Pharmaceutical formulations comprising anti-VEGFA and VEGFC bispecific binding proteins

Abstract

The present application provides pharmaceutical formulations, uses thereof, and kits containing the same, wherein the pharmaceutical formulations comprise (1) an anti-VEGFA and VEGFC bispecific binding protein, (2) a buffering agent, (3) a stabilizer, and (4) a surfactant, and wherein the pH of the pharmaceutical formulation is 5.5 to 7.0. The pharmaceutical formulations exhibit long-term stability and angiogenesis inhibitory effects.

Description

[Technical Field]

[0001] The present application is in the field of pharmaceutical formulations, and specifically relates to pharmaceutical formulations comprising anti-VEGFA and VEGFC bispecific binding proteins that exhibit superior stability. [Background technology]

[0002] Vascular endothelial growth factor (VEGF) is a key regulator of vascular development and blood and lymphatic vascular function in healthy and diseased adults and is overexpressed in many human tumors. The VEGF family is currently known to consist of five structurally related factors: VEGFA (vascular endothelial growth factor A, also known as VEGF-A or VEGFA165), VEGFB, VEGFC (vascular endothelial growth factor C, also known as VEGF-C), VEGFD, and placental growth factor (PLGF). VEGF family members exist primarily as homodimeric polypeptides and induce signal transduction by binding to their associated VEGF receptors, resulting in corresponding biological effects. VEGFA binds to the receptors VEGFR1 (also known as FLT1) and VEGFR2 (also known as KDR), while VEGFC binds to the receptors VEGFR2 (also known as KDR) and VEGFR3 (also known as FLT4).

[0003] Given the roles of VEGF-A and VEGF-C in the development of blood and lymphatic vessels, researchers have suggested that VEGF-A and VEGF-C may have a synergistic promoting effect on each other in angiogenesis-related diseases such as tumor growth and metastasis. Therefore, bispecific molecules that can simultaneously target both VEGF-A and VEGF-C may be useful in suppressing the progression of angiogenesis-related diseases by blocking both vascular endothelial growth factors A and C. Such bispecific molecules also offer advantages when administered compared with the separate combinations of anti-VEGF-A and anti-VEGF-C molecules.

[0004] Bispecific antibodies are protein molecules, and as biopolymers, proteins present challenges during long-term storage. Proteins are known to be unstable (e.g., bispecific antibodies are prone to aggregation and degradation during storage, reducing the safety and efficacy of the antibody). Therefore, the development of stable bispecific antibody formulations is essential to ensure in vivo therapeutic efficacy and clinical safety. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above-mentioned technical problems existing in the prior art, it is necessary to develop and provide a formulation of the bispecific antibody of the present application that has long-term stability in order to facilitate effective clinical administration of the antibody. [Means for solving the problem]

[0006] The present application provides a pharmaceutical formulation comprising: (1) an anti-VEGFA and VEGFC bispecific binding protein; (2) a buffering agent; (3) a stabilizer; and (4) a surfactant, wherein the pH of the pharmaceutical formulation is 5.5 to 7.0.

[0007] In some embodiments, the bispecific binding protein comprises: (i) a first antigen-binding moiety that specifically binds to human VEGFC, wherein the first antigen-binding moiety consists of an anti-VEGFC single domain antibody or an anti-VEGF C VHH domain, and the single domain antibody or VHH domain comprises a CDR1 having the amino acid sequence set forth in SEQ ID NO:1, a CDR2 having the amino acid sequence set forth in SEQ ID NO:2, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:3; and (ii) comprises a second antigen-binding moiety that specifically binds to human VEGFA.

[0008] In some embodiments, the first antigen-binding component (also referred to as an anti-VEG Fc component) may be a polypeptide / protein comprising a single-domain antibody or a VHH domain thereof described herein, for example, a fusion protein (e.g., a VHH-Fc polypeptide) comprising the VHH domain and an immunoglobulin constant region (e.g., an Fc portion, e.g., a human IgG Fc region, preferably a human IgG1 Fc region). In certain embodiments, the VHH-Fc polypeptide comprises a single-domain antibody or a VHH domain thereof and an Fc portion located at the C-terminus thereof. In certain embodiments, the human IgG1 Fc region is an Fc region comprising the amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. In certain embodiments, the VHH-Fc polypeptides described herein comprise an Fc portion fused via a C-peptide (eg, hinge region) to the C-terminus of said single domain antibody or its VHH domain.

[0009] As used herein, the VHH domain included in the anti-VEGF-C single domain antibody may have the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In some embodiments, the VHH domain comprises (i) the amino acid sequence set forth in SEQ ID NO:4, or (ii) an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of (i), or (iii) an amino acid sequence having at least 1 to 30, or 1 to 20, or 1 to 15, or 1 to 10, or 1 to 5 amino acid changes (e.g., substitutions, deletions, and / or insertions, preferably substitutions, more preferably conservative substitutions) relative to the amino acid sequence of (i). In certain preferred embodiments, the VHH domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, an anti-VEGFA / VEGFC bispecific binding molecule according to the present application comprises at least one, e.g., two or more (preferably two) anti-VEGFC single domain antibodies or VHH domains according to the present application.

[0010] The second antigen-binding moiety (also referred to as an anti-VEGFA moiety) described herein may include any molecule with a VEGFA-binding domain, for example, an anti-VEGFA Trap molecule, for example, a Trap molecule comprising the extracellular domain of the VEGFA receptor VEGFR1, a Trap molecule comprising the extracellular domain of the VEGFA receptor VEGFR2, or a Trap molecule comprising the extracellular domains of VEGFR1 and VEGFR2, as well as a fusion protein or chimeric polypeptide of a VEGFA-binding domain and the Fc portion of an immunoglobulin.

[0011] In some embodiments, the anti-VEGFA component of the present application is provided by an Fc chimeric polypeptide comprising a VEGFA binding domain, whereby the anti-VEGFA component is a fusion protein comprising an Fc portion and a VEGFA binding domain.

[0012] In certain embodiments, the VEGFA-binding domain is a VEGFA-binding polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. Preferably, the VEGFA-binding polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:6.

[0013] In a specific embodiment, the Fc portion is an Fc region of a human IgG, such as a human IgG1 Fc region, and comprises or consists of the amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0014] In certain embodiments, the anti-VEGFA component comprising a VEGFA-binding domain and an Fc portion comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. In some preferred embodiments, the second antigen-binding component that binds to VEGFA in a dimeric form with high affinity and blocks activation of the VEGFA-induced VEGFR signaling pathway can be VEGF-Trap (Aflibercept, Eylea), a recombinant fusion protein (SEQ ID NO:5) comprising a VEGF-A-binding domain portion (SEQ ID NO:6) derived from human VEGF receptors 1 and 2 fused to a human IgG1 Fc region (SEQ ID NO:7) at the C-terminus.

[0015] In the bispecific binding proteins described herein, the anti-VEGFA component is linked or fused to the C-terminus or N-terminus of the anti-VEGFA component. In some embodiments, the anti-VEGFA component can be covalently attached to the N-terminus or C-terminus, preferably the C-terminus, of the anti-VEGFA component, either directly or via a linker sequence. In some embodiments, the anti-VEGFA component binds to human VEGFA and inhibits binding of VEGFA to its receptors VEGFR1 and / or VEGFR2 and the signal transduction induced thereby.

[0016] In embodiments in which the anti-VEGFA component is an Fc fusion protein, preferably the VEGFA binding domain may be located at the N-terminus or C-terminus of the Fc portion, and the anti-VEGFA component is attached at the opposite end of the Fc portion.

[0017] As will be understood by those skilled in the art, each component of the above-described fusion protein can be operably linked so as to perform its intended function. In certain embodiments, the components of the fusion protein can be directly linked via a C-peptide or a linker. In some embodiments, the linker comprises an amino acid sequence derived from an immunoglobulin hinge region or a flexible amino acid sequence (e.g., a sequence consisting of glycine and serine, such as (G4S)n or G(G4S)n, where n is 1, 2, 3, 4, 5, 6, or 7, preferably 2, 3, or 4).

[0018] In some embodiments, the bispecific binding proteins described herein can include a linker connecting the anti-VEGF A component and the anti-VEGF C component. In certain embodiments, the linker is a peptide about 6 to about 30 amino acids in length, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Preferably, the amino acid sequence of the linker is (G4S)n or G(G4S)n, where n is 1, 2, 3, 4, or 5, preferably 2, 3, or 4.

[0019] In some embodiments, the present application provides anti-VEGFA / VEGFC bispecific binding proteins comprising an anti-VEGFA component and an anti-VEGFC component in the form of an Fc fusion protein. In some embodiments, the anti-VEGFA / VEGFC bispecific binding proteins of the present application form dimers by dimerization of the Fc regions. In certain preferred embodiments, a VEGFA-binding polypeptide described herein and an anti-VEGFC VHH domain described herein are fused to the N-terminus and C-terminus, respectively, of an immunoglobulin Fc region, and the resulting fusion polypeptide chains form dimers by dimerization of the Fc regions.

[0020] In certain preferred embodiments, the bispecific binding proteins described herein comprise a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain comprise a fusion polypeptide formed by fusing a VEGFA-binding polypeptide and an anti-VEGFC VHH domain, respectively, to the N-terminus and C-terminus of an immunoglobulin Fc region, and wherein the first polypeptide chain and the second polypeptide chain can be the same or different. Preferably, the first polypeptide chain and the second polypeptide chain are the same, and the bispecific binding proteins described herein are homodimeric proteins comprising the first polypeptide chain and the second polypeptide chain.

[0021] In some embodiments, the first and second polypeptide chains are the same and are VEGFA-binding polypeptides each comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. In other embodiments, the first and second polypeptide chains are the same and each comprise an anti-VEGF C VHH domain comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1-3, and particularly preferably, the VHH domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. In another specific embodiment, the first polypeptide chain and the second polypeptide chain are the same and each comprise an Fc region derived from human IgG1. In another specific embodiment, the first polypeptide chain and the second polypeptide chain are the same and the anti-VEGFC VHH domain is fused to the C-terminus of the immunoglobulin Fc region via a linker.

[0022] In some embodiments, the first polypeptide chain and the second polypeptide chain are the same and each comprise the amino acid sequence set forth in SEQ ID NO:8.

[0023] In some embodiments, the concentration of the bispecific binding protein in the pharmaceutical formulation is less than or equal to 80 mg / mL and greater than 0, preferably 20 mg / mL to 80 mg / mL, 30 mg / mL to 80 mg / mL, 40 mg / mL to 80 mg / mL, 50 mg / mL to 80 mg / mL, 20 mg / mL to 70 mg / mL, 30 mg / mL to 70 mg / mL, 40 mg / mL to 70 mg / mL, 20 mg / mL to 60 mg / mL, 30 mg / mL to 60 mg / mL, 40 mg / mL to 60 mg / mL, or 45 mg / mL to 65 mg / mL.

[0024] In some embodiments, the buffer is a phosphoric acid or phosphate buffer system, or a histidine buffer system.

[0025] In some preferred embodiments, the phosphate or phosphate buffer system comprises phosphoric acid, sodium dihydrogen phosphate, potassium dihydrogen phosphate.

[0026] In some preferred embodiments, the histidine buffer system comprises histidine, or histidine and L-histidine hydrochloride, preferably histidine and L-histidine hydrochloride, hi some embodiments, the histidine buffer system comprises 1.0 mg / mL to 2.0 mg / mL, 1.2 mg / mL to 1.8 mg / mL, 1.3 mg / mL to 1.7 mg / mL, 1.4 mg / mL to 1.7 mg / mL, 1.4 mg / mL to 1.65 mg / mL, 1.4 mg / mL to 1.6 mg / mL, 1.45 mg / mL to 1.6 mg / mL, or 1.5 mg / mL to 1.6 mg / mL of histidine. In some further embodiments, the histidine buffer system has a concentration of 0.80 mg / mL to 1.5 mg / mL, 0.80 mg / mL to 1.3 mg / mL, 0.80 mg / mL to 1.2 mg / mL, 0.80 mg / mL to 1.1 mg / mL, 0.80 mg / mL to 1.0 mg / mL, 0.80 to 0.95 mg / mL, 0.85 mg / mL to 1.5 mg / mL, 0.85 mg / mL to 1.3 mg / mL, 0.85 mg / mL to 1.2 mg / mL, 0.85 mg / mL to 1.1 mg / mL, 0.85 mg / mL to 1.0 mg / mL, or 0.85 mg / mL to 0.95 mg / mL (preferably 0.90 mg / mL). L-histidine and 0.80 mg / mL to 1.5 mg / mL, 0.80 mg / mL to 1.3 mg / mL, 0.80 mg / mL to 1.2 mg / mL, 0.80 mg / mL to 1.1 mg / mL, 0.80 mg / mL to 1.0 mg / mL, 0.80 to 0.95 mg / mL, 0.85 mg / mL to 1.5 mg / mL, 0.85 mg / mL to 1.3 mg / mL, 0.85 mg / mL to 1.2 mg / mL, 0.85 mg / mL to 1.1 mg / mL, 0.85 mg / mL to 1.0 mg / mL, or 0.85 mg / mL to 0.95 mg / mL (preferably, 0.90 mg / mL) of L-histidine hydrochloride.

[0027] In some embodiments, the stabilizer is a sugar, sugar alcohol, or basic amino acid capable of stabilizing proteins. In some preferred embodiments, the sugar is trehalose, sucrose, or the like, for example, at a concentration of 50 mg / mL to 100 mg / mL, 60 mg / mL to 100 mg / mL, 70 mg / mL to 100 mg / mL, 80 mg / mL to 100 mg / mL, 85 mg / mL to 100 mg / mL, 50 mg / mL to 95 mg / mL, 60 mg / mL to 95 mg / mL, 70 mg / mL to 95 mg / mL, 80 mg / mL to 95 mg / mL, 85 mg / mL to 100 mg / mL. 95 mg / mL, 50 mg / mL to 90 mg / mL, 60 mg / mL to 90 mg / mL, 70 mg / mL to 90 mg / mL, 80 mg / mL to 90 mg / mL, or 85 mg / mL to 90 mg / mL of sugar (for example, trehalose with a concentration of 80 mg / mL to 100 mg / mL, 85 mg / mL to 100 mg / mL, 85 mg / mL to 95 mg / mL, 80 mg / mL to 90 mg / mL, or 85 mg / mL to 90 mg / mL). In some preferred embodiments, the sugar alcohol is sorbitol or mannitol, for example, a sugar alcohol having a concentration of 30 mg / mL to 80 mg / mL, 35 mg / mL to 70 mg / mL, 40 mg / mL to 60 mg / mL, 45 mg / mL to 55 mg / mL, or 50 mg / mL to 55 mg / mL (e.g., sorbitol having a concentration of 40 mg / mL to 60 mg / mL, 45 mg / mL to 55 mg / mL, or 50 mg / mL to 55 mg / mL).In some preferred embodiments, the basic amino acid is arginine or lysine, for example, at a concentration of 5 mg / mL to 30 mg / mL, 8 mg / mL to 25 mg / mL, 9 mg / mL to 25 mg / mL, 10 mg / mL to 25 mg / mL, 11 mg / mL to 25 mg / mL, 12 mg / mL to 25 mg / mL, 8 mg / mL to 20 mg / mL, 9 mg / mL to 20 mg / mL, 10 mg / mL to 20 mg / mL, 11 mg / mL to 25 mg / mL, 10 mg / mL to 15 mg / mL, 11 mg / mL to 15 mg / mL, 12 mg / mL to 15 mg / mL, or 13 mg / mL to 15 mg / mL of basic amino acids (e.g., arginine at a concentration of 10 mg / mL to 15 mg / mL, 11 mg / mL to 15 mg / mL, 12 mg / mL to 15 mg / mL, or 13 mg / mL to 15 mg / mL).

[0028] In some embodiments, the surfactant is a nonionic surfactant, preferably a polysorbate such as polysorbate 20 or polysorbate 80, with preferred concentrations of polysorbate being 0.1 mg / mL to 0.5 mg / mL, 0.2 mg / mL to 0.5 mg / mL, 0.25 mg / mL to 0.5 mg / mL, 0.2 mg / mL to 0.45 mg / mL, 0.25 mg / mL to 0.45 mg / mL, 0.2 mg / mL to 0.4 mg / mL, or 0.25 mg / mL to 0.4 mg / mL, preferably 0.3 mg / mL to 0.4 mg / mL (e.g., polysorbate 80 at a concentration of 0.2 mg / mL to 0.4 mg / mL, 0.25 mg / mL to 0.4 mg / mL, or preferably 0.3 mg / mL to 0.4 mg / mL).

[0029] In some embodiments, the pharmaceutical formulation is a liquid injectable solution (eg, a liquid in a vial, an ampoule, or a prefilled syringe) or a lyophilized powder.

[0030] In some embodiments, the pH of the pharmaceutical formulation is 5.8 to 6.5, 5.9 to 6.5, 6.0 to 6.5, 6.0 to 6.4, 6.0 to 6.3, or 6.0 to 6.2, for example, the pH is 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0031] In some embodiments, the pharmaceutical formulation comprises 40 mg / mL to 80 mg / mL of the anti-VEGFA and VEGFC bispecific binding protein, 0.80 mg / mL to 1.0 mg / mL of histidine, 0.80 mg / mL to 1.0 mg / mL of L-histidine hydrochloride, 80 mg / mL to 100 mg / mL of the sugar, and 0.2 mg / mL to 0.5 mg / mL of polysorbate, wherein the pH of the pharmaceutical formulation is 6.0 to 6.5.

[0032] In some embodiments, the pharmaceutical formulation comprises 40 mg / mL to 80 mg / mL of the anti-VEGFA and VEGFC bispecific binding protein, 0.80 mg / mL to 1.0 mg / mL histidine, 0.80 mg / mL to 1.0 mg / mL L-histidine hydrochloride, 80 mg / mL to 100 mg / mL trehalose, 0.2 mg / mL to 0.5 mg / mL polysorbate 80, wherein the pH of the pharmaceutical formulation is 6.0 to 6.5.

[0033] In some embodiments, the pharmaceutical formulation comprises 40 mg / mL to 60 mg / mL of the anti-VEGFA and VEGFC bispecific binding protein, 0.85 mg / mL to 0.95 mg / mL histidine, 0.85 mg / mL to 0.95 mg / mL L-histidine hydrochloride, 85 mg / mL to 100 mg / mL trehalose, and 0.2 mg / mL to 0.4 mg / mL polysorbate 80, wherein the pH of the pharmaceutical formulation is 6.0 to 6.2.

[0034] In certain embodiments, the pharmaceutical formulation comprises 50 mg / mL of anti-VEGFA and VEGFC bispecific binding protein, 0.90 mg / mL histidine, 0.90 mg / mL L-histidine hydrochloride, 90.00 mg / mL trehalose, 0.3 mg / mL polysorbate 80, wherein the pH of the pharmaceutical formulation is 6.2.

[0035] In some embodiments, the osmolality of the pharmaceutical formulation is 280 mOsmol / kg or more, preferably 300 mOsmol / kg or more, for example, 300 to 350 mOsmol / kg.

[0036] In some embodiments, the pharmaceutical formulation, in liquid form or lyophilized powder form, is stable at 30°C to 50°C for at least 2 weeks, preferably at least 4 weeks. Herein, the stability of the pharmaceutical formulation can be determined by, but is not limited to, measuring the protein content using a UV method, measuring the purity using a SEC-HPLC method or a non-reducing CE-SDS method, visually inspecting for foreign matter using a visual foreign matter inspection method, visual inspection using an observation method, and / or measuring the pH of the formulation using a pH measurement method. In some embodiments, when the pharmaceutical formulation is stored in liquid form at 30°C to 50°C for at least 2 weeks, the purity measured by a SEC-HPLC method remains at 55% or more, 65% or more, 70% or more, 75% or more, or 80% or more.

[0037] In another aspect, the present application relates to use of the pharmaceutical preparation in the preparation of a medicament for treating an angiogenesis-related disease or a diagnostic reagent for an angiogenesis-related disease. Alternatively, the present application relates to the pharmaceutical preparation for use in treating or diagnosing an angiogenesis-related disease. Alternatively, the present application relates to a method for treating or diagnosing an angiogenesis-related disease, comprising administering the pharmaceutical preparation to a subject in need thereof. Alternatively, the present application relates to a medicament comprising the pharmaceutical preparation for use in treating or diagnosing an angiogenesis-related disease.

[0038] In some embodiments, the disease is a solid tumor or an ocular disease. In some preferred embodiments, the disease is a solid tumor, preferably melanoma, wherein the pharmaceutical preparation can be used to inhibit neovascularization and / or tumor growth within the tumor. In other preferred embodiments, the disease is an ocular disease, preferably age-related macular degeneration, diabetic retinopathy, retinal vascular occlusion, and corneal neovascularization.

[0039] In another aspect, the present application relates to a kit comprising said pharmaceutical formulation.

[0040] In some embodiments, the kit further comprises one or more additional active agents that can be co-administered with the pharmaceutical formulation of the present application. The co-administration can be in the form of simultaneous, concurrent, or sequential administration in any order.

[0041] In some embodiments, the active agent may be any one of a chemotherapeutic agent, a radiotherapeutic agent, an anti-angiogenic agent, an immunosuppressant, an anti-fibrotic agent, a neuroprotective agent, and an agent with tumor suppressive effects.

[0042] In some embodiments, the kit further comprises a device for administration to a subject, hi some embodiments, the device for administration to a subject is a syringe (e.g., a prefilled syringe, a needleless syringe, an injection pen), an inhalation device, an implantable device, or an infusion device.

[0043] In some embodiments, the kit may further comprise, for example, instructions, other reagents such as markers or coupling reagents, and / or a pharmaceutically acceptable carrier.

[0044] In some embodiments, the bispecific binding protein comprising the pharmaceutical formulation, medicament, or kit of the present application comprises: (i) inhibiting the activation of the VEGFR2 signaling pathway induced by hVEGFA alone, preferably by measuring the IC50 value of the inhibitory activity using a KDR reporter molecule assay test, wherein the IC50 value is, for example, 0.1 to 10 nM, for example, about 0.2 to 1 nM, for example, 0.6 nM, or about 1 to 3 nM, for example, about 1 nM; (ii) inhibiting the activation of the VEGFR2 signaling pathway induced by hVEGFC alone, preferably by measuring the IC50 value of the inhibitory activity using a KDR reporter molecule assay test, wherein the IC50 value is 0.1 to 1 nM, for example, about 0.2 to 0.6 nM; (iii) inhibiting hVEGFC-induced lymphatic cell proliferation, preferably determined by measuring the inhibitory activity IC50 value using a cell proliferation assay test, wherein the IC50 value is 0.01 to 0.5 nM, for example, about 0.1 to 0.5 nM; (iv) inhibiting hVEGFC and hVEGFA co-induced endothelial cell survival and proliferation at an inhibition level of at least 80%, 85%, 90%, 95%, or about 100%, preferably determined by measuring the inhibitory activity IC50 value using a cell proliferation assay test, wherein the IC50 value is 0.1 to 0.5 nM, for example, about 0.2 nM; (v) inhibiting the formation of angiogenic structures co-induced by hVEGFC and hVEGFA at an inhibition level of at least 80%, 85%, 90%, 95%, or about 100%, preferably as determined by measuring inhibitory activity using an endothelial cell tube formation assay, e.g., the method described in Example 13; (vi) inhibiting angiogenesis in tumors (e.g., solid tumors, e.g., melanoma); (vii) inhibiting the growth of tumors (e.g., solid tumors, e.g., melanoma), for example, in tumor-bearing animal models, the tumor inhibition rate by single-agent administration reaches 50% or more; (viii) inhibiting the onset and / or progression of angiogenesis-associated ocular diseases, for example, reducing the level of angiogenesis, reducing extravasation, inhibiting angiogenesis-induced retinal edema and / or fundus thickening; The compound may achieve one or more biological activities selected from the following:

[0045] The pharmaceutical formulations, medicaments and kits described herein may be used in vivo and / or in vitro for the following purposes: binding to VEGFA and / or VEGFC antigens, Blocking the binding of VEGFA and / or VEGFC to their associated receptors, such as VEGFR2 and / or VEGFR3; Inhibition of VEGFA and / or VEGFC-induced activation of VEGFR2 and / or VEGFR3 cell signaling, Inhibition of vascular endothelial cell survival, proliferation and / or migration induced by VEGFA and / or VEGFC, Inhibition of lymphocyte survival and / or proliferation induced by VEGFC, Inhibition of VEGFC-induced lymphangiogenesis and lymphatic endothelial cell proliferation and migration, Inhibition of angiogenesis and / or extravasation induced by VEGFA and / or VEGFC, · Inhibition of tumor angiogenesis and / or growth and / or migration. [Brief explanation of the drawings]

[0046] [Figure 1] 1 shows a VEGFR3 blocking experiment of anti-VEGFC affinity matured VHHs. [Figure 2] 1 shows a blocking experiment of affinity-matured anti-VEGFC VHH antibody on VEGFC-induced activation of HEK 293 KDR reporter. [Figure 3] 1 shows a blocking experiment of affinity matured anti-VEGFC VHH antibodies against VEGF-C-induced proliferation of BaF3-FLT4 cells. [Figure 4] 1 shows a blocking experiment of bispecific antibody molecules on VEGFA-induced activation of the HEK 293 KDR reporter. [Figure 5] 1 shows a blocking experiment of bispecific antibody molecules on VEGFC-induced activation of the HEK 293 KDR reporter. [Figure 6] 1 shows an experiment of inhibiting the proliferation of BaF3-FLT4 cells induced by VEGFC using a bispecific antibody molecule. [Figure 7] 1 shows an experiment of inhibiting bispecific antibody molecules against VEGFA+C-induced HUVEC proliferation. [Figure 8A] 8A and 8B show an experiment of inhibiting VEGFA+C-induced HUVEC tube formation by bispecific antibody molecules. Figure 8A shows tube formation images, and Figure 8B shows tube formation statistics. [Figure 8B] 8A and 8B show an experiment of inhibiting VEGFA+C-induced HUVEC tube formation by bispecific antibody molecules. Figure 8A shows tube formation images, and Figure 8B shows tube formation statistics. [Figure 9] Figures 9A-9C show an experiment of inhibiting neovascularization of bispecific antibody molecules in an A375 subcutaneous tumor model. Figure 9A: A375 tumor mass statistics, Figure 9B: A375 tumor volume statistics, Figure 9C: A375 tumor CD31 staining image. [Figure 10] 1 shows an experiment of inhibiting bispecific antibody molecules against laser-induced CNV. [Figure 11] 1 shows fundus angiographic images of the inhibition of laser-induced choroidal neovascularization by anti-VEGFA / VEGFC bispecific antibodies. [Figure 12] 1 shows inhibition images of anti-VEGFA / VEGFC bispecific antibody on laser-induced retinal thickening OCT. [Figure 13A] 1 shows the inhibition of pathological changes in a laser-induced choroidal neovascularization model by an anti-VEGFA / VEGFC bispecific antibody. [Figure 13B]1 shows the inhibition of pathological changes in a laser-induced choroidal neovascularization model by an anti-VEGFA / VEGFC bispecific antibody. [Figure 14] Figure 1 shows a schematic diagram of the structure of a bispecific antibody constructed using a VEGF-A binding domain-Fc fusion protein and an anti-VEGF-C VHH antibody as building blocks. [Figure 15] SEC-HPLC raw spectra of samples left at different pH levels for two weeks are shown. [Figure 16-1] Non-reduced CE-SDS raw spectra of samples left at different pH levels for two weeks are shown. [Figure 16-2] Non-reduced CE-SDS raw spectra of samples left at different pH levels for two weeks are shown. [Figure 17] The graph shows the change in the purity (SEC-HPLC method) of the monomers and aggregates of samples with different formulations. [Figure 18] The graph shows the change over time in the purity of monomers, aggregates, and fragments of samples with different formulations (non-reducing CE-SDS method).

[0047] Specific Embodiments The present application will be described in detail below with reference to examples, but this does not mean that there are any adverse limitations on the present application. The present application has been described in detail herein, and specific embodiments thereof have been disclosed herein. Various changes and modifications to the specific embodiments of the present application that are within the scope of protection of those skilled in the art without departing from the spirit and scope of the present application are also included in the scope of protection of the present application.

[0048] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which this disclosure belongs.See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Perbal, A Practical Guide to Molecular Cloning (1984).

[0049] As used herein, unless the context clearly indicates otherwise, the word "or" is intended to include "and," and vice versa. As used herein, singular terms include plural references and vice versa, unless otherwise stated.

[0050] As used herein, unless otherwise specified, the terms "comprise," "comprises," and "comprising" or equivalents (e.g., contain, containing, include, including) are open-ended and should be understood to mean "including, but not limited to," meaning that other unspecified elements, components, and steps may be covered in addition to those listed.

[0051] The term "about," when used in conjunction with a numerical value, is meant to encompass numerical values ​​within a range having a lower limit of 5% below the specified numerical value and an upper limit of 5% above the specified numerical value.

[0052] As used herein, the terms "fusion protein" and "chimeric polypeptide" are used interchangeably to refer to a larger polypeptide formed by the fusion of at least two heterologous polypeptide sequences, optionally via a linker. Fusion proteins can be produced by recombinant expression.

[0053] As used herein, the term "ligand trap" molecule refers to a fusion protein comprising the ligand-interacting extracellular domain of a receptor and a human IgG Fc region. Currently, trap molecules have been developed that "trap" various VEGF ligands, including VEGFA and VEGFC. These trap molecules can be used to bind to and reduce the concentration of the corresponding ligand in the extracellular environment. In certain embodiments, the anti-VEGFA component of the present application comprises a trap molecule in the form of an Fc fusion protein that "traps" VEGFA. The trap molecule can inhibit VEGFA-induced signal transduction by competing with natural VEGFA cells for VEGFA binding. In certain embodiments, the trap molecule in the form of an Fc fusion protein comprises the amino acid sequence of SEQ ID NO:5. The VEGFA-trap molecule is formed by fusing the extracellular ligand-binding domains from VEGF receptors 1 and 2 to the Fc portion of human IgG1, and contains all human amino acids, thereby minimizing the immunogenicity of the molecule in vivo.

[0054] As used herein, the term "VHH domain" refers to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain (sometimes referred to herein as an HcAb antibody), also known as a single variable domain fragment or nanobody. Thus, unlike the VH domains of conventional four-chain immunoglobulins, VHH domains do not need to pair with a light chain variable domain to form an antigen-binding site. Such VHH domain molecules can be derived from antibodies produced in camelids (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Heavy chain antibodies naturally lacking light chains can also be produced in species other than camelids, and such VHHs are also within the scope of the present invention. In certain cases, reducing the immunogenicity of VHH domains is desirable for therapeutic applications. Therefore, preferably, in certain embodiments, the VHH domains used herein are humanized VHH domains or further sequence-optimized forms thereof (e.g., affinity-matured forms with increased binding affinity).

[0055] As used herein, the term "single domain antibody" or "sdAb" refers to an antibody polypeptide that recognizes and binds to a target antigen via a single variable antibody domain (e.g., a VHH, a single VH, or a single VL). The single variable antibody domain of a single domain antibody can recognize and bind to a target antigen without being paired with other antibody variable domains. As used herein, a single domain antibody comprising a heavy chain variable domain (VHH) of a heavy chain antibody is also referred to as a VHH single domain antibody. The VHH single domain antibody used herein is preferably derived from a camelid, such as an alpaca, or is a humanized or sequence-optimized form thereof. In some embodiments, the VHH single domain antibody of the present application is a monovalent, monospecific polypeptide molecule consisting of, or consisting essentially of, a single VHH domain.

[0056] The "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" of an antibody refers to the region of the variable domain (VH or VHH) of an antibody that is highly variable in sequence, forms structurally determined loops ("hypervariable loops"), and / or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to an antigen epitope. The CDRs of heavy and light chains are generally referred to as CDR1, CDR2, and CDR3, and are numbered from the N-terminus. CDRs located in an antibody heavy chain variable domain are also referred to as HCDR1, HCDR2, and HCDR3, while CDRs located in an antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. For a given amino acid sequence of a light chain variable region or heavy chain variable region, its CDR sequences can be determined using various schemes known in the art. For example, there are Chothia, which is based on the three-dimensional structure and CDR loop topology of an antibody; Kabat, which is based on the variability of antibody sequences (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)); AbM (University of Bath); Contact (University College London); International Immunogenetics Database (IMGT) (International Immunogenetics Information System, www.imgt.cines.fr / ); and North CDR definition, which is based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

[0057] Below are the region boundaries of the CDRs as defined by the Kabat, AbM, Chothia, Contact and IMGT schemes.

[0058] [Table 0]

[0059] Unless otherwise stated, in this application the term "CDR" or "CDR sequence" includes CDR sequences determined in any of the above ways.

[0060] CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence. Unless otherwise specified, in this application, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), it refers to numbering positions based on the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0061] "Percent identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific amino acid sequence set forth herein when comparing the candidate sequence to the specific amino acid sequence set forth herein, introducing null positions as necessary to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0062] The present application contemplates variants of the antibody molecules of the present application, said variants having substantial identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more identity, to the antibody molecules and sequences specifically disclosed herein. Said variants may include conservative modifications.

[0063] With respect to polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions to a polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often involve replacing a particular amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists eight amino acid groups that contain conservative substitutions: 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the desired antigen-binding properties of the antibody molecule or binding protein molecule of the present application that contains the amino acid sequence. For example, conservatively modified variants maintain at least 80%, 85%, 90%, 95%, 98%, 99% or more, e.g., 100%-110% or more, of the binding affinity for the target antigen relative to the parent antibody or binding protein.

[0064] The terms "individual" and "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Individual specifically refers to humans.

[0065] The term "treatment" refers to a clinical intervention intended to alter the natural history of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or amelioration of the disease state, and amelioration or improvement of prognosis. In some embodiments, the antibody molecules of the present application are used to slow or delay disease progression.

[0066] The term "anti-tumor effect" or "tumor suppressor effect" refers to a biological effect that can be demonstrated by various means, including, but not limited to, a reduction in tumor size, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival. As used herein, the terms "tumor" and "cancer" are used interchangeably and include various solid tumors.

[0067] As used herein, the term "angiogenesis-associated disease" refers to a disease, condition, or pathology whose onset, progression, and / or course involves neovascularization (including neovascularization, neolymphatic vessel formation, and / or both), and which is ameliorated by inhibiting the biological activity of VEGF-C or VEGF-A, or both.

[0068] As used herein, FR1 to FR4 refer to framework regions 1 to 4, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0069] The bispecific binding proteins or variants thereof described herein have dual antagonistic activity, inhibiting the binding of VEGFA to a VEGF receptor and inhibiting the binding of VEGFC to a VEGF receptor.

[0070] Drug stability is one of the most important indicators of drug efficacy and safety. Obtaining a formulation that provides good drug stability is crucial for maintaining the drug's safety and efficacy throughout its shelf life. To obtain a formulation with good stability, the formulation is then evaluated and determined through high-temperature stability experiments, screening buffer pH and additives, and measuring appearance, visible foreign matter, protein content, purity, charge variants, polysorbate 80 content, and biological activity.

[0071] Example In the following examples, experimental methods for which no specific conditions are given are carried out according to conventional conditions. Unless otherwise specified, the reagents, materials, or equipment used in the following examples are commercially available conventional products unless the manufacturer is specifically stated.

[0072] Example 1: Expression and purification of anti-VEGFC humanized VHH antibody-like proteins Plasmids containing nucleic acids encoding the anti-VEGF C antibody am63G12.1-5G8-18B9 (VHH sequence SEQ ID NO:4, CDR1: SEQ ID NO:1; CDR2: SEQ ID NO:2; CDR3: SEQ ID NO:3, where CDR1 is defined according to the Kabat and Chothia combination, and CDR2 and CDR3 are defined according to Kabat) were prepared for transfection of 293 cells and expression of VHH single domain antibodies or VHH-Fc antibodies. Expi-293 cells (Invitrogen) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5 × 10 one day before transfection. 6 The cell density on the day of transfection was adjusted to approximately 3 × 10 cells / mL. 6The transfection volume was 1 / 10 of the final volume of F17 medium (Gibco, A13835-01) was used as the transfection buffer, and an appropriate amount of plasmid was added and mixed thoroughly. An appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid (the ratio of plasmid to PEI in 293F cells was 1:3), mixed thoroughly, and incubated at room temperature for 10 minutes to obtain a DNA / PEI mixture. The cells were resuspended in the DNA / PEI mixture and incubated at 36.5°C and 8% CO2 for 24 hours. Then, FEED (Sigma) equivalent to 2% of the transfection volume was added and cultured at 36.5°C, 120 rpm, and 8% CO2. Culture was continued until day 6 or cell viability reached 60% or less. The cell supernatant was collected and purified.

[0073] The collected medium prior to purification was centrifuged at 4500 rpm for 30 minutes, and the cells were discarded. The supernatant was then filtered through a 0.22 μm filter. A Protein A column (Hitrap Mabselect Sure 5*5 ml, GE, 11-0034-95) was equilibrated with 10 mL of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). The filtered supernatant was loaded onto the purification column and re-equilibrated with 15 mL of binding buffer. 5 mL of elution buffer (citric acid + 0.1 M sodium citrate, pH 3.5) was added, and the eluate was collected. 80 μL of Tris-HCl was added per mL of eluate. The collected antibody was ultrafiltered, concentrated, and exchanged into PBS (Gibco, 70011-044) for concentration measurement.

[0074] Example 2 Measurement of antigen binding kinetics of the present antibody by biofilm thin layer interferometry The equilibrium dissociation constant (K D) was measured by biofilm thin layer interferometry (ForteBio). ForteBio affinity measurements were performed using a conventional method (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): P270-8).

[0075] Thirty minutes before the start of the experiment, an appropriate number of AMQ (Pall, 1506091) (for sample measurement) or AHQ (Pall, 1502051) (for positive control measurement) sensors were immersed in SD buffer (PBS 1x, BSA 0.1%, Tween-20 0.05%) depending on the quantity of sample (anti-VEGF C antibody am63G12.1-5G8-18B9).

[0076] 100 μL of SD buffer, antibody, and antigen (human VEGFC (Beijing Yiqiao)) were added to a 96-well black polystyrene half-volume microplate (Greiner, 675076). The sample position was plated and the sensor position selected. The instrument settings were as follows: Run sequence: Baseline, Loading ~ 1 nm, Baseline, Association, and Dissociation. The run time for each sequence depended on the binding and dissociation rates of the sample. The rotation speed was 400 rpm, and the temperature was 30°C. D Values ​​were analyzed using ForteBio analysis software.

[0077] In the experiments using the above-mentioned measurement method, the affinity of the antibodies is shown in Table 1.

[0078] [Table 1]

[0079] In the above test, the binding affinity of the affinity matured antibody Am63G12.1-5G8-18B9 showed further increase in binding affinity, and monovalent K D The value was 5.70E-09M.

[0080] Example 3 Anti-VEGFC VHH antibody ELISA blocking experiment The inhibitory effect of the present antibody on hVEGFC binding to the receptor KDR was measured.

[0081] Streptavidin (SA) was diluted to 1 μg / mL and plated at 100 μL / well on an ELISA plate and incubated overnight at 4°C. After washing three times with PBST, the plate was sealed with 3% BSA for 1.5 hours. After washing three times with PBST, 50 ng / mL of VEGFC-biotin (biotin-labeled human VEGFC) was added and incubated for 1.5 hours. 50 μL of antibody and VEGFR2-Fc (final concentration 0.2 μg / mL, Beijing Yiqiao) or VEGFR3-Fc (final concentration 0.2 μg / mL, Beijing Yiqiao) were added to the plate for 20 minutes. After washing three times with PBST, anti-human FcHRP antibody (diluted 1:10,000 in BETHYL) was added and incubated for 30 minutes. After washing six times with PBST, the plate was developed with TMB (SOLARBIO) for 5 minutes, and the OD450nm value was read after the development.

[0082] The blocking results of the anti-VEGF C VHH antibodies obtained in this study are shown in Figure 1. The candidate molecule Am63G12.1-5G8-18B9 antibody was able to block VEGFC and VEGFR3 binding, with an inhibitory IC50 value comparable to that of the positive control molecule OPT-302 (VEGF-C-trap, SEQ ID NO:9).

[0083] Example 4: Experiment to demonstrate that anti-VEGFC VHH antibodies block VEGFC-induced activation of HEK293 KDR reporter VEGFC binds to the related receptor VEGFR2 (KDR), activates the VEGFR2 signaling pathway, and induces vascular endothelial cell survival, proliferation, migration, and other functions. In this study, we used a KDR reporter experimental system to measure the inhibitory effect of gradient-diluted antibodies on VEGFC-induced activation of the related receptor signaling pathway using NFAT-RE-luc2P / KDR HEK293 cells (Promega Cat CS181401).

[0084] The experimental method was based on the supplier's instructions.

[0085] NFAT-RE-luc2P / KDR HEK293 cells were extracted from the experimental medium (DMEM medium containing 10% FBS) three days prior, the old medium was aspirated, and the cells were washed once with PBS. Afterwards, they were digested with 1 mL of Accutase solution (Sigma) until the cells became round and separated from the cell wall. The reaction was terminated with 5 mL of diluted medium (DMEM medium containing 10% FBS). The cells were aspirated into a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The medium was discarded, and 10 mL of diluted medium was added to resuspend the cells. After mixing well, the cells were counted and cell viability was 90% or higher. The cell density in the diluted medium was adjusted to 0.8 x 10 6 The solution was adjusted to cells / mL and added at 50 μL / well to a 96-well white cell culture plate according to the experimental layout.

[0086] A mixture of 200 ng / mL hVEGFC (R&D) and gradient-diluted test antibodies (including the present VHH antibody, positive control molecule (OPT-302), and negative control antibody (IgG isotype control antibody)) was prepared and allowed to stand for 30 minutes. After that, 50 μL / well of the mixture was added to a 96-well white cell culture plate containing the cells and incubated for 6 hours at 37°C in a 5% CO incubator. A blank control group without any antibody or VEGFC and a VEGF-C experimental group without any antibody but with VEGFC alone were also set up.

[0087] After 6 hours of incubation, the 96-well white cell culture plate was removed from the CO2 incubator and allowed to equilibrate to room temperature for 10-15 minutes. According to the experimental layout, 100 μL of Bio-Glo Luciferase Assay System (Promega), which had been removed and equilibrated to room temperature, was added to the 96-well white cell culture plate at room temperature in the dark for 5 minutes.

[0088] The fluorescence value was read using a multi-function microplate reader, the plate reading mode was set to chemiluminescence mode, the plate reading type was set to end point method, the wavelength was set to all wavelengths, the fluorescence was collected row by row, and the collection time for each row was 1000 ms.

[0089] As shown in Figure 2, the Am63G12.1-5G8-18B9 antibody was able to completely inhibit the activation of the KDR signaling pathway induced by VEGFC, with an IC50 of approximately 4.457 nM, which was lower than that of its parent antibody (data not shown).

[0090] Example 5: Inhibitory experiment of anti-VEGFC VHH antibody on VEGFC-induced BaF3-FLT4 cell proliferation In this study, BaF3 cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) overexpressing FLT4 (VEGFR3), BaF3-FLT4, were co-cultured with antibodies and recombinant human VEGFR protein. The number of viable cells was detected using a CCK-8 kit (Dojindo Laboratories) to reflect the inhibitory effect of different antibodies on VEGFR-induced BaF3-FLT4 proliferation.

[0091] BaF3 cells were infected with a lentivirus carrying the FLT4 gene to obtain BaF3 cells, BaF3-FLT4, that overexpress FLT4.

[0092] A cell growth inhibition test was performed according to the instructions in the CCK-8 kit. Experimental medium was prepared using 1640 medium containing 10% FBS. The highest final concentration of the test antibodies (including the present VHH antibody, positive control molecule (OPT-302), and negative control antibody (IgG isotype control antibody)) was 20 μg / mL, and serial dilutions were performed at a ratio of 1:3. A blank control group (blank) was also set up, in which no antibody or VEGFC was added, and a VEGF-C experimental group was also set up, in which only VEGFC was added without any antibody. In the assay system, the final concentration of hVEGFC (R&D) was 20 ng / mL, and the final concentration of BaF3-FLT4 cells was 2*10 5 100 μL of the culture solution in each well of a 96-well plate was incubated in a CO 2 incubator at 37°C for 72 hours.

[0093] Then, 15 μL of CCK-8 was added to each well and incubated for 4 hours in a CO2 incubator at 37°C. The absorbance was measured at dual wavelengths, with a detection wavelength of 450 nm and a reference wavelength of 620 nm. OD450-OD620 values ​​were measured.

[0094] The experimental results are shown in Figure 3. The present Am63G12.1-5G8-18B9 antibody was able to effectively inhibit the survival and proliferation of BaF3-FLT4 cells induced by hVEGFC in vitro, with an IC50 of 0.7917 nM, which was lower than that of its parent antibody (data not shown).

[0095] Example 6 Construction of anti-VEGFA / VEGF C bispecific antibodies Using am63G12.1-5G8-18B9, an anti-VEGFA / VEGFC bispecific antibody was constructed and eukaryotic protein expression was performed in 293 cells according to Example 1. A bispecific antibody comprising a first polypeptide chain and a second polypeptide chain, as shown in Figure 14, was constructed by linking a VHH single-domain antibody to the C-terminus of the Fc region of VEGF-Trap (Aflibercept, Eylea) via a linker. VEGF-Trap, which binds to VEGF-A with high affinity in the form of a dimer and blocks activation of the VEGF-A-induced VEGFR signaling pathway, is a recombinant fusion protein (SEQ ID NO: 5) in which the VEGF-A-binding domain (SEQ ID NO: 6) derived from human VEGF receptors 1 and 2 is fused to the C-terminus of a human IgG1 Fc region (SEQ ID NO: 7).

[0096] Table 2 below shows the constructed bispecific antibodies and their configurations.

[0097] [Table 2]

[0098] Example 7 Blocking experiment of anti-VEGFA / VEGF C bispecific antibody against VEGFA or C-induced activation of HEK293 KDR reporter VEGFA or VEGFC binds to the related receptor VEGFR2 (KDR), activates the VEGFR2 signaling pathway, and induces effects such as survival, proliferation, and migration of vascular endothelial cells. In this study, we used a KDR reporter experimental system and NFAT-RE-luc2P / KDR HEK293 cells (Promega Cat CS181401) to measure the inhibitory effect of gradient-diluted antibodies on the activation of the related receptor signaling pathway by VEGFA and VEGFC. The experimental method was almost the same as that described in Example 4. In summary, NFAT-RE-luc2P / KDR HEK293 cells (0.8 × 10 6A 96-well white cell culture plate containing 100 ng / mL hVEGFA (R&D) or 200 ng / mL hVEGFC (R&D) was prepared. A gradient dilution of the test antibody mixture was prepared and incubated for 30 minutes. 50 μL of the mixture was added to the 96-well white cell culture plate containing NFAT-RE-luc2P / KDR HEK293 cells and incubated for 6 hours at 37°C in a 5% CO2 incubator. A blank control group (no antibody or VEGFC), an experimental group (no antibody but VEGFC) and an experimental group (VEGFC and an IgG isotype control antibody) were also set up. As controls, the VEGF-A signaling pathway inhibitory activity of the anti-VEGF A molecule IBI304 (SEQ ID NO:10) and the bispecific antibody Faricimab (anti-Ang-2 / anti-VEGF-A), and the VEGF-C signaling pathway inhibitory activity of the anti-VEGF-C molecule OPT-302 were also measured.

[0099] After 6 hours of incubation, the 96-well white cell culture plate was removed from the CO2 incubator and allowed to equilibrate to room temperature for 10-15 minutes. According to the experimental layout, 100 μL of Bio-Glo Luciferase Assay System (Promega), which had been removed and equilibrated to room temperature, was added to the 96-well white cell culture plate at room temperature in the dark for 5 minutes.

[0100] The fluorescence value was read using a multi-function microplate reader, the plate reading mode was set to chemiluminescence mode, the plate reading type was set to end point method, the wavelength was set to all wavelengths, the fluorescence was collected row by row, and the collection time for each row was 1000 ms.

[0101] The measurement results are shown in Figures 4 and 5. All of the measured anti-VEGFA and anti-VEGFC bispecific antibodies were able to block activation of the KDR signaling pathway induced by VEGFA (Figure 4) or VEGFC (Figure 5).

[0102] Example 8 Inhibitory experiment of anti-VEGFA / VEGFC bispecific antibody against VEGFC-induced proliferation of BaF3-FLT4 The bispecific antibody of the present application was applied to a BaF3-FLT4 proliferation experimental system to measure the effect of the antibody on VEGFC-induced BaF3-FLT4 proliferation. The experimental method was almost the same as that described in Example 5.

[0103] Experimental medium was prepared using 1640 medium containing 10% FBS. The highest final concentration of the test antibody was 10 nM, and serial dilutions were performed at a ratio of 1:3. In the assay system, the final concentration of hVEGFC (R&D) was 20 ng / mL, and the final concentration of BaF3-FLT4 cells was 2*10 5 Expressed as cells / mL.

[0104] The following experimental groups were set up: a blank control group (no antibody or VEGFC added), an experimental group (no antibody added but VEGFC added), and an experimental group (VEGF-C added with an IgG isotype control antibody). The inhibitory activity of the anti-VEGF-C molecule OPT-302 on VEGFC-induced proliferation of BaF3-FLT4 cells was also measured.

[0105] The measurement results are shown in FIG. 6, and all of the measured anti-VEGFA / VEGFC bispecific antibodies are capable of inhibiting the proliferation of BaF3-FLT4 cells induced by VEGFC.

[0106] Example 9 Inhibitory experiment of anti-VEGFA / VEGFC bispecific antibody against VEGFA+VEGFC-induced HUVEC proliferation VEGFA and VEGFC act on VEGFR and other related receptors in vascular endothelial cells, promoting their survival, proliferation, and migration, and inducing neovascularization. In this experiment, VEGFA and VEGFC were used to induce human umbilical vein endothelial cell (HUVEC) survival and proliferation, and the inhibitory effects of antibodies on VEGFA- and VEGFC-induced primary cell survival and proliferation were measured. In this example, CCK-8 was used to measure HUVEC survival and proliferation. The specific method is as follows: Cells were treated one day in advance, plated at 2,000 cells / well in a 96-well culture plate, and incubated for 24 hours at 37°C in a 5% CO2 incubator. After the cells adhered to the wall, experimental medium (DMEM medium) containing or not containing final concentrations of 5 ng / mL VEGFA and 50 ng / mL VEGFC and / or gradient-diluted antibodies was prepared, and the endothelial cell medium in the 96-well plate was replaced with this medium and incubated at 37°C in a 5% CO2 incubator for 72 hours. Experimental groups were divided as follows:

[0107] Blank group: DMEM medium VEGFA group: DMEM medium + 5ng / mL VEGFA VEGFC group: DMEM medium + 50ng / mL VEGFC VEGFA + VEGFC group: DMEM medium + 5 ng / mL VEGFA + 50 ng / mL VEGFC IgG group: DMEM medium + 5ng / mL VEGFA + 50ng / mL VEGFC + isotype control IgG IBI304 + OPT-302 group: DMEM medium + 5 ng / mL VEGFA + 50 ng / mL VEGFC + IBI304 + OPT-302 (IBI304 and OPT-302 were combined at a molar ratio of 1:1) Bispecific antibody group: DMEM medium + 5ng / mL VEGFA + 50ng / mL VEGFC + test bispecific antibody After incubating the cells in experimental medium, 10 μL / well of CCK-8 detection solution (Dojindo Laboratories) was added and the cells were incubated in a 37°C, 5% CO2 incubator for 12 to 24 hours. The absorbance (OD) was measured using a multifunctional microplate reader. 450 -OD 620 The value was read.

[0108] In the experiment using the above assay method, the measurement results are shown in Figure 7. The measured anti-VEGFA and anti-VEGFC bispecific antibodies were able to completely inhibit the proliferation and survival of HUVEC cells induced by VEGFA+VEGFC.

[0109] Example 10: Inhibitory experiment of anti-VEGFA / VEGFC bispecific antibody on HUVEC tube formation induced by VEGFA+VEGFC VEGFA and VEGFC act on vascular endothelial cells to promote the formation of tube-like structures in vascular endothelial cells, inducing the formation of neovascular structures. In this experiment, VEGFA and VEGFC were used to induce human umbilical vein endothelial cell (HUVEC) tube formation, and the inhibitory effect of antibodies on VEGFA- and VEGFC-induced primary cell tube formation was measured.

[0110] In this example, we performed a HUVEC tube formation assay to measure the inhibitory effect of anti-VEGFA / VEGFC bispecific antibodies on HUVEC tube formation. The specific method was as follows: Matrigel (BD) was thawed on ice one day prior to the experiment, added to a 96-well plate at 100 μL per well, and allowed to harden in a 37°C, 5% CO2 incubator for 30 minutes. Accutase solution-treated cells were plated into a 96-well culture plate at 20,000 cells / well and incubated for 24 hours in a 37°C, 5% CO2 incubator. Experimental media (EGM2) were prepared with or without a final concentration of 5 ng / mL VEGFA and 50 ng / mL VEGFC and / or gradient-diluted antibodies. HUVEC cells were resuspended in different experimental media, plated into a 96-well culture plate at 20,000 cells / well, and incubated for 24 hours in a 37°C, 5% CO2 incubator. Experimental groups were divided as follows:

[0111] Blank group: EGM-2 medium VEGFA group: EGM-2 medium + 5ng / mL VEGFA VEGFC group: EGM-2 medium + 50 ng / mL VEGFC VEGFA + VEGFC group: EGM-2 medium + 5 ng / mL VEGFA + 50 ng / mL VEGFC IgG group: EGM-2 medium + 5 ng / mL VEGFA + 50 ng / mL VEGFC + 20 nM isotype control IgG IBI304 + OPT-302 group: EGM-2 medium + 5 ng / mL VEGFA + 50 ng / mL VEGFC + 20 nM IBI304 + 20 nM OPT-302 IEX04-056 group: EGM-2 medium + 5ng / mL VEGFA + 50ng / mL VEGFC + 20nM IEX04-056.

[0112] Images were taken under a microscope, and the number of tubes was calculated. The measurement results are shown in Figures 8A and 8B. Figure 8A shows the tube formation images, and Figure 8B shows the tube formation statistics. The anti-VEGFA and anti-VEGFC bispecific antibodies completely inhibited the tube formation of HUVEC cells induced by VEGFA + VEGFC.

[0113] Example 11: Inhibition experiment of anti-VEGFA / VEGFC bispecific antibody against A375 tumor angiogenesis Overexpression of VEGFA and VEGFC in tumor cells can induce neovascularization in vivo and promote tumor growth.

[0114] In this example, nude mice were used, and A375 human melanoma cells were inoculated at 3*10 cells per mouse. 6 The anti-angiogenic and anti-tumor effects of the anti-VEGFA / VEGFC antibodies of the present invention were measured by cell inoculation.

[0115] Humanized nude mice: Female BALB / c background nude mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. and were SPF grade. The mice were housed for 7 days after arrival, after which the study began.

[0116] cell: Human A375 cells were purchased from ATCC (CAT#: CRL-1619) and subcultured according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in sterile PBS to a cell density of 1.5 × 10 7 On day 0, 0.2 mL of the cell suspension was collected and subcutaneously inoculated into the axilla of a human mouse to create an A375 tumor-bearing mouse model.

[0117] Dosage: Mice were randomly divided into groups (6 mice per group). Tumor volume was measured for each mouse on days 7 and 21 after tumor cell inoculation. The doses and administration methods are shown in Table 3. PBS (purchased from Gibco) was administered as a negative control on days 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 after inoculation, respectively. Tumor volume and body weight of the mice were monitored twice weekly. Body weight and tumor volume were measured before each administration, and the relative tumor inhibition rate (TGI%) was calculated on day 21 after inoculation using the following formula: TGI% = 100% * (tumor volume in the control group - tumor volume in the treatment group) / (tumor volume in the control group - tumor volume before administration in the control group). Tumor volume measurement: The longest axis (L) and widest axis (W) of the tumor were measured using calipers, and tumor volume was calculated using the following formula: V = L × W. 2 / 2. Weight was measured using an electronic balance.

[0118] [Table 3]

[0119] The results of tumor inhibition rates are shown in Figures 9A and 9B and Table 4. At 21 days after inoculation, tumor inhibition was observed at each dose of IEX04-056 alone. At 21 days after inoculation, the tumor inhibition rates were 54% at 1 mg / kg, 71% at 5 mg / kg, and 77% at 25 mg / kg. Therefore, the bispecific binding protein of the present application targeting VEGFA and VEGFC has a significant tumor-inhibitory effect on tumors.

[0120] [Table 4]

[0121] Tumor tissues were sectioned on days 7 and 21 after inoculation and stained for CD31 (a vascular endothelial marker), demonstrating microvascularization in the tumor tissue. The wax blocks embedded in OCT compound were placed in a freezing microtome and cut into 6-μm-thick sections.

[0122] 1. The sections were left at room temperature for 30 minutes, fixed with acetone pre-cooled to 4°C, and air-dried at room temperature.

[0123] 2. Immerse in PBST and rinse three times for 5 minutes each (PBST: 1x PBS + 0.05% Tween 20).

[0124] 3. A circle was drawn using an immunohistochemical staining pen, and then the tissue was sealed with 10% goat serum (diluted in PBST), 100 μL of which was added dropwise to each tissue, and the tissue was sealed at room temperature.

[0125] 4. The primary antibody PE-anti-mouse CD31 (100 μL) was added dropwise directly and incubated at 4°C overnight.

[0126] 5. The next day, soak in PBST and rinse three times for 5 minutes each time.

[0127] 6. Cell nuclei were stained with DAPI, and 100 μl of the stain was added dropwise to each tissue and incubated for 5 minutes in the dark.

[0128] 7. Add DAPI staining solution, immerse in PBST, and rinse 3 times for 5 minutes each time.

[0129] Sections were blocked with anti-fluorescence quenching blocker (prolonged diamond antifade mountant, Invitrogen) and then scanned using a fully automated quantitative analysis scanner. The results are shown in Figure 9C. After IEX04-056 treatment, A375 tumor neovascularization was significantly reduced.

[0130] Example 12. Pharmacodynamic study of laser-induced choroidal neovascularization In this experiment, the inhibitory effect of the present anti-VEGFA / VEGFC bispecific antibody on angiogenesis was measured using a cynomolgus monkey model of laser-induced choroidal neovascularization.

[0131] Species: Cynomolgus monkey, Grade: Normal, Age: 2.5-5 years, Weight: 2.45-5.55 kg, average 4.02 kg, Weight at modeling 3.35-4.35 kg.

[0132] In this experiment, to establish an animal model similar to human choroidal neovascularization, laser photocoagulation was performed around the central macula of the fundus of rhesus monkeys to induce choroidal neovascularization. Fluorescein fundus angiography was performed before and 20 days after photocoagulation to assess the modeling status. Twelve rhesus monkeys (half male and half female) that successfully modeled were divided into three groups (four monkeys per group): the model control group, the IEX04-056 group, and the Eylea + OPT-302 group. On day 21 after photocoagulation, IEX04-056 or Eylea + OPT-302 was injected into the vitreous of both eyes at the doses shown in Table 5 below, and the model control group received an equal volume of 0.9% sodium chloride injection. The animals in each group were subjected to color fundus photography, fluorescein fundus angiography, and optical coherence tomography examination on days 7, 14, 21, and 28 after administration to observe the inhibition of choroidal neovascularization by the test product. On day 29 after administration, the animals were euthanized, and both eyes were collected for histological examination with HE staining.

[0133] [Table 5]

[0134] Color fundus photography and fluorescein angiography Evaluation metrics: (1) Fluorescent spot evaluation Grading criteria for fluorescent spots captured by fluorescein angiography after modeling: Grade 1: Fluorescent spots do not show hyperfluorescence. Grade 2: The fluorescent spots show hyperfluorescence but there is no leakage of fluorescein. Grade 3: The fluorescent spot is hyperfluorescent, showing mild fluorescein leakage, but the leakage does not extend beyond the edge of the spot. Grade 4: The fluorescent spot was hyperfluorescent, exhibiting significant fluorescein leakage, with leakage beyond the edge of the spot.

[0135] Fluorescence spots of grades 1 to 4 were statistically analyzed, and the grade of fundus laser spots was recorded for each examination.

[0136] (2) Improvement rate of fluorescein leakage area Fluorescein leakage area improvement rate (%) = (fluorescein leakage area before administration - fluorescein leakage area after administration) / fluorescein leakage area before administration * 100%

[0137] (3) Reduction in fluorescein leakage area Reduction in fluorescein leakage area = fluorescein leakage area before administration - fluorescein leakage area after administration Optical Coherence Tomography (OCT) Evaluation metrics: 1) Improvement rate of fundus retinal thickening

number

[0138] (2) Reduction in fundus retinal thickness Reduction in fundus retinal thickness = fundus retinal thickness before administration - fundus retinal thickness after administration The results of color fundus photography and fluorescence angiography are shown in Figures 10 and 11. The present antibody showed a significant angiogenesis inhibitory effect 28 days after administration, demonstrating that the present antibody has a significant inhibitory effect (P<0.001) on laser-induced fundus neovascularization while also protecting the integrity of blood vessels. The OCT results are shown in Figure 12. The present antibody significantly inhibited retinal thickening 14 to 28 days after administration (P<0.05), demonstrating that the present antibody has the function of inhibiting retinal edema and thickening caused by neovascularization.

[0139] Histopathological examination Twenty-nine days after administration, rhesus monkeys were anesthetized with pentobarbital sodium (approximately 30 mg / kg by intravenous injection, the dose could be adjusted depending on the health condition of the animals) according to their body weight, euthanized by exsanguination from the abdominal aorta or femoral artery, macroscopic observation was performed, and both eyeballs were enucleated.

[0140] In some animals, both eyes were fixed in modified Davidson's fixative, embedded in paraffin, sectioned, and the laser modeling area was selected for routine histopathological examination, including HE staining.

[0141] In pathological sections, the antibody of the present application was able to better improve pathological changes such as edema, hyperplasia, and fibrosis at the laser-injured site compared to the combination of the anti-VEGFA molecule Eylea and the anti-VEGFC molecule OPT-032, and showed superior retinal morphological improvement (Figures 13A and 13B).

[0142] Next, high-temperature experiments were conducted to investigate the effects of different pH and different additives (such as sorbitol, sucrose, trehalose, arginine, sodium chloride, polysorbate 20, and polysorbate 80) on protein stability.

[0143] Example 13: Preliminary screening of low concentration formulations - Preformulation testing Buffer solutions containing 1.55 mg / mL histidine and 50.00 mg / mL sorbitol were prepared and adjusted to pH 5.0, 5.5, 6.0, 6.5, and 7.0 with dilute hydrochloric acid. The ultrafiltrated anti-VEGFA and VEGFC bispecific binding proteins prepared as described above were replaced with buffers of different pH values, and polysorbate 80 was added to adjust the protein content to approximately 120 mg / mL, resulting in a final concentration of 0.2 mg / mL. The samples were filtered, dispensed into vials, stoppered, and capped with rolling screws. The samples were subjected to stability testing at 40°C ± 2°C. The specific testing protocol is shown in Table 6.

[0144] [Table 6]

[0145] Based on an understanding of the sample being tested and the accuracy of the equipment and method, we established criteria for determining whether the sample measured value shows no change in quality compared to the initial value. Since standard testing methods for purity, charge isomers, activity, etc. have not yet been established for pre-formulation testing, these criteria are not currently included in the criteria for determining whether product quality has not changed. Specific criteria are shown in Table 7.

[0146] [Table 7]

[0147] Experimental results Pre-formulation testing showed that the pH 5.0 sample exhibited a cloudy appearance after two weeks at 40°C ± 2°C, while the samples at other pH conditions passed the test in terms of both appearance and visible impurities. As shown in Figures 15 and 16, the peak shapes of the purity (SEC-HPLC and non-reduced CE-SDS) of the samples at different pH conditions were significantly different. The purity of the sample at pH 6.0 was superior to that of the samples at other pH conditions. Subsequent formulation experiments will be conducted at pH 6.2.

[0148] Example 14 Screening of low-concentration formulations - formulation determination experiment This study mainly investigated the effect of excipients (histidine, sodium dihydrogen phosphate, sorbitol, sucrose, trehalose, arginine, sodium chloride, polysorbate 20, and polysorbate 80) on protein stability. Detailed prescribing information is shown in Table 8.

[0149] A buffer solution for each formulation was prepared according to Table 8, and the protein was ultrafiltered and replaced with the respective formulation solution. After the replacement was completed, the protein content of Formulations 1 to 4 (F1 to F4) was adjusted to 50 mg / mL. Polysorbate 80 or polysorbate 20 was added to the solution to a final concentration of 0.3 mg / mL. The solution was filtered, dispensed into vials, and then sealed with caps by rolling. The samples were subjected to stability testing at 40°C ± 2°C.

[0150] [Table 8]

[0151] The stability test plan is shown in Table 9, the evaluation criteria in Table 10, and the test results in Table 11.

[0152] [Table 9]

[0153] [Table 10] [Table 11-1] [Table 11-2]

[0154] The results in Table 11 show that after two weeks at 40°C ± 2°C, the appearance and visible foreign matter of the samples with different formulations all passed the test. No significant changes were observed in protein content or pH value. The purity of samples from all formulations (SEC-HPLC and non-reduced CE-SDS methods) changed significantly, with the purity of the sample from formulation 4 showing the least decrease (SEC-HPLC and non-reduced CE-SDS methods). Specific time series are shown in Figures 17 and 18.

[0155] To avoid pH adjustment with hydrochloric acid during manufacturing, the buffer system was adjusted to a mixed solution of histidine and L-histidine hydrochloride. At the same time, because the osmolality of Formulation 4 was low (262 mOsmol / kg), the trehalose concentration was increased to 90 mg / mL (the osmolality of the formulation at this point was 323 mOsmol / kg) to ensure isotonicity of the formulation buffer. The final formulation consisted of 50.0 mg / mL recombinant anti-vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor C (VEGFC) bispecific binding protein, 0.90 mg / mL histidine, 0.90 mg / mL L-histidine hydrochloride, 90.00 mg / mL trehalose, 0.3 mg / mL polysorbate 80, and pH 6.2.

[0156] Based on the above experimental results, the final formulation was determined as follows: recombinant anti-vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor C (VEGFC) bispecific binding protein 50.0 mg / mL, histidine 0.90 mg / mL, L-histidine hydrochloride 0.90 mg / mL, trehalose 90.00 mg / mL, polysorbate 80 0.3 mg / mL, pH 6.2. When this formulation is used, the anti-VEGFA and VEGFC bispecific binding protein of the present application can exhibit good stability and can demonstrate the desired safety and good efficacy in clinical settings.

[0157] It should be understood by those skilled in the art to which this application pertains that the description of any of the above embodiments is merely illustrative and does not mean that the scope of protection of the present disclosure is limited to these embodiments. Under the concept of the present disclosure, the technical features of the above embodiments or different embodiments can be combined, procedures can be performed in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure described above, which are not described in detail for the sake of brevity. The disclosed embodiments are intended to encompass all such alternatives, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any changes, equivalent substitutions, improvements, etc. made within the spirit and principles of the disclosed embodiments are included in the scope of protection of the present invention.

[0158] All patents, patent applications, and other publications are expressly incorporated herein by reference for purposes of explanation and disclosure. These publications are provided for their disclosure prior to the filing date of this application. Any statement as to the date of these documents or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any reference to these publications herein does not constitute an admission that the publications form part of the common general knowledge in the art in any country.

Claims

1. 1. A pharmaceutical formulation comprising: (1) an anti-VEGFA and VEGFC bispecific binding protein; (2) a buffering agent; (3) a stabilizer; and (4) a surfactant, wherein the pH of the pharmaceutical formulation is 5.5 to 7.

0.

2. the concentration of said bispecific binding protein is less than or equal to 80 mg / mL and greater than 0; Alternatively, the pharmaceutical formulation of claim 1, wherein the concentration of the bispecific binding protein is 20 mg / mL to 80 mg / mL, 50 mg / mL to 80 mg / mL, 40 mg / mL to 60 mg / mL, or 45 mg / mL to 65 mg / mL.

3. the buffer is a phosphoric acid or phosphate buffer system, or a histidine buffer system; Preferably, the histidine buffer system comprises histidine, or histidine and L-histidine hydrochloride; 3. The pharmaceutical formulation of claim 1, wherein the histidine buffer system comprises 1.0 mg / mL to 2.0 mg / mL, or 1.5 mg / mL to 1.6 mg / mL of histidine, or 0.80 mg / mL to 1.5 mg / mL, 0.80 mg / mL to 1.0 mg / mL, 0.80 mg / mL to 0.95 mg / mL, 0.85 mg / mL to 1.0 mg / mL, or 0.85 mg / mL to 0.95 mg / mL of histidine and 0.80 mg / mL to 1.5 mg / mL, 0.80 mg / mL to 1.0 mg / mL, 0.80 mg / mL to 0.95 mg / mL, 0.85 mg / mL to 1.0 mg / mL, or 0.85 mg / mL to 0.95 mg / mL of L-histidine hydrochloride.

4. the stabilizer is a sugar, a sugar alcohol, or a basic amino acid capable of stabilizing a protein; Preferably, the sugar is trehalose or sucrose; the sugar alcohol is sorbitol or mannitol, or The pharmaceutical formulation according to any one of claims 1 to 3, wherein the basic amino acid is arginine or lysine.

5. the sugar has a concentration of 50 mg / mL to 100 mg / mL, 80 mg / mL to 100 mg / mL, 85 mg / mL to 100 mg / mL, 80 mg / mL to 95 mg / mL, 85 mg / mL to 95 mg / mL, 80 mg / mL to 90 mg / mL, or 85 mg / mL to 90 mg / mL; the sugar alcohol has a concentration of 30 mg / mL to 80 mg / mL, 40 mg / mL to 60 mg / mL, 45 mg / mL to 55 mg / mL, or 50 mg / mL to 55 mg / mL; or 5. The pharmaceutical formulation of claim 4, wherein the concentration of the basic amino acid is 5 mg / mL to 30 mg / mL, 10 mg / mL to 20 mg / mL, 10 mg / mL to 15 mg / mL, 11 mg / mL to 15 mg / mL, 12 mg / mL to 15 mg / mL, or 13 mg / mL to 15 mg / mL.

6. 6. The pharmaceutical formulation of claim 4, wherein the sugar is trehalose at a concentration of 80 mg / mL to 100 mg / mL, 85 mg / mL to 100 mg / mL, 85 mg / mL to 95 mg / mL, 80 mg / mL to 90 mg / mL, or 85 mg / mL to 90 mg / mL.

7. the surfactant is a nonionic surfactant, Preferably, the surfactant is a polysorbate; Particularly preferably, the surfactant is polysorbate 20 or polysorbate 80; Alternatively, preferably, the surfactant is a polysorbate at a concentration of 0.1 mg / mL to 0.5 mg / mL, 0.25 mg / mL to 0.45 mg / mL, 0.2 mg / mL to 0.4 mg / mL, 0.25 mg / mL to 0.4 mg / mL, or 0.3 mg / mL to 0.4 mg / mL; or Particularly preferably, the surfactant is polysorbate 80 at a concentration of 0.2 mg / mL to 0.4 mg / mL, 0.25 mg / mL to 0.4 mg / mL, or 0.3 mg / mL to 0.4 mg / mL.

8. the pharmaceutical preparation is a liquid injection or a lyophilized powder; The pharmaceutical formulation according to any one of claims 1 to 7, wherein the pH of the pharmaceutical formulation is preferably 5.8 to 6.5, 6.0 to 6.5, or 6.0 to 6.

2.

9. The bispecific binding protein comprises: (i) a first antigen-binding moiety that specifically binds to human VEGFC, wherein the first antigen-binding moiety consists of an anti-VEGFC single domain antibody or an anti-VEGFC VHH domain, and the single domain antibody or VHH domain comprises a CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and 9. The pharmaceutical formulation of any one of claims 1 to 8, comprising (ii) a second antigen-binding moiety that specifically binds to human VEGFA.

10. The first antigen-binding component is a fusion protein comprising the anti-VEGFC single domain antibody or its VHH domain and an Fc portion thereof. Preferably, the fusion protein comprises the single domain antibody or its VHH domain and its C-terminal Fc portion. Particularly preferably, the VHH domain comprises (i) the amino acid sequence shown in SEQ ID NO: 4, (ii) an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of (i), or (iii) an amino acid sequence having at least 1 to 30, or 1 to 20, or 1 to 15, or 1 to 10, or 1 to 5 amino acid changes relative to the amino acid sequence of (i). Particularly preferably, the VHH domain comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 1 to 30, or 1 to 20, or 1 to 15, or 1 to 10, or 1 to 5 amino acid changes relative to the amino acid sequence of (i). It consists of the amino acid sequence shown in NO: 4, or, 10. The pharmaceutical formulation of claim 9, wherein the second antigen-binding component is a fusion protein comprising an Fc portion and a VEGFA-binding domain, and preferably the VEGFA-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and particularly preferably the VEGFA-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

6.

11. 11. The pharmaceutical formulation of claim 10, wherein the Fc portion is a human IgG Fc region, and the human IgGl Fc region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

12. 12. The pharmaceutical formulation of any one of claims 9 to 11, wherein the second antigen-binding moiety comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

13. the first antigen-binding moiety is linked or fused to the C-terminus or N-terminus of the second antigen-binding moiety; 13. The pharmaceutical formulation of any one of claims 9 to 12, wherein the bispecific binding protein comprises a linker connecting the first antigen-binding moiety to the second antigen-binding moiety, and the amino acid sequence of the linker is (GS)n or G(GS)n, where n is 1, 2, 3, 4, or 5.

14. 14. The pharmaceutical formulation of any one of claims 1 to 13, wherein the bispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are the same and each comprise the amino acid sequence shown in SEQ ID NO:

8.

15. the pharmaceutical formulation comprises 40 mg / mL to 80 mg / mL of the anti-VEGFA and VEGFC bispecific binding protein, 0.80 mg / mL to 1.0 mg / mL histidine, 0.80 mg / mL to 1.0 mg / mL L-histidine hydrochloride, 80 mg / mL to 100 mg / mL sugar, and 0.2 mg / mL to 0.5 mg / mL polysorbate, wherein the pH of the pharmaceutical formulation is 6.0 to 6.5; Alternatively, the pharmaceutical formulation comprises 40 mg / mL to 80 mg / mL of the anti-VEGFA and VEGFC bispecific binding protein, 0.80 mg / mL to 1.0 mg / mL histidine, 0.80 mg / mL to 1.0 mg / mL L-histidine hydrochloride, 80 mg / mL to 100 mg / mL trehalose, and 0.2 mg / mL to 0.5 mg / mL polysorbate 80, wherein the pH of the pharmaceutical formulation is 6.0 to 6.5; Alternatively, the pharmaceutical formulation comprises 40 mg / mL to 60 mg / mL of the anti-VEGFA and VEGFC bispecific binding protein, 0.85 mg / mL to 0.95 mg / mL histidine, 0.85 mg / mL to 0.95 mg / mL L-histidine hydrochloride, 85 mg / mL to 100 mg / mL trehalose, and 0.2 mg / mL to 0.4 mg / mL polysorbate 80, wherein the pH of the pharmaceutical formulation is 6.0 to 6.2; 15. Alternatively, the pharmaceutical formulation of any one of claims 1-14, comprising 50 mg / mL of the anti-VEGFA and VEGFC bispecific binding protein, 0.90 mg / mL histidine, 0.90 mg / mL L-histidine hydrochloride, 90.00 mg / mL trehalose, and 0.3 mg / mL polysorbate 80, wherein the pH of the pharmaceutical formulation is 6.

2.

16. 16. The pharmaceutical formulation according to any one of claims 1 to 15, wherein the osmolarity of the pharmaceutical formulation is 280 mOsmol / kg or more, 300 mOsmol / kg or more, or 300 to 350 mOsmol / kg.

17. The pharmaceutical formulation is stable in liquid form or freeze-dried powder form at 30°C to 50°C for at least 2 weeks or at least 4 weeks; The pharmaceutical formulation according to any one of claims 1 to 16, wherein the purity measured by SEC-HPLC is maintained at 55% or more, 65% or more, 70% or more, 75% or more, or 80% or more when the pharmaceutical formulation is left in liquid form at 30°C to 50°C for 2 weeks or more.

18. A pharmaceutical preparation for use in the treatment or diagnosis of angiogenesis-related diseases, comprising: Preferably, the disease is a solid tumor or an eye disease; Particularly preferred is the pharmaceutical preparation according to any one of claims 1 to 17, wherein the disease is melanoma, age-related macular degeneration, diabetic retinopathy, retinal vascular occlusion or corneal neovascularization.

19. A kit comprising the pharmaceutical formulation of any one of claims 1 to 17.

20. The kit may comprise one or more additional active agents to be administered in combination with the pharmaceutical formulation; a device for administering to a subject, and / or further comprising instructions, a marker or coupling reagent, and / or a pharmaceutically acceptable carrier; 20. The kit of claim 19, wherein the active agent is preferably any one of a chemotherapeutic agent, a radiotherapeutic agent, an anti-angiogenic agent, an immunosuppressant, an anti-fibrotic agent, a neuroprotective agent, and an agent having tumor-suppressing effects, or wherein the device for administration to a subject is preferably a syringe, an inhalation device, an implantable device, or an infusion device.

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