Stable high-concentration anti-pd-1 antibody pharmaceutical formulations

Abstract

The present application provides a stable high-concentration anti-PD-1 antibody drug preparation, comprising anti-PD-1 antibody, histidine-histidine hydrochloride, proline, methionine, polysorbate 20 or polysorbate 80, optional arginine or arginine hydrochloride, and the pH is 5.0-6.0. The present application also provides the use of the anti-PD-1 antibody drug preparation in the preparation of a medicament for treating cancer.

Description

Stable high-concentration anti-PD-1 antibody drug formulation

[0001] This application claims priority to Chinese patent application CN202311861803.1 filed on December 29, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present application provides a stable, high-concentration anti-PD-1 antibody pharmaceutical formulation comprising an anti-PD-1 antibody, histidine-histidine hydrochloride, proline, methionine, polysorbate 20 or polysorbate 80, and optionally arginine or arginine hydrochloride, at a pH of about 5.0 to about 6.0. The present application also provides a method for treating cancer in a subject using the anti-PD-1 antibody pharmaceutical formulation, comprising subcutaneously administering the pharmaceutical formulation of the present invention to the subject. Background Art

[0003] Programmed cell death receptor 1 (PD-1), also known as CD279, is an important immunosuppressive molecule. It is a member of the CD28 family of the immunoglobulin superfamily and a type I transmembrane glycoprotein with a molecular weight of 50-55 kDa. PD-1 is constitutively expressed on the surface of T cells, B cells, natural killer cells, monocytes, and myeloid cells. PD-1 has two known ligands, PD-L1 and PD-L2. Studies have shown that binding of PD-1 to its ligand, PD-L1, transmits inhibitory signals, reducing the proliferation of CD8+ T cells in lymph nodes. PD-1 can also control the accumulation of antigen-specific T cells in lymph nodes by regulating the Bcl-2 gene. Blocking the binding of PD-1 to PD-L1 effectively prevents the generation of inhibitory signals by T lymphocytes, thereby breaking the immune system's peripheral immune tolerance to self-tissues and promoting T lymphocyte activation and proliferation, as well as cytokine expression.

[0004] Immune checkpoint therapy targeting the PD-1 axis has led to breakthrough improvements in clinical responses in a variety of human cancers (Brahmer et al., N Engl J Med 2012, 366:2455-65; Garon et al., N Engl J Med 2015, 372:2018-28; Hamid et al., N Engl J Med 2013, 369:134-44; Robert et al., Lancet 2014, 384:1109-17; Robert et al., N Engl J Med 2015, 372:2521-32; Robert et al., N Engl J Med 2015, 372:320-30; Topalian et al., N Engl J Med 2012, 366:2443-54; Topalian et al., J Clin Oncol 2014, 32:1020-30; Wolchok et al., N Engl J Med 2013,369:122-33). Immunotherapies targeting the PD-1 axis include monoclonal antibodies against the PD-1 receptor (KEYTRUDA). TM (pembrolizumab),Merck and Co., Inc.,OPDIVO TM (nivolumab), Bristol-Myers Squibb Company, and Hans- TM (serplulimab, Shanghai Henlius Biotech, etc.) and monoclonal antibodies that bind to PD-L1 ligands (MPDL3280A; TECENTRIQ TM (atezolizumab), Genentech; IMFINZI TM (durvalumab),AstraZeneca Pharmaceuticals LP; BAVENCIO TM (avelumab, Merck KGaA). Both treatments have demonstrated anti-tumor effects in multiple cancer types.

[0005] Currently, the most common route of administration for these antibody drugs is intravenous (IV). This is due to the low bioavailability of most other routes of administration, the need for more monitoring during clinical administration, and the faster drug development process. For products that require frequent and long-term administration, the alternative subcutaneous (SC) route of administration is more attractive. When used in conjunction with prefilled syringes and autoinjector technology, SC administration allows for home administration and improves compliance.

[0006] High-dose treatments, such as those with a dose greater than 1 mg / kg or 100 mg / dose, require the development of formulations with concentrations exceeding 100 mg / ml, as small volumes (<1.5 ml) are required for SC administration. For antibody drugs that tend to aggregate at higher concentrations, achieving such high concentrations is a significant development challenge.

[0007] The principle of controlling the solubility of antibody drugs is more complex than that of small molecules, so different strategies are needed to overcome the solubility problem of antibody drugs. Operationally, the solubility of antibody molecules can be described as the maximum amount of antibody molecules when the solution remains clear (e.g., without precipitation, crystallization or gelation of antibody molecules) in the presence of cosolutes. The dependence of the solubility of antibody molecules on ionic strength, salt form, pH, temperature and certain excipients can be mechanically explained by the change in the surface tension of bulk water and the comparison of the binding of antibody molecules to water and ions with their own binding, see Arakawa et al., Theory of protein solubility, Methods of Enzymology, 114: 49-77, 1985; Schein Solubility as a function of protein structure and solvent components, Biotechnology 8 (4): 308-317, 1990; Jenkins Three solutions of the protein solubility problem, Protein Science 7 (2): 376-382, 1998; and others. The binding of antibody molecules to certain excipients or salts can affect solubility by changing the conformation of the antibody molecule or masking certain amino acids involved in self-binding. Antibody molecules can also be preferentially hydrated (and stabilized to form a more compact conformation) by certain salts, amino acids, and sugars, leading to changes in their solubility.

[0008] Aggregation requires collision between two molecules and is the main degradation pathway in antibody molecule solution. The relationship between concentration and formation aggregation depends on the size and binding mechanism of aggregates. Antibody molecule aggregation can cause covalent (such as disulfide bond connection) or non-covalent (reversible or irreversible) binding. The irreversible aggregation caused by non-covalent binding usually occurs via the hydrophobic region exposed by temperature, mechanical or chemical process that can change the native conformation of antibody molecule. Antibody molecule aggregation can affect antibody molecule activity, pharmacokinetics and safety, such as due to immunogenicity.

[0009] Therefore, there is an urgent need in the art for a stable, high-concentration anti-PD-1 antibody pharmaceutical formulation with lower viscosity, less antibody molecule aggregation, and therefore less degradation. Summary of the Invention

[0010] The present application provides a stable high-concentration anti-PD-1 antibody pharmaceutical formulation comprising an anti-PD-1 antibody, histidine-histidine hydrochloride, proline, methionine, polysorbate 20 or polysorbate 80, with a pH of about 5.0 to about 6.0. The present application also provides a method for treating cancer in a subject using the anti-PD-1 antibody pharmaceutical formulation, comprising subcutaneously administering the pharmaceutical formulation of the present invention to the subject. The present application is based in part on this new stable high-concentration anti-PD-1 antibody pharmaceutical formulation, which has higher thermal stability, longer shelf life, lower viscosity, less antibody molecule aggregation, and therefore less degradation.

[0011] Specifically, one aspect of the present application relates to a stable, high-concentration anti-PD-1 antibody pharmaceutical formulation comprising: an anti-PD-1 antibody, histidine-histidine hydrochloride, proline, methionine, polysorbate 20 or polysorbate 80, with a pH of about 5.0 to about 6.0.

[0012] In certain embodiments, the pharmaceutical formulation comprises 120-200 mg / mL of anti-PD-1 antibody.

[0013] In certain embodiments, the pharmaceutical formulation comprises 20-50 mmol / L histidine-histidine hydrochloride.

[0014] In certain embodiments, the pharmaceutical formulation comprises 150-200 mM proline.

[0015] In certain embodiments, the pharmaceutical formulation comprises 0.1-0.3% (w / v) methionine.

[0016] In certain embodiments, the pharmaceutical formulation comprises 0.01-0.03% (w / v) polysorbate 20 or polysorbate 80.

[0017] Another aspect of the present application relates to a pharmaceutical formulation for subcutaneous administration, comprising: 200 mg / mL-220 mg / mL anti-PD-1 antibody, 20 mmol / L histidine-histidine hydrochloride, 40-50 mM proline, 20-70 mM histidine hydrochloride, 20-70 mM arginine, pH 5.0-6.0.

[0018] In certain embodiments, the pharmaceutical formulation comprises an optional hyaluronidase.

[0019] In certain embodiments, the pharmaceutical formulations of the present invention are used to inhibit the interaction of PD-1 and PD-L1 in a patient.

[0020] In certain embodiments, the pharmaceutical formulations of the present invention are used to treat cancer in a patient.

[0021] Yet another aspect of the present application relates to a method of treating cancer in a patient, comprising subcutaneously administering to the patient a therapeutically effective amount of the pharmaceutical formulation of the present invention.

[0022] Yet another aspect of the present application relates to use of the pharmaceutical formulation of the present invention in the preparation of a medicament for treating cancer in a subject.

[0023] Yet another aspect of the present application relates to a pharmaceutical product comprising a sterile container containing a therapeutically effective amount of the pharmaceutical formulation of the present invention.

[0024] The present application also relates to a device comprising a therapeutically effective amount of the pharmaceutical formulation of the present invention.

[0025] Surprisingly, the present application provides an anti-PD-1 antibody pharmaceutical formulation for subcutaneous administration, which has higher thermal stability, longer shelf life, lower viscosity, less antibody molecule aggregation and therefore less degradation.

[0026] The details of the present application are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are now described. Other features, objects and advantages of the present application will be apparent from the specification and claims. In the specification and the appended claims, the singular also includes the plural, unless the context clearly provides otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present application belongs. All patents and publications cited in this specification are incorporated herein by reference in their entirety.

[0027] The contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 shows the DoE interactions of stabilizer, antioxidant, and surfactant screening for h1G4;

[0029] Figure 2 shows the DoE prediction profilers for stabilizer, antioxidant, and surfactant screening. DETAILED DESCRIPTION

[0030] The present application provides a stable high-concentration anti-PD-1 antibody pharmaceutical formulation comprising an anti-PD-1 antibody, histidine-histidine hydrochloride, proline, methionine, polysorbate 20 or polysorbate 80, with a pH of about 5.0 to about 6.0. The present application also provides a method for treating cancer in a subject using the anti-PD-1 antibody pharmaceutical formulation, comprising subcutaneously administering the pharmaceutical formulation of the present invention to the subject. The present application is based in part on this new stable high-concentration anti-PD-1 antibody pharmaceutical formulation, which has higher thermal stability, longer shelf life, lower viscosity, less antibody molecule aggregation, and therefore less degradation.

[0031] For the sake of clarity and not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0032] 1. Definition;

[0033] 2. Anti-PD-1 antibodies;

[0034] 3. Pharmaceutical preparations and their preparation methods;

[0035] 4. Administration and treatment methods; and

[0036] 5. Drug products and devices.

[0037] 1. Definition

[0038] As used herein, the term "antibody" includes full-length antibodies and any antigen-binding fragments thereof (i.e., antibody fragments). An "antibody" can be a part of an independent molecule or an antibody derivative. Exemplary antibody derivatives include, but are not limited to, multifunctional antibodies (e.g., multispecific antibodies (e.g., bispecific antibodies)), antigen recognition receptors (e.g., chimeric antigen receptors), antibody conjugates comprising additional proteins or non-protein moieties (e.g., antibody-drug conjugates or polymer-coated antibodies), and other multifunctional molecules comprising antibodies.

[0039] As used herein, "full-length antibody," "intact antibody," and "whole antibody" refer to antibodies that are structurally similar to natural antibodies or have heavy chains that comprise an Fc region as defined herein. In certain embodiments, a full-length antibody comprises two heavy chains and two light chains. In certain embodiments, the variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the heavy and light chains may be referred to as "VH" and "VL," respectively. The variable regions in both the heavy and light chains typically contain three highly variable loops, known as complementarity determining regions (CDRs) (light chain (LC) CDRs, including LCDR1, LCDR2, and LCDR3; heavy chain (HC) CDRs, including HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by well-known conventions, for example, the conventions of Kabat, Chothia, MacCallum, IMGT, and AHo as described below. The three CDRs of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In certain embodiments, full-length antibodies are glycosylated. In certain embodiments, full-length antibodies contain glycans attached to their Fc region. In certain embodiments, full-length antibodies contain branched glycans.

[0040] As used herein, the terms "antigen-binding portion," "antibody fragment," and "antibody portion" of an antibody refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFv-Fc), single domain antibodies, VHH, VHH-Fc, nanobodies, domain antibodies, bivalent domain antibodies, or any other fragments of an antibody that bind an antigen, or a combination thereof.

[0041] As used herein, the term "CDR" or "complementarity determining region" is intended to refer to the non-contiguous antigen binding sites within the variable region of the heavy and / or light chain. These specific regions have been described by: Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., US Pat. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), wherein the definitions include amino acid residues or subsets of amino acid residues that overlap when compared to each other. However, the application of any definition to refer to the CDRs of an antibody or transplanted antibody or variant thereof is intended to fall within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs defined in each of the references cited above are listed in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and may be included in one or more claims herein.

[0042] Table: CDR Definition 1 The residue numbering follows the nomenclature of Kabat et al. (see above) 2 The residue numbering follows the nomenclature of Chothia et al. (see above) 3 Residue numbering follows the nomenclature of MacCallum et al. (see above)4 Residue numbering follows the nomenclature of Lefranc et al. (see above) 5 Residue numbering follows the nomenclature of Honegger and Plückthun (see above)

[0043] As used herein, a "stable" formulation or drug product is one in which the anti-PD-1 antibody molecule substantially maintains its physical and chemical stability and integrity during storage. The stability of an anti-PD-1 antibody molecule formulation can be determined after a selected period of time at a selected temperature. For example, an increase in aggregate formation after storage at a specific temperature (e.g., storage at 40°C or 25°C (e.g., 2 weeks or 4 weeks) or storage at 2-8°C (3 months, 6 months, 12 months, 18 months, or up to 24 months)) or under specific conditions (e.g., exposure to light, shaking, or freeze-thaw) can be used as an indicator of instability of an anti-PD-1 antibody molecule formulation. In addition to aggregate formation, the retention of original clarity, color, and odor during storage can be used as an indicator to monitor the stability of an anti-PD-1 antibody molecule solution. HMW species are multimers (e.g., tetramers, heptamers, etc.). Generally, a "stable" drug product is one in which the increase in aggregation (as measured by the increase in high molecular weight species (% HMW)) is less than about 5% and preferably 3% after storage at 2-8°C for one year.

[0044] Monomers, dimers, and HMW species of anti-PD-1 antibody molecules can be separated by size exclusion chromatography (SEC). SEC separates molecules based on molecular size. Separation is achieved by differential molecular exclusion or entry as the molecules migrate along the length of the column. Therefore, the resolution increases with increasing column length. A sample of anti-PD-1 antibody molecules can be separated using a 2695 Alliance HPLC (Waters, Milford, MA) connected in series with a TSK GelG3000SWXL (300 mm × 7.8 mm) and a TSK GelG3000SWXL (40 mm × 6.0 mm) column (Tosoh Bioscience, Montgomery, PA). Sample absorbance is measured using a dual-wavelength detector. The area under each peak is integrated. The % HMW species is calculated by dividing the peak area of ​​the HMW species by the total peak area.

[0045] The term "buffer" refers to one or more components that, when added to an aqueous solution, prevents changes in the pH of the solution upon addition of an acid or base or upon dilution with a solvent.

[0046] A "surfactant" is a surface-active molecule containing a hydrophobic portion (eg, an alkyl chain) and a hydrophilic portion (eg, carboxyl and carboxylic acid groups). Surfactants can be added to the formulations of the present invention. Suitable surfactants for use in the formulations of the present invention include, but are not limited to, polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); sorbitan esters and their derivatives; Triton; sodium lauryl sulfate; sodium octylglucoside; lauryl, myristyl, linoleoyl-, or stearoyl-sulfobetaine; lauryl, myristyl, linoleoyl, or stearoyl-sarcosine; linoleoyl-, myristyl, or hexadecyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitamidopropyl-, or isostearamidopropyl betaine (e.g., lauramidopropyl); myristamidopropyl-, palmitamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleoyl taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.).

[0047] As used herein, an "effective amount" refers to an amount sufficient to achieve a desired effect. In certain embodiments, an effective amount is a therapeutically effective amount, i.e., an amount sufficient to achieve a desired therapeutic effect in a subject. Some examples of desired therapeutic effects include, but are not limited to, inhibiting or reducing the binding or interaction of PD-1 with its ligand PD-L1 in a subject, preventing, treating, or ameliorating cancer in a subject.

[0048] As used herein, "subject" generally refers to a mammal. In certain embodiments, the subject is a mammal other than a human or non-human primate. In certain embodiments, the subject is a human or non-human primate. In certain embodiments, the subject is a human. In certain embodiments, the subject is an adult, i.e., a human who is at least 18 years old. In certain embodiments, the subject is a human who is less than 18 years old.

[0049] As used herein, the term "about" refers to amounts within ±10% of any given specified amount. For example, about 200 mg / mL encompasses 90% to 110% of 200 mg / mL, ie, 180 to 220 mg / mL.

[0050] 2. Anti-PD-1 antibodies

[0051] In certain embodiments, the anti-PD-1 antibody of the present invention comprises: a heavy chain variable region (VH) comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and a light chain variable region (VL) comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0052] In certain embodiments, the anti-PD-1 antibody of the present invention comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:8.

[0053] In certain embodiments, the anti-PD-1 antibody of the present invention comprises a heavy chain (HC) of SEQ ID NO: 9 and a light chain (LC) of SEQ ID NO: 10.

[0054] In certain embodiments, the anti-PD-1 antibodies of the present invention comprise a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, a scFv-Fc fusion, a scFv-Fv fusion, or a diabody. In certain embodiments, the anti-PD-1 antibodies of the present invention comprise an Fc region. In further embodiments, the Fc region is a human Fc region.

[0055] In certain embodiments, the Fc region comprises an IgG1 Fc region or an IgG4 Fc region.

[0056] In a preferred embodiment, the Fc region is an IgG4 Fc region.

[0057] In certain embodiments, the Fc region comprises a C-terminal lysine.

[0058] In certain embodiments, the Fc region comprises a deletion of the C-terminal lysine.

[0059] In certain embodiments, the pharmaceutical formulations and compositions of the invention are substantially homogeneous with respect to the anti-PD-1 antibodies of the invention.

[0060] 3. Pharmaceutical preparations and their preparation methods

[0061] 3.1. Pharmaceutical preparations

[0062] The pharmaceutical formulations of the present invention may be used to treat any disease or condition in which it is desirable to reduce the binding or interaction of PD-1 and its ligand PD-L1 in a subject, such as cancer.

[0063] One aspect of the present invention is a pharmaceutical formulation for subcutaneous administration comprising an anti-PD-1 antibody, histidine-histidine hydrochloride, proline, methionine, polysorbate 20 or polysorbate 80, at a pH of about 5.0 to about 6.0. In a further embodiment, the pharmaceutical formulation comprises about 120 mg / mL to about 200 mg / mL of the anti-PD-1 antibody. In a further embodiment, the pharmaceutical formulation comprises about 20 mmol / L to about 50 mmol / L of histidine-histidine hydrochloride. In a further embodiment, the pharmaceutical formulation comprises about 20 mmol / L of histidine-histidine hydrochloride. In a further embodiment, the pharmaceutical formulation comprises about 150 mM to about 200 mM proline. In a further embodiment, the pharmaceutical formulation comprises about 0.1% to about 0.3% methionine. In a further embodiment, the pharmaceutical formulation comprises about 0.01% to about 0.03% polysorbate 20 or polysorbate 80. In a further embodiment, the pharmaceutical formulation comprises about 0.01%-0.03% polysorbate 20. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0064] Another aspect of the present invention is a pharmaceutical formulation comprising about 120 mg / mL to about 200 mg / mL of an anti-PD-1 antibody, about 20 mmol / L to about 50 mmol / L histidine-histidine hydrochloride, about 150 mM to about 200 mM proline, about 0.1% to about 0.3% methionine, about 0.01% to about 0.03% polysorbate 20, and a pH of about 5.0 to about 6.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0065] Another aspect of the present invention is a pharmaceutical formulation comprising about 120 mg / mL to about 200 mg / mL of an anti-PD-1 antibody, about 20 mmol / L histidine-histidine hydrochloride, about 150 mM to about 200 mM proline, about 0.1% to about 0.3% methionine, about 0.01% to about 0.03% polysorbate 20, and a pH of about 5.0 to about 6.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0066] In certain embodiments, the pharmaceutical formulation comprises the following amounts of anti-PD-1 antibody: about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about 124 mg / mL, about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about 128 mg / mL, about 129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 mg / mL, about 133 mg / mL, about 134 mg / mL, about 135 mg / mL, about 136 mg / mL, about 137 mg / mL, about 138 mg / mL, about 139 mg / mL, about 200 mg / mL, about 210 mg / mL, about 221 mg / mL, about 222 mg / mL, about 223 mg / mL, about 224 mg / mL 8mg / mL, about 139mg / mL, about 140mg / mL, about 141mg / mL, about 142mg / mL, about 143mg / mL, about 144mg / mL, about 145mg / mL, about 146mg / mL, about 147mg / mL, about 148mg / mL, about 149mg / mL, about 150mg / mL, about 151mg / mL, about 152mg / mL, about 153mg / mL, about 154mg / mL, about 155mg / mL, about 156mg / mL, about 157mg / mL, about 158mg / mL, about 159mg / mL g / mL, about 160 mg / mL, about 161 mg / mL, about 162 mg / mL, about 163 mg / mL, about 164 mg / mL, about 165 mg / mL, about 166 mg / mL, about 167 mg / mL, about 168 mg / mL, about 169 mg / mL, about 170 mg / mL, about 171 mg / mL, about 172 mg / mL, about 173 mg / mL, about 174 mg / mL, about 175 mg / mL, about 176 mg / mL, about 177 mg / mL, about 178 mg / mL, about 179 mg / mL, about 180 mg / mL

[0015] In some embodiments, the present invention provides an aqueous solution of at least about 1% tantalum acetate (AOC) ...

[0067] In certain embodiments, the pharmaceutical formulation comprises proline in an amount of about 150 mM, about 151 mM, about 152 mM, about 153 mM, about 154 mM, about 155 mM, about 156 mM, about 157 mM, about 158 ​​mM, about 159 mM, about 160 mM, about 161 mM, about 162 mM, about 163 mM, about 164 mM, about 165 mM, about 166 mM, about 167 mM, about 168 mM, about 169 mM, about 170 mM, about 171 mM, about 172 mM, about 173 mM, about 174 mM, about 175 mM, about 176 mM, about 177 mM, about 178 mM, about 179 mM, about 200 mM, about 201 mM, about 202 mM, about 203 mM, about 204 mM, about 205 mM, about 206 mM, about 207 mM, about 208 mM, about 209 mM, about 210 mM, about 211 mM, about 212 mM, about 213 mM, about 214 mM 188mM, about 189mM, about 190mM, about 191mM, about 192mM, about 193mM, about 194mM, about 195mM, about 196mM, about 197mM, about 198mM, about 199mM, about 200mM, or a value therebetween, or any value therebetween.

[0068] In certain embodiments, the pharmaceutical formulation comprises methionine in an amount of about 0.1%, about 0.2%, or about 0.3%, or any value therebetween.

[0069] In certain embodiments, the pharmaceutical formulation comprises polysorbate 20 in an amount of about 0.01%, about 0.02%, or about 0.03%, or any value therebetween.

[0070] In certain embodiments, the pH of the pharmaceutical formulation is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0, or any value therebetween.

[0071] Another aspect of the present invention is a pharmaceutical formulation comprising about 120 mg / mL of an anti-PD-1 antibody, about 20 mmol / L of histidine-histidine hydrochloride, about 200 mM proline, about 0.1% methionine, about 0.02% polysorbate 20, and a pH of about 5.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, LCDR3 of SEQ ID NO: 3, HCDR1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 5, and HCDR3 of SEQ ID NO: 6.

[0072] Another aspect of the present invention is a pharmaceutical formulation comprising about 120 mg / mL of an anti-PD-1 antibody, about 20 mmol / L of histidine-histidine hydrochloride, about 200 mM proline, about 0.1% methionine, about 0.02% polysorbate 20, and a pH of about 5.5. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0073] Another aspect of the present invention is a pharmaceutical formulation comprising about 120 mg / mL of an anti-PD-1 antibody, about 20 mmol / L of histidine-histidine hydrochloride, about 200 mM proline, about 0.1% methionine, about 0.02% polysorbate 20, and a pH of about 6.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0074] Another aspect of the present invention is a pharmaceutical formulation comprising about 120 mg / mL of an anti-PD-1 antibody, about 20 mmol / L histidine-histidine hydrochloride, about 200 mM proline, about 10 mM methionine, about 0.02% polysorbate 20, and a pH of about 5.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0075] Another aspect of the present invention is a pharmaceutical formulation comprising about 120 mg / mL of an anti-PD-1 antibody, about 20 mmol / L histidine-histidine hydrochloride, about 200 mM proline, about 10 mM methionine, about 0.02% polysorbate 20, and a pH of about 5.5. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0076] Another aspect of the present invention is a pharmaceutical formulation comprising about 120 mg / mL of an anti-PD-1 antibody, about 20 mmol / L histidine-histidine hydrochloride, about 200 mM proline, about 10 mM methionine, about 0.02% polysorbate 20, and a pH of about 6.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0077] Another aspect of the present invention is a pharmaceutical formulation comprising about 150 mg / mL of an anti-PD-1 antibody, about 20 mmol / L of histidine-histidine hydrochloride, about 200 mM proline, about 0.1% methionine, about 0.02% polysorbate 20, and a pH of about 5.5. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0078] Another aspect of the present invention is a pharmaceutical formulation comprising about 150 mg / mL of an anti-PD-1 antibody, about 20 mmol / L of histidine-histidine hydrochloride, about 200 mM proline, about 0.1% methionine, about 0.02% polysorbate 20, and a pH of about 6.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0079] Another aspect of the present invention is a pharmaceutical formulation comprising about 200 mg / mL of an anti-PD-1 antibody, about 20 mmol / L of histidine-histidine hydrochloride, about 200 mM proline, about 0.1% methionine, about 0.02% polysorbate 20, and a pH of about 5.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0080] Another aspect of the present invention is a pharmaceutical formulation comprising about 200 mg / mL of an anti-PD-1 antibody, about 20 mmol / L of histidine-histidine hydrochloride, about 200 mM proline, about 0.1% methionine, about 0.02% polysorbate 20, and a pH of about 5.5. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0081] Another aspect of the present invention is a pharmaceutical formulation comprising about 200 mg / mL of an anti-PD-1 antibody, about 20 mmol / L of histidine-histidine hydrochloride, about 200 mM proline, about 0.1% methionine, about 0.02% polysorbate 20, and a pH of about 6.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0082] Another aspect of the present invention is a pharmaceutical formulation comprising about 200 mg / mL to about 220 mg / mL of an anti-PD-1 antibody, about 20 mmol / L histidine-histidine hydrochloride, about 40 mM to about 50 mM proline, about 20 mM to about 70 mM histidine hydrochloride, about 20 mM to about 70 mM arginine, about 0.01-0.03% polysorbate 20 or polysorbate 80, and a pH of about 5.0 to about 6.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0083] Another aspect of the present invention is a pharmaceutical formulation comprising about 200 mg / mL to about 220 mg / mL of an anti-PD-1 antibody, about 20 mmol / L histidine-histidine hydrochloride, about 40 mM to about 50 mM proline, about 70 mM histidine hydrochloride, about 40 mM to about 50 mM arginine, about 0.01-0.03% polysorbate 20 or polysorbate 80, and a pH of about 5.0 to about 6.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0084] Another aspect of the present invention is a pharmaceutical formulation comprising about 200 mg / mL of an anti-PD-1 antibody, about 20 mmol / L histidine-histidine hydrochloride, about 50 mM proline, about 70 mM histidine hydrochloride, about 50 mM arginine, about 0.02% polysorbate 20, and a pH of about 6.0. In a further embodiment, the anti-PD-1 antibody comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0085] In certain embodiments, the anti-PD-1 antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, a scFv-Fc fusion, a scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

[0086] In certain embodiments, the anti-PD-1 antibody comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:8.

[0087] In certain embodiments, the anti-PD-1 antibody comprises an Fc region.

[0088] In certain embodiments, the Fc region is a human Fc region.

[0089] In certain embodiments, the Fc region is an IgG4 Fc region.

[0090] In certain embodiments, the Fc region comprises a C-terminal lysine.

[0091] In certain embodiments, the Fc region comprises a deletion of the C-terminal lysine.

[0092] In certain embodiments, the anti-PD-1 antibody comprises a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO:10.

[0093] In certain embodiments, the pharmaceutical formulations of the present invention comprise an optional hyaluronidase.

[0094] In certain embodiments, the pharmaceutical formulation comprises hyaluronidase in an amount of about 50 Units / mL, 100 Units / mL, 200 Units / mL, 300 Units / mL, 400 Units / mL, 500 Units / mL, 600 Units / mL, 700 Units / mL, 800 Units / mL, 900 Units / mL, 1000 Units / mL, 1500 Units / mL, 2000 Units / mL, 2500 Units / mL, 3000 Units / mL, 3500 Units / mL, 4000 Units / mL, 4500 Units / mL, 5000 Units / mL, or any value therebetween.

[0095] In certain embodiments, the pharmaceutical formulation comprises hyaluronidase in an amount of about 50 units / mL to 5000 units / mL, preferably 1000 units / mL to 5000 units / mL, more preferably 1000 units / mL to 4000 units / mL, and most preferably 1000 units / mL to 3000 units / mL.

[0096] In certain embodiments, the pharmaceutical formulations of the present invention are suitable for subcutaneous use.

[0097] In certain embodiments, the pharmaceutical preparations of the present invention are substantially free of dissolved oxygen. As used herein, the term "substantially free" refers to at least 95% free of dissolved oxygen. For example, in certain embodiments, the pharmaceutical preparation is at least 95% free of dissolved oxygen. In certain embodiments, the pharmaceutical preparation is at least 96%, at least 97%, at least 98%, at least 99% or 100% free of dissolved oxygen. Assuming that water is usually balanced with air (which is 20% oxygen), in certain embodiments, the pharmaceutical preparation that is substantially free of dissolved oxygen comprises less than or equal to 1% dissolved oxygen. In certain embodiments, the pharmaceutical preparation is 100% free of dissolved oxygen.

[0098] In certain embodiments, the pharmaceutical formulation is used to inhibit the binding or interaction of PD-1 and PD-L1 in a patient.

[0099] In certain embodiments, the pharmaceutical preparation is used to treat cancer in a patient. In a further embodiment, the cancer is selected from: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, breast cancer, gastrointestinal cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, primary mediastinal large B cell lymphoma, kidney cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, endometrial cancer, skin squamous cell carcinoma, thyroid cancer, prostate cancer, glioblastoma, Merkel cell carcinoma and salivary gland cancer. In a further embodiment, the patient suffers from a tumor with a high mutation load. In a further embodiment, the patient has microsatellite instability high (MSI-H) or mismatch repair defective solid tumor. In a further embodiment, the cancer is an unresectable or metastatic melanoma. In a further embodiment, the cancer is metastatic non-small cell lung cancer (NSCLC).

[0100] In certain embodiments, the pharmaceutical preparation is suitable for in vivo use. For example, in such embodiments, the pharmaceutical preparation is sterile and does not contain pharmaceutically unacceptable amounts of toxic substances, such as endotoxins. Such pharmaceutical preparations can meet, for example, Good Manufacturing Process (GMP) quality standards according to regulations issued by the U.S. Food and Drug Administration (FDA).

[0101] 3.2. Methods for preparing the formulation

[0102] The preparation according to the present invention can be prepared using any suitable method. Typically, the anti-PD-1 antibody of the present invention is prepared by eukaryotic cells comprising an expression vector or a nucleic acid sequence encoding the antibody. For example, eukaryotic cells can be: Chinese hamster ovary (CHO) cells, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR-), HELA (human cervical cancer), CVI (monkey kidney line), COS (derivatives of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblasts) BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / 0 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney) or NS0 cells. In one embodiment, the eukaryotic cell for expressing the anti-PD-1 antibody of the present invention is a CHO cell. See, for example, WO 2018052818A1, the entire contents of which are incorporated herein by reference. The anti-PD-1 antibodies of the present invention are generally expressed as secreted proteins, which can be isolated from cells using techniques known in the art. Typically, the isolated and unconcentrated protein product is then placed in a sterile aqueous solution.

[0103] This initial product is then concentrated and buffer exchanged as appropriate to obtain a concentrated protein solution comprising an anti-PD-1 antibody of the invention at a concentration equal to or exceeding the target final concentration.

[0104] Any suitable method known in the art can be used for concentration. Such methods may include, but are not limited to, tangential flow filtration (TFF), dialysis, ultrafiltration, and freeze drying. For commercial production purposes, TFF can generally be used.

[0105] Additional components may then be added to obtain the desired final formulation. For example, additional components (e.g., NaCl, arginine HCl, sucrose, and / or polysorbate) may be added from concentrated stock solutions of each of the additional components, and, if necessary, water may be added to obtain the desired final formulation. In a specific embodiment, polysorbate 20 (PS20) or polysorbate 80 (PS80) is added as the last excipient of the formulation to achieve the correct pH (due to the molecular weight of polysorbate together with the anti-PD-1 antibodies of the present invention, adding polysorbate at the end avoids concentration).

[0106] In certain embodiments, the intermediate solution and additional components are degassed or otherwise treated to reduce or eliminate dissolved oxygen. For example, the intermediate solution and components can be balanced with argon or nitrogen.

[0107] In certain embodiments, the final pharmaceutical formulation is degassed or otherwise treated to reduce or eliminate dissolved oxygen. For example, argon or nitrogen can be used to equilibrate the final pharmaceutical formulation by bubbling the gas through the final pharmaceutical formulation for a time sufficient to reduce or eliminate dissolved oxygen from the formulation. In certain embodiments, the final pharmaceutical formulation is then stored under a nitrogen atmosphere.

[0108] The pharmaceutical preparations thus prepared will typically be sterile filtered and then aliquoted and stored in sterile containers or devices as described herein.

[0109] 4. Administration and Treatment Methods

[0110] 4.1. Route of administration

[0111] The pharmaceutical preparations of the present invention are suitable for parenteral administration. In certain embodiments, the pharmaceutical preparations of the present invention are suitable for subcutaneous administration. In certain embodiments, the pharmaceutical preparations of the present invention are suitable for intravenous administration. In certain embodiments, the pharmaceutical preparations of the present invention are suitable for intraperitoneal administration.

[0112] 4.2. Treatment methods and uses

[0113] The anti-PD-1 antibody formulations provided herein can be administered to a subject (e.g., a mammal such as a human) to treat diseases and disorders involving aberrant PD-1 activity, including, for example, cancer.

[0114] Another aspect of the present invention is a method of treating cancer in a subject, comprising subcutaneously administering to the subject a therapeutically effective amount of a pharmaceutical formulation of the present invention.

[0115] Yet another aspect of the present invention is the use of a pharmaceutical formulation of the present invention in the preparation of a medicament for treating cancer in a subject.

[0116] In certain embodiments, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, breast cancer, gastrointestinal cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, endometrial cancer, squamous cell carcinoma of the skin, thyroid cancer, prostate cancer, glioblastoma, Merkel cell carcinoma, and salivary gland cancer.

[0117] Many diagnostic methods are known in the art for cancer (such as melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, breast cancer, gastrointestinal cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, endometrial cancer, cutaneous squamous cell carcinoma, thyroid cancer, prostate cancer, glioblastoma, Merkel cell carcinoma and salivary gland cancer) or any other disease showing aberrant PD-1 activity, and the clinical description of such diseases. These methods include, but are not limited to, for example, immunohistochemistry, PCR, fluorescence in situ hybridization (FISH). Additional details regarding diagnostic methods for aberrant PD-1 activity or expression are reported, for example, in Gupta et al., (2009) Mod Pathol. 22(1):128-133; Lopez-Rios et al., (2013) J Clin Pathol. 66(5):381-385; Ellison et al., (2013) J Clin Pathol 66(2):79-89; and Guha et al., (2013) PLoS ONE 8(6):e67782.

[0118] In certain embodiments, the subject has a tumor with a high mutational burden.

[0119] In certain embodiments, the subject has a microsatellite instability-high (MSI-H) or mismatch repair-deficient solid tumor.

[0120] In certain embodiments, the cancer is unresectable or metastatic melanoma.

[0121] In certain embodiments, the cancer is metastatic non-small cell lung cancer (NSCLC). In a further embodiment, the subject has a tumor that expresses PD-L1 as measured by a tumor proportion score (TPS) of ≥1% and has not been previously treated with platinum-containing chemotherapy. In a further embodiment, the subject has a tumor that expresses PD-L1 as measured by a tumor proportion score (TPS) of ≥1% and has been previously treated with platinum-containing chemotherapy. In a further embodiment, the subject's tumor does not have EGFR or ALK genomic aberrations. In a further embodiment, the method further comprises administering pemetrexed and platinum chemotherapy to the subject. In a further embodiment, the NSCLC is squamous and the subject is also treated with carboplatin and paclitaxel or albumin-bound paclitaxel.

[0122] In certain embodiments, the cancer is recurrent or metastatic head and neck squamous cell carcinoma (HNSCC).

[0123] In certain embodiments, the cancer is locally advanced or metastatic urothelial carcinoma.

[0124] In certain embodiments, the cancer is locally advanced or metastatic gastric cancer or gastroesophageal junction adenocarcinoma.

[0125] In certain embodiments, the cancer is cervical cancer.In further embodiments, the cervical cancer is recurrent or metastatic cervical cancer and the subject has disease progression during or after chemotherapy.

[0126] In certain embodiments, the cancer is primary mediastinal large B-cell lymphoma (PMBCL).In further embodiments, the subject has refractory PMBCL or has relapsed after 2 or more prior therapies.

[0127] In certain embodiments, the cancer is resected stage IIB, IIC, or III melanoma.

[0128] In certain embodiments, the cancer is hepatocellular carcinoma.

[0129] In certain embodiments, the cancer is renal cell carcinoma (RCC).

[0130] In certain embodiments, the cancer is advanced clear cell RCC.

[0131] In certain embodiments, the cancer is recurrent, locally advanced, or metastatic Merkel cell carcinoma (MCC).

[0132] In certain embodiments, the cancer is small cell lung cancer.

[0133] In certain embodiments, the anti-PD-1 antibody pharmaceutical formulations provided herein are administered in combination with a second, third, or fourth agent (including, for example, an anti-tumor agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent) to treat a disease or disorder involving aberrant PD-1 activity. Such agents include, for example, docetaxel, gefitinib, FOLFIRI (irinotecan, 5-fluorouracil, and leucovorin), irinotecan, cisplatin, carboplatin, paclitaxel, bevacizumab (anti-VEGF antibody), FOLFOX-4, infusional fluorouracil, and leucovorin. fluorouracil), leucovorin, and oxaliplatin, afatinib, gemcitabine, capecitabine, pemetrexed, tivantinib, everolimus, CpG-ODN, rapamycin, lenalidomide, vemurafenib, endostatin, lapatinib, PX-866, Imprime PGG, and erlotinib.

[0134] In certain embodiments, the anti-PD-1 antibody pharmaceutical preparations provided herein are used in combination with one or more additional treatments, such as radiotherapy, surgery, chemotherapy and / or targeted therapy. In certain embodiments, the anti-PD-1 antibody pharmaceutical preparations provided herein are used in combination with radiotherapy. In certain embodiments, the anti-PD-1 antibody pharmaceutical preparations provided herein are used in combination with radiotherapy to treat a cancer selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, breast cancer, gastrointestinal cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, endometrial cancer, squamous cell carcinoma of the skin, thyroid cancer, prostate cancer, glioblastoma, Merkel cell carcinoma, and salivary gland cancer.

[0135] Depending on the condition to be treated and factors related to administration familiar to physicians in the field, the anti-PD-1 antibody pharmaceutical formulations provided herein will be administered at a dose that effectively treats the condition while minimizing toxicity and side effects. The therapeutic or preventive efficacy can be monitored by regular assessment of treated patients. In terms of repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired inhibitory effect of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the present invention.

[0136] Cancer treatment can be assessed by, for example, but not limited to, tumor regression, reduction in tumor weight or volume, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and / or activity. Methods for determining the efficacy of treatment can be employed, including, for example, measuring response by radiographic imaging.

[0137] 5. Drug products and devices

[0138] 5.1. Pharmaceutical Products

[0139] One aspect of the present invention is a packaged pharmaceutical product comprising a sterile container containing a therapeutically effective amount of a pharmaceutical formulation of the present invention. In various embodiments, the packaged pharmaceutical product can be presented as a single-use vial, a multiple-use vial, or a prefilled syringe.

[0140] 5.2. Device

[0141] One aspect of the invention is a device comprising a therapeutically effective amount of a pharmaceutical formulation of the invention.

[0142] In certain embodiments, the device comprises a syringe containing a pharmaceutical formulation. Such a syringe may optionally be equipped with a needle suitable for administering at least a portion of the aqueous solution contained in the syringe to a subject. Fine gauge needles (small diameter) provide less pain for the patient, but require low viscosity medications. The needle gauge is preferably 27 or higher (i.e., a smaller diameter). The outer diameter of the needle may be 0.413 mm, 0.41 mm, or less.

[0143] In certain embodiments, the syringe is presented as a pre-filled syringe. Such a pre-filled syringe may be suitable for single use, or alternatively, for multiple (two or more) uses. Such a pre-filled syringe may optionally be equipped with a needle suitable for administering at least a portion of the aqueous solution contained in the pre-filled syringe to a subject. In certain embodiments, the pre-filled syringe is presented in a single-unit package.

[0144] In certain embodiments, the prefilled syringe is substantially free of air. That is, in such embodiments, the pharmaceutical formulation contained within the prefilled syringe is substantially free of dissolved oxygen. For example, the pharmaceutical formulation contained within the prefilled syringe can be prepared as described herein in the presence of nitrogen and then placed within the syringe and sealed under a nitrogen atmosphere to exclude atmospheric air. In certain such embodiments, the prefilled syringe can be present in airtight packaging.

[0145] In a specific embodiment, the present invention is a pre-filled syringe filled with an anti-PD-1 antibody pharmaceutical formulation described herein. Alternatively, the present invention is a vial filled with an anti-PD-1 antibody pharmaceutical formulation described herein. Such a vial can be present in a kit together with a needle suitable for administering at least a portion of the aqueous solution contained in the vial to a subject.

[0146] The pharmaceutical formulation can be administered as a subcutaneous injection by the patient himself, for example, by using a pre-filled syringe with 2 mL-5 mL of the pharmaceutical formulation according to the present invention in one go (one shot). Such a "push" subcutaneous administration takes about 12 to 20 seconds or at most 1 minute. In comparison: it can take several minutes to several hours for a nurse or caregiver to infuse a subject; an IV (intravenous) infusion of the anti-PD-1 antibody formulation of the present invention takes about 60 minutes. The pre-filled syringe provides an advantage for the patient because it can be used as a subcutaneous maintenance dose by self-administration.

[0147] In addition, this application also relates to the following implementation schemes:

[0148] 1. A pharmaceutical preparation of an anti-PD-1 antibody, comprising:

[0149] a. 120 mg / mL to 200 mg / mL of an anti-PD-1 antibody, wherein the anti-PD-1 antibody comprises:

[0150] a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and

[0151] a light chain variable region comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0152] b. 20mmol / L-50mmol / L histidine-histidine hydrochloride,

[0153] c.150mM-200mM proline,

[0154] d. 0.1%-0.3% (w / v) methionine,

[0155] e. 0.01%-0.03% (w / v) nonionic surfactant, wherein the nonionic surfactant is selected from polysorbate 20 and polysorbate 80,

[0156] pH 5.0-6.0.

[0157] 2. The pharmaceutical formulation according to embodiment 1, wherein the formulation comprises 20 mmol / L histidine-histidine hydrochloride.

[0158] 3. The pharmaceutical formulation according to embodiment 1 or 2, wherein the formulation comprises 0.01%-0.03% (w / v) polysorbate 20.

[0159] 4. The pharmaceutical formulation according to any one of the preceding embodiments, further comprising hyaluronidase.

[0160] 5. The pharmaceutical formulation according to embodiment 4, wherein the pharmaceutical formulation comprises 50 U / ml to 5000 U / ml of hyaluronidase.

[0161] 6. A pharmaceutical formulation of an anti-PD-1 antibody for subcutaneous administration, comprising:

[0162] 1) 200 mg / mL-220 mg / mL anti-PD-1 antibody, wherein the anti-PD-1 antibody comprises:

[0163] a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and

[0164] a light chain variable region comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0165] 2) 20mmol / L histidine-histidine hydrochloride,

[0166] 3) 40-50 mM proline,

[0167] 4) 20-70 mM histidine hydrochloride,

[0168] 5) 20-70 mM arginine,

[0169] 6) 0.01-0.03% polysorbate 20 or polysorbate 80,

[0170] pH 5.0-6.0.

[0171] 7. The pharmaceutical formulation of embodiment 6, wherein the formulation comprises 70 mM histidine hydrochloride.

[0172] 8. The pharmaceutical formulation according to embodiment 6 or 7, wherein the formulation comprises 40 mM-50 mM arginine.

[0173] 9. The pharmaceutical formulation of any one of embodiments 6-8, wherein the formulation comprises 0.01-0.03% polysorbate 20.

[0174] 10. The pharmaceutical formulation of any one of embodiments 6-9, comprising hyaluronidase.

[0175] 11. The pharmaceutical formulation of any one of embodiments 6-9, wherein the concentration of the hyaluronidase is about 50 U / ml to 5000 U / ml.

[0176] 12. The pharmaceutical formulation according to any one of the preceding embodiments, wherein the anti-PD-1 antibody comprises the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 8.

[0177] 13. The pharmaceutical formulation of any one of the preceding embodiments, wherein the anti-PD-1 antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

[0178] 14. The pharmaceutical formulation according to any one of the preceding embodiments, wherein the anti-PD-1 antibody comprises an Fc region.

[0179] 15. The pharmaceutical formulation of embodiment 14, wherein the Fc region is a human Fc region.

[0180] 16. The pharmaceutical formulation of embodiment 14 or 15, wherein the Fc region is an IgG4 Fc region.

[0181] 17. The pharmaceutical formulation of any one of embodiments 13-16, wherein the Fc region comprises a C-terminal lysine.

[0182] 18. The pharmaceutical formulation of any one of embodiments 13-17, wherein the Fc region comprises a deletion of the C-terminal lysine.

[0183] 19. The pharmaceutical formulation according to any one of the preceding embodiments, wherein the anti-PD-1 antibody comprises a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10.

[0184] 20. The pharmaceutical formulation according to any one of embodiments 1-19, comprising 120 mg / mL anti-PD-1 antibody, 20 mmol / L histidine-histidine hydrochloride, 200 mM proline, 0.1% (w / w) methionine, 0.02% (w / v) polysorbate 20, pH 5.0-5.5.

[0185] 21. The pharmaceutical formulation of any one of embodiments 1-19, comprising 150 mg / mL anti-PD-1 antibody, 20 mmol / L histidine-histidine hydrochloride, 200 mM proline, 0.1% (w / w) methionine, 0.02% (w / v) polysorbate 20, pH 5.5-6.0.

[0186] 22. The pharmaceutical formulation of any one of embodiments 1-19, comprising 200 mg / mL anti-PD-1 antibody, 20 mmol / L histidine-histidine hydrochloride, 200 mM proline, 0.1% (w / w) methionine, 0.02% (w / v) polysorbate 20, pH 5.0-5.5.

[0187] 23. The pharmaceutical formulation according to any one of embodiments 1-19, comprising: 200 mg / mL anti-PD-1 antibody, 20 mmol / L histidine-histidine hydrochloride, 50 mM proline, 70 mM histidine hydrochloride, 50 mM arginine, 0.02% (w / v) polysorbate 20, pH 6.0.

[0188] 24. The pharmaceutical formulation according to any one of the preceding embodiments, wherein the pharmaceutical formulation is substantially free of dissolved oxygen.

[0189] 25. The pharmaceutical formulation according to any one of the preceding embodiments, wherein the pharmaceutical formulation is suitable for subcutaneous administration.

[0190] 26. The pharmaceutical formulation according to any one of the preceding embodiments, for use in inhibiting the interaction of PD-1 and PD-L1 in a patient.

[0191] 27. The pharmaceutical formulation according to any one of the preceding embodiments, for use in treating cancer in a patient.

[0192] 28. The pharmaceutical formulation of embodiment 27, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, breast cancer, gastrointestinal cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, endometrial cancer, squamous cell carcinoma of the skin, thyroid cancer, prostate cancer, glioblastoma, Merkel cell carcinoma, and salivary gland cancer.

[0193] 29. The pharmaceutical formulation of embodiment 27 or 28, wherein the patient has a tumor with a high mutational load.

[0194] 30. The pharmaceutical formulation of embodiment 27 or 28, wherein the patient has a microsatellite instability-high (MSI-H) or mismatch repair-deficient solid tumor.

[0195] 31. The pharmaceutical formulation of embodiment 27 or 28, wherein the cancer is unresectable or metastatic melanoma.

[0196] 32. The pharmaceutical formulation of embodiment 27 or 28, wherein the cancer is metastatic non-small cell lung cancer (NSCLC).

[0197] 33. A method of treating cancer in a patient comprising subcutaneously administering to said patient a therapeutically effective amount of the pharmaceutical formulation of any one of embodiments 1-23.

[0198] 34. The method of embodiment 33, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, breast cancer, gastrointestinal cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, endometrial cancer, squamous cell carcinoma of the skin, thyroid cancer, prostate cancer, glioblastoma, Merkel cell carcinoma, and salivary gland cancer.

[0199] 35. The method of embodiment 33 or 34, wherein the patient has a tumor with a high mutational load.

[0200] 36. The method of embodiment 33 or 34, wherein the patient has a microsatellite instability-high (MSI-H) or mismatch repair-deficient solid tumor.

[0201] 37. The method of embodiment 33 or 34, wherein the cancer is unresectable or metastatic melanoma.

[0202] 38. The method of embodiment 33 or 34, wherein the cancer is metastatic non-small cell lung cancer (NSCLC).

[0203] 39. The method of embodiment 38, wherein the patient has a tumor that expresses PD-L1 as measured by a tumor proportion score (TPS) of ≥1% and has not been previously treated with platinum-containing chemotherapy.

[0204] 40. The method of embodiment 38, wherein the patient has a tumor that expresses PD-L1 as measured by a tumor proportion score (TPS) of ≥1% and was previously treated with platinum-containing chemotherapy.

[0205] 41. The method of any one of embodiments 38-40, wherein the patient's tumor does not have EGFR or ALK genomic aberrations.

[0206] 42. The method of any one of embodiments 38-41, wherein the method further comprises administering pemetrexed and platinum chemotherapy to the patient.

[0207] 43. The method of embodiment 38, wherein the NSCLC is squamous and the patient is also treated with carboplatin and paclitaxel or nab-paclitaxel.

[0208] 44. The method of embodiment 33 or 34, wherein the cancer is recurrent or metastatic head and neck squamous cell carcinoma (HNSCC).

[0209] 45. The method of embodiment 33 or 34, wherein the cancer is locally advanced or metastatic urothelial carcinoma.

[0210] 46. ​​The method of embodiment 33 or 34, wherein the cancer is locally advanced or metastatic gastric cancer or gastroesophageal junction adenocarcinoma.

[0211] 47. The method of embodiment 33 or 34, wherein the cancer is cervical cancer.

[0212] 48. The method of embodiment 47, wherein the cervical cancer is recurrent or metastatic cervical cancer and the patient has disease progression during or after chemotherapy.

[0213] 49. The method of embodiment 33 or 34, wherein the cancer is primary mediastinal large B-cell lymphoma (PMBCL).

[0214] 50. The method of embodiment 49, wherein the patient has refractory PMBCL or has relapsed after 2 or more prior therapies.

[0215] 51. The method of embodiment 33 or 34, wherein the cancer is resected stage IIB, IIC, or III melanoma.

[0216] 52. The method of embodiment 33 or 34, wherein the cancer is hepatocellular carcinoma.

[0217] 53. The method of embodiment 33 or 34, wherein the cancer is renal cell carcinoma (RCC).

[0218] 54. The method of embodiment 53, wherein the cancer is advanced clear cell RCC.

[0219] 55. The method of embodiment 33 or 34, wherein the cancer is recurrent, locally advanced or metastatic Merkel cell carcinoma (MCC).

[0220] 56. The method of embodiment 33 or 34, wherein the cancer is small cell lung cancer.

[0221] 57. A pharmaceutical product comprising a sterile container containing a therapeutically effective amount of the pharmaceutical formulation according to any one of embodiments 1 to 23.

[0222] 58. A device comprising a therapeutically effective amount of the pharmaceutical formulation according to any one of embodiments 1 to 23.

[0223] 59. The device of embodiment 58, wherein the device comprises a syringe containing the drug formulation.

[0224] 60. The device of embodiment 58 or 59, wherein the syringe is a pre-filled syringe.

[0225] 61. Use of the pharmaceutical formulation according to any one of embodiments 1-23 in the preparation of a medicament for treating cancer.

[0226] 62. The use according to embodiment 61, wherein the pharmaceutical preparation is administered subcutaneously or intravenously.

[0227] 63. The use according to embodiment 61 or 62, wherein the cancer is selected from the group consisting of: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, breast cancer, gastrointestinal cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, endometrial cancer, squamous cell carcinoma of the skin, thyroid cancer, prostate cancer, glioblastoma, Merkel cell carcinoma, and salivary gland cancer.

[0228] Sequence Listing

[0229] The following examples are merely illustrative of the subject matter of the present invention and should not be considered limiting in any way.

[0230] Example

[0231] Determination method

[0232] Appearance and visible foreign matter

[0233] Visual inspection was used to inspect the appearance of the samples. Wipe the sample bottle clean and place it in a darkroom under a Shanghai Huanghai Drug Inspection SC-4000A Clarity Tester. Adjust the illumination to 2000-3750 lux. Hold the sample bottle at the edge of the light shield (25 cm) and hold the neck of the test bottle. Visually inspect the sample for color, clarity, and visible foreign matter against both black and white backgrounds.

[0234] Protein concentration (A 280 )

[0235] Using a Trinean Dropsense 16 protein analyzer, add 2 μL of sample and measure the absorbance at 280 nm to calculate the concentration. The extinction coefficient is 1.50 AU*mL*mg-1*cm-1.

[0236] pH

[0237] The pH meter was calibrated with three standard solutions (pH values ​​of the standard solutions were 4.01, 7.00, and 9.21, respectively) to keep the electrode slope within the range of 95% to 105%. A 50 μL sample was taken to determine the pH value.

[0238] Osmotic pressure

[0239] The osmotic pressure was measured using an Advanced Osmo PRO osmometer in accordance with General Chapter 0632 “Osmolality Determination Method” of the Chinese Pharmacopoeia (2015 edition). 20 μL of sample and two 290 mOsmol / kg osmotic pressure standards were taken, and the osmotic pressure values ​​of the sample and standard were determined by the freezing point method.

[0240] Viscosity

[0241] Rotational viscometer

[0242] A Brookfield DV27 rotational viscometer with a CPA-40Z rotor was used, with the torque set at 40-60% and the stop time at 1 minute. 500 μL of sample was added for detection.

[0243] High-throughput microrheometer

[0244] Use the RheoSense VROC initium high-throughput microrheometer with E02 or B05 chip to add 60-100 μL of sample to the liner tube of the sample bottle for detection.

[0245] Size Exclusion Chromatography (SEC)

[0246] An Agilent 1260 HPLC was used with a TOSOH TSK G3000 (300 x 7.8 mm) column. The column temperature was room temperature, the flow rate was 0.5 mL / min, and the detection wavelength was 280 nm. The sample was diluted to approximately 1 mg / mL with the mobile phase, and 50 μL was injected with a 20-min dwell time.

[0247] Cation exchange chromatography (CEX)

[0248] Agilent 1260 high performance liquid chromatography, ThermoPropac TM A WCX-10 BioLC (4 × 250 mm) column was used with a column temperature of 35°C, a flow rate of 1.0 mL / min, and a detection wavelength of 280 nm. The sample was diluted to approximately 1 mg / mL with mobile phase, and 20 μL was injected. The elution time was 45 min.

[0249] Capillary gel electrophoresis (CE-SDS)

[0250] A Beckman PA800 plus capillary electrophoresis instrument with Beckman Coulter non-coated capillaries (total length 67 cm, inner diameter 50 μm, catalog number: 390953 / A10663 / 338451) was used. The capillary temperature was set at 25°C, the sample chamber temperature was 8°C, the detection wavelength was 220 nm, the injection time was 20.0 s, and the sample separation time was 35 min.

[0251] Prepare the alkylation reagent with ultrapure water to a concentration of 46 mg / mL, and dilute the sample to 4 mg / mL with ultrapure water for later use. Prepare the test solution according to the following table (Table 1). Heat the test sample at 70°C for 10 minutes and then cool to room temperature.

[0252] Table 1 Capillary gel electrophoresis sample preparation

[0253] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

[0254] Dilute the sample to 2.0 mg / mL with ultrapure water and prepare a 12.5 mg / mL alkylation protecting agent (NEM) solution. Treat according to Table 2.

[0255] Table 2 SDS-PAGE sample processing

[0256] The treated samples were heated at 70°C for 10 minutes. Protein purity and molecular weight were determined by SDS-PAGE on reduced and non-reduced forms using an Invitrogen PS0091 electrophoresis instrument and NuPAGE 4-12% Bis-Tris gels. A sample load of 10 μg / well was used, the voltage was set at 200 V, and the electrophoresis time was approximately 35 minutes (the electrophoresis time could be adjusted based on the band migration speed).

[0257] After electrophoresis, rinse the gel with water, stain it with staining solution, and decolorize it with ultrapure water after the bands are clear. Scan the completely destained gel using a BIO-RAD GS-900 gel scanner and analyze the sample purity.

[0258] Dynamic particle morphology analysis (FlowCam)

[0259] The morphology and number of subvisible particles in the samples were determined using a FlowCam 8100 particle analyzer, and the sample was injected manually once (Table 3).

[0260] Table 3 Subvisible particle detection parameter settings

[0261] Dynamic Light Scattering (DLS)

[0262] The particle size and size distribution of the samples were determined using a WYATT DynaProPlateReader-III high-throughput dynamic and static light scattering instrument. Within a clean bench, 25 μL of sample was added to a 384-well microplate to avoid the introduction of extraneous particles that could affect the assay (Table 4).

[0263] Table 4 Parameter settings for measuring sample particle size using high-throughput dynamic and static light scattering instrument

[0264] Aggregation temperature (T agg )

[0265] The sample aggregation temperature T was determined using a WYATT DynaProPlateReader-Ⅲ high-throughput dynamic and static light scattering instrument. agg 25 μL of sample was added to a 384-well microplate in a clean bench to avoid the introduction of exogenous particles that could affect the assay (Table 5).

[0266] Table 5 Parameter settings for the detection of sample aggregation temperature by high-throughput dynamic and static light scattering instrument

[0267] Denaturation temperature (T m )

[0268] The initial denaturation temperature T of the sample was determined using a UNcle differential fluorescence scanner. m onset and denaturation temperature T m . 9 μL of sample was taken and added to the Uni tube in the ultra-clean workbench to avoid the introduction of exogenous particles that would affect the detection (Table 6).

[0269] Table 6 Parameter settings for sample denaturation temperature determination using differential fluorescence scanner

[0270] Diffusion interaction parameter (k D )

[0271] Diffusion interaction parameter (k D ) describes the diffusion coefficient (D t ) with concentration, including all intermolecular forces, such as charge interaction, hydrophobic interaction and hydrogen bond. This experiment uses the WayytDynaPro Plate Reader-III high-throughput dynamic and static light scattering instrument to measure the D of samples with different concentrations. t The linear fitting of Dt and protein concentration (c) is performed as follows: t =D0(1+k D c)

[0272] Among them D t is the diffusion coefficient at different protein concentrations, D0 is the diffusion coefficient when the protein concentration is 0, k D is the diffusion interaction parameter, c is the protein concentration. The slope of the fitted line / D0 is the k of the sample. D .

[0273] The test process is as follows: dilute the sample with placebo to 2-20 mg / mL in a biosafety cabinet, dilute 5-10 concentration gradients, take 100 μL and add it to a 96-well plate, try to avoid impurities in the air from mixing into the solution and sample pool to affect the test. D Analysis fitted the protein k D Specific parameter settings are shown in Table 7.

[0274] Table 7 k D Detection parameter settings

[0275] Example 1. Preparation of antibodies

[0276] The anti-PD-1 antibody h1G4 is disclosed in patent application WO2018052818A1, and its preparation method is as described in the patent application.

[0277] Example 2. Effect of protein concentration

[0278] The basic physicochemical properties and stability of h1G4 at different protein concentrations were investigated (see Table 8).

[0279] Table 8 Composition of candidate formulations for h1G4 drugability study

[0280] Filter the sample with a 0.22 μm disposable sterile filter in a biosafety cabinet, aseptically dispense 1.0 mL of the sample into a 2 mL sterile vial, add a 13 mm rubber stopper and a 13 mm aluminum-plastic combination cap, and sample and test according to the experimental plan (Table 9).

[0281] Table 9 Investigation conditions and detection methods for the drugability study of h1G4

[0282] Initial protein quality results showed that all formulations F1_10 to F4_200 were free of visible foreign matter, with protein concentration, pH, and osmotic pressure meeting target values. The formulations appeared as a light yellow, slightly opalescent liquid, with color and opalescence increasing with increasing protein concentration. The four formulations demonstrated good colloidal and conformational stability, with viscosity increasing exponentially with increasing protein concentration, albeit at a relatively low value. While initial h1G4 purity (SEC, CEX, non-reduced CE-SDS, and reduced CE-SDS) remained unchanged with increasing protein concentration, the aggregate content, as measured by DLS and subvisible particle size, increased.

[0283] Drugability studies showed that after four weeks of stabilization at 40°C, none of the four formulations produced visible particles, but aggregates of other sizes increased with increasing protein concentration. Degradation and charge isoforms were independent of protein concentration and were better than the average levels of marketed drugs and the company's pipeline. After two weeks of stabilization at 25°C, the aggregate content and number of subvisible particles increased with increasing protein concentration, while DLS-sized aggregates showed no significant changes. Degradation and charge isoforms were independent of protein concentration and showed no significant changes after two weeks of accelerated stabilization at room temperature. After two weeks of stabilization under illumination (25°C, 5000 lx), the four formulations did not produce visible particles, but aggregates of other sizes increased with increasing protein concentration. Higher protein concentrations were associated with more pronounced changes in degradation and charge isoforms. After 10 cycles of freezing and thawing from -20°C to room temperature, the four formulations readily formed subvisible particles, but were less likely to form soluble aggregates and DLS-sized particles. Repeated freeze-thaw cycles were less likely to induce protein degradation and charge isoform changes.

[0284] Example 3. Determination of Stable Formulations at 120-150 mg / mL

[0285] Screen stable preparations based on buffer type, pH, ionic strength, and excipients.

[0286] 3.1 Effect of buffer type and pH

[0287] Various formulations were prepared for screening as shown in the table below. Formulations were prepared and characterized as described in the previous examples.

[0288] Table 10 h1G4 formulations with various buffers and pH

[0289] Table 11 Summary of buffer type and pH screening data

[0290] Initial protein quality results showed that the viscosity of the formulation decreased with increasing pH in the citric acid and acetate buffer systems, while it initially decreased and then increased in the histidine buffer system, reaching its lowest value at pH 6.0. The colloidal and conformational stability of the high-concentration h1G4 formulation increased with increasing pH, with histidine and acetate providing greater colloidal stability and acetate providing the best conformational stability. The citric acid system exhibited a heavier opalescence and larger particle size, while the histidine and acetate systems exhibited lighter opalescence and smaller particle size. These values ​​were similar at the same pH and increased with increasing pH. There was no significant difference in initial purity between the different buffer systems.

[0291] As shown in Table 11, formulations containing a citrate buffer system exhibited heavier opalescence, larger particle size, poor colloidal and conformational stability, and a rapid increase in SEC aggregate content and CEX acid peak. Therefore, this buffer system was discontinued. Formulations containing an acetic acid buffer system exhibited lighter opalescence, smaller particle size, and good colloidal and conformational stability. However, the SEC aggregate content and CEX acid peak increased more rapidly than those obtained with histidine, so this buffer system was discontinued.

[0292] For preparations containing a histidine buffer system, the opalescence is lighter, the particle size is smaller, the colloidal stability is good, and the SEC aggregates, DLS average diameter, PdI, and CEX acid peaks all perform well. In preparations containing a histidine buffer system, the protein viscosity first decreases and then increases with increasing pH, with the lowest viscosity at pH 6.0. The higher the pH, the stronger the colloidal stability and conformational stability, but the larger the initial particle size. After acceleration, the preparations containing a histidine buffer system have higher pH, higher SEC aggregate content, faster CEX acid peak growth, and lower SDS-PAGE reduction purity. Combined with the initial protein quality of the histidine buffer system and the protein stability results after acceleration, at pH 5.0-6.0, the viscosity is lower, the colloidal stability and conformational stability are better, and the accelerated stability is good. Therefore, the histidine buffer system was selected for subsequent development.

[0293] 3.2 Effects of stabilizers, antioxidants and surfactants

[0294] Various formulations were prepared for screening as shown in the table below. Formulations were prepared and characterized as described in the previous examples.

[0295] Table 12 Formulations containing various stabilizers, antioxidants, and surfactants

[0296] The inspection conditions and test methods are shown in the following table.

[0297] Table 13 Investigation conditions and detection methods

[0298] Table 14 Summary of screening data for stabilizers, antioxidants and surfactants

[0299] As shown in Table 14, the viscosity, T agg The results showed that the SEC main peak content, CEX main peak content, DLS average diameter and PdI, and FlowCam subvisible particles all performed well. Regarding antioxidants, methionine's SEC aggregates and FlowCam subvisible particles performed better than EDTA. Regarding surfactants, polysorbate 20's subvisible particles performed better. Therefore, hydrophobic amino acids will be selected as stabilizers, methionine as an antioxidant, and polysorbate 20 as a surfactant in the future.

[0300] Based on these results, proline was selected as a stabilizer, methionine as an antioxidant, and polysorbate 20 as a surfactant. This round of custom DoEs was conducted to screen for appropriate excipient concentrations (Table 15). Protein concentration and ionic strength were also considered as factors in this round of research (Table 15). Ultimately, 21 candidate formulations were designed (Table 16).

[0301] Table 15 Stabilizer, antioxidant, and surfactant concentrations designed for DoE screening

[0302] Table 16 h1G4 formulations containing stabilizers, antioxidants, and surfactants designed for DoE screening

[0303] Table 17 Investigation conditions and detection methods

[0304] All preparations were light yellow slightly opalescent liquids with no visible particles. The basic physicochemical properties of the protein (pH, protein concentration, osmotic pressure) all met the target values ​​(Table 21).

[0305] The initial protein properties and stability results (viscosity, osmotic pressure, Tagg, Tm, SEC initial main peak content, DLS initial average diameter and PdI, initial FlowCam subvisible particle count) and accelerated protein stability results (SEC main peak change slope, DLS average diameter change slope, accelerated FlowCam subvisible particle count) were imported into JMP 15 software, and the standard least squares method was used to perform multiple linear regression fitting for each factor (Table 18). If the model P value is ≤ 0.05, it means that the model is significant, and the regression model can be used to analyze the experimental results instead of the actual test points. The model R 2 ≥0.9, indicating that the model fits well and can be used for prediction. The model P value of viscosity, osmotic pressure, Tm, SEC initial main peak content, DLS initial average diameter and PdI, SEC main peak change slope, DLS average diameter change slope and the number of sub-visible particles in FlowCam after acceleration is ≤0.05, and R 2 ≥0.9 (Table 18). There were no significant differences in Tagg, SEC initial main peak content, and initial FlowCam subvisible particle count for all preparations, so the model P values ​​for these test items were all greater than 0.05, and R 2 Less than 0.9 (Table 18).

[0306] Table 18 Summary of DoE models for screening of stabilizers, antioxidants, and surfactants for h1G4

[0307] For well-fitting models, if the significance probability of a model factor is less than 0.05, the factor is a main effect of the model, indicating that changes in the factor have a significant impact on the model response. If the significance probability of two factors acting together is less than 0.05, it indicates that there is an interaction effect between the two factors. A change in the value of one factor may cause a change in the impact of the other factor on the model response. If the significance probability of the quadratic term of a factor is less than 0.05, it indicates that the factor's impact on the model response has reached an inflection point.

[0308] The viscosity model showed that protein concentration and ionic strength were the primary effects: higher protein concentrations were associated with greater viscosity, while greater ionic strengths were associated with lower viscosity (Tables 19 and 21, Figure 2). There was an interaction between protein concentration and ionic strength: at low protein concentrations, ionic strength had little effect on viscosity, while at high protein concentrations, higher ionic strengths significantly reduced protein viscosity (Tables 19 and 21, Figure 1).

[0309] The Tm model showed that higher ionic strength correlated with lower Tm, while higher proline content correlated with higher Tm (Tables 19, 21, and Figure 1). Although methionine and polysorbate 20 were primary effects, with changes in excipient content affecting Tm, the predictive profiler results showed that changes in Tm with methionine and polysorbate 20 content were minor, albeit with minor, changes (Tables 19, 21, and Figure 1). There was an interaction between ionic strength and proline content, but this interaction did not alter the direction of their effects on Tm. That is, regardless of the interaction, higher ionic strength correlated with lower Tm, while higher proline content correlated with higher Tm (Tables 19, 21, and Figure 1).

[0310] The initial DLS mean diameter model showed that higher ionic strength and protein concentration led to larger mean diameters. Polysorbate 20 and methionine, while dominant, had minor influences (Tables 19 and 21, and Figure 1). There was an interaction between protein concentration and polysorbate 20, but this interaction did not alter the direction of the effects of either factor on mean diameter (Tables 19 and 21, and Figure 1).

[0311] The DLS initial PdI model showed that higher protein concentration and higher polysorbate 20 content were associated with greater initial PdI. Although proline was a major effect, its influence was not significant (Tables 19 and 21, Figure 1). There was an interaction between protein concentration and polysorbate 20: at low protein concentrations, polysorbate 20 had little effect on PdI. However, at high protein concentrations, higher polysorbate 20 content was associated with greater initial PdI (Tables 19 and 21, Figure 1).

[0312] The SEC main peak change slope model showed that the higher the protein concentration, the faster the SEC main peak decreased (Table 19, Table 21, Figure 2).

[0313] The DLS mean diameter change slope model showed that both proline and polysorbate 20 content were main effects, with proline and methionine quadratic terms being the main effects (Tables 19 and 21, Figures 2 and 3). When the proline content was less than 150 mM and the methionine content was less than 0.35%, higher proline content was associated with slower mean diameter growth. When the proline content was greater than 150 mM and the methionine content was greater than 0.35%, higher proline content was associated with faster mean diameter growth (Tables 19 and 21, Figures 1 and 2). Protein concentration interacted with ionic strength and polysorbate 20 content. At low protein concentrations, ionic strength and polysorbate 20 content had little effect on mean diameter. At high protein concentrations, higher ionic strength and higher polysorbate 20 content were associated with faster mean diameter growth (Tables 19 and 21, Figures 1 and 2).

[0314] Because there was no significant difference in the initial number of subvisible particles on the FlowCam, the number of subvisible particles on the FlowCam after acceleration was used to represent the increase in the number of insoluble protein particles. Higher methionine content, polysorbate 20 content, and protein concentration were associated with more subvisible particles on the FlowCam after acceleration. A quadratic inflection point was observed for protein concentration, methionine content, and ionic strength. The number of subvisible particles was lowest at a protein concentration of approximately 150 mg / mL and a methionine content of approximately 0.2%, and was highest at an ionic strength of approximately 30 mM (Table 19, Table 21, Figure 2). There were interactions between protein concentration and ionic strength, and between ionic strength and proline content. At low protein concentrations, higher ionic strength resulted in more particles, while at high protein concentrations, higher ionic strength resulted in fewer particles. At an ionic strength of 20 mM, proline content had little effect on particle number. At an ionic strength of 50 mM, higher proline content resulted in fewer particles (Table 19, Table 21, Figure 2).

[0315] By analyzing the DoE significance model, factor interactions, and DoE prediction profilers, the optimal ranges of factors under each model were screened out (Table 20). When proline was 150-200 mM, methionine was 0.1-0.3% (7-21 mM), and polysorbate 20 was 0.01-0.03%, the protein viscosity was lower and different aggregate sizes performed well.

[0316] Table 19 DoE model analysis of h1G4 stabilizers, antioxidants and surfactants

[0317] Table 20 Suitable content range of stabilizers, antioxidants and surfactants for screening excipients

[0318] Table 21 Summary of screening data for stabilizers, antioxidants and surfactants

[0319] Validation of 150 mg / mL Antibody Concentration

[0320] The antibody concentration was adjusted to 150 mg / mL to verify whether the above-screened formulations were equally effective.

[0321] Table 22 h1G4 formulations validated at 150 mg / mL

[0322] The inspection conditions and test methods are shown in the following table.

[0323] Table 23 Investigation conditions and detection methods

[0324] The formulation and characterization methods were as described in the previous examples.

[0325] Table 24 Summary of initial protein quality data for 150 mg / mL h1G4 formulation

[0326] Note: *In the actual production process, protein concentration allows a certain production error. Therefore, during the stability study, the protein concentration is set according to the upper limit of the production error.

[0327] Table 25 Summary of accelerated stability data for 150 mg / mL h1G4 formulation

[0328] Results showed that when the antibody concentration was adjusted to 150 mg / mL, the formulation exhibited excellent colloidal and conformational stability and low viscosity. Initial aggregates and particles were minimal. While aggregates increased slightly during the accelerated phase, DLS and subvisible aggregates showed no significant increase. Degradation and charge isoform variation were also well characterized. Therefore, the formulation composition was effective even at an antibody concentration of 150 mg / mL.

[0329] 3.4. Validation of 200 mg / mL Antibody Concentration

[0330] The antibody concentration was adjusted to 200 mg / mL to verify whether the above-screened formulations were equally effective.

[0331] Table 26 h1G4 formulations validated at 200 mg / mL

[0332] The inspection conditions and test methods are shown in the following table.

[0333] Table 27 Investigation conditions and detection methods

[0334] The initial protein quality results showed that 200 mg / mL h1G4 protein had no visible foreign matter in the above-screened preparations, with a protein concentration of 209 mg / mL, a pH of 6.0, an osmotic pressure of 382 mOsm / kg, a light yellow slightly opalescent liquid, a viscosity of 41.2 cP, and a T agg is 63.2℃, T monset The temperature of the prepared polymer was 60.0 °C (Table 28), indicating that both the colloidal stability and conformational stability were good.

[0335] The results showed that when the antibody concentration was adjusted to 200 mg / mL, the colloidal stability and conformational stability of the formulation were good. Initial aggregates and particles were relatively few, and aggregates increased during the acceleration process (Table 29). Degradation and charge isomer changes were also good (Table 29). Therefore, the above formulation composition is also effective for 200 mg / mL antibody concentration.

[0336] Table 28 Summary of initial protein mass data for 200 mg / mL h1G4 formulation

[0337] Table 29 Summary of accelerated stability data for 200 mg / mL h1G4 formulations

[0338] Example 4. Optimization of 200 mg / mL Antibody Concentration Formulation

[0339] The purpose of this example is to further optimize a stable formulation suitable for use at an antibody concentration of 200 mg / mL.

[0340] 4.1. Optimization plan 1: screening of excipient types

[0341] The purpose of this study was to investigate the effects of various excipients on the protein viscosity and stability of h1G4 formulations.

[0342] Table 30 200 mg / mL h1G4 formulations containing various excipients

[0343] The inspection conditions and test methods are shown in the following table.

[0344] Table 31 Investigation conditions and test methods for 200 mg / mL h1G4 preparation

[0345] The excipients were divided into charged excipients (F1_Arg~F10_Tau), hydrophobic excipients (F11_Ile~F13_G), a combination of hydrophobic and charged excipients (F14_PH, proline as a hydrophobic excipient and histidine as a charged excipient), and a hydrophobic control (F15_control, proline and methionine are both hydrophobic excipients).

[0346] Table 32 Summary of data for 200 mg / mL h1G4 formulations used for excipient screening

[0347] The results of the initial protein quality study showed that all preparations had no visible particles on the surface, and appeared as a light yellow slightly opalescent liquid. The basic physical and chemical properties (protein concentration, pH value, osmotic pressure) all met the target values.

[0348] When the protein concentration was 200 mg / mL, the viscosities of F1_Arg, F4_NH4Cl, and F14_PH were less than 20 cP, and the viscosities of F3_Lys, F6_MgCl2, F7_CaCl2, and F8_Na2SO4 were less than 22 cP (Table 32). According to experience, when the protein viscosity is less than 20 cP, the syringe sliding force is smaller during clinical administration, making it more convenient to use. The smaller the protein viscosity, the smaller the sliding force. The difference in viscosity reflects the different forces between protein molecules. When the hydrophobic interaction force of the protein is stronger or the electrostatic interaction force is weaker, the protein tends to undergo reversible self-association, which easily causes an increase in protein viscosity. In this round of research, the formulations with lower viscosity all contained charged amino acids or charged salts, so it is reasonable to speculate that excipients with charged properties can change the electrostatic interactions between h1G4 proteins and reduce protein viscosity.

[0349] and F15_control T agg In comparison, the preparations containing charged excipients (F1_Arg~F10_Tau, F_PH)T agg The values ​​were approximately 10-15°C lower (Table 32). Furthermore, the average diameters of formulations containing charged excipients (F1_Arg, F10_Tau, and F_PH) were 4-9 nm higher than those of the F15_control. There was no significant difference in the average diameters between the formulations containing uncharged excipients (F11_Ile, F12_Phe, and F13_G) and the F15_control (Table 32). This suggests that charged excipients alter protein electrostatic interactions, potentially leading to decreased colloidal stability. However, the colloidal stability was still good.

[0350] After 4 weeks at 40°C, the basic physicochemical properties of the protein (appearance, protein concentration, pH, and osmotic pressure) remained unchanged. The SEC main peak content, except for F5_NaCl to F9_MgSO4, was greater than 96% (Table 32). The SEC main peak decline rate was less than 0.1% / day, which is within the range of the SEC main peak decline rate for marketed drugs and the company's research projects in the stability database. For the average DLS diameter, only F1_Arg, F4_NH4Cl, F5_NaCl, and F6_Mg2Cl showed no significant increase. The average diameters of F7_CaCl2, F12_Phe, and F13_G increased rapidly, exceeding 5 nm over 4 weeks (Table 32). When the DLS PdI is less than 0.15, the protein particles are relatively uniformly dispersed. After 4 weeks of accelerated growth at 40°C, only F1_Arg, F2_Lys, and F3_NH4Cl showed a relatively uniform particle size distribution. The PdI of F7_CaCl2, F12_Phe, and F13_G showed significant changes (Table 32). Compared with the F15_control, the number of particles with all charged excipients increased significantly, while the number of subvisible particles with the hydrophobic excipient did not increase significantly (Table 32). Combined with the analysis results of the content of aggregates of different sizes, formulations containing charged excipients generally inhibit the growth of the DLS mean diameter and PdI, but are prone to the production of subvisible particles. Formulations containing hydrophobic excipients, while less likely to produce subvisible particles, have a faster growth rate of the DLS mean diameter and PdI.

[0351] In summary, charged excipients alter the electrostatic interactions of h1G4 protein, reducing protein viscosity but also decreasing its colloidal stability. Charged excipients inhibited the growth of the average DLS diameter and PdI, but were prone to producing subvisible particles. Hydrophobic excipients failed to significantly reduce h1G4 protein viscosity and were less likely to produce subvisible particles, but exhibited rapid increases in the average DLS diameter and PdI.

[0352] Charged and hydrophobic excipients each offer advantages in terms of viscosity and aggregate control. Arginine hydrochloride is a polar amino acid that exhibits both charge and hydrophobic properties. The combination of proline and histidine also provides both hydrophobic and charged excipients. Both formulations, F1_Arg and F14_PH, significantly reduced protein viscosity, but exhibited a high prevalence of subvisible particles. Therefore, future plans aim to balance the advantages and disadvantages of these two excipient types by varying the excipient content.

[0353] Optimization Plan 2: Excipient Concentration Investigation

[0354] The purpose of this study was to investigate the effects of the concentrations of the aforementioned charged and hydrophobic excipients on the viscosity and stability of the h1G4 protein.

[0355] Table 33 200 mg / mL h1G4 formulations containing various excipients

[0356] The inspection conditions and test methods are shown in the following table.

[0357] Table 34 Investigation conditions and detection methods of h1G4 preparations

[0358] The results of the initial protein quality study showed that all preparations had no visible particles and appeared as a light yellow, slightly opalescent liquid. The basic physicochemical properties (protein concentration, pH value, and osmotic pressure) all met the target values ​​(Table 35).

[0359] The viscosity of the control formulation, F20_PT, was 27 cP (Table 35). The viscosity of the protein in the arginine hydrochloride-containing formulations (F1_Arg20 to F7_Arg320) decreased significantly, decreasing from 21.6 cP to 16.4 cP with increasing arginine hydrochloride content. The viscosity reached a plateau of approximately 17 cP when the arginine hydrochloride content reached 170 mM (Table 35). The viscosity of the protein in the proline-containing formulations (F8_Pro20 to F14_Pro320) did not decrease significantly. When the proline content increased to 70 mM, the viscosity reached its first plateau of approximately 24 cP. There was no significant difference in viscosity between 70 and 270 mM proline content, and the viscosity further decreased to approximately 20 cP when the proline content increased to 320 mM (Table 35). The viscosity of the histidine-containing formulations (F15_His20 to F19_Pro220) reached its lowest point of approximately 20 cP at a histidine content of 170 mM (Table 35). Considering the convenience of clinical use, the viscosity of high-concentration preparations needs to be less than 20 cP. Changing the excipient content with proline and histidine cannot reduce the viscosity of 200 mg / mL h1G4 protein to less than 20 cP.

[0360] The Tagg of the control formulation F20_PT was 73°C (Table 35). The Tagg results for F1_Arg20 to F7_Arg320 showed that arginine hydrochloride reduced the Tagg by approximately 15°C (Table 35), with no significant differences between different excipient contents. The Tagg results for F8_Pro20 to F14_Pro320 showed that proline did not reduce the protein Tagg (Table 35). The Tagg results for F15_His20 to F19_Pro220 showed that when the histidine content was 20 mM, the Tagg was higher, approximately 65°C. When the histidine content increased to 70 to 220 mM, the Tagg decreased to 60°C, with no significant differences between different histidine contents (Table 35).

[0361] After being placed at 40°C for 4 weeks, both F13_Pro270 and F14_Pro320 became turbid and had visible particles. The turbidity of F14_Pro320 was higher than that of F13_Pro270. The other preparations had no visible particles and were light yellow slightly opalescent liquids.

[0362] After 4 weeks at 40°C, the main SEC peak change was primarily due to aggregate growth. All formulations showed a trend of decreasing aggregate growth with increasing excipient content. When the excipient content was constant, the aggregate content showed little difference (Table 35). DLS results showed no significant increase in the average diameter and PdI of the arginine hydrochloride-containing formulations (F1_Arg20 to F7_Arg320). However, the average diameter and PdI of the proline-containing formulations (F8_Pro20 to F14_Pro320) and histidine-containing formulations (F15_His20 to F19_Pro220) increased significantly, with average diameters increasing by approximately 1 to 2 nm and PdIs increasing by approximately 0.1 to 0.2 (Table 35). The MFI sub-visible particle results showed that when the arginine hydrochloride content was 20 mM, the number of sub-visible particles was low, and when the content exceeded 70 mM, the number of sub-visible particles was high. When the proline content was 20-220 mM, the sub-visible content was low and had no obvious relationship with the excipient content. When the histidine content was less than 120 mM, the sub-visible particle content was low, and when it was greater than 120 mM, the sub-visible particle content was high (Table 35).

[0363] In summary, it is impossible to balance protein viscosity and stability by changing the excipient content. When the arginine hydrochloride content is low, the viscosity is higher, and when the content is higher, more subvisible particles are produced. Increasing the excipient content of proline and histidine (less than 220mM) does not significantly reduce viscosity. When the proline content is increased to 320mM, the viscosity decreases significantly, but visible particles appear on the surface. When the histidine content is increased to 170mM, the viscosity decreases significantly, but more subvisible particles are produced.

[0364] Table 35 Summary of data for 200 mg / mL h1G4 formulations used for excipient screening

[0365] 4.3. Optimization plan 3: Excipient combination screening

[0366] The results of optimization scheme 1 showed that it was impossible to balance the viscosity and stability of h1G4 high-concentration protein by changing the excipient concentration. For arginine hydrochloride and histidine excipients, the number of subvisible particles in the preparation was large, and proline was not effective in reducing viscosity. Therefore, this round of planning aimed to improve the viscosity and stability of h1G4 by combining excipients and changing the surfactant content, and designed 6 alternative preparations (Table 36). To address the problem of many subvisible particles with arginine hydrochloride, F1 and F2 changed the surfactant content, and F3 combined proline and histidine. To address the problem of poor viscosity reduction with proline and many subvisible particles when the histidine content was high, F4 and F5 combined proline and histidine to adjust the histidine content. F6 combined proline, arginine, and histidine hydrochloride.

[0367] Table 36 Various h1G4 preparations

[0368] The inspection conditions and test methods are shown in the following table.

[0369] Table 37 Investigation conditions and detection methods of h1G4 preparations

[0370] The results of the initial protein quality study showed that all preparations had no visible particles and appeared as a light yellow, slightly opalescent liquid. The basic physicochemical properties (protein concentration, pH value, and osmotic pressure) all met the target values ​​(Table 38).

[0371] Viscosity results showed that all formulations had viscosities less than 20 cP. However, when the histidine content was low (F5_ProHis20) or the arginine hydrochloride content was low (F3_ArgPro), the viscosity remained high, at 19.5 cP and 19.6 cP, respectively. The three-excipient combination of F6_ProArgHis achieved the best results, with the lowest protein viscosity at 17.4 cP (Table 38). Tagg results showed that F5_ProHis20 had the highest Tagg of 70.2°C. When the formulation contained arginine or had a high histidine content, the Tagg values ​​were approximately 60°C lower (Table 38).

[0372] After 4 weeks at 40°C, the SEC main peak decreased due to increased aggregate content, with no significant differences in SEC aggregate growth between the different formulations (Table 38). DLS average diameters showed no significant increase (Table 38). DLS PdI showed that F6_ProArgHis performed better, with a DLS PdI of 0.081 after 4 weeks of acceleration (Table 38). MFI subvisible particle results showed that increasing the surfactant content (F2_0.1PS20) increased the number of subvisible particles in F1_0.02PS20, F2_0.1PS20, and F3_ArgPro. The combination of arginine hydrochloride and proline (F3_ArgPro) did not reduce the number of subvisible particles caused by the arginine hydrochloride excipient. Compared with F4_ProHis and F5_ProHis20, decreasing the histidine content reduced the number of subvisible particles. The F6_ProArgHis excipient combination had the lowest number of subvisible particles (Table 38).

[0373] In summary, neither altering the surfactant content nor adjusting the excipient combination can improve the problem of high subvisible particle counts at high arginine HCl levels. Combining proline and histidine resulted in higher viscosity at low histidine levels and more subvisible particles at high histidine levels. The F6_ProArgHis excipient combination achieved the lowest viscosity, and despite a lower Tagg, aggregates of varying sizes performed well after acceleration.

[0374] Table 38 Summary of data for various h1G4 preparations

[0375] The above results show that the combination of arginine hydrochloride, proline and histidine can balance protein viscosity and stability. This round of DoE experiments was used to screen the optimal range of combined excipients.

[0376] Table 39 Component concentration ranges of h1G4 formulations designed for DoE screening

[0377] Table 40 h1G4 formulations designed for DoE screening

[0378] The inspection conditions and testing methods are as follows:

[0379] Table 41 Investigation conditions and detection methods of h1G4 preparations

[0380] All preparations were light yellow slightly opalescent liquids with no visible particles, and the basic physicochemical properties of the protein (pH, protein concentration, osmotic pressure) all met the target values ​​(Table 42).

[0381] Table 42 Summary of data for various h1G4 preparations

[0382] Initial protein properties and stability results (viscosity, osmotic pressure, T agg , SEC initial main peak content, DLS initial average diameter and PdI, initial MFI subvisible particle number) and accelerated protein stability results (SEC main peak change slope, DLS average diameter and PdI change slope, MFI subvisible particle number change slope, accelerated protein color score) were imported into JMP 15 software, and multiple linear regression fitting was performed on each factor using standard least squares method or stepwise method (Table 43). If the model P value is ≤0.1, it means that the model is significant, and the regression model can be used to analyze the experimental results instead of the actual test points. The model R 2 ≥0.7, indicating that the model fits well and can be used for prediction. agg , osmotic pressure, SEC initial main peak content, DLS PdI change slope model P value ≤ 0.1, and R 2 ≥0.7 (Table 43). The model P values ​​of the slope of the SEC main peak change, the initial average diameter of DLS and PdI, and the number of subvisible particles at the initial and after acceleration of MFI were all greater than 0.1, and R 2 All were less than 0.7 (Table 43). Accelerated protein color scoring model R 2Although it is less than 0.7, the model P value is less than 0.1 (Table 43), which means that the regression model can be used to replace the real test points to analyze the experimental results, but the model cannot be used for prediction.

[0383] Table 43 Summary of DoE model data for h1G4 formulation used for excipient combination screening

[0384] For well-fitting models, if the probability of significance for a model factor is less than 0.05, the factor is considered a main effect of the model, indicating that changes in that factor have a significant impact on the model response. If the probability of significance for the combined effect of two factors is less than 0.05, this indicates an interaction effect between the two factors; changes in one factor may cause changes in the impact of the other factor on the model response. If the probability of significance for the quadratic term of a factor is less than 0.05, this indicates that there is an inflection point in the effect of that factor on the model response. The factor levels for histidine hydrochloride and proline content ranged from 10 to 90 mM, and the factor level for arginine content ranged from 10 to 50 mM. This analysis was limited to results within this factor level range.

[0385] The viscosity model and prediction profiler showed that the higher the histidine hydrochloride and arginine content, the lower the protein viscosity. However, after the histidine hydrochloride content exceeded 70 mM, the protein viscosity did not decrease significantly when the histidine hydrochloride content continued to increase (Table 42, Table 44).

[0386] T agg The model shows that the higher the histidine hydrochloride content, the higher the T agg The lower the content, the more interactive effect there is between histidine hydrochloride and arginine. When histidine hydrochloride is 10 mM, the arginine content has a significant effect on T agg There is no significant effect. When the histidine hydrochloride content is 90mM, the higher the arginine content, the higher the T agg When the arginine content is 10mM, the higher the histidine hydrochloride content, the higher the T agg The lower the arginine content, the lower the effect of histidine hydrochloride content on T agg There was no significant effect (Table 42, Table 44).

[0387] The SEC initial main peak content model showed that the higher the histidine hydrochloride content, the higher the SEC initial main peak content (Table 42, Table 44).

[0388] The DLS mean diameter change slope model shows that histidine hydrochloride content of about 70mM and arginine content of about 40mM are quadratic inflection points. At this point, the mean diameter change slope is minimum, and the protein mean diameter after acceleration is smaller (Table 42, Table 44). The DLS PdI model shows that the higher the histidine hydrochloride and arginine content, the smaller the PdI change slope, and the smaller the protein PdI after acceleration. There is an interaction between histidine hydrochloride and arginine. When the two factors are at a high level, the higher the other factor content, the larger the PdI change slope. When the two factors are at a low level, the higher the other factor content, the smaller the PdI change slope. This shows that for PdI, histidine hydrochloride and arginine cannot be high levels at the same time (Table 42, Table 44). There is an inflection point in proline content. When the content is less than 40mM, the higher the proline content, the slower the PdI growth. When the content is greater than 40mM, the higher the proline content, the faster the PdI growth (Table 42, Table 44).

[0389] The MFI subvisible particle number change model showed that the higher the histidine hydrochloride content, the greater the number of subvisible particles (Table 42, Table 44).

[0390] The accelerated protein color model showed that proline had a quadratic inflection point, and when the proline content was approximately 50 mM, the protein color after acceleration was the lightest. The higher the histidine content, the darker the protein color after acceleration (Table 42, Table 44).

[0391] By analyzing the DoE significance model, factor interactions, and DoE prediction profilers, we screened for optimal factor ranges for each model (Table 45). At 40-50 mM proline, 70 mM histidine hydrochloride, and 40-50 mM arginine, protein viscosity was low, aggregate sizes were well-defined, and protein color did not change significantly after acceleration. Within this excipient range, the protein osmotic pressure was predicted to be 370 mOsm / kg. Literature reports indicate that the optimal osmotic pressure range for high-concentration subcutaneous preparations is 240-600 mOsm / kg. The osmotic pressure of the selected excipients met the requirements.

[0392] Table 44 DoE model analysis of h1G4 formulations used for excipient combination screening

[0393] Table 45 h1G4 excipient combination screening (200 mg / mL) suitable excipient content range

[0394] The results showed that when proline was 40-50 mM, histidine hydrochloride was 70 mM, and arginine was 40-50 mM, the protein viscosity was low, the sizes of different aggregates were well behaved, the protein color did not change significantly after acceleration, and the osmotic pressure was appropriate.

[0395] 4.4. Validation of representative solutions

[0396] The purpose of this study was to verify the availability of a representative formulation.

[0397] Prepare the following preparation:

[0398] Table 46 h1G4 formulations validated at 200 mg / mL

[0399] The inspection conditions and testing methods are as follows:

[0400] Table 47 Investigation conditions and detection methods of h1G4 preparations

[0401] Initial protein quality studies revealed that the target formulation had no visible particles and appeared as a light yellow, slightly opalescent liquid. Basic physical and chemical properties (protein concentration, pH, and osmotic pressure) all met target values. Viscosity was 15.9 cP, and Tagg was 57.2°C (Table 48).

[0402] Table 48 Data summary of h1G4 preparation

[0403] The results of 4 weeks of acceleration at 40°C showed that there were no visible particles on the protein surface, and the MFI subvisible particle results showed that there were no particles larger than 50μm. The number of subvisible particles increased after 1 week of acceleration, but the number of subvisible particles did not increase further from 2 to 4 weeks. The final number of subvisible particles was approximately 5000 particles / mL, 69 particles / mL of particles larger than 10μm, and 8 particles / mL of particles larger than 25μm (Table 48) (The 2020 edition of the "Chinese Pharmacopoeia" stipulates that the number of insoluble particles larger than 10μm should be less than 6000 particles / bottle, and particles larger than 25μm should be less than 600 particles / bottle). There was no significant change in the DLS average diameter, and the DLS PdI increased, but after 4 weeks of acceleration, the PdI was still less than 0.2 (Table 48). The change in the SEC main peak content was mainly due to the growth of SEC aggregates. The main peak content decreased from 97.9% to 95.4%, and the SEC aggregate content increased from 2.1% to 4.3% (Table 48).

[0404] In summary, the 200 mg / mL h1G4 formulation had low viscosity, and aggregates of different sizes performed well, which was able to balance protein viscosity and stability.

[0405] 4.5. Validation of 220 mg / mL Antibody Concentration

[0406] The antibody concentration was adjusted to 220 mg / mL to verify whether the above-screened formulations were equally effective.

[0407] Prepare the following preparation:

[0408] Table 49 h1G4 formulations validated at 220 mg / mL

[0409] The inspection conditions and testing methods are as follows:

[0410] Table 50 Investigation conditions and detection methods of h1G4 preparations

[0411] The initial protein quality results showed that there was no visible foreign matter in the 220mg / mL h1G4 preparation, and the appearance was a light yellow slightly opalescent liquid with a viscosity of 27.4cP and T agg The viscosity of the mixture was 57.6°C (Table 51), indicating that both the colloidal stability and conformational stability were good, and the viscosity was feasible for both the preparation production process and clinical subcutaneous administration.

[0412] The results showed that when the antibody concentration was adjusted to 220 mg / mL, the colloidal stability and conformational stability of the formulation were good, and the viscosity was high. Initial aggregates and particles were relatively few, and the increase in aggregates during the acceleration process was relatively small (Table 52). Degradation and charge isomer changes were also good (Table 52). Therefore, the above formulation composition was equally effective for a 220 mg / mL antibody concentration.

[0413] Table 51 Summary of initial protein quality data for h1G4 preparation

[0414] Table 52 Summary of accelerated stability data of h1G4 formulation

[0415] Example 5. h1G4 preparation containing hyaluronidase

[0416] Based on the h1G4 preparations screened in Example 3 above, h1G4 preparations containing hyaluronidase were designed with 120 mg / mL as an exemplary, but not limiting, antibody concentration.

[0417] Table 53 h1G4 preparations containing hyaluronidase * Hyaluronidase was produced by Shanghai Hanlin Biotech Co., Ltd., and its amino acid sequence is SEQ ID NO: 11. 2000 units / mL was selected here as an exemplary, but not limiting, concentration of hyaluronidase. # The methionine content was screened with reference to the stabilizers, antioxidants, and surfactants in Example 3. The suitable range of methionine content was 0.1-0.3% (i.e., 7-21 mM) (Table 20). Due to the low solubility of methionine, the methionine content of the F1 formulation was selected to be 10 mM.

[0418] Table 54 Investigation conditions and detection methods of h1G4 preparations containing hyaluronidase

[0419] Initial research results showed that the addition of hyaluronidase to the h1G4 formulation resulted in a higher Tagg, lower viscosity, good colloidal stability, initial hyaluronidase activity consistent with the theoretical addition value, and fewer initial aggregates and particles (Table 55).

[0420] Results from high-temperature acceleration and light exposure showed that the h1G4 protein formulation exhibited excellent stability when hyaluronidase was added at 2000 units / mL. Aggregates of all sizes grew minimally during the accelerated process, and degradation and charge isoform changes were well-characterized. Hyaluronidase activity remained unchanged after 8 weeks at 30°C (Table 56). Therefore, the above formulation composition is also effective even with the addition of hyaluronidase.

[0421] Table 55 Summary of initial protein quality data for h1G4 formulations containing hyaluronidase

[0422] Table 56 Summary of protein stability data for h1G4 formulations containing hyaluronidase

[0423] In addition to the various embodiments depicted and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, the specific features presented herein can be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The above descriptions of specific embodiments of the disclosed subject matter have been presented for purposes of illustration and description. The above descriptions are not intended to be exhaustive or to limit the disclosed subject matter to those disclosed embodiments.

[0424] It will be apparent to those skilled in the art that various modifications and variations can be made to the composition and method of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Therefore, it is intended that the disclosed subject matter include modifications and variations within the scope of the appended claims and their equivalents.

[0425] Various publications, patents, and patent applications are cited herein, the contents of which are hereby incorporated by reference in their entirety.

Claims

1. A pharmaceutical preparation of an anti-PD-1 antibody, comprising: a. 120 mg / mL - 200 mg / mL anti-PD-1 antibody, wherein the anti-PD-1 antibody comprises: a heavy chain variable region comprising HCDR1 of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, and HCDR3 of SEQ ID NO:3; and a light chain variable region comprising LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO:6; b. 20 mmol / L - 50 mmol / L histidine - histidine hydrochloride, c. 150 mM - 200 mM proline, d. 0.1% - 0.3% (w / v) methionine, e. 0.01% - 0.03% (w / v) nonionic surfactant selected from polysorbate 20 and polysorbate 80, pH 5.0 - 6.

0.

2. The pharmaceutical preparation according to claim 1, wherein the preparation comprises 20 mmol / L histidine - histidine hydrochloride.

3. The pharmaceutical preparation according to claim 1 or 2, wherein the preparation comprises 0.01% - 0.03% (w / v) of polysorbate 20.

4. The pharmaceutical preparation according to any one of the preceding claims, further comprising hyaluronidase.

5. The pharmaceutical preparation according to claim 4, wherein the pharmaceutical preparation comprises 50 U / ml to 5000 U / ml of hyaluronidase.

6. A pharmaceutical preparation of an anti-PD-1 antibody for subcutaneous administration, comprising: 1) 200 mg / mL - 220 mg / mL anti-PD-1 antibody, wherein the anti-PD-1 antibody comprises: a heavy chain variable region comprising HCDR1 as in SEQ ID NO:1, HCDR2 as in SEQ ID NO:2, and HCDR3 as in SEQ ID NO:3; and a light chain variable region comprising LCDR1 as in SEQ ID NO:4, LCDR2 as in SEQ ID NO:5, and LCDR3 as in SEQ ID NO:6; 2) 20 mmol / L histidine - histidine hydrochloride, 3) 40 - 50 mM proline, 4) 20 - 70 mM histidine hydrochloride, 5) 20 - 70 mM arginine, 6) 0.01 - 0.03% (w / v) polysorbate 20 or polysorbate 80, pH 5.0 - 6.

0.

7. The pharmaceutical preparation according to claim 6, wherein the preparation comprises 70 mM histidine hydrochloride.

8. The pharmaceutical preparation according to claim 6 or 7, wherein the preparation comprises 40 mM - 50 mM arginine.

9. The pharmaceutical preparation according to any one of claims 6 - 8, wherein the preparation comprises 0.01 - 0.03% polysorbate 20.

10. The pharmaceutical preparation according to any one of claims 6 - 9, comprising hyaluronidase.

11. The pharmaceutical preparation according to claim 10, wherein the concentration of the hyaluronidase is about 50 U / ml to 5000 U / ml.

12. The pharmaceutical preparation according to any one of the preceding claims, wherein the anti-PD-1 antibody comprises VH of SEQ ID NO:7 and VL of SEQ ID NO:

8.

13. The pharmaceutical preparation according to any one of the preceding claims, wherein the anti-PD-1 antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, F(ab’)2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

14. The pharmaceutical preparation according to any one of the preceding claims, wherein the anti-PD-1 antibody comprises an Fc region.

15. The pharmaceutical preparation according to claim 14, wherein the Fc region is a human Fc region.

16. The pharmaceutical preparation according to claim 14 or 15, wherein the Fc region is an IgG4 Fc region.

17. The pharmaceutical preparation according to any one of claims 14-16, wherein the Fc region comprises a C-terminal lysine.

18. The pharmaceutical preparation according to any one of claims 14-17, wherein the Fc region comprises a deletion of the C-terminal lysine.

19. The pharmaceutical preparation according to any one of the preceding claims, wherein the anti-PD-1 antibody comprises a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO:

10.

20. The pharmaceutical preparation according to any one of claims 1-19, which comprises 120 mg / mL anti-PD-1 antibody, 20 mmol / L histidine-hydrochloride histidine, 200 mM proline, 0.1% (w / w) methionine, 0.02% (w / v) polysorbate 20, pH 5.0-5.

5.

21. The pharmaceutical preparation according to any one of claims 1-19, which comprises 150 mg / mL anti-PD-1 antibody, 20 mmol / L histidine-hydrochloride histidine, 200 mM proline, 0.1% (w / w) methionine, 0.02% (w / v) polysorbate 20, pH 5.5-6.

0.

22. The pharmaceutical preparation according to any one of claims 1-19, which comprises 200 mg / mL anti-PD-1 antibody, 20 mmol / L histidine-hydrochloride histidine, 200 mM proline, 0.1% (w / w) methionine, 0.02% (w / v) polysorbate 20, pH 5.0-5.

5.

23. The pharmaceutical preparation according to any one of claims 1-19, which comprises: 200 mg / mL anti-PD-1 antibody, 20 mmol / L histidine-hydrochloride histidine, 50 mM proline, 70 mM histidine hydrochloride, 50 mM arginine, 0.02% (w / v) polysorbate 20, pH 6.

0.

24. The pharmaceutical preparation according to any one of the preceding claims, wherein the pharmaceutical preparation is substantially free of dissolved oxygen.

25. The pharmaceutical preparation according to any one of the preceding claims, wherein the pharmaceutical preparation is suitable for subcutaneous administration.

26. The pharmaceutical preparation according to any one of the preceding claims, which is used to inhibit the interaction between PD-1 and PD-L1 in a patient.

27. The pharmaceutical preparation according to any one of the preceding claims, which is used to treat cancer in a patient.

28. The pharmaceutical preparation according to claim 27, wherein the cancer is selected from: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, breast cancer, gastrointestinal cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, endometrial cancer, cutaneous squamous cell carcinoma, thyroid cancer, prostate cancer, glioblastoma, Merkel cell carcinoma, and salivary gland carcinoma.

29. The pharmaceutical preparation according to claim 27 or 28, wherein the patient has a tumor with a high mutation burden.

30. The pharmaceutical preparation according to claim 27 or 28, wherein the patient has a high microsatellite instability (MSI-H) or mismatch repair-deficient solid tumor.

31. The pharmaceutical preparation according to claim 27 or 28, wherein the cancer is unresectable or metastatic melanoma.

32. The pharmaceutical preparation according to claim 27 or 28, wherein the cancer is metastatic non-small cell lung cancer (NSCLC).

33. A method for treating cancer in a patient, which comprises subcutaneously administering to the patient a therapeutically effective amount of the pharmaceutical preparation according to any one of claims 1-23.

34. The method according to claim 33, wherein the cancer is selected from: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, breast cancer, gastrointestinal cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, endometrial cancer, cutaneous squamous cell carcinoma, thyroid cancer, prostate cancer, glioblastoma, Merkel cell carcinoma, and salivary gland carcinoma.

35. The method according to claim 33 or 34, wherein the patient has a tumor with a high mutation burden.

36. The method according to claim 33 or 34, wherein the patient has a high microsatellite instability (MSI-H) or mismatch repair-deficient solid tumor.

37. The method according to claim 33 or 34, wherein the cancer is unresectable or metastatic melanoma.

38. The method according to claim 33 or 34, wherein the cancer is metastatic non-small cell lung cancer (NSCLC).

39. The method according to claim 38, wherein the patient has a tumor with PD-L1 expression as measured by a tumor proportion score (TPS) of ≥ 1% and has not been previously treated with platinum-containing chemotherapy.

40. The method according to claim 38, wherein the patient has a tumor with PD-L1 expression as measured by a tumor proportion score (TPS) of ≥ 1% and has been previously treated with platinum-containing chemotherapy.

41. The method according to any one of claims 38-40, wherein the patient's tumor does not have EGFR or ALK genomic aberrations.

42. The method according to any one of claims 38-41, wherein the method further comprises administering pemetrexed and platinum chemotherapy to the patient.

43. The method according to claim 38, wherein the NSCLC is squamous and the patient is also treated with carboplatin and paclitaxel or albumin-bound paclitaxel.

44. The method according to claim 33 or 34, wherein the cancer is recurrent or metastatic head and neck squamous cell carcinoma (HNSCC).

45. The method according to claim 33 or 34, wherein the cancer is locally advanced or metastatic urothelial carcinoma.

46. The method according to claim 33 or 34, wherein the cancer is locally advanced or metastatic gastric cancer or gastroesophageal junction adenocarcinoma.

47. The method according to claim 33 or 34, wherein the cancer is cervical cancer.

48. The method according to claim 47, wherein the cervical cancer is recurrent or metastatic cervical cancer and the patient has disease progression during or after chemotherapy.

49. The method according to claim 33 or 34, wherein the cancer is primary mediastinal large B-cell lymphoma (PMBCL).

50. The method according to claim 49, wherein the patient has refractory PMBCL or relapses after two or more prior therapies.

51. The method according to claim 33 or 34, wherein the cancer is resected stage IIB, IIC or III melanoma.

52. The method according to claim 33 or 34, wherein the cancer is hepatocellular carcinoma.

53. The method according to claim 33 or 34, wherein the cancer is renal cell carcinoma (RCC).

54. The method according to claim 53, wherein the cancer is advanced clear cell RCC.

55. The method according to claim 33 or 34, wherein the cancer is recurrent, locally advanced or metastatic Merkel cell carcinoma (MCC).

56. The method according to claim 33 or 34, wherein the cancer is small cell lung cancer.

57. A pharmaceutical product comprising a sterile container containing a therapeutically effective amount of the pharmaceutical formulation according to any one of claims 1 to 23.

58. A device comprising a therapeutically effective amount of the pharmaceutical formulation according to any one of claims 1 to 23.

59. The device according to claim 58, wherein the device comprises a syringe containing the pharmaceutical formulation.

60. The device according to claim 58 or 59, wherein the syringe is a pre-filled syringe.

61. Use of the pharmaceutical preparation according to any one of claims 1 - 23 in the manufacture of a medicament for the treatment of cancer.

62. The use according to claim 61, wherein the pharmaceutical preparation is administered subcutaneously or intravenously.

63. The use according to claim 61 or 62, wherein the cancer is selected from: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, breast cancer, gastrointestinal cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, endometrial cancer, cutaneous squamous cell carcinoma, thyroid cancer, prostate cancer, glioblastoma, Merkel cell carcinoma, and salivary gland carcinoma.