Bispecific binding molecules that specifically bind to VEGF and PKa

Abstract

The present disclosure provides bispecific binding molecules that specifically bind to VEGF and PKa. The bispecific binding molecules of the present disclosure comprise a first binding module that specifically binds to VEGF, and a second binding module (especially an anti-PKa antibody or an antigen binding fragment thereof) that specifically binds to PKa, wherein the anti-PKa antibody or the antigen binding fragment thereof is directly or indirectly linked to the first binding module. The invention also provides a preparation method of the bispecific binding molecule and application of the bispecific binding molecule in treating diseases.

Description

[0001] This application claims priority to Chinese patent application 2023113225623 filed on October 12, 2023. Technical Field

[0002] The present disclosure belongs to the field of biopharmaceuticals. Specifically, the present disclosure relates to a first binding module that specifically binds to anti-plasma kallikrein (PKa) and a second binding module that specifically binds to VEGF, as well as a bispecific binding molecule formed by the fusion of the first binding module and the second binding module, and its preparation method and application. In particular, the bispecific binding molecule is used to treat or prevent diseases mediated by high levels of VEGF protein and PKa protein, such as microangiopathy-related diseases (such as but not limited to proliferative retinopathy, neovascular glaucoma, diabetic macular edema (DME) and age-related macular degeneration (AMD)). Background Art

[0003] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0004] The VEGF-VEGFR family is the most studied angiogenic regulatory factor, which induces endothelial cell differentiation and angiogenesis. There are five main receptors that bind to VEGF ligands: VEGFR-1, VEGFR-2, VEGFR-3, neuropilin-1 (NRP-1) and NRP-2, of which three vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2, VEGFR-3) are receptor tyrosine kinases.

[0005] Under normal physiological conditions, angiogenesis is in a relatively dynamic equilibrium state due to the strict regulation of pro-angiogenic factors (VEGF) and anti-angiogenic factors (anti-VEGF). Under pathological conditions, the balance of pro-angiogenic and anti-angiogenic factors is broken, and pro-angiogenic factors play a dominant role. They can cause the proliferation of vascular endothelial cells, promote the formation of new blood vessels, and increase vascular permeability. The level of VEGF in the eyes of patients with diabetic macular edema (DME) and age-related macular degeneration (AMD) will increase abnormally, which will lead to the formation of dysplastic and fragile new blood vessels in the choroid of the eye. This abnormal new blood vessel will quickly enter between the retina and the choroid, causing fundus hemorrhage and fluid leakage, further damaging photoreceptor cells and retinal pigment epithelial cells, and causing severe vision loss in patients.

[0006] The onset of diabetic macular edema (DME) and age-related macular degeneration (AMD) is also related to the kallikrein-kinin system (KKS) signaling pathway. Plasma prekallikrein (PPK) in the KKS signaling pathway can be converted into catalytically active plasma kallikrein (PKa) through factor XIIa (FXIIa) and participate in inflammatory responses. PKa cleaves kininogen to produce bradykinin (BK). BK is a strong inflammatory factor that stimulates the release of mediators such as eicosanoids, endothelial-derived hyperpolarizing factor, NO, allyl alcohol, tissue-type plasminogen activator (tPA), and glucose transporter-1 / 2 (glu1 / 2) by activating BK receptors, further mediating a variety of signal cascade reactions. These mediators, together with inflammation, angiogenesis, vasodilation, and increased vascular permeability, can induce the formation of DME.

[0007] Currently, simple anti-VEGF treatment has limited efficacy in more than 50% of patients, especially in some subtypes of patients, where there is no significant difference in the efficacy of different single drugs. Therefore, the combination therapy of drugs targeting new targets and anti-VEGF and the development of VEGF-independent pathway drugs are extremely promising solutions. Multi-target drug design or multi-drug combination is also a hot development direction in recent years.

[0008] The marketed humanized VEGFR antibodies include ranibizumab (trade name Lucentis), a monoclonal antibody fragment that is derived from the same mouse antibody as bevacizumab. Ranibizumab is much smaller than the parent antibody molecule, has a more stable affinity, and has a stronger ability to bind to VEGF-A (WO98 / 45331). As an anti-angiogenic drug, ranibizumab has been approved for the treatment of "wet" age-related macular degeneration (ARMD), a common age-related vision loss disease. Aflibercept is a recombinant fusion protein composed of the VEGF binding portion of the extracellular domain of human VEGFR-1 and VEGFR-2, fused to the human IgG1 immunoglobulin Fc fragment. The marketed PKa humanized antibody Lanadelumab is an investigational fully human monoclonal antibody that specifically binds to and inhibits plasma kallikrein as a treatment for preventing angioedema attacks in HAE patients.

[0009] There is no technical solution for the combination of VEGF and PKa at present. There is still a need in the art to provide a VEGF and PKa bispecific binding molecule to provide an effective treatment solution for diseases such as proliferative retinopathy, neovascular glaucoma, diabetic macular edema (DME) and age-related macular degeneration (AMD) caused by retinal microvascular disorders. Summary of the invention

[0010] In view of the above-mentioned needs in the art, according to some embodiments, the present disclosure provides a bispecific binding molecule comprising:

[0011] at least one first binding module that specifically binds to VEGF,

[0012] at least one second binding moiety that specifically binds PKa; and

[0013] • At least one Fc region.

[0014] In some embodiments, a bispecific binding molecule is provided, comprising:

[0015] at least one first binding module that specifically binds to VEGF,

[0016] at least one second binding moiety that specifically binds PKa; and

[0017] at least one Fc region;

[0018] The first binding module comprises the extracellular domain of human VEGF receptor 1 and the extracellular domain of human VEGF receptor 2;

[0019] The second binding module comprises a heavy chain variable region PKa-VH and a light chain variable region PKa-VL; or the second binding module comprises a PKa heavy chain and a PKa light chain.

[0020] In some embodiments of the aforementioned bispecific binding molecules, when the heavy chain variable region PKa-VH and the light chain variable region PKa-VL are in the same chain, the two can be interchanged in order. For example, PKa-VH is at the N-terminus of PKa-VL, or PKa-VH is at the C-terminus of PKa-VL. PKa-VH and PKa-VL can be connected by a linker.

[0021] In some embodiments of the aforementioned bispecific binding molecules, the first binding module is a fusion protein formed by the extracellular domain of human VEGF receptor 1 and the extracellular domain of human VEGF receptor 2. The extracellular domain of human VEGF receptor 1 and the extracellular domain of human VEGF receptor 2 can be interchanged in order. For example, the extracellular domain of human VEGF receptor 1 is at the N-terminus of the extracellular domain of human VEGF receptor 2; or the extracellular domain of human VEGF receptor 1 is at the C-terminus of the extracellular domain of human VEGF receptor 2. The extracellular domain of human VEGF receptor 1 and the extracellular domain of human VEGF receptor 2 are directly or indirectly connected.

[0022] In some embodiments of the foregoing bispecific binding molecules, the Fc region is an IgG Fc region.

[0023] In some specific embodiments of the aforementioned bispecific binding molecules, the Fc region is an IgG1 Fc region.

[0024] In some specific embodiments of the aforementioned bispecific binding molecules, the Fc region has a LALA mutation.

[0025] In other embodiments of the aforementioned bispecific binding molecules, the Fc region comprises a first subunit and a second subunit. The first subunit comprises a protruding structure of Knob-Hole technology, and the second subunit comprises a pore structure of Knob-Hole; or vice versa, the second subunit comprises a protruding structure of Knob-Hole technology, and the first subunit comprises a pore structure of Knob-Hole.

[0026] It should be understood that the first subunit and the second subunit herein function to form a dimer and are therefore interchangeable.

[0027] In some specific embodiments of the foregoing bispecific binding molecules, the bispecific binding molecules are dimers (homodimers or heterodimers).

[0028] In some specific embodiments of the foregoing bispecific binding molecules, the bispecific binding molecule is a trimer (homotrimer or heterotrimer).

[0029] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0030] Two identical polypeptides of formula 1, in order from N to C-terminus:

[0031] First binding module+Fc region+first linker+PKa-VH+second linker+PKa-VL (Formula 1).

[0032] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0033] Two identical polypeptides of formula 2, in order from N to C-terminus:

[0034] First binding module+Fc region+first linker+PKa-VL+second linker+PKa-VH (Formula 2).

[0035] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0036] A first chain of formula 3, and a second chain, wherein:

[0037] Formula 3 In order from N to C terminus: PKa heavy chain + linker + first binding module (Formula 3);

[0038] The second chain is a PKa light chain.

[0039] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0040] A first chain of formula 4, and a second chain, wherein:

[0041] Formula 4 In order from N to C terminus: first binding module + linker + PKa heavy chain (Formula 4);

[0042] The second chain is a PKa light chain.

[0043] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0044] A second chain of formula 5, and a first chain, wherein:

[0045] Formula 5 in order from N to C terminus: first binding module + linker + PKa light chain (Formula 5);

[0046] The first chain is the PKa heavy chain.

[0047] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0048] A second chain of formula 6, and a first chain, wherein:

[0049] Formula 6 In order from N to C terminus: PKa light chain + linker + first binding module (Formula 6);

[0050] The first chain is the PKa heavy chain.

[0051] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0052] A first chain shown in formula 4, and a second chain shown in formula 5.

[0053] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0054] A first chain, a second chain, and a third chain:

[0055] The first chain is in the order from N to C terminus: the first binding module + the first subunit (Formula 7);

[0056] The second chain, in N- to C-terminal order: the PKa heavy chain containing the second subunit;

[0057] The third chain is in order from N to C terminus: PKa light chain.

[0058] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0059] A first chain, a second chain, and a third chain:

[0060] The first chain is in the order from N to C terminus: first binding module + second subunit (Formula 8);

[0061] The second chain, in N- to C-terminal order: the PKa heavy chain containing the first subunit;

[0062] The third chain is in order from N to C terminus: PKa light chain.

[0063] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0064] A first chain, a second chain, and a third chain:

[0065] The first chain is in the order from N to C terminus: first binding module + linker + first binding module + first subunit (Formula 9);

[0066] The second chain, in N- to C-terminal order: the PKa heavy chain containing the second subunit;

[0067] The third chain is in order from N to C terminus: PKa light chain.

[0068] In some specific embodiments of the aforementioned bispecific binding molecules, the structure is shown in the following formula:

[0069] A first chain, a second chain, and a third chain:

[0070] The first chain is in the order from N to C terminus: first binding module + linker + first binding module + second subunit (Formula 10);

[0071] The second chain, in N- to C-terminal order: the PKa heavy chain containing the first subunit;

[0072] The third chain is in order from N to C terminus: PKa light chain.

[0073] In some specific embodiments of the aforementioned bispecific binding molecules, when the bispecific binding molecule comprises at least two first binding moieties, each first binding moiety is the same or different.

[0074] In some specific embodiments of the aforementioned bispecific binding molecules, the first linker and the second linker are the same or different.

[0075] In some specific embodiments of the aforementioned bispecific binding molecules, the linker, the first linker, and the second linker are each independently (GGGGS) n , n is an integer from 3 to 6.

[0076] In some specific embodiments of the foregoing bispecific binding molecules, "+" indicates a covalent link.

[0077] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises: PKa-HCDR1, PKa-HCDR2 and PKa-HCDR3 in SEQ ID NO: 7; and the PKa-VL comprises: PKa-LCDR1, PKa-LCDR2 and PKa-LCDR3 in SEQ ID NO: 8; HCDR and LCDR are according to Kabat, IMGT, Chothia, AbM or Contact coding rules.

[0078] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises: PKa-HCDR1 shown in SEQ ID NO: 1, PKa-HCDR2 shown in SEQ ID NO: 2, and PKa-HCDR3 shown in SEQ ID NO: 3; the PKa-VL comprises: PKa-LCDR1 shown in SEQ ID NO: 4, PKa-LCDR2 shown in SEQ ID NO: 5, and PKa-LCDR3 shown in SEQ ID NO: 6; HCDR and LCDR are according to the Kabat coding rules.

[0079] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 7, 9, and 11, and the PKa-VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 8, 10, and 12.

[0080] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises the amino acid sequence shown in SEQ ID NO:7, and the PKa-VL comprises the amino acid sequence shown in SEQ ID NO:8.

[0081] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises the amino acid sequence shown in SEQ ID NO:7, and the PKa-VL comprises the amino acid sequence shown in SEQ ID NO:10.

[0082] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises the amino acid sequence shown in SEQ ID NO:7, and the PKa-VL comprises the amino acid sequence shown in SEQ ID NO:12.

[0083] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises the amino acid sequence shown in SEQ ID NO:9, and the PKa-VL comprises the amino acid sequence shown in SEQ ID NO:8.

[0084] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises the amino acid sequence shown in SEQ ID NO:9, and the PKa-VL comprises the amino acid sequence shown in SEQ ID NO:10.

[0085] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises the amino acid sequence shown in SEQ ID NO:9, and the PKa-VL comprises the amino acid sequence shown in SEQ ID NO:12.

[0086] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises the amino acid sequence shown in SEQ ID NO:11, and the PKa-VL comprises the amino acid sequence shown in SEQ ID NO:8.

[0087] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises the amino acid sequence shown in SEQ ID NO:11, and the PKa-VL comprises the amino acid sequence shown in SEQ ID NO:10.

[0088] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa-VH comprises the amino acid sequence shown in SEQ ID NO:11, and the PKa-VL comprises the amino acid sequence shown in SEQ ID NO:12.

[0089] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa light chain comprises the amino acid sequence shown in SEQ ID NO:14; and the PKa heavy chain comprises the amino acid sequence shown in SEQ ID NO:13.

[0090] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa light chain comprises the amino acid sequence shown in SEQ ID NO:14; and the PKa heavy chain comprises the amino acid sequence shown in SEQ ID NO:45.

[0091] In some specific embodiments of the aforementioned bispecific binding molecules, the PKa light chain comprises the amino acid sequence shown in SEQ ID NO:14; and the PKa heavy chain comprises the amino acid sequence shown in SEQ ID NO:46.

[0092] In some specific embodiments of the aforementioned bispecific binding molecules, the human VEGF receptor 1 extracellular domain comprises the amino acid sequence shown in SEQ ID NO:18.

[0093] In some specific embodiments of the aforementioned bispecific binding molecules, the human VEGF receptor 2 extracellular domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, and 17.

[0094] In some specific embodiments of the aforementioned bispecific binding molecules, the first binding moiety comprises an amino acid sequence selected from any one of SEQ ID NOs: 19-22.

[0095] In some specific embodiments of the aforementioned bispecific binding molecules, the first binding module is shown in SEQ ID NO:19.

[0096] In some specific embodiments of the aforementioned bispecific binding molecules, the first binding module is shown in SEQ ID NO:20.

[0097] In some specific embodiments of the aforementioned bispecific binding molecules, the first binding module is shown in SEQ ID NO:21.

[0098] In some specific embodiments of the aforementioned bispecific binding molecules, the first binding module is shown in SEQ ID NO:22.

[0099] In some specific embodiments of the aforementioned bispecific binding molecules, the Fc region comprises an amino acid sequence selected from any one of SEQ ID NOs: 25-28.

[0100] In some specific embodiments of the aforementioned bispecific binding molecules, the first subunit is shown in SEQ ID NO:26, and the second subunit is shown in SEQ ID NO:28.

[0101] In some specific embodiments of the aforementioned bispecific binding molecules, the first subunit is shown in SEQ ID NO:28, and the second subunit is shown in SEQ ID NO:26.

[0102] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises two amino acid sequences shown in SEQ ID NO:31.

[0103] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises two amino acid sequences shown in SEQ ID NO:32.

[0104] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises two amino acid sequences shown in SEQ ID NO:33.

[0105] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises two amino acid sequences shown in SEQ ID NO:34.

[0106] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises two amino acid sequences shown in SEQ ID NO:35.

[0107] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises two amino acid sequences shown in SEQ ID NO:36.

[0108] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises two amino acid sequences shown in SEQ ID NO:37.

[0109] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:38, and a second chain shown in SEQ ID NO:14.

[0110] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:39, and a second chain shown in SEQ ID NO:14.

[0111] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:40, and a second chain shown in SEQ ID NO:14.

[0112] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:50, and a second chain shown in SEQ ID NO:14.

[0113] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:13, and a second chain shown in SEQ ID NO:41.

[0114] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:13, and a second chain shown in SEQ ID NO:48.

[0115] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:13, and a second chain shown in SEQ ID NO:49.

[0116] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:42, and a second chain shown in SEQ ID NO:43.

[0117] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:44, a second chain shown in SEQ ID NO:45 and a third chain shown in SEQ ID NO:14.

[0118] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:51, a second chain shown in SEQ ID NO:45 and a third chain shown in SEQ ID NO:14.

[0119] In a specific embodiment of the aforementioned bispecific binding molecule, it comprises a first chain shown in SEQ ID NO:46, a second chain shown in SEQ ID NO:47 and a third chain shown in SEQ ID NO:14.

[0120] According to some embodiments, the present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of any of the aforementioned bispecific binding molecules; one or more pharmaceutically acceptable carriers, diluents, buffers or excipients; and optionally a second therapeutic agent.

[0121] In some embodiments of the pharmaceutical composition, the second therapeutic agent is selected from: anti-diabetic agents, agents for treating overweight, agents for treating obesity, agents for treating hypertension, agents for treating pulmonary hypertension, agents for treating heart failure, agents for treating atherosclerosis, and agents for treating eye diseases.

[0122] In some embodiments of the pharmaceutical composition, the antidiabetic agent is selected from: GK agonists (GLP-1 receptor agonists, exenatide, liraglutide, semaglutide, dulaglutide, loxenatide, albiglutide), α-glucosidase inhibitors (voglibose, acarbose), SGLT-2 inhibitors canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, luseogliflozin and tofogliflozin), DPP-4 inhibitors (sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, gemigliptin, teneligliptin, metformin).

[0123] In some embodiments of the pharmaceutical composition, the drug for treating hypertension is selected from: angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, calcium channel blockers, beta-receptor blockers, diuretics, and alpha-receptor blockers; preferably, the drug for treating hypertension is selected from: felodipine, nifedipine, nitrendipine, nisoldipine, nicardipine, lacidipine, lercanidipine, amlodipine, levamlodipine, verapamil, diltiazem, captopril, enalapril, benazepril, lislopril, ramipril, fosinopril, cilazapril, perindopril, quinapril, imidapril, zofenopril, losartan, valsartan, irbesartan, irbesartan, candesartan cilexetil, telmisartan, and olmesartan medoxomil.

[0124] In some embodiments of the pharmaceutical composition, the agent for treating pulmonary arterial hypertension is selected from: bosentan, riociguat, macitentan, and selexipag.

[0125] In some embodiments of the pharmaceutical composition, the agent for treating heart failure and / or atherosclerosis is selected from: mevastatin, lovastatin, pravastatin, simvastatin, fluvastatin, cholestyramine, colestipol, aspirin, clopidogrel, cilostazol, ω-3 polyunsaturated fatty acids, ω-6 monounsaturated fatty acids, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, finerenone, evolocumab (Rebai'an, Evolocumab), alirocumab (Alirocumab), Inclisiran (Leqvio).

[0126] In some embodiments of the pharmaceutical composition, the agent for treating eye diseases is selected from: corticosteroids, complement factor 3 inhibitors, complement factor 5 inhibitors, complement factor B inhibitors, complement factor D inhibitors, anti-PDGF antibodies, VAP-1 inhibitors, bradykinin receptor 1 antagonists, Tie-2 agonists, and CCR-2 antagonists.

[0127] In some embodiments of the pharmaceutical composition, the aforementioned pharmaceutical composition can be used to prevent, slow down the progression of, or treat one or more metabolic disorders selected from the following: type I diabetes, type II diabetes, impaired glucose tolerance, hyperglycemia, postprandial hyperglycemia, impaired fasting blood glucose, overweight, obesity, hypertension, insulin resistance, and / or metabolic syndrome. Or improve blood sugar control and / or reduce fasting plasma glucose. Or prevent, slow, delay, or reverse diabetic complications.

[0128] According to some embodiments, the present disclosure also provides an isolated nucleic acid encoding any of the aforementioned bispecific binding molecules.

[0129] According to some embodiments, the present disclosure also provides a host cell comprising the aforementioned isolated nucleic acid.

[0130] According to some embodiments, the present disclosure also provides the use of the aforementioned bispecific binding molecules or the aforementioned pharmaceutical compositions in the preparation of a medicament for preventing or treating a disease selected from the following: diabetic retinopathy, diabetic macular edema, retinal vein occlusion, central retinal vein occlusion, macular degeneration, wet age-related macular degeneration, retinopathy of prematurity, neovascular glaucoma, retinitis pigmentosa, proliferative retinopathy, non-proliferative retinopathy, retinal angiomatous proliferation, macular telangiectasia, ischemic retinopathy, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, polypoidal choroidal vasculopathy, retinal degeneration, hereditary angioedema, brain edema after stroke, and retinal vascular permeability diabetic complications.

[0131] According to some embodiments, the present disclosure also provides a method for preventing or treating a disease, the method comprising: administering a preventively or therapeutically effective amount of the aforementioned bispecific binding molecule or the aforementioned pharmaceutical composition to a subject.

[0132] In some embodiments of preventing or treating a disease, the disease is selected from any one of the following: diabetic retinopathy, diabetic macular edema, retinal vein occlusion, central retinal vein occlusion, macular degeneration, wet age-related macular degeneration, retinopathy of prematurity, neovascular glaucoma, retinitis pigmentosa, proliferative retinopathy, non-proliferative retinopathy, retinal angiomatous proliferation, macular telangiectasia, ischemic retinopathy, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, polypoidal choroidal vasculopathy, retinal degeneration, hereditary angioedema, brain edema after stroke, and retinal vascular permeability diabetic complications.

[0133] In some embodiments of preventing or treating a disease, the disease is selected from any one of the following: diabetic retinopathy, diabetic macular edema, retinal vein occlusion, central retinal vein occlusion, wet age-related macular degeneration, non-exudative choroidal neovascularization, hereditary angioedema, brain edema after stroke.

[0134] The binding molecules of the present disclosure (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-term or long-term. The present disclosure contemplates a variety of dosing schedules, including but not limited to, single or multiple administrations at multiple time points, push administration and pulse infusion.

[0135] The bispecific binding molecules according to the present disclosure can be used to prevent or treat ocular vascular diseases. To this end, the pharmaceutical composition containing the bispecific binding molecules for intravitreal administration is provided as a liquid isotonic preparation with a viscosity of 20 mPas or less (preferably 15 mPas or less), a turbidity of 30 FTU or less (preferably 25 FTU or less), an osmotic pressure of 300 mOsm / kg ± 50 mOsm / kg, and substantially no visible particles. To this end, the liquid isotonic pharmaceutical composition of the bispecific binding molecule is provided in a glass vial, or in the form of a prefilled syringe, particularly in the form of a prefilled glass syringe.

[0136] In another aspect of the present disclosure, a product (such as a medicine box) is provided, which contains materials that can be used to treat, prevent and / or diagnose the above-mentioned conditions. The product comprises a container and a label or package insert. Suitable containers include, for example, bottles, vials, syringes. The container contains the binding molecules of the present disclosure. As an example, the product may include: a first container, which contains the binding molecules of the present disclosure; and a second container, which contains other therapeutic agents. The product further includes other materials, such as buffers, diluents, filters, needles and syringes. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figures 1 to 18 : Exemplary structures of bispecific binding molecules. DETAILED DESCRIPTION

[0138] The terms used in the present disclosure are only for the purpose of describing the embodiments and are not intended to be limiting. Unless otherwise defined, all technical terms and scientific terms used in the present disclosure have the same meaning as commonly understood by ordinary technicians in the field to which the present disclosure belongs.

[0139] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising", "having", "including", etc. should be understood to have an inclusive meaning, that is, the meaning of "including but not limited to". Unless otherwise stated, "comprising" includes "consisting of".

[0140] The ordinal numbers "first", "second", "third", etc. in the present disclosure do not limit the quantity, sequence, or level, but are only used to distinguish different chemical entities, features, steps, or elements.

[0141] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0142] The term "and / or", such as "X and / or Y" should be understood to mean "X and Y" or "X or Y".

[0143]

[0063] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0144] The term "amino acid mutation" includes amino acid substitution, deletion, insertion and modification. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, as long as the final construct has the desired properties. Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and / or carboxyl terminus of the polypeptide chain. Specific amino acid mutations can be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, that is, an amino acid is replaced with another amino acid having different structural and / or chemical properties. Various names can be used in the present disclosure to indicate the same amino acid mutation. The amino acid residue at a specific site can be represented by the position + amino acid residue, for example, 366W means that the amino acid residue at the 366th site is W. T366W means that the amino acid residue at the 366th site has mutated from the original T to W.

[0145] "VEGF" should be understood broadly and is intended to cover various forms of VEGF molecules at various stages in the mammalian body, such as but not limited to molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification of the VEGF gene (e.g., precursor VEGF, mature VEGF, membrane-expressed VEGF, VEGF splice variants, modified VEGF, or fragments thereof); the term also covers artificially prepared or in vitro expressed VEGF, and VEGF expressed on the cell surface.

[0146] "PKa" should be understood broadly and is intended to cover various forms of PKa molecules at various stages in mammals, such as but not limited to molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification of the BCMA gene (e.g., precursor PKa, mature PKa, membrane-expressed PKa, PKa splice variants, modified PKa, or fragments thereof); the term also covers artificially prepared or in vitro expressed PKa, and PKa expressed on the cell surface.

[0147] The term "binding molecule" is used in the broadest sense and covers various molecules that specifically bind to a target, including but not limited to antibodies, other polypeptides with target binding activity, and fusion proteins formed by the fusion of the two, as long as they exhibit the desired target binding activity.

[0148] The term "bispecific binding molecule" as used in the present disclosure is a molecule that has binding activity to two different targets (or different epitopes of the same antigen).

[0149] The bispecific binding molecules of the present disclosure are specific for two different targets, namely VEGF as a first target and PKa as a second target. The binding molecules in the present disclosure are bispecific binding molecules.

[0150] The bispecific binding molecules of the present disclosure comprise two binding moieties, a first binding moiety that specifically binds to VEGF and a second binding moiety that specifically binds to PKa.

[0151] The term "binding moiety" refers to a polypeptide molecule that specifically binds to a target or antigen (or epitope). Exemplary binding moieties include the antigen binding domain of an antibody, for example, comprising a heavy chain variable region and a light chain variable region.

[0152] The first binding module is not an antibody or an antigen-binding fragment thereof, but a domain in VEGFR or a fusion protein thereof.

[0153] The second binding moiety is an antibody or an antigen-binding fragment thereof, which comprises a variable region (VH) and a variable region (VL), which together constitute the binding domain for the antigen (PKa).

[0154] The term "binding module that specifically binds to PKa" refers to a module that can bind to PKa or its epitope with sufficient affinity so that the molecule containing the module can be used as a diagnostic agent and / or therapeutic agent targeting PKa. For example, a binding module that specifically binds to PKa has the following equilibrium dissociation constant (KD): < about 1×10 -8 M or less as measured by surface plasmon resonance assay.Antigen binding moieties include antibody fragments as defined in the present disclosure, such as Fab (such as substituted Fab) or scFv.

[0155] The bispecific binding molecules disclosed herein are not limited to a specific molecular structure, as long as they have the desired antigen binding function. For example, the bispecific binding molecules disclosed herein may be bivalent, trivalent or quadrivalent. The binding module in the binding molecule may be any antibody fragment with target binding activity, which is fused through a peptide linker. The peptide linker disclosed herein may be any suitable peptide chain, as long as the binding molecule can exhibit the desired target binding activity. For example, the peptide linker may be a flexible peptide of 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of L1-(GGGGS)n-L2, wherein L1 is a bond, A, GS, GGS, GGGS, SGGGGS, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and L2 is a bond, G, GG, GGG or GGGG. In some embodiments, the length of the peptide linker is 3-15 amino acid residues. In some embodiments, the peptide linkers each independently have the structure of (GGGGS)n, wherein n is 3-6.

[0156] In order to recombinantly produce binding molecules, the nucleic acid encoding the protein is separated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can be specifically bound to the genes encoding the heavy and light chains of antibodies), or produced by recombinant methods or obtained by chemical synthesis.

[0157] The term "antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, specific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments), so long as they exhibit the desired target binding activity.

[0158] Natural IgG antibodies are heterotetrameric proteins, consisting of two light chains and two heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as the heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as the light chain variable domain, followed by a constant light domain (light chain constant region, CL). VH and VL each contain four conserved framework regions (FR) and three complementary determining regions (CDR). Among them, the term "complementarity determining region" or "CDR" refers to the region within the variable domain that mainly contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than CDR residues. VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3; VL contains 3 CDR regions: LCDR1, LCDR2 and LCDR3.

[0159] The term "variable region" or "variable domain" refers to a domain in a binding molecule that binds to a target. In the present disclosure, the heavy chain variable region in the binding module that specifically binds to PKa is labeled PKa-VH, and the light chain variable region is labeled PKa-VL. In the present disclosure, the three CDR regions in PKa-VH are labeled PKa-HCDR1, PKa-HCDR2, and PKa-HCDR3, respectively; the three CDR regions in PKa-VL are labeled PKa-LCDR1, PKa-LCDR2, and PKa-LCDR3, respectively. Each VH and VL is sequentially: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus.

[0160] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "Kabat" numbering rule, "Chothia" numbering rule, "ABM" numbering rule, "contact" numbering rule and ImMunoGenTics (IMGT) numbering rule, etc. The correspondence between various numbering systems is well known to those skilled in the art.

[0161] Unless otherwise stated, the variable region and CDR sequences in the embodiments of the present disclosure are subject to the "Kabat" numbering convention. Although in a specific embodiment, a numbering system (such as Kabat) is used to define amino acid residues, the corresponding technical solutions of other numbering systems will be regarded as equivalent technical solutions.

[0162] The term "antigen-binding fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that retains the antigen-binding ability of the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g., scFv).

[0163] The term "Fc region" is used to define the C-terminal region of an antibody heavy chain, including a native Fc region and a modified Fc region. In some embodiments, the Fc region comprises two identical or different subunits.

[0164] In some embodiments, a suitable native sequence Fc region comprises human IgGl. Unless otherwise indicated, the numbering convention for Fc regions is the EU index.

[0165] The Fc region of the binding molecules of the present disclosure comprises one or more amino acid substitutions that reduce its binding to Fc receptors (e.g., Fcγ receptors) and reduce or eliminate effector functions. Native IgG Fc regions, specifically IgG1 Fc regions, may cause the binding molecules of the present disclosure to target cells expressing Fc receptors rather than cells expressing target / antigen.

[0166] The engineered Fc region of the present disclosure exhibits reduced binding affinity to Fc receptors and / or reduced effector function. As an example, the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index.

[0167] As another example, when the binding molecule comprises different binding modules fused to two subunits in the Fc region, undesirable homodimerization may result. In order to improve yield and purity, it is therefore advantageous to introduce modifications that promote heterodimerization in the Fc region of the binding molecule of the present disclosure. In some embodiments, the Fc region of the present disclosure comprises the transformation according to the knob-into-hole (KIH) technology, which involves introducing a protrusion structure (knob) at the interface of the first subunit and introducing a hole structure (hole) at the interface of the second subunit. The protrusion structure is positioned in the hole structure, promoting the formation of heterodimers and inhibiting the generation of homodimers. The protrusion structure is constructed by replacing the small amino acid side chains from the interface of the first subunit with larger side chains (such as tyrosine or tryptophan). And the hole structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller amino acid side chains (such as alanine or threonine).

[0168] The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; or it can be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues are removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG.

[0169] The term "affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). The affinity of a molecule for its ligand can generally be represented by an equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art. The term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). The KD value of an antibody can be measured using methods known in the art, such as surface plasmon resonance.

[0170] The term "antigen" refers to a molecule or part thereof that can be selectively recognized or bound by a binding molecule (eg, an antibody). An antigen may have one or more epitopes that can interact with different binding molecules (eg, antibodies).

[0171] The term "specific binding" means that the bispecific binding molecule can bind to a target or antigen (or epitope) with a higher affinity than other targets or antigens (or epitopes). -7 M or less (e.g., about 1×10 -8The bispecific binding molecules specifically binding to a target or antigen (or epitope) do not exclude cross-reactivity to targets or antigens (or epitopes) related to other species, for example, cross-reactivity to corresponding targets or antigens (or epitopes) from other species (homologous) such as humans or monkeys.

[0172] The term "does not bind" means that the bispecific binding molecule is unable to bind to a target or antigen (or epitope) in the above-mentioned specific binding manner.

[0173] The term "fused" or "linked" means that the components (eg, binding moieties, Fc regions) are covalently linked directly or via a linker.

[0174] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector, in which additional DNA segments can be ligated into the viral genome.

[0175] The term "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, without considering the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells.

[0176] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (eg, mammals and non-mammals), such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles.

[0177] Unless expressly indicated otherwise, the terms "patient" or "subject" are used interchangeably in this disclosure. In certain embodiments, the individual or subject is a human.

[0178] The term "pharmaceutically acceptable" as used in this disclosure refers to compounds, materials, compositions and / or dosage forms that are, within the scope of reasonable medical judgment, suitable for use in contact with subject samples without excessive toxicity, irritation, allergic response or other problems or complications, have a reasonable benefit / risk ratio, and are effective for the intended use.

[0179] "Treatment" refers to a clinical intervention attempted to be applied to the individual being treated, and can be performed for preventive purposes, or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. In some embodiments, the molecules of the present disclosure are used to delay the development of a disease or slow the progression of a disease.

[0180] The term "therapeutically effective amount" refers to an amount of a drug or composition sufficient to achieve or at least partially achieve the desired effect. The determination of a therapeutically effective amount varies from person to person, depending on the age and general condition of the recipient, and also on the specific active substance. The appropriate therapeutically effective amount in an individual can be determined by a person skilled in the art based on routine tests.

[0181] As used in this disclosure, the singular forms "a," "an," and "the" include the corresponding plural forms and vice versa, unless the context clearly dictates otherwise.

[0182] It should be noted that, unless explicitly stated otherwise, the description method “…independently selected” used in the present disclosure should be understood in a broad sense, meaning that each individual described is selected independently of each other.

[0183] Example

[0184] The following examples are only used to illustrate the present disclosure, but are not used to limit the present disclosure. Modifications, changes, variations, etc. made within the scope of protection of the present disclosure are all within the scope of protection of the present disclosure. Experimental methods without specific conditions in the examples of the present disclosure are usually carried out under conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual; or according to the conditions recommended by the raw material or commodity manufacturer. Reagents without specific sources are conventional reagents purchased from the market.

[0185] The compounds provided by the present disclosure can be prepared using the specific synthesis schemes described below, from readily available starting materials, which can be synthesized using or according to methods known in the art, or can be purchased commercially. The experimental methods for which specific conditions are not indicated in the following examples can be determined by conventional methods and conditions by those skilled in the art through conventional optimization procedures.

[0186] Embodiment 1: First combination module, second combination module

[0187] 1. Binding module specific to PKa (refer to US20120201756)

[0188] (1) VH+VL

[0189] PKa-VH (SEQ ID NO: 7)

[0190] EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVS GIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIG VPRRDEFDIWGQGTMVTVSS; The bold is CDR.

[0191] PKa-VL (SEQ ID NO: 8)

[0192] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKA STLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEI K; The bold is CDR.

[0193] (2) VH + VL

[0194] PKa-VH with cys mutation (SEQ ID NO: 9)

[0195] EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGK C LEWVS GIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIGV PRRDEFDIWGQGTMVTVSS.

[0196] PKa-VL with cys mutation (SEQ ID NO: 10)

[0197] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKA STLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFG C GTKVEIK.

[0198] (3) VH + VL

[0199] PKa-VH with cys mutation (SEQ ID NO: 11)

[0200] EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIGVPRRDEFDIWG C GTMVTVSS

[0201] PKa-VL with cys mutations (SEQ ID NO: 12)

[0202] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGK C PKLLIYKA STLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK。

[0203] (4) Heavy chain, light chain

[0204] PKa heavy chain (SEQ ID NO: 13)

[0205] EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIGVPRRDEFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK ; Bold is CDR, underlined is Fc-3;

[0206] PKa light chain (SEQ ID NO: 14)

[0207] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; Bold is CDR.

[0208] PKa-HCDR1: HYIMM (SEQ ID NO: 1)

[0209] PKa-HCDR2: GIYSSGGITVYADSVKG (SEQ ID NO: 2)

[0210] PKa-HCDR3:RRIGVPRRDEFDI (SEQ ID NO: 3)

[0211] PKa-LCDR1: RASQSISSWLA (SEQ ID NO: 4)

[0212] PKa-LCDR2: KASTLES (SEQ ID NO: 5)

[0213] PKa-LCDR3: QQYNTYWT (SEQ ID NO: 6).

[0214] 2. The first binding module specific to VEGF

[0215] (1) Human VEGF receptor 2 extracellular domain

[0216] or

[0217] or

[0218] VLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKEPKS SDKTHTCP (SEQ ID NO: 17).

[0219] (2) Extracellular domain of human VEGF receptor 1

[0220] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGK RIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDV (SEQ ID NO: 18).

[0221] (3) Extracellular domain of human VEGF receptor 1 + extracellular domain of human VEGF receptor 2:

[0222] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCP (SEQ ID NO: 19); or

[0223] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO: 20);

[0224] SDTGPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHG IELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKEPKSSDKTHTCP(SEQID NO: 21).

[0225] (4) Human VEGF receptor 2 extracellular domain + human VEGF receptor 1 extracellular domain (SEQ ID NO: 22):

[0226] VLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDV.

[0227] (5) Fusion protein formed by human VEGF receptor 1 extracellular domain + human VEGF receptor 2 extracellular domain + IgG1Fc:

[0228] SDTGPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVL SPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCP PCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23); (the last K can be added or omitted).

[0229] (6) Fusion protein formed by human VEGF receptor 1 extracellular domain + human VEGF receptor 2 extracellular domain + IgG1FcLALA:

[0230] SDTGPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVL SPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCP PCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24, Fc with LALA mutation), the underline is Fc; (the last K can be added or omitted).

[0231] 3. Fc region sequence:

[0232] Fc-1 (SEQ ID NO: 25, with LALA mutation) (the last K can be omitted)

[0233] PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0234] Fc-2 (SEQ ID NO: 26) (the last K can be omitted) with knob

[0235] PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0236] Fc-3 (SEQ ID NO: 27) (the last K can be omitted)

[0237] PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0238] Fc-4 (SEQ ID NO: 28) (the last K can be omitted) with hole mutation

[0239] PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0240] 4. Linker: (GGGGS)3 (SEQ ID NO: 29) or (GGGGS)4 (SEQ ID NO: 30). Example 2: Preparation of bispecific binding molecules

[0241] 1. Sample No. P216280:

[0242] Homodimer, two identical strands of formula 1,

[0243] First binding module + Fc + linker + PKa-VH + linker + PKa-VL (Formula 1) (see Figure 1 )

[0244]

[0245]

[0246] Italics: SEQ ID NO: 19;

[0247] Bold: PKa-VH

[0248] Dashed line: PKa-VL

[0249] Wavy line: connector

[0250] Underlined: Fc-1 (Fc region with LALA mutation).

[0251] 2. Sample No. P216281:

[0252] Homodimer, two identical strands of formula 2,

[0253] First binding module + Fc + linker + PKa-VL + linker + PKa-VH (Formula 2) Figure 2 )

[0254]

[0255] Italics: SEQ ID NO: 19;

[0256] Bold: PKa-VH

[0257] Dashed line: PKa-VL

[0258] Wavy line: connector

[0259] Underlined: Fc-1.

[0260] 3. Sample No. P216282:

[0261] Homodimer, two identical strands of formula 1,

[0262] First binding module + Fc++ linker + PKa-VH + linker + PKa-VL (Formula 1) Figure 3 )

[0263]

[0264] Italics: SEQ ID NO: 19;

[0265] Bold: PKa-VH with Cys mutation

[0266] Dashed line: PKa-VL with Cys mutation

[0267] Wavy line: connector

[0268] Underlined: Fc-1.

[0269] 4. Sample No. P216283:

[0270] Homodimer, two identical strands of formula 2,

[0271] First binding module + Fc++ linker + PKa-VL + linker + PKa-VH (Formula 2) Figure 4 )

[0272]

[0273] Italics: SEQ ID NO: 19;

[0274] Bold: PKa-VH with Cys mutation

[0275] Dashed line: PKa-VL with Cys mutation

[0276] Wavy line: connector

[0277] Underlined: Fc-1.

[0278] 5. Sample No. P216284:

[0279] Homodimer, two identical formula 1,

[0280] First binding module + Fc + linker + PKa-VH + linker + PKa-VL (Formula 1) Fig.14 )

[0281]

[0282] Italics: SEQ ID NO: 19;

[0283] Bold: PKa-VH with mutations

[0284] Dashed line: PKa-VL with mutation

[0285] Wavy line: connector

[0286] Underlined: Fc-1.

[0287] 6. Sample No. P216285:

[0288] Homodimer, two identical strands of formula 2,

[0289] First binding module + linker + PKa-VL + linker + PKa-VH (Formula 2) Fig.15 )

[0290]

[0291]

[0292] Italics: SEQ ID NO: 19;

[0293] Bold: PKa-VH with mutations

[0294] Dashed line: PKa-VL with mutation

[0295] Wavy line: connector

[0296] Underlined: Fc-1.

[0297] 7. Sample No. P216286:

[0298] Homodimer, two identical formula 1,

[0299] First binding module + Fc + linker + PKa-VH + linker + PKa-VL (Formula 1) Fig.16 )

[0300]

[0301] Italics: SEQ ID NO: 19

[0302] Bold: PKa-VH

[0303] Dashed line: PKa-VL

[0304] Wavy line: connector (( GGGGS)4 )

[0305] Underlined: Fc-1.

[0306] 8. Sample No. P216287:

[0307] Heterodimer,

[0308] First chain: PKa heavy chain (with R / K mutation) + linker + first binding module (Formula 3) ( Figure 5 )

[0309]

[0310]

[0311] Italics: SEQ ID NO: 20;

[0312] Bold: PKa-VH

[0313] Wavy line: connector

[0314] Underline: Fc-1

[0315] Second chain: PKa light chain (SEQ ID NO: 14)

[0316] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0317] 9. Sample No. P216288:

[0318] First chain: PKa heavy chain + linker + first binding module (Formula 3) ( Figure 6 ),

[0319]

[0320] Italics: first binding module (human VEGF receptor 2 extracellular domain + human VEGF receptor 1 extracellular domain) (SEQ ID NO: 22)

[0321] Bold: PKa-VH

[0322] Wavy line: connector

[0323] Underline: Fc-1

[0324] Second chain: PKa light chain (SEQ ID NO: 14)

[0325]

[0326] 10. Sample No. P216289:

[0327] First chain: first binding module + linker + heavy chain (Formula 4) ( Figure 7 )

[0328]

[0329] Italics: SEQ ID NO: 20;

[0330] Underline: Fc-1

[0331] Second chain: PKa light chain (SEQ ID NO: 14)

[0332]

[0333] 11. Sample No. P216290:

[0334] First chain: PKa heavy chain (SEQ ID NO: 13) ( Figure 8 )

[0335]

[0336]

[0337] Bold: PKa-VH

[0338] Underline: Fc-3

[0339] Second chain: first binding module + linker + PKa light chain (Formula 5)

[0340]

[0341] Italics: SEQ ID NO: 20;

[0342] The dashed line is the PKa light chain (SEQ ID NO: 14).

[0343] 12. Sample No. P216291:

[0344] First chain: first binding module + linker + PKa heavy chain ( Fig. 9 )

[0345]

[0346] Italics: SEQ ID NO: 20;

[0347] Bold: PKa-VH

[0348] Wavy line: connector

[0349] Underline: Fc-1

[0350] Second chain: first binding module + linker + PKa light chain

[0351]

[0352] Italics: SEQ ID NO: 20;

[0353] The dashed line is the PKa light chain (SEQ ID NO: 14).

[0354] 13. Sample No. P216292:

[0355] Three-chain structure,

[0356] First chain: first binding module + HC-knob (Formula 7) ( Fig.10 )

[0357]

[0358] Italics: first binding module (SEQ ID NO: 21)

[0359] Fc-2 is underlined;

[0360] Second chain sequence: PKa heavy chain with HC-hole modification

[0361]

[0362] Bold: PKa-VH

[0363] Fc-4 is underlined;

[0364] Third chain: PKa light chain (SEQ ID NO: 14)

[0365] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0366] 14. Sample No. P216293:

[0367] Three-chain structure ( Fig.11 ),

[0368] First chain: PKa heavy chain with HC-knob

[0369]

[0370] Bold: PKa-VH

[0371] Underline: Fc-2;

[0372] Second chain: first binding module + HC-hole (Formula 8)

[0373]

[0374] Italics: first binding module (SEQ ID NO: 21)

[0375] Fc-4 is underlined

[0376] Third chain: PKa light chain (SEQ ID NO: 14)

[0377] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0378] 15. Sample No. P216294:

[0379] First chain: PKa heavy chain (SEQ ID NO: 13) ( Fig.12 )

[0380]

[0381] Bold: PKa-VH

[0382] Underline: Fc-3

[0383] Second chain: PKa light chain + linker + first binding module (Formula 6)

[0384]

[0385] Dashed line: PKa light chain SEQ ID NO: 14

[0386] Wavy line: connector

[0387] Italics: SEQ ID NO:20.

[0388] 16. Sample No. P216295:

[0389] First chain: PKa heavy chain (SEQ ID NO: 13) ( Fig.13 )

[0390]

[0391] Bold: PKa-VH

[0392] Underline: Fc-3

[0393] Second chain: PKa light chain + linker + first binding module (Formula 6)

[0394]

[0395] Italics: first binding module (human VEGF receptor 2 extracellular domain + human VEGF receptor 1 extracellular domain) (SEQ ID NO: 22)

[0396] Wavy line: connector

[0397] The dashed line is the PKa light chain (SEQ ID NO: 14).

[0398] 17. Sample No. P216296:

[0399] First chain: PKa heavy chain + linker + first binding module ( Fig.17 )(Formula 3)

[0400]

[0401] Bold: PKa-VH

[0402] Italics: SEQ ID NO: 20;

[0403] Fc-1 is underlined;

[0404] Second chain: PKa light chain (SEQ ID NO: 14)

[0405] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0406] 18. Sample No. P216297:

[0407] First chain: first binding module + linker + first binding module + HC-knob ( Fig.18 )(Formula 9 or Formula 10)

[0408]

[0409] Italics: first binding module SEQ ID NO: 20;

[0410] Boxed: first binding module SEQ ID NO: 21;

[0411] Fc-2 is underlined;

[0412] Second chain: PKa heavy chain with HC-hole

[0413]

[0414] Fc-4 is underlined;

[0415] Third chain: PKa light chain (SEQ ID NO: 14)

[0416]

[0417] Bispecific binding molecules are prepared according to routine methods in the art.

[0418] Test Case

[0419] Test Example 1: In vitro VEGFR1 and VEGFR2 inhibitory activity test of bispecific binding molecules

[0420] The IC50 value was determined using 33P isotope-labeled kinase activity assay (Reaction Biology Corp) to evaluate the inhibitory ability of the test compounds on human VEGFR1 and human VEGFR2.

[0421] Buffer conditions: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EDTA, 0.02% Brij35, 0.02 mg / mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO.

[0422] Experimental steps: At room temperature, the test compound was dissolved in DMSO to prepare a 10 mM solution for standby use. The substrate was dissolved in a newly prepared buffer, and the VEGFR1 or VEGF2 kinase to be tested was added thereto and mixed evenly. The DMSO solution containing the test compound was added to the above mixed reaction solution using acoustic technology (Echo 550). The concentrations of the compounds in the reaction solution were 3μM, 1μM, 0.333μM, 0.111μM, 37.0nM, 12.3nM, 4.12nM, 1.37nM, 0.457nM, and 0.152nM. After incubation for 15 minutes, 33P-ATP (activity 0.0lμCi / μL) was added to start the reaction. After the reaction was carried out at room temperature for 120 minutes, the reaction solution was spotted on P81 ion exchange filter paper (Whatman#3698-915). After repeatedly washing the filter paper with 0.75% phosphoric acid solution, the radioactivity of the phosphorylated substrate remaining on the filter paper was measured. The kinase activity data were expressed as the comparison of the kinase activity of the test compound and the kinase activity of the blank group (containing only DMSO), and the IC was obtained by curve fitting using Prism4 software (GraphPad). 50 value.

[0423] The bispecific binding molecules of the present application can inhibit the activities of VEGFR1 and VEGF2 in vitro.

[0424] Table 1. In vitro VEGFR1 and VEGFR2 inhibitory activity of bispecific antibodies

[0425]

[0426] The above results indicate that the bispecific binding molecules disclosed herein have strong inhibitory activity against VEGFR1 and VEGFR2.

[0427] Test Example 2: Bispecific binding molecules inhibit VEGF-induced proliferation of HUVEC

[0428] VEGF binds to VEGFR on HUVEC, causing phosphorylation of VEGFR intracellular kinase and promoting HUVEC proliferation. This experiment is used to identify bispecific binding molecules that can prevent VEGF-induced HUVEC proliferation.

[0429] The specific method is as follows:

[0430] HUVEC were collected at 5×10 4 / ml density was inoculated into a T75 cell bottle, and the cells grew to the logarithmic phase in about 2-3 days. The HUVEC cells in the logarithmic growth phase were digested with 0.08% trypsin, about 1-2min at room temperature, and 10% FBS was added to terminate. The HUVEC after the above digestion was collected, centrifuged at 800rpm / min for 5min, and washed three times with PBS to remove the cytokines that stimulate HUVEC proliferation in the culture medium (800rpm / min, centrifuged for 5min). The HUVEC cells were resuspended in 6% FBS culture medium, and after cell counting, they were inoculated in a white 96-well cell culture plate at 4000 cells / 50μl / well and cultured in an incubator for 2h. The starting concentration of VEGF was adjusted to 300ng / ml, and 120μl / well was added to a sterile 96-well plate. The bispecific binding molecules to be tested were gradiently diluted: the starting concentration was 600nM, and the gradient dilution was performed at a 4-fold ratio. The diluted bispecific binding molecules were added to the above 96-well plate in equal volumes and incubated at room temperature for 30min. The incubated bispecific binding molecules and antigen mixture were added to the adhered HUVEC cells at 100 μl / well and cultured in an incubator for 5 days. After the culture was completed, 50 μl / well of (G7573, PROMEGA) was added and incubated at room temperature in the dark for 10 minutes. The cells were detected using the Luminescence program of the Cytation5 cell imager.

[0431] The results showed that the bispecific binding molecules of the present application can significantly inhibit the proliferation of HUVECs caused by VEGF.

[0432] Table 2. Bispecific antibodies inhibit VEGF-induced HUVEC cell proliferation

[0433] Sample No. <![CDATA[EC 50 (nM)]]> P216280 0.10 P216281 0.03 P216282 0.88 P216283 0.80 P216284 0.11 P216285 0.02 P216286 0.02 P216287 0.02 P216288 0.02 P216289 0.03 P216290 0.07 P216291 0.05 P216292 0.05 P216293 0.06 P216294 0.42 P216295 0.03 P216296 0.04 P216297 0.03 Aflibercept positive control 0.11

[0434] The above results indicate that the bispecific binding molecules disclosed herein have strong inhibitory activity on VEGF-induced HUVEC cell proliferation.

[0435] Test Example 3: In vitro and in vivo assays to evaluate the efficacy of plasma kallikrein binding proteins

[0436] There are proteins such as PPK and PK in human plasma, but the detection found that PK in human plasma stock solution or its dilution did not show activity, which may be an equilibrium state of enzyme inhibition. If additional factor XIIa is added, the balance will be broken and PK protein with enzyme activity will be produced. Based on this principle, the activity of PK inhibitors can be detected.

[0437] First, prepare the antibody sample, dilute the bispecific antibody to 200 μg / ml as the starting concentration with a detection buffer (20 mM Tris-HCl, PH 7.50, 150 mM NaCl, 1 mM EDTA, 0.1% PEG-8000 and 0.1% Triton X-100), perform a 4-fold gradient dilution, a total of 11 concentration gradients, and set aside after dilution. Dilute human plasma 40 times with a detection buffer, dilute factor XIIa to 100 ng / ml, and dilute the substrate peptide (H-Pro-Phe-Arg-AMC) to 1000 μM for use. 50 μl of detection buffer and each diluted concentration gradient bispecific solution were added to a 96-well light-proof ELISA plate; 20 μl of 40-fold dilution of human plasma was added to the above 96-well plate and incubated at 37°C for 30 min; 20 μl of 100 ng / ml factor XIIa was added and incubated at 37°C for 45 min; finally, 10 μl of 1000 μM substrate peptide was added to each well of the above 96-well light-proof ELISA plate. The prepared 96-well plate was placed in a multifunctional microplate reader (Spectra Max i3X) for detection. The detection parameters were: excitation light 360 nm, emission light 480 nm, reading every 60 s, and reading for a total of 10 min. The enzyme reaction rate (slope) was normalized and fitted with the concentration (nM) for a four-parameter curve to calculate the IC50 (nM). The specific results are shown in Table 3.

[0438] Table 3. Results of human plasma PK activity test of bispecific antibodies

[0439] Sample No. <![CDATA[IC 50 (nM)]]> P216280 42.3 P216281 28.5 P216282 36.4 P216283 51.8 P216284 53.3 P216285 18.6 P216286 28.5 P216287 33.8 P216288 27.4 P216289 16.6 P216290 12.5 P216291 46.9 P216292 45.3 P216293 65.2 P216294 78.5 P216295 66.3 P216296 21.7 P216297 18.3 Lanadelumab* 34.1

[0440] *Lanadelumab, a monoclonal antibody against plasma kallikrein, was used as a positive control.

[0441] The above results indicate that the bispecific binding molecules disclosed herein have strong inhibitory activity against PK in whole human plasma.

Claims

1. A bispecific binding molecule comprising: at least one first binding module that specifically binds to VEGF, at least one second binding moiety that specifically binds PKa; and at least one Fc region; The first binding module comprises the extracellular domain of human VEGF receptor 1 and the extracellular domain of human VEGF receptor 2; The second binding module comprises a heavy chain variable region PKa-VH and a light chain variable region PKa-VL; or the second binding module comprises a PKa heavy chain and a PKa light chain; When the heavy chain variable region PKa-VH and the light chain variable region PKa-VL are on the same chain, the order can be interchanged; Preferably, the first binding module is a fusion protein formed by the extracellular domain of human VEGF receptor 1 and the extracellular domain of human VEGF receptor 2, and the order of the extracellular domain of human VEGF receptor 1 and the extracellular domain of human VEGF receptor 2 can be interchanged; Preferably, the Fc region is an IgG Fc region, more preferably an IgG1 Fc region; Preferably, the Fc region comprises a first subunit and a second subunit; The first subunit comprises a protruding structure of Knob-Hole technology, and the second subunit comprises a hole structure of Knob-Hole; or The second subunit comprises a protruding structure of Knob-Hole technology, and the first subunit comprises a hole structure of Knob-Hole.

2. The bispecific binding molecule of claim 1, wherein: The bispecific binding molecule is a dimer or trimer; The bispecific binding molecules are homomers or heteromers.

3. The bispecific binding molecule according to claim 1 or 2, which is selected from any one of the structures 1) to 11) below: 1) Two identical polypeptides of formula 1, in order from N to C-terminus: First binding module + Fc region + first linker + PKa-VH + second linker + PKa-VL (Formula 1); 2) Two identical polypeptides of formula 2, in order from N to C-terminus: First binding module + Fc region + first linker + PKa-VL + second linker + PKa-VH (Formula 2); 3) a first chain of formula 3, and a second chain, wherein: Formula 3 In order from N to C terminus: PKa heavy chain + linker + first binding module (Formula 3); The second chain is a PKa light chain; 4) a first chain of formula 4, and a second chain, wherein: Formula 4 In order from N to C terminus: first binding module + linker + PKa heavy chain (Formula 4); The second chain is a PKa light chain; 5) a second chain of formula 5, and a first chain, wherein: Formula 5 in order from N to C terminus: first binding module + linker + PKa light chain (Formula 5); The first chain is a PKa heavy chain; 6) a second chain of formula 6, and a first chain, wherein: Formula 6 In order from N to C terminus: PKa light chain + linker + first binding module (Formula 6); The first chain is a PKa heavy chain; 7) a first chain of formula 4, and a second chain of formula 5; 8) A first chain, a second chain and a third chain: The first chain is in the order from N to C terminus: the first binding module + the first subunit (Formula 7); The second chain, in N- to C-terminal order: the PKa heavy chain containing the second subunit; The third chain is in the order from N to C terminus: PKa light chain; 9) A first chain, a second chain and a third chain: The first chain is in the order from N to C terminus: first binding module + second subunit (Formula 8); The second chain, in N- to C-terminal order: the PKa heavy chain containing the first subunit; The third chain is in the order from N to C terminus: PKa light chain; 10) A first chain, a second chain and a third chain: The first chain is in the order from N to C terminus: first binding module + linker + first binding module + first subunit (Formula 9); The second chain, in N- to C-terminal order: the PKa heavy chain containing the second subunit; The third chain is in the order from N to C terminus: PKa light chain; 11) A first chain, a second chain and a third chain: The first chain is in the order from N to C terminus: first binding module + linker + first binding module + second subunit (Formula 10); The second chain, in N- to C-terminal order: the PKa heavy chain containing the first subunit; The third chain is in the order from N to C terminus: PKa light chain; in; When the bispecific binding molecule comprises at least two first binding moieties, each first binding moiety is the same or different; The first connector and the second connector are the same or different; The joint, the first joint, and the second joint are each independently (GGGGS) n , n is an integer from 3 to 6; "+" indicates a covalent bond.

4. The bispecific binding molecule according to any one of claims 1 to 3, wherein: The PKa-VH comprises: PKa-HCDR1, PKa-HCDR2 and PKa-HCDR3 in SEQ ID NO: 7, The PKa-VL comprises: PKa-LCDR1, PKa-LCDR2 and PKa-LCDR3 in SEQ ID NO: 8; HCDR and LCDR are coded according to Kabat, IMGT, Chothia, AbM or Contact; The human VEGF receptor 1 extracellular domain comprises the amino acid sequence shown in SEQ ID NO: 18; The human VEGF receptor 2 extracellular domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, and 17.

5. The bispecific binding molecule according to any one of claims 1 to 4, wherein: The PKa-VH comprises: PKa-HCDR1 shown in SEQ ID NO: 1, PKa-HCDR2 shown in SEQ ID NO: 2, and PKa-HCDR3 shown in SEQ ID NO: 3, The PKa-VL comprises: PKa-LCDR1 shown in SEQ ID NO:4, PKa-LCDR2 shown in SEQ ID NO:5, and PKa-LCDR3 shown in SEQ ID NO:6; HCDR and LCDR are numbered according to Kabat.

6. The bispecific binding molecule according to any one of claims 1 to 5, wherein: The Fc region comprises an amino acid sequence selected from any one of SEQ ID NOs: 25-28.

7. The bispecific binding molecule according to any one of claims 1 to 6, wherein: The PKa-VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 7, 9, and 11, and The PKa-VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 8, 10, and 12.

8. The bispecific binding molecule according to any one of claims 1 to 7, wherein: The first subunit is shown in SEQ ID NO: 26, and the second subunit is shown in SEQ ID NO: 28; or The first subunit is shown in SEQ ID NO:28, and the second subunit is shown in SEQ ID NO:

26.

9. The bispecific binding molecule according to any one of claims 1 to 8, wherein: The first binding module comprises an amino acid sequence selected from any one of SEQ ID NOs: 19-22.

10. The bispecific binding molecule according to any one of claims 1 to 9, wherein: The PKa light chain comprises the amino acid sequence shown in SEQ ID NO: 14; The PKa heavy chain comprises an amino acid sequence selected from any one of SEQ ID NOs: 13, 45, and 46.

11. The bispecific binding molecule according to any one of claims 1 to 10, comprising any one selected from the group consisting of: 1) two amino acid sequences shown in SEQ ID NO: 31; 2) two amino acid sequences shown in SEQ ID NO: 32; 3) two amino acid sequences shown in SEQ ID NO: 33; 4) two amino acid sequences shown in SEQ ID NO: 34; 5) two amino acid sequences shown in SEQ ID NO: 35; 6) two amino acid sequences shown in SEQ ID NO: 36; 7) two amino acid sequences shown in SEQ ID NO: 37; 8) a first strand represented by SEQ ID NO:38, and a second strand represented by SEQ ID NO:14; 9) a first strand represented by SEQ ID NO:39, and a second strand represented by SEQ ID NO:14; 10) a first strand represented by SEQ ID NO:40, and a second strand represented by SEQ ID NO:14; 11) a first strand represented by SEQ ID NO:50, and a second strand represented by SEQ ID NO:14; 12) a first strand represented by SEQ ID NO: 13, and a second strand represented by SEQ ID NO: 41; 13) a first strand represented by SEQ ID NO: 13, and a second strand represented by SEQ ID NO: 48; 14) a first strand represented by SEQ ID NO: 13, and a second strand represented by SEQ ID NO: 49; 15) a first strand represented by SEQ ID NO:42, and a second strand represented by SEQ ID NO:43; 16) a first strand shown by SEQ ID NO:44, a second strand shown by SEQ ID NO:45, and a third strand shown by SEQ ID NO:14; 17) a first strand shown by SEQ ID NO:51, a second strand shown by SEQ ID NO:45, and a third strand shown by SEQ ID NO:14; 18) a first chain shown by SEQ ID NO:46, a second chain shown by SEQ ID NO:47, and a third chain shown by SEQ ID NO:

14.

12. A pharmaceutical composition comprising: A therapeutically effective amount of the bispecific binding molecule according to any one of claims 1 to 11, one or more pharmaceutically acceptable carriers, diluents, buffers or excipients, and Optionally, a second therapeutic agent; The second therapeutic agent is selected from the group consisting of antidiabetic agents, agents for treating overweight, agents for treating obesity, agents for treating hypertension, agents for treating pulmonary hypertension, agents for treating heart failure, agents for treating atherosclerosis, and agents for treating eye diseases; The antidiabetic agent is selected from: GK agonists (GLP-1 receptor agonists, exenatide, liraglutide, semaglutide, dulaglutide, loxenatide, albiglutide), α-glucosidase inhibitors (voglibose, acarbose), SGLT-2 inhibitors (canagliflozin, dapagliflozin, empagliflozin, empagliflozin, rupagliflozin, tolagliflozin), DPP-4 inhibitors (sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, gemagliptin, tenegliptin, metformin); The drug for treating hypertension is selected from the group consisting of angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, calcium channel blockers, beta-receptor blockers, diuretics, and alpha-receptor blockers; preferably, the drug for treating hypertension is selected from the group consisting of felodipine, nifedipine, nitrendipine, nisoldipine, nicardipine, lacidipine, lercanidipine, amlodipine, levoamlodipine, verapamil, diltiazem, captopril, enalapril, benazepril, lislopril, ramipril, fosinopril, cilazapril, perindopril, quinapril, imidapril, zofenopril, losartan, valsartan, irbesartan, irbesartan, candesartan cilexetil, telmisartan, and olmesartan medoxomil; The drug for treating pulmonary hypertension is selected from the group consisting of: bosentan, riociguat, macitentan, and selexipag; The heart failure treatment agent and / or the atherosclerosis treatment agent is selected from the group consisting of mevastatin, lovastatin, pravastatin, simvastatin, fluvastatin, cholestyramide, colestipol, aspirin, clopidogrel, cilostazol, ω-3 polyunsaturated fatty acids, ω-6 monounsaturated fatty acids, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, finerenone, eloumab, alirocumab, and inxilan; The drug for treating eye diseases is selected from: corticosteroids, complement factor 3 inhibitors, complement factor 5 inhibitors, complement factor B inhibitors, complement factor D inhibitors, anti-PDGF antibodies, VAP-1 inhibitors, bradykinin receptor 1 antagonists, Tie-2 agonists, and CCR-2 antagonists.

13. An isolated nucleic acid encoding the bispecific binding molecule of any one of claims 1 to 11.

14. A host cell comprising the isolated nucleic acid of claim 13.

15. Use of the bispecific binding molecule according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12 in the preparation of a medicament, wherein: The drug is used to prevent or treat a disease; The disease is selected from any one of the following: diabetic retinopathy, diabetic macular edema, retinal vein occlusion, central retinal vein occlusion, macular degeneration, wet age-related macular degeneration, retinopathy of prematurity, neovascular glaucoma, retinitis pigmentosa, proliferative retinopathy, non-proliferative retinopathy, retinal angiomatous proliferation, macular telangiectasia, ischemic retinopathy, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, polypoidal choroidal vasculopathy, retinal degeneration, hereditary angioedema, brain edema after stroke, retinal vascular permeability diabetic complications; Preferably, the disease is selected from any one of the following: diabetic retinopathy, diabetic macular edema, retinal vein occlusion, central retinal vein occlusion, wet age-related macular degeneration, non-exudative choroidal neovascularization, hereditary angioedema, brain edema after stroke.

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