CN112741804A

CN112741804A - Stable formulations containing anti-PD-L1 antibodies

Info

Publication number: CN112741804A
Application number: CN201911053752.3A
Authority: CN
Inventors: 刘洪川; 刘沛想; 张静; 孟琴; 姚盛; 冯辉; 武海
Original assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Current assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2021-05-04
Also published as: WO2021083271A1; CN114616249B; CN114616249A

Abstract

The invention provides a stable anti-PD-L1 (programmed cell death protein ligand 1) antibody pharmaceutical preparation and application thereof in medicine. The preparation contains anti-PD-L1 antibody, buffer solution, at least one stabilizer, and optionally surfactant. The pharmaceutical preparation provided by the invention can effectively inhibit the aggregation of the antibody and prevent the degradation of the antibody product, and has high stability.

Description

Stable formulations containing anti-PD-L1 antibodies

Technical Field

The present invention relates to the field of therapeutic pharmaceutical formulations. In particular, the invention relates to the field of pharmaceutical preparations comprising a humanized antibody that specifically binds programmed cell death protein ligand 1 (PD-L1).

Background

Programmed death protein ligand 1(PD-L1), also known as cluster of differentiation 274 (CD 274) or B7 homologous protein 1(B7homolog1, B7-H1), belongs to the tumor necrosis factor superfamily, is a type I transmembrane glycoprotein consisting of 290 amino acid residues, comprises an IgV-like region, an IgC-like region, a transmembrane hydrophobic region and a 30 amino acid intracellular tail, and has a complete molecular weight of 40 kDa. PD-L1 mRNA is expressed in almost all tissues, but PD-L1 protein is persistently expressed in only a small proportion of tissues, including the liver, lungs, tonsils, and immune privileged tissues such as the eye, placenta, etc. PD-L1 is also expressed on activated T cells, B cells, monocytes, dendritic cells, macrophages and the like.

The receptor of PD-L1 is PD-1, and is mainly expressed on the surfaces of immune cells such as CD4+ T cells, CD8+ T cells, NKT cells, B cells, activated monocytes and the like. Binding of PD-L1 to PD-1 initiates phosphorylation of tyrosine residues in the cytoplasmic ITIM (immunoreceptor tyrosine inhibition module) of PD-1, promotes binding of tyrosine phospholipase to SHP2, activates SHP2, dephosphorylates downstream Syk and PI3K to deliver a termination signal, and limits interaction of antigen presenting cells or dendritic cells with T cells. The combination can further inhibit the metabolism of T cells, inhibit the secretion of anti-apoptotic protein Bcl-X2, reduce the secretion of effector cytokines IL-2 and IFN-r, and induce the exhaustion and apoptosis of the T cells, thereby reducing the immune response involved by immune T cells and performing negative regulation function.

T cells recognize antigen and, upon activation, secrete IFN-r. T cell-derived IFN-r will expand and maintain T cell functions such as up-regulation of MHC molecules, enhancing antigen processing and presentation by target cells, promoting T cell differentiation. IFN-r can also induce the expression of PD-L1 of tissues at immune inflammation sites, and prevent damage to tissues caused by excessive immunity. IFN-r can induce the expression of PD-L1 on the cell surface of conventional epithelial cells, vascular endothelial cells, myeloid cells, naive T cells and the like. IFN-r-induced interferon regulatory factor 1(IRF-1) can also bind to interferon regulatory factor binding sites 200bp and 320bp before the transcription start site of PD-L1 to regulate PD-L1 at the transcription level. PD-L1 may exert a negative regulatory function in conjunction with PD-1 on the surface of T cells, thereby protecting inflammatory sites.

The negative regulatory function of PD-L1 plays an important role in tumor immunity. In 2004, Konishi et al first found expression of PD-L1 in a tissue sample of a patient with non-small cell lung cancer, and subsequently, PD-L1 was found to be expressed in tissues of various tumor patients, including stomach cancer, lung cancer, liver cancer, intrahepatic bile duct cancer, colon cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, bladder cancer, renal cell carcinoma, skin cancer, oral squamous cell carcinoma, and the like. In the process of cell malignant transformation, new protein molecules can be generated due to gene mutation, exogenous gene (virus) expression or resting gene activation and the like, and after the proteins are degraded in cells, certain degraded peptide segments can be expressed on the surfaces of the cells to become tumor antigens. The immune system recognizes tumor antigens and eliminates tumor cells by immune surveillance, which escape immune attack using PD-L1.

Expression of PD-L1 at the tumor site can protect tumor cells from damage in a variety of ways. The secretion of IFN-r by Tumor Infiltrating Lymphocytes (TIL) can induce the expression of PD-L1 by tumor cells and peripheral stromal cells. While PD-L1 of tumor cells can bind to PD-1 on TIL, inhibit activation of TIL cells and further cause apoptosis. In vitro experiments demonstrated that tumor cell-associated PD-L1 increased apoptosis of tumor-specific T cells, while the PD-L1 monoclonal antibody attenuated this effect. The tumor-associated PD-L1 can promote the expression of IL-10 by T cells and further inhibit immune response. PD-L1 is not only a ligand for PD-1, but also can be used as a receptor to transmit reverse signals to protect tumor cells from apoptosis induced by other anti-tumor pathways such as FAS-FASL.

A variety of chronic and acute viruses also use the PD-L1 signal to evade human immunodetection. Wang et al found that the expression of PD-L1 on dendritic cells derived from bone marrow of HIV-infected persons is obviously up-regulated, and after HIV replication is inhibited through antiviral infection, the expression of PD-L1 is down-regulated, and the number of T cells is up-regulated at the same time; chen et al found that expression of PD-L1 was also up-regulated on T lymphocytes and dendritic cells from chronic HBV infected patients. Viral infection induces high expression of PD-L1 in infected cells, and simultaneously induces expression of PD-1 in CD8+ T cells, thereby inhibiting the action of the T cells and leading to exhaustion of effector T cells.

Thus, there is a need in the art for protein formulations with high stability.

Disclosure of Invention

The present invention provides a pharmaceutical preparation comprising a human antibody that specifically binds to programmed cell death protein ligand 1 (PD-L1).

In one aspect, the invention provides an anti-PD-L1 antibody pharmaceutical formulation comprising: (1) a buffer solution; (2) a stabilizer; (3) an anti-PD-L1 antibody or antigen-binding fragment thereof.

Preferably, the pharmaceutical preparation may further comprise a non-ionic surfactant.

In some embodiments, the buffer is one of an acetate buffer, a citrate buffer, a histidine buffer, or a combination thereof. In some embodiments, the buffer is a histidine buffer. In some embodiments, the histidine buffer is made from L-histidine and L-histidine monohydrochloride. In an embodiment of the invention, the buffer is present at a concentration of about 1mM to 50mM, preferably 5mM to 40mM, more preferably 10mM to 30 mM. In some embodiments, the histidine buffer is at a concentration of about 10mM to 30 mM. In some embodiments, the histidine buffer is at a concentration of about 10 mM. In some embodiments, the histidine buffer is at a concentration of about 20 mM. In some embodiments, the histidine buffer is at a concentration of about 30 mM.

In some embodiments, the stabilizer comprises one or a combination of sodium chloride, arginine hydrochloride, mannitol, sorbitol, sucrose, and trehalose. In some embodiments, wherein the stabilizing agent is trehalose. In an embodiment of the invention, the concentration of the stabilizer is about 50mM to 300mM, preferably 100mM to 300mM, more preferably 200mM to 250 mM. In some embodiments, the trehalose is at a concentration of about 200mM to about 250 mM. In some specific embodiments, the trehalose is at a concentration of about 200mM, 120mM, or 240 mM.

In some embodiments, wherein the pharmaceutical formulation has a pH of about 5.0 to 6.5. In some embodiments, wherein the pharmaceutical formulation has a pH of about 5.0. In some embodiments, wherein the pharmaceutical formulation has a pH of about 5.5. In some embodiments, wherein the pharmaceutical formulation has a pH of about 6.0. In some embodiments, wherein the pharmaceutical formulation has a pH of about 6.5.

In some embodiments, wherein the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 1; the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2; the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3; LCDR1 has an amino acid sequence shown in SEQ ID NO. 4; LCDR2 has an amino acid sequence shown in SEQ ID NO. 5; and LCDR3 has an amino acid sequence shown in SEQ ID NO 6. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH has an amino acid sequence shown in SEQ ID NO: 7; and VL has the amino acid sequence shown in SEQ ID NO 8. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a Heavy Chain (HC) and a Light Chain (LC), wherein the HC has an amino acid sequence as set forth in SEQ ID NO. 9; and LC has an amino acid sequence shown in SEQ ID NO 10. In some embodiments, the antibody or antigen-binding fragment thereof is present at a concentration of about 30mg/mL to about 80 mg/mL. In some embodiments, the antibody or antigen-binding fragment thereof is present at a concentration of about 40mg/mL to about 60 mg/mL. In a specific embodiment, wherein the antibody or antigen-binding fragment thereof is present at a concentration of about 40 mg/mL. In a specific embodiment, wherein the concentration of said antibody or antigen-binding fragment thereof is about 50 mg/mL. In a specific embodiment, wherein the antibody or antigen-binding fragment thereof is present at a concentration of about 60 mg/mL.

In some embodiments, the pharmaceutical formulation provided by the present invention further comprises a surfactant selected from one of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a combination thereof. In some embodiments, the pharmaceutical formulations provided herein comprise polysorbate 20. In a specific embodiment, the pharmaceutical formulations provided herein comprise polysorbate 20 at a concentration of about 0.01% to about 0.05%. In a specific embodiment, the pharmaceutical formulation provided by the present invention comprises polysorbate 20 at a concentration of about 0.02%.

In some embodiments, the pharmaceutical formulations provided herein comprise: (1) about 10-30 mM histidine buffer; (2) about 200mM to about 250mM of a trehalose stabilizer; (3) about 30mg/mL to about 80mg/mL of an anti-PD-L1 antibody or antigen-binding fragment thereof; and (4) about 0.01% to about 0.05% polysorbate 20; wherein the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 1; the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2; the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3; LCDR1 has an amino acid sequence shown in SEQ ID NO. 4; LCDR2 has an amino acid sequence shown in SEQ ID NO. 5; and LCDR3 has an amino acid sequence shown in SEQ ID NO 6.

In another aspect, the invention provides the use of any one of the pharmaceutical preparations described above in the preparation of a medicament for the treatment of a disease associated with PD-L1.

Drawings

FIG. 1: binding of humanized antibody to human PD-L1.

FIG. 2: binding of the humanized antibody to PD-L1 on 293F cells.

FIG. 3: the humanized antibody inhibits the binding of human PD-L1 to PD-1 on 293F cells.

FIG. 4: jurkat fluorescein analysis of humanized antibodies.

FIG. 5: the in vivo experiment detects the tumor growth inhibition effect of the humanized antibody.

Detailed Description

Definitions and explanations

The invention features stable aqueous liquid pharmaceutical formulations comprising an anti-PD-L1 antibody, or antigen-binding portion thereof, having improved properties compared to art-recognized formulations. The preparation provided by the invention has high concentration and high stability.

It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

As used herein, "about" when referring to a measurable value (e.g., amount, duration, etc.) is intended to encompass variations of ± 20% or ± 10% from the particular value, including ± 5%, ± 1% and ± 0.1%, as such variations are suitable for performing the disclosed methods.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those disclosed herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

By "therapeutically active antibody" or "therapeutic antibody" is meant an antibody that can be used for therapeutic purposes, i.e., for treating a disorder in a subject. It should be noted that although therapeutic proteins may be used for therapeutic purposes, the invention is not limited to such use as the proteins may also be used for in vitro studies.

The term "pharmaceutical formulation" or "formulation" is an article of manufacture in a form such that the biological activity of the active ingredient is effective and free of other ingredients having unacceptable toxicity to the subject to which the formulation is administered. The formulation is sterile.

The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation. The liquid formulations of the present invention are stable upon storage and their stability is independent of lyophilization (or other state change methods, such as spray drying).

The term "aqueous liquid formulation" refers to a liquid formulation that uses water as a solvent. In one embodiment, the aqueous liquid formulation is one that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).

The term "excipient" refers to an agent that can be added to a formulation to provide a desired characteristic (e.g., consistency, improved stability) and/or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

Herein, the term "buffer of pH of about 5.0 to about 6.5" refers to an agent that by the action of its acid/base conjugate components makes a solution containing the agent resistant to pH changes. The buffer used in the formulation of the present invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.0. In some embodiments, the pH is about 6.0.

Examples of "buffers" that control the pH within this range herein include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconic acid, histidine, methionine, citrate, phosphate, citrate/phosphate, imidazole, acetic acid, acetates, citrate, combinations thereof, and other organic acid buffers. In some embodiments, the buffer is not a protein. In some embodiments, the buffer is histidine. In embodiments, the buffer has a concentration of about 5-100mM, such as 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, or 100mM, or ranges formed by any two of these ranges as endpoints. In some embodiments, the buffer concentration is about 20 mM. In some embodiments, the buffer concentration is about 30 mM.

Herein, a "histidine buffer" is a buffer comprising histidine. Examples of histidine buffers include salts of histidine and histidine, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. In some embodiments, the histidine buffer is: histidine buffer made from 1-20mM L-histidine and 1-20mM L-histidine monohydrochloride. In some embodiments, the histidine buffer consists of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1: 4. In some embodiments, the histidine buffer consists of 1 histidine and histidine hydrochloride in a 1: molar ratio. In some embodiments, the histidine buffer consists of histidine and histidine hydrochloride in a molar ratio of 1: 3. In some embodiments, the histidine preparation is: histidine buffer pH 5.5 was prepared from 4.5mM L-histidine and 15.5mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: histidine buffer pH6.0 was prepared from 15mM histidine and 15mM histidine hydrochloride.

As used herein, the term "surfactant" generally includes agents that protect proteins, such as antibodies, from air/solution interface-induced stress, solution/surface-induced stress to reduce aggregation of the antibodies or minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronics), Sodium Dodecyl Sulfate (SDS). In some embodiments, the nonionic surfactant is polysorbate 20. In some embodiments, the concentration of polysorbate 20 is about 0 to 0.1% (w/v). In some embodiments, the concentration of polysorbate 20 is 0.01% to about 0.05% (w/v). In some embodiments, the concentration of polysorbate 20 is about 0.02% (w/v).

As used herein, the term "stabilizer" can reduce aggregation of antibodies and other proteins. Exemplary stabilizers include, but are not limited to: human serum albumin (hsa), bovine serum albumin (bsa), alpha-casein, globulin, alpha-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAaseA. Stabilizers also include amino acids, sugars, polyols, and their metabolites, such as: sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, arginine, glycine, alanine (α -alanine, β -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ -aminobutyric acid (GABA), opioids (opioids) (alaninoids, octopine, glycopyrrolate (strombine)), and N-oxide of Trimethylamine (TMAO). The concentration of the stabilizing agent in the formulation of the invention may be in the range 20-300mM, such as 50-300mM or 50-250 mM. In some embodiments, the stabilizing agent is a sugar. In some embodiments, the stabilizing agent is trehalose. In some embodiments, the trehalose concentration is about 20 to 300 mM. In some embodiments, the trehalose concentration is about 100 to 250 mM. In some embodiments, the trehalose concentration is about 200 to 250 mM. In some embodiments, the trehalose is at a concentration of about 200mM, 220mM, 240mM, or 250 mM. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., may also act to control osmotic pressure. In some embodiments, such stabilizers are included in the formulations of the present invention, preferably in combination with trehalose, such as sodium chloride in combination with trehalose. In some embodiments, the stabilizers of the present invention are sodium chloride and trehalose, wherein the concentration of sodium chloride in the formulation is 30-80mM, preferably 40-70mM, and the concentration of trehalose is 100-200mM, preferably 100-150 mM. It is to be understood that when trehalose is used in combination with such a stabilizer having an osmotic pressure controlling function, the concentration of trehalose may be in the range of 100-. In some embodiments, the formulations of the present invention do not contain such stabilizing agents that control osmotic pressure.

By "isotonic" is meant that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using an osmometer of the vapor pressure or subfreezing type.

A "stable" formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. The pharmaceutical preparation may be stable even if the contained antibody fails to maintain 100% of its chemical structure or biological function after storage over a certain period of time. In certain instances, an antibody structure or function that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage over a period of time may also be considered "stable". Various analytical techniques for measuring Protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-: 29-90 (both incorporated by reference).

After storage of the formulation at a temperature and for a period of time, its stability can be measured by determining the percentage of native antibody remaining therein (among other methods). The percentage of native antibody can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SEC-HPLC ]), among other methods, "native" referring to unaggregated and undegraded. In some embodiments, the stability of a protein is determined as a percentage of monomeric protein in a solution having a low percentage of degraded (e.g., fragmented) and/or aggregated protein. In one embodiment, the formulation is stable for storage at room temperature, about 25-30 ℃, or 40 ℃ for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, up to no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.

Stability can be measured by determining (among other methods) the percentage of antibody ("acidic form") that migrates during ion exchange in a fraction that is acidic relative to the main fraction of antibody ("predominantly charged form"), where stability is inversely proportional to the percentage of acidic form antibody. The percentage of "acidified" antibody can be measured by, among other methods, ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [ CEX-HPLC ]). In some embodiments, an acceptable degree of stability means that no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in acidic form can be detected in the formulation after storage of the formulation at a temperature and for a time. The certain time period of storage prior to measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When evaluating stability, a temperature that allows for storage of the pharmaceutical formulation can be any temperature in the range of about-80 ℃ to about 45 ℃, e.g., storage at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 2-8 ℃, about 5 ℃, about 25 ℃, or about 40 ℃.

An antibody "retains its physical stability" in the pharmaceutical formulation if it shows substantially no signs of, for example, aggregation, precipitation and/or denaturation when visually inspected for color and/or clarity or measured by UV light scattering or by pore size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate, covalently or non-covalently, to form aggregates. Aggregation may proceed to the extent that a visible precipitate is formed.

Stability, e.g., physical stability, of a formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements correlate with the turbidity of the formulation. Turbidity of a formulation is, in part, an inherent property of proteins dissolved in solution and is typically measured by nephelometry and is measured in Nephelometric Turbidity Units (NTU).

The level of turbidity which varies with, for example, the concentration of one or more components in the solution (e.g., protein and/or salt concentration) is also referred to as the "opacification" or "opacified appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using suspensions of known turbidity. Reference standards for determining the turbidity level of a pharmaceutical composition may be based on the "European Pharmacopoeia" standards (European Pharmacopoeia), fourth edition, "European commission for Quality of Medicine instructions" (EDQM), Strasbourg, France). According to the european pharmacopoeia standard, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension according to the european pharmacopoeia standard having a turbidity of about 3. Nephelometric turbidity measurements can detect rayleigh scattering in the absence of association or non-ideal effects, which typically varies linearly with concentration. Other methods for assessing physical stability are well known in the art.

An antibody "retains its chemical stability" in a pharmaceutical formulation if its chemical stability at a given point in time is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be assessed, for example, by detecting or quantifying chemically altered forms of the antibody. Chemical changes may include size changes (e.g., clipping), which may be assessed using, for example, pore size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

An antibody in a pharmaceutical formulation "retains its biological activity" if it is biologically active for its intended purpose. For example, a formulation of the invention may be considered stable if, after storage of the formulation for a certain period of time (e.g., 1 to 12 months) at a temperature, e.g., 5 ℃, 25 ℃, 45 ℃, etc., the formulation comprises an anti-PD-L1 antibody that binds PD-L1 with an affinity that is at least 90%, 95% or more of the binding affinity of the antibody prior to said storage. Binding affinity can also be determined using, for example, ELISA or plasmon resonance techniques.

In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody, in a pharmacological sense, refers to an amount effective in the prevention or treatment or alleviation of the symptoms of the disorder that the antibody is effective to treat.

The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects/patients include human and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a particular embodiment of the invention, the subject is a human.

anti-PD-L1 antibody

The term "antibody" as used herein is to be understood as including whole antibody molecules and antigen-binding fragments thereof. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to human PD-L1 or an epitope thereof.

The term "full length antibody", as used herein, refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70kDa in total length) and two light (L) chains (about 25kDa in total length) linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) with high variability and regions that are spaced apart to be more conserved, called Framework Regions (FRs). Each VH or VL region is formed by, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable sequence. There are 3 CDRs in each variable region of the heavy and light chains, designated HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each heavy and light chain variable region. The exact boundaries of these CDRs are defined differently for different systems. The system described by Kabat (supra) provides not only a clear residue numbering system applicable to any variable region of an antibody, but also provides an accurate residue boundary defining 3 CDRs. These CDRs may be referred to as Kabat CDRs. Chothia et al found that certain subsections within the Kabat CDRs adopt nearly identical peptide backbone configurations despite large diversity at the amino acid sequence level (Chothia et al, (1987) mol.biol.196: 901-883; Chothia et al, (1989) Nature 342: 877-883). Other boundaries defining CDRs which overlap with the Kabat CDRs have been described by Padlan (1995) FASEB J.9: 133-. Other CDR boundary definitions may not strictly follow one of the systems described herein, but still overlap with the Kabat CDRs, although they may be shortened or lengthened, since specific residues or groups of residues or even the entire CDR are not found to significantly affect antigen binding according to prediction or experimentation. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs.

The anti-PD-L1 antibodies or antigen-binding fragments thereof described herein include any of the anti-PD-L1 antibodies described in international publication No. WO2018153320a1, the entire disclosure of which is incorporated herein by reference. In one embodiment, the antibody used in the methods and compositions of the invention comprises a CDR sequence from JS 003.

As used herein, "antigen-binding fragment" includes fragments or derivatives of an antibody, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antigen binding fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as sc-Fv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments. When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not significantly alter its biological activity.

In some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: the amino acid sequence of HCDR1 is shown as SEQ ID NO. 1, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 2, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 6.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO. 7 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO. 8.

The non-limiting, exemplary antibody used in the examples herein is referred to as "JS 003", which is a humanized complete antibody that specifically binds to human PD-L1 and comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is SEQ ID NO. 9 and the amino acid sequence of the light chain is SEQ ID NO. 10.

In some embodiments, the CDR sequences of the heavy and light chains of clone 30(JS003) and clone 38 are shown in the table below; further preferably, the light and heavy chain sequences of clone or antibody 38 are shown as SEQ ID NOS 46 and 48 in WO2018153320A1, respectively.

LCDR1	SEQ ID NO：1
		LCDR2	SEQ ID NO：2
LCDR3	SEQ ID NO：3
		HCDR1	SEQ ID NO：4
HCDR2	SEQ ID NO：5
		HCDR3	SEQ ID NO：6

Pharmaceutical preparation

The formulation of the present invention is a liquid formulation comprising an antibody at a high concentration and having high stability. In particular, the invention finds that the addition of a single trehalose can obviously improve the stability of the preparation.

In some embodiments, the formulations of the present invention comprise: (1) an antibody or antigen-binding fragment thereof against PD-L1; (2) a buffer at a pH of about 5.0 to about 6.5; and (3) a stabilizer. Typically, the pH of the formulations of the present invention is about 5.0 to 6.5.

The anti-PD-L1 antibody contained in the preparation of the present invention is an anti-PD-L1 antibody according to any one of the embodiments of the present invention, and is preferably a humanized antibody comprising SEQ ID NOS 1-3 as LCDR1, LCDR2 and LCDR3, respectively, and SEQ ID NOS 4-6 as HCDR1, HCDR2 and HCDR3, respectively; more preferably a humanized antibody having the amino acid sequence of VH of SEQ ID NO. 7 and the amino acid sequence of VL of SEQ ID NO; further preferably, the antibody has the amino acid sequence of the heavy chain shown in SEQ ID NO. 9 and the amino acid sequence of the light chain shown in SEQ ID NO. 10. The concentration of the antibody or antigen-binding fragment thereof in the formulation may be in the range of 30mg/mL to 80mg/mL, preferably 40-60 mg/mL.

The buffer contained in the preparation can be selected from one of acetic acid buffer, citric acid buffer and histidine buffer or the combination thereof. The pH of the buffer may be in the range of 5.0-6.5, such as in the range of 5.5-6.5, or in the range of 5.5-6.0. Preferred buffers contain histidine with acetic acid or a histidine salt.

In some embodiments, the buffer in the formulations of the invention comprises histidine and acetic acid; optionally or preferably, the molar ratio of the two is 1:1 to 1.5: 1; optionally or preferably, such buffers have a pH of 5.5 ± 0.3, preferably about 5.5; optionally or preferably, such buffers contain 15-20mM histidine and 12-15mM acetic acid.

In some embodiments, the buffer in the formulations of the invention comprises histidine and a histidine salt (also referred to herein as such buffer as a histidine buffer), with a preferred histidine salt being histidine hydrochloride, such as histidine monohydrochloride. In such buffers, the molar ratio of histidine to histidine salt is from 1:1 to 1: 4. Preferably, such a buffer contains 1-20mM histidine and 1-20mM histidine monohydrochloride.

Typically, the buffer is present in the formulations of the invention at a concentration of about 10 to about 30 mM.

The stabilizer in the formulation of the present invention may be one of sodium chloride, arginine hydrochloride, mannitol, sorbitol, sucrose, trehalose, or a combination thereof, as described above. The concentration of the stabilizer in the formulation may be in the range 50-300mM, such as 50-250 mM. Preferably, the stabilizer in the formulation of the present invention is trehalose and/or sodium chloride. When only sodium chloride is present as a stabilizer, it is preferred that the concentration of sodium chloride in the formulation does not exceed 140mM, preferably in the range of 40-70 mM. When both sodium chloride and trehalose are present as stabilizers, it is preferred that the concentration of sodium chloride in the formulation is in the range of 40-70mM and the concentration of trehalose is in the range of 100-200 mM. When only trehalose is contained as a stabilizer, it is preferable that the concentration of trehalose in the preparation is 200 to 250 mM.

The formulations of the present invention may also include a nonionic surfactant. Examples of the nonionic surfactant include, but are not limited to, one of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 or a combination thereof, preferably polysorbate 20. The concentration of the surfactant in the formulation is from about 0.01% to about 0.05%.

In some embodiments, the formulations of the present invention comprise: (1) about 30mg/mL to about 80mg/mL of an antibody described herein or an antigen-binding fragment thereof directed to PD-L1; (2) about 10-25mM histidine buffer as described herein, at a pH of about 5.0-6.5; (3) about 100mM to about 250mM trehalose; and (4) from about 0% to about 0.1% of a nonionic surfactant as described herein.

In some embodiments, the formulations of the present invention comprise: (1) about 30mg/mL to about 80mg/mL of an anti-PD-L1 antibody or antigen-binding fragment thereof described herein; (2) about 20mM histidine buffer as described herein, at a pH of about 5.5-6.5; (3) about 100mM to about 250mM trehalose; (4) about 20mM to about 200mM sodium chloride; and (5) from about 0% to about 0.1% of a nonionic surfactant as described herein.

In some embodiments, the formulations of the present invention comprise: (1) about 50mg/mL of an anti-PD-L1 antibody or antigen-binding fragment thereof described herein; (2) about 20mM histidine buffer as described herein, at a pH of about 5.5-6.5; (3) about 220mM trehalose; and (4) about 0.02% polysorbate 20.

In some embodiments, the formulations of the present invention comprise: (1) about 60mg/mL of an anti-PD-L1 antibody or antigen-binding fragment thereof described herein; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 240mM trehalose; and (4) about 0.02% polysorbate 20.

In some embodiments, the formulations of the present invention comprise: (1) about 60mg/mL of an anti-PD-L1 antibody or antigen-binding fragment thereof described herein; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 135mM trehalose; (4) about 59mM sodium chloride; and (5) about 0.02% polysorbate 80.

In some embodiments, the formulations of the present invention comprise: (1) about 50mg/mL of an antibody described herein or an antigen-binding fragment thereof directed against PD-L1, wherein the antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 220mM trehalose; and (4) about 0.02% polysorbate 20.

In some embodiments, the formulations of the present invention have a pH of 5.5 to 6.5 and comprise: (1) about 50mg/mL of an anti-PD-L1 antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH has an amino acid sequence as set forth in SEQ ID No. 7; and VL amino acid sequence SEQ ID NO 8; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 220mM trehalose; and (4) about 0.02% polysorbate 20.

In some embodiments, the formulations of the present invention comprise: (1) about 50mg/mL of an antibody or antigen-binding fragment thereof to PD-L1, wherein the antibody is a full-length antibody in which the amino acid sequence of the heavy chain is SEQ ID No. 9 and the amino acid sequence of the light chain is SEQ ID No. 10; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 220mM arginine; and (4) about 0.02% polysorbate 20.

Therapeutic use of pharmaceutical preparations

The pharmaceutical preparation of the present invention can be used for preventing or treating a disease or condition mediated by PD-L1, preferably cancer; more preferably a cancer expressing PD-L1. Exemplary PD-L1-mediated cancers include breast, lung, stomach, intestinal, kidney and melanoma, preferably non-small cell lung, melanoma and kidney.

The pharmaceutical formulation of the present invention may be used for the preparation of a medicament for the prevention or treatment of a PD-1 mediated disease or condition, preferably cancer; more preferably a cancer expressing PD-L1. Exemplary PD-L1-mediated cancers include breast, lung, stomach, intestinal, kidney and melanoma, preferably non-small cell lung, melanoma and kidney.

The present invention will be illustrated below by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise indicated, conventional in the art.

Example 1: buffer system and pH screening assay

In the liquid type pharmaceutical formulation, the buffer system and pH closely affect the stability of the antibody, and each antibody having unique physicochemical properties has the optimum kind and pH of the buffer. This example is directed to screening an optimal buffer system and pH for optimal stability of the anti-PD-L1 antibodies disclosed herein for clinical use.

This example was performed with JS003 at a concentration of about 40mg/mL and about 60 mg/mL. And (4) carrying out dialysis liquid change by using a dialysis bag so that the JS003 protein is in the corresponding prescription, and placing the sample in a sealed centrifugal tube for buffer liquid screening. We screened sodium acetate buffer, citric acid buffer and histidine buffer at pH from 5.0 to 6.5 (as shown in table 1). The samples were placed at 40 ℃ and removed for assay at weeks 0, 2 and 4, respectively. The major pathways for protein degradation are the formation of aggregates, cleavage products and charged variants. The percentage of the natural form (protein monomer) to the aggregated form of JS003 was determined by size exclusion chromatography (SEC-HPLC) and the percentage of the acidic and basic forms of mAb by cation exchange chromatography (CEX-HPLC). The effect of different buffer systems and pH on the stability of JS003 antibody was examined by fitting a straight line and calculating the slope of decline (%/week) with the SEC-HPLC monomer content and CEX-HPLC main peak content at the start of the experiment (0W), at two weeks (2W) and at four weeks (4W), and the results are summarized in tables 2 and 3.

Table 1: buffer system and prescription information in pH screening experiments

Note: "-" indicates no addition.

Table 2: buffer system and rate of degradation of monomer content in pH screening experiments

Table 3: degradation rate of charge variant content in buffer system and pH screening experiment

As shown in tables 2 and 3, in CEX-HPLC experimental detection, the antibody can be kept relatively stable within the pH range of 5.5-6.5, and after the antibody is placed at the high temperature of 40 ℃ for 4 weeks, the monomer content reduction rate of a sample is basically below 3%/week; the decrease rate of the main charge of the sample in the detection of SEC-HPLC experiments is below 6 percent/week. When the buffer system was histidine buffer and the pH was 6.0 (recipe Nos. 7 and 8), the average rate of decrease in monomer purity of the samples after 4 weeks at high temperature 40 ℃ was only 0.29%/week, which is about 10% of that of sodium acetate buffer (recipe 2). Based on these results, histidine buffer at pH 5.5-6.0 was selected for further study.

Example 2: stabilizer screening experiments

In order to further explore the influence of different auxiliary materials on the stability of the antibody, a preparation of one or a combination of sodium chloride, arginine hydrochloride, mannitol, sorbitol, sucrose or trehalose is selected for comparative testing. The different adjuvants or their combination are added into 20mM histidine buffer containing JS003 of about 60mg/mL (molar ratio of histidine to histidine salt is 1:3 or 1:1, pH is 5.5 or 6.0), and the specific prescription information is shown in Table 4. The preparations of all the prescriptions are subpackaged and placed at 40 ℃, and are taken out for analysis and detection at the 0 th week and the 2 nd week respectively. And detecting the content change of the JS003 monomer by using a molecular exclusion high performance liquid chromatography (SEC-HPLC) and detecting the content of the JS003 charge main peak by using a weak cation high performance liquid chromatography (CEX-HPLC). The results are shown in Table 5.

According to the stability investigation result, after the preparation prescription samples of different auxiliary materials are placed for 2 weeks at the high temperature of 40 ℃, the antibody has stronger thermal stability.

By combining various data analyses, the recipe 18 containing trehalose as an adjuvant is most stable under the condition of histidine buffer system of pH6.0. Specifically, after being placed at the high temperature of 40 ℃ for 2 weeks, the preparation only contains trehalose accessories: (1) for the stability of the antibody structure, the reduction rate of the monomer purity of the antibody is obviously lowest, as low as 1.2%/week, about 30-40% of the highest sucrose group, and the monomer purity of the antibody reaches 97.60%; (2) for stability of e.g. antibody charge, the rate of decrease of the antibody's main charge is low, 10.75%/week, with a main charge of up to 78.5%.

Table 4: prescription information in adjuvant screening experiments

Note: "-" indicates no addition.

Table 5: summary of findings from the adjuvant screening experiment

Example 3: surfactant and adjuvant screening experiments

The addition of surfactants to liquid formulations is commonly used as an agent to protect proteins such as antibodies from air/solution interface-induced stress, solution/surface-induced stress during storage to reduce aggregation of the antibodies or minimize the formation of particulate matter in the formulation, which facilitates the stabilization of the physicochemical properties of the antibodies. Different concentrations of excipients, (0-0.5%) polysorbate 20 or polysorbate 80 were added to the formulations containing 20mM histidine buffer (histidine to histidine salt molar ratio 1:1, pH 6.0) and 50mg/ml JS003, respectively, and were assayed after standing at 40 ℃ for 4 weeks. The results are shown in Table 6.

Comprehensive analysis shows that when the auxiliary material is trehalose and the surfactant is polysorbate 20 with the concentration of 0.02%, the stability of the antibody JS003 in the preparation is optimal, the SEC-HPLC monomer content reduction rate is lowest and is as low as 0.43%/week, and the CEX-HPLC main peak content reduction rate is lower and is 7.75%/week.

Table 6: results of surfactant screening

Example 4: ELISA for detection of binding of humanized antibody to human PD-L1

The 96-well enzyme label plate is plated, coated with PD-L1, and incubated at 37 ℃ for 60 minutes. The well solution was then discarded, washed 3 times with wash buffer, and blocked for 60 minutes by adding 2% BSA in PBS. After washing 3 times with the washing buffer, 100. mu.l of each well was added with biotin-labeled IgG4 antibody, incubated at 37 ℃ for 30 minutes, washed 3 times with the washing buffer, then humanized antibodies 30 and 38 (recipe No.: 30) were added at different dilution ratios, respectively, incubated at 37 ℃ for 1 hour, washed three times with the washing buffer, and then mixed with the washing buffer at a ratio of 1: HPR-labeled mouse anti-human IgG (H + L) was diluted 10000 times, incubated at room temperature for 1 hour, washed 3 times with washing buffer, then developed by adding 100. mu.l of TMB substrate solution, reacted at room temperature for 30 minutes, then quenched with 100. mu.l of 2M hydrochloric acid solution and the absorbance read at 450 nm.

As shown in FIG. 1, humanized antibodies bound to EC of PD-L1₅₀Values were 558pg/mL and 837pg/mL, respectively.

Example 5: binding of humanized antibodies to PD-L1 on 293F cells

CHO cells expressing PD-L1 were digested and then centrifuged to resuspend them in FACS buffer at 2.5X 10 cell counts⁴Adding 50ul of diluted solution of 50ul of antibody (prescription number: 30) with different concentrations into a 1.5ml EP tube, mixing, and incubating at room temperature for 30 min; washing the cells twice by using FACS buffer solution, adding 100ul goat anti-human IgG-PE antibody, and incubating for 30min in a dark place; FACS detection was performed after two washes with FACS buffer.

As shown in fig. 2, humanized

antibodies

30 and 38 can specifically bind to PD-L1 on 293F cells.

Example 6: humanized antibodies inhibit the binding of human PD-L1 to PD-1 on 293F cells

The humanized antibody (10ug/ml, prepared in recipe 30) was mixed with biotin-labeled human PD-L1(1ug/ml) and incubated at room temperature for 30 minutes. The mixture was then mixed with 293F PD-1 stable transgenic cell line (1.5X 10)⁵Cells) were incubated at 37 ℃ for 15 min, eluted 3 times with PBS, and SA-APC was added at 5. mu.g/ml and uptake at 4-Incubate at room temperature for 15 minutes. After 3 elution with PBS, flow cytometry was performed to verify whether the humanized antibody could inhibit the binding of human PD-L1 to PD-1 on the surface of 293F cells.

As shown in FIG. 3, the humanized antibody specifically inhibited the binding of human PD-L1 to PD-1 on the surface of 293F cells.

Example 7: jurkat fluorescein analysis of humanized antibodies

CHO cells expressing PD-L1 were plated in 96-well plates at a cell count of 5X 10 per well⁴，37℃，7％CO₂Overnight incubation, cell supernatants were removed, 40ul dilutions of antibodies 30 and 38 (prepared according to prescription 30) were added to each well (initial concentration 60ug/ml, 3-fold concentration gradient dilution), 40ul Jurkat reporter cells were added which expressed both PD-1 and NFAT-luciferase reporter genes continuously, and the total cell count was 1 × 10⁵Cell, 37 ℃, 7% CO₂Culturing for 6 hours, adding luciferase reagent, and detecting the luminescence value by an enzyme-labeling instrument.

As shown in FIG. 4, humanized

antibodies

30 and 38 specifically inhibited the binding of human PD-L1 to PD-1, promoting the expression of the reporter gene.

Example 8: inhibition of tumor growth in mice by humanized antibodies

36 female C57BL/6J mice 6-8 weeks old were injected right underarm with 1X 10⁶(50. mu.L) MC38-B7H1 colon carcinoma cells, tumor volume was measured in mice after tumor formation could be palpable on days 5-7. Divided into 3 groups of 5 pieces. Group 1 injection of 100 μ L KLH per peritoneal cavity; group 2 was administered by intraperitoneal injection of 150. mu.g/100. mu.L of antibody 30 (prepared in prescription No. 30); group 3 was administered with 150. mu.g/100. mu.L of antibody 38 (prepared in prescription No. 30) intraperitoneally. 2 injections per week for 3 weeks; measurements were taken 3 times per week. According to length x width²The tumor volume was calculated 2.

As shown in FIG. 5, the humanized antibody significantly inhibited the growth of MC38-B7H 1-induced tumors.

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SUZHOU JUNMENG BIOSCIENCES Co.,Ltd.

<120> Stable formulation containing anti-PD-L1 antibody

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Claims

1. An anti-PD-L1 antibody pharmaceutical formulation comprising:

(a) a buffer solution;

(b) a stabilizer;

(c) an antibody or antigen-binding fragment thereof that specifically binds human PD-L1, wherein the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein:

the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 1;

the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2;

the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3;

LCDR1 has an amino acid sequence shown in SEQ ID NO. 4;

LCDR2 has an amino acid sequence shown in SEQ ID NO. 5; and

LCDR3 has an amino acid sequence shown in SEQ ID NO. 6; and is

The pH of the pharmaceutical formulation is about 5.0 to 6.5.

2. The pharmaceutical formulation of claim 1, wherein the buffer is one of acetate buffer, citrate buffer, histidine buffer, or a combination thereof.

3. The pharmaceutical formulation of claim 2, wherein the buffer is a histidine buffer.

4. A pharmaceutical formulation according to any one of claims 1 to 3, wherein the buffer is present in a concentration of about 10 to 30 mM.

5. The pharmaceutical formulation of claim 1, wherein the stabilizer is one of sodium chloride, arginine hydrochloride, mannitol, sorbitol, sucrose, trehalose, or a combination thereof.

6. The pharmaceutical formulation of claim 5, wherein the stabilizer is trehalose.

7. Pharmaceutical formulation according to any one of claims 1, 5 or 6, characterized in that the stabilizer concentration is about 30-300mM, preferably 200-250 mM.

8. The pharmaceutical formulation of claim 1, further comprising a surfactant, wherein the surfactant is selected from one of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a combination thereof.

9. The pharmaceutical formulation of claim 1, wherein the surfactant is polysorbate 20.

10. The pharmaceutical formulation of claim 1, 8 or 9, wherein the surfactant concentration is from about 0.01% to about 0.05%.

11. The pharmaceutical formulation of claim 1, wherein the pH is about 6.0.

12. The pharmaceutical formulation of claim 1, comprising:

(a) about 10-30 mM histidine buffer;

(b) about 200mM to 250mM trehalose;

(c) about 0.01% to about 0.05% polysorbate 20; and

(d) about 30mg/mL to about 80mg/mL of the antibody or antigen-binding fragment thereof that specifically binds human PD-L1.

13. The pharmaceutical formulation of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH has the amino acid sequence set forth in SEQ ID No. 7; and VL has the amino acid sequence shown in SEQ ID NO 8.

14. The pharmaceutical formulation of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a Heavy Chain (HC) and a Light Chain (LC), wherein HC has an amino acid sequence as set forth in SEQ ID No. 9 and LC has an amino acid sequence as set forth in SEQ ID No. 10.

15. Use of a pharmaceutical formulation according to any one of claims 1 to 14 in the manufacture of a medicament for the treatment of a disease associated with PD-L1.