WO2021083271A1 - Stable preparation containing anti-pd-l1 antibody - Google Patents

Abstract

The present invention provides a stable pharmaceutical preparation containing an anti-PD-L1 (Programmed death-ligand 1) antibody and a medical application thereof. The preparation contains an anti-PD-L1 antibody and a buffer solution, may further contain at least one stabilizer, and optionally may further contain a surfactant. The pharmaceutical preparation provided by the present invention can effectively inhibit the aggregation of antibodies and prevent the degradation of antibody products, and has high stability.

Description

Stable formulation containing anti-PD-L1 antibody Technical field

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

Background technique

Programmed cell death protein ligand 1 (Programmed death-ligand 1, PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7 homolog1, B7-H1), It belongs to the tumor necrosis factor superfamily. It is a type I transmembrane glycoprotein composed of 290 amino acid residues, including an IgV-like region, an IgC-like region, a transmembrane hydrophobic region, and an intracellular tail of 30 amino acids. The molecular weight is 40kDa. PD-L1 mRNA is expressed in almost all tissues, but PD-L1 protein is only continuously expressed in a small number of tissues, including liver, lungs, tonsils, and immune amnesty tissues such as eyes and placenta. PD-L1 is also expressed on activated T cells, B cells, monocytes, dendritic cells, macrophages, etc.

The receptor of PD-L1 is PD-1, which is mainly expressed on the surface of immune cells such as CD4+ T cells, CD8+ T cells, NKT cells, B cells and activated monocytes. The combination of PD-L1 and PD-1 can initiate the phosphorylation of ITIM (immunoreceptor tyrosine inhibitory action module) tyrosine residues in the cytoplasm of PD-1, which promotes the binding of tyrosine phospholipase to SHP2 and activates SHP2. The downstream Syk and PI3K are dephosphorylated to transmit the termination signal and limit the interaction between antigen-presenting cells or dendritic cells and T cells. This 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, induce T cell exhaustion and apoptosis, thereby reducing immune T cells Participating in the immune response exercises a negative regulatory function.

After T cells recognize the antigen and activate, they secrete IFN-r. IFN-r derived from T cells can expand and maintain T cell functions, such as up-regulating MHC molecules, enhancing the antigen processing and presentation of target cells, and promoting T cell differentiation. IFN-r also induces the expression of PD-L1 in the tissues of immune inflammation, preventing excessive immunity from causing damage to the tissues. IFN-r can induce the expression of PD-L1 on the surface of conventional epithelial cells, vascular endothelial cells, myeloid cells, and naive T cells. The interferon regulatory factor 1 (IRF-1) induced by IFN-r can also bind to the 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 can combine with PD-1 on the surface of T cells to exercise a negative regulatory function, thereby protecting the inflammatory site.

The negative regulatory function of PD-L1 plays an important role in tumor immunity. In 2004, Konishi et al. were the first to find the expression of PD-L1 in tissue samples from patients with non-small cell lung cancer. Subsequently, PD-L1 was found to be expressed in the tissues of various tumor patients, including gastric cancer, lung cancer, liver cancer, and intrahepatic cholangiocarcinoma. , 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, etc. In the process of cell malignant transformation, new protein molecules will be produced due to gene mutation, foreign gene (virus) expression or quiescent gene activation. After these proteins are degraded in the cell, certain degraded peptides can be expressed on the cell surface and become Tumor antigen. The immune system can recognize tumor antigens and eliminate tumor cells through immune surveillance, and tumor cells use PD-L1 to escape immune attack.

The expression of PD-L1 at the tumor site can protect tumor cells from harm through various ways. Tumor infiltrating lymphocytes (TIL) secreting IFN-γ can induce tumor cells and surrounding stromal cells to express PD-L1. The PD-L1 of tumor cells can bind to PD-1 on TIL, inhibit the activation of TIL cells, and further lead to their apoptosis. In vitro experiments have shown that tumor cell-related PD-L1 can increase tumor-specific T cell apoptosis, and PD-L1 monoclonal antibody can attenuate this effect. Tumor-related PD-L1 can promote the expression of IL-10 by T cells and further suppress the immune response. PD-L1 is not only a ligand for PD-1, it can also act as a receptor to transmit reverse signals to protect tumor cells from apoptosis induced by FAS-FASL and other anti-tumor pathways.

Many chronic and acute viruses also use PD-L1 signals to evade human immune detection. Wang et al. found that the expression of PD-L1 on bone marrow-derived dendritic cells of HIV-infected patients was significantly up-regulated. After anti-viral infection and inhibition of HIV replication, the expression of PD-L1 was down-regulated, accompanied by an up-regulation of the number of T cells; Chen et al. research It was found that the expression of PD-L1 on T lymphocytes and dendritic cells of patients with chronic HBV infection was also up-regulated. Virus infection can induce high expression of PD-L1 in infected cells, and at the same time induce CD8+ T cells to express PD-1, thereby inhibiting the effect of T cells and leading to depletion of effector T cells.

Therefore, there is a need for high-stability protein formulations in the art.

Summary of the invention

The present invention provides pharmaceutical preparations containing human antibodies that specifically bind to programmed cell death protein ligand 1 (PD-L1).

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

As a preferred mode, the pharmaceutical preparation may also contain a non-ionic surfactant.

In some embodiments, the buffer is one of acetate buffer, citrate buffer, histidine buffer, or a combination thereof.

In some embodiments, the buffer is a histidine buffer; preferably, the histidine buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer , Preferably histidine-hydrochloride buffer.

In some embodiments, the histidine buffer is made of L-histidine and L-histidine monohydrochloride.

In some embodiments, the concentration of the buffer is about 1 mM to 50 mM, preferably 5 mM to 40 mM, more preferably 10 mM to 30 mM.

In some embodiments, the buffer is a histidine buffer, wherein the concentration of the histidine buffer is about 10 mM to 30 mM, preferably 20 mM to 30 mM. In some embodiments, the concentration of the histidine buffer is about 10 mM. In some embodiments, the concentration of the histidine buffer is about 20 mM. In some embodiments, the concentration of the histidine buffer is about 30 mM.

In some schemes, the above-mentioned histidine buffer is a histidine-acetate buffer, preferably, the molar ratio of the two is 1:1 to 1.5:1, and preferably, the pH of such a buffer is 5.5 ±0.3, preferably about 5.5. Preferably, this type of buffer contains 15-20 mM histidine and 12-15 mM acetic acid.

In some solutions, the above-mentioned buffer is an acetate buffer. Preferably, the acetate buffer is an acetic acid-sodium acetate buffer or an acetic acid-potassium acetate buffer, preferably an acetic acid-sodium acetate buffer.

In some solutions, the above-mentioned buffer is a citric acid buffer, and preferably, the citric acid buffer is a citric acid-sodium citrate buffer.

In some solutions, the above-mentioned buffer is a succinic acid buffer, and preferably, the succinic acid buffer is a succinic acid-sodium succinate buffer.

In some embodiments, the pH of the aforementioned buffer is about 5.0-6.5, preferably about 5.0-6.0, preferably about 5.5-6.5, preferably about 5.0-5.5, preferably about 5.5-6.0, preferably about 6.0-6.5 , A non-limiting example of the pH of the above buffer is about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably about 5.0, 5.5 or 6.0.

In some embodiments, the stabilizer includes one or a combination selected from sodium chloride, arginine hydrochloride, mannitol, sorbitol, sucrose, and trehalose. In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is a combination of trehalose and sodium chloride.

In an embodiment of the present invention, the concentration of the stabilizer is about 50 mM to 300 mM, preferably 100 mM to 300 mM, more preferably 200 mM to 250 mM.

In some embodiments, the stabilizer is sodium chloride at a concentration of about 30-200 mM; or the stabilizer is mannitol at a concentration of about 100-300 mM; or the stabilizer is sorbitol at a concentration of about 100-300 mM Or said stabilizer is sucrose with a concentration of about 100-300mM; or said stabilizer is trehalose with a concentration of about 100-300mM; or said stabilizer is arginine hydrochloride with a concentration of about 30-200mM; or said The stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol; or the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose; or the stabilizer The agent is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose; or the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM trehalose; or the stabilizer It is a combination of about 30-200 mM arginine hydrochloride and about 30-200 mM sucrose.

In some embodiments, the stabilizer is about 100-300 mM trehalose.

In some aspects, the aforementioned stabilizer is sodium chloride. In some embodiments, the aforementioned stabilizer is sodium chloride at a concentration of about 30-200 mM, and the concentration of the aforementioned sodium chloride is preferably about 50-190 mM, preferably about 100-180 mM, preferably about 120-170 mM, preferably about 130. -150mM, non-limiting examples of the above sodium chloride concentration are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140mM .

In some aspects, the aforementioned stabilizer is mannitol. In some embodiments, the aforementioned stabilizer is mannitol at a concentration of about 100-300 mM, the concentration of the aforementioned mannitol is preferably about 150-300 mM, preferably about 200-280 mM, and a non-limiting example of the aforementioned mannitol concentration is about 200 mM. , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240mM.

In some aspects, the aforementioned stabilizer is sorbitol. In some embodiments, the aforementioned stabilizer is sorbitol at a concentration of about 100-300 mM, the concentration of the aforementioned sorbitol is preferably about 150-300 mM, preferably about 200-280 mM, and a non-limiting example of the aforementioned sorbitol concentration is about 200 mM. , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240mM.

In some aspects, the aforementioned stabilizer is sucrose. In some embodiments, the aforementioned stabilizer is sucrose at a concentration of about 100-300 mM. The concentration of the aforementioned sucrose is preferably about 150-300 mM, preferably about 200-280 mM. A non-limiting example of the aforementioned sucrose concentration is about 200 mM, 210 mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240mM.

In some aspects, the aforementioned stabilizer is trehalose. In some embodiments, the aforementioned stabilizer is trehalose at a concentration of about 100-300 mM, the concentration of the aforementioned trehalose is preferably about 150-300 mM, preferably about 200-250 mM, and a non-limiting example of the aforementioned trehalose concentration is about 180 mM. , 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 200mM, 220mM or 240mM.

In some aspects, the aforementioned stabilizer is arginine hydrochloride. In some embodiments, the aforementioned stabilizer is arginine hydrochloride at a concentration of about 30-200 mM, and the concentration of the aforementioned arginine hydrochloride is preferably about 50-190 mM, preferably about 100-180 mM, preferably about 120-170 mM, preferably about It is 130-150mM, non-limiting examples of the above-mentioned arginine hydrochloride concentration are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140mM.

In some aspects, the aforementioned stabilizer is a combination of sodium chloride and mannitol. In some aspects, the aforementioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol, preferably a combination of about 40-150 mM sodium chloride and about 40-180 mM mannitol, preferably about A combination of 40-120 mM sodium chloride and about 40-150 mM mannitol, a non-limiting example of the above stabilizer is a combination of about 59 mM sodium chloride and about 135 mM mannitol, and about 50 mM sodium chloride and A combination of about 135 mM mannitol, a combination of about 101 mM sodium chloride and about 60 mM mannitol, a combination of about 28 mM sodium chloride and about 190 mM mannitol.

In some aspects, the aforementioned stabilizer is a combination of sodium chloride and sucrose. In some aspects, the aforementioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose, preferably a combination of about 30-150 mM sodium chloride and about 50-190 mM sucrose, preferably about 40-200 mM sodium chloride and about 50-190 mM sucrose. A combination of 100 mM sodium chloride and about 60-150 mM sucrose. A non-limiting example of the above stabilizer is a combination of about 59 mM sodium chloride and about 135 mM sucrose, and about 28 mM sodium chloride and about 190 mM sucrose. The combination of about 101mM sodium chloride and about 60mM sucrose.

In some aspects, the aforementioned stabilizer is a combination of sodium chloride and trehalose. In some aspects, the aforementioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM trehalose, preferably about 30-150 mM sodium chloride and about 50-190 mM trehalose, preferably about A combination of 40-100 mM sodium chloride and about 60-150 mM trehalose, a non-limiting example of the above stabilizer is about a combination of 59 mM sodium chloride and about 135 mM sucrose.

In some aspects, the aforementioned stabilizer is a combination of arginine hydrochloride and sucrose. In some embodiments, the aforementioned stabilizer is a combination of about 30-200 mM arginine hydrochloride and about 30-200 mM sucrose, preferably a combination of about 40-150 mM arginine hydrochloride and about 40-150 mM sucrose, preferably about 40-150 mM sucrose. A combination of 40-100 mM arginine hydrochloride and about 80-150 mM sucrose. A non-limiting example of the above stabilizer is a combination of about 59 mM arginine hydrochloride and about 135 mM sucrose.

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

In some embodiments, the concentration of the antibody or antigen-binding fragment thereof is about 10-200 mg/mL, preferably about 20-100 mg/mL, more preferably about 30-80 mg/mL; more preferably, the above-mentioned anti-PD- The concentration of the L1 antibody or its antigen-binding fragment is about 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL or 80 mg/mL, preferably about 40 mg/mL, 50 mg/mL or 60 mg/mL.

In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: the amino acid sequence of HCDR1 is as shown in SEQ ID NO:1; the amino acid sequence of HCDR2 is As shown in SEQ ID NO: 2; HCDR3 has an amino acid sequence as SEQ ID NO: 3; LCDR1 has an amino acid sequence as SEQ ID NO: 4; LCDR2 has an amino acid sequence as SEQ ID NO: 5 ; And the amino acid sequence of LCDR3 is 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 the amino acid sequence of the VH is as shown in SEQ ID NO: 7. Show; and the amino acid sequence of VL is 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 amino acid sequence of HC is shown in SEQ ID NO: 9; and The amino acid sequence is shown in SEQ ID NO: 10. In some embodiments, the concentration of the antibody or antigen-binding fragment thereof is about 30 mg/mL to about 80 mg/mL. In some embodiments, the concentration of the antibody or antigen-binding fragment thereof is about 40 mg/mL to about 60 mg/mL. In a specific embodiment, the concentration of the antibody or antigen-binding fragment thereof is about 40 mg/mL. In a specific embodiment, the concentration of the antibody or antigen-binding fragment thereof is about 50 mg/mL. In a specific embodiment, the concentration of the antibody or antigen-binding fragment thereof is about 60 mg/mL.

In some embodiments, the pharmaceutical preparation provided by the present invention further comprises a surfactant, which is selected from one of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. Or a combination. In some embodiments, the pharmaceutical formulation provided by the present invention contains polysorbate 20.

In some solutions, calculated by w/v, the concentration of the above-mentioned surfactant is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.01%-0.05%; as a non-limiting example, the above-mentioned surfactant concentration is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.01%-0.05%; The concentration of the surfactant is about 0.02%, 0.04% or 0.08%, preferably 0.02%.

In a specific embodiment, the pharmaceutical preparation provided by the present invention contains polysorbate 20 at a concentration of about 0.01% to about 0.05%. In a specific embodiment, the pharmaceutical preparation provided by the present invention contains polysorbate 20 at a concentration of about 0.02%.

In some embodiments, the pharmaceutical preparations provided by the present invention comprise: (1) about 10-30 mM histidine buffer; (2) about 200 mM to about 250 mM trehalose stabilizer; (3) about 30 mg/mL to about About 80 mg/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, where: the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2; the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 4; the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5; and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6.

In some embodiments, the pharmaceutical preparation contains the following components shown in any one of (1) to (5):

(1) (a) about 30mg/mL to about 80mg/mL anti-PD-L1 antibody or its antigen-binding fragment; (b) about 10-25mM histidine buffer, pH about 5.0-6.5; (c) about 100 mM to about 250 mM trehalose; and (d) about 0% to about 0.1% nonionic surfactant;

(2) (a) About 30 mg/mL to about 80 mg/mL anti-PD-L1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.5-6.5; (c) about 100 mM To about 250 mM trehalose; (d) about 20 mM to about 200 mM sodium chloride; and (e) about 0% to about 0.1% nonionic surfactant;

(3) (a) about 50 mg/mL anti-PD-L1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.5-6.5; (c) about 220 mM trehalose; and ( d) about 0.02% polysorbate 20;

(4) (a) about 60 mg/mL anti-PD-L1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.5-6.5; (c) about 240 mM trehalose; and ( d) about 0.02% polysorbate 20;

(5) (a) about 60mg/mL anti-PD-L1 antibody or its antigen-binding fragment; (b) about 20mM histidine buffer, pH about 5.5-6.5; (c) about 135mM trehalose; (d) ) About 59 mM sodium chloride; and (e) about 0.02% polysorbate 20.

In some embodiments, the pharmaceutical preparation comprises:

(1) (a) about 50 mg/mL anti-PD-L1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 6.0; (c) about 220 mM trehalose; and (d) About 0.02% polysorbate 20;

(2) (a) about 60 mg/mL anti-PD-L1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 6.0; (c) about 240 mM trehalose; and (d) About 0.02% polysorbate 20; or

(3) (a) about 60 mg/mL anti-PD-L1 antibody or its antigen-binding fragment; (b) about 20 mM histidine buffer, pH about 6.0; (c) about 135 mM trehalose; (d) about 59 mM sodium chloride; and (e) about 0.02% polysorbate 20;

Wherein, the amino acid sequence of the heavy chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 9 and the amino acid sequence of the light chain is shown in SEQ ID NO: 10.

In some aspects, the pharmaceutical formulation is a liquid formulation or a lyophilized formulation.

In some aspects, the pharmaceutical formulation is a liquid formulation.

In some scenarios, the above-mentioned liquid formulation or lyophilized formulation is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

In some scenarios, the above-mentioned liquid formulation or lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.

On the other hand, the present invention provides the application of any of the above-mentioned pharmaceutical preparations in the preparation of drugs for the treatment of PD-L1 related diseases; preferably, the diseases include breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, and melanoma Tumors are preferably non-small cell lung cancer, melanoma and kidney cancer.

Description of the drawings

Figure 1: Binding of humanized antibody to human PD-L1.

Figure 2: Binding of humanized antibody to PD-L1 on 293F cells.

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

Figure 4: Jurkat fluorescein analysis of humanized antibodies.

Figure 5: In vivo experiments to detect the inhibitory effect of humanized antibodies on tumor growth.

Detailed ways

Definition and description

The present invention is characterized by a stable aqueous liquid pharmaceutical formulation containing an anti-PD-L1 antibody or an antigen-binding portion thereof, which has improved properties compared with formulations recognized in the art. The preparation provided by the invention has high concentration and high stability.

It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in this application are only used to describe specific implementations and are not intended to be limiting. Unless the content is clearly stated otherwise, the singular forms "a", "an" and "the" used in this specification and the appended claims include plural references. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

The "about" used in this application when referring to a measurable value (such as amount, duration, etc.) is intended to cover a variation of ±20% or ±10% relative to a specific value, including ±5%, ±1%, and ±0.1 %, because these changes are suitable for carrying out the disclosed method.

Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or identical to those disclosed in this application can be used in the practice of testing the present invention, this application describes preferred materials and methods. When describing and claiming rights to the present invention, the following terms will be used.

"Therapeutically active antibody" or "therapeutic antibody" refers to an antibody that can be used for therapeutic purposes, that is, to treat a disorder in a subject. It should be noted that although therapeutic proteins can be used for therapeutic purposes, the present invention is not limited to such uses, as the proteins can also be used in in vitro studies.

The term "pharmaceutical preparation" or "preparation" is a product that adopts a form that makes the biological activity of the active ingredient effective and does not contain other ingredients that have unacceptable toxicity to the subject to which the formulation is administered. The preparation is sterile.

The term "liquid formulation" refers to a formulation in a liquid state, and is not intended to refer to a lyophilized formulation that is resuspended by weight. The liquid formulation of the present invention is stable during storage, and its stability does not depend on lyophilization (or other state change methods, such as spray drying).

The term "aqueous liquid preparation" refers to a liquid preparation using water as a solvent. In one embodiment, the aqueous liquid formulation is a formulation 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 desired characteristics (e.g., consistency, increased 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.

As used herein, the term "buffer with a pH of about 5.0 to about 6.5" refers to a reagent whose acid/base conjugated component acts to make a solution containing the reagent 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 6.5, or a pH in the range of about 5.5 to about 6.0. In some embodiments, the pH is about 6.0.

Herein, examples of the "buffer" for controlling the pH within this range include acetate (e.g. sodium acetate), succinate (e.g. sodium succinate), gluconic acid, histidine, methionine, lemon Acid salts, phosphates, citrate/phosphate, imidazole, acetic acid, acetate, 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 an embodiment, the concentration of the buffer is about 5-100 mM, such as 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM or any two values within these ranges are used as the range formed by the endpoints. In some embodiments, the buffer concentration is about 20 mM. In some embodiments, the buffer concentration is about 30 mM.

Here, "histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. The present invention preferably uses a histidine buffer with a pH of about 5.5-6.0. In some embodiments, the histidine buffer is a histidine buffer made of 1-20 mM L-histidine and 1-20 mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4. In some embodiments, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1. In some embodiments, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:3. In some embodiments, the histidine preparation is: a histidine buffer with a pH of 5.5 made of 4.5 mM L-histidine and 15.5 mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: a histidine buffer with a pH of 6.0 made of 15 mM histidine and 15 mM histidine hydrochloride. In some embodiments, the histidine preparation is: a histidine buffer with a pH of 6.0 made of 10 mM histidine and 10 mM histidine hydrochloride.

As used herein, the term "surfactant" generally includes protection of proteins such as antibodies from stresses induced by the air/solution interface, solution/surface-induced stresses to reduce antibody aggregation or minimize the formation of particulates in the formulation Chemical reagents. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, poly Ethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene- Polyoxypropylene copolymer (Pluronic), 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), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, egg white Protein and RNAaseA. Stabilizers also include amino acids, sugars, polyhydric alcohols and their metabolites, such as: sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, arginine, glycine, propyl Acid (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine , Γ-aminobutyric acid (GABA), opines (alanine, octopine, glycinine (strombine)) and trimethylamine N-oxide (TMAO). In the formulation of the present invention, the concentration of the stabilizer may be in the range of 20-300 mM, such as 50-300 mM or 50-250 mM. In some embodiments, the stabilizer is a sugar. In some embodiments, the stabilizer 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 concentration of trehalose is about 200 mM, 220 mM, 240 mM, or 250 mM. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc. can also play a role in controlling osmotic pressure. In some embodiments, the formulation of the present invention contains such stabilizers, preferably a combination of such stabilizers and trehalose, such as a combination of sodium chloride and trehalose. In some embodiments, the stabilizer of the present invention is sodium chloride and trehalose, wherein the concentration of sodium chloride in the formulation is 30-80 mM, preferably 40-70 mM, and the concentration of trehalose is 100-200 mM, preferably 100- 150mM. It should be understood that when trehalose is used in combination with such a stabilizer having the function of controlling osmotic pressure, the concentration of trehalose may be in the range of 100-200mM, preferably 100-150mM, and the concentration of the stabilizer having the function of controlling osmotic pressure may be In the range of 30-80 mM, preferably 40-70 mM. In some embodiments, the formulations of the present invention do not contain such stabilizers that can control osmotic pressure.

"Isotonic" means that the preparation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. The isotonicity can be measured using a vapor pressure or freezing point drop osmometer.

A "stable" formulation is one in which the antibody substantially maintains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or during storage. Even if the contained antibody fails to maintain 100% of its chemical structure or biological function after a certain period of storage, the pharmaceutical preparation can be stable. In some cases, after a certain period of storage, it can maintain about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure or function of the antibody, which can also be considered as " stable". Various analytical techniques for measuring protein stability are available in this technical field, and are reviewed in "Peptide and Protein Drug Delivery" 247-301, Vincent Lee, Editor-in-Chief, Marcel Dekker, Inc. ., New York, NY, Pubs. (1991)), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both are incorporated by reference).

After the preparation has been stored at a certain temperature for a certain period of time, its stability can be measured by determining the percentage of natural antibodies remaining in it (and other methods). Among other methods, the percentage of natural antibodies can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SEC-HPLC]), where "natural" refers to unaggregated and undegraded. In some embodiments, the stability of the protein is determined by the percentage of monomeric protein in a solution that has a low percentage of degradation (eg, fragmentation) and/or aggregated protein. In one embodiment, the formulation can be stored stably at room temperature, about 25-30°C or 40°C 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, no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregate form at most.

By determining the percentage (and other methods) of the antibody ("acid form") that migrates in a fraction that is more acidic than the antibody main fraction ("mainly charged form") during ion exchange, stability can be measured, where stability is related to The percentage of acidic form of antibody is inversely proportional. Among other methods, the percentage of "acidified" antibodies can be measured by ion exchange chromatography (eg, cation exchange high performance liquid chromatography [CEX-HPLC]). In some embodiments, an acceptable degree of stability means that when the formulation is stored at a certain temperature for a certain period of time, the acidic form of the antibody that can be detected therein does not exceed about 49%, 45%, 40%, 35. %, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%. The certain period of storage before 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 certain temperature that allows the storage of the pharmaceutical preparation can be any temperature in the range of about -80°C to about 45°C, for example, storage at about -80°C, about -30°C, about -20°C, or about 0°C , About 2-8°C, about 5°C, about 25°C, or about 40°C.

If the antibody does not substantially show signs of aggregation, precipitation, and/or denaturation during visual inspection of color and/or clarity or by UV light scattering or measurement by pore exclusion chromatography, the antibody is in the pharmaceutical preparation "Maintain its physical stability". Aggregation is the process by which single molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the point where visible precipitates are formed.

The stability of the formulation, such as physical stability, can be evaluated by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurement is related to the turbidity of the formulation. The turbidity of a formulation is partly an inherent property of the protein dissolved in the solution, and is usually measured by turbidimetry and measured in turbidity units (NTU).

The level of turbidity that varies with, for example, the concentration of one or more components in the solution (eg, protein and/or salt concentration) is also referred to as the "opacity" or "opaque appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using a suspension of known turbidity. The reference standard used to determine the turbidity level of the pharmaceutical composition can be based on the "European Pharmacopoeia" standard ("European Pharmacopoeia", fourth edition, "Directorate for the Quality of Medicine" of the Council of Europe (EDQM), Strasbourg, France). According to the "European Pharmacopoeia" standard, a clear solution is defined as a solution with a turbidity lower than or equal to the turbidity of a reference suspension of about 3 according to the "European Pharmacopoeia" standard. Turbidity measurement by turbidimetry can detect Rayleigh scattering in the absence of association or non-ideal effects, which usually varies linearly with concentration. Other methods for assessing physical stability are well known in the art.

If the chemical stability of the antibody at a given point in time is such that the antibody is considered to retain its biological activity as defined below, the antibody "retains its chemical stability" in the pharmaceutical formulation. The chemical stability can be assessed by, for example, detecting or quantifying the form of chemical changes in the antibody. Chemical changes can include size changes (e.g., cropping), which can be assessed using, for example, 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 (for example, occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

If the antibody in the pharmaceutical formulation is biologically active for its intended purpose, the antibody "retains its biological activity" in the pharmaceutical formulation. For example, if the preparation is stored at a temperature such as 5°C, 25°C, 45°C for a certain period of time (for example, 1 to 12 months), the binding affinity of the anti-PD-L1 antibody contained in the preparation to PD-L1 is the storage If the binding affinity of the previous antibody is at least 90%, 95% or more, it can be considered that the formulation of the present invention is stable. The binding affinity can also be determined by techniques such as ELISA or plasmon resonance.

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

The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects/patients include humans 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 specific embodiment of the invention, the subject is a human.

Anti-PD-L1 antibody

The term "antibody" as used herein should be understood to include intact antibody molecules and antigen-binding fragments thereof. The term “antigen-binding portion” or “antigen-binding fragment” (or simply “antibody portion” or “antibody fragment”) of an antibody as used herein refers to the antibody that retains the ability to specifically bind to human PD-L1 or its epitope One or more fragments of.

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 full length) and two light (L) chains (about 50-70kDa in full length). 25kDa) are connected to each other through disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region (abbreviated as CH herein). The heavy chain constant region is composed of three structural domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region consists of a domain CL. The VH and VL regions can be further subdivided into complementarity determining regions (CDR) with high variability and more conservatively spaced regions called framework regions (FR). Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors (including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system).

As used herein, the term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. There are 3 CDRs in each variable region of the heavy chain and light chain, which are named HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 for each variable region of the heavy chain and light chain. The exact boundaries of these CDRs have different definitions according to different systems.

The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the present invention can be determined using any of many well-known schemes, including Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273,927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College of London), International ImmunoGeneTics database (IMGT) (1999Nucle) Research, 27,209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. The CDR of the antibody of the present invention can be determined by those skilled in the art according to any scheme in the art (for example, different assignment systems or combinations).

The anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention includes any one of the anti-PD-L1 antibodies described in the international publication number WO2018153320A1, and the entire content disclosed herein is incorporated herein by way of introduction. In one embodiment, the antibodies used in the methods and compositions of the invention include CDR sequences from JS003.

As used herein, "antigen-binding fragment" includes fragments or derivatives of antibodies, usually including at least one fragment of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody, which retains at least some of the binding of the parent antibody Specificity. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments And multispecific antibodies. When the antigen-binding activity is expressed on a molar concentration basis, the binding fragment or derivative usually 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 expected that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibody).

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

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH has SEQ ID NO: 7 The amino acid sequence of VL has the amino acid sequence shown in SEQ ID NO: 8.

The non-limiting, exemplary antibody used in the examples herein is called "JS003", which is a humanized fully humanized antibody that specifically binds to human PD-L1, which comprises a heavy chain and a light chain, where the heavy chain The amino acid sequence is SEQ ID NO: 9, and the light chain amino acid sequence 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 following table; further preferably, the light chain variable region and heavy chain variable region sequences of clone or antibody 30 are respectively As shown in SEQ ID NOs: 42 and 44 in WO2018153320A1; the light chain variable region and heavy chain variable region sequences of clone or antibody 38 are shown in SEQ ID NO: 46 and 48 in WO2018153320A1, respectively. The amino acid sequence boundaries of the CDRs in the table below adopt the IMGT scheme.

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

Medicinal preparations

The preparation of the present invention is a liquid preparation containing a high concentration of antibodies and having high stability. In particular, the present invention found that the addition of a single trehalose can significantly improve the stability of the formulation.

In some embodiments, the formulation of the present invention comprises: (1) an anti-PD-L1 antibody or antigen-binding fragment thereof; (2) a buffer with a pH of about 5.0-6.5; and (3) a stabilizer. Generally, the pH of the formulation 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 the anti-PD-L1 antibody according to any embodiment of the present invention, and preferably contains SEQ ID NO: 1-3 as LCDR1, LCDR2, LCDR3 and SEQ ID NO: 4, respectively -6 as a humanized antibody of HCDR1, HCDR2, and HCDR3; more preferably, a humanized antibody with the amino acid sequence of VH as SEQ ID NO: 7, and the amino acid sequence of VL as SEQ ID NO:; further preferably a heavy chain antibody The amino acid sequence is shown in SEQ ID NO: 9 and the amino acid sequence of the light chain is 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 30 mg/mL to 80 mg/mL, preferably 40-60 mg/mL.

The buffer contained in the preparation of the present invention may be selected from one of acetate buffer, citrate buffer, and histidine buffer, or a 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. A preferred buffer contains histidine and acetic acid or histidine salt.

In some embodiments, the buffer in the formulation of the present invention contains histidine and acetic acid; optionally or preferably, the molar ratio of the two is 1:1 to 1.5:1; optionally or preferably, such The pH of the buffer is 5.5±0.3, preferably about 5.5; optionally or preferably, this type of buffer contains 15-20 mM histidine and 12-15 mM acetic acid.

In some embodiments, the buffer in the formulation of the present invention contains histidine and histidine salt (this type of buffer is also referred to as histidine buffer in the present invention), and the preferred histidine salt is histidine salt. Acid salts, such as histidine monohydrochloride. In this type of buffer, the molar ratio of histidine to histidine salt is 1:1 to 1:4. Preferably, such a buffer contains 1-20 mM histidine and 1-20 mM histidine monohydrochloride.

Generally, the concentration of the buffer in the formulation of the present invention is about 10-30 mM.

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

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

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

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

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

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

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

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

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

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

Therapeutic use of medicinal preparations

The pharmaceutical preparations of the present invention can be used to prevent or treat PD-L1 mediated diseases or conditions, and the disease is preferably cancer; more preferably, PD-L1 expressing cancer. Exemplary PD-L1 mediated cancers include breast cancer, lung cancer, gastric cancer, bowel cancer, kidney cancer, and melanoma, preferably non-small cell lung cancer, melanoma, and kidney cancer.

The pharmaceutical preparations of the present invention can be used to prepare medicines for preventing or treating PD-1 mediated diseases or disorders. The disease is preferably cancer; more preferably, it is a cancer that expresses PD-L1. Exemplary PD-L1 mediated cancers include breast cancer, lung cancer, gastric cancer, bowel cancer, kidney cancer, and melanoma, preferably non-small cell lung cancer, melanoma, and kidney cancer.

Hereinafter, the present invention will be explained by means of specific embodiments. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. Unless otherwise specified, the methods and materials used in the examples are conventional methods and materials in the art.

Example 1: Anti-PD-L1 antibody prescription buffer system and pH screening experiment

In liquid pharmaceutical preparations, the buffer system and pH closely affect the stability of the antibody. Each antibody with unique physical and chemical properties has the most suitable buffer type and pH. This example aims to screen an optimal buffer system and pH, so that the anti-PD-L1 antibody disclosed in the present invention has the best stability for clinical application.

This example was performed with JS003 at a concentration of about 40 mg/mL and about 60 mg/mL. Use a dialysis bag for dialysis exchange to make the JS003 protein in the corresponding prescription, and place the sample in a closed centrifuge tube for buffer screening. We screened sodium acetate buffer, citrate buffer and histidine buffer with pH from 5.0 to 6.5 (as shown in Table 1). The samples were placed in an environment of 40°C, and were taken out in the 0th week, the 2nd week and the 4th week for analysis and testing. The main pathways of protein degradation are the formation of aggregates, lysate products and charged variants. Size exclusion chromatography (SEC-HPLC) was used to determine the percentage of natural form (protein monomer) and aggregate form of JS003, and cation exchange chromatography (CEX-HPLC) was used to determine the percentage of acid and basic forms of mAb. SEC-HPLC monomer content and CEX-HPLC main peak content at the beginning of the test (0W), two weeks (2W) and four weeks (4W) were used to fit a straight line and calculate the falling slope (%/week) to investigate different buffer liquids The effect of system and pH on the stability of JS003 antibody, the results are summarized in Table 2 and Table 3.

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

Prescription number pH Buffer system Stabilizer 1 Stabilizer 2 1 5.0 22mM sodium acetate+8mM acetic acid 50mM sodium chloride 135mM mannitol 2 5.0 22mM sodium acetate+8mM acetic acid 140mM sodium chloride - 3 5.5 16.5mM histidine+13.5mM acetic acid 50mM sodium chloride 135mM mannitol 4 5.5 16.5mM histidine+13.5mM acetic acid 140mM sodium chloride - 5 5.5 27mM sodium acetate+3mM acetic acid 50mM sodium chloride 135mM mannitol 6 5.5 27mM sodium acetate+3mM acetic acid 140mM sodium chloride - 7 6.0 15mM histidine+15mM histidine hydrochloride 50mM sodium chloride 135mM mannitol 8 6.0 15mM histidine+15mM histidine hydrochloride 140mM sodium chloride -

9 6.0 3mM citrate+27mM sodium citrate 50mM sodium chloride 135mM mannitol 10 6.0 3mM citrate+27mM sodium citrate 140mM sodium chloride - 11 6.5 1mM citrate+29mM sodium citrate 50mM sodium chloride 135mM mannitol 12 6.5 1mM citrate+29mM sodium citrate 140mM sodium chloride -

Note: "-" means not added.

Table 2: Degradation rate of monomer content in buffer system and pH screening experiment

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

As shown in Table 2 and Table 3, in the CEX-HPLC test, the antibody remained relatively stable within the pH range of 5.5 to 6.5. After being placed at a high temperature of 40°C for 4 weeks, the monomer content of the sample decreased at a rate of 3%/ Less than a week: In the SEC-HPLC test, the main charge of the sample decreases at a rate of less than 6%/week. When the buffer system is histidine buffer and the pH is 6.0 (prescription numbers 7 and 8), the average drop rate of monomer purity of the sample after being placed at high temperature of 40°C for 4 weeks is only 0.29%/week, which is approximately sodium acetate buffer (Prescription 2) 10%. Based on these results, a histidine buffer with a pH of 5.5-6.0 was selected for further research.

Example 2: Anti-PD-L1 antibody stabilizer screening experiment

In order to further explore the influence of different stabilizers on antibody stability, we selected one or a combination of sodium chloride, arginine hydrochloride, mannitol, sorbitol, sucrose, or trehalose for comparative testing. That is, the above-mentioned different stabilizers or their combinations are added to 20mM histidine buffer containing about 60mg/mL JS003 (the molar ratio of histidine to histidine salt is 1:3 or 1:1, and the pH is 5.5 or 6.0. ), the specific prescription information is shown in Table 4. Each prescription preparation was placed in 40°C after being subpackaged, and then taken out in the 0th week and the 2nd week for analysis and testing. The JS003 monomer content change was detected by size exclusion high performance liquid chromatography (SEC-HPLC), and the main charge peak content of JS003 was detected by weak cation high performance liquid chromatography (CEX-HPLC). The results are shown in Table 5.

According to the results of the stability investigation, after the formulation samples of different stabilizers are placed at 40°C for 2 weeks, the antibodies have strong thermal stability.

Based on the analysis of various data, under the condition of the histidine buffer system of pH 6.0, the prescription 18 containing trehalose is the most stable. Specifically, in the formulations 13-18 containing only one stabilizer, the formulation group containing only trehalose after being placed at a high temperature of 40°C for 2 weeks (prescription number 18): (1) For the stability of the antibody structure, the antibody monomer The rate of decrease in purity is obviously the lowest, as low as 1.2%/week, which is about 49% of the sucrose group (prescription 17), and the purity of antibody monomer is 97.60%; (2) For the stability of antibody charge, the main charge of antibody The rate of decline is relatively low, 10.75%/week, and the main charge is 78.5%. Among the formulations 19-27 using two stabilizers, the formulation group containing trehalose and sodium chloride (prescription 22) also had excellent stability, and the rate of decrease in antibody monomer purity was about 1.5%/week, approximately It was 51% of the highest sucrose + arginine hydrochloride group (prescription 23), and the decline rate of the main charge of the antibody was only 10.05%/week.

Table 4: Prescription information in the stabilizer screening experiment

Note: "-" means not added.

Table 5: Summary of the results of the stabilizer screening experiment

Prescription number SEC-HPLC CEX-HPLC

To Monomer decline rate (%/week) Main peak decline rate (%/week) 13 2.50 10.45 14 1.95 10.45 15 2.25 10.20 16 2.15 10.35 17 2.45 10.85 18 1.20 10.75 19 2.60 10.60 20 2.20 10.40 twenty one 2.70 10.8 twenty two 1.50 10.05 twenty three 2.90 11.00 twenty four 2.60 10.35 25 2.75 11.15 26 2.45 10.70 27 2.70 10.80 28 3.35 11.45 29 3.05 10.90

Example 3: Screening experiment of surfactants and excipients

Surfactants added to liquid formulations are often used to protect proteins such as antibodies from the stress induced by the air/solution interface and the stress induced by the solution/surface during storage to reduce the aggregation of antibodies or minimize the formation of particulate matter in the formulation. Reagent, which is beneficial to the stability of the physical and chemical properties of the antibody. In the preparation containing 20mM histidine buffer (the molar ratio of histidine to histidine salt is 1:1, pH is 6.0) and 50mg/ml of JS003, different concentrations of stabilizers, (0-0.5 %) Polysorbate 20 or Polysorbate 80, placed at 40°C for 4 weeks and then analyzed and tested. The results are shown in Table 6.

Comprehensive analysis shows that when the stabilizer is trehalose and the surfactant is polysorbate 20 with a concentration of 0.02%, the stability of the antibody JS003 in the formulation is the best, and the SEC-HPLC monomer content decline rate is the lowest, as low as 0.43%/ Weekly, the decrease rate of the main peak content of CEX-HPLC was lower, which was 7.75%/week.

Table 6: Screening results of surfactants

Example 4: Evaluation of anti-PD-L1 antibody formulation stability

In this example, the stability evaluation of the JS003 antibody prescription (prescription number 30) was carried out for 24 months. The anti-PD-L1 antibody prescription contains 20mM histidine buffer (the molar ratio of histidine to histidine salt is 1:1, and the pH is 6.0), 50mg/ml JS003 antibody, 220mM trehalose and 0.02% poly Sorbate 20.

Evaluation indicators include: 1. Appearance (visual inspection); 2. Visible foreign matter (visual inspection); 3. Protein content (ultraviolet spectrophotometry); 4. Size exclusion chromatography (SEC-HPLC) to measure antibody monomer and polymer Content of body and fragment; 5. Cation exchange chromatography (CEX-HPLC) to measure the content of antibody main peak, acidic peak and basic peak; 6. Non-reducing/reducing capillary gel electrophoresis (NR/R-CE-SDS); 7 . Insoluble particles (photoresistance method); 8. ELISA method to detect the relative binding activity and relative blocking activity of the antibody.

The results are shown in Table 7. The above JS003 antibody preparation prescription is stored at 2-8°C for 0-24 months. The appearance, insoluble particles, protein content, and purity (SEC-HPLC, CEX-HPLC, NR/R-CE -SDS) and biological activity have no significant changes, with very good stability.

Table 7: Results of long-term stability of anti-PD-L1 antibody formulations

Note: / = not tested.

Example 5: ELISA to detect the binding of humanized antibody to human PD-L1

Plate the 96-well microtiter plate, coat with PD-L1, and incubate at 37°C for 60 minutes. Then the solution in the well was discarded, washed 3 times with washing buffer, and blocked with PBS solution containing 2% BSA for 60 minutes. After washing 3 times with washing buffer, add 100μl of biotin-labeled IgG4 antibody to each well, incubate at 37 degrees Celsius for 30 minutes, rinse with washing buffer 3 times, and then add humanized antibodies 30 and 38 of different dilutions (prescription number) : 30), incubate at 37 degrees Celsius for 1 hour, wash with washing buffer three times, dilute HPR-labeled mouse anti-human IgG (H+L) with washing buffer 1:10000, incubate at room temperature for 1 hour, wash buffer After rinsing the solution 3 times, add 100μl TMB substrate solution for color development. After reacting at room temperature for 30 minutes, stop the reaction with 100μl 2M hydrochloric acid solution and read the absorbance at 450nm.

As shown in Figure 1, the EC ₅₀ values of humanized antibody binding to PD-L1 were 558 pg/mL and 837 pg/mL, respectively.

Example 6: Binding of humanized antibody to PD-L1 on 293F cells

The CHO cells expressing PD-L1 were digested and resuspended in FACS buffer by centrifugation ^{, and added to a 1.5ml EP tube with a cell mass of ~2.5×10 4} and a volume of 50ul. Add 50ul of antibodies of different concentrations (prescription number: 30) Mix the diluent and incubate at room temperature for 30 minutes; wash the cells twice with FACS buffer, add 100ul goat anti-human IgG-PE antibody, and incubate for 30 minutes in the dark; wash twice with FACS buffer and perform FACS detection.

As shown in Figure 2, humanized antibodies 30 and 38 can specifically bind to PD-L1 on 293F cells.

Example 7: Humanized antibody inhibits the binding of human PD-L1 to PD-1 on 293F cells

Take humanized antibody (10ug/ml, prepared with prescription number 30) and mix with biotin-labeled human PD-L1 (1ug/ml), and incubate at room temperature for 30 minutes. Then the mixture was incubated with the stable 293F PD-1 cell line (1.5×10 ⁵ cells) at 37 degrees Celsius for 15 minutes, eluted with PBS three times, and 5 μg/ml SA-APC was added and incubated at 4 degrees Celsius for 15 minutes. After eluting with PBS three times, it was tested by flow cytometry to verify whether the humanized antibody can inhibit the binding of human PD-L1 to PD-1 on the surface of 293F cells.

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

Example 8: Jurkat fluorescein analysis of humanized antibodies

CHO cells expressing PD-L1 were plated on a 96-well plate, with a cell volume of 5×10 ⁴ per well, cultured overnight at 37°C and 7% CO ₂ , the cell supernatant was removed, and 40 ul of antibody 30 and 38 ( Prepared with prescription number 30) (initial concentration 60ug/ml, 3 times concentration gradient dilution), add 40ul Jurkat reporter cells that can continuously express PD-1 and NFAT-luciferase reporter gene, the total cell number is Incubate 1×10 ⁵ cells at 37° C., 7% CO _{2 for} 6 hours, add luciferase reagent, and detect the luminescence value with a microplate reader.

As shown in Figure 4, humanized antibodies 30 and 38 can specifically inhibit the binding of human PD-L1 to PD-1 and promote the expression of reporter genes.

Example 9: Inhibition of humanized antibody on tumor growth in mice

Take 36 female C57BL/6J mice aged 6-8 weeks and inject 1×10 ⁶ (50μL) MC38-B7H1 colon cancer cells into the right armpit. After the tumor is palpable on day 5-7, measure the tumor volume of. Divide into 3 groups, 5 in each group. Group 1 was intraperitoneally injected with 100 μL KLH; group 2 was intraperitoneally injected with 150 μg/100 μL antibody 30 (prescription number 30); group 3 was intraperitoneally injected with 150 μg/100 μL antibody 38 (prescription number 30). Inject twice a week for 3 weeks; measure 3 times a week. The tumor volume was calculated according to length×width ^{2 /2.}

As shown in Figure 5, humanized antibodies can significantly inhibit the growth of tumors induced by MC38-B7H1.

Claims (15)

An anti-PD-L1 antibody pharmaceutical preparation, comprising: (a) Buffer; (b) Stabilizer; and (c) An antibody or antigen-binding fragment thereof that specifically binds to 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 HCDR1 is shown in SEQ ID NO:1; The amino acid sequence of HCDR2 is shown in SEQ ID NO: 2; The amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 4; The amino acid sequence of LCDR2 is shown in SEQ ID NO: 5; and The amino acid sequence of LCDR3 is shown in SEQ ID NO: 6; and The pH of the pharmaceutical preparation is about 5.0-6.5, preferably about 5.5-6.5, more preferably about 6.0; Preferably, the pharmaceutical preparation is a liquid preparation or a lyophilized preparation. The pharmaceutical preparation according to claim 1, wherein the buffer is one or a combination of acetate buffer, citrate buffer, and histidine buffer; preferably, the buffer is a combination of Acid buffer. The pharmaceutical preparation according to claim 2, characterized in that the concentration of the buffer is about 10-30 mM; preferably, the pH of the buffer is about 5.0-6.5. The pharmaceutical preparation according to any one of claims 1 to 3, wherein the stabilizer is one of sodium chloride, arginine hydrochloride, mannitol, sorbitol, sucrose, trehalose, or a combination thereof; Preferably, the stabilizer is trehalose or a combination of trehalose and sodium chloride. The pharmaceutical preparation according to claim 4, characterized in that the concentration of the stabilizer is about 30-300 mM; preferably about 200-250 mM. The pharmaceutical preparation according to any one of claims 1 to 3, wherein the stabilizer is sodium chloride with a concentration of about 30-200 mM; or the stabilizer is mannitol with a concentration of about 100-300 mM; or The stabilizer is sorbitol at a concentration of about 100-300 mM; or the stabilizer is sucrose at a concentration of about 100-300 mM; or the stabilizer is trehalose at a concentration of about 100-300 mM; or the stabilizer is at a concentration of about 100-300 mM. About 30-200mM arginine hydrochloride; Or the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol; or the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose; or The stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose; or the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM trehalose; or The stabilizer is a combination of about 30-200 mM arginine hydrochloride and about 30-200 mM sucrose; preferably, the stabilizer is about 100-300 mM trehalose. The pharmaceutical preparation according to any one of claims 1 to 3, which is characterized by further comprising a surfactant, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, and polysorbate 60. One of polysorbate 80 or a combination thereof; preferably, the surfactant is polysorbate 20. The pharmaceutical formulation of claim 7, wherein the concentration of the surfactant is about 0.01% to about 0.05%. The pharmaceutical preparation according to any one of claims 1-8, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH has The amino acid sequence is shown in SEQ ID NO: 7; and the amino acid sequence of VL is shown in SEQ ID NO: 8. The pharmaceutical preparation according to any one of claims 1-8, characterized in that 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 such as SEQ ID NO As shown in :9, the amino acid sequence of LC is as shown in SEQ ID NO: 10. The pharmaceutical preparation according to any one of claims 1-10, wherein the concentration of the antibody or antigen-binding fragment thereof is about 10-200 mg/mL, preferably about 20-100 mg/mL, more preferably about 30-80 mg/mL mL. The pharmaceutical preparation according to claim 1, which is characterized by comprising: (a) About 10-30mM histidine buffer; (b) about 200 mM to 250 mM trehalose; (c) about 0.01% to about 0.05% polysorbate 20; and (d) About 30 mg/mL to about 80 mg/mL of the antibody or antigen-binding fragment thereof that specifically binds to human PD-L1. The pharmaceutical preparation according to claim 1, characterized in that the pharmaceutical preparation comprises any one of the following components (1)-(5): (1) (a) about 30mg/mL to about 80mg/mL anti-PD-L1 antibody or its antigen-binding fragment; (b) about 10-25mM histidine buffer, pH about 5.0-6.5; (c) about 100 mM to about 250 mM trehalose; and (d) about 0% to about 0.1% nonionic surfactant; (2) (a) About 30 mg/mL to about 80 mg/mL anti-PD-L1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.5-6.5; (c) about 100 mM To about 250 mM trehalose; (d) about 20 mM to about 200 mM sodium chloride; and (e) about 0% to about 0.1% nonionic surfactant; (3) (a) about 50 mg/mL anti-PD-L1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.5-6.5; (c) about 220 mM trehalose; and ( d) about 0.02% polysorbate 20; (4) (a) about 60 mg/mL anti-PD-L1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.5-6.5; (c) about 240 mM trehalose; and ( d) about 0.02% polysorbate 20; (5) (a) about 60mg/mL anti-PD-L1 antibody or its antigen-binding fragment; (b) about 20mM histidine buffer, pH about 5.5-6.5; (c) about 135mM trehalose; (d) ) About 59 mM sodium chloride; and (e) about 0.02% polysorbate 20. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation comprises: (1) (a) about 50 mg/mL anti-PD-L1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 6.0; (c) about 220 mM trehalose; and (d) About 0.02% polysorbate 20; (2) (a) about 60 mg/mL anti-PD-L1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 6.0; (c) about 240 mM trehalose; and (d) About 0.02% polysorbate 20; or (3) (a) about 60 mg/mL anti-PD-L1 antibody or its antigen-binding fragment; (b) about 20 mM histidine buffer, pH about 6.0; (c) about 135 mM trehalose; (d) about 59 mM sodium chloride; and (e) about 0.02% polysorbate 20; Wherein, the amino acid sequence of the heavy chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 9 and the amino acid sequence of the light chain is shown in SEQ ID NO: 10. The use of the pharmaceutical preparation according to any one of claims 1-14 in the preparation of a medicine for treating PD-L1 related diseases, preferably, the diseases include breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, and melanoma Tumors are preferably non-small cell lung cancer, melanoma and kidney cancer.

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