Anti-PD-1 antibody formulation, preparation method therefor and use thereof

Abstract

The present invention provides an anti-PD-1 antibody formulation, a preparation method therefor and a use thereof. The anti-PD-1 antibody comprises heavy chain CDRs as shown in SEQ ID NOs: 1-3 and light chain CDRs as shown in SEQ ID NOs: 4-6.

Description

An anti-PD-1 antibody formulation and methods of making and uses thereof TECHNICAL FIELD

[0001] The present invention relates to an antibody formulation comprising an anti-PD-1 antibody, methods of making and uses thereof. BACKGROUND

[0002] Programmed death receptor-1 (PD-1) is an immunoinhibitory receptor expressed on activated T cells, B cells and myeloid cells, and is a member of the CD28 immunoglobulin superfamily. PD-1 is a 55 kDa type I transmembrane glycoprotein that contains an Ig variable-type domain that binds ligand and a cytoplasmic tail that is responsible for binding signal transducing molecules. The PD-1 cytoplasmic tail contains two tyrosine-based signaling motifs, ITIM (immunoreceptor tyrosine-based inhibitory motif) and ITSM (immunoreceptor tyrosine-based switch motif).

[0003] Anti-PD-1 antibodies can be used in the manufacture of a medicament for treating cancer, and there is a need to develop stable pharmaceutical formulations with longer shelf life and storage and transportation stability to meet the needs of clinical use. SUMMARY

[0004] The present invention provides an antibody formulation comprising an anti-PD-1 antibody comprising a heavy chain variable region comprising HCDR1-3 set forth in SEQ ID NOs: 1-3 and a light chain variable region comprising LCDR1-3 set forth in SEQ ID NOs: 4-6, the antibody formulation having a pH of about 4.5-6.1. In some embodiments, the antibody formulation has a pH of about 4.5-6.0. In some embodiments, the antibody formulation has a pH of about 5.0-6.0. In some embodiments, the antibody formulation has a pH of about 5.1-5.9. In some embodiments, the antibody formulation has a pH of about 5.2-5.8.

[0005] In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 8. In some embodiments, the anti-PD-1 antibody comprises a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10. In some embodiments, the anti-PD-1 antibody has a concentration of about 5-50 mg / mL, or about 10-40 mg / mL, or about 20-30 mg / mL or about 5, 10, 20, 25, 30, 40, 50 mg / mL, or a range between any two of these values (including the endpoints) or any value therein. In some embodiments, the anti-PD-1 antibody has a concentration of about 25 mg / mL.

[0006] In some embodiments, the antibody formulation further comprises one or more of a buffer, a stabilizer, and a surfactant. In some embodiments, the antibody formulation comprises a buffer. In some embodiments, the antibody formulation comprises a buffer and a surfactant. In some embodiments, the antibody formulation comprises a buffer, a stabilizer, and a surfactant.

[0007] In some embodiments, the buffer is selected from the group consisting of an acetate buffer, a citrate buffer, a phosphate buffer, a succinate buffer, and a histidine buffer, or a combination thereof. In some embodiments, the buffer is selected from the group consisting of a citrate buffer, a phosphate buffer, a phosphate and citrate buffer, a histidine buffer, a succinate buffer, and an acetate buffer. In some embodiments, the buffer is selected from the group consisting of an acetate buffer, a citrate buffer, a succinate buffer, and a histidine buffer. In some embodiments, the buffer is a phosphate / citrate buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer concentration is about 5-20 mM, or about 8-15 mM, or about 5, 8, 10, 12, 15, 18, 20 mM, or a range between any two of these values, inclusive of the endpoints, or any value therein. In some embodiments, the buffer concentration is about 10 mM.

[0008] In some embodiments, the stabilizer is selected from one or more of an inorganic salt, a sugar, a polyol, or an amino acid. In some embodiments, the stabilizer is about 160-300 mM sugar or polyol, or about 5-100 mM amino acid. In some embodiments, the stabilizer is about 200-250 mM sugar or polyol. In some embodiments, the stabilizer is about 20-70 mM amino acid. In some embodiments, the sugar is sucrose or trehalose. In some embodiments, the sugar is at a concentration of about 60-100 mg / mL, or about 70-90 mg / mL, or about 60, 62, 65, 68, 70, 72, 75, 78, 80, 82, 85, 88, 90, 92, 95, 98, or 100 mg / mL, or a range between any two of these values (including endpoints) or any value therein. In some embodiments, the trehalose is trehalose dihydrate. In some embodiments, the polyol is sorbitol or mannitol. In some embodiments, the polyol is at a concentration of about 30-60 mg / mL, or about 35-50 mg / mL, or about 40-45 mg / mL, or about 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 mg / mL, or a range between any two of these values (including endpoints) or any value therein. In some embodiments, the amino acid is arginine or a salt thereof (e.g., arginine hydrochloride). In some embodiments, the amino acid is at a concentration of about 1-20 mg / mL, or about 5-15 mg / mL, or about 1, 5, 8, 10, 12, 15, 18, 20 mg / mL, or a range between any two of these values (including endpoints) or any value therein. In some embodiments, the inorganic salt is sodium chloride. In some embodiments, the inorganic salt is at a concentration of about 100-200 mM, or about 140-160 mM or about 154 mM, or about 100, 120, 130, 140, 150, 160, 170, 180, 200 mM, or a range between any two of these values (including endpoints) or any value therein.

[0009] In some embodiments, the surfactant is selected from one or more of polysorbate and poloxamer. In some embodiments, the surfactant is selected from one or more of polysorbate 20, polysorbate 80, and poloxamer 188. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the concentration of the surfactant is about 0.01-2 mg / mL, or about 0.1-1 mg / mL, or about 0.2-0.7 mg / mL, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.5, 1.8, or 2 mg / mL, or a range between any two of these values, inclusive of the endpoints, or any value therein.

[0010] In some embodiments, the antibody formulation comprises: about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM buffer, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80, the buffer selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, succinate buffer, and histidine buffer, or a combination thereof.

[0011] In some embodiments, the antibody formulation comprises: about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM buffer, about 160-300 mM sugar or polyol or 5-100 mM amino acid, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80, the buffer selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, succinate buffer, and histidine buffer, or a combination thereof.

[0012] In some embodiments, the antibody formulation comprises: about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM buffer, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80, the buffer selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, succinate buffer, and histidine buffer, or a combination thereof.

[0013] In some embodiments, the antibody formulation comprises: about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM buffer, about 200-250 mM sugar or polyol or 20-70 mM amino acid, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80; the sugar is sucrose or trehalose, the polyol is sorbitol or mannitol, the amino acid is arginine hydrochloride, the buffer selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, succinate buffer, and histidine buffer, or a combination thereof.

[0014] In some embodiments, the antibody formulation comprises:

[0015] 1) about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM histidine acetate, citrate, phosphate / citrate, or succinate buffer, about 160-300 mM sugar or polyol or 5-100 mM amino acid, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80; or

[0016] 2) about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM histidine acetate, citrate, phosphate / citrate, or succinate buffer, about 200-250 mM sugar or polyol or 20-70 mM amino acid, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80, the sugar being sucrose or trehalose, the polyol being sorbitol or mannitol, the amino acid being arginine hydrochloride; or

[0017] 3) about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM histidine acetate, citrate, phosphate / citrate, or succinate buffer, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80; or

[0018] 4) about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM histidine acetate, citrate, phosphate / citrate, or succinate buffer, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80.

[0019] In some embodiments, the antibody formulation comprises:

[0020] 1) about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM histidine acetate, citrate, phosphate / citrate, or succinate buffer, about 60-100 mg / mL trehalose dihydrate or sucrose, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80; or

[0021] 2) about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM histidine acetate, citrate, phosphate / citrate, or succinate buffer, about 30-60 mg / mL sorbitol or mannitol, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80; or

[0022] 3) about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM histidine acetate, citrate, phosphate / citrate, or succinate buffer, about 1-20 mg / mL arginine hydrochloride, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80; or

[0023] 4) about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM histidine, acetate, citrate, phosphate / citrate, or succinate buffer, about 70-90 mg / mL trehalose dihydrate or sucrose, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80; or

[0024] 5) about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM histidine, acetate, citrate, phosphate / citrate, or succinate buffer, about 40-45 mg / mL sorbitol or mannitol, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80; or

[0025] 6) about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM histidine, acetate, citrate, phosphate / citrate, or succinate buffer, about 5-15 mg / mL arginine hydrochloride, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80.

[0026] In some embodiments, the antibody formulation comprises about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine, acetate, citrate, phosphate / citrate, or succinate buffer, about 70-90 mg / mL trehalose dihydrate or sucrose, about 0.1-1 mg / mL polysorbate 80.

[0027] In some embodiments, the antibody formulation comprises

[0028] 1) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 90 mg / mL trehalose dihydrate, about 0.2-0.7 mg / mL polysorbate 80; or

[0029] 2) about 25 mg / mL anti-PD-1 antibody, about 10 mM succinate buffer, about 90 mg / mL trehalose dihydrate, about 0.2-0.7 mg / mL polysorbate 80; or

[0030] 3) about 25 mg / mL anti-PD-1 antibody, about 10 mM acetate buffer, about 90 mg / mL trehalose dihydrate, about 0.2-0.7 mg / mL polysorbate 80; or

[0031] 4) about 25 mg / mL anti-PD-1 antibody, about 10 mM citrate buffer, about 90 mg / mL trehalose dihydrate, about 0.2-0.7 mg / mL polysorbate 80; or

[0032] 5) about 25 mg / mL anti-PD-1 antibody, about 10 mM phosphate / citrate buffer, about 90 mg / mL trehalose dihydrate, about 0.2-0.7 mg / mL polysorbate 80.

[0033] In some embodiments, the antibody formulation comprises

[0034] 1) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 75 mg / mL trehalose dihydrate, about 0.2-0.7 mg / mL polysorbate 80; or

[0035] 2) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 70 mg / mL sucrose, about 0.2-0.7 mg / mL polysorbate 80; or

[0036] 3) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 42 mg / mL sorbitol or mannitol, about 0.2-0.7 mg / mL polysorbate 80; or

[0037] 4) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 10 mg / mL arginine hydrochloride, about 0.2-0.7 mg / mL polysorbate 80; or

[0038] 5) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 0.2-0.7 mg / mL polysorbate 80.

[0039] In some embodiments, the antibody formulation has a pH of about 4.5-6.1, or about 5.0-6.0, or about 5.2-5.8, or about 5.3, or about 5.5. In some embodiments, the antibody formulation has a pH of about 4.5, about 4.9, 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, about 6.0, about 6.1, or a range between any two of these values, inclusive of the endpoints, or any value therein.

[0040] In some embodiments, the antibody formulation comprises

[0041] 1) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 90 mg / mL trehalose dihydrate, about 0.2 mg / mL polysorbate 80, at a pH of about 5.5; or

[0042] 2) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 75 mg / mL trehalose dihydrate, about 0.7 mg / mL polysorbate 80, at a pH of about 5.5; or

[0043] 3) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 75 mg / mL trehalose dihydrate, about 0.4 mg / mL polysorbate 20, at a pH of about 5.5; or

[0044] 4) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 70 mg / mL sucrose, about 0.7 mg / mL polysorbate 80, at a pH of about 5.5; or

[0045] 5) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 70 mg / mL sucrose, about 0.4 mg / mL polysorbate 20, at a pH of about 5.5; or

[0046] 6) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 70 mg / mL sucrose, about 0.4 mg / mL polysorbate 20, at a pH of about 5.3.

[0047] In some embodiments, the antibody formulation is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water, such as ultrapure water or water for injection.

[0048] In some embodiments, the antibody formulation is an injection formulation. In some embodiments, the antibody formulation is an intravenous injection formulation.

[0049] In some embodiments, the present application provides a method for preparing the antibody formulation, comprising: dissolving and mixing each ingredient in water in a formulation amount, adjusting the pH value, to obtain the antibody formulation.

[0050] In some embodiments, the present application provides a method for preparing the antibody formulation, comprising: preparing a buffer, ultrafiltration of anti-PD-1 antibody into the buffer, adding excipients, to obtain the antibody formulation.

[0051] In some embodiments, the antibody formulation is a lyophilized formulation, which is obtained by freeze-drying the liquid formulation described herein.

[0052] In some embodiments, the antibody formulation is a reconstituted formulation, which is obtained by reconstituting the lyophilized formulation described herein.

[0053] In some embodiments, the antibody formulation is used for intravenous injection after dilution with an intravenous injection solvent (such as 0.9% normal saline solution or 5% glucose solution). In some embodiments, the concentration after dilution is about 1-10 mg / mL.

[0054] The antibody formulations of the present application have good stability under conditions of shaking, high temperature, light, acceleration, long-term storage, etc. In some embodiments, the antibody formulations of the present application can be stably stored for at least 4 weeks under high temperature conditions, at least 14 days under light conditions, at least 3 months under 25°C acceleration conditions, and at least 33 months under 2-8°C conditions. In some embodiments, the formulations of the present application have good compatibility stability.

[0055] In another aspect, the present application provides a pharmaceutical preparation comprising the antibody formulation as described above and a container for storing the formulation. In some embodiments, the pharmaceutical preparation further comprises instructions for use. The container can be any container conventionally used in the art for storing pharmaceutical products, vials (such as a

[0056] In another aspect, the present application provides the use of the above-mentioned anti-PD-1 antibody formulation in the preparation of a medicament for treating cancer or for stimulating or enhancing an immune response. The cancer includes, but is not limited to, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), pancreatic cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), gastric cancer, gastroesophageal junction cancer, esophageal cancer, head and neck tumor (e.g., head and neck squamous cell carcinoma), liver cancer (e.g., hepatocellular carcinoma (HCC)), ovarian cancer, cervical cancer, endometrial cancer, thyroid cancer, glioblastoma, bladder cancer, glioma, leukemia, lymphoma, and other malignancies.

[0057] Definitions

[0058] Unless otherwise defined, the technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0059] It should be noted that the term "a" or "an" entity refers to one or more than one of that entity; for example, "an antibody" should be understood to refer to one or more antibodies, and, accordingly, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably in this document.

[0060] The terms "comprising" or "including" or "having" as used herein, mean that the compositions and methods include the recited elements, e.g., ingredients or steps, but do not exclude others. "Consisting essentially of, when used to define compositions and methods, excludes other elements of any essential significance to the combination, but does not exclude elements that do not pertain to the combination. "Consisting of, when used to define compositions and methods, excludes the elements specifically recited. Embodiments defined by each of these transition terms are within the scope of the present application. For example, when a composition is described as including components A, B, and C, a composition consisting essentially of A, B, and C, and a composition consisting of A, B, and C are independently within the scope of the present application.

[0061] "About" means a range of values that a person of ordinary skill in the art would readily know to be a normal error range for a value in question. In some embodiments, "about" refers to a range of values ±10%, ±5%, or ±1% of the value being described.

[0062] It is noted that in the present application, "%" referring to components of a formulation means weight by volume (w / v) percentage, such as 1% stabilizer in a formulation means 1 g of stabilizer in 100 mL of the formulation, or 0.01 g / mL of stabilizer.

[0063] The term "buffer" is also known as a buffer system, which includes, but is not limited to, organic acids and their salts, such as succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, or phthalic acid and their salts; Tris, or inorganic acids and their salts, such as phosphate buffers. In addition, amino acids can also be used as buffers. Such amino acids include, but are not limited to, glycine, histidine, arginine, lysine, ornithine, isoleucine, leucine, alanine, glutamic acid, or aspartic acid and their salts. In some embodiments, the buffer is a histidine buffer. In some embodiments, the acetic acid buffer is an acetic acid-sodium acetate buffer. In some embodiments, the succinic acid buffer is a succinic acid-sodium succinate buffer. In some embodiments, the citric acid buffer is a citric acid-sodium citrate buffer. In some embodiments, the histidine buffer is a histidine-histidine hydrochloride buffer. In some embodiments, the phosphate / citrate buffer is a phosphate / citrate buffer.

[0064] The amount of a buffer in the present invention refers to the total amount of the buffer pair in the buffer system that makes up the buffer. In some embodiments, molarity is used as the unit of amount of the buffer, and the value refers to the molarity of the buffer pair in the buffer system of the buffer. For example, a given concentration of a histidine buffer (e.g., 10 mM) is the combined concentration of histidine and histidine hydrochloride. In some embodiments, the histidine buffer comprises histidine and histidine hydrochloride. In some embodiments, a 10 mM histidine buffer comprises about 0.4 g / L histidine and about 1.55 g / L histidine hydrochloride monohydrate.

[0065] The term "stabilizer" includes, but is not limited to, polyols such as mannitol, sorbitol, glycerol, arabitol, propylene glycol, polyethylene glycol, and the like; sugars such as monosaccharides, fructose, maltose, galactose, glucose, sorbose, and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; ionic stabilizers such as sodium chloride, potassium chloride, magnesium chloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride, and / or sodium acetate, and the like inorganic salts; amino acids such as arginine, arginine hydrochloride, proline, glycine, methionine, or salts thereof.

[0066] The term "surfactant" includes, but is not limited to, polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; octyl glucoside sodium salt; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine, or cetyl betaine; lauryl amidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, or isostearamidopropyl betaine (e.g., lauryl amidopropyl); myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, and the like); and the like.

[0067] The formulations described herein can be formulated with the excipients or hydrates thereof. For example, trehalose can be formulated with anhydrous trehalose or with trehalose dihydrate. The trehalose dihydrate described herein can also be replaced with a corresponding amount of trehalose.

[0068] "Stable", "stability" herein refers to the absence, or only minimal, aggregation, degradation or fragmentation of an antibody (including antibody fragments thereof) in a formulation comprising the antibody under given production, preparation, transport and / or storage conditions. A "stable" formulation retains biological activity under given production, preparation, transport and / or storage conditions. The stability of the antibody can be assessed by measuring the extent of aggregation, degradation or fragmentation of the formulation, e.g., by size exclusion chromatography (SEC or SEC-HPLC), ion exchange chromatography (IEC or IEC-HPLC), reducing (R) or non-reducing (NR) capillary electrophoresis (CE), e.g., CE-SDS (R) or CE-SDS (NR), visual inspection and turbidity, insoluble particulates, DLS detection particle size, etc.

[0069] "Water for injection" as described herein is water obtained by distillation of purified water.

[0070] Instability can be: aggregation (e.g., non-covalent soluble aggregation (caused by hydrophobic or charge interactions), covalent soluble aggregation (e.g., disulfide rearrangement / heterogeneity), insoluble aggregation (caused by protein denaturation at liquid / air and liquid / solid interfaces), etc.

[0071] "Aggregate", "SEC multimer" or "soluble aggregate" refers to more than one and less than or equal to ten antibody proteins and / or fragments associated together by covalent, ionic or hydrophobic interactions to form a larger protein body.

[0072] The term "antibody" encompasses a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that the class of heavy chains includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses among them (e.g., γ1-γ4). The "class" of an antibody is determined by the nature of the constant domains of its heavy chains. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and functional specificities conferred are known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is of the IgG class. The four chains are connected through disulfide bonds in a "Y" configuration, with the light chain branching from the "Y" mouth and continuing around the heavy chain through the variable region.

[0073] Light chains can be of either kappa (K) or lambda (l) type. Each heavy chain can be combined with either K or l light chain. Generally, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, its light and heavy chains are bound, by covalent disulfide bonds, at their headpiece portions, and the "tail" portions of the two heavy chains are bound, by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y-shaped endpiece to the C-terminus at the bottom of each chain. The immunoglobulin K light chain variable region is V λ .

[0074] The variable regions of light (VL) and heavy (VH) chains determine antigen recognition and specificity. The constant regions of the light (CL) and heavy (CH) chains confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant regions increases as they become more distal from the antigen binding site or amino-terminal end of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains comprise the carboxy terminus of the heavy and light chains, respectively.

[0075] “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, slow down, ameliorate, and stop undesirable physiological changes or disorders, such as the progress of a disease, including but not limited to alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment or eradication of disease state (whether partial or total), prolonging of survival to that expected in the absence of treatment, and the like. Patients in need of treatment include those already with the condition or disorder, those likely to develop the condition or disorder, or those in need of a preventative measure against the condition or disorder, those who can or are expected to benefit from administration of the antibodies or compositions disclosed herein for detection, diagnostic procedures, and / or treatment.

[0076] DNA encoding the antibodies can be designed and synthesized according to the amino acid sequences of the antibodies described herein using conventional methods, inserted into an expression vector, and then transfected into host cells, which are cultured in a medium to produce the monoclonal antibodies. In some embodiments, the expression vector for the antibodies includes at least one promoter element, the antibody coding sequence, a transcription termination signal and a poly A tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the insert. Efficient transcription can be obtained by the early and late promoters of SV40, the long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIV1, and the early promoter of cytomegalovirus, as well as other promoters of some cells such as the actin promoter. Suitable expression vectors can include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, or pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), pSVL, pMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, murine L cells, and CHO cells, etc. BRIEF DESCRIPTION OF DRAWINGS

[0077] Figure 1 SEC monomer (left) and SEC multimer (right) content of samples at different pHs under high temperature conditions as a function of time;

[0078] Figure 2 IEC main peak (left) and IEC acid region (right) content of samples at different pHs under high temperature conditions as a function of time;

[0079] Figure 3 SEC monomer (left) and IEC main peak (right) content of samples at different pHs under light conditions as a function of time;

[0080] Figure 4 SEC monomer (left) and SEC multimer (right) content of samples containing different stabilizers under high temperature conditions as a function of time;

[0081] Figure 5 SEC monomer (left) and SEC multimer (right) content of samples containing different stabilizers under light conditions as a function of time;

[0082] Figure 6 IEC main peak content of samples containing different stabilizers under light conditions as a function of time;

[0083] Figure 7 Turbidity change trend of samples containing different types and contents of Tween after shaking;

[0084] Figure 8 SEC monomer (left) and IEC main peak (right) content of samples under high temperature conditions as a function of time;

[0085] Figure 9 SEC monomer (left) and SEC multimer (right) content of each sample over time under light conditions;

[0086] Figure 10 nrCE-SDS main peak content of each sample over time under high temperature conditions;

[0087] Figure 11 SEC monomer (left) and SEC multimer (right) content of each sample over time under accelerated conditions;

[0088] Figure 12 IEC main peak (left) and nrCE-SDS main peak (right) content of each sample over time under accelerated conditions. DETAILED DESCRIPTION

[0089] The technical solutions of the present application are further illustrated by specific examples below, which do not represent a limitation on the scope of protection of the present application. Some non-essential modifications and adjustments made by others according to the concept of the present application still fall within the scope of protection of the present application.

[0090] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified. In the following examples, SEC-HPLC, IEC-HPLC, capillary electrophoresis (such as CE-SDS) detection, etc. are carried out according to conventional methods.

[0091] The preparation method of antibody A can refer to WO2020207432A1 patent application; the amino acid sequence of antibody A is shown in Table 1.

[0092] Table 1 Amino acid sequence of antibody A

[0093] Example 1.

[0094] Prepare 10 mM anhydrous disodium hydrogen phosphate solution and 10 mM citric acid solution according to conventional methods. The prepared 10 mM anhydrous disodium hydrogen phosphate solution is adjusted to pH with 10 mM citric acid solution to obtain phosphate / citrate (PB+CB) buffer solutions with pH 4.5 and pH 6.5, respectively. Antibody A is diluted with PB+CB buffer solutions with pH 4.5 and pH 6.5, respectively, and the concentration of antibody A is 25 mg / mL to obtain formulation samples with pH 4.5 and pH 6.5, and then the pH 4.5 and pH 6.5 samples are mixed to obtain formulation samples with pH 5.0, pH 5.2, pH 5.4, pH 5.6, pH 5.8, and pH 6.0, respectively. The samples are filtered with sterile filters (0.22 μm) respectively. The stability of each sample under high temperature (40°C, avoiding light) or light (4000lx, 25°C) conditions is detected.

[0095] From the results of Table 2, Figure 1 and Figure 2, it can be seen that under high temperature conditions, the SEC-HPLC monomer content of the 8 groups of samples showed a downward trend, the SEC-HPLC monomer content of the pH 5.2-pH 6.5 group of samples decreased the least, which was better than the samples at pH 5.0 and pH 4.5, and the SEC-HPLC multimers of the pH 5.2-pH 6.5 group of samples increased slowly. The IEC-HPLC main peak content of the pH 4.5-pH 6.0 group decreased the least, the IEC-HPLC main peak content of the pH 6.5 group decreased the most, and the IEC-HPLC acid region of the pH 6.5 group increased the most. The samples were relatively stable in the pH 5.0-pH 6.0 range.

[0096] From the results of Table 3 and Figure 3, it can be seen that under light conditions, the SEC-HPLC monomer content of the pH 4.5-pH 5.8 group of samples decreased the least, and the pH 6.5 group of samples decreased the most. The IEC-HPLC main peak content of the pH 4.5-pH 6.0 group of samples decreased the least, and the pH 6.5 group of samples decreased the most. The samples were relatively stable in the pH 4.5-pH 6.0 range.

[0097] Table 2 SEC-HPLC and IEC-HPLC of different pH samples under high temperature (40°C, dark) conditions over time

[0098] Table 3 SEC-HPLC and IEC-HPLC of different samples under light (4000lx, 25°C) conditions over time

[0099] Seven pH gradient samples containing 10mM histidine (His) buffer were prepared, and the preparation information is shown in Table 4. The samples were filtered with sterile filters (0.22μm). The divided samples were placed under high temperature (40°C, dark) and light (4000lx, 25°C) conditions to investigate the sample stability.

[0100] Table 4 Preparation information

[0101] The results are shown in Table 5. The change trends of the 7 groups of samples were basically consistent. The SEC-HPLC monomer content decreased slowly with the increase of time. After being placed at high temperature for 4 weeks, the monomer content of the samples decreased obviously. At the same time point, the SEC monomer content of the sample at pH 4.9 decreased the most. The SEC multimers and SEC fragments of the sample at pH 4.9 increased the fastest. The IEC-HPLC main peak of the sample at pH 4.9-5.9 decreased slowly, and the change in the alkaline region was not obvious. The samples at pH 5.1-5.9 remained stable.

[0102] Table 5 Change of SEC-HPLC and IEC-HPLC of samples at different pH with time under high temperature (40°C, light shielding) condition

[0103] The results are shown in Table 6. Under light condition, the SEC-HPLC monomer content of the sample at pH 4.9-5.9 decreased the least. The IEC-HPLC main peak content of the sample at pH 4.9-5.9 decreased the least. It is shown that the protein of the sample is relatively stable at pH 4.9-5.9.

[0104] Table 6 Change of SEC-HPLC and IEC-HPLC of samples at different pH with time under light (4000lx, 25°C) condition

[0105] Example 2.

[0106] Five different buffers were prepared by a conventional method: succinic acid / sodium succinate buffer (Sua), citric acid / sodium citrate buffer (CB), phosphoric acid / citrate buffer (PB+CB), histidine / histidine hydrochloride buffer (His), and acetic acid / sodium acetate buffer (NaAc). Fucose was weighed and dissolved in the corresponding buffer, and polysorbate 80 was added to prepare a mother liquor. The five prepared samples were added with stabilizers and surfactants, and the antibody concentration was 25 mg / mL. The compositions of the preparations are shown in Table 7. The divided samples were placed under high temperature (40°C, light shielding) and light (4000lx, 25°C) conditions, respectively, to investigate their stability.

[0107] Table 7 Preparation information

[0108] From the data of Table 8, under high temperature conditions, SEC-HPLC results showed that the 5 groups of samples remained stable, with 1-His having the best stability. IEC-HPLC results of the 5 groups of samples showed that the 5 different buffers had little effect on the IEC main peak and acid region content, and the samples remained stable.

[0109] Table 8 SEC-HPLC and IEC-HPLC of different buffer samples under high temperature (40°C, light-protected) conditions over time

[0110] From the data of Table 9, under light conditions, the 5 groups of samples remained stable, with 4-Sua, 1-His and 2-NaAc having the best stability.

[0111] Table 9 SEC-HPLC and IEC-HPLC of different buffer samples under light (4000lx, 25°C) conditions over time

[0112] Example 3.

[0113] A 10mM His buffer at pH 5.5 was prepared, and the corresponding amount of stabilizer and surfactant was added to prepare the formulations shown in Table 10. The formulation samples were filtered through sterile filters (0.22μm). The stability of each sample under high temperature (40°C, light-protected) or light (4000lx, 25°C) conditions was detected.

[0114] Table 10 Formulation information

[0115] The results, as shown in Table 11 and Figure 4, were that the 7 groups of samples remained stable.

[0116] Table 11 SEC-HPLC and IEC-HPLC of different stabilizer samples under high temperature (40°C, light-protected) conditions over time

[0117] The results, as shown in Table 12 and Figures 5-6, were that the 7 groups of samples remained stable under light conditions, with ArgHCl, Suc and NaCl groups having the best stability.

[0118] Table 12 SEC-HPLC and IEC-HPLC of different stabilizer samples under light (4000lx, 25°C) conditions over time

[0119] Example 4.

[0120] The formulations in Table 13 were prepared, and each sample was filtered through a sterile filter (0.22 μm). The stability of each sample under the conditions of shaking, freeze-thawing, etc. was detected.

[0121] Table 13 Formulation Information

[0122] As shown in Figure 7, after being placed under shaking conditions for 2 h, the blank control sample with only 9% trehalose dihydrate began to become turbid, and the sample with polysorbate remained clear until 72 h. The sample with 0.02% polysorbate 20 had relatively high turbidity, and the turbidity of the other groups was low.

[0123] After the samples were repeatedly freeze-thawed 5 times, the SEC monomer purity of the blank control and the samples with different Tween contents did not show a significant downward trend, and there was no significant difference between polysorbate 20 and polysorbate 80 in the SEC-HPLC monomer purity after freeze-thawing, indicating that freeze-thawing had little effect on the SEC-HPLC monomer purity of the samples, and the samples had good stability.

[0124] Example 5.

[0125] The formulations in Table 14 were prepared, and each sample was filtered through a sterile filter (0.22 μm). The stability of each sample under the conditions of high temperature (40°C, avoiding light), light (4000 lx, 25°C), acceleration (25°C, avoiding light), etc. was detected.

[0126] Table 14 Formulation Information

[0127] Note: TW20 is polysorbate 20, and TW80 is polysorbate 80.

[0128] As shown in Figures 8 and 10, after being placed at high temperature of 40°C for 4 weeks, the SEC-HPLC monomer purity, IEC-HPLC main peak content, and nrCE-SDS main peak content of the 6 groups of samples did not differ much under high temperature conditions. The relative binding activity remained within the quality standard range. The stability of the 6 groups of samples did not differ much under high temperature conditions, and all had good stability.

[0129] As shown in Figure 9, after being placed under light for 14 days, the SEC-HPLC monomer purity of the 6 groups of samples decreased consistently, indicating that the stability of the 6 groups of samples did not differ much under light conditions, and all had good stability.

[0130] As shown in Figure 11, after being placed under acceleration conditions at 25°C for 3 months, the SEC-HPLC monomer purity of the 6 groups of samples decreased consistently, and all had good stability.

[0131] The results are shown in Figure 12. After 6 groups of samples were placed at 25°C for 3 months, the content of the main peak of IEC decreased consistently, and the content of the main peak of nrCE-SDS decreased consistently. After 6 groups of samples were placed at 25°C for 3 months, the relative binding activity remained within the quality standard range. After 3 months of placement under accelerated conditions at 25°C, polysorbate 80 in each sample did not show significant degradation. The 6 groups of preparation samples all had good stability.

[0132] After 33 months of placement at 2-8°C, the 6 groups of preparation samples still had excellent stability and binding activity, and had superior long-term stability.

[0133] Example 6.

[0134] The 6 groups of samples in Example 5 were mixed with 0.9% saline solution to a concentration of about 1 mg / mL. At 0h, 2h, 6h, and 24h, the insoluble particles were sampled and measured. It was found that after preparation 1 was mixed, flocculent visible foreign matter was slowly found in the infusion bag, and preparations 2-6 were not. After the solution was filtered using a filter with a pore size of 5 μm, the particles were significantly reduced, and most of the particle data was qualified, with only a few points having particles exceeding 25 / mL, and the range of exceeding was not large. After the solution was filtered using a filter with a pore size of 0.22 μm, the particles were significantly reduced, and all particle data was qualified, not exceeding 25 / mL. It was indicated that the sample of preparation 1 should avoid being mixed with saline, and if the particles of preparations 2-6 were slightly high after being mixed with saline, a clinical infusion tube with a pore size of 0.2-5 μm (such as 0.22 μm) filter could be used for infusion.

[0135] The sample of preparation 4 was mixed with 5% glucose solution to a concentration of 1 mg / mL, 2 mg / mL, 3 mg / mL, and 10 mg / mL. At 0h, 2h, 8h, and 24h, the insoluble particles were sampled and measured. It was found that after preparation 4 was mixed, no flocculent visible foreign matter was found in the infusion bag, and after the solution was filtered using a 0.22 μm filter, the particles were significantly reduced, and all particle data was qualified, not exceeding 25 / mL. A clinical infusion tube with a 0.2-5 μm (such as 0.22 μm) filter could be used for infusion.

Claims

1. An anti-PD-1 antibody formulation comprising an anti-PD-1 antibody, pH is about 4.5-6.1; the anti-PD-1 antibody comprises a heavy chain variable region comprising HCDR1-3 set forth in SEQ ID NOs: 1-3 and a light chain variable region comprising LCDR1-3 set forth in SEQ ID NOs: 4-6.

2. The antibody formulation of claim 1, the anti-PD-1 antibody comprises a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 8; or, the anti-PD-1 antibody comprises a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO:

10.

3. The antibody formulation of claim 1 or 2, the concentration of the anti-PD-1 antibody is about 5-50 mg / mL.

4. The antibody formulation of any one of claims 1-3, the pH is about 5.0-6.

0.

5. The antibody formulation of any one of claims 1-4, the formulation further comprises one or more of a buffer, a stabilizer, and / or a surfactant.

6. The antibody formulation of any one of claims 1-5, comprising a buffer selected from one or more of a histidine buffer, an acetate buffer, a citrate buffer, a phosphate buffer, and a succinate buffer.

7. The antibody formulation of claim 5 or 6, the buffer is selected from a citrate buffer, a phosphate buffer, a phosphate and citrate buffer, a histidine buffer, a succinate buffer, and an acetate buffer.

8. The antibody formulation of any one of claims 5-7, the concentration of the buffer is about 5-20 mM, or about 8-15 mM, or about 10 mM.

9. The antibody formulation of any one of claims 1-8, comprising a stabilizer selected from one or more of an inorganic salt, a sugar, a polyol, and an amino acid.

10. The antibody formulation of claim 9, the stabilizer is about 160-300 mM sugar or polyol, or about 200-250 mM sugar or polyol; or the stabilizer is about 5-100 mM amino acid, or about 20-70 mM amino acid; or, the stabilizer is about 100-200 mM inorganic salt.

11. The antibody formulation of claim 9, the sugar is sucrose or trehalose, the polyol is sorbitol or mannitol, the amino acid is arginine hydrochloride, and the inorganic salt is sodium chloride.

12. The antibody formulation of any one of claims 9-11, the stabilizer is about 60-100 mg / mL, or about 70-90 mg / mL sugar.

13. The antibody formulation of any one of claims 9-11, the stabilizer is about 30-60 mg / mL, or 35-50 mg / mL, or 40-45 mg / mL polyol.

14. The antibody formulation of any one of claims 9-11, the stabilizer is about 1-20 mg / mL, or 5-15 mg / mL amino acid.

15. The antibody formulation of any of claims 9-11, wherein the stabilizer is about 100-200 mM, or about 140-160 mM, of an inorganic salt.

16. The antibody formulation of any of claims 1-15, comprising a surfactant selected from one or more of a polysorbate and a poloxamer.

17. The antibody formulation of claim 16, wherein the surfactant is at a concentration of 0.01-2 mg / mL, or 0.1-1 mg / mL, or 0.2-0.7 mg / mL, and the surfactant is selected from one or more of polysorbate 20, polysorbate 80, and poloxamer 188.

18. The antibody formulation of any of claims 1-4, comprising: 1) about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM histidine buffer, acetate buffer, citrate buffer, phosphate / citrate buffer, or succinate buffer, about 160-300 mM sugar or polyol or 5-100 mM amino acid, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80; or 2) about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM histidine buffer, acetate buffer, citrate buffer, phosphate / citrate buffer, or succinate buffer, about 200-250 mM sugar or polyol or 20-70 mM amino acid, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80; or 3) about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM histidine buffer, acetate buffer, citrate buffer, phosphate / citrate buffer, or succinate buffer, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80; or 4) about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM histidine buffer, acetate buffer, citrate buffer, phosphate / citrate buffer, or succinate buffer, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80.

19. The antibody formulation of any of claims 1-4, comprising: 1) about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM histidine buffer, acetate buffer, citrate buffer, phosphate / citrate buffer, or succinate buffer, about 60-100 mg / mL trehalose dihydrate or sucrose, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80; or 2) about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM histidine buffer, acetate buffer, citrate buffer, phosphate / citrate buffer, or succinate buffer, about 30-60 mg / mL sorbitol or mannitol, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80; or 3) about 5-50 mg / mL anti-PD-1 antibody, about 5-20 mM histidine buffer, acetate buffer, citrate buffer, phosphate / citrate buffer, or succinate buffer, about 1-20 mg / mL arginine hydrochloride, about 0.01-2 mg / mL polysorbate 20 or polysorbate 80.

20. The antibody formulation of any of claims 1-4, comprising: 1) about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM histidine buffer, acetate buffer, citrate buffer, phosphate / citrate buffer, or succinate buffer, about 70-90 mg / mL trehalose dihydrate or sucrose, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80; or 2) about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM histidine buffer, acetate buffer, citrate buffer, phosphate / citrate buffer, or succinate buffer, about 40-45 mg / mL sorbitol or mannitol, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80; or 3) about 10-40 mg / mL anti-PD-1 antibody, about 8-15 mM histidine buffer, acetate buffer, or succinate buffer, about 5-15 mg / mL arginine hydrochloride, about 0.1-1 mg / mL polysorbate 20 or polysorbate 80.

21. The antibody formulation of any of claims 1-4, comprising: 1) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, acetate buffer, citrate buffer, phosphate / citrate buffer, or succinate buffer, about 70-90 mg / mL trehalose dihydrate or sucrose, about 0.1-1 mg / mL polysorbate 80; or 2) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, succinate buffer, acetate buffer, citrate buffer, or phosphate / citrate buffer, about 90 mg / mL trehalose dihydrate, about 0.2-0.7 mg / mL polysorbate 80.

22. The antibody formulation of any of claims 1-4, comprising: 1) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 75 mg / mL trehalose dihydrate, about 0.2-0.7 mg / mL polysorbate 80; or 2) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 70 mg / mL sucrose, about 0.2-0.7 mg / mL polysorbate 80; or 3) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 42 mg / mL sorbitol or mannitol, about 0.2-0.7 mg / mL polysorbate 80; or 4) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 10 mg / mL arginine hydrochloride, about 0.2-0.7 mg / mL polysorbate 80; 5) about 25 mg / mL anti-PD-1 antibody, about 10 mM histidine buffer, about 0.2-0.7 mg / mL polysorbate 80.

23. The antibody formulation of any one of claims 17-22, wherein the concentration of polysorbate 80 is about 0.7 mg / mL.

24. The antibody formulation of any one of claims 1-23, which is a liquid formulation or a lyophilized formulation.

25. The antibody formulation of any one of claims 1-24, which is an injectable formulation, or is an intravenous injectable formulation.

26. A method of preparing an antibody formulation, comprising: The antibody formulation of any one of claims 1-25 is prepared by formulating a buffer, ultrafiltrating the anti-PD-1 antibody into the buffer, and optionally adding a stabilizer and / or a surfactant.

27. Use of the antibody formulation of any one of claims 1-25 in the manufacture of a medicament for treating a cancer, including but not limited to, melanoma, renal cancer, prostate cancer, pancreatic cancer, breast cancer, colorectal cancer, lung cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, head and neck tumor, liver cancer, ovarian cancer, cervical cancer, endometrial cancer, thyroid cancer, glioblastoma, bladder cancer, glioma, leukemia, lymphoma, and other malignancies, or for stimulating or enhancing an immune response.

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