below.

[0099] Protein concentration Variable path length and Lambert-Beer's law (A=cle, where A=absorbance, c=concentration, l=path length, e=1.5 mg ml -1 cm -1 Protein concentrations are determined spectrophotometrically using SoloVPE (C Technologies Inc.) based on the FTIR spectrophotometer. Samples are equilibrated to ambient temperature, added to sample containers (C Tech Inc. #OC0009-1-P50) and loaded into the container holder of the detection window platform. For each sample, a clean fiberlet (#OF0002-P50) is attached to the fiberlet coupler and the slope is measured at 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 μL microVISC™ disposable pipettes and coupled 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] 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.

[0102] SEC-HPLC The formation of soluble aggregates is analyzed by size exclusion chromatography (SEC) on a Waters HPLC system. 100 μg of protein is separated based on molecular size on a TSKgel G3000™ SWXL column maintained at 37±5° C. using an isocratic gradient method. 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.

[0103] CZE The charge heterogeneity of the 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 consisting mainly of caproic acid. Samples are analyzed in their native state 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.

[0104] 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.

[0105] Example 1: Preparation of high-concentration anti-PD-1 antibody formulations The anti-human PD-1 antibody, tislelizumab (BGB-A317, Table 1), was prepared and purified.

[0106] Preparation of Formulation 1 (F1): The tislelizumab antibody was buffer exchanged into 20 mM histidine-histidine HCl (pH 6.0) and then concentrated to approximately 200 mg / mL with a 30 kDa Amicon Ultra™ centrifugal filter. Samples of various concentrations were made by dilution with 20 mM histidine-histidine HCl (pH 6.0) buffer.

[0107] Preparation of Formulations 2 to 7 (F2 to F7) and Formulations 10 to 12 (F10 to F12): To prepare stock solutions of tislelizumab, tislelizumab was buffer exchanged into 20 mM histidine-histidine HCl, pH 6.0, using a 10 kDa MWCO dialysis cassette. Stock solutions of ArgGlu (a mixture of L-arginine and L-glutamic acid), ArgHCl, ArgAsp (a mixture of L-arginine and L-aspartic acid), proline, LysHCl, NaGlu (an equimolar mixture of L-glutamic acid and sodium hydroxide), a combination of ArgGlu and trehalose, a combination of ArgHCl and trehalose, and a combination of ArgHCl and proline, all dissolved in 20 mM histidine-histidine HCl, pH 6.0, were prepared. All stock solutions were adjusted to pH 6.0 with hydrochloric acid. All stock solutions were spiked into the tislelizumab stock solution to achieve the desired excipient concentrations shown in Table 2. Samples were then concentrated to approximately 200 mg / mL with a 30 kDa Amicon Ultra™ centrifugal filter. Formulations of various protein concentrations were made by dilution with 1× stock solution.

[0108] Preparation of formulations 8 and 9 (F8 and F9): To prepare a stock solution of tislelizumab, tislelizumab was buffer exchanged into 20 mM histidine-acetate, pH 6.0 and 20 mM histidine-citrate, pH 6.0 using a 10 kDa MWCO dialysis cassette. A stock solution of 500 mM (5x) arginine in 20 mM histidine-acetate, pH 6.0 and 20 mM histidine-citrate, pH 6.0 was prepared. The pH was adjusted to 6.0 using acetic acid and citric acid. The 5x arginine stock solution was spiked into a tislelizumab stock solution of 20 mM histidine-acetate, pH 6.0 and 20 mM histidine-citrate, pH 6.0. Samples were then concentrated to approximately 200 mg / mL using a 30 kDa Amicon Ultra™ centrifugal filter. Formulations of various protein concentrations were made by dilution with a 100 mM (1×) arginine stock solution.

[0109] This preparation method can also be applied to the preparation of other high-concentration anti-PD-1 antibody formulations. [Table 2]

[0110] Example 2: Viscosity-reducing effect of arginine salt in high-concentration PD-1 antibody formulation In this series of experiments, we determined the viscosity results for formulations 1-12 with various protein concentrations as described in Example 1. In this example, we investigated the viscosity reducing effect of arginine salt, proline, a combination of arginine salt and polyol, a combination of arginine and proline, and other organic electrolytes on viscosity and antibody stability variables.

[0111] Viscosity analysis, flow rate 500S -1 , at a temperature of about 25° C. The results are shown in Table 3. The data showed that the viscosity of the tislelizumab formulation was highly dependent on the protein concentration. In the absence of a viscosity-lowering agent, the viscosity increased exponentially (F1) when the protein concentration was increased from 160.42 mg / mL to 199.29 mg / mL. In the absence of a viscosity-lowering agent, the viscosity values ​​were 29.69 cP at a tislelizumab protein concentration of 173.08 mg / mL and 53.29 cP at an antibody concentration of 199.29 mg / mL. In an unexpected result, the addition of arginine salts (e.g., ArgGlu, ArgHCl, and ArgAsp) significantly reduced the viscosity of the high concentration tislelizumab antibody formulation. In the presence of arginine salt, the viscosity values ​​were 22.88 cP (189.47 mg / mL) and 30.62 cP (197.92 mg / mL) for F2 (ArgGlu), 21.24 cP (185.60 mg / mL) and 27.95 cP (191.70 mg / mL) for F3 (ArgHCl), and 20.70 cP (186.87 mg / mL) and 40.99 cP (211.33 mg / mL) for F4 (ArgAsp). At a tislelizumab concentration of approximately 200 mg / mL, the viscosity was reduced by approximately 42.5% with ArgGlu. The results for these formulations are summarized in Table 3 below.

[0112] We also evaluated the effect of proline (F5) as well as other organic electrolytes (e.g., NaGlu (F7) and LysHCl (F6)) in various tislelizumab formulations. Results showed that proline (F5) and NaGlu (F7) did not significantly reduce the viscosity, while a decrease in viscosity was observed with LysHCl (F6). However, the viscosity-reducing effect of LysHCl was less significant than ArgGlu, ArgHCl, and ArgAsp, as seen in F2-F4. The results for these formulations are summarized in Table 3 below.

[0113] Formulations 8 and 9 evaluated the viscosity-lowering effect of arginine in the presence of acetate counterions (e.g., arginine acetate (F8)) or citrate counterions (e.g., arginine citrate (F9)). Both arginine acetate and arginine citrate caused a decrease in viscosity of the tislelizumab formulation, but arginine acetate caused a greater decrease in viscosity than arginine citrate. However, the viscosity reduction caused by the presence of arginine acetate or arginine citrate was not as pronounced as that seen with ArgGlu, ArgHCl, and ArgAsp in F2-F4. The results for these formulations are summarized in Table 3 below.

[0114] Formulations 10, 11, and 12 investigated the effect of arginine salts and neutral organic combinations (e.g., trehalose and proline). Results showed that significant viscosity reductions were observed with the combination of 100 mM ArgHCl and 70 mM trehalose (F11), 100 mM ArgGlu and 70 mM trehalose (F10), and 100 mM ArgHCl and 100 mM proline (F12). In summary, the viscosity reduction caused by the combination of 100 mM arginine salt and neutral organics seen in formulations F10-F12 was slightly smaller than that caused by 140 mM arginine salt alone.

[0115] In conclusion, the excipients that had the greatest impact on the viscosity of high-concentration tislelizumab formulations included ArgGlu-, ArgHCl-, and ArgAsp-based formulations found in F2–F4. [Table 3-1] [Table 3-2]

[0116] Example 3: Effect of pH and buffer on the viscosity of high concentration PD-1 antibody formulations This example evaluated the effect of pH and various buffers on the viscosity of high-concentration tislelizumab formulations containing arginine salts. The formulations are summarized in Table 4. The preparation of high-concentration tislelizumab antibody was described in Example 1 above. Briefly, tislelizumab antibody stock solutions in 20 mM histidine-histidine HCl (pH 6.5, pH 6.0, and pH 5.5) and 20 mM acetic acid (pH 5.5 and pH 5.0) were prepared by dialysis through a 10 kDa MWCO dialysis cassette. A stock solution of viscosity-lowering agent (no polysorbate 80 (PS80)) was spiked into the tislelizumab stock solution, and the sample was then concentrated to approximately 200 mg / mL with a 30 kDa Amicon Ultra™ centrifugal filter. A high-concentration PS80 solution was then added to achieve a target concentration of 0.02%. Finally, formulations of various protein concentrations were made by dilution with a 1x stock solution of viscosity-lowering agent containing 0.02% PS80.

[0117] The viscosities of formulations F13-F18 containing ArgGlu and ArgHCl at a wide range of tislelizumab concentrations are summarized in Table 5. The results showed that for formulations containing arginine salt, the viscosity was significantly reduced when the pH was decreased from 6.5 to 5.0. At pH 5.5, the viscosity value of formulation F17 was 15.61 cP at an antibody concentration of 183.05 mg / mL, F18 was 16.32 cP at an antibody concentration of 183.17 mg / mL, and F19 was 16.68 cP at an antibody concentration of 178.60 mg / mL. At pH 5.0, the viscosity value of F20 was 18.66 cP at an antibody concentration of 185.68 mg / mL and 24.74 cP at an antibody concentration of 199.95 mg / mL. Notably, no significant change was observed in the viscosity of formulations containing acetate buffer compared to histidine buffer. In other words, changing the buffer may not affect the viscosity of a tislelizumab antibody formulation containing an arginine salt.

[0118] 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). Specifically, the viscosity generated by high-concentration tislelizumab formulations has shown good compatibility with syringes equipped with 23-gauge or 25-gauge needles typically used for subcutaneous administration. Thus, the protein concentrations of the high tislelizumab antibody formulations F17, F18, F19, and F20 can be 180-200 mg / mL, which is an acceptable viscosity for subcutaneous administration by syringe. [Table 4] [Table 5]

[0119] Example 4: Effect of changing arginine salt concentration on the viscosity of high-concentration PD-1 antibody formulations These experiments examined the effect of various concentrations of arginine salt on the viscosity of high concentration anti-PD-1 antibody formulations. To evaluate the effect of arginine salt concentration on viscosity, tislelizumab antibody was formulated in 20 mM histidine (pH 5.5), 0 / 50 / 100 / 280 mM arginine salt, and 0.2 mg / ml polysorbate 80 (PS80). The preparation methods described in Examples 1 and 3 were used.

[0120] Viscosity and osmolality were measured. The results are summarized in Table 6. The viscosity and osmolality of tislelizumab antibody formulations containing 0, 50, 100, 140, and 280 mM ArgHCl or ArgGlu at various antibody concentrations were tested. The results showed that the addition of 50 mM ArgHCl or ArgGlu significantly reduced the viscosity of the tislelizumab antibody formulation. Furthermore, gradually increasing the concentration of ArgHCl or ArgGlu up to 280 mM further reduced the viscosity.

[0121] The experimentally determined osmolality values ​​for each of these formulations are also shown in Table 6. The osmolality values ​​of the 140 mM ArgGlu and 140 mM ArgHCl formulations were 360-390 mOsmol / kg, which was slightly hypertonic. Ideally, injections should be formulated as isotonic solutions (osmolality approximately 300 mOsm / kg). However, for subcutaneous (SC) injections, strict isotonicity is not absolutely necessary. To minimize pain induced by hyperosmolality, it is recommended that the osmolality upper limit be controlled below 600 mOsmol / kg for pharmaceuticals for subcutaneous injection. Thus, administration of formulations containing the high concentration of arginine salt (140 mM) presented herein to reduce the viscosity of the formulation does not appear to present a risk of tissue damage at the injection site. [Table 6]

[0122] Example 5: Evaluation of the stability of PD-1 antibodies in low-viscosity and high-concentration formulations Further studies were conducted to evaluate the stability of formulations containing approximately 180-200 mg / mL of tislelizumab antibody. In this study, arginine hydrochloride (ArgHCl), arginine glutamate (ArgGlu), and arginine aspartate (ArgAsp) were used as viscosity-reducing agents to evaluate their effect on stress and storage stability.

[0123] In this study, tislelizumab antibody was formulated in 20 mM histidine / acetic acid (pH 5.5), 140 mM arginine salt in preparation of formulations F2-F7 as shown in Example 1. Subsequently, high concentration PS80 solution was added to achieve a target concentration of 0.05%. Each of the formulated solutions was filtered using Millex™ GP 0.22 μm PES 33 mm filters and filled into 2 mL ready-to-use glass vials (Schott) with a drug product fill volume of 0.5 mL. Samples were staged, protected from light, and placed in environmental stability chambers at 2-8°C, 25°C, and 40°C. Freeze-thaw stability was determined by subjecting the formulations to three freeze-thaw cycles.

[0124] For stability, the turbidity of the drug products was determined by measuring the optical density at 350 nm. There was no significant change in any of the formulations at 5° C. and 40° C. under freeze-thaw conditions.

[0125] The purity of the formulations was determined by SEC-HPLC. At 40°C, an increase in aggregates and a corresponding decrease in monomers was observed. Formulation F23 showed the greatest changes compared to the other formulations. However, these reductions were considered acceptable for a liquid formulation at the intended storage condition of 5°C. There was no change in SEC purity at 5°C and after freeze-thaw stress. An increase of approximately 0.4% to 0.6% in aggregates was detected in formulations F21 to F23 at 25°C for 6 months.

[0126] Charge heterogeneity was detected by CZE and assessed by monitoring the main peak along with the acidic and basic species. For any of the formulations, no measurable changes were seen in any of the individual peaks, including the main peak, for up to 6 months at 5°C. At 25°C for 6 months, only a slight decrease in the main peak was observed. At 40°C for 4 weeks, the main peak showed a significant decrease (indicating decreased stability of the antibody). Similar to the results observed at 25°C, an increase was observed in the acidic variants and a decrease in the basic peak was observed (data not shown).

[0127] Purity by CE-SDS was measured under non-reduced conditions. There was no measurable change in purity as a function of time for any of the formulations up to 6 months at 5° C. The % purity at 5° C., 25° C., and 40° C. were all >96%, within the clinical acceptance criteria of >90.0% for non-reduced CE-SDS.

[0128] The results showed that these formulations, containing approximately 180-200 mg / mL of tislelizumab antibody, were stable after three freeze-thaw cycles and after six months of storage at 5° C. and 25° C. These are summarized in Tables 7-9. [Table 7] [Table 8] [Table 9]

Claims

1. 1. A low viscosity pharmaceutical formulation comprising: about 10 mg / mL to about 200 mg / mL of an anti-Programmed Death Receptor 1 (PD-1) antibody, or antigen-binding fragment thereof; a formulation buffer providing a pH of about 5.0 to about 7.0; Viscosity reducers, and a non-ionic surfactant, The low viscosity pharmaceutical formulation, wherein the formulation has a viscosity of 35 cP or less and an osmolality of about 200 mOsmol / kg to about 400 mOsmol / kg.

2. 2. The formulation of claim 1, wherein the PD-1 antibody or antigen-binding fragment thereof 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.

3. 3. The formulation of claim 2, 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.

4. 4. The formulation of claim 3, wherein the formulation buffer is histidine.

5. 4. The formulation of claim 3, wherein the formulation buffer is acetate.

6. The formulation of claim 4 or 5, wherein the buffer concentration is from 15 mM to 25 mM.

7. 6. The formulation of claim 4 or 5, wherein the formulation comprises a 20 mM histidine buffer or a 20 mM acetate buffer.

8. The formulation according to claim 5 or 6, wherein the pH is from 5.0 to 6.

0.

9. The anti-human PD-1 antibody formulation of claim 1, wherein the viscosity-lowering agent is an arginine salt.

10. 10. The formulation of claim 9, wherein the arginine salt is an equal mixture of 50 mM to 280 mM L-arginine and L-glutamic acid (ArgGlu).

11. 10. The formulation of claim 9, wherein the arginine salt is 50 mM to 280 mM of a complex salt of L-arginine and L-glutamic acid (ArgGlu).

12. 10. The formulation of claim 9, wherein the arginine salt is an equal mixture of 50 mM to 280 mM L-arginine and L-aspartic acid (ArgAsp).

13. 10. The formulation of claim 9, wherein the arginine salt is 50 mM to 280 mM of a complex salt of L-arginine and L-aspartic acid (ArgAsp).

14. 10. The formulation of claim 9, wherein the arginine salt is 50 mM to 280 mM L-arginine hydrochloride (ArgHCl).

15. The formulation of any one of claims 1 to 14, wherein the non-ionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, or poloxamer 188.

16. 16. The formulation of claim 15, wherein the polysorbate 80 concentration is from 0.02% to 0.08%.

17. 17. The formulation of claim 16, wherein the polysorbate 20 concentration is 0.05%.

18. 2. The formulation of claim 1, wherein the formulation comprises 20 mM Histidine-Histidine HCl, 140 mM ArgGlu, 0.05% Polysorbate 80, and has a pH of pH 5.

5.

19. 2. The formulation of claim 1, wherein the formulation comprises 20 mM acetate, 140 mM ArgAsp, 0.05% polysorbate 80, and has a pH of pH 5.

5.

20. 2. The formulation of claim 1, wherein the formulation comprises 20 mM Histidine-Histidine HCl, 140 mM ArgHCl, 0.05% Polysorbate 80, and has a pH of pH 5.

5.

21. 21. The formulation of any one of claims 1 to 20, wherein the concentration of the anti-human PD-1 antibody or antigen-binding fragment thereof is about 100 mg / mL to 200 mg / mL.

22. 13. A method for treating cancer in a human patient in need of such treatment, comprising subcutaneously administering an effective amount of the anti-human PD-1 antibody formulation of claim 1.

23. The method of claim 22, wherein the anti-human PD-1 antibody formulation is administered at a dose of about 100 mg to about 1000 mg.

24. The method of claim 23, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 200 mg.

25. 24. The method of claim 23, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 300 mg.

26. 24. The method of claim 23, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 400 mg.

27. 24. The method of claim 23, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 500 mg.

28. The method of claim 23, wherein the anti-human PD-1 antibody formulation is administered subcutaneously once every three weeks.

29. The method of claim 23, wherein the anti-human PD-1 antibody formulation is administered once a week.

30. The method of claim 23, wherein the anti-human PD-1 antibody formulation is administered once every two weeks.

31. The method of claim 23, wherein the anti-human PD-1 antibody formulation is administered once every three weeks.

32. 24. The method of claim 23, 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.

33. 24. The method of claim 23, wherein the human patient is administered at least one other therapeutic agent.

34. 34. The method of claim 33, wherein the at least one other therapeutic agent 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 second PD-1 antibody, a CD40 agonist, a TLR agonist, a CAR-T cell, or a chemotherapeutic agent.

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