Stable high concentration arginine formulations containing PD-1 antibodies and methods of use thereof

Abstract

The present invention relates generally to the field of pharmaceutical formulations of antibodies or antigen-binding fragments thereof directed against the human programmed death receptor PD-1. These formulations may further contain a formulation buffer, a viscosity reducer, and a nonionic surfactant. The pharmaceutical formulations of the invention exhibit a low viscosity and a comparable degree of antibody stability upon exposure to heat and other physical stresses. Methods of making such antibody formulations and methods of using such antibody formulations are also provided.

Description

Technical Field

[0002] Disclosed herein are stable, high-concentration formulations comprising antibodies or antigen-binding fragments thereof that bind to human programmed death receptor 1 (PD-1). Also disclosed herein are methods of preparing these formulations and treating cancer with the formulations of the disclosure. Background Art

[0004] There has been an increase in the use of antibodies as therapeutic agents in the clinic. However, while antibodies generally have similar structures, they differ in primary amino acid sequence, 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 main determinants of the solubility and / or stability properties of the antibody in different formulations. An antibody formulation that provides solubility and stability for one antibody may perform poorly for another, resulting in antibody precipitation or fragmentation. This is particularly true when a subcutaneous antibody formulation is desired.

[0005] Subcutaneous injection has received increasing attention in the delivery of protein therapeutics because it provides patients with the potential for self-administration. Through rapid, low-volume injections, patients can administer antibody therapeutics without the need for intravenous infusions, which typically require a visit to the hospital. However, many antibodies require a certain dose to be effective, and the antibodies usually need to be concentrated into a small volume. The volume limitation of the subcutaneous administration route is a key factor to be considered for subcutaneous administration, resulting in the need for high-concentration antibody doses. In turn, this has created challenges related to the solubility of proteins, physical and chemical stability, and the manufacture, storage and delivery difficulties of subcutaneous antibody formulations. For example, antibodies lose solubility during processing and / or storage and form particles in certain formulations, which makes subcutaneous administration less effective. Due to the concentrated nature of antibodies in subcutaneous formulations, high viscosity is another problem to be overcome because it limits the injectability of the product. In addition, during the manufacturing process, high-viscosity antibody formulations have difficulties in processing, especially in ultrafiltration and sterile filtration. Finally, subcutaneous antibody formulations need to maintain the structure and function of antibodies. Subcutaneous antibody formulations that result in proteolysis or structural degradation of the antibody will reduce efficacy, as well as impair the ability of the antibody to bind to its target protein.

[0006] Therefore, there has long been a need in the art for subcutaneous antibody formulations of anti-human PD-1 antibodies for the treatment of various cancers and infectious diseases. Such formulations can have good antibody solubility, stability, long shelf life, and are easy to administer at high concentrations. Summary of the invention

[0008] The present invention provides a stable, low-viscosity and high-concentration antibody formulation. A low-viscosity pharmaceutical formulation comprising:

[0009] About 10 mg / mL to about 200 mg / mL of an anti-programmed death receptor 1PD-1 antibody, or

[0010] antigen-binding fragments thereof;

[0011] a formulation buffer providing a pH of about 5.0 to about 7.0;

[0012] Viscosity reducers; and

[0013] Nonionic surfactants,

[0014] The viscosity of the formulations mentioned therein does not exceed 35 cP and the osmolarity is from about 200 mOsmol / kg to about 400 mOsmol / kg.

[0015] The formulation, wherein the PD-1 antibody or antigen-binding fragment thereof comprises (a) HCDR1 (heavy chain complementary 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, the light chain variable region comprising: (d) LCDR1 (light chain complementary determining region 1) of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

[0016] The formulation, wherein the formulation buffer is selected from the group consisting of: histidine, acetate, citrate, succinate, phosphate, a mixture of histidine and acetic acid, or a mixture of histidine and citric acid.

[0017] The formulation, wherein the formulation buffer is histidine.

[0018] The formulation, wherein the formulation buffer is acetate.

[0019] The formulation, wherein the concentration of the buffer is 15 mM to 25 mM.

[0020] The formulation, wherein the formulation comprises 20 mM histidine buffer or 20 mM acetate buffer.

[0021] The formulation, wherein the pH is 5.0-6.0.

[0022] The anti-human PD-1 antibody formulation, wherein the viscosity reducing agent is an arginine salt.

[0023] The formulation, wherein the arginine salt is an equal mixture of 50 mM to 280 mM L-arginine and L-glutamic acid (ArgGlu).

[0024] The preparation, wherein the arginine salt is a 50mM to 280mM L-arginine and L-glutamic acid complex salt (ArgGlu).

[0025] The formulation, wherein the arginine salt is an equal mixture of 50 mM to 280 mM L-arginine and L-aspartic acid (ArgAsp).

[0026] The preparation, wherein the arginine salt is a 50 mM to 280 mM complex salt of L-arginine and L-aspartic acid (ArgAsp).

[0027] The formulation, wherein the arginine salt is 50 mM to 280 mM L-arginine hydrochloride (ArgHCl).

[0028] The formulation, wherein the non-ionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80 or poloxamer 188.

[0029] The formulation, wherein the concentration of polysorbate 80 is 0.02% to 0.08%.

[0030] The formulation, wherein the concentration of polysorbate 20 is 0.05%.

[0031] The formulation, wherein the formulation comprises 20 mM histidine-histidine HCl, 140 mM ArgGlu, 0.05% polysorbate 80, has a pH of pH 5.5.

[0032] The formulation, wherein the formulation comprises 20 mM acetate, 140 mM ArgAsp, 0.05% polysorbate 80, and its pH is pH 5.5.

[0033] The formulation, wherein the formulation comprises 20 mM histidine-histidine HCl, 140 mM ArgHCl, 0.05% polysorbate 80, has a pH of pH 5.5.

[0034] 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.

[0035] A method for treating cancer in a human patient in need thereof, the method comprising subcutaneously administering an effective amount of an anti-human PD-1 antibody formulation.

[0036] 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.

[0037] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 200 mg.

[0038] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 300 mg.

[0039] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 400 mg.

[0040] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 500 mg.

[0041] The method, wherein the anti-human PD-1 antibody formulation is administered subcutaneously once every three weeks.

[0042] The method, wherein the anti-human PD-1 antibody formulation is administered once a week.

[0043] The method, wherein the anti-human PD-1 antibody formulation is administered once every two weeks.

[0044] The method, wherein the anti-human PD-1 antibody formulation is administered once every three weeks.

[0045] The method, wherein the cancer is 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, 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.

[0046] The method, wherein at least one other therapeutic agent is administered to the human patient.

[0047] The method, 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, a second PD-1 antibody, a CD40 agonist, a TLR agonist, CAR-T cells, or a chemotherapeutic agent.

[0048] In some embodiments, the antibody formulation comprises an anti-PD-1 antibody, or an antigen-binding fragment thereof, a formulation buffer, a viscosity reducer, and a nonionic surfactant. In some embodiments, the formulation buffer provides a pH range between 5.0 and 7.0. In some embodiments, the viscosity of the antibody formulation does not exceed 50 centipoise (cP). In some embodiments, the osmotic pressure of the antibody formulation is about 200mOsmol / kg to about 400mOsmol / kg. In some embodiments, the antibody formulation is stable after freeze-thaw stress and storage for 3 months at 5°C and 25°C.

[0049] In some embodiments, the antibody formulation may comprise or consist essentially of an anti-PD-1 antibody or antigen-binding fragment thereof at between about 75 mg / mL to about 200 mg / mL, a formulation buffer, a viscosity reducer, and a nonionic surfactant, and a pH of about 5.5±0.5. In some embodiments, the antibody formulation may consist essentially of an anti-PD-1 antibody at between about 100 mg / mL to about 200 mg / mL, a formulation buffer, a viscosity reducer, and a nonionic surfactant, and a pH of 5.5±0.5.

[0050] In some embodiments, the formulation buffer is selected from the group consisting of: histidine, acetate, citrate, succinate, phosphate, a mixture of histidine and acetic acid, a mixture of histidine and citric acid. In some embodiments, the formulation buffer can be a histidine buffer. In some embodiments, the concentration of the histidine buffer is about 10mM to about 30mM. In some embodiments, the concentration of the histidine buffer is about 20mM histidine.

[0051] In other embodiments, the formulation buffer is an acetate buffer. In some embodiments, the concentration of the acetate buffer is about 5 mM to about 30 mM, preferably about 20 mM.

[0052] In certain embodiments, the viscosity reducing agent is an amino acid or a derivative thereof. In certain 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 certain 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.

[0053] In some embodiments, the concentration of the amino acid or its derivative is about 25mM to about 400mM. In some embodiments, the concentration of the amino acid or its derivative is about 50mM to about 280mM, preferably about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, about 125mM, about 130mM, about 135mM and about 140mM.

[0054] 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.

[0055] In some embodiments, the nonionic surfactant is selected from the group consisting of polysorbate 80 (PS80), polysorbate 20 (PS20) or poloxamer 188. In some embodiments, the concentration of the nonionic surfactant can be about 0.01 to about 1 mg / mL. In some embodiments, the concentration of 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.

[0056] 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 an antigen-binding fragment thereof.

[0057] 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 an 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; d) about 0.2 to about 0.5 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition mentioned is about 5.5±0.5. In a specific embodiment, arginine hydrochloride is a complex salt of arginine hydrochloride or a mixture of L-arginine and hydrochloric acid.

[0058] 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 an 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; d) about 0.2 to about 0.5 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition mentioned is about 5.5±0.5. In a specific embodiment, arginine glutamate is a complex salt of arginine glutamate or a mixture of L-arginine and L-glutamic acid.

[0059] 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 an 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; d) about 0.2 to about 0.5 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition mentioned is about 5.5±0.5. In a specific embodiment, arginine aspartate is a complex salt of arginine aspartate or a mixture of L-arginine and L-aspartic acid.

[0060] In some embodiments, the composition can have a viscosity of less than about 50 cP, less than about 40 cP, preferably less than about 35 cP at 25°C.

[0061] In some embodiments of the present invention, the PD-1 antibody is tislelizumab (BGB-A317) or an antigen-binding fragment of tislelizumab.

[0062] Also provided herein is a method of treating cancer in a human patient having cancer, the method comprising subcutaneously administering to the patient an effective amount of an antibody formulation as described herein.

[0063] Provided herein are methods of treating cancer in a human patient having a cancer that expresses PD-L1, the method comprising subcutaneously administering to the patient an effective amount of an antibody formulation as described herein.

[0064] In some embodiments, the present disclosure provides a method for treating cancer with a combination of an anti-PD-1 subcutaneous antibody formulation and other therapeutic agents. Other therapeutic agents are, for example, zanubrutinib, pamiparib, sitrutinib, anti-CTLA4 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-TIGIT antibody, 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

[0065] Figure 1 shows the amount of aggregates (Figure 1A) and monomers (Figure 1B) for each formulation as measured by SEC-HPLC. "T0" refers to the initial point of the sample. "40C4W" refers to the sample stored at 40°C for 4 weeks. "3FT" refers to the sample undergoing three 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.

[0066] Figure 2 The results of the CZE study of the formulations are shown. "T0" refers to the initial 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.

[0067] Figure 3 The purity of the formulations as measured by CE-SDS under non-reducing conditions is shown. "T0" refers to the initial 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

[0069] definition

[0070] Unless explicitly 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.

[0071] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0072] Unless the context clearly indicates otherwise, the term "or" is used to mean and is interchangeable with the term "and / or."

[0073] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to implicitly include the stated amino acid sequence, DNA sequence, steps or groups thereof, but not to exclude any other amino acid sequence, DNA sequence, step. When used herein, the term "comprising" may be replaced with the terms "containing", "including", or sometimes "having".

[0074] The terms "administration / administering" and "treating / treatment" herein, when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids, mean that an exogenous drug, therapeutic agent, diagnostic agent, or antibody formulation is in contact with the animal, human, subject, cell, tissue, organ, or biological fluid. The treatment of cells encompasses the contact of reagents with cells and the contact of reagents with fluids, wherein the fluid is in contact with cells. The terms "administration" and "treatment" also refer to in vitro and ex vivo treatments, for example, cells are treated by reagents, diagnostic agents, binding compounds or by another cell. The term "subject" herein includes any organism, preferably an animal, more preferably a mammal (e.g., rats, mice, dogs, cats, rabbits), and most preferably a human. On the one hand, treating any disease or disorder refers to improving the disease or disorder (i.e., slowing down or preventing or reducing the development of a disease or at least one clinical symptom thereof). On the other hand, "treat, treating or treatment" refers to alleviating or improving at least one physical parameter, including those that the patient may not be able to discern. In yet another aspect, "treat," "treating," or "treatment" refers to modulating the disease or disorder physically (eg, stabilization of a discernible symptom), physiologically (eg, stabilization of a physical parameter), or both.

[0075] As used herein, the term "therapeutically effective amount" refers to an amount of an anti-PD-1 antibody sufficient to affect the treatment of a disease, disorder, or symptom when administered to a subject to treat a disease, or at least one clinical symptom of a disease or disorder. The "therapeutically effective amount" may vary with 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 one skilled in the art or may be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combination subject used to effectively treat a disease, disorder, or condition. In some embodiments of the present disclosure, the subject is a human.

[0076] "Pharmaceutical formulation" or "formulation" refers to a preparation of an antibody that is in a form that permits the active ingredients to be effective and that contains no additional components that are toxic to a subject to which the formulation is administered.

[0077] A "stable" formulation is a formulation of an antibody that is prepared in such a way that the physical stability and / or chemical stability and / or biological activity of the antibody is maintained over time. Various analytical techniques for measuring protein stability are available in the art and are reviewed in the following literature: Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York City, New York, published in (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature over a selected time period.

[0078] The term "antibody" is used in the broadest sense herein and specifically encompasses antibodies (including full-length monoclonal antibodies) and antibody fragments, as long as they recognize an antigen, such as PD-1. Antibodies are typically monospecific, but may also be described as heterospecific, heterospecific, or multispecific. Antibody molecules bind to specific antigenic determinants or epitopes on an antigen through specific binding sites.

[0079] The term "monoclonal antibody" or "mAb" or "Mab" herein refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules contained in the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically include a variety of different antibodies with different amino acid sequences in their variable domains, particularly their complementary determining regions (CDRs), which are typically specific for different epitopes. The modifier "monoclonal" indicates the characteristics of antibodies obtained from a substantially homogeneous antibody population and should not be construed as requiring antibodies to be produced by any 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; U.S. 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. The hybridoma producing the mAb can be cultured in vitro or in vivo. High titer mAbs can be obtained by in vivo production, in which cells from a single hybridoma are injected intraperitoneally into mice, such as primary primed Balb / c mice, to produce ascites fluid containing high concentrations of the desired mAb. MAbs of isotype IgM or IgG can be purified from such ascites fluid, or from culture supernatants, using column chromatography methods well known to those skilled in the art.

[0080] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair having a "light chain" (about 25 kDa) and a "heavy chain" (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of the heavy chain can be defined as a constant region that is primarily responsible for effector function. Typically, human light chains are classified as κ and λ light chains. In addition, human heavy chains are typically classified as α, δ, ε, γ or μ, and the isotype of the antibody is defined as IgA, IgD, IgE, IgG and IgM, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids.

[0081] The variable regions of each light chain / heavy chain (VL / VH) pair form an antibody binding site. Therefore, in general, a complete antibody has two binding sites. Except for bifunctional or bispecific antibodies, in general, the two binding sites are identical.

[0082] Typically, the variable domains of both the heavy and light chains contain three hypervariable regions, also referred to as "complementarity determining regions (CDRs)", which are located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions to enable binding to specific epitopes. In general, from N-terminus to C-terminus, both the light and heavy chain variable domains contain 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 order. In general, the amino acid assignments for each domain conform to the definition of an immunologically interesting protein sequence, Kabat et al., National Institutes of Health, Bethesda, Maryland, 5th edition; NIH Publication 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.

[0083] The term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "CDRs" (i.e., VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable domain and VH-CDR1, VH-CDR2, and VH-CDR3 in the heavy chain variable domain). See Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Maryland (defining the CDR regions of antibodies by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (defining the CDR regions of antibodies by structure). The term "framework" or "FR" residues is intended to refer to those variable domain residues other than the hypervariable region residues herein defined as CDR residues.

[0084] 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 a full-length antibody, such as 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.

[0085] Antibodies that specifically bind to a particular target protein are also described as specifically binding to a particular target protein. This means that compared to other proteins, the antibody shows preferential binding to the target protein, but this specificity does not require absolute binding specificity. If the binding of the antibody determines the presence of the target protein in the sample, for example, no undesirable results, such as false positives, are produced, the antibody is considered to be "specific" for its intended target. Antibodies or binding fragments thereof that can be used in the present invention will bind to the target protein with an affinity that is at least twice higher than that of the non-target protein, preferably at least 10 times higher, more preferably at least 20 times higher, and most preferably at least 100 times higher. The antibodies herein are referred to as specifically binding to a polypeptide comprising a given amino acid sequence.

[0086] The term "human antibody" as used herein means an antibody that comprises only human immunoglobulin protein sequences. If produced in mice, mouse cells, or hybridomas derived from mouse cells, human antibodies may contain murine carbohydrate chains. Similarly, "mouse antibody" or "rat antibody" means an antibody that comprises only mouse or rat immunoglobulin protein sequences, respectively.

[0087] The term "humanized antibody" means an antibody form containing sequences from non-human (e.g., mouse) antibodies and human antibodies. Such antibodies contain the minimum sequence derived from non-human immunoglobulins. Generally, humanized antibodies will include substantially at least one and typically all of two variable domains, all or substantially all of its hypervariable loops corresponding to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin sequences. Humanized antibodies will also optionally include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. When it is necessary to distinguish humanized antibodies from parent rodent antibodies, prefixes "hum", "hu", "Hu" or "h" are added to the antibody clone name. The rodent antibody of humanized form will generally include the same CDR sequence of the parent rodent antibody, but some amino acid substitutions may be included to increase affinity, increase the stability of the humanized antibody, or for other reasons.

[0088] The antibodies of the present application have potential therapeutic uses in treating cancer. The term "cancer" or "tumor" herein refers to or describes a physiological condition in which a mammal is typically characterized by dysregulated 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, 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.

[0089] In addition, the antibodies of the present application have potential therapeutic uses in controlling viral infections and other human diseases that are mechanistically related to immune tolerance or "exhaustion." In the context of the present application, the term "exhaustion" refers to a process that leads to a weakening of the ability of immune cells to respond to cancer or chronic viral infections.

[0090] Anti-PD-1 antibodies

[0091] The present disclosure provides anti-PD-1 antibodies and subcutaneous formulations thereof. For example, tislelizumab (BGB-A317) is an anti-PD-1 antibody disclosed in US Pat. No. 8,735,553 having the sequence provided in Table 1 below.

[0092] Table 1

[0093] Anti-PD1 antibodies may include, but are not limited to, tislelizumab, pembrolizumab or nivolumab. Tislelizumab is disclosed in US 8,735,553. Pembrolizumab (formerly known as MK-3475) disclosed by Merck in US 8,354,509 and US 8,900,587 is a humanized IgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is being studied clinically for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin's lymphoma (cHL). Nivolumab (as disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Pat. No. US 8,008,449 and WO 2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin's lymphoma.

[0094] Further changes to the Fc region framework

[0095] In other aspects, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues so that the antibody has a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand with changed affinity can be, for example, an Fc receptor or the C1 component of complement. This method is described in, for example, U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al.

[0096] In another aspect, one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered CIq binding and / or reduced or abolished complement dependent cytotoxicity (CDC).This approach is described, for example, in U.S. Patent No. 6,194,551 to Idusogie et al.

[0097] On the other hand, one or more amino acid residues are changed to change the ability of the antibody to fix complement. The method is described in, for example, PCT Publication WO 94 / 29351 of Bodmer et al. In a specific aspect, one or more amino acids of the antibody or its antigen-binding fragment of the present disclosure are replaced by one or more allotype amino acid residues of IgG1 subclass and κ isotype. Allotype amino acid residues also include but are not limited to the constant region of the heavy chain of IgG1, IgG2 and IgG3 subclasses and the constant region of the light chain of κ isotype, as described in Jefferis et al., MAbs [monoclonal antibodies] 1: 332-338 (2009).

[0098] In another aspect, the Fc region is modified by modifying one or more amino acids to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. This approach is described, for example, in PCT Publication WO 00 / 42072 by Presta. In addition, binding sites for FcγRI, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. [Journal of Biological Chemistry] 276: 6591-6604, 2001).

[0099] On the other hand, the glycosylation of the antibody is modified. For example, aglycosylated antibodies (i.e., antibodies lacking or having reduced glycosylation) can be prepared. For example, glycosylation can be changed to increase the affinity of the antibody to the "antigen". This carbohydrate modification can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, which lead to the elimination of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation of the site. This glycosylation can increase the affinity of the antibody to the antigen. This method is described in, for example, U.S. Patent Nos. 5,714,350 and 6,350,861 of Co et al.

[0100] Additionally or alternatively, antibodies with altered glycosylation types can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art, and these cells can be used as host cells in which recombinant antibodies are expressed to thereby produce 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 a cell line exhibit hypofucosylation. PCT Publication WO 03 / 035835 to Presta describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 to Umana et al. describes a cell line engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosamine transferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0101] On the other hand, if the desired ADCC is reduced, many previous reports show that human antibody subclass IgG4 has only moderate ADCC and almost no CDC effector function (Moore GL et al., 2010 MAbs [monoclonal antibodies], 2: 181-189). On the other hand, it was found that native IgG4 is less stable under stress conditions (such as in acidic buffers or at elevated temperatures) (Angal, S. 1993 Mol Immunol [molecular immunology], 30: 105-108; Dall'Acqua, W. et al., 1998 Biochemistry [biochemistry], 37: 9266-9273; Aalberse et al., 2002 Immunol [immunology], 105: 9-19). Reduced ADCC can be achieved by operably linking the antibody to an IgG4 engineered with a combination of alterations having reduced or ineffective FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector functions. Considering the physicochemical properties of antibodies as biopharmaceuticals, one of the less desirable inherent properties of IgG4 is that its two heavy chains dynamically separate in solution to form half antibodies, which leads to the generation of bispecific antibodies in vivo through a process called "Fab arm exchange" (Van der Neut Kolfschoten M et al., 2007 Science, 317: 1554-157). The mutation of serine at position 228 (EU numbering system) to proline exhibits an inhibitory effect on the separation of IgG4 heavy chains (Angal, S. 1993 Mol Immunol, 30: 105-108; Aalberse et al., 2002 Immunol, 105: 9-19). It has been reported that some amino acid residues in the hinge region and the γFc region have an influence on the interaction of antibodies with Fcγ receptors (Chappel SM et al., 1991 Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J [Journal of the Federation of American Societies for Experimental Biology], 9:115-119; Armour, KL et al., 1999 Eur J Immunol [European Journal of Immunology], 29:2613-2624; Clynes, RA et al., 2000 Nature Medicine [Nature Medicine], 6:443-446; Arnold JN, 2007 Annu Rev immunol [Annual Review of Immunology], 25:21-50).In addition, some rare IgG4 isotypes in the population can also cause different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet [European Journal of Immunogenetics], 25: 349-55; Aalberse et al., 2002 Immunol [Immunology], 105: 9-19). In order to produce PD-1 antibodies with low ADCC, CDC and instability, the hinge region and Fc region of human IgG4 can be modified and many changes can be introduced. These modified IgG4 Fc molecules can be found in SEQ ID NO: 83-88, U.S. Patent No. 8,735,553.

[0102] Treatment

[0103] The antibodies or antigen-binding fragments of the present disclosure can be used in a variety of applications, including but not limited to methods of treating PD-1-related disorders or diseases. In one aspect, the PD-1-related disorder or disease is cancer.

[0104] In one aspect, the present disclosure provides a method for treating cancer. In some aspects, the method comprises administering an effective amount of an anti-PD-1 antibody or antigen binding fragment to a patient in need. 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, 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.

[0105] Antibodies or antigen-binding fragments of the present invention can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if necessary for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-lived or long-term. Various dosing regimens are contemplated herein, including but not limited to single administration or multiple administrations at different time points, push injection administration and pulse infusion.

[0106] The antibody or antigen-binding fragment of the present invention can be formulated, administered and used in a manner consistent with good medical practice. Factors to be considered in this regard include the specific obstacle for treatment, the specific mammal for treatment, the clinical condition of the individual patient, the cause of the obstacle, the delivery site of the agent, the method of administration, the administration regimen and other factors known to medical practitioners. The antibody does not need to be formulated with one or more agents currently used to prevent or treat the obstacle under study. The effective amount of such other agents depends on the amount of the antibody present in the formulation, the type of obstacle or treatment and other factors discussed above. These are usually used with the same dosage and route of administration as described herein, or with about 1%-99% of the dosage described herein, or with any dosage and any route determined to be suitable by experience / clinical use.

[0107] For the prevention or treatment of disease, the appropriate dosage of the antibodies or antigen-binding fragments 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 preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0108] Antibodies related to PD-1 have been shown to be safe when applied to human cancer patients in various dosage ranges and administration cycles. The subcutaneous antibody formulation disclosed herein can be applied at 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg. Subcutaneous antibody formulations can be applied twice a day, once a day, once a week, twice a week, three times a week, four times a week, five times a week, once every two weeks, once every three weeks, once a month, 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 applying tislelizumab once every three weeks at 100mg. In some embodiments, the dosing regimen includes applying tislelizumab once every three weeks at 200mg. In some embodiments, the dosing regimen includes applying tislelizumab once every three weeks at 300mg. In some embodiments, the dosing regimen includes applying tislelizumab once every three weeks at 400mg. In some embodiments, the dosing regimen comprises administering tislelizumab at 500 mg once every three weeks. In some embodiments, the dosing regimen comprises administering tislelizumab at 600 mg once every three weeks.

[0109] In certain embodiments, tislelizumab can be administered in combination with other therapies, e.g., 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.

[0110] Pharmaceutical compositions and formulations

[0111] Compositions comprising anti-PD-1 antibodies or antigen-binding fragments thereof or polynucleotides comprising sequences encoding anti-PD-1 antibodies or antigen-binding fragments are also provided, including pharmaceutical formulations. In certain embodiments, the composition comprises one or more antibodies or antigen-binding fragments that bind to PD-1, or one or more polynucleotides comprising sequences 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 pharmaceutically acceptable excipient comprising a buffer.

[0112] Pharmaceutical formulations of the anti-PD-1 antibodies or antigen-binding fragments described herein are prepared by mixing such antibodies or antigen-binding fragments having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)), in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinyl pyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG), polysorbate 20 or polysorbate 80.

[0113] Sustained release preparations may 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, eg films, or microcapsules.

[0114] Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. Examples

[0115] The description of examples and certain embodiments should be considered illustrative, rather than limiting, of the present invention as defined by the claims. As will be readily appreciated, many variations and combinations of the features set forth above may be used without departing from the present invention as set forth in the claims. All such variations are intended to be included within the scope of the present invention. All references cited are incorporated herein by reference in their entirety.

[0116] Analytical methods

[0117] The Methods section provides a summary of the methods used in Examples 1-5 below.

[0118] Protein concentration

[0119] Protein concentration was determined spectroscopically using SoloVPE (C Technologies Inc.) based on variable path length and the Beer-Lambert law (A = cle, where A = absorbance, c = concentration, l = path length, and e = 1.5 mg ml -1 cm -1 ). The samples were equilibrated to ambient temperature, added to a sample container (C Technology #OC0009-1-P50), and then loaded into the container holder of the detection window platform. For each sample, a clean fiber (#OF0002-P50) was installed in the fiber coupler and the slope was measured at 280 nm.

[0120] Viscosity

[0121] Viscosity of Antibody Formulations in Chip-Based MicroVISC TM The pressure difference is related to the dynamic viscosity of the solution. The sample volume is approximately 70-100 μL. An aliquot is loaded onto a 400 μL microVISC TM A disposable pipette was placed in the microarray and attached to the chip. -1 The measurements were performed in triplicate at a shear rate of 1.54°C and a temperature of approximately 25°C.

[0122] Turbidity

[0123] Using 96-well plate molecular device M2e TM The reader measures UV absorbance at 350 nm as an indication of turbidity. The absorbance readings are controlled against blank well readings and normalized to the sample path length.

[0124] SEC-HPLC

[0125] The formation of soluble aggregates was analyzed by size exclusion chromatography (SEC) on a Waters HPLC system. An isocratic gradient was used on a TSKgel G3000 maintained at 37°C ± 5°C. TM 100 μg of protein was separated by molecular size on a SWXL column. Molecular weight species were eluted and detected by UV absorption at 280 nm. The distribution of aggregates, monomers and fragments was quantified via peak areas of standards and samples.

[0126] CZ

[0127] Using PA800 Plus TM (Beckman) The charge heterogeneity of the samples was determined by capillary zone electrophoresis (CZE), also known as free solution capillary electrophoresis. The samples were separated according to their electrophoretic mobilities, which are caused by differences in charge and hydrodynamic radius of the analytes in a capillary filled with a buffer solution composed mainly of hexanoic acid. When an external electric field is applied, the samples are analyzed in their native state, resulting in a specific peak pattern showing the various charge variants of the antibody (acidic, basic and primary charge variants). The samples were injected by pressure and the mobile proteins were detected by UV absorbance at 214 nm.

[0128] CE-SDS(NR)

[0129] Using PA800 Plus TM (Beckman) The purity of the sample was determined by capillary gel electrophoresis (CE) method. The sample was denatured with sodium dodecyl sulfate (SDS) and separated according to size in a capillary filled with gel, which served as a sieving medium. In non-reduced (NR) samples, an alkylating agent N-ethylmaleimide (NEM) was added to avoid any fragmentation induced by the sample preparation and to ensure that the main IgG peak remained intact. The sample was injected electrically and the mobile protein was detected by UV absorbance at 200nm using a UV detector. The reportable value of the non-reduced sample is the time-corrected area percentage (TCA%) of the IgG main peak.

[0130] Example 1: Preparation of high concentration anti-PD-1 antibody formulation

[0131] The anti-human PD-1 antibody, tislelizumab (BGB-A317, Table 1), was prepared and purified.

[0132] Formula 1 (F1) preparation:

[0133] Tislelizumab antibody was buffer exchanged into 20 mM histidine-histidine HCl (pH 6.0) and then quantified using a 30 kDa Amicon Ultra TMThe centrifugal filter was concentrated to approximately 200 mg / mL. Samples of varying concentrations were prepared by dilution with 20 mM histidine-histidine HCl pH 6.0 buffer.

[0134] Formula 2-7 (F2-F7) and Formula 10-12 (F10-F12) are prepared:

[0135] To prepare tislelizumab stock solutions, 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 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 tislelizumab stock solutions to obtain the desired excipient concentrations shown in Table 2. Then, 30 kDa Amicon Ultra TM Centrifugal filters concentrated the samples to approximately 200 mg / mL. Formulations with varying protein concentrations were prepared by dilution from 1x stock solutions.

[0136] Formula 8 and 9 (F8 and F9) are prepared:

[0137] To prepare a stock solution of tislelizumab, buffer exchange tislelizumab into 20 mM histidine-acetic acid, pH 6.0, and 20 mM histidine-citric acid, pH 6.0, using a 10 kDa MWCO dialysis cassette. Prepare a stock solution of 500 mM (5x) arginine dissolved in 20 mM histidine-acetic acid, pH 6.0, and 20 mM histidine-citric acid, pH 6.0. Adjust the pH to 6.0 using acetic acid and citric acid. Spike the 5x arginine stock solution into the tislelizumab stock solution in 20 mM histidine-acetic acid, pH 6.0, and 20 mM histidine-citric acid, pH 6.0. Subsequently, dialysis was performed using a 30 kDa Amicon Ultra TM Centrifugal filters concentrated the samples to approximately 200 mg / mL. Formulations with varying protein concentrations were prepared by dilution with a 100 mM (1x) arginine stock solution.

[0138] This preparation method can also be applied to the preparation of other high-concentration anti-PD-1 antibody preparations.

[0139] Table 2. Formulation composition

[0140] Example 2: Viscosity reduction effect of arginine salt in high concentration PD-1 antibody formulation

[0141] This set of experiments determined the viscosity results for Formulations 1-12 with varying protein concentrations as described in Example 1. In this example, we investigated the viscosity reducing effects of arginine salts, proline, combinations of arginine salts and polyols, combinations of arginine and proline, and other organic electrolytes on viscosity and antibody stability variables.

[0142] Viscosity analysis at 500S -1 The flow rate and the temperature of about 25 ℃ were carried out. The results are shown in Table 3. The data prove that the viscosity of the tislelizumab formulation is highly dependent on the protein concentration. In the absence of a viscosity reducer (F1), the viscosity increases exponentially when the protein concentration increases from 160.42 mg / mL to 199.29 mg / mL. In the absence of a viscosity reducer, the viscosity value is 29.69 cP at a tislelizumab protein concentration of 173.08 mg / mL, and the viscosity value is 53.29 cP at an antibody concentration of 199.29 mg / mL. In an unexpected result, the addition of arginine salts such as ArgGlu, ArgHCl and ArgAsp significantly reduces the viscosity of high-concentration tislelizumab antibody formulations. When arginine salts are present, the viscosity values ​​in F2 (ArgGlu) are 22.88 cP (189.47 mg / mL) and 30.62 cP (197.92 mg / mL), the viscosity values ​​in F3 (ArgHCl) are 21.24 cP (185.60 mg / mL) and 27.95 cP (191.70 mg / mL) and the viscosity values ​​in F4 (ArgAsp) are 20.70 cP (186.87 mg / mL) and 40.99 cP (211.33 mg / mL). At a concentration of about 200 mg / ml of tislelizumab, ArgGlu reduces viscosity by about 42.5%. The results of these formulations are summarized in Table 3 below.

[0143] We also evaluated the effect of proline (F5) and other organic electrolytes such as NaGlu (F7) and LysHCl (F6) in various tislelizumab formulations. The results showed that proline (F5) and NaGlu (F7) did not significantly reduce viscosity, while for LysHCl (F6), a reduction in viscosity was observed. However, the viscosity reduction effect of LysHCl was significantly lower than that of ArgGlu, ArgHCl and ArgAsp, as found in F2-F4. The results of these formulations are summarized in Table 3 below.

[0144] Formulations 8 and 9 evaluated the viscosity reducing 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 produced a decrease in viscosity in the tislelizumab formulations, with arginine acetate producing a greater decrease in viscosity than arginine citrate. However, the viscosity decrease produced by the presence of arginine acetate or arginine citrate was not as significant as that of ArgGlu, ArgHCl, and ArgAsp, as found in F2-F4. The results of these formulations are summarized in Table 3 below.

[0145] Formulations 10, 11 and 12 investigated the effects of arginine salts and neutral organic combinations (e.g., trehalose and proline). The results showed that significant reductions in viscosity 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 summarizing these results, the combination of 100 mM arginine salts and neutral organics found in formulations F10-F12 caused a slightly lower reduction in viscosity than 140 mM arginine salt alone.

[0146] In summary, the excipients with the greatest impact on the viscosity of high-concentration tislelizumab formulations included ArgGlu, ArgHCl, and ArgAsp based formulations found in F2-F4.

[0147] Table 3. Viscosity of high concentration PD-1 antibody formulations containing different excipients

[0148] Example 3: Effect of pH and buffer on viscosity of high concentration PD-1 antibody formulations

[0149] This example evaluates the effect of pH and different 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 antibodies is described in Example 1 above. Briefly, tislelizumab antibody stock solutions in 20 mM histidine-histidine HCl at pH 6.5, pH 6.0, and pH 5.5 and in 20 mM acetate at pH 5.5 and pH 5.0 were prepared by dialysis with a 10 kDa MWCO dialysis cassette. A stock solution of a viscosity reducer (without polysorbate 80 (PS80)) was added to the tislelizumab stock solution and then dialyzed with a 30 kDa Amicon Ultra TMThe centrifugal filter concentrated the sample to approximately 200 mg / mL. A high concentration PS80 solution was then added to obtain a target concentration of 0.02%. Finally, formulations with different protein concentrations were prepared by diluting with a 1x viscosity reducer stock solution containing 0.02% PS80.

[0150] The viscosity of formulations F13-F18 containing ArgGlu and ArgHCl over a range of tislelizumab concentrations is summarized in Table 5. The results show that in formulations containing arginine salts, lowering the pH from 6.5 to 5.0 significantly reduced the viscosity. At pH 5.5, at an antibody concentration of 183.05 mg / mL, formulation F17 had a viscosity value of 15.61 cP, at an antibody concentration of 183.17 mg / mL, F18 had a viscosity value of 16.32 cP, and at an antibody concentration of 178.60 mg / ml, F19 had a viscosity value of 16.68 cP. At pH 5.0, at an antibody concentration of 185.68 mg / mL, F20 had a viscosity value of 18.66 cP, and at an antibody concentration of 199.95 mg / mL, F20 had a viscosity value of 24.74 cP. Notably, no significant changes in the viscosity of formulations containing acetate buffer were observed compared to histidine buffer. In other words, changing the buffer may not affect the viscosity of tislelizumab antibody formulations containing arginine salt.

[0151] The subcutaneous formulations have the element of "syringability", i.e., the ability of the subcutaneous formulation to be administered through a syringe (e.g., 20-25 gauge) needle. In particular, the viscosity produced by the high concentration tislelizumab formulations shows good compatibility with syringes containing 23-gauge or 25-gauge needles typically used for subcutaneous administration. Thus, the tislelizumab antibody formulations of F17, F18, F19, and F20 can have a high protein concentration of up to 180-200 mg / mL with acceptable viscosities for subcutaneous delivery by syringe.

[0152] Table 4. Formulation composition

[0153] Table 5. Solution viscosity of PD-1 antibody formulations with different pH and buffers

[0154] Example 4: Effect of different concentrations of arginine salt on the viscosity of high concentration PD-1 antibody formulations

[0155] These experiments tested the effect of different concentrations of arginine salts on the viscosity of high concentration PD-1 antibody formulations. In order to evaluate the effect of the concentration of arginine salts on viscosity, the tislelizumab antibody was formulated in 20mM histidine (pH5.5), 0 / 50 / 100 / 280mM arginine salts and 0.2mg / ml polysorbate 80 (PS80). The preparation method is as described in Examples 1 and 3.

[0156] Viscosity and osmotic pressure were measured. The results are summarized in Table 6. The viscosity and osmotic pressure of the tislelizumab antibody formulations with 0, 50, 100, 140 and 280 mM ArgHCl or ArgGlu were tested at a range of antibody concentrations. The results showed that the addition of 50 mM ArgHCl or ArgGlu significantly reduced the viscosity of the tislelizumab antibody formulation. In addition, gradually increasing the concentration of ArgHCl or ArgGlu to 280 mM further reduced the viscosity.

[0157] The osmotic pressure value of each experimental determination of these preparations is also listed in Table 6.The osmotic pressure value of 140mM ArgGlu and 140mM ArgHCl preparations is between 360 and 390mOsmol / kg, and it has hypertonicity slightly.Ideally, the injectable product should be formulated into isotonic solution (osmotic pressure is about 300mOsm / kg).However, strict isotonicity is not absolutely necessary for subcutaneous (SC) injection.It is suggested that the osmotic pressure upper limit of the drug product intended for subcutaneous injection should be controlled under 600mOsmol / kg, to minimize the pain of hypertonic induction.Therefore, the preparation containing the higher concentration arginine salt (140mM) proved herein for reducing the viscosity of the preparation does not seem to have the risk of tissue damage at the injection site.

[0158] Table 6. Viscosity of high concentration PD-1 antibody formulations containing different concentrations of arginine salts

[0159] Example 5: Stability evaluation of PD-1 antibodies in low viscosity and high concentration formulations

[0160] Further studies were conducted to evaluate the stability of formulations containing about 180-200 mg / mL tislelizumab antibody. In this study, arginine hydrochloride (ArgHCl), arginine glutamate (ArgGlu), and arginine aspartate (ArgAsp) were used as viscosity reducing agents, and the effects on stress and storage stability were evaluated.

[0161] For this study, tislelizumab antibody was formulated in 20 mM histidine / acetate (pH 5.5), 140 mM arginine salt as shown in Example 1 in the preparation of Formulations F2-F7. A high concentration PS80 solution was then added to obtain a target concentration of 0.05%. Millex TM Each formulated solution was filtered through a GP 0.22 μm PES 33 mm filter and filled into 2 mL ready-to-use glass vials (Schott) with a fill volume of 0.5 mL drug product. The samples were staged, protected from light, and placed in environmental stability chambers at 2°C-8°C, 25°C, and 40°C. Freeze-thaw stability was determined by subjecting the formulations to three freeze-thaw cycle stresses.

[0162] The turbidity stability of the drug product was determined by measuring the optical density at 350 nm. There was no significant change in turbidity for any of the formulations at 5°C and 40°C and under freeze-thaw conditions.

[0163] The purity of the formulations was determined by SEC-HPLC. At 40°C, an increase in aggregates was observed, with a corresponding decrease in monomers. Formulation F23 showed the greatest change compared to the other formulations. However, these decreases are considered acceptable for a liquid formulation with an expected storage condition of 5°C. There was no change in SEC purity at 5°C and after freeze-thaw stress. At 25°C, an increase of approximately 0.4%-0.6% in aggregates was detected for formulations F21-F23 over a 6-month period.

[0164] Charge heterogeneity was detected by CZE and evaluated by monitoring the main peak as well as acidic and basic substances. No measurable changes were found in any single peak (including the main peak of any formulation) for up to 6 months at 5°C. Only a slight decrease was observed in the main peak at 25°C for 6 months. At 40°C for 4 weeks, the main peak showed a significant decrease, indicating that the antibody stability was reduced. Similar to the results observed at 25°C, an increase in acidic variants was observed, and a decrease in basic peaks was observed (data not shown).

[0165] Purity by CE-SDS was measured under non-reducing conditions. There was no measurable change in purity over time for any formulation at 5°C for up to 6 months. Purity % at 5°C, 25°C, and 40°C were all >96% and within the clinical acceptance criteria of >90.0% for non-reducing CE-SDS.

[0166] These results indicate that the formulations containing approximately 180-200 mg / mL tislelizumab antibody were stable after 3 freeze-thaw cycles and after 6 months of storage at 5°C and 25°C, and are summarized in Tables 7-9.

[0167] Table 7. Composition of formulations used in stability studies

[0168] Table 8. Stability study plan

[0169] Table 9. Turbidity data for formulations F21-F23

[0170] Note: 40C4W means the sample was stored at 40°C for 4 weeks. 3FT means the sample was subjected to 3 freeze-thaw cycles. 25C3M and 25C6M mean the sample was stored at 25°C for 3 months and 6 months, respectively. 5C3M and 5C6M mean the sample was stored at 5°C for 3 months and 6 months, respectively.

Claims

1. A low viscosity pharmaceutical formulation comprising: About 10 mg / mL to about 200 mg / mL of an anti-programmed death receptor 1PD-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 Nonionic surfactants, The viscosity of the formulations mentioned therein does not exceed 35 cP and the osmolarity is from about 200 mOsmol / kg to about 400 mOsmol / kg.

2. The formulation of claim 1, wherein the PD-1 antibody or antigen-binding fragment thereof comprises (a) HCDR1 (heavy chain complementary 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 complementary 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 of claim 2, wherein the formulation buffer is selected from the group consisting of histidine, acetate, citrate, succinate, phosphate, a mixture of histidine and acetic acid, or a mixture of histidine and citric acid.

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

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

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

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

8. The formulation of claim 5 or 6, wherein the pH is 5.0-6.

0.

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

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

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

14. 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 1-14, wherein the non-ionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80 or poloxamer 188.

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

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

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

5.

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

5.

20. The formulation of claim 1, wherein the formulation comprises 20 mM histidine-histidine HCl, 140 mM Arg HCl, 0.05% polysorbate 80, and its pH is pH 5.

5.

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. A method for treating cancer in a human patient in need thereof, the method 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. The method of claim 23, wherein the anti-human PD-1 antibody formulation is administered subcutaneously at a dose of 300 mg.

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

27. 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. 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, stomach 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.

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

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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