WO2023030433A1 - Anti-alk-1/anti-vegf bispecific antibody and use thereof - Google Patents

Abstract

A bispecific antibody specifically binding to ALK-1 and VEGF, a pharmaceutical composition comprising the antibody, and the use thereof. The bispecific antibody against ALK-1 and VEGF comprises a first antigen binding region (ALK-1 binding region) specifically binding to ALK-1 and a second antigen binding region (VEGF binding region) specifically binding to VEGF, and can inhibit angiogenesis and/or treat tumors and has a good prospect for application.

Description

Anti-ALK-1/anti-VEGF bispecific antibody and its application

This application claims the priority of the Chinese patent application with the application number 202111032288.7 and the invention title "anti-ALK-1/anti-VEGF bispecific antibody and its application" submitted to the China Patent Office on September 3, 2021, the entire content of which Incorporated in this application by reference.

technical field

The present invention relates to a bispecific antibody specifically binding to ALK-1 and VEGF, a pharmaceutical composition containing the antibody, and applications thereof.

Background technique

ALK-1 is a type I cell surface receptor for transforming growth factor-beta-1 (TGF-beta-1). In general, ligands of the TGF-β superfamily exert biological activity through binding to heteroreceptor complexes formed by two classes (type I and type II) of serine/threonine kinases. Type II receptors are constitutively active kinases that phosphorylate type I receptors upon ligand binding. Thereafter, activated type I kinases phosphorylate downstream signaling molecules, including various Smads, which enter the nucleus and initiate transcriptional responses. Heldin et al. Nature, 1997, vol.390, pp.465-471. For ALK-1, we have found that Smad1 is specifically phosphorylated and enters the nucleus to directly regulate the expression of Smad1-responsive genes Id1 and EphB2.

ALK-1 is selectively overexpressed in endothelial cells and other highly vascularized tissues such as placenta or brain. Through Affymetrix analysis and real-time RT-PCR, we have found that ALK-1 is significantly expressed in endothelial cells over its co-receptor activin type II and endoglin, its ligands TGF-β-1 or ALK-5. Mutations in ALK-1 are associated with hereditary hemorrhagic telangiectasia (HHT), suggesting an important role for ALK-1 in regulating angiogenesis or repair. Abdalla et al. J. Med. Genet, 2003, vol.40, pp.494-502; Sadick et al. Hematologica/The Hematology J., 2005, vol.90, 818-828. Furthermore, two independent experiments in ALK-1 knockout mice provided important in vivo evidence for the role of ALK-1 in angiogenesis. Oh et al. Proc Natl Acad Sci U S A, 2000, vol.97, pp.2626-2631; Urness et al. Nature Genetics, 2000, vol.26, pp.328-331.

Angiogenesis is involved in the pathogenesis of various diseases, therefore, drugs and methods capable of inhibiting angiogenesis have been clinically urgently needed.

Contents of the invention

After a lot of experiments, the inventors of the present invention unexpectedly obtained a bispecific antibody that specifically binds ALK-1 and VEGF, which shows good affinity for both ALK-1 and VEGF, and has good druggability.

In a first aspect, the present invention provides a bispecific antibody comprising a first antigen-binding region specifically binding to ALK-1 (ALK-1 binding region) and a second antigen-binding region specifically binding to VEGF (VEGF binding region), the first antigen-binding region specifically binding to ALK-1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the second antigen-binding region specifically binding to VEGF comprises A heavy chain variable region (VH) and a light chain variable region (VL) or a VEGF receptor fragment that specifically binds VEGF.

In some embodiments of the invention, the heavy chain variable region of the first antigen binding region comprises

a) CDR1H as set forth in SEQ ID NO: 2, CDR2H as set forth in SEQ ID NO: 3 and CDR3H as set forth in SEQ ID NO: 4; or

b) CDR1H with the same function as SEQ ID NO: 2 obtained by adding, deleting, or replacing one or more amino acids from the sequence shown in SEQ ID NO: 2, and adding, deleting, or replacing the sequence shown in SEQ ID NO: 3 The CDR2H with the same function as SEQ ID NO: 3 obtained by replacing one or more amino acids and the function of SEQ ID NO: 4 obtained by adding, deleting, or replacing one or more amino acids in the sequence shown in SEQ ID NO: 4 Same CDR3H.

In some embodiments of the invention, the light chain variable region of the first antigen binding region comprises

a) CDR1L as set forth in SEQ ID NO: 6, CDR2L as set forth in SEQ ID NO: 7 and CDR3L as set forth in SEQ ID NO: 8; or

b) CDR1L obtained from the sequence shown in SEQ ID NO: 6 by adding, deleting, or replacing one or more amino acids with the same function as SEQ ID NO: 6, by adding, deleting, or replacing the sequence shown in SEQ ID NO: 7 The CDR2L with the same function as SEQ ID NO: 7 obtained by replacing one or more amino acids and the function of SEQ ID NO: 8 obtained by adding, deleting, or replacing one or more amino acids from the sequence shown in SEQ ID NO: 8 Same CDR3L.

In some embodiments of the present invention, the heavy chain variable region of the first antigen-binding region comprises the following CDR combination: CDR1H as shown in SEQ ID NO: 2, CDR2H as shown in SEQ ID NO: 3 and CDR3H as shown in SEQ ID NO:4.

In some embodiments of the invention, the light chain variable region of the first antigen binding region comprises a combination of CDRs selected from:

a) CDR1L as set forth in SEQ ID NO: 6, CDR2L as set forth in SEQ ID NO: 7 and CDR3L as set forth in SEQ ID NO: 8; and

b) CDR1L as shown in SEQ ID NO:6, CDR2L as shown in SEQ ID NO:7 and CDR3L as shown in SEQ ID NO:16.

In some embodiments of the invention, the heavy chain variable region and the light chain variable region of the first antigen binding region comprise a combination of CDRs selected from:

a) said heavy chain variable region comprises CDR1H as shown in SEQ ID NO: 2, CDR2H as shown in SEQ ID NO: 3 and CDR3H as shown in SEQ ID NO: 4, and

The light chain variable region comprises CDR1L shown in SEQ ID NO: 6, CDR2L shown in SEQ ID NO: 7 and CDR3L shown in SEQ ID NO: 8;

b) said heavy chain variable region comprises CDR1H as shown in SEQ ID NO: 2, CDR2H as shown in SEQ ID NO: 3 and CDR3H as shown in SEQ ID NO: 4, and

The light chain variable region comprises CDR1L as shown in SEQ ID NO:6, CDR2L as shown in SEQ ID NO:7 and CDR3L as shown in SEQ ID NO:16.

In some embodiments of the present invention, the heavy chain variable region of the first antigen-binding region comprises the sequence shown in SEQ ID NO:1.

In some embodiments of the present invention, the light chain variable region of the first antigen-binding region comprises a sequence selected from SEQ ID NO: 5 or SEQ ID NO: 15:

In some embodiments of the invention, the heavy chain variable region and the light chain variable region of the first antigen binding region comprise a combination selected from:

a) a heavy chain variable region sequence as shown in SEQ ID NO: 1 and a light chain variable region sequence as shown in SEQ ID NO: 5; and

b) the heavy chain variable region sequence as shown in SEQ ID NO: 1 and the light chain variable region sequence as shown in SEQ ID NO: 15.

In some embodiments of the invention, the bispecific antibody comprises an IgG heavy chain constant region, preferably an IgG1 or IgG2 heavy chain constant region, more preferably an IgG1 heavy chain constant region, most preferably Specifically, comprising a heavy chain constant region as shown in SEQ ID NO:9.

In some embodiments of the present invention, the second antigen-binding region that specifically binds to VEGF comprises a heavy chain variable region and a light chain variable region,

The heavy chain variable region comprises CDR1H as shown in SEQ ID NO: 19, CDR2H as shown in SEQ ID NO: 20 and CDR3H as shown in SEQ ID NO: 21; and

The light chain variable region comprises CDR1L as shown in SEQ ID NO: 23, CDR2L as shown in SEQ ID NO: 24 and CDR3L as shown in SEQ ID NO: 25.

In some embodiments of the present invention, the heavy chain variable region comprises a sequence as shown in SEQ ID NO: 18, and the light chain variable region comprises a sequence as shown in SEQ ID NO: 22.

In some embodiments of the present invention, the second antigen-binding region that specifically binds VEGF comprises a VEGF receptor fragment that specifically binds VEGF, and the VEGF receptor fragment comprises the extracellular domain of VEGF receptor-1 2 and the extracellular domain 3 of VEGF receptor-2,

Preferably, the VEGF receptor fragment comprises the sequence shown in SEQ ID NO: 17 or the sequence shown in SEQ ID NO: 17 through addition, deletion, replacement of one or more amino acids obtained with the function of SEQ ID NO: 17 the same sequence,

More preferably, the VEGF receptor fragment comprises the sequence shown in SEQ ID NO: 17.

In some embodiments of the invention, said first antigen binding region or said second antigen binding region is in the form of a scFv.

In some embodiments of the present invention, the first antigen-binding region is connected to the second antigen-binding region through a Linker,

Preferably, the Linker includes (G4S)n, where n is an integer greater than 1,

More preferably, the Linker is composed of (G4S)n, where n is an integer of 2-10,

More preferably, the Linker is composed of (G4S)n, n is 2, 3 or 4, for example, the Linker is GGGGSGGGGS (SEQ ID NO: 12) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 11).

In some embodiments of the present invention, the scFv comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are connected by a Linker,

Preferably, the Linker includes (G4S)n, where n is an integer greater than 1,

More preferably, the Linker is composed of (G4S)n, where n is an integer of 2-10,

More preferably, the Linker is composed of (G4S)n, where n is 2, 3 or 4.

In some embodiments of the present invention, the bispecific antibody consists of 4 peptide chains, 2 identical first chains and 2 identical second chains,

The first chain sequentially comprises VH of the VEGF binding region, CH of the VEGF binding region, Linker, VH of the ALK-1 binding region, Linker, and VL of the ALK-1 binding region from the N-terminal to the C-terminal, and

The second chain comprises the VL and CL of the VEGF binding domain sequentially from N-terminus to C-terminus.

In some embodiments of the present invention, the specific sequence contained in the bispecific antibody is as follows:

The VH sequence of the VEGF binding region is shown in SEQ ID NO: 18,

The CH sequence of the VEGF binding region is shown in SEQ ID NO: 9,

The VL sequence of the VEGF binding region is shown in SEQ ID NO: 22,

The CL sequence of the VEGF binding region is shown in SEQ ID NO: 10,

The Linker sequence is shown in SEQ ID NO: 11,

The VH sequence of the ALK-1 binding region is shown in SEQ ID NO: 13, and

The VL sequence of the ALK-1 binding region is shown in SEQ ID NO:14.

In some embodiments of the invention, the bispecific antibody consists of 4 peptide chains, 2 identical first chains and 2 identical second chains,

The first chain sequentially comprises the VH of the ALK-1 binding region, the CH of the ALK-1 binding region, the Linker and the VEGF binding region from the N-terminal to the C-terminal, and

The second chain sequentially comprises VL and CL of the ALK-1 binding region from N-terminus to C-terminus.

In some embodiments of the invention, the bispecific antibody consists of 4 peptide chains, 2 identical first chains and 2 identical second chains,

The first strand sequentially comprises VH and CH of the ALK-1 binding region from the N-terminus to the C-terminus, and

The second chain includes VEGF binding region, Linker, VL and CL of ALK-1 binding region sequentially from N-terminal to C-terminal.

In some embodiments of the present invention, the bispecific antibody is composed of two identical peptide chains, and the peptide chains sequentially include VEGF binding region, IgG1-Fc, Linker, ALK-1 binding region from N-terminal to C-terminal VH of the region, Linker and VL of the ALK-1 binding region. Herein, IgG1-Fc means the Fc region of IgG1, preferably the Fc region of human IgG1.

In some embodiments of the invention, the bispecific antibody consists of 4 peptide chains, 2 identical first chains and 2 identical second chains,

The first chain sequentially comprises the VH of the ALK-1 binding region, the CH of the ALK-1 binding region, the Linker, the VH of the VEGF binding region, the Linker, the VL of the VEGF binding region from the N-terminal to the C-terminal, and

The second chain sequentially comprises VL and CL of the ALK-1 binding region from N-terminus to C-terminus.

In a second aspect, the present invention provides a polynucleotide encoding the bispecific antibody or fragment thereof in the first aspect. The bispecific antibody fragments referred to herein refer to the full length or fragments of the polypeptide chains constituting the bispecific antibody.

In the third aspect, the present invention provides an expression vector capable of expressing the bispecific antibody or fragment thereof described in the first aspect.

In the fourth aspect, the present invention provides an engineered cell comprising the vector described in the third aspect.

In the fifth aspect, the present invention provides a pharmaceutical composition comprising the bispecific antibody described in the first aspect, the polynucleotide described in the second aspect, the carrier described in the third aspect, or the bispecific antibody described in the second aspect. The cell described in the four aspects, and a pharmaceutically acceptable carrier.

In the sixth aspect, the present invention provides the bispecific antibody described in the first aspect, the polynucleotide described in the second aspect, the vector described in the third aspect, the cell or cell described in the fourth aspect The use of the pharmaceutical composition described in the fifth aspect in the preparation of drugs for inhibiting angiogenesis, such as inhibiting tumors or angiogenesis in some ocular diseases, such as retinal detachment, vitreoretinopathy, premature birth Pediatric retinopathy, glaucoma, synovitis, proliferative diabetic retinopathy, branch retinal vein occlusion, etc.

In the seventh aspect, the present invention provides the bispecific antibody described in the first aspect, the polynucleotide described in the second aspect, the vector described in the third aspect, the cell or cell described in the fourth aspect Use of the pharmaceutical composition described in the fifth aspect in the preparation of a drug for treating tumors, which includes solid tumors and non-solid tumors, such as advanced or refractory hepatocellular carcinoma (HCC), colorectal cancer ( RCC), non-small cell lung cancer (NSCLC), triple negative breast cancer, gastric cancer (GC), gastroesophageal junction (GEJ) adenocarcinoma, cholangiocarcinoma, urothelial carcinoma (UC), esophageal square cell carcinoma (ESCC), Brain tumor, lung cancer, breast cancer, ovarian cancer, fallopian tube cancer, glioblastoma, colorectal adenocarcinoma, pituitary tumor, pituitary adenoma, pituitary macroadenoma, gestational trophoblastic tumor, choriocarcinoma, placental site trophoblastic tumor , epithelioid trophoblastic tumor, renal cell carcinoma, lung adenocarcinoma, and gliosarcoma.

In an eighth aspect, the present invention provides a method for inhibiting angiogenesis, comprising administering an effective dose of the bispecific antibody described in the first aspect, the multinuclear antibody described in the second aspect to a subject in need thereof. Nucleic acid, the carrier described in the third aspect, the cell described in the fourth aspect or the pharmaceutical composition described in the fifth aspect, such as inhibiting tumor or angiogenesis in some ocular diseases, the ocular diseases Such as retinal detachment, vitreoretinopathy, retinopathy of prematurity, glaucoma, synovitis, proliferative diabetic retinopathy, branch retinal vein occlusion, etc.

In the ninth aspect, the present invention provides a method for treating tumors, comprising administering an effective dose of the bispecific antibody described in the first aspect and the polynucleoside described in the second aspect to a subject in need thereof. acid, the carrier of the third aspect, the cell of the fourth aspect or the pharmaceutical composition of the fifth aspect, the tumor includes solid tumors and non-solid tumors, such as advanced or refractory liver cells Carcinoma (HCC), colorectal cancer (RCC), non-small cell lung cancer (NSCLC), triple negative breast cancer, gastric cancer (GC), gastroesophageal junction (GEJ) adenocarcinoma, cholangiocarcinoma, urothelial carcinoma (UC) , Esophageal square cell carcinoma (ESCC), brain tumors, lung cancer, breast cancer, ovarian cancer, fallopian tube cancer, glioblastoma, colorectal adenocarcinoma, pituitary tumor, pituitary adenoma, pituitary macroadenoma, gestational trophoblastic tumor , choriocarcinoma, placental site trophoblastic tumor, epithelioid trophoblastic tumor, renal cell carcinoma, lung adenocarcinoma, and gliosarcoma.

In the tenth aspect, the present invention provides a composition for inhibiting angiogenesis, which comprises the bispecific antibody described in the first aspect, the polynucleotide described in the second aspect, and the polynucleotide described in the third aspect. The carrier described in the fourth aspect or the cell and the pharmaceutically acceptable carrier described in the fourth aspect.

In the eleventh aspect, the present invention provides a composition for treating tumors, which comprises the bispecific antibody described in the first aspect, the polynucleotide described in the second aspect, and the polynucleotide described in the third aspect. The carrier described in the fourth aspect or the cell and the pharmaceutically acceptable carrier described in the fourth aspect.

Description of drawings

Figure 1 shows the affinity distribution of the antibody obtained in Example 3 binding to ALK-1, the triangles are GT90001m, and the dots are mutants.

Fig. 2 shows the affinity distribution of the antibody obtained in Example 5 binding to ALK-1, the triangles are GT90001m, and the dots are mutants.

Fig. 3 shows a schematic diagram of GT90001m-scFv (ScFv end: GT90001m or GT90001mut).

Figure 4 shows a schematic diagram of Bi804-GT90001LC (Fab end: GT90001m; IgG1 C-terminal connected to Elyea) and Bi804-GT90001mutVL-8 (Fab end: GT90001mutVL-8; IgG1 C-terminal connected to Eylea).

Figure 5 shows a schematic diagram of Bi805-GT90001HC (Fab end: GT90001m; the N-terminal of the Fab light chain is connected to Eylea

Figure 6 shows the schematic diagram of Bi807-26 and Bi811-26 (Fab end: Bevacizumab; scFv end: GT90001m or GT90001mutVL-8).

Fig. 7 shows a schematic diagram of Bi808 (scFv end: GT90001m; IgG1 N-terminal connected to Elyea).

Fig. 8 shows a schematic diagram of Bi809-GT90001LC (Fab end: GT90001m; scFv end: Bevacizumab).

Figure 9 shows the binding activity of the bispecific antibody of the present invention to CHO-ALK-1 cells.

Figure 10 shows the binding activity of the bispecific antibody of the present invention to HUVEC.

Figure 11 shows that the bispecific antibody of the present invention blocks the binding activity of VEGF165 protein to human VEGFRII.

Fig. 12 shows the experiment of blocking VEGF/VEGFRII-NFAT signaling pathway by the bispecific antibody of the present invention.

Figure 13 shows that the bispecific antibody of the present invention blocks BMP9-induced phosphorylation of Smad1.

Detailed ways

the term

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Notwithstanding that the numerical ranges and parameter approximations setting forth the broad scope of the invention, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective measurements. Additionally, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between a minimum value of 1 and a maximum value of 10, inclusive; that is, all subranges beginning with a minimum value of 1 or greater , such as 1 to 6.1, and subranges that end at a maximum value of 10 or less, such as 5.5 to 10. Additionally, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

It should also be noted that, as used in this specification, a singular form includes a plural form of its referent unless clearly and definitely limited to one referent. The term "or" is used interchangeably with the term "and/or" unless the context clearly dictates otherwise.

As used herein, the term "antibody" encompasses full-length antibodies (e.g., IgG1 or IgG4 antibodies), various functional fragments thereof (e.g., may only comprise an antigen-binding portion, such as a Fab, F(ab') ₂ or scFv fragment), and Antibodies that have been modified (eg, humanized, glycosylated, etc.). In some applications, it may be useful to modify antibodies to remove undesired glycosylation sites, or the absence of fucose moieties on the oligosaccharide chain, for example to enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be made to alter complement dependent cytotoxicity (CDC).

As used herein, the term "bispecific antibody" refers to an antibody that has the ability to bind at least two different antigens or two different epitopes of the same antigen.

The term "CDR region" or "CDR" as used herein refers to the hypervariable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., U.S. Department of Health and Human Services, NIH, 1991, and later editions). There are three heavy chain CDRs and three light chain CDRs. The term CDR or CDRs is used herein, as the case may be, to denote one of these regions, or several or even all of these regions, which comprise the majority of the amino acid residues responsible for binding by the antibody's affinity for the antigen or the epitope it recognizes. base.

The term "Fc region" or "Fc portion" as used herein is a term well known to those skilled in the art.

As used herein, the term "Fab region" refers to an immunoglobulin composed of the VH and CH1 domains of a heavy chain ("Fab heavy chain") or the VL and CL domains of a light chain ("Fab light chain"), or both. By.

As used herein, the term "scFv" or "single-chain antibody fragment" means an antibody heavy chain variable region and an antibody light chain variable region that are linearly linked together by a linker (eg, a short peptide of 10-25 amino acids). A single chain that exhibits specific binding to the antigen.

The term "peptide linker" as used in the present invention denotes a method for linking different antigen-binding sites and/or antibody fragments (such as single-chain Fv, full-length antibodies, VH domains and/or VL domain, Fab, (Fab) ₂ and Fc part) linked together, preferably it has an amino acid sequence of synthetic origin. A peptide linker may comprise one or more of the amino acid sequences listed in the Examples, as well as other arbitrarily selected amino acids.

Fortebio)测定研究抗体与抗原或FcyRIII的结合。结合亲和力通过术语ka(抗体/抗原复合物中抗体的结合速率常数)、kD(解离常数)和KD(kD/ka)定义。 As used herein, the term "binding" or "specific binding" refers to the binding of an antibody to an antigenic epitope in an in vitro assay (ELISA). It can be obtained by using molecular interaction instrument (

Fortebio) assay to study antibody binding to antigen or FcyRIII. Binding affinity is defined by the terms ka (rate constant for association of an antibody in an antibody/antigen complex), kD (dissociation constant) and KD (kD/ka).

As used herein, "therapeutically effective amount" or "effective amount" refers to a dose sufficient to demonstrate a benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend upon the individual condition and severity of the individual being treated. The prescribing of treatment (e.g. decisions on dosage, etc.) is ultimately the responsibility of and relies on general practitioners and other medical practitioners to make decisions, usually taking into account the disease being treated, the individual patient's condition, the site of delivery, the method of administration and what has become known to the physician. other known factors.

As used herein, the term "subject" refers to mammals, such as humans, but may also be other animals, such as wild animals (such as herons, storks, cranes, etc.), domestic animals (such as ducks, geese, etc.) or experimental animals (such as orangutans, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).

Compositions of the invention can be administered by a variety of methods known in the art. The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the desired result. In order to administer a compound of the invention by a particular route of administration, it may be necessary to cover the compound with, or co-administer the compound with, a material that prevents its inactivation. For example, compounds can be administered to a subject in a suitable carrier, such as liposomes or diluents. Pharmaceutically acceptable diluents include saline solutions and aqueous buffered solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. The presence of microorganisms can be avoided both by the sterilization procedure hereinabove and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. Additionally, prolonged absorption of the injectable pharmaceutical forms can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

Bevacizumab (trade name Avastin) is a recombinant humanized anti-VEGF monoclonal antibody. Approved by the FDA on February 26, 2004, it is the first drug to inhibit tumor angiogenesis approved for marketing in the United States. It is confirmed by in vivo and in vitro detection systems that the antibody can combine with human vascular endothelial growth factor (VEGF) and block its biological activity.

Aflibercept (trade name Eylea, Zaltrap), was first approved by the US Food and Drug Administration (FDA) on November 18, 2011, approved by the European Medicines Agency (EMA) on November 22, 2012, and approved by the European Medicines Agency (EMA) on September 28, 2012 Japan Pharmaceuticals and Medical Devices Administration (PMDA) approved a soluble decoy receptor jointly developed by Regeneron (Regeneron) and Bayer (Bayer), which can bind to vascular endothelial growth factor VEGF-A, VEGF-B and placental growth factor (PIGF), its affinity is higher than the natural receptor of human body. VEGF does not bind to its original receptor on the cell, but instead binds to Aflibercept by mistake, thereby reducing the activity of VEGF. It is suitable for the treatment of neovascular (Wet) age-related macular degeneration, macular edema following retinal vein occlusion and diabetic macular edema.

Bispecific Antibody (Bispecific Antibody), also known as bifunctional antibody, is a specific drug that targets two different antigens at the same time, which can be produced by immuno-sorting and purification. In addition, it can also be obtained through genetic engineering. Genetic engineering methods have corresponding flexibility in terms of binding site optimization, synthetic form considerations, and yields, so they have certain advantages. Currently, more than 45 forms of it have been proven (Dafne Müller, Kontermann R E. 2010, BioDrugs, 24(2):89-98). A variety of bispecific antibodies that have been developed are in the form of IgG-scFv, that is, the Morrison model (Coloma MJ, Morrison SL.1997, Nat Biotechnol.15:159-163). The advantages of antibody engineering, expression and purification have been proved to be one of the ideal forms of bifunctional antibodies (Miller BR, Demarest SJ, et al., 2010, Protein Eng Des Sel; 23:549-57; Fitzgerald J, Lugovskoy A. 2011. MAbs; 3:299-309).

Example

Some preferred embodiments and aspects of the present invention will be further described below in conjunction with specific examples, which should not be construed as limiting the scope thereof.

The antibody mutants involved in the examples of the present invention were constructed based on GT90001m. GT90001 is an anti-ALK-1 monoclonal antibody of human IgG2 subtype (CN101517068A). Compared with GT90001, GT90001m is an anti-ALK-1 monoclonal antibody of human IgG1 subtype, with the same CDR region. The mutant after affinity maturation is numbered GT90001mut, which does not distinguish between GT90001m and GT90001.

Example 1 Construction of ALK-1 antibody affinity maturation library

Using the GT90001m gene sequence as a template, the heavy chain variable region (Variable region of heavy chain, VH) and light chain variable region (Variable region of light) of GT90001m were amplified by Polymerase Chain Reaction (PCR). chain, VL), and then according to the instructions of the ClonExpress Ultra One Step Cloning Kit (Vazyme Biotech, C115-02), the VH and VL of the amplified GT90001m were respectively constructed into the yeast expression vector (name: pYD-1 ).

GT90001m sequence as template:

Heavy Chain Variable Region (VH)

3 CDRs of VH:

CDR1H: GGSISSGEYY (SEQ ID NO: 2)

CDR2H:IYYSGST (SEQ ID NO:3)

CDR3H:ARESVAGFDY (SEQ ID NO:4)

Light chain variable region (VL)

3 CDRs of VL:

CDR1L:QSVSSSY (SEQ ID NO:6)

CDR2L:GTS (SEQ ID NO:7)

CDR3L:QQYGSSPIT (SEQ ID NO:8)

Heavy chain constant region (CH)

Light chain constant region (CL)

20828)中，分别构建GT90001m重链突变、轻链突变、重轻链混合突变的抗体文库，并测定文库大小。文库大小分别为4×10 ⁷、4.5×10 ⁷、4×10 ⁷。 Mutation and amplification of the variable region gene of the GT90001m antibody were carried out according to conventional methods of the prior art, and the linearized yeast display vector and the amplified PCR product were mixed and electrotransformed into Saccharomyces cerevisiae (saccharomyces cerevisiae,

20828), the antibody libraries of GT90001m heavy chain mutation, light chain mutation, and heavy and light chain mixed mutation were respectively constructed, and the size of the library was determined. The library sizes were 4×10 ⁷ , 4.5×10 ⁷ , and 4×10 ⁷ , respectively.

Example 2 Screening of ALK-1 antibody

1. Human ALK-1 protein biotinylation

Dissolve human ALK-1 protein (Acro biosystems, AL1-H5227) in an appropriate amount of double-distilled water, dissolve the biotin and mix it with the protein solution according to the product instructions of the biotin labeling kit (purchased from Thermo), and incubate at 4°C 2 hours. A desalting column (purchased from Thermo) was used to remove excess biotin, and the pretreatment of the desalting column and the collection of samples were performed according to the steps in the product manual.

2. Using MACS to enrich yeast single clones that can specifically bind to ALK-1

Inoculate the yeast library constructed in Example 1 into SD-CAA expansion medium (1L SD-CAA expansion medium contains 6.7g YNB, 5g casamino acids, 13.62g Na ₂ HPO ₄ ·I ₂ H ₂ O, In 7.44g NaH ₂ PO ₄ and 2% glucose), the number of inoculated yeast cells was greater than 10 times the library capacity (the initial amplification concentration was 0.5OD ₆₀₀ /ml), and cultured overnight at 30°C and 225rpm. Take 10 times the capacity of the yeast cell library, centrifuge at 3000rpm for 5 minutes (the following centrifugation is the same) to remove the medium, resuspend the yeast cells with SD-CAA induction medium, the initial adjustment concentration is 0.5OD ₆₀₀ /ml, and induce overnight. Determine the library concentration after induction, take 10 times the capacity of the yeast cell library, and centrifuge to remove the medium. Yeast cells were resuspended with 50ml wash buffer (PBS+0.5%BSA+2mM EDTA) and centrifuged to remove the supernatant. Yeast cells were resuspended with 10 ml wash buffer.

Add biotin-labeled ALK-1 protein (final concentration 100mM), incubate at room temperature for 30 minutes, centrifuge, collect yeast cells, and wash 3 times with 50ml washing buffer. Yeast cells were resuspended with 5ml washing buffer, 200ml SA magnetic beads (purchased from Miltenyi) were added, and the cells were incubated upside down for 10 minutes. The mixture of yeast and magnetic beads was washed 3 times with washing buffer, and then added to an LS column (purchased from Miltenyi). Place the LS column on a magnetic stand and wash with wash buffer to remove non-specifically bound yeast cells. Remove the column from the magnetic stand and add wash buffer to elute the yeast. The eluted yeast was centrifuged and transferred to 200ml SD-CAA expansion medium for amplification.

3. Flow cytometric sorting to obtain high-affinity yeast cells

The yeast cells enriched by MACS were inoculated into the SD-CAA expansion medium, and the initial expansion concentration was 0.5OD ₆₀₀ /ml. Shake flask cultures were performed overnight at 30°C and 225 rpm. Yeast cells were resuspended in SD-CAA induction medium (1L SD-CAA induction medium contained 6.7g YNB, 5g casamino acids, 13.62g Na ₂ HPO ₄ ·I ₂ H ₂ O, 7.44g NaPbPCF, 2% galactose , 2% raffinose and 0.1% glucose), the initial concentration was 0.5OD ₆₀₀ /ml, and induced overnight. Add 100 nM biotinylated ALK-1 antigen and incubate at room temperature for 20 min. The yeast was washed 3 times with PBS, FITC-labeled c-Myc antibody and streptavidin APC-conjugated fluorescent antibody (purchased from Invitrogen) diluted at 1:500 were added and incubated at 4°C in the dark for 15 minutes. Add 2ml of PBS to resuspend the cells, and use a BD FACSArial II instrument to sort the yeast with high binding affinity to the ALK-1 antigen.

4. Obtain the gene sequence of the ALK-1 antibody candidate molecule

The yeast liquid enriched by MACS and FACS with high binding ability to ALK-1 antigen was cultured overnight in SD-CAA expansion medium at 30°C and 225rpm. The yeast plasmid was extracted according to the operation of the yeast plasmid extraction kit (purchased from Tiangen, Cat. No. DP112-02). The plasmid was electrotransferred to Top 10 competent cells (purchased from Tiangen, Cat. No. CB104-02), coated on an ampicillin-resistant plate, and cultured overnight at 37°C. Single clones were picked and sequenced to obtain the gene sequence of the ALK-1 antibody candidate molecule.

Example 3 Construction and expression purification of ALK-1 antibody

1. Antibody gene construction into pCDNA3.1 expression vector

The GT90001m heavy chain mutant gene sequence and light chain mutant gene sequence were respectively constructed into pCDNA3.1-constant heavy chain and pCDNA3.1-constant light chain vectors according to the instructions of the rapid cloning kit (Vazyme, C115-02). The homologous recombination product was transformed into Top 10 competent cells, coated on an ampicillin-resistant plate, cultured overnight at 37°C, and single clones were picked and sequenced.

2. Cell transfection and antibody purification

The plasmid was transformed into Expi-293F cells using the Expi293F ^TM expression system kit (GibcoTM, A14528), and the transfection method was performed according to the product instructions. Subculture HEK293 cells according to the required transfection volume, and adjust the cell density to 2.5-3×10 ⁶ cells/ml one day before transfection; count the cells to be transfected, and use preheated OPM-293CD05 Medium medium to Adjust the cell density to 3×106 cells/ml; take one-tenth of the cell transfection volume MEM medium as the transfection buffer, and add the plasmid according to the concentration of 1 mg plasmid for 1L cells (the ratio of heavy chain to light chain is H:L=1:1), mix well, and filter to sterilize; add PEI according to the mass ratio PEI:plasmid=3:1, mix well and incubate at room temperature for 10-20min, gently add the mixture into HEK293F cells, 36.5℃ , 8% CO ₂ , cultured in a shaker at 130 rpm, and 20-22 hours after transfection, 5% of the transfection volume of the cells was added to the Opmai Profeed feed. After the Expi-293 cells were cultured for 3 days, the supernatant was collected, and Fortebio was used to detect whether the supernatant was bound to the ALK-1 protein.

ForteBio affinity was determined according to existing methods (Estep P, Reid F, Nauman C, Liu Y, Sun T, Sun J, Xu Y. High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs. 2013 Mar- Apr;5(2):270-8.doi:10.4161/mabs.23049.PMID:23575269;PMCID:PMC3893237.) carried out. Briefly, the sensor was equilibrated offline for 30 min in assay buffer, then tested online for 60 s to establish a baseline, and the purified antibody obtained as described above was loaded onto the AHQ sensor online. Then the sensor was reacted in 100nM ALK-1 antigen for 5 minutes, and then transferred to PBS to dissociate for 5 minutes. Dynamic analysis The 1:1 combined model is used for dynamic analysis.

According to the results of affinity determination, the plasmids of the clones that can specifically bind to ALK-1 were selected and transfected into Expi-293F cells. After the cells were cultured for 6 days, the cell supernatant was collected, and rProtein A Beads agarose gel affinity resin was used to (purchased from Changzhou Tiandi Renhe, product number: SA015005) according to its instructions to purify the target protein. Fortebio was used to detect the binding affinity of the protein to ALK-1, and at the same time to detect the druggability.

Example 4 Detection of affinity and druggability of ALK-1 antibody mutants

Fortebio was used to detect the binding affinity of the antibody obtained in Example 3 to ALK-1. The binding activity of 17 anti-ALK-1 monoclonal antibodies to human ALK-1 protein was determined, and the results showed that 16 of them could bind to human ALK-1 at the protein level, and their affinity was higher than that of GT90001m and ALK-1 The binding, monovalent affinity ranges are shown in Figure 1 and Table 1. mutVH indicates heavy chain variable region mutants, and mutVL indicates light chain variable region mutants.

Table 1. Antibody affinity test results

According to existing methods (Jain T, Sun T, Durand S, Hall A, Houston NR, Nett JH, Sharkey B, Bobrowicz B, Caffry I, Yu Y, Cao Y, Lynaugh H, Brown M, Baruah H, Gray LT ,Krauland EM,Xu Y,Vásquez M,Wittrup KD.Biophysical properties of the clinical-stage antibody landscape.Proc Natl Acad Sci U S A.2017Jan 31;114(5):944-949.doi:10.1073/pnas.16146408 .Epub 2017 Jan 17.PMID:28096333; PMCID:PMC5293111.) The druggability of these 17 antibodies was tested.

Thermal Stability (Differential Scanning Fluorescence, DSF)

Take 50 μg of the sample to be tested and mix it with 10 μl 20× sypro orange (Biorad) dye, transfer the sample into a PCR tube and put it into a PCR instrument, set the temperature rise program to 25°C as the initial temperature, and a temperature rise rate of 0.01°C per second. to 95°C. After the experiment, the Tm value was determined according to the curve. Tm1 is the temperature value when the Fab segment of the antibody elutes; Tm2 and Tm3 are the temperature values when the Fc segment of the antibody elutes; the higher the Tm, the better the thermal stability of the antibody.

Colloidal stability (capturing self-interaction-nanoparticle spectroscopy, AC-SINS)

Coupling the capture antibody on the gold nanoparticles, concentrating the gold nanoparticles after reacting at room temperature for 30 minutes, taking the concentrated gold nanoparticles and the antibody to be tested for 2 hours incubation at room temperature, scanning the absorbance value (OD) at 510nm to 570nm with a microplate reader value), the smaller the OD value, the more stable the antibody.

Purity - Molecular Hydrophobicity (SEC-HIC)

The sample to be tested was put on a Zenix SEC-300 chromatographic column, the injection volume was 50 μg, the mobile phase was phosphate buffer, and the flow rate was 0.5ml/min. The hydrophobicity of the sample was determined according to the peak elution time. The smaller the retention time (RT) of the peak, the smaller its hydrophobicity.

Nonspecific adsorption (Cross Interaction Chromatography, CIC)

A CIC column was prepared by coupling approximately 30 mg of human serum polyclonal antibody (Sigma, 14506) to a 1 mL HiTrap column (GE Healthcare, 17-0716-01), followed by inactivation with ethanolamine. On the Agilent 1100 series high-performance liquid chromatography system, PBS was used as the mobile phase and the flow rate was 0.1ml/min to detect the candidate antibodies. The injection volume of the candidate antibody was 5 μg. The smaller the retention time (RT) value of the peak, the smaller the non-specific adsorption of the antibody.

The druggability results are shown in Table 2. Among the 16 candidate molecules, mutVL-4, mutVL-7, mutVL-8, and mutVL-12 have excellent druggability.

Table 2. Druggability test results of 16 antibodies

Compared with GT90001m, the thermal stability, non-specific adsorption and purity of the 16 mutants were similar, and the thermal stability of the antibody was not affected by the amino acid mutation, nor did it increase the non-specific adsorption of the antibody. However, amino acid mutations affect the molecular interaction of the antibody itself, and compared with GT90001m, the stability becomes worse, such as: mutVH-7, mutVH-8, mutVH-13. The best colloidal stability is the light chain mutants, such as: mutVL-1, mutVL-4, mutVL-7, mutVL-8, mutVL-11, mutVL-12. In addition, compared with GT90001m, the light chain mutants mutVL-4, mutVL-7, mutVL-8, and mutVL-12 had better hydrophobicity.

Example 5 Heavy Chain Mutation and Light Chain Mutation of Cross-Paired ALK-1 Antibody

Plasmids with 9 heavy chain mutations and 7 light chain mutations that can bind to ALK-1 with high affinity were paired in pairs, transiently expressed in Expi-293F cells, and Fortebio was used to detect the binding of 63 mutants to ALK-1 Among them, 19 mutants have higher affinity with human ALK-1 protein, and the monovalent affinity range is shown in Figure 2 and Table 3. And carry out druggability detection simultaneously, detection method is the same as embodiment 4.

Table 3. Affinity test results of 19 antibodies

Sample ID Response KD(M) kon(1/Ms) kdis(1/s) GT90001mutVL-1GT90001mutVH-5 0.2024 4.08E-09 2.28E+05 9.31E-04 GT90001mutVL-2GT90001mutVH-8 0.1626 3.89E-09 2.85E+05 1.11E-03 GT90001mutVL-8GT90001mutVH-1 0.2286 3.09E-09 2.48E+05 7.64E-04 GT90001mutVL-8GT90001mutVH-2 0.2069 2.32E-09 2.48E+05 5.76E-04 GT90001mutVL-8GT90001mutVH-4 0.2185 2.66E-09 2.76E+05 7.35E-04 GT90001mutVL-8GT90001mutVH-5 0.2031 2.99E-09 2.46E+05 7.35E-04 GT90001mutVL-8GT90001mutVH-7 0.1639 3.92E-09 3.02E+05 1.18E-03 GT90001mutVL-8GT90001mutVH-8 0.1845 3.53E-09 2.60E+05 9.18E-04 GT90001mutVL-8GT90001mutVH-9 0.2114 2.45E-09 2.53E+05 6.21E-04 GT90001mutVL-11GT90001mutVH-1 0.199 2.25E-09 2.56E+05 5.76E-04 GT90001mutVL-11GT90001mutVH-4 0.1963 1.92E-09 3.01E+05 5.76E-04 GT90001mutVL-11GT90001mutVH-5 0.2009 7.37E-10 2.61E+05 1.92E-04 GT90001mutVL-11GT90001mutVH-9 0.2041 8.15E-10 2.84E+05 2.31E-04 GT90001mutVL-12GT90001mutVH-1 0.1858 8.02E-10 2.78E+05 2.23E-04 GT90001mutVL-12GT90001mutVH-2 0.1694 3.50E-10 2.86E+05 1.00E-04 GT90001mutVL-12GT90001mutVH-5 0.1743 3.53E-10 2.83E+05 1.00E-04 GT90001mutVL-12GT90001mutVH-7 0.1868 5.47E-10 2.83E+05 1.55E-04 GT90001mutVL-12GT90001mutVH-9 0.182 3.38E-10 2.96E+05 1.00E-04 GT90001mutVL-2GT90001mutVH-7 0.1045 1.98E-09 4.01E+05 7.94E-04 GT90001m 0.1633 6.09E-09 3.03E+05 1.85E-03

The druggability results are shown in Table 4. Among the 19 candidate molecules, GT90001mutVL-2GT90001mutVH-7, GT90001mutVL-2GT90001mutVH-8, GT90001mutVL-8GT90001mutVH-7, GT90001mutVL-8GT90001mutVH-8 had poor druggability.

Table 4. Druggability of 19 antibodies

From the test results of affinity and druggability, the mutants with double mutations in light and heavy chains did not have higher affinity and better druggability than mutants with single mutations in light chains. Therefore, the subsequent bispecific antibody preparation uses the GT90001m mutant with light chain mutation.

Example 6 Different scFv formats of ALK-1 antibody

Different ALK-1 antibody scFv structures (Bi800, Bi801, Bi802, Bi803) were constructed, see Figure 3 for a schematic diagram. Explore the influence of scFv with different structures on the affinity KD value. The difference between Bi800, Bi801, Bi802 and Bi803 lies in the length of Linker and the relative position of VH and VL.

Table 5. Constructed GT90001m-scFv

Antibody number structure Bi800 Fc-(G ₄ S) ₄ -GT90001mVH-(G ₄ S) ₄ -GT90001mVL Bi801 Fc-(G ₄ S) ₂ -GT90001mVH-(G ₄ S) ₄ -GT90001mVL Bi802 Fc-(G ₄ S) ₄ -GT90001mVL-(G ₄ S) ₄ -GT90001mVH Bi803 Fc-(G ₄ S) ₂ -GT90001mVL-(G ₄ S) ₄ -GT90001mVH

The sequence is as follows:

Fc

Linker 1 is (G ₄ S) ₄

Linker 2 is (G ₄ S) ₂

VH

CDR1H: GGSISSGEYY (SEQ ID NO: 2)

CDR2H:IYYSGST (SEQ ID NO:3)

CDR3H:ARESVAGFDY (SEQ ID NO:4)

VL

The results are shown in Table 6. The different Linker lengths of GT90001m-scFv and the connection sequence of VH and VL had little effect on the affinity. Compared with Fab, the affinity of scFv was lower. In the later stage, the scFv mode of Bi800 was selected for bispecific antibody construction.

Table 6. Affinity detection results of GT90001m-scFv

sample Response KD(M) kon(1/Ms) kdis(1/s) Bi800 0.2475 1.24E-08 3.10E+05 3.85E-03 Bi801 0.2322 1.41E-08 2.97E+05 4.18E-03 Bi802 0.2459 1.37E-08 2.93E+05 4.01E-03 Bi803 0.2133 1.52E-08 2.66E+05 4.06E-03 GT90001m 0.2422 8.98E-09 4.37E+05 3.93E-03

Example 7 Construction of different forms of anti-ALK-1/anti-VEGF bispecific antibodies

The bispecific antibody was constructed, the ALK-1 binding region was derived from GT90001m or GT90001mutVL-8, and the VEGF binding region was derived from Bevacizumab or Aflibercept. The sequences involved are as follows:

Compared with GT90001m, GT90001mutVL-8 only has 2 amino acid site mutations (underlined positions) in CDR3L of VL, and the sequences of other regions are the same.

VL of GT90001mutVL-8

The CDR1L sequence is shown in SEQ ID NO:6, and the CDR2L sequence is shown in SEQ ID NO:7.

CDR3L

QLYGSFPIT (SEQ ID NO: 16)

The sequence of Aflibercept used in the examples (VEGF binding region, HC-free)

The VH and VL sequences of Bevacizumab used in the examples

VH

CDR1H

GYTFTNYG (SEQ ID NO:19)

CDR2H

INTYTGEP (SEQ ID NO: 20)

CDR3H

AKYPHYYGSSHWYFDV (SEQ ID NO:21)

VL

CDR1L

QDISNY (SEQ ID NO:23)

CDR2L

FTS (SEQ ID NO:24)

CDR3L

QQYSTVPWT (SEQ ID NO: 25)

Table 7: Anti-ALK-1/anti-VEGF bispecific antibody constructed in the example

name Source of ALK-1 binding domain Source of VEGF-binding domain structure Bi804-GT90001 LC GT90001m Aflibercept Figure 4 Bi804-GT90001mutVL-8 GT90001mutVL-8 Aflibercept Figure 4 Bi805-GT90001 HC GT90001m Aflibercept Figure 5 Bi807-26 GT90001m Bevacizumab Figure 6 Bi811-26 GT90001mutVL-8 Bevacizumab Figure 6 Bi808 GT90001m Aflibercept Figure 7 Bi809-GT90001LC GT90001m Bevacizumab Figure 8

Bi807-26

The sequence of the VEGF binding region is as follows:

The VH sequence is shown in SEQ ID NO: 18.

The CH sequence is shown in SEQ ID NO:9.

The VL sequence is shown in SEQ ID NO:22.

The CL sequence is shown in SEQ ID NO:10.

The Linker sequence is shown in SEQ ID NO:11. Linker is located between the VEGF binding region and the ALK-1 binding region.

The ALK-1 binding region is in the form of scFv, and the sequence is as follows:

The VH sequence is shown in SEQ ID NO:13.

The VL sequence is shown in SEQ ID NO:14.

The Linker sequence is shown in SEQ ID NO:11. Linker is located between VH and VL of scFv.

Bi807-26 consists of 4 peptide chains, 2 identical first chains and 2 identical second chains. The first chain sequentially comprises VH of the VEGF binding region, CH of the VEGF binding region, Linker, VH of the ALK-1 binding region, Linker and VL of the ALK-1 binding region from N-terminal to C-terminal. The second chain comprises the VL and CL of the VEGF binding domain sequentially from N-terminus to C-terminus. The four peptide chains can be linked by disulfide bonds.

Bi807-26 first strand sequence:

Bi807-26 second strand sequence:

Bi811-26

Compared with Bi807-26, only the CDR3L of the VL sequence of the ALK-1 binding region has 2 amino acid differences, and the sequences of other regions are the same.

VL of the ALK-1 binding region of Bi811-26:

Bi811-26 first strand sequence:

Bi811-26 second strand sequence:

Bi804-GT90001LC

The sequence of the ALK-1 binding region is as follows:

The VH sequence is shown in SEQ ID NO:1.

The CH sequence is shown in SEQ ID NO:9.

The VL sequence is shown in SEQ ID NO:5.

The CL sequence is shown in SEQ ID NO:10.

The Linker sequence is shown in SEQ ID NO:11. Linker is located between the VEGF binding region and the ALK-1 binding region.

The sequence of the VEGF binding region is shown in SEQ ID NO:17.

Bi804-GT90001LC consists of 4 peptide chains, 2 identical first chains and 2 identical second chains. The first chain sequentially comprises the VH of the ALK-1 binding region, the CH of the ALK-1 binding region, the Linker and the VEGF binding region from the N-terminal to the C-terminal. The second chain sequentially comprises VL and CL of the ALK-1 binding region from N-terminus to C-terminus. The four peptide chains can be linked by disulfide bonds.

Bi804-GT90001LC first strand sequence:

Bi804-GT90001LC second strand sequence:

Bi804-GT90001mutVL-8

Compared with Bi804-GT90001LC, only the CDR3L of the VL sequence of the ALK-1 binding region has 2 amino acid differences, and the VL sequence is shown in SEQ ID NO:15.

Bi804-GT90001mutVL-8 first strand sequence:

Bi804-GT90001mutVL-8 second strand sequence:

Bi805-GT90001HC

Each partial sequence is the same as that of Bi804-GT90001LC, the difference lies in the position of the VEGF binding region. In Bi804-GT90001LC, the VEGF binding region is connected to the Fc end of the ALK-1 binding region; in Bi805-GT90001LC, the VEGF binding region is connected to the VL end of the ALK-1 binding region.

Bi805-GT90001LC consists of 4 peptide chains, 2 identical first chains and 2 identical second chains. The first chain comprises VH and CH of the ALK-1 binding region sequentially from the N-terminus to the C-terminus. The second chain includes VEGF binding region, Linker, VL and CL of ALK-1 binding region sequentially from N-terminal to C-terminal. The four peptide chains can be linked by disulfide bonds.

Bi805-GT90001LC first strand sequence:

Bi805-GT90001LC second strand sequence:

Bi808

The sequence of the VEGF binding region is shown in SEQ ID NO:17, and the sequence of the Fc region is shown in SEQ ID NO:9.

The VH sequence of the ALK-1 binding region is shown in SEQ ID NO:13, and the VL sequence is shown in SEQ ID NO:14. The ALK-1 binding domain is in scFv format.

The Linker sequence is shown in SEQ ID NO:11. Linker is located between VEGF binding region and ALK-1 binding region, and Linker is located between VH and VL of scFv.

Bi808 consists of 2 identical peptide chains. The peptide chain comprises VEGF binding region, IgG1-Fc, Linker, VH of ALK-1 binding region, Linker and VL of ALK-1 binding region sequentially from N terminal to C terminal.

Bi808 sequence:

Bi809-GT90001LC

ALK-1 binding domain:

The VH sequence is shown in SEQ ID NO:1.

The CH sequence is shown in SEQ ID NO:9.

The VL sequence is shown in SEQ ID NO:5.

The CL sequence is shown in SEQ ID NO:10.

The Linker sequence is shown in SEQ ID NO:11. Linker is located between VEGF binding region and ALK-1 binding region, and Linker is located between VH and VL of scFv.

VEGF binding region (scFv format):

VH

VL

Bi809-GT90001LC second strand sequence:

The affinities of bispecific antibodies binding to ALK-1 and VEGF were detected by Fortebio, and the results are shown in Table 7 and Table 8.

Table 7. Affinity of GT90001m-based bispecific antibodies to ALK-1

sample Response KD(M) kon(1/Ms) kdis(1/s) GT90001m 0.2166 5.51E-09 3.32E+05 1.83E-03 Bi804-GT90001LC 0.0592 3.55E-09 6.33E+05 2.25E-03 Bi805-GT90001HC 0.164 5.43E-09 2.52E+05 1.37E-03 Bi807-26 0.1604 1.06E-08 2.84E+05 3.01E-03 Bi808 0.1336 9.90E-09 2.86E+05 2.83E-03 Bi809-GT90001LC 0.1461 4.07E-09 4.06E+05 1.65E-03

Table 8. Affinity of GT90001m-based bispecific antibodies to VEGF

sample Response KD(M) kon(1/Ms) kdis(1/s) Aflibercept 0.2573 1.79E-09 2.09E+05 3.75E-04 Bi804-GT90001LC 0.325 4.10E-09 7.42E+04 3.04E-04 Bi805-GT90001HC 0.2859 2.01E-09 1.30E+05 2.62E-04

Bi809-GT90001LC consists of 4 peptide chains, 2 identical first chains and 2 identical second chains. The first chain sequentially comprises VH of the ALK-1 binding region, CH of the ALK-1 binding region, Linker, VH of the VEGF binding region, Linker, and VL of the VEGF binding region from N-terminal to C-terminal. The second chain sequentially comprises VL and CL of the ALK-1 binding region from N-terminus to C-terminus. The four peptide chains can be linked by disulfide bonds.

Bi809-GT90001LC first strand sequence:

Bi808 0.3026 3.19E-09 1.15E+05 3.68E-04 Bevacizumab 0.3545 8.33E-10 1.20E+05 1.00E-04 Bi807-26 0.3002 6.44E-10 1.55E+05 1.00E-04 Bi809-GT90001LC 0.2145 1.01E-09 9.86E+04 1.00E-04

Table 9. Affinity of GT90001mutVL-8-based bispecific antibodies to ALK-1

sample Response KD(M) kon(1/Ms) kdis(1/s) Bi807-26 0.1769 9.63E-09 2.82E+05 2.71E-03 Bi811-26 0.173 7.89E-10 2.52E+05 1.99E-04 Bi804-mutVL-8 0.165 6.18E-10 3.25E+05 2.01E-04 GT90001mutVL-8 0.211 6.89E-10 2.67E+05 1.84E-04 GT90001m 0.2136 5.59E-09 3.03E+05 1.69E-03

Table 10. Affinity of GT90001mutVL-8-based bispecific antibodies to VEGF

sample Response KD(M) kon(1/Ms) kdis(1/s) Bevacizumab 0.2882 9.54E-10 1.05E+05 1.00E-04 Bi807-26 0.3068 8.19E-09 1.22E+04 1.00E-04 Bi811-26 0.2706 3.48E-09 2.87E+04 1.00E-04 Bi804-mutVL-8 0.2632 1.62E-10 6.16E+05 1.00E-04 Aflibercept 0.3193 3.03E-09 2.66E+05 8.06E-04

Example 8 Expanded expression and purification of bispecific antibodies

1. Bispecific antibody preparation

With embodiment 3.

2. Affinity purification

On the 6th day of transfection, the cells were centrifuged to collect the supernatant, and the sample was filtered through a 0.22 μm vacuum filtration system; the MabSelect SuReTM LX chromatography column was equilibrated with 5-10CV (column volume) of equilibration buffer until the conductivity and pH of the effluent were consistent. Change. Load the sample at a flow rate of 5ml/min. After the sample is loaded, continue to rinse the chromatography column with equilibration buffer until the penetration is complete and the UV value no longer drops. Elute with elution buffer and collect the effluent. After elution, the collected antibody solution should be neutralized to the stable pH of the antibody with the neutralization adjusting solution immediately.

3. Fine purification of protein samples

Select the buffer solution 20Mm PB6.0, 20mM PB6.0+500mM NaCl whose pH is 1-3 pH units lower than the theoretical isoelectric point of the antibody to be purified for this purification. Dilute the antibody conductance to <5mS/cm using buffer 20Mm PB6.0. Load the diluted sample into the cationic purification column (cation filler Capto TM S ImpAct) at a flow rate of 2-3ml/min; use 5CV of balance solution A to maintain a flow rate of 2-3ml/min to wash the purification column until it flows out The conductance and pH of the solution are consistent with the balance solution A, and the absorbance of A280 is stable; the salt concentration is used for linear elution, and the elution conditions are set: flow rate 1-2ml/min, running for 150min to reach 100% B, collecting eluted samples in separate tubes, and according to Elution peaks were selected and samples were sent to SEC.

Example 9 Detection of Antigen-Antibody Cell Level Combination by Flow Cytometry

The binding activity of the bispecific antibody to CHOK1-ALK1 cells was detected by flow cytometry.

Adjust the overexpression cells of the expanded culture to a suitable cell density and add them to a 96-well flow plate. For the detection of CHO-ALK1 cells, after centrifugation, add serially diluted samples to be tested, and incubate at 4°C for 1 hour. Wash twice with PBS, add correspondingly diluted fluorescent secondary antibody (Abcam, ab98596) to an appropriate concentration, incubate at 4°C for 30 min, wash twice with PBS. Add PBS to resuspend cells, detect on CytoFlex flow cytometer and calculate corresponding MFI. For the detection of HUVEC cells, the cells need to be fixed with 4% formaldehyde solution first, then centrifuged and then added with serially diluted samples to be tested, and incubated at 4°C for 1 hour or at room temperature for 2 hours. Wash twice with PBS, add fluorescent secondary antibody correspondingly diluted to an appropriate concentration, incubate at 4°C for 30 minutes or at room temperature for 1 hour, and wash twice with PBS. Add PBS to resuspend cells, detect on CytoFlex flow cytometer and calculate corresponding MFI.

The results are shown in Figure 9, the bispecific antibody of the present invention has binding activity to CHOK1-ALK-1 cells, wherein the ALK-1 affinity matured molecule GT90001mutVL-8 (also represented as GT90001-VL8 in the figure) is compatible with the parent The binding activity of molecule GT90001m is equivalent; the binding ability of bispecific antibody molecules Bi804-mutVL-8 (also represented as Bi804 in the accompanying drawings) and Bi811-26 (also represented as Bi811 in the accompanying drawings) based on GT90001mutVL-8 is comparable to that of Bispecific antibody engineered molecule Bi807-26 (also denoted as Bi807 in the figure) based on GT90001m has comparable binding activity.

Since CHOK1-ALK-1 cells are overexpressed cells, in order to further verify the binding ability of bispecific antibody molecules and cells at normal physiological levels, we selected HUVEC, which expresses ALK-1 endogenously, for cell binding activity detection. The results are shown in Figure 10, the cell binding activity of GT90001mutVL-8 (also indicated as GT90001-VL8 in the accompanying drawings) is superior to that of GT90001m, and at the same time the bispecific antibody molecule Bi804-mutVL-8 constructed with GT90001mutVL-8 (in the accompanying drawings Also indicated as Bi804), Bi811-26 (also indicated as Bi811 in the accompanying drawings) have better cell binding activity at the ALK-1 end than bispecific antibody molecule Bi807-26 based on GT90001m structure (also indicated as Bi807 in the accompanying drawings) .

Compared with other bispecific antibodies, Bi804-mutVL-8 and Bi811-26 showed excellent binding ability to ALK-1 on HUVEC, with the smallest EC ₅₀ .

Example 10 Detection of Binding Blocking of VEGF/VEGFR Protein Levels by ELISA

Based on the structural design of the bispecific antibody of the present invention, the binding activity of the bispecific antibody of the present invention to block human VEGF165 protein and human VEGFRII was detected by ELISA method.

Human VEGFRII protein was diluted to an appropriate concentration with ELISA coating solution, added to the ELISA plate, and coated overnight at 4°C. Block with 5% BSA for 1 hour at room temperature. The samples to be tested were serially diluted and co-incubated with biotinylated human VEGF165 protein for 1 hour at room temperature. Add the incubated samples to the sealed ELISA plate and react at room temperature for 2 hours. Wash 3 times with PBS washing solution, add diluted Streptavidin (HRP) to react at room temperature for 1 hour, wash 3 times with PBS washing solution, add ELISA chromogenic solution, let stand at room temperature for 3 minutes, add ELISA stop solution, and read the absorbance value at 450nm.

The results are shown in Figure 11, all bispecific antibodies can well block the binding of human VEGF165 protein to human VEGFRII at the ELISA level, among which Bi807-26 (also represented as Bi807 in the accompanying drawing) and Bi811-26 (in the accompanying drawing The blocking level of the molecule also represented as Bi811 in the figure is equivalent to that of the positive control molecule Bevacizumab, and the molecule Bi804-mutVL-8 (also represented as Bi804 in the figure) is slightly weaker than that of the positive control molecule Aflibercept.

The above-mentioned bispecific antibody molecules can all block the binding of VEGF165 and VEGFRII, and the blocking activity is similar.

Example 11 VEGFRII/NFAT luciferase reporter gene detection

The previous experiments showed that the bispecific antibody molecule of the present invention can effectively block the binding of human VEGF protein and human VEGFRII protein at the ELISA level. We further used VEGFRII-NFAT fluorescent reporter cells to detect the bispecific antibody molecule blocking VEGF/ Cellular activity of VEGFRII-NFAT signaling.

Dilute VEGF165 to 400ng/ml with 0.5% FBS/DMEM reaction medium, mix evenly with the test product 1:1 with the same reaction medium 4 times the final concentration gradient dilution, and incubate in a carbon dioxide incubator for 60 minutes. Take the 293-VEGFRII/NFAP effector cells, resuspend the cells with the reaction medium to the appropriate cell density, inoculate 50 μl/well of 96-well cell culture white bottom plate, then add 50 μl/well of the pre-incubated test product/VEGF165 mixture, place in 37°C, 5% CO2 incubator for 6 hours, during which the Bio-Glo™ reagent was returned to room temperature. After the culture was completed, the cells were taken out, equilibrated at room temperature for 5 minutes, 100 μl/well of Bio-Glo ^TM reagent was added, and the fluorescent signal value was read with a multi-functional microplate reader.

The results are shown in Figure 12, the bispecific antibody molecules of the present invention can effectively block the VEGF signaling pathway in vitro, wherein Bi807-26 (also represented as Bi807 in the accompanying drawings) and Bi811-26 (also represented as Bi807 in the accompanying drawings) Bi811) was slightly weaker than its corresponding positive control molecules Bevacizumab and Aflibercept; Bi804-mutVL-8, Bi807-26 and Bi811-26 had similar blocking activities.

All of the above bispecific antibody molecules can block the VEGF/VEGFRII-NFAT signaling pathway, and the blocking activity is similar to that of Bevacizumab and Aflibercept.

Example 12 Detection of BMP9-induced ALK-1 downstream Smad1 phosphorylation blocking

As the ligand of ALK-1, BMP9 molecule can activate the ALK-1 receptor by binding to the ALK-1 protein on the surface of HUVEC cells, thereby mediating the phosphorylation of the downstream protein Smad1. Therefore, we designed an experiment to detect the blocking effect of the bispecific antibody of the present invention on BMP9-induced Smad1 phosphorylation.

HUVEC cells were seeded on a 96-well plate, and 2×10 ⁴ cells per well were placed in ECM (Sciencell, 1001) medium containing 5% FBS and ECG. Incubate overnight in a 37°C, 5% CO ₂ incubator. Remove medium from cell culture plates and wash twice with 200 μl PBS. Add 100 μl of ECM without FBS and ECG, then starve the cells for 4 hours. Remove the medium from the cell culture plate, add 100uL of the test substance diluted in a certain concentration gradient, and treat for 1.5 hours, and then add BMP9 at a final concentration of 1ng/mL to the medium to treat the cells for 45 minutes. The medium was removed, and the phosphorylation level of Smad1 was determined using an ELISA kit (Invitrogen, 85-86182-11).

The results are shown in Figure 13, the ALK-1 monoclonal antibody GT90001mutVL-8 after affinity maturation (also indicated as GT90001-VL8 in the figure) has the strongest blocking effect, and the bispecific antibody constructed based on GT90001mutVL-8 among the bispecific antibodies Antibody molecules Bi804-mutVL-8 (also indicated as Bi804 in the accompanying drawings), Bi811-26 (also indicated as Bi811 in the accompanying drawings) are significantly better than Bi807-26 (also indicated as Bi807 in the accompanying drawings) constructed based on the parent GT90001m.

All of the above bispecific antibodies can block the phosphorylation of Smad1 protein in the BMP9-induced TGFb pathway on HUVEC cells. And the blocking activity was greatly improved compared with GT90001m.

Example 13 Detection of druggability of ALK-1/VEGF bispecific antibody

The druggability results of ALK-1/VEGF bispecific antibody are shown in Table 11.

Table 11. Druggability test results of bispecific antibodies

The thermal stability of Bi804-GT90001LC, Bi800, Bi804-GT90001mutVL-8 is not as good as Bi810-26, Bi811-26, Bi807-26; but the colloidal stability and non-specific adsorption of Bi810-26 and Bi807-26 are not as good as Bi811-26 .

The applicant conducted an accelerated stability assessment of the Bi811-26 antibody, respectively, under (1) high temperature conditions (solution buffer: 4% sucrose, 0.02% Tween 20, 20mM His-HCl, pH 6.0, concentration: 10mg/mL; temperature: 40°C; placed for 0 weeks, 2 weeks, 4 weeks), (2) low pH conditions (pH=3.5; placed for 0h, 2h, 4h) and (3) repeated freeze-thaw conditions (temperature: -20°C; repeated freeze-thaw 0, 3, 5 cycles) to test. The test results showed that the antibody was stable under the above test conditions, and the sample concentration and purity did not decrease significantly.

All publications and patents mentioned in this application are incorporated herein by reference. Various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in terms of specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims (30)

A bispecific antibody comprising a first antigen-binding region (ALK-1 binding region) that specifically binds ALK-1 and a second antigen-binding region (VEGF-binding region) that specifically binds VEGF, the specific binding The first antigen-binding region of ALK-1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the second antigen-binding region that specifically binds VEGF comprises a heavy chain variable region (VH) and a light chain variable region (VL). The light chain variable region (VL) or VEGF receptor fragment specifically binds VEGF. The bispecific antibody according to claim 1, wherein the heavy chain variable region of the first antigen-binding region comprises a) CDR1H as set forth in SEQ ID NO: 2, CDR2H as set forth in SEQ ID NO: 3 and CDR3H as set forth in SEQ ID NO: 4; or b) CDR1H with the same function as SEQ ID NO: 2 obtained by adding, deleting, or replacing one or more amino acids from the sequence shown in SEQ ID NO: 2, and adding, deleting, or replacing the sequence shown in SEQ ID NO: 3 The CDR2H with the same function as SEQ ID NO: 3 obtained by replacing one or more amino acids and the function of SEQ ID NO: 4 obtained by adding, deleting, or replacing one or more amino acids in the sequence shown in SEQ ID NO: 4 Same CDR3H. The bispecific antibody according to claim 1 or 2, wherein the light chain variable region of the first antigen-binding region comprises a) CDR1L as set forth in SEQ ID NO: 6, CDR2L as set forth in SEQ ID NO: 7 and CDR3L as set forth in SEQ ID NO: 8; or b) CDR1L obtained from the sequence shown in SEQ ID NO: 6 by adding, deleting, or replacing one or more amino acids with the same function as SEQ ID NO: 6, by adding, deleting, or replacing the sequence shown in SEQ ID NO: 7 The CDR2L with the same function as SEQ ID NO: 7 obtained by replacing one or more amino acids and the function of SEQ ID NO: 8 obtained by adding, deleting, or replacing one or more amino acids from the sequence shown in SEQ ID NO: 8 Same CDR3L. The bispecific antibody according to any one of claims 1 to 3, wherein the heavy chain variable region of the first antigen-binding region comprises CDR1H as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3 CDR2H and CDR3H as shown in SEQ ID NO:4. The bispecific antibody according to any one of claims 1 to 4, wherein the light chain variable region of the first antigen-binding region comprises a combination of CDRs selected from the following: a) CDR1L as set forth in SEQ ID NO: 6, CDR2L as set forth in SEQ ID NO: 7 and CDR3L as set forth in SEQ ID NO: 8; and b) CDR1L as shown in SEQ ID NO:6, CDR2L as shown in SEQ ID NO:7 and CDR3L as shown in SEQ ID NO:16. The bispecific antibody according to any one of claims 1 to 5, wherein the heavy chain variable region and the light chain variable region of the first antigen-binding region comprise a combination of CDRs selected from the following: a) said heavy chain variable region comprises CDR1H as shown in SEQ ID NO: 2, CDR2H as shown in SEQ ID NO: 3 and CDR3H as shown in SEQ ID NO: 4, and The light chain variable region comprises CDR1L as shown in SEQ ID NO: 6, CDR2L as shown in SEQ ID NO: 7 and CDR3L as shown in SEQ ID NO: 8; and b) said heavy chain variable region comprises CDR1H as shown in SEQ ID NO: 2, CDR2H as shown in SEQ ID NO: 3 and CDR3H as shown in SEQ ID NO: 4, and The light chain variable region comprises CDR1L as shown in SEQ ID NO:6, CDR2L as shown in SEQ ID NO:7 and CDR3L as shown in SEQ ID NO:16. The bispecific antibody according to any one of claims 1 to 6, wherein the heavy chain variable region of the first antigen-binding region comprises the sequence shown in SEQ ID NO:1. The bispecific antibody according to any one of claims 1 to 7, wherein the light chain variable region of the first antigen-binding region comprises a sequence selected from SEQ ID NO: 5 or SEQ ID NO: 15. The bispecific antibody according to any one of claims 1 to 8, wherein the heavy chain variable region and the light chain variable region of the first antigen-binding region comprise a combination selected from the following: a) a heavy chain variable region sequence as shown in SEQ ID NO: 1 and a light chain variable region sequence as shown in SEQ ID NO: 5; and b) the heavy chain variable region sequence as shown in SEQ ID NO: 1 and the light chain variable region sequence as shown in SEQ ID NO: 15. The bispecific antibody according to any one of claims 1 to 9, wherein the bispecific antibody comprises an IgG heavy chain constant region, preferably, an IgG1 or IgG2 heavy chain constant region, more preferably, an IgG1 The heavy chain constant region, most preferably, comprises the heavy chain constant region shown in SEQ ID NO:9. The bispecific antibody according to any one of claims 1 to 10, wherein the second antigen-binding region that specifically binds to VEGF comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises CDR1H as shown in SEQ ID NO: 19, CDR2H as shown in SEQ ID NO: 20 and CDR3H as shown in SEQ ID NO: 21; and The light chain variable region comprises CDR1L as shown in SEQ ID NO: 23, CDR2L as shown in SEQ ID NO: 24 and CDR3L as shown in SEQ ID NO: 25. The bispecific antibody according to claim 11, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 18, and the light chain variable region comprises the sequence shown in SEQ ID NO: 22 . The bispecific antibody according to any one of claims 1 to 10, wherein the second antigen-binding region that specifically binds VEGF comprises a VEGF receptor fragment that specifically binds VEGF, and the VEGF receptor fragment comprises a VEGF receptor -1 extracellular domain 2 and VEGF receptor-2 extracellular domain 3, Preferably, the VEGF receptor fragment comprises the sequence shown in SEQ ID NO: 17 or the sequence shown in SEQ ID NO: 17 through addition, deletion, replacement of one or more amino acids obtained with the function of SEQ ID NO: 17 the same sequence, More preferably, the VEGF receptor fragment comprises the sequence shown in SEQ ID NO: 17. The bispecific antibody according to any one of claims 1 to 13, wherein the first antigen-binding region or the second antigen-binding region is in the form of scFv. The bispecific antibody according to any one of claims 1 to 14, wherein the first antigen-binding region and the second antigen-binding region are connected by a Linker, Preferably, the Linker comprises (G ₄ S)n, where n is an integer greater than 1, More preferably, the Linker is composed of (G ₄ S)n, n is an integer of 2-10, More preferably, the Linker is composed of (G ₄ S)n, where n is 2, 3 or 4. The bispecific antibody according to any one of claims 14 or 15, wherein said scFv comprises a heavy chain variable region and a light chain variable region, and said heavy chain variable region and light chain variable region are connected by a Linker connect, Preferably, the Linker comprises (G ₄ S)n, where n is an integer greater than 1, More preferably, the Linker is composed of (G ₄ S)n, n is an integer of 2-10, More preferably, the Linker is composed of (G ₄ S)n, where n is 2, 3 or 4. The bispecific antibody according to any one of claims 1 to 16, which consists of 4 peptide chains, 2 identical first chains and 2 identical second chains, The first chain sequentially comprises VH of the VEGF binding region, CH of the VEGF binding region, Linker, VH of the ALK-1 binding region, Linker, and VL of the ALK-1 binding region from the N-terminal to the C-terminal, and The second chain comprises the VL and CL of the VEGF binding domain sequentially from N-terminus to C-terminus. The bispecific antibody according to claim 17, wherein The VH sequence of the VEGF binding region is shown in SEQ ID NO: 18, The CH sequence of the VEGF binding region is shown in SEQ ID NO: 9, The VL sequence of the VEGF binding region is shown in SEQ ID NO: 22, The CL sequence of the VEGF binding region is shown in SEQ ID NO: 10, The Linker sequence is shown in SEQ ID NO: 11, The VH sequence of the ALK-1 binding region is shown in SEQ ID NO: 13, and The VL sequence of the ALK-1 binding region is shown in SEQ ID NO: 14 or SEQ ID NO: 26; Preferably, the first chain sequence of the bispecific antibody is shown in SEQ ID NO: 29, the second chain sequence is shown in SEQ ID NO: 30; or the bispecific antibody The first chain sequence is shown in SEQ ID NO:31, and the second chain sequence is shown in SEQ ID NO:32. The bispecific antibody according to any one of claims 1 to 16, which consists of 4 peptide chains, 2 identical first chains and 2 identical second chains, The first chain sequentially comprises the VH of the ALK-1 binding region, the CH of the ALK-1 binding region, the Linker and the VEGF binding region from the N-terminal to the C-terminal, and The second chain sequentially comprises VL and CL of the ALK-1 binding region from N-terminus to C-terminus. The bispecific antibody according to any one of claims 1 to 16, which consists of 4 peptide chains, 2 identical first chains and 2 identical second chains, The first chain comprises the VH and CH of the ALK-1 binding region sequentially from the N-terminus to the C-terminus, and The second chain includes VEGF binding region, Linker, VL and CL of ALK-1 binding region sequentially from N-terminal to C-terminal. The bispecific antibody according to any one of claims 1 to 16, which consists of two identical peptide chains, and the peptide chains sequentially comprise a VEGF binding region, IgG1-Fc, Linker, ALK- 1 VH of the binding region, Linker and VL of the ALK-1 binding region. The bispecific antibody according to any one of claims 1 to 16, which consists of 4 peptide chains, 2 identical first chains and 2 identical second chains, The first chain sequentially comprises the VH of the ALK-1 binding region, the CH of the ALK-1 binding region, the Linker, the VH of the VEGF binding region, the Linker, the VL of the VEGF binding region from the N-terminal to the C-terminal, and The second chain sequentially comprises VL and CL of the ALK-1 binding region from N-terminus to C-terminus. A polynucleotide encoding the bispecific antibody or fragment thereof according to any one of claims 1 to 22. An expression vector capable of expressing the bispecific antibody or fragment thereof according to any one of claims 1-22. An engineered cell comprising the carrier according to claim 24. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 22, the polynucleotide according to claim 23, the carrier according to claim 24 or the vector according to claim 25 cells, and a pharmaceutically acceptable carrier. The bispecific antibody according to any one of claims 1 to 22, the polynucleotide according to claim 23, the vector according to claim 24, the cell according to claim 25 or the cell according to claim 26 Use of the pharmaceutical composition in the preparation of medicines for inhibiting angiogenesis. The bispecific antibody according to any one of claims 1 to 22, the polynucleotide according to claim 23, the vector according to claim 24, the cell according to claim 25 or the cell according to claim 26 The use of the pharmaceutical composition in the preparation of medicines for treating tumors, Preferably, the tumor is selected from advanced or refractory hepatocellular carcinoma, colorectal cancer, non-small cell lung cancer, triple-negative breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, urothelial carcinoma, and esophageal squamous cell carcinoma. Cell carcinoma, brain tumor, lung cancer, breast cancer, ovarian cancer, fallopian tube cancer, glioblastoma, colorectal adenocarcinoma, pituitary tumor, pituitary adenoma, pituitary macroadenoma, gestational trophoblastic tumor, choriocarcinoma, placental site Trophoblastic tumor, epithelioid trophoblastic tumor, renal cell carcinoma, lung adenocarcinoma, and gliosarcoma. A method for inhibiting angiogenesis, comprising administering to a subject in need thereof an effective dose of the bispecific antibody according to any one of claims 1 to 22, the polynucleotide according to claim 23, according to The carrier according to claim 24, the cell according to claim 25 or the pharmaceutical composition according to claim 26. A method for treating tumors, comprising administering an effective dose of the bispecific antibody according to any one of claims 1 to 22, the polynucleotide according to claim 23, and the polynucleotide according to claim 23 to a subject in need thereof. The carrier according to claim 24, the cell according to claim 25 or the pharmaceutical composition according to claim 26, Preferably, the tumor is selected from advanced or refractory hepatocellular carcinoma, colorectal cancer, non-small cell lung cancer, triple-negative breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, urothelial carcinoma, and esophageal squamous cell carcinoma. Cell carcinoma, brain tumor, lung cancer, breast cancer, ovarian cancer, fallopian tube cancer, glioblastoma, colorectal adenocarcinoma, pituitary tumor, pituitary adenoma, pituitary macroadenoma, gestational trophoblastic tumor, choriocarcinoma, placental site Trophoblastic tumor, epithelioid trophoblastic tumor, renal cell carcinoma, lung adenocarcinoma, and gliosarcoma.

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