Pharmaceutical compositions containing anti-pd-1 / vegf bispecific antibodies and uses thereof

Abstract

The present application provides pharmaceutical compositions containing anti-PD-1 / VEGF bispecific antibodies and uses thereof. The pharmaceutical compositions of the present application comprise anti-PD-1 / VEGF bispecific antibodies, buffers, stabilizers and surfactants. The pharmaceutical compositions of the present application have good stability and can be used for preparing drugs for treating and / or preventing diseases or disorders mediated by PD-1 and / or VEGF.

Description

Technical Field

[0001] This application relates to a pharmaceutical preparation containing an antibody, specifically to a pharmaceutical composition containing an anti-PD-1 / VEGF bispecific antibody and its use. Background Technology

[0002] Antibody formulations must not only be suitable for patient administration but also maintain stability during storage and subsequent use. Inappropriate formulations can lead to degradation and aggregation of therapeutic antibodies in liquid formulations. The stability of antibodies in liquid formulations depends not only on the type of excipients used but also on their concentration and proportion. Furthermore, antibody formulations require appropriate antibody concentrations, preservation of antibody activity, and a good appearance.

[0003] Bispecific antibody drugs have large molecular weights and complex structures, making them prone to degradation, aggregation, or unwanted chemical modifications. This leads to antibody heterogeneity (including size and charge heterogeneity), as well as aggregates and fragments, affecting the quality of antibody formulations, especially under high-temperature or transport conditions. To ensure bispecific antibodies are suitable for production and administration, and to maintain their biological activity and stability during storage and subsequent use, stable bispecific antibody drug formulations need to be developed. Various anti-PD-1 / VEGF bispecific antibodies exist in the current technology, with different amino acid sequences and structures; therefore, for each specific antibody, it remains necessary to develop suitable formulation types and formulations.

[0004] Anti-PD-1 / VEGF bispecific antibodies have drawbacks such as low solubility and poor sample stability. Currently, there is a need to develop an anti-PD-1 / VEGF bispecific antibody formulation that can ensure drug quality during long-term storage, have a safe and reliable formulation process, a simple formulation, and convenient injection. Summary of the Invention

[0005] This application provides a pharmaceutical composition containing an anti-PD-1 / VEGF bispecific antibody and its use. By selecting an appropriate buffer system and pH, and optimizing the stabilizer and surfactant, this application obtains a pharmaceutical composition containing an anti-PD-1 / VEGF bispecific antibody with good stability, which can be used to prepare drugs for treating and / or preventing PD-1 and / or VEGF-mediated diseases or conditions.

[0006] The first aspect of this application provides a pharmaceutical composition comprising an anti-PD-1 / VEGF bispecific antibody, a buffer, a stabilizer, and a surfactant; wherein the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable region comprising amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; the anti-PD-1 / VEGF bispecific antibody comprises a first protein functional region and a second protein functional region; the first protein functional region is an anti-PD-1 antibody, and the second protein functional region is an anti-VEGF single-domain antibody; the anti-VEGF single-domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. NO:9 shows HCDR1, HCDR2 and HCDR3; the anti-VEGF single-domain antibody consists of two antibodies, the N-terminus of each single-domain antibody is connected to the C-terminus of the CH1 region of the heavy chain constant region of the anti-PD-1 antibody, and the C-terminus of each single-domain antibody is connected to the N-terminus of the CH2 region of the heavy chain constant region of the anti-PD-1 antibody.

[0007] The buffer solution is selected from histidine buffer, the stabilizer is selected from sucrose, and the surfactant is selected from polysorbate 80(II).

[0008] In the pharmaceutical composition, the concentration of the anti-PD-1 / VEGF bispecific antibody is 8 mg / mL to 40 mg / mL; the concentration of the buffer is 8 mM to 25 mM; the concentration of the stabilizer is 170 mM to 470 mM or 6% to 16% (w / v); and the concentration of the surfactant is 0.01% to 0.06% (w / v).

[0009] The pH value of the pharmaceutical composition is 4.5 to 8.0.

[0010] The second aspect of this application provides the use of the pharmaceutical composition described in the first aspect of this application in the preparation of a medicament for treating and / or preventing PD-1 and / or VEGF-mediated diseases or conditions.

[0011] Beneficial effects:

[0012] The pharmaceutical composition of this application exhibits good stability, maintaining excellent protein content, appearance, purity, antigen-binding activity, affinity, and cellular activity even under high temperature, accelerated and long-term conditions, and repeated freeze-thaw cycles, with no significant changes in insoluble microparticles. Furthermore, the pharmaceutical composition of this application can be used to prepare drugs for the treatment and / or prevention of PD-1 and / or VEGF-mediated diseases or conditions.

[0013] Of course, implementing any product or method of this application does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these accompanying drawings.

[0015] Figure 1 This is a schematic diagram of one of the structures of an anti-PD-1 / VEGF bispecific antibody;

[0016] Figure 2 To block the VEGF and VEGFR2 signaling pathway with a drug composition (reporter gene method);

[0017] Figure 3 The drug composition blocks the PD-1 and PD-L1 signaling pathways (reporter gene method). Detailed Implementation

[0018] The technical solutions of this application will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0019] definition

[0020] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. It should be understood that this application is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. It should also be understood that the terminology used in this application is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0021] Unless otherwise expressly stated, the singular forms “a,” “an,” and “the” used in this specification and the appended claims include plural references. Thus, for example, reference to “a pharmaceutical composition” includes two or more specific pharmaceutical compositions.

[0022] The terms "pharmaceutical composition" or "formulation" refer to a mixture containing one or more antibodies described in this application along with other components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0023] The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a lyophilized formulation that is suspended by weight. The liquid formulation of this application is stable during storage, and its stability is independent of lyophilization (or other state-changing methods, such as spray drying).

[0024] The term "aqueous liquid formulation" refers to a liquid formulation that uses water as a solvent. In some formulations, aqueous liquid formulations are formulations that do not require lyophilization, spray drying, and / or freezing to maintain stability (e.g., chemical and / or physical stability and / or biological activity).

[0025] The term "lyophilized formulation," also known as lyophilized powder for injection, refers to sterile powder for injection prepared by freeze-drying. The lyophilized formulation dosage form of this application is also referred to as lyophilized powder for injection.

[0026] The term "reconstituted preparation" refers to a solution or suspension prepared by adding a specific solvent, such as water for injection, physiological saline, or glucose solution, to a lyophilized drug preparation before use, to dissolve or dilute it so that it can be used by injection, oral administration, or other routes.

[0027] The term "excipient" refers to an agent that can be added to a formulation to provide desired properties (such as consistency, improved stability) and / or adjust osmotic pressure. Common examples of excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

[0028] The term “about” as used in this application, when referring to measurable values ​​(such as quantity, duration, etc.), is intended to cover variations of ±20% or ±10% relative to a specific value, including ±5%, ±4%, ±3%, ±2%, ±1%, and ±0.1%, because such variations are suitable for carrying out the disclosed methods.

[0029] The term "buffer solution with a pH of about 4.5 to 8.0" refers to a reagent that, through the action of its acid / base conjugate components, enables a solution containing the reagent to resist pH changes. The buffer solution used in the pharmaceutical compositions of this application may have a pH in the range of about 4.5 to about 8.0, or a pH in the range of about 5.0 to about 7.5, or a pH in the range of about 6.0 to about 7.0.

[0030] In this document, examples of “buffers” that control the pH of a pharmaceutical composition within this range include acetates (e.g., sodium acetate), histidine, histidine hydrochloride, citrate, phosphate, citrate / phosphate, acetic acid, acetate, citrate, combinations thereof, and other organic acid buffers.

[0031] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate, and L-histidine hydrochloride; histidine can be L-histidine; specifically, histidine buffers containing histidine and histidine hydrochloride, and L-histidine-L-histidine hydrochloride buffers; the histidine buffers of this application also include histidine buffers containing histidine and acetate (such as sodium or potassium salts).

[0032] "Citrate buffer," also known as citrate ion buffer, is a buffer solution containing citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, and magnesium citrate. The preferred citrate buffer is sodium citrate buffer.

[0033] Acetate buffer, also known as acetate salt buffer, is a buffer solution containing acetate ions. Examples of acetate buffers include acetate-sodium acetate, acetate-potassium acetate, acetate-calcium acetate, and acetate-magnesium acetate. The preferred acetate buffer is acetate-sodium acetate buffer.

[0034] The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacturing, storage, and application. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or its salts (e.g., arginine hydrochloride), glycine, and alanine (α-alanine, β-alanine). Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, and sucrose, may also function to control osmotic pressure. The stabilizers specifically used in this application are selected from one or more of polyols, amino acids, salts, and sugars. Preferred salts are sodium chloride, preferred sugars are sucrose and trehalose, and preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine or its salts (e.g., arginine hydrochloride), glycine, and proline. Preferred stabilizers are mannitol, sucrose, glycine, sucrose + mannitol, and sucrose + mannitol + glycine.

[0035] The term "surfactant" generally refers to agents that protect proteins, such as antibodies, from air / solution interface-induced stress and solution / surface-induced stress to reduce antibody aggregation or minimize particulate formation in formulations. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 80, polysorbate 80(II)), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (pluronic), and sodium dodecyl sulfate (SDS).

[0036] The term "isotonic" means that the formulation has an osmotic pressure that is essentially the same as that of human blood. Isotonic formulations generally have an osmotic pressure of approximately 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmoremeter.

[0037] The term "stable formulation" refers to a formulation in which the antibody substantially retains its physical and / or chemical stability and / or biological activity during the manufacturing process and / or storage. A pharmaceutical formulation may be considered stable even if the contained antibody fails to retain 100% of its chemical structure or biological function after a certain period of storage. In some cases, maintaining approximately 90%, 95%, 96%, 97%, 98%, or 99% of the antibody structure or function after a certain period of storage can also be considered "stable." Various analytical techniques for measuring protein stability are available in this field and are reviewed in *Peptide and Protein Drug Delivery* 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both incorporated herein by reference).

[0038] After a formulation 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 (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 non-aggregated and non-degraded antibodies. In some formulations, protein stability is determined by the percentage of monomeric proteins in a solution with a low percentage of degradation (e.g., fragmentation) and / or aggregated proteins. In some formulations, 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, with a maximum of about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of aggregated antibodies.

[0039] Stability can be measured (and by other methods) by determining the percentage of antibody (“acidic form”) that migrates in the more acidic fraction of the main antibody fraction (“major charged form”) during ion exchange, wherein stability is inversely proportional to the percentage of acidic antibody. Among other methods, the percentage of “acidified” antibody can be measured by ion exchange chromatography (e.g., cation exchange high-performance liquid chromatography [CEX-HPLC]). In some embodiments, acceptable stability means that after storage at a certain temperature for a certain period of time, the detectable acidic antibody form does not exceed approximately 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. The storage period prior to stability measurement may 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 assessing stability, the permissible storage temperature for the pharmaceutical preparation may be any temperature within the range of about -80°C to about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 2-8°C, about 5°C, about 25°C, or about 40°C.

[0040] If an antibody does not show substantially no signs of aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity, or upon measurement by UV light scattering or size exclusion chromatography, then the antibody “maintains its physical stability” in the pharmaceutical composition. Aggregation is the process by which individual molecules or complexes covalently or nonvalently associate to form aggregates. Aggregation can proceed to the extent that visible precipitates are formed.

[0041] The stability of a formulation, such as its physical stability, can be assessed using methods known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurements are related to the turbidity of the formulation. The turbidity of a formulation is partly an inherent property of proteins dissolved in solution and is typically measured by turbidimetry and quantified in turbidimetric units (NTU).

[0042] The turbidity level, which varies with the concentration of one or more components in the solution (e.g., protein and / or salt concentration), is also referred to as the “emulsification” or “emulsive appearance” of the formulation. The turbidity level can be calculated by referring to a standard curve generated using a suspension of known turbidity. Reference standards used to determine the turbidity level of a pharmaceutical composition can be based on the European Pharmacopoeia (European Pharmacopoeia, 4th edition, “Directorate for the Quality of Medicine of the Council of Europe” (EDQM), Strasbourg, France). According to the European Pharmacopoeia, a clear solution is defined as a solution with a turbidity less than or equal to that of a reference suspension having a turbidity of approximately 3 according to the European Pharmacopoeia. Turbidimetric 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.

[0043] An antibody is considered to retain its biological activity as defined below if its chemical stability at a given time point is sufficient to ensure that the antibody retains its chemical stability in the pharmaceutical composition. Chemical stability can be assessed, for example, by detecting or quantifying chemical changes in the antibody. Chemical changes can include size changes (e.g., shortening), 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 (e.g., occurring as a result of deamidation or oxidation), which can be assessed, for example, by ion exchange chromatography.

[0044] If the antibody in the pharmaceutical composition is biologically active for its intended purpose, then the antibody “retains its biological activity” in the pharmaceutical composition. For example, if, after storage at temperatures such as 5°C, 25°C, and 45°C for a certain period of time (e.g., 1 to 12 months), the anti-PD-1 / VEGF bispecific antibody contained in the formulation has an affinity for binding to the PD-1 or VEGF antigen that is at least 90%, 95%, or greater than the antibody binding affinity before the storage, then the formulation of this application can be considered stable. Binding affinity can also be determined using, for example, ELISA or plasmon resonance techniques.

[0045] In the context of this application, in a pharmacological sense, a "therapeutic effective amount" or "effective amount" of an antibody refers to an amount effective in preventing, treating, or alleviating symptoms of an impairment for which the antibody can be effectively treated. In this application, a "therapeutic effective amount" or "therapeutic effective dose" of a drug is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from disease onset or promotes disease regression, said disease regression being demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of injury or disability caused by disease suffering. The ability of a drug to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting human efficacy, or by measuring the activity of the drug in an in vitro assay. A therapeutically effective amount of a drug includes a "preventatively effective amount," which is any amount of the drug that, when given alone or in combination with other therapeutic agents to a subject at risk of disease or a subject experiencing disease relapse, inhibits the development or recurrence of the disease.

[0046] The terms "subject" or "patient" are 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 some embodiments of this application, the subject is a human.

[0047] The terms “administration,” “giving,” and “treatment” refer to the introduction of a composition containing a therapeutic agent into a subject using any of the various methods or delivery systems known to those skilled in the art. Routes of administration for anti-PD-1 / VEGF antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal, or other parenteral routes, such as injection or infusion. “Parenteral administration” refers to administration by injection other than enteral or local administration, including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intra-tracheal, intracapsular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, intra-articular, and intrasternal injections and infusions, as well as intracorporeal electroporation.

[0048] The term "antibody" as used herein should be understood to include both the complete antibody molecule and its antigen-binding fragment. The term "antigen-binding portion" or "antigen-binding fragment" (or simply "antibody portion" or "antibody fragment") of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to human PD-1 or VEGF or their epitopes.

[0049] As used herein, the term "full-length antibody" or "complete antibody molecule" refers to an immunoglobulin molecule containing four peptide chains: two heavy (H) chains (approximately 50-70 kDa in full length) and two light (L) chains (approximately 25 kDa in full length) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region (abbreviated as CH in this document). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this document) 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 highly variable complementarity-determining regions (CDRs) and regions separated by more conserved regions called framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors (including various cells of the immune system, such as effector cells) and the first component (Clq) of the classical complement system.

[0050] The term "single-domain antibody" (sdAb), also known as "nanobody," "single-domain antibody," or "VHH," refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region).

[0051] When used herein, the term "CDR" refers to the complementarity-determining region within the variable sequence of the antibody. Three CDRs exist in each variable region of the heavy and light chains, which are named HCDR1, HCDR2, and HCDR3, or LCDR1, LCDR2, and LCDR3, for each heavy and light chain variable region. The precise boundaries of these CDRs are defined differently according to different systems. The CDRs of the antibody in this application can be determined according to well-known definitions in the art, such as those from Kabat, AbM, Chothia, Contact, and IMGT.

[0052] The term "bispecific antibody" refers to an antibody molecule that can bind to two independent antigens or has binding specificity to different epitopes within the same antigen. For example, the bispecific antibody of this application can simultaneously bind to VEGF and PD-1, competitively blocking the interaction between VEGFA, PD-1 and their ligands, thereby exerting antitumor activity. The structures of bispecific antibodies are diverse.

[0053] In this application, VEGF specifically refers to VEGFA. The anti-VEGF antibody in this application specifically binds to VEGFA and can very effectively block the binding of VEGFA to VEGFR2.

[0054] Bispecific antibodies

[0055] The pharmaceutical composition provided in this application includes an anti-PD-1 / VEGF bispecific antibody, which is also referred to herein as the bispecific antibody, or recombinant humanized anti-VEGF and PD-1 bispecific antibody, or JS207.

[0056] The bispecific antibody is the anti-PD-1-anti-VEGF bispecific antibody disclosed in patent application PCT / CN2024 / 094648, the entire contents of which are incorporated herein by reference.

[0057] The bispecific antibody comprises a first protein functional region and a second protein functional region, wherein: the first protein functional region is an anti-PD-1 antibody or its antigen-binding fragment, the second protein functional region is an anti-VEGF antibody or its antigen-binding fragment, the anti-VEGF antibody or its antigen-binding fragment comprises a heavy chain variable region, the heavy chain variable region comprising amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 respectively, namely HCDR1, HCDR2 and HCDR3; the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, namely HCDR1, HCDR2 and HCDR3, the light chain variable region comprising amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively, namely LCDR1, LCDR2 and LCDR3.

[0058] Specifically, the first protein functional region of the anti-PD-1 / VEGF bispecific antibody includes a heavy chain variable region and a light chain variable region as shown in SEQ ID NO: 10 and 11, respectively; the second protein functional region includes the heavy chain variable region shown in SEQ ID NO: 12.

[0059] More specifically, the anti-PD-1 / VEGF bispecific antibody is antibody 23, antibody 24, antibody 27 or antibody 28 as described in patent application PCT / CN2024 / 094648, preferably antibody 27, and the amino acid sequence of each antibody is shown in Table 1 below.

[0060] Table 1. Amino acid sequences contained in anti-PD-1 / VEGF bispecific antibody molecules.

[0061] Antibody 23 Antibody 24 Antibody 27 Antibody 28 First polypeptide chain SEQ ID NO: 13 SEQ ID NO: 13 SEQ ID NO: 13 SEQ ID NO: 17 Second polypeptide chain SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 18

[0062] The structural schematic diagram of antibody 27 is shown in this application. Figure 1 As shown in the figure, DotAb is a single-domain antibody (VHH) that specifically binds to VEGF (anti-VEGF).

[0063] Furthermore, the anti-PD-1 / VEGF bispecific antibody comprises an immunoglobulin as the first protein functional region and a single-domain antibody or a single-chain antibody as the second protein functional region.

[0064] In some embodiments, the first protein functional region of the anti-PD-1 / VEGF bispecific antibody is an anti-PD-1 antibody, and the second protein functional region is an anti-VEGF single-domain antibody. The single-domain antibody consists of two antibodies, with the N-terminus of each antibody connected to the C-terminus of the heavy chain constant region CH1 of the immunoglobulin, and the C-terminus of each antibody connected to the N-terminus of the heavy chain constant region CH2 of the immunoglobulin.

[0065] In some implementations, the specific anti-PD-1 / VEGF bispecific antibody uses a PD-1 monoclonal antibody (such as toripalimab) as its backbone, with two anti-VEGF single-domain antibody DotAbs inserted between its Fab and Fc domains. A schematic diagram of the specific structure is attached. Figure 1 As shown.

[0066] Furthermore, the anti-PD-1 / VEGF bispecific antibody is a recombinant humanized antibody.

[0067] Pharmaceutical Composition

[0068] A first aspect of this application provides a pharmaceutical composition comprising an anti-PD-1 / VEGF bispecific antibody, a buffer solution, a stabilizer, and a surfactant, wherein...

[0069] The anti-PD-1 / VEGF bispecific antibody comprises a first protein functional region and a second protein functional region; the first protein functional region is an anti-PD-1 antibody, and the second protein functional region is an anti-VEGF single-domain antibody; the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 (HCDR1, HCDR2, and HCDR3 respectively), and the light chain variable region comprising amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 (LCDR1, LCDR2, and LCDR3 respectively); the anti-VEGF single-domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 respectively. NO:9 shows HCDR1, HCDR2 and HCDR3; the anti-VEGF single-domain antibody consists of two antibodies, the N-terminus of each single-domain antibody is connected to the C-terminus of the CH1 region of the heavy chain constant region of the anti-PD-1 antibody, and the C-terminus of each single-domain antibody is connected to the N-terminus of the CH2 region of the heavy chain constant region of the anti-PD-1 antibody.

[0070] The buffer solution is selected from at least one of acetate buffer, citrate buffer, histidine buffer, and phosphate buffer; the stabilizer is selected from at least one of sucrose, mannitol, and glycine; and the surfactant is selected from polysorbate 80(II).

[0071] In the pharmaceutical composition, the concentration of the anti-PD-1 / VEGF bispecific antibody is 5 mg / mL to 90 mg / mL; the concentration of the buffer is 5 mM to 50 mM; the concentration of the stabilizer is 100 mM to 500 mM or 3% to 16% (w / v); and the concentration of the surfactant is 0.01% to 0.06% (w / v).

[0072] The pH value of the pharmaceutical composition is 4.5 to 8.0.

[0073] It should be noted that, for example, the concentration of the stabilizer is 170mM to 470mM or 6% to 16% (w / v), which are just different changes in units, but the actual concentration is the same.

[0074] In the pharmaceutical composition, the concentration of the anti-PD-1 / VEGF bispecific antibody can be 5 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, or a range of any two of these values; the concentration of the buffer solution can be 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or a range thereof. The concentration of the stabilizer can be 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, or any two of these values; or the concentration of the stabilizer can be 3% (w / v), 5% (w / v), 8% (w / v), 10% (w / v), 13% (w / v), 16% (w / v), or any two of these values; the concentration of the surfactant can be 0.01% (w / v), 0.02% (w / v), 0.03% (w / v), 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), or any two of these values.

[0075] In this application, the pH value of the pharmaceutical composition can be 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or a range of any two values ​​therein.

[0076] In some embodiments, the concentration of the anti-PD-1 / VEGF bispecific antibody is 8 mg / mL to 60 mg / mL, preferably 8 mg / mL to 40 mg / mL, more preferably 8 mg / mL to 32 mg / mL, even more preferably 10 mg / mL to 30 mg / mL, and even more preferably 10 mg / mL to 20 mg / mL.

[0077] In some embodiments, the concentration of the buffer solution is 8 mM to 25 mM, preferably 8 mM to 22 mM, more preferably 10 mM to 20 mM, and even more preferably 9 mM to 16 mM.

[0078] In some embodiments, the concentration of the stabilizer is 170 mM to 470 mM, preferably 210 mM to 370 mM, more preferably 230 mM to 370 mM, and even more preferably 245 mM to 368 mM; or, the concentration of the stabilizer is 5% to 16% (w / v), preferably 6% to 16% (w / v), more preferably 8% to 13% (w / v), and even more preferably 8.4% to 12.6% (w / v).

[0079] In some embodiments, the concentration of the surfactant is 0.01% to 0.05% (w / v), preferably 0.01% to 0.04% (w / v).

[0080] In some embodiments, the pH value of the pharmaceutical composition is 5.0 to 7.5, preferably 5.5 to 6.5.

[0081] In some embodiments, the concentration of the buffer solution is about 10 mM, 10.5 mM, 12 mM, 15 mM, 15.73 mM, 16 mM, or 20 mM; in some embodiments, the buffer solution is about 20 mM histidine buffer; in some embodiments, the buffer solution is about 10 mM histidine buffer; in some embodiments, the buffer solution is about 20 mM acetate buffer, or about 20 mM citrate buffer, or about 20 mM phosphate buffer; here, "about" means that the millimolecular concentration value may fluctuate within 1, 2, or 3 above or below a specific numerical point.

[0082] In some embodiments, the pH value of the pharmaceutical composition of this application is about 5.0, about 5.5, about 6.0, about 6.5, about 7.0 or about 7.5; where “about” means that the pH value may fluctuate within 0.2 or 0.1 above or below a specific value.

[0083] In some embodiments, the stabilizer of this application is sucrose. In some embodiments, the stabilizer of this application is sucrose + mannitol. In some embodiments, the stabilizer of this application is sucrose + mannitol + glycine. In some embodiments, the stabilizer of this application is sucrose + glycine.

[0084] In some embodiments, the concentration of the stabilizer is about 175 mM, 210 mM, about 220 mM, about 230 mM, about 234 mM, about 245 mM, about 250 mM, about 280 mM, about 300 mM, about 320 mM, about 350 mM, 368 mM, about 467 mM, about 470 mM, or about 490 mM, where “about” means that the millimolecular concentration value can fluctuate within 5, 10, or 15 millimoles above or below the specific numerical point.

[0085] In some embodiments, the concentration of the stabilizer is about 3%, about 4%, about 5%, about 6%, about 8%, about 8.4%, about 9%, about 11%, about 12%, about 12.6%, about 13%, about 14%, or about 16% (w / v), where “about” means that the w / v concentration value may fluctuate within 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% above or below a specific numerical point.

[0086] The concentration of the stabilizer, when the stabilizer consists of multiple components, refers to the sum of the individual concentrations of the various stabilizers in the stabilizer.

[0087] In some embodiments, the stabilizer is about 230 mM sucrose.

[0088] In some embodiments, the stabilizer is about 8% (w / v) sucrose.

[0089] In some embodiments, the stabilizer is about 234 mM sucrose.

[0090] In some embodiments, the stabilizer is about 8.4% (w / v) of sucrose.

[0091] In some embodiments, the stabilizer is about 245 mM sucrose.

[0092] In some embodiments, the stabilizer is about 12.6 (w / v) of sucrose.

[0093] In some embodiments, the stabilizer is approximately 370 mM sucrose.

[0094] In some embodiments, the stabilizer is approximately 368 mM sucrose.

[0095] In some embodiments, the stabilizer consists of about 2% sucrose (w / v) + about 1.2% mannitol (w / v) + about 1.2% glycine (w / v).

[0096] In some embodiments, the stabilizer consists of about 3% sucrose (w / v) + about 3% mannitol (w / v).

[0097] In some embodiments, the concentration of the surfactant is about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, or about 0.06% (w / v); here, "about" means that the w / v concentration value can fluctuate within 0.001%, 0.002%, or 0.003% above or below the specific numerical point.

[0098] In some embodiments, the concentration of the bispecific antibody is approximately 10 mg / mL, approximately 15 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, or approximately 40 mg / mL, where “approximately” means that the concentration value in mg / mL may fluctuate within 1, 2, or 3 above or below the specific value.

[0099] In some embodiments, the pharmaceutical composition comprises 8 mg / mL to 40 mg / mL of anti-PD-1 / VEGF bispecific antibody, 8 mM to 25 mM of histidine buffer, 170 mM to 470 mM or 6% to 16% (w / v) of sucrose, and 0.01% to 0.06% (w / v) of polysorbate 80(II), and the pH of the pharmaceutical composition is 4.5 to 8.0.

[0100] In some embodiments, the pharmaceutical composition comprises 8 mM to 25 mM histidine buffer, 170 mM to 470 mM sucrose, 0.01% to 0.06% (w / v) polysorbate 80(II), 8 mg / mL to 40 mg / mL anti-PD-1 / VEGF bispecific antibody, and a pH of 4.5 to 8.0.

[0101] In some embodiments, the pharmaceutical composition comprises 8 mg / mL to 32 mg / mL of anti-PD-1 / VEGF bispecific antibody, 8 mM to 22 mM of histidine buffer, 230 mM to 370 mM or 8% to 13% (w / v) of sucrose, and 0.01% to 0.05% (w / v) of polysorbate 80(II), and the pH of the pharmaceutical composition is 5.0 to 7.5.

[0102] In some embodiments, the pharmaceutical composition comprises 8 mM to 22 mM histidine buffer, 230 mM to 370 mM sucrose, 0.01% to 0.05% (w / v) polysorbate 80(II), 8 mg / mL to 32 mg / mL anti-PD-1 / VEGF bispecific antibody, and a pH of 5.0 to 7.5.

[0103] In some embodiments, the pharmaceutical composition comprises 8 mg / mL to 32 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM to 20 mM of histidine buffer, 230 mM to 370 mM or 8% to 13% (w / v) of sucrose, and 0.01% to 0.04% (w / v) of polysorbate 80(II), and the pH of the pharmaceutical composition is 5.5 to 6.5.

[0104] In some embodiments, the pharmaceutical composition comprises 10 mM to 20 mM histidine buffer, 230 mM to 370 mM sucrose, 0.01% to 0.04% (w / v) polysorbate 80(II), 8 mg / mL to 32 mg / mL anti-PD-1 / VEGF bispecific antibody, and a pH of 5.5 to 6.5.

[0105] In some embodiments, the pharmaceutical composition comprises 10 mM to 20 mM histidine buffer, 245 mM to 368 mM sucrose, 0.01% to 0.04% (w / v) polysorbate 80(II), 8 mg / mL to 32 mg / mL anti-PD-1 / VEGF bispecific antibody, and a pH of 5.5 to 6.5.

[0106] In some embodiments, the pharmaceutical composition comprises 10 mM to 20 mM histidine buffer, 8% to 13% (w / v) mM sucrose, 0.01% to 0.04% (w / v) polysorbate 80(II), 8 mg / mL to 32 mg / mL anti-PD-1 / VEGF bispecific antibody, and a pH of 5.5 to 6.5.

[0107] In some embodiments, the pharmaceutical composition comprises 10 mM to 20 mM histidine buffer, 8% to 12.6% (w / v) mM sucrose, 0.01% to 0.04% (w / v) polysorbate 80(II), 8 mg / mL to 32 mg / mL anti-PD-1 / VEGF bispecific antibody, and a pH of 5.5 to 6.5.

[0108] In some embodiments, the pharmaceutical composition comprises 10 mM to 20 mM histidine buffer, 8% to 12.6% (w / v) mM sucrose, 0.01% to 0.04% (w / v) polysorbate 80(II), 8 mg / mL to 30 mg / mL anti-PD-1 / VEGF bispecific antibody, and a pH of 5.5 to 6.5.

[0109] In some embodiments, the pharmaceutical composition comprises 10 mg / mL to 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM to 20 mM of histidine buffer, 230 mM to 370 mM or 8% to 13% (w / v) of sucrose, and 0.01% to 0.04% (w / v) of polysorbate 80(II), and the pH of the pharmaceutical composition is 5.5 to 6.5.

[0110] In some embodiments, the pharmaceutical composition comprises 10 mM to 20 mM histidine buffer, 8% to 13% (w / v) mM sucrose, 0.01% to 0.04% (w / v) polysorbate 80(II), 8 mg / mL to 30 mg / mL anti-PD-1 / VEGF bispecific antibody, and a pH of 5.5 to 6.5.

[0111] In some embodiments, the pharmaceutical composition comprises 10 mM to 20 mM histidine buffer, 8% to 12.6% (w / v) mM sucrose, 0.01% to 0.04% (w / v) polysorbate 80(II), 10 mg / mL to 30 mg / mL anti-PD-1 / VEGF bispecific antibody, and a pH of 5.5 to 6.5.

[0112] In some embodiments, the pharmaceutical composition comprises 10 mM to 20 mM histidine buffer, 8% to 12.6% (w / v) mM sucrose, 0.01% to 0.03% (w / v) polysorbate 80(II), 10 mg / mL to 30 mg / mL anti-PD-1 / VEGF bispecific antibody, and a pH of 5.5 to 6.5.

[0113] In some embodiments, the pharmaceutical composition comprises 10 mg / mL to 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM to 20 mM of histidine buffer, 245 mM to 368 mM or 8.4% to 12.6% (w / v) of sucrose, and 0.01% to 0.04% (w / v) of polysorbate 80(II), and the pH of the pharmaceutical composition is 5.5 to 6.5.

[0114] In some embodiments, the pharmaceutical composition comprises 10 mg / mL to 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM histidine buffer, 234 mM or 8% (w / v) sucrose, and 0.01% to 0.03% (w / v) polysorbate 80(II), and the pharmaceutical composition has a pH of 6.0.

[0115] In some embodiments, the pharmaceutical composition comprises 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM histidine buffer, 234 mM or 8% (w / v) sucrose, and 0.02% (w / v) polysorbate 80(II), and the pharmaceutical composition has a pH of 6.0.

[0116] In some embodiments, the pharmaceutical composition comprises 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM histidine buffer, 245 mM or 8.4% (w / v) sucrose, and 0.02% (w / v) polysorbate 80(II), and the pharmaceutical composition has a pH of 6.0.

[0117] In some embodiments, the pharmaceutical composition comprises 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10.5 mM histidine buffer, 245 mM or 8.4% (w / v) sucrose, and 0.01% to 0.03% (w / v) polysorbate 80(II), and the pharmaceutical composition has a pH of 6.0.

[0118] In some embodiments, the pharmaceutical composition comprises 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10.5 mM histidine buffer, 245 mM or 8.4% (w / v) sucrose, and 0.02% (w / v) polysorbate 80(II), and the pharmaceutical composition has a pH of 6.0.

[0119] In some embodiments, the pharmaceutical composition comprises 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, 16 mM histidine buffer, 380 mM or 13% (w / v) sucrose, and 0.03% (w / v) polysorbate 80(II), and the pharmaceutical composition has a pH of 6.0.

[0120] In some embodiments, the pharmaceutical composition comprises 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, 15.73 mM histidine buffer, 368 mM or 12.6% (w / v) sucrose, and 0.03% (w / v) polysorbate 80(II), and the pharmaceutical composition has a pH of 6.0.

[0121] In some embodiments, the pharmaceutical composition comprises 10 mg / mL of anti-PD-1 / VEGF bispecific antibody, 20 mM acetate buffer, 230 mM sucrose, and 0.04% (w / v) polysorbate 80(II), and the pH of the pharmaceutical composition is 5.0 to 5.5.

[0122] In some embodiments, the pharmaceutical composition comprises about 10 mM histidine buffer, about 8% (w / v) sucrose, about 0.01 to 0.03% (w / v) polysorbate 80(II), about 10 mg / mL to 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0123] In some embodiments, the pharmaceutical composition comprises about 10 mM histidine buffer, about 8% (w / v) sucrose, about 0.02% (w / v) polysorbate 80(II), about 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0124] In some embodiments, the pharmaceutical composition comprises 10.5 mM histidine buffer, about 8% (w / v) sucrose, about 0.02% (w / v) polysorbate 80(II), about 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0125] In some embodiments, the pharmaceutical composition comprises 10.5 mM histidine buffer, about 234 mM sucrose, about 0.02% (w / v) polysorbate 80(II), about 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0126] In some embodiments, the pharmaceutical composition comprises about 10 mM histidine buffer, about 8.4% (w / v) sucrose, about 0.02% (w / v) polysorbate 80(II), about 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0127] In some embodiments, the pharmaceutical composition comprises about 10 mM histidine buffer, about 245 mM sucrose, about 0.02% (w / v) polysorbate 80(II), about 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0128] In some embodiments, the pharmaceutical composition comprises 10.5 mM histidine buffer, about 8.4% (w / v) sucrose, about 0.01 to 0.03% (w / v) polysorbate 80(II), about 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0129] In some embodiments, the pharmaceutical composition comprises 10.5 mM histidine buffer, about 8.4% (w / v) sucrose, about 0.02% (w / v) polysorbate 80(II), about 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0130] In some embodiments, the pharmaceutical composition comprises 10.5 mM histidine buffer, about 245 mM sucrose, about 0.02% (w / v) polysorbate 80(II), about 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0131] In some embodiments, the pharmaceutical composition comprises about 16 mM histidine buffer, about 13% (w / v) sucrose, about 0.03% (w / v) polysorbate 80(II), about 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0132] In some embodiments, the pharmaceutical composition comprises 15.73 mM histidine buffer, about 12.6% (w / v) sucrose, about 0.03% (w / v) polysorbate 80(II), about 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0133] In some embodiments, the pharmaceutical composition comprises 15.73 mM histidine buffer, about 368 mM sucrose, about 0.03% (w / v) polysorbate 80(II), about 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, at a pH of about 6.0.

[0134] In some embodiments, the pharmaceutical composition comprises about 20 mM of acetate buffer, about 230 mM of sucrose, about 0.04% (w / v) of polysorbate 80(II), about 10 mg / mL of anti-PD-1 / VEGF bispecific antibody, and a pH of about 5.0 to 5.5.

[0135] In some embodiments, the histidine buffer is a histidine-histidine hydrochloride buffer; preferably, it is an L-histidine-L-histidine hydrochloride buffer.

[0136] In some embodiments, the pharmaceutical composition is formulated as a pharmaceutically acceptable preparation, preferably a lyophilized preparation, a reconstituted preparation, or a liquid preparation.

[0137] In some embodiments, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.

[0138] Medical Use

[0139] The second aspect of this application provides the use of the pharmaceutical composition described in the first aspect of this application in the preparation of a medicament for treating and / or preventing PD-1 and / or VEGF-mediated diseases or conditions.

[0140] In some implementations, the disease or condition is a malignant tumor.

[0141] In some implementations, the disease or condition is a malignant solid tumor.

[0142] In some implementations, the disease or condition is a gynecological tumor.

[0143] In some implementations, the disease or condition includes at least one of lung cancer, non-small cell lung cancer (NSCLC), squamous non-small cell lung cancer, non-squamous non-small cell lung cancer, colorectal cancer, triple-negative breast cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma (HCC), pancreatic cancer, and clear cell renal cell carcinoma (ccRCC).

[0144] In some implementations, the anti-PD-1 / VEGF bispecific antibody of this application has shown tolerable safety and encouraging remissions in non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), and clear renal cell carcinoma (ccRCC).

[0145] In some implementations, the anti-PD-1 / VEGF bispecific antibody of this application has shown tolerable safety and encouraging remissions, particularly against first-line PD-L1-positive non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), and clear renal cell carcinoma (ccRCC).

[0146] A third aspect of this application provides a method for treating and / or preventing PD-1 and / or VEGF-mediated diseases or conditions, comprising administering to a patient in need a therapeutically effective amount of the pharmaceutical composition described in the first aspect of this application.

[0147] In some embodiments, the PD-1 and / or VEGF-mediated disease or condition is a malignant tumor, preferably a malignant solid tumor; more preferably a gynecological tumor.

[0148] In some embodiments, the malignant tumor includes at least one of lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer, non-squamous non-small cell lung cancer, colorectal cancer, triple-negative breast cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, pancreatic cancer, and clear renal cell carcinoma; preferably, the malignant tumor includes at least one of PD-L1 positive non-small cell lung cancer, hepatocellular carcinoma, and clear renal cell carcinoma.

[0149] The pharmaceutical composition of this application exhibits good stability, maintaining excellent protein content, appearance, purity, antigen-binding activity, affinity, and cellular activity even under high temperature, accelerated and long-term conditions, and repeated freeze-thaw cycles, with no significant changes in insoluble microparticles. The pharmaceutical composition of this application can be used to prepare drugs for the treatment and / or prevention of PD-1 and / or VEGF-mediated diseases or conditions.

[0150] Example

[0151] The following examples illustrate the implementation of this application in more detail. Various tests and evaluations were performed according to the methods described below. Unless otherwise specified, "parts" and "%" refer to mass. The anti-PD-1 / VEGF bispecific antibodies used in the following examples are all antibody 27 as described above. The components of the pharmaceutical composition, except for the buffer system, stabilizer, surfactant, and bispecific antibody, consist of water.

[0152] Example 1: Screening of Drug Compositions - pH Buffer Screening

[0153] 1.1 Experimental Samples

[0154] The recombinant humanized anti-VEGF and PD-1 bispecific antibody JS207 was replaced with ultrafiltration membrane in a pH 5.5 histidine buffer system (FS1-6). The protein was then replaced with different buffer systems by dialysis. The final concentration was then diluted to the corresponding concentration with the appropriate buffer and filled into vials (2.0 mL / vial) in a clean bench. The stability of the aqueous injection samples was directly sampled and tested, while the stability of the lyophilized samples was sampled and tested after lyophilization.

[0155] The components of different test drug compositions are shown in Table 2, where: acetate buffer refers to acetate-sodium acetate buffer; histidine buffer refers to L-histidine-L-histidine hydrochloride buffer; citrate buffer refers to citrate-sodium citrate buffer; "FS" represents liquid samples of different formulations, and "Lyo" represents lyophilized samples of different formulations.

[0156] Table 2 Components of different test drug compositions

[0157]

[0158]

[0159] 1.2 Experimental Methods and Results

[0160] The stability test conditions include:

[0161] (1) Placed in an environment of 5±3℃ for 1W (week), 2W or 4W, hereinafter referred to as "long-term (5±3℃)";

[0162] (2) Placed in an environment of 25±2℃ for 1W (week), 2W or 4W, hereinafter referred to as "acceleration (25±2℃)".

[0163] (3) Placed in an environment of 40±2℃ for 1W (week), 2W or 4W, hereinafter referred to as "high temperature (40±2℃)".

[0164] 1.2.1 Thermal stability test

[0165] Test method: Take 200 μL of sample and place it in a centrifuge tube, then centrifuge at 10,000 rpm for 5 min. Take 150 μL of supernatant and add it to a 96-well plate, centrifuge at 3,000 rpm for 5 min to remove air bubbles from the sample plate, and use a multi-well plate direct-reading dynamic light scattering instrument (DynaPro Plate Reader III) to analyze the initial aggregation temperature (Tagg) value.

[0166] Test results: Tagg levels for FS1-1 and FS1-3 were lower than those for other formulations. See Table 3 for details.

[0167] Table 3 Results of thermal stability study

[0168] Sample number Tagg (°C) FS1-1 54.92 FS1-2 57.83 FS1-3 54.12 FS1-4 57.00 FS1-5 57.75 FS1-6 57.39 FS1-7 58.14 FS1-8 59.40

[0169] 1.2.2 Protein content test

[0170] Test method: Protein concentration was detected using a NanoDrop one ultra-micro spectrophotometer, and the percentage extinction coefficient (E1%) was set at 14.56 (g / 100mL). -1 cm -1 Clean the test wells with ultrapure water and perform blank calibration with ultrapure water. Perform each sample measurement twice in parallel, cleaning the test wells with ultrapure water before each measurement; record the protein concentration for each measurement (Note: This method is used for the first round of formulation screening, i.e., FS1).

[0171] Test results: After being placed under high temperature, accelerated, and long-term conditions for 4 weeks, and subjected to 3 freeze-thaw cycles (3C) and 5 freeze-thaw cycles (5C), the protein content of all samples did not show significant changes. See Table 4 for details.

[0172] Table 4. Protein content results (mg / mL)

[0173]

[0174]

[0175] 1.2.3 Appearance Test

[0176] Test Method: Appearance is inspected visually. Ensure the light intensity of the clarity meter is maintained between 1000 and 1500 lux. Hold the sample at eye level and gently shake or invert it to avoid air bubbles. Perform visual inspection against both black and white backgrounds. Record results for color, opalescence, and visible foreign matter.

[0177] Test results: According to the appearance results in Table 5, the opalescence intensity of the FS1-6-20mg / mL sample is relatively stronger than that of other formulations; after being placed under high temperature conditions, the opalescence intensity of the FS1-3, FS1-4, FS1-6 and FS1-6-20mg / mL formulations all increased, while the appearance of other formulation samples did not change significantly.

[0178] Table 5 Appearance Results

[0179]

[0180] Note: " / " indicates that it was not detected.

[0181] 1.2.4 SEC-HPLC (Size Exclusion High Performance Liquid Chromatography) Purity Test

[0182] SEC-HPLC purity was determined using a column equipped with an SEC column ( BEH Detection was performed using a Waters e2695 HPLC system (7.8 mm × 30 cm, 3.5 μm). The mobile phase consisted of 50 mM phosphate, 300 mM sodium chloride, pH 6.8 ± 0.2. The peak area percentages of monomers, polymers, and fragments were calculated using the area normalization method. See Table 6 for details.

[0183] Table 6. SEC-HPLC Purity Test Parameters

[0184]

[0185] Test Results: According to the SEC-HPLC purity results in Table 7, after 4 weeks of high-temperature storage, the SEC-HPLC purity of Lyo-1 to Lyo-3 samples showed no significant change. The fragment and polymer ratios of FS1-1 to FS1-6-20 mg / mL samples all increased slightly, while the monomer purity ratio decreased. The rate of decrease in monomer purity was relatively slower in FS1-8 compared to other systems. The monomer purity decreased most rapidly in FS1-6-20 mg / mL and FS1-3. After 4 weeks of accelerated or long-term storage under FS2-1 to FS2-3 conditions, the fragment and polymer ratios of all samples increased, and the monomer purity decreased to varying degrees. The rate of decrease in monomer content was slower in FS2-2 (10 mg / mL). After 3 and 5 cycles of repeated freeze-thaw cycles and 4 weeks of long-term storage, the SEC-HPLC purity of FS1-1 to FS1-6-20 mg / mL samples showed no significant change.

[0186]

[0187]

[0188] 1.2.5 ICIF Purity Test

[0189] Test method:

[0190] (1) Prepare the reagent according to the proportions in the table below (this volume can be injected multiple times for one sample). If there are multiple samples, multiply the volume of each reagent by the corresponding factor. Add the reagent to the centrifuge tube according to the calculated volume, and vortex for at least 15 seconds to ensure complete mixing. Store at 2–8℃ and discard after use on the same day.

[0191] Table 8. Dilution Ratio of ICIF Purity Samples

[0192] Reagent Name Required volume (μL) for each sample 1% MC 35 Pharmalyte 3-10 4 pI Marker 5.85 0.5 pI Marker 9.46 0.5 500mM Arg 2 <![CDATA[H2O]]> 38

[0193] (2) Dilute the sample (reference / test) to 1 mg / mL with ultrapure water.

[0194] (3) Take 20 μL of system suitability control solution (1 mg / mL) and mix it with 80 μL of premixed solution. Centrifuge at 10000 rpm for 5 min. Take 75 μL of supernatant and add it to a 96-well plate. Be careful to avoid generating air bubbles. Then centrifuge the 96-well plate at 1000 g for 5 min to remove air bubbles.

[0195] (4) Detection was performed using a capillary electrophoresis apparatus (Maurice).

[0196] Test results: According to the icIEF purity results in Table 9, after being placed under high temperature conditions for 4 weeks, the acid peaks of samples FS1-2 to FS1-6-20 mg / mL all increased to varying degrees; after being placed under long-term conditions for 4 weeks, the icIEF purity of all samples did not change significantly.

[0197] Table 9. icIEF Detection Results

[0198]

[0199]

[0200] Note: " / " indicates that it was not detected.

[0201] 1.2.6rCE-SDS Purity Test

[0202] Test Method: rCE-SDS purity was determined using a capillary electrophoresis system (Maurice instrument). Based on a final volume of 100 μL, 1% SDS, 40 mM phosphate buffer (pH 6.5), reference or sample, and 5 μL β-mercaptoethanol were added sequentially to a 1.5 mL EP tube to achieve a final concentration of 1.0 mg / mL for the reference or sample. The mixture was thoroughly mixed; centrifuged at 3000 rpm for 30 seconds at room temperature, followed by incubation at 70°C for 15 min. After incubation, the mixture was cooled to room temperature and centrifuged at 12000 rpm for 5 min at room temperature. 75 μL of sample solution was then transferred from each sample tube to a 96-well plate, avoiding air bubbles, and centrifuged at 1000 g for 10 min at room temperature. The sample was then analyzed using a capillary electrophoresis system (Maurice).

[0203] Test Results: According to the rCE-SDS results (%) in Table 10, the purity of formulations FS1-1 to FS1-6 (20 mg / mL) decreased slightly after 4 weeks of storage under high temperature conditions. Formulations FS1-3 and FS1-8 showed the worst performance, with a significantly higher rate of purity decrease than other formulations. Sample FS1-8 showed a decrease in purity after 4 weeks of storage under both accelerated and long-term conditions, while other samples showed no significant changes. Formulations FS2-1 to FS2-3 and Lyo-1 to Lyo-3 showed no significant change in purity after 4 weeks of storage under long-term, accelerated, or high temperature conditions.

[0204] Table 10 rCE-SDS test results

[0205]

[0206]

[0207] Note: " / " indicates that it was not detected.

[0208] 1.2.7nrCE-SDS Purity Test

[0209] Test Method: The purity of nrCE-SDS was determined using a capillary electrophoresis system (Maurice instrument). Based on a final volume of 100 μL, Maurice CE-SDS 1×Sample Buffer PLUS, 5 μL of 0.25 MNEM, and the sample or reference were added sequentially to a 1.5 mL EP tube to achieve a final concentration of 1.0 mg / mL. The mixture was thoroughly mixed, centrifuged at 3000 rpm for 30 s at room temperature, and then incubated at 70 °C for 5 min. After incubation, the mixture was cooled to room temperature and centrifuged at 12000 rpm for 5 min at room temperature. 75 μL of sample solution was then transferred from each sample tube to a 96-well plate, avoiding air bubbles, and centrifuged at 1000 g for 10 min at room temperature. The sample was then analyzed using a capillary electrophoresis system (Maurice).

[0210] Test Results: According to the nrCE-SDS results (%) in Table 11, after 4 weeks of storage under high temperature conditions, the purity of formulations FS1-1 to FS1-6 (20 mg / mL) all showed a slight decrease. Formulations FS1-3 and FS1-8 performed worse, with a significantly higher rate of decrease than other formulations. Sample FS1-8 showed a decrease in purity after 4 weeks of storage under both accelerated and long-term conditions. Formulations FS2-1 to FS2-3 and Lyo-1 to Lyo-3 showed no significant change in purity after 4 weeks of storage under long-term, accelerated, or high temperature conditions.

[0211] Table 11 nrCE-SDS Data

[0212]

[0213] Note: " / " indicates that it was not detected.

[0214] 1.2.8 Combination activity test

[0215] Test Method: This method employs an indirect approach. Human PD-1 and human VEGF are used as antigens to coat 96-well plates. JS207 binds to Human PD-1 and Human VEGF. Mouse anti-Human IgG4 Fc-HRP specifically binds to JS207 bound to the solid-phase antigens (Human PD-1 and Human VEGF). Under hydrogen peroxide, JS207 catalyzes the formation of TMB, turning blue. The intensity of the blue color is positively correlated with the amount of Mouse Anti-Human IgG4 Fc-HRP bound. The color turns yellow after termination with 2M hydrochloric acid. The absorbance (OD value) is measured using a microplate reader at 450nm / 620nm wavelengths. The effective binding activity (EC50) is determined by passing a standard curve. 50 To examine the ability of JS207 bispecific antibodies to bind to HumanPD-1 and Human VEGF, respectively.

[0216] See the table below for details:

[0217] Table 12 Experimental methods for JS207 combined with PD-1 ELISA

[0218]

[0219] Table 13 JS207 combined with VEGF ELISA experimental method

[0220]

[0221] Test Results: Based on the binding activity test results in Table 14, the activity of all samples showed no significant change after 4 weeks of long-term, accelerated, or high-temperature storage.

[0222] Table 14 shows the results of activity detection.

[0223]

[0224]

[0225] Note: " / " indicates that it was not detected.

[0226] 1.2.9 Cell viability test

[0227] Test method: 1) VEGF end

[0228] Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a highly specific pro-vascular endothelial cell growth factor that promotes increased vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and angiogenesis. The high-affinity receptors that specifically bind to VEGF are called vascular endothelial growth factor receptors (VEGFRs), mainly classified into three types: VEGFR-1, VEGFR-2, and VEGFR-3. VEGFR-1 and VEGFR-2 are primarily distributed on the surface of tumor vascular endothelium, regulating tumor angiogenesis; VEGFR-3 is mainly distributed on the surface of lymphatic endothelium, regulating tumor lymphatic vessel formation.

[0229] This study utilized target cells 293VEGFR2 cells overexpressing VEGFR2 and a luciferase reporter gene system, along with recombinant VEGF (R&D systems, cat#11066-HNAB) protein, to detect the biological activity of JS207 cells. Target cells 293VEGFR2 were seeded at a density of 50,000 cells per well in 96-well flat-bottomed white plates (corning, cat#3917), and serially diluted test antibodies were added. Then, VEGF protein diluted to a working concentration of 25 ng / mL was added, and the cells were co-incubated at 37°C for 5–6 h. Finally, the luciferase substrate One-Lite (Vazyme, cat#DD1203-03-AB) was added, and chemiluminescence was detected.

[0230] 2) PD-1 terminal

[0231] NFAT is a key transcription factor for T cell receptor activation of downstream signals and is crucial for IL-2 secretion and T cell proliferation responses. The JS207 antibody can specifically bind to PD-1 on the cell surface, blocking PD-1 / PD-L1 interaction, thereby activating the downstream NFAT signaling pathway.

[0232] The system used in this study included two cell lines: Jurkat PD-1 effector cells, which stably express human PD-1 and an NFAT-coupled luciferase reporter gene on Jurkat cells; and CHO PDL1 target cell line, which stably expresses CD3 scFv and human PDL1 on CHO-K1 cells. When PD-1 on the effector cell surface binds to PDL1 on the target cell surface, the NFAT signaling pathway is inhibited, and the expression of the NFAT-coupled luciferase reporter gene is also inhibited. JS207 can specifically bind to PD-1, blocking the PD-1 / PDL1 interaction, thereby activating the downstream NFAT signaling pathway. The experimental protocol was as follows: On day 1, target cells PD-L1 aAPC / CHO-K1 were plated and cultured overnight in a cell culture incubator; on day 2, the experimental antibody was prepared and then added to the effector cells; the cells were cultured at 37°C in a 5% CO2 incubator for 4–6 hours; luciferase substrate was added, and the plates were read and data analyzed using a multi-mode microplate reader. Test Results: Based on the cell biological activity test results in Table 15, no significant changes in the activity of any samples were observed after 4 weeks of long-term, accelerated, or high-temperature storage.

[0233] Table 15 Results of Cell Biology Detection

[0234]

[0235] Note: " / " indicates that it was not detected.

[0236] 1.2.10 Insoluble Particle Test

[0237] Test method: The HIAC insoluble particle counter was used as the detection instrument. This instrument uses the optical obscuration method to statistically determine insoluble particles. The Run counter mode was used, and the parameters were set as shown in Table 16.

[0238] Table 16 Operating parameters of the insoluble particle counter

[0239]

[0240]

[0241] Test results: According to the insoluble particulate matter test results in Table 17, the number of particles in FS1-3 and FS1-4 formulations increased significantly after being placed under high temperature conditions for 4 weeks. The number of insoluble particulate matter in the other formulations did not change significantly after being placed under high temperature, accelerated, and long-term conditions for 4 weeks and after 3 and 5 freeze-thaw cycles.

[0242] Table 17 Results of insoluble particulate matter detection (light obscuration method, particles / mL)

[0243]

[0244] 1.2.11 RP-HPLC Purity Test

[0245] Test Method: RP-HPLC purity was determined using an HPLC system (Waters E2695 instrument) equipped with an Agilent column (AdvanceBio RP-mAb, 4.6 × 150 mm). The mobile phase composition was: Phase A: 0.1% TFA water; Phase B: 0.1% TFA ACN. Peak area normalization was used to calculate the percentage of the main peak and impurities. If automatic integration could not obtain reasonable integration results, manual integration was used. Detailed chromatographic parameters are shown in the table below:

[0246] Table 18 RP-HPLC Purity Test Parameters

[0247]

[0248]

[0249] Test Results: According to the RP-HPLC purity results in Table 19, after 4 weeks of storage under high temperature and accelerated conditions, the fragment ratio of all samples increased, while the purity ratio decreased. The purity decrease rate was relatively slower in FS1-5 and FS1-7 compared to other systems; FS1-8 showed the fastest purity decrease rate. After 4 weeks of storage under long-term conditions, the purity decrease rate was faster in FS1-3 and FS1-8, while the remaining samples showed no significant changes. After 3 and 5 freeze-thaw cycles, the purity of sample FS1-8 decreased slightly, while the purity of the remaining samples showed no significant changes.

[0250] Table 19 RP-HPLC Detection Results

[0251]

[0252] In summary, based on the aggregation temperature data (Tagg), the initial aggregation temperature of FS1-6 was higher than that of other formulations. From the appearance data, the opalescence intensity of the citrate buffer systems (FS1-3, FS1-4, FS1-5) and the FS1-6-20 mg / mL formulation was relatively stronger than other formulations. According to RP-HPLC purity results, at pH 6.0 (FS1-5, FS1-7), the rate of purity decrease in the samples was relatively slower than other systems; the purity decrease rate was fastest in the pH 7.5 phosphate buffer system (FS1-8). SEC-HPLC, icIEF, and CE results showed that the proteins in the pH 5.0 citrate buffer system (FS1-3) and the pH 7.5 phosphate buffer system (FS1-8) were relatively less stable than other formulations. Under accelerated and long-term conditions, the purity of the SEC monomers in the FS2-1 to FS2-3 formulations decreased significantly with increasing protein concentration, while different Tween concentrations had no significant difference in their effect on stability. After being placed under high temperature or long-term conditions for 4W, the appearance, SEC-HPLC purity, AEX-HPLC purity, rCE-SDS purity, nrCE-SDS purity, and biological activity of the Lyo-1 to Lyo-3 formulation samples showed no significant changes, and no significant differences were observed between the groups.

[0253] There were no significant differences in the protein concentration, binding activity, and cell biological activity results among all samples.

[0254] Based on the principle of simple formulation composition, FS2-1 (i.e., pH 6.0, 10mM histidine buffer system, 8% (w / v) sucrose as lyophilization protectant, 0.02% (w / v) polysorbate 80(II), and recombinant humanized anti-VEGF and PD-1 bispecific antibody 20 mg / mL) was finally selected. In clinical use, hemolysis is prone to occur when the osmotic pressure is low. Therefore, the sucrose content in the final formulation was increased from 8% to 8.4% to achieve isotonicity. Thus, the final formulation components are: pH 6.0, 10mM histidine buffer system, 8.4% (w / v) sucrose, 0.02% (w / v) polysorbate 80(II), and recombinant humanized anti-VEGF and PD-1 bispecific antibody 20 mg / mL.

[0255] The stock solution was prepared as follows: 20 mg / mL of recombinant humanized anti-VEGF and PD-1 bispecific antibody, 0.76 mg / mL of histidine, 1.07 mg / mL of histidine hydrochloride, 84.00 mg / mL of sucrose, and 0.20 mg / mL of polysorbate 80(II).

[0256] Formulation stock solution 2: Recombinant humanized anti-VEGF and PD-1 bispecific antibody 30mg / mL, histidine 1.14mg / mL, histidine hydrochloride 1.605mg / mL, sucrose 126.00mg / mL, polysorbate 80(II) 0.30mg / mL.

[0257] After the stock solution is prepared, it is lyophilized. The lyophilized finished product is reconstituted with an appropriate amount of water for injection, and the concentration of each component is kept the same as the concentration of each component in the final prescription.

[0258] The pharmaceutical composition used in the following examples consists of: 10 mM histidine buffer system, 8.4% (w / v) sucrose, 0.02% (w / v) polysorbate 80 (II), 20 mg / mL recombinant humanized anti-VEGF and PD-1 bispecific antibody, and pH 6.0.

[0259] Example 2: Determination of antigen affinity of pharmaceutical composition

[0260] 2.1 Experimental Methods

[0261] The binding affinity of injectable JS207 to human PD-1 and human VEGFA was determined using a Biacore T200 molecular interaction analyzer from GE Healthcare Life Sciences. 40 μg / mL of goat anti-human Fc fragment antibody was conjugated to the surface of a CM5 chip. JS207 samples were then captured, followed by the injection of serially diluted human PD-1 and human VEGFA proteins, and the binding signal was detected. The serial dilutions of human PD-1 protein were 140 nM, 70 nM, 35 nM, 17.5 nM, 8.75 nM, and 4.375 nM, with 140 nM being a replicate. The serial dilutions of human VEGFA protein were 6 nM, 3 nM, 1.5 nM, 0.75 nM, 0.375 nM, and 0.188 nM, with 6 nM being a replicate. Kinetic model analysis using Biacore T200 Evaluation Software 3.0 was employed to obtain the affinity KD values.

[0262] 2.2 Experimental Results

[0263] Biacore detected that JS207 has binding activity with both human PD-1 and human VEGFA.

[0264] Table 20 Summary of Affinity of Antibody JS207 with Human PD-1 and Human VEGFA

[0265] antigen ka(1 / Ms) kd(1 / s) KD(M) Human PD-1 4.37E+04 2.01E-05 4.60E-10 Human VEGFA 1.12E+06 1.01E-05 9.00E-12

[0266] The results in Table 20 show that JS207 has binding activity with both human PD-1 and human VEGFA.

[0267] Example 3: Antigen-binding activity of the pharmaceutical composition

[0268] 3.1 Experimental Methods

[0269] 3.1.1JS207 combined with Human PD-1

[0270] This experiment used a Thermo Scientific or Molecular Devices microplate reader. The plates were coated with 0.3 μg / mL Human PD-1, blocked with 2% BSA, and then serially diluted JS207 antibody and negative control antibody (starting at 1.0 μg / mL, with 12 concentrations obtained through 2.5-fold serial dilutions). Mouse Anti-Human IgG4 Fc-HRP diluted 5000-fold was used as the detection antibody. The reaction was then developed with 0.1 mg / mL TMB, and finally terminated with 2M HCl. Plate readings were performed at 450 nm and 620 nm. A four-parameter logarithmic regression (4PL) model was used to fit the EC50 values.

[0271] 3.1.2JS207 combined with Human VEGF

[0272] This experiment used a Thermo Scientific or Molecular Devices microplate reader. The plates were coated with 0.5 μg / mL Human VEGF and blocked with 2% BSA. Serially diluted JS207 antibody and negative control antibody (starting at 1.0 μg / mL, with 12 concentrations obtained through 3-fold serial dilutions) were added. Mouse Anti-Human IgG4 Fc-HRP diluted 5000-fold was used as the detection antibody. The reaction was then developed with 0.1 mg / mL TMB, and finally terminated with 2M HCl. Plate readings were performed at 450 nm and 620 nm. A four-parameter logarithmic regression (4PL) model was used to fit the EC50 values.

[0273] 3.2 Experimental Results

[0274] The experimental results are shown in Table 21. The binding of this drug composition and the negative control antibody (anti-KLH hIgG4) to Human PD-1 and Human VEGF was measured, respectively. This drug composition specifically binds to both Human PD-1 and Human VEGF, and the binding EC50 of Human PD-1 is [missing value]. 50 The value was 9.2 ng / mL, and it bound to Human VEGF EC. 50 The value was 4.8 ng / mL, and the negative control showed no binding.

[0275] Table 21 Summary of ELISA results for binding of drug compositions to recombinant Human PD-1 and Human VEGF proteins

[0276]

[0277] Example 4: Cellular activity of the pharmaceutical composition

[0278] 4.1 Experimental Methods

[0279] 4.1.1 VEGF end

[0280] Biological activity of JS207 cells was detected using target cells 293VEGFR2 overexpressing VEGFR2 and the luciferase reporter gene system, and recombinant human / cynomolgus VEGF / VEGFA / VEGF165 (Sino Biological, cat#11066-HNAB) protein. 293VEGFR2 cells were seeded at 50,000 cells per well in 96-well flat-bottomed white plates (corning, cat#3917). Serially diluted test antibody (JS207 batch number: JS207-IRS-01) was added, along with a working concentration of 25 ng / mL VEGF protein. The cells and antibody were incubated at 37°C for 5–6 h. Finally, the luciferase substrate One-Lite (Vazyme, cat#DD1203-03) was added, and chemiluminescence was detected. A four-parameter regression curve was fitted using GraphPad Prism software, and the IC50 was calculated. 50 value.

[0281] 4.1.2 PD-1 terminal

[0282] NFAT is a key transcription factor for T cell receptor activation of downstream signaling, and is crucial for IL-2 secretion and T cell proliferation responses. The JS207 antibody specifically binds to PD-1 on the cell surface, blocking PD-1 / PD-L1 interaction and thereby activating the downstream NFAT signaling pathway. This experiment investigated the biological activity of JS207 by blocking PD-1 / PD-L1 interaction at the cellular level, thereby activating the downstream NFAT signaling pathway. Target cells, CHO PD-L1 cells (stable expression of human PD-L1 and CD3scFv), were cultured in 96-well white flat-bottomed plates at 5 × 10⁶ cells per well. 4The cells were seeded overnight; the next day, after aspirating the culture medium from the wells, serially diluted test antibody (JS207 batch number: JS207-IRS-01) was added to the target cells and incubated at 37°C for 30 minutes. Then, effector cells Jurkat PD-1 (stable expression of human PD-1 and luc2P / NFAT-RE) were added at a rate of 1 × 10⁶ cells per well. 5 Cells were added to cell culture plates and incubated at 37°C for 6 hours. Finally, ONE-Lite luciferase assay reagent (Vazyme, Cat#DD1203-03) was added to the cell-antibody mixture, and the chemiluminescence signal was detected using a multi-functional microplate reader (TECANM1000 pro). A four-parameter regression curve was fitted using GraphPad Prism software, and EC50 was calculated. 50 value.

[0283] 4.2 Experimental Results

[0284] 4.2.1 VEGF end

[0285] like Figure 2 As shown, JS207 can specifically bind to VEGF antigen, thereby blocking the binding of VEGF protein and its receptor, thus blocking downstream signaling pathways, inhibiting tumor-induced angiogenesis, and IC50. 50 The value is 0.7726 nM.

[0286] 4.2.2 PD-1 terminal

[0287] like Figure 3 As shown, JS207 exhibits strong T-cell activation activity in the aforementioned reporter gene system, EC 50 It is 7.13 nM.

[0288] Example 5: Study of JS207 in patients with advanced malignant tumors

[0289] An ongoing first-in-human study of JS207 by the applicant explored the safety and preliminary clinical activity of the bispecific antibody of this application as a monotherapy in patients with advanced malignant tumors.

[0290] This study was divided into two parts. Part A consisted of patients with advanced malignant tumors who had failed standard therapy or had no standard therapy. Dose escalation and expansion were performed in five dose groups (1-20 mg / kg, specifically 1 mg / kg, 3 mg / kg, 10 mg / kg, 15 mg / kg, and 20 mg / kg), with JS207 administered intravenously every 3 weeks. Accelerated titration and Bayesian optimal interval (BOIN) design guided dose escalation. Part B consisted of first-line patients with PD-L1 positive (TPS ≥ 1%) NSCLC, HCC, ccRCC, etc. The primary endpoint was safety and the recommended dose for a phase II clinical trial (RP2D). JS207 is an injectable formulation prepared using the preferred formulation of the composition described in this application.

[0291] As of December 20, 2024, a total of 23 patients were enrolled in Part A, including 17 patients on dose escalation and 6 patients on a 20 mg / kg extension dose. The main tumor types were gynecologic tumors, colorectal cancer, and NSCLC (N = 5, 21.7%). 15 patients (65.2%) had received ≥2 lines of prior systemic anti-tumor therapy, 3 patients (13.0%) had received immunotherapy, and 10 patients (43.5%) had received bevacizumab therapy. Part B enrolled 43 patients at a dose of 15 mg / kg, and no dose-limiting toxicities (DLTs) were observed. The most common adverse events (AEs) (>20%) were hypoalbuminemia and proteinuria (N = 25, 37.9%, each), hypertension (N = 24, 36.4%), and anemia (N = 18, 27.3%). The median follow-up time was 8.3 months for Part A and 2.9 months for Part B. In Part A, among the 21 evaluable patients, the objective response rate (ORR) was 9.5%, with 2 partial responses (PR) (1 patient at 10 mg / kg and 1 patient at 20 mg / kg). Part B enrolled 36 evaluable patients, with ORR and disease control rate (DCR) of 51.9% (14 PRs out of 27 patients) and 96.3% (27 PRs out of 26 patients), respectively. In PD-L1-positive NSCLC, these figures were 33.3% (1 PR out of 3 patients) and 100.0%, respectively; in HCC, they were 83.3% (5 PRs out of 6 patients); and in ccRCC, they were 100.0% (1 PR out of 1 patient).

[0292] Results showed that JS207 demonstrated tolerable safety and encouraging responses in advanced solid tumors, particularly in first-line PD-L1 positive NSCLC, HCC, and ccRCC patients.

[0293] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A pharmaceutical composition comprising an anti-PD-1 / VEGF bispecific antibody, a buffer solution, a stabilizer, and a surfactant, wherein, The anti-PD-1 / VEGF bispecific antibody comprises a first protein functional region and a second protein functional region; the first protein functional region is an anti-PD-1 antibody, and the second protein functional region is an anti-VEGF single-domain antibody; the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 (HCDR1, HCDR2, and HCDR3 respectively), and the light chain variable region comprising amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 (LCDR1, LCDR2, and LCDR3 respectively); the anti-VEGF single-domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 respectively. NO:9 shows HCDR1, HCDR2 and HCDR3; the anti-VEGF single-domain antibody consists of two antibodies, the N-terminus of each single-domain antibody is connected to the C-terminus of the CH1 region of the heavy chain constant region of the anti-PD-1 antibody, and the C-terminus of each single-domain antibody is connected to the N-terminus of the CH2 region of the heavy chain constant region of the anti-PD-1 antibody. The buffer solution is selected from histidine buffer, the stabilizer is selected from sucrose, and the surfactant is selected from polysorbate 80(II). In the pharmaceutical composition, the concentration of the anti-PD-1 / VEGF bispecific antibody is 8 mg / mL to 40 mg / mL, the concentration of the buffer is 8 mM to 25 mM, the concentration of the stabilizer is 170 mM to 470 mM or 6% to 16% (w / v), and the concentration of the surfactant is 0.01% to 0.06% (w / v). The pH value of the pharmaceutical composition is 4.5 to 8.

0.

2. The pharmaceutical composition according to claim 1, wherein, In the pharmaceutical composition, the concentration of the anti-PD-1 / VEGF bispecific antibody is 8 mg / mL to 32 mg / mL, preferably 10 mg / mL to 30 mg / mL, more preferably 10 mg / mL to 20 mg / mL; and / or, The concentration of the buffer solution is 8 mM to 22 mM, preferably 10 mM to 20 mM, and more preferably 10 mM to 16 mM; and / or, The concentration of the stabilizer is 210 mM to 370 mM, preferably 230 mM to 370 mM, more preferably 245 mM to 368 mM, or the concentration of the stabilizer is 8% to 13% (w / v), more preferably 8.4% to 12.6% (w / v); and / or, The concentration of the surfactant is 0.01% to 0.05% (w / v), preferably 0.01% to 0.04% (w / v); and / or, The pH value of the pharmaceutical composition is 5.0 to 7.5, preferably 5.5 to 6.

5.

3. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition comprises 8 mg / mL to 32 mg / mL of anti-PD-1 / VEGF bispecific antibody, 8 mM to 22 mM of histidine buffer, 230 mM to 370 mM or 8% to 13% (w / v) of sucrose, and 0.01% to 0.05% (w / v) of polysorbate 80(II), and the pH of the pharmaceutical composition is 5.0 to 7.5; Preferably, the pharmaceutical composition comprises 8 mg / mL to 32 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM to 20 mM of histidine buffer, 230 mM to 370 mM or 8% to 13% (w / v) of sucrose, and 0.01% to 0.04% (w / v) of polysorbate 80(II), and the pH of the pharmaceutical composition is 5.5 to 6.5; More preferably, the pharmaceutical composition comprises 10 mg / mL to 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM to 20 mM of histidine buffer, 230 mM to 370 mM or 8% to 13% (w / v) of sucrose, and 0.01% to 0.04% (w / v) of polysorbate 80(II), and the pH of the pharmaceutical composition is 5.5 to 6.5; More preferably, the pharmaceutical composition comprises 10 mg / mL to 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM to 20 mM of histidine buffer, 245 mM to 368 mM or 8.4% to 12.6% (w / v) of sucrose, and 0.01% to 0.04% (w / v) of polysorbate 80(II), and the pH of the pharmaceutical composition is 5.5 to 6.

5.

4. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition comprises 10 mg / mL to 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM histidine buffer, 234 mM or 8% (w / v) sucrose, and 0.01% to 0.03% (w / v) polysorbate 80(II), and the pH of the pharmaceutical composition is 6.0; Preferably, the pharmaceutical composition comprises 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM histidine buffer, 234 mM or 8% (w / v) sucrose, and 0.02% (w / v) polysorbate 80(II), and the pH of the pharmaceutical composition is 6.

0.

5. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition comprises 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10 mM histidine buffer, 245 mM or 8.4% (w / v) sucrose, and 0.02% (w / v) polysorbate 80(II), and the pH of the pharmaceutical composition is 6.

0.

6. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition comprises 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10.5 mM histidine buffer, 245 mM or 8.4% (w / v) sucrose, and 0.01% to 0.03% (w / v) polysorbate 80(II), and the pH of the pharmaceutical composition is 6.

0.

7. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition comprises 20 mg / mL of anti-PD-1 / VEGF bispecific antibody, 10.5 mM histidine buffer, 245 mM or 8.4% (w / v) sucrose, and 0.02% (w / v) polysorbate 80(II), and the pH of the pharmaceutical composition is 6.

0.

8. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition comprises 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, 16 mM histidine buffer, 380 mM or 13% (w / v) sucrose, and 0.03% (w / v) polysorbate 80(II), and the pH of the pharmaceutical composition is 6.

0.

9. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition comprises 30 mg / mL of anti-PD-1 / VEGF bispecific antibody, 15.73 mM histidine buffer, 368 mM or 12.6% (w / v) sucrose, and 0.03% (w / v) polysorbate 80(II), and the pH of the pharmaceutical composition is 6.

0.

10. The pharmaceutical composition according to any one of claims 1-9, wherein, The histidine buffer is a histidine-histidine hydrochloride buffer; preferably an L-histidine-L-histidine hydrochloride buffer.

11. The pharmaceutical composition according to any one of claims 1-10, wherein, The pharmaceutical composition is a liquid formulation or a lyophilized formulation.

12. The pharmaceutical composition according to any one of claims 1-11, wherein, The anti-PD-1 / VEGF bispecific antibody comprises a first protein functional region including a heavy chain variable region and a light chain variable region as shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and a second protein functional region including a heavy chain variable region as shown in SEQ ID NO:

12. Preferably, the amino acid sequence of the first peptide chain of the anti-PD-1 / VEGF bispecific antibody is shown in SEQ ID NO: 13, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO:

16.

13. Use of the pharmaceutical composition according to any one of claims 1-12 in the preparation of a medicament for treating and / or preventing PD-1 and / or VEGF-mediated diseases or conditions; Preferably, the PD-1 and / or VEGF-mediated disease or condition is a malignant tumor; More preferably, the malignant tumor includes at least one of lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer, non-squamous non-small cell lung cancer, colorectal cancer, triple-negative breast cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, pancreatic cancer, and renal cell clear carcinoma; More preferably, the malignant tumor includes at least one of PD-L1 positive non-small cell lung cancer, hepatocellular carcinoma, and clear renal cell carcinoma.

Images