Anti-VEGFA and VEGFC Bispecific Antibodies and Uses Thereof

Abstract

The present invention relates to single domain antibody polypeptides and constructs thereof that specifically bind to VEGFC, in particular to anti-VEGFA / VEGFA bispecific binding proteins. The present invention also relates to polynucleotides encoding said polypeptides and proteins, expression vectors and host cells, and pharmaceutical compositions thereof, as well as methods and uses for treating angiogenesis-related diseases.

Description

[Technical field]

[0001] The present invention relates to single domain antibody polypeptides and constructs thereof that specifically bind to VEGFC, in particular to anti-VEGFA / VEGFA bispecific binding proteins. The present invention also relates to polynucleotides encoding said polypeptides and proteins, expression vectors and host cells, and pharmaceutical compositions thereof, as well as methods and uses for treating angiogenesis-related diseases. [Background technology]

[0002] Vascular endothelial growth factors (VEGFs) are key regulators of vascular development and blood and lymphatic vascular function in adults in health and disease. Currently, the VEGF family is known to consist of five structurally related factors: VEGFA (also called VEGFA165), VEGFB, VEGFC, VEGFD, and placental growth factor (PlGF). VEGF family members exist primarily in the form of homodimeric polypeptides and induce signal transduction to elicit the corresponding biological effects by binding to the associated VEGF receptor.

[0003] Vascular endothelial growth factor A (VEGF-A), as an angiogenic cytokine, is involved in normal and abnormal angiogenesis processes through its interaction with two high-affinity transmembrane tyrosine kinase receptors (VEGFR-1 and VEGFR-2). Currently, various methods have been proposed to inhibit the VEGF-A pathway so that angiogenesis-related diseases can be improved. Proposed VEGF-A inhibitors / antagonists include neutralizing antibodies that target VEGF-A, and soluble decoy receptors and trap molecules that prevent the binding of VEGF-A to its normal receptors. For example, bevacizumab (trade name Avastin), a humanized monoclonal anti-VEGF-A antibody, has been approved to treat colorectal cancer, breast cancer, and lung cancer. Ranibizumab (trade name Lucentis), a monoclonal antibody fragment derived from the same parent murine antibody as bevacizumab, is much smaller than the parent molecule and provides stronger VEGF-A binding properties through affinity maturation (WO98 / 45331). Ranibizumab has been approved to treat wet age-related macular degeneration. Aflibercept (also called VEGFA-trap, trade name Elyea) is a recombinant fusion protein formed by fusing the ligand-binding domains from human VEGF receptors 1 and 2 with the human IgG1 Fc region, and is approved to treat angiogenesis-associated retinal diseases such as age-related macular degeneration, and is being used in clinical trials for the treatment of solid tumors.

[0004] Vascular endothelial growth factor C (VEGF-C) has been identified as a lymphangiogenesis-related cytokine, which acts through the tyrosine kinase receptors VEGFR2 and VEGFR3. VEGF-C is involved in the angiogenesis process by binding to VEGFR2 on vascular endothelial cells. In addition, VEGF-C stimulates lymphangiogenesis and lymphatic endothelial cell proliferation and migration by binding to its receptor VEGFR3. VEGFR3 is structurally similar to VEGFR1 and 2, but does not bind to VEGF-A. VEGFR3 was found to be highly expressed in vascular endothelial cells in addition to lymphatic cells. Inhibition of the VEGF-C / VEGFR3 signaling pathway was proposed to suppress tumor lymphangiogenesis and metastasis in various metastatic tumor models. Furthermore, VEGF-C trap molecules such as OPT-032 (a VEGFC / D inhibitor constructed from the ligand extracellular domain derived from VEGFR3) developed by Opthea are currently being developed for the treatment of neovascularization-associated retinopathy.

[0005] Many disease processes have been identified that are associated with the occurrence of angiogenesis, including, for example, cancer, autoimmune diseases, and retinopathies. Given the role of VEGF-A and VEGF-C in the growth of blood and lymphatic vessels, it has been proposed that VEGF-A and VEGF-C may have a synergistic promoting effect on each other in angiogenesis-related diseases, such as tumor growth and metastasis. Thus, there is a need in the art to develop new anti-angiogenic molecules, especially bispecific molecules that can simultaneously target both VEGF-A and VEGF-C. Such bispecific molecules would simultaneously inhibit both vascular endothelial growth factors A and C, contributing to suppress the progression of angiogenesis-related diseases. Compared to the combined use of independent anti-VEGF-A and anti-VEGF-C molecules, such bispecific molecules would also provide advantages in administration. Currently, in the treatment of eye diseases, many independent anti-VEGF-A molecules such as aflibercept, ranibizumab and conbercept have limitations, namely, they need to be administered intravitreally as injection solutions, which are difficult to administer, which leads to poor compliance and high treatment burden for patients.Therefore, instead of this combination drug, it is advantageous to design a bispecific molecule that can simultaneously bind and neutralize VEGF-A and VEGF-C with strong and specificity. Summary of the Invention

[0006] In order to meet the above demand, the present inventor has, after extensive research, provided a new high-affinity anti-VEGF-C single domain scAb antibody polypeptide through humanization and affinity maturation based on the screening of a phage display library, and used the above single domain antibody polypeptide as a component to construct and produce a bispecific molecule with excellent VEGF-A and VEGF-C dual antagonistic activity and anti-angiogenic effect by binding with an anti-VEGF-A molecule.

[0007] Thus, in one aspect, the invention provides a single domain antibody (sdAb) that specifically binds to human VEGF-C. In one embodiment, the anti-VEGF-C single domain antibody of the invention comprises a VHH domain having the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where CDR1-3 are, respectively: (1) the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3; (2) the amino acid sequences shown in SEQ ID NOs: 9, 10 and 11; (3) The amino acid sequences shown in SEQ ID NOs: 17, 18 and 19, and (4) Amino acid sequences represented by SEQ ID NOs: 21, 22 and 23; The amino acid sequence may be selected from the group consisting of:

[0008] In a further aspect the present invention provides a protein comprising at least a single domain antibody of the invention, preferably said protein is a fusion protein or a chimeric polypeptide, such as a VHH-Fc antibody.

[0009] In a further aspect, the present invention provides a method for producing a method for the treatment of a cancer (i) a first antigen-binding moiety that specifically binds human VEGFC; (ii) a second antigen-binding moiety that specifically binds human VEGFA; and The present invention provides a bispecific binding protein comprising: wherein the first antigen binding moiety comprises a single domain antibody polypeptide of the invention; and The bispecific binding protein has dual antagonistic activity, inhibiting the binding of VEGFA to its VEGF receptor and inhibiting the binding of VEGFC to its VEGF receptor.

[0010] In further aspects, the present invention provides polynucleotides encoding the molecules of the invention (single domain antibody polypeptides, proteins or bispecific binding proteins), vectors, host cells, and pharmaceutical compositions, drug combinations and reagent kits comprising the molecules of the invention.

[0011] In a further aspect, the present invention provides the use of the molecules (single domain antibody polypeptides, proteins or bispecific binding proteins) of the present invention for the prevention and / or treatment of diseases, said diseases being, for example, angiogenesis-related diseases such as tumors and eye diseases.

[0012] In a further aspect, the present invention also provides diagnostic uses of the molecules of the invention (single domain antibody polypeptides, proteins or bispecific binding proteins). [Brief description of the drawings]

[0013] [Figure 1-1] ELISA experiments showing inhibition of binding of VEGFC to receptors VEGFR2 / VEGFR3 by anti-VEGFC VHH antibodies. A: VEGFR2 inhibition experiment by anti-VEGFC VHH, B: VEGFR3 inhibition experiment by anti-VEGFC VHH. [Figure 1-2] ELISA experiment showing the inhibition of binding of VEGFC to the receptors VEGFR2 / VEGFR3 by anti-VEGFC VHH antibody. C: VEGFR3 inhibition experiment of anti-VEGFC humanized VHH, D: VEGFR3 inhibition experiment of anti-VEGFC affinity matured VHH. [Diagram 2] 1 shows an experiment in which anti-VEGFCVHH antibody inhibits VEGFC-induced activation of HEK293 KDR reporter molecule. [Diagram 3] 1 shows an experiment in which anti-VEGFCVHH antibody inhibits VEGFC-induced proliferation of Baf3-FLT4. [Figure 4] 1 shows an experiment in which humanized anti-VEGFC VHH antibody inhibits VEGFC-induced activation of HEK293 KDR reporter molecule. [Diagram 5] 1 shows an experiment in which humanized anti-VEGFC VHH antibodies inhibit VEGFC-induced Baf3-FLT4 proliferation. [Figure 6]1 shows an experiment in which the affinity matured molecule anti-VEGFC VHH antibody inhibits VEGFC-induced activation of the HEK293 KDR reporter molecule. [Figure 7] 1 shows an experiment in which affinity matured anti-VEGFC VHH antibodies inhibit VEGF-C induced Baf3-FLT4 proliferation. [Figure 8] 1 shows an experiment in which a bispecific antibody molecule inhibits VEGFA-induced activation of a HEK293 KD reporter molecule. [Figure 9] 1 shows an experiment in which a bispecific antibody molecule inhibits VEGFC-induced activation of a HEK293 KDR reporter. [Figure 10] 1 shows an experiment in which a bispecific antibody molecule inhibits VEGFC-induced proliferation of BaF3-FLT4. [Figure 11] 1 shows an experiment in which a bispecific antibody molecule inhibits VEGFA+C-induced proliferation of HUVEC. [Figure 12] 1 shows an experiment in which a bispecific antibody molecule inhibits VEGFA+C-induced HUVEC tube formation. A: Images of tube formation, B: Statistics of tube formation. [Figure 13] 1 shows an experiment on the inhibition of angiogenesis in A375 subcutaneous tumor model by bispecific antibody molecules. A: A375 tumor mass statistics, B: A375 tumor volume statistics, C: A375 tumor CD31 staining image. [Figure 14] 1 shows an experiment in which a bispecific antibody molecule suppresses laser-induced CNV. [Figure 15] 1 shows fundus angiography images of the inhibition of laser-induced choroidal neovascularization by anti-VEGFA / VEGFC bispecific antibody. [Figure 16] OCT images showing inhibition of laser-induced retinal thickening by anti-VEGFA / VEGFC bispecific antibody. [Figure 17] This shows that anti-VEGFA / VEGFC bispecific antibody suppresses pathological changes in a laser-induced choroidal neovascularization model. [Figure 18]FIG. 1 shows schematic diagrams of the structures of two types of bispecific antibodies constructed by combining a VEGF-A binding domain-Fc fusion protein and an anti-VEGF-A Fab antibody with an anti-VEGF-C VHH antibody as a component. [Figure 19] 1 shows the CDR1 to 3 sequences and the VHH sequence of the anti-VEGF-C VHH single domain antibody of the present invention. Detailed Description of the Invention

[0014] Unless otherwise specified, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art of the present invention. All publications, patent applications, patents or other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and not limiting. Other features, objects and advantages of the present invention will become apparent from the specification and drawings, and from the appended claims.

[0015] definition The term "about," when used in conjunction with a number or numerical value, is meant to cover a range of numbers or numerical values ​​that is 5% less than the number or numerical value specified as the lower limit and 5% more than the number or numerical value specified as the upper limit.

[0016] As used herein, the term "comprising" or "including" means the inclusion of the stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.

[0017] The term "antibody" is used herein in the broadest sense to mean a protein that contains an antigen-binding site and includes natural and artificial antibodies of various structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, single domain antibodies, full length antibodies and antibody fragments.

[0018] The terms "complete antibody" or "full-length antibody" are used interchangeably herein and refer to an antibody molecule having a natural immunoglobulin molecular structure. In the case of a typical four-chain IgG antibody, a full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of a heavy-chain antibody that has only heavy chains and lacks light chains, a full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds.

[0019] In a typical four-chain IgG antibody, the full-length antibody heavy chain generally consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, where the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain generally consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region, where the light chain constant region comprises one domain CL. Each heavy chain variable region VH and each light chain variable region comprises three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0020] In heavy chain antibodies, the full-length antibody heavy chain generally consists of a heavy chain variable region (abbreviated as VHH herein) and a heavy chain constant region. The heavy chain constant region consists of domains CH2 and CH3. Each VHH consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0021] The term "antigen-binding fragment" of an antibody refers to a portion or fragment of a complete or full-length antibody that contains fewer amino acid residues than the complete or full-length antibody, but is capable of binding to an antigen or competing with the complete antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) to bind to an antigen. An antigen-binding fragment can be prepared by cleaving a complete antibody by recombinant DNA technology or by enzymatic or chemical means. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single domain antibody (sdAb), and nanobody. For example, a Fab fragment can be obtained by digesting a complete antibody with papain. Also, F(ab')2 produced by digesting a complete antibody below the disulfide bond in the hinge region with pepsin is a dimer of Fab' and is a bivalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bond in the hinge region, which converts the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region. The Fv fragment consists of the single arm VL and VH domains of an antibody. The two domains of the Fv fragment, VL and VH, can be encoded by independent genes, or they can be recombinantly linked using a synthetic linking peptide to produce a single protein chain, in which the VL and VH regions pair to form a single chain Fv (scFv).

[0022] The term "variable region" or "variable domain" of an antibody refers to the domain of the antibody heavy or light chain that is involved in the binding of the antibody to an antigen. For example, if a heavy chain antibody is derived from a camelid heavy chain antibody, a single VH domain can be sufficient to confer antigen binding specificity. The VHH of a natural heavy chain antibody has a similar structure to the heavy chain variable region VH of a natural IgG antibody, i.e., it contains four conserved framework regions (FR) and three complementarity determining regions (CDRs), and has the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0023] The "complementarity determining region", "CDR region", "CDR" or "hypervariable region" of an antibody is the region in the antibody variable domain (VH or VHH) that is hypervariable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are numbered sequentially from the N-terminus and are commonly referred to as CDR1, CDR2 and CDR3. The CDRs located in the heavy chain variable domain of an antibody are also referred to as HCDR1, HCDR2 and HCDR3, and the CDRs located in the light chain variable domain of an antibody are referred to as LCDR1, LCDR2 and LCDR3. For a given light chain variable region or heavy chain variable region amino acid sequence, the CDR sequence can be determined by various methods known in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat based on the variability of the antibody sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (International Immunogenetics Information System, World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 2009, 144:131-135). Biology, 406, 228-256 (2011)).

[0024] Below are the CDR region ranges defined in the Kabat, AbM, Chothia, Contact and IMGT methods. [Table 1]

[0025] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined by any one of the above methods.

[0026] CDRs may be determined based on having the same Kabat numbering position as the reference CDR sequence. Unless otherwise specified, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0027] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains, namely, a CH2 domain, a CH3 domain and an optional CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) derived from the two heavy chains of IgG, IgA and IgD class antibodies, or the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) derived from the two heavy chains of IgM and IgE class antibodies. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or heavy chain constant region is based on the EU numbering system (also called the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the terms "Fc region," "Fc portion," and "Fc fragment" do not include the heavy chain variable region VH and light chain variable region VL, and the heavy chain constant region CH1 and light chain constant region CL of an immunoglobulin, but optionally include the hinge region at the N-terminus of the heavy chain constant region.

[0028] The term "chimeric antibody" refers to an antibody molecule in which (a) the constant region or a portion thereof has been altered, replaced or exchanged so that the antigen binding site is linked to a constant region having a different or altered type, effector function and / or species origin, or to an entirely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug), etc., that confers new properties to the chimeric antibody, or (b) the variable region or a portion thereof has been altered, replaced or exchanged by a variable region having a different or altered antigen specificity. For example, a camelid heavy chain antibody can be modified by changing its constant region to a constant region derived from a human immunoglobulin. Due to the alteration to a human constant region, the chimeric antibody can retain its specificity in antigen recognition and have reduced antigenicity in humans compared to the original camelid antibody.

[0029] As used herein, a "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., an alpaca monoclonal antibody) and is less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their corresponding human-derived portions (i.e., replacing the portions of the constant and variable regions that are not involved in binding with the corresponding portions of a human antibody).

[0030] As used herein, the terms "fusion protein" and "chimeric polypeptide" can be used interchangeably and refer to a larger polypeptide formed by fusing at least two heterologous polypeptide sequences, optionally via a linker. Fusion proteins can be produced by recombinant expression.

[0031] The term "heterologous" when referring to a fusion protein or chimeric polypeptide means that the fusion protein or chimeric polypeptide comprises two or more subsequences that are not found in the same protein or polypeptide in nature.

[0032] The term "recombinant" as used herein refers to a polypeptide or protein produced by a living host. The host may be selected from, but is not limited to, a mammalian expression system, an insect cell expression system, a yeast expression system, and a bacterial expression system. In one embodiment, the polypeptide / protein of the present invention is a recombinant polypeptide / protein produced by expression in a prokaryotic (e.g., E. coli cell) or eukaryotic host cell (e.g., a mammalian host cell).

[0033] As used herein, the term "monospecific" refers to a polypeptide / protein molecule having one or more antigen-binding sites, each of which binds to the same epitope of the same antigen. As used herein, the term "multispecific" refers to a polypeptide / protein molecule having at least two antigen-binding sites that bind to different epitopes (different epitopes on the same antigen or different epitopes on different antigens). In some embodiments, the invention provides a monospecific binding molecule comprising a single domain antibody of the invention, in some embodiments, the molecule is monovalent, e.g., a single VHH-Fc polypeptide having only a single VHH domain, and in some other embodiments, the molecule is multivalent, e.g., a heavy chain antibody formed from two VHH-Fc polypeptides by dimerization of the Fc regions. In some embodiments, the invention provides a bispecific binding molecule, the two antigen-binding specificities of which target the antigens VEGF-A and VEGF-C, in particular human VEGFA and human VEGFC, respectively, and preferably at least one anti-VEGF-C specificity therein is provided by a single domain antibody of the invention.

[0034] As used herein, the terms "antigen-binding site" and "antigen-binding domain" can be used interchangeably and refer to the region of a molecule that actually binds to a target antigen. Examples of antigen-binding sites include, but are not limited to, for example, the variable domain of an antibody, the extracellular ligand-binding domain of a receptor, and the like. Preferably, the VEGFC antigen-binding site for the bispecific binding protein of the invention is provided by the variable domain of an anti-VEGFC heavy chain antibody (i.e., "VHH"), and the VEGFA antigen-binding site for the bispecific binding protein of the invention is provided by the paired heavy chain variable region (VH) and light chain variable region (VL) of an anti-VEGFA antibody, or by a polypeptide fragment of the VEGF receptor extracellular domain that specifically binds to VEGFA, or by an artificial ligand "trap" molecule.

[0035] As used herein, the term ligand "trap" molecule is a fusion protein comprising the ligand-interacting extracellular domain of the receptor and the human IgG Fc region. Currently, various trap molecules have already been developed that "trap" VEGF ligands, including VEGFA and VEGFC. These trap molecules can be used to bind and reduce the concentration of the corresponding ligand in the extracellular environment. In one embodiment, the anti-VEGFA component of the present invention comprises a trap molecule in the form of an Fc fusion protein that "traps" VEGFA. The trap molecule can inhibit VEGFA-induced signal transduction by competing with the native VEGFA cell receptor to bind to VEGFA. In one embodiment, the trap molecule in the form of an Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 25. The VEGFA-trap molecule is formed by fusing the extracellular ligand-binding domains from VEGF receptors 1 and 2 to the Fc portion of human IgG1, and has amino acids of completely human origin, minimizing the immunogenicity of the molecule in the human body.

[0036] As used herein, the term "VEGFA" or "VEGF-A" refers to vascular endothelial growth factor A (e.g., human VEGFA protein with accession number UniProt P15692). VEGFA binds to the receptors VEGFR1 (also known as FLT1) and VEGFR2 (also known as KDR). As used herein, "antigen-binding specificity for VEGFA" refers to a binding site or domain in a molecule that specifically binds to human VEGFA.

[0037] The term "VEGFC" or "VEGF-C" refers to vascular endothelial growth factor C (e.g., human VEGFC protein at accession number UniProt P49767). VEGFC binds to receptors VEGFR2 (also called KDR) and VEGFR3 (also called FLT4). As used herein, "antigen binding specificity for VEGFC" refers to a binding site or binding domain in a molecule that specifically binds to human VEGFC. In one embodiment, when detected by biolayer interference technology, the VHH antigen binding site that binds to VEGFC in the molecule of the present invention has high affinity binding activity for human VEGFC, for example, a monovalent binding affinity KD value of about 1 nM to 10 nM, or a bivalent binding affinity of about 0.1 nM to 0.7 nM.

[0038] An "anti-VEGF-C moiety" as used herein refers to a polypeptide capable of binding to a VEGF-C protein. For example, an anti-VEGF-C moiety may be a polypeptide comprising a VHH domain of the invention. In one embodiment, an anti-VEGF-C moiety is a single domain antibody that binds to VEGF-C. In a further embodiment, an anti-VEGF-C moiety comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24. Preferably, the anti-VEGF-C component comprises three CDR sequences of a VHH domain selected from the amino acid sequences set forth in SEQ ID NOs: 4, 8, 12, 16, 20 and 24, wherein the CDR sequences are defined according to Kabat or defined according to Chothia, or wherein CDR1 is defined according to a combination of Kabat and Chothia and CDR2 and CDR3 are defined according to Kabat.

[0039] "Anti-VEGF-A component" refers herein to a polypeptide capable of binding to VEGF-A protein. For example, the anti-VEGF-A component may be a chimeric polypeptide / fusion protein comprising a VEGFA receptor extracellular domain polypeptide and an Fc portion fused to its C-terminus, or may be an anti-VEGFA antibody comprising a heavy chain and a light chain, such as a Fab antibody or other full-length antibody fragment. In one embodiment, the anti-VEGF-A component is provided by a trap molecule in the form of an Fc fusion protein, and forms a dimer by dimerization of the Fc region. In another embodiment, the anti-VEGF-A component is provided by an anti-VEGFA antibody in the form of a Fab, where the heavy chain of the Fab (i.e., the polypeptide chain comprising the heavy chain constant region) and the light chain of the Fab (i.e., the polypeptide chain comprising the light chain constant region) are paired to form a dimer.

[0040] The terms "Fab fragment", "Fab" or "Fab antibody" are used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains and including an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL and a light chain constant domain CL, where one polypeptide chain includes, from N-terminus to C-terminus, a VH and one constant domain selected from CH1 and CL, and the other polypeptide chain includes, from N-terminus to C-terminus, a VL and another constant domain selected from CL and CH1, where the VH and VL domains pair to form an antigen-binding site. Herein, when one polypeptide chain of a Fab includes a VH linked to CL and the other polypeptide chain includes a VL linked to CH1, the Fab is also referred to as a crossFab.

[0041] Herein, a Fab polypeptide chain comprising a heavy chain constant region CH1 is also referred to as a Fab heavy chain, and a Fab polypeptide chain comprising a light chain constant region CL is also referred to as a Fab light chain accordingly. Thus, in one Fab antibody, in the Fab heavy chain, CH1 can be linked to a VH or a CL, and in the Fab light chain, CL can be linked to a VL or a VH, provided that the VH and VL can pair to form an antigen-binding site that specifically binds to a target antigen.

[0042] The term "VHH domain" is used herein to refer to a heavy chain variable domain derived from a heavy chain antibody devoid of light chains (sometimes referred to herein as HcAb antibody), also referred to as a single variable domain fragment or nanobody. Thus, unlike the common VH domains of four-chain immunoglobulins, VHH domains do not need to pair with a light chain variable domain to form an antigen-binding site. Such VHH domain molecules may be derived from antibodies produced in Camelidae species (e.g. camel, alpaca, dromedary, llama, guanaco). Species other than Camelidae can also produce natural light chain-devoid heavy chain antibodies, and such VHHs are also within the scope of the present invention. In some cases, it is desirable for the therapeutic use of a VHH domain to reduce its immunogenicity. Thus, preferably, in one embodiment, the VHH domain used in the present invention is a humanized VHH domain or a further sequence-optimized form thereof (e.g., an affinity-matured form to increase binding affinity).

[0043] "Single domain antibody" or "sdAb" as used herein refers to an antibody polypeptide that recognizes and binds to a target antigen by a single variable antibody domain, e.g., a VHH or a single VH or a single VL. The single variable antibody domain of a single domain antibody can recognize and bind to a target antigen without pairing with another antibody variable domain. Herein, a single domain antibody comprising a heavy chain variable domain (VHH) of a heavy chain antibody is also referred to as a VHH single domain antibody. The VHH single domain antibody used in the present invention is preferably derived from a camelid, e.g., an alpaca, or a humanized or sequence-optimized form thereof. In some embodiments, the VHH single domain antibody of the present invention is a monovalent, monospecific polypeptide molecule that is composed or essentially composed of a single VHH domain.

[0044] The single domain antibody or VHH domain of the present invention may be included in a larger polypeptide / protein. Examples of polypeptides / proteins that include the single domain antibody of the present invention that may be mentioned include, but are not limited to, heavy chain antibodies (HcAbs), such as heavy chain antibodies with framework regions and / or heavy chain constant regions derived from camelids (llamas, camels, especially alpacas), humanized forms thereof or sequence-optimized forms thereof (affinity matured forms), or fragments thereof (e.g., fragments containing at least a part of the constant region); fusion proteins formed with the VHH domain derived from the heavy chain antibody as a component, such as fusion proteins with (a part of) immunoglobulin constant regions (e.g., Fc regions). In one embodiment, the VHH domain of the present invention is fused with an Fc region, such as a human IgG1 Fc region, to form a VHH-Fc antibody.

[0045] As used herein, the term "flexible connecting peptide", "connecting peptide" or "linker" refers to a short amino acid sequence consisting of amino acids, e.g., glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or a hinge region derived from an immunoglobulin. In one embodiment, the connecting peptide has a length of 5 to 50 amino acids, e.g., 10, 15, 20, 25 or 30 amino acids. In one embodiment, the connecting peptide comprises the amino acid sequence (G4S)n, where n is an integer equal to or greater than 1, e.g., n is an integer equal to or greater than 2, 3, 4, 5, 6 or 7. In one embodiment, the connecting peptide comprises the amino acid sequence TS(G4S)n, where n is an integer equal to or greater than 1, e.g., n is an integer equal to or greater than 2, 3, 4, 5, 6 or 7. In one embodiment, the connecting peptide comprises the amino acid sequence G(G4S)n, where n is an integer equal to or greater than 1, e.g., n is an integer equal to or greater than 2, 3, 4, 5, 6 or 7. In a further embodiment, the linking peptide is a hinge region derived from an immunoglobulin, for example, the amino acid sequence of the hinge region containing "CPPC", for example, the amino acid sequence "EPKSCDKTHTCPPCP" or "EPKSSDKTHTCPPCP". Linking peptides that can be used to link to each domain of the antibody molecule of the present invention may be, for example, but are not limited to, the amino acid sequences GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LQRDGERP, LRQKDGGGSERP, and GSTSGSGKPGSGEGSTKG. Alternatively, a computer program may be used to simulate the three-dimensional structure of proteins and peptides, or a suitable flexible linking peptide may be rationally designed by phage display methods.

[0046] As used herein, the terms "binding" or "specifically binding" mean that the binding action is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The binding ability of an antigen-binding site for a particular antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other common binding assays known in the art.

[0047] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X to partner Y can generally be expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0048] "Percentage identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are the same as those in the specific amino acid sequences shown herein, after aligning the candidate sequence with the specific amino acid sequences shown herein and introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and where any conservative substitutions are not considered as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention, which have a comparable identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity, to the antibody molecules and sequences specifically disclosed herein. The variants may include conservative modifications.

[0049] With respect to a polypeptide sequence, "conservative modifications" include substitutions, deletions, or additions to the polypeptide sequence, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following eight groups of amino acids are conservative substitutes for each other: 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or change the target antigen binding properties of the antibody or binding protein molecule of the invention that contains the amino acid sequence. For example, conservatively modified variants retain at least 80%, 85%, 90%, 95%, 98%, 99% or more, e.g., 100%-110% or more, of the binding affinity to the target antigen relative to the parent antibody or binding protein.

[0050] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells", including the originally transformed cell and their derived progeny. Host cells may be any type of cell line that can be used to produce the antibody molecule of the invention, and include eukaryotic cells, such as mammalian cells, insect cells, yeast cells, and prokaryotic cells, such as E. coli cells. Host cells include cultured cells, including cells within transgenic animals, transgenic plants, or cultured plant or animal tissue.

[0051] The term "expression vector" refers to a vector containing a recombinant polynucleotide, including an expression control sequence operatively linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; additional elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., nude or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) into which a recombinant polynucleotide has been incorporated.

[0052] The terms "individual" or "subject" can be used interchangeably and refer to a mammal. Mammals include, but are not limited to, domestic animals (e.g., dairy cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual refers to a human.

[0053] The term "treatment" refers to a clinical intervention that seeks to alter the natural course of a disease in an individual undergoing treatment. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of progression of the disease, amelioration or alleviation of the morbidity, and alleviation or improvement of the prognosis. In some embodiments, the antibody molecule of the present invention is for delaying the onset of the disease or for reducing the progression of the disease.

[0054] The term "anti-tumor effect" or "tumor suppressor effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell viability. The terms "tumor" and "cancer" are used interchangeably herein and encompass a variety of solid tumors.

[0055] As used herein, the term "angiogenesis-associated disease" refers to a disease, disorder, and / or condition whose onset, progression, and / or course involve vasculogenesis (including angiogenesis, lymphangiogenesis, and / or both). Such a disease, disorder, or condition would benefit from inhibition of the biological activity of VEGF-C or VEGF-A, or both.

[0056] Each aspect of the present invention will be described in detail below. I. Single Domain Antibody Polypeptides and Derivatives Thereof Single domain antibodies or VHH domains have a molecular weight approximately ten times smaller than that of a human IgG molecule and a physical diameter of only a few nanometers. Due to their small molecular size, single domain monoclonal antibodies have the advantage of higher stability, solubility, and the ability to recognize hidden antigenic sites compared to common four-chain antibodies. Furthermore, single domain antibodies are cheaper to prepare than common four-chain antibodies and can be easily expressed and purified in E. coli at higher expression levels. In addition to their use as separate molecules, single domain antibodies are also suitable building blocks for the construction of multispecific molecules.

[0057] As a result of intensive research, the present invention provides, in one aspect, a single domain antibody and its derivatives. The single domain antibody of the present invention specifically binds to human VEGFC and preferably inhibits its binding to the receptor and the signal transduction induced thereby. In some embodiments, the single domain antibody of the present invention is (i) binds to human VEGF-C (hVEGF-C) with high affinity, preferably with a monovalent binding affinity KD value of about 1 nM to 10 nM, e.g., about 5 nM to 6 nM, and a bivalent binding affinity KD value of between about 0.1 nM to 1 nM, e.g., about 0.6 nM, as measured by biolayer interference technology, e.g., as measured by the method described in Example 3; (ii) inhibiting the binding of hVEGF-C to the receptor VEGFR2, preferably having an IC50 value of about 0.1 nM to 0.5 nM, more preferably about 0.01 nM to 0.05 nM, as measured by an ELISA method, for example, the method described in Example 4; (iii) inhibiting the binding of hVEGF-C to the receptor VEGFR3, preferably having an IC50 value of about 0.1 nM to 0.5 nM, more preferably about 0.01 nM to 0.05 nM, as measured by an ELISA method, for example, the method described in Example 4; (iv) inhibiting activation of a signal transduction pathway mediated by the binding of VEGFC-C to VEGFR2, preferably measured by a receptor-reporter molecule system, for example, the method described in Example 5; (v) inhibiting activation of signaling pathways mediated by binding of VEGFC-C to VEGFR3; (vi) inhibiting vascular endothelial-related activities induced by the binding of VEGFC to VEGFR2, such as vascular endothelial cell survival, proliferation and / or migration, angiogenesis and / or vascular leakage; and (vii) inhibiting the survival and / or proliferation of vascular endothelial cells and / or lymphatic endothelial cells induced by the binding of VEGFC to VEGFR3; The compound has one or more biological activities selected from the following:

[0058] Structurally, like other conventional antibodies, the sdAbs of the present invention have a modular structure, in which the VHH variable region sequence comprises three CDRs and four highly conserved framework regions, and generally has a structure represented by the formula FR1-CDR-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3. The CDR sequences in the VHH variable region can be determined according to any of the CDR definition schemes described in the "Definition" section, and preferably the boundaries of the three CDRs in the sdAb variable region sequence can be defined by Kabat, Chothia, or a combination thereof.

[0059] In some embodiments, the invention provides single domain antibodies that are expressed and isolated in prokaryotic host cells, such as E. coli, hi still some further embodiments, the invention provides single domain antibodies that are expressed and isolated in eukaryotic host cells, such as mammalian cells, such as CHO cells or 293 cells.

[0060] In some embodiments, the single domain antibodies of the invention comprise CDR amino acid sequences and / or framework region (FR) amino acid sequences derived from a camelid heavy chain antibody produced by immunization of a camelid (e.g., an alpaca). In some embodiments, the single domain monoclonal antibodies of the invention derived from a camelid heavy chain antibody may be engineered to include, for example, human amino acid sequences (i.e., a human antibody) or framework region and / or constant region sequences derived from other non-camelid mammalian species. For example, the antibody may include a framework region, some or all of the heavy chain constant region (e.g., separate CH1, CH2 or CH3 regions, or any combination thereof, e.g., CH2-CH3 regions), an immunoglobulin Fc region or a fragment thereof, and / or a hinge region or a portion of a hinge region derived from a human or non-human primate. In one embodiment, to further improve the properties (e.g., affinity) of the engineered antibody, camelid amino acid residues at the corresponding positions in the parent camelid antibody may be introduced by back mutation at one or more positions (e.g., framework regions) in the engineered antibody.

[0061] In one embodiment, the single domain antibody or VHH domain of the invention is humanized. Humanization can be achieved by replacing one or more amino acid residues, particularly framework region sequences, in a natural VHH sequence of non-human origin (e.g., VHH sequence of camelid or alpaca immunity origin), with residues at the corresponding positions of the heavy chain VH of a conventional antibody of human origin. Methods for humanizing single domain monoclonal antibodies are well known in the art. Typically, humanizing substitutions are made such that the favorable binding properties of the single domain antibody are retained. Tests for ascertaining the biological properties, such as binding affinity, of humanized single domain antibodies in order to determine and select suitable humanizing residue mutations or combinations of mutations are well known in the art.

[0062] In some embodiments, the humanized single domain antibody of the invention comprises: (1) determining the CDR loop structure of a parent single domain antibody (e.g., a camelid VHH antibody screened from a phage display library); (2) finding the closest homologous sequence for each V / J region in a human germline sequence database; (3) screening for the closest matching human germline heavy chain and the lowest amount of back mutations; (4) constructing the CDR regions of the chimeric antibody onto human framework regions; (5) using the sequence and structural features to determine amino acid positions in the framework region that function to maintain the CDRs; (6) backmutating (reverting to the input amino acid type) at sequence positions determined to be important; (7) optimizing the amino acids at the risk sites; (8) obtaining humanized antibodies and optionally sequencing the antibody VHH sequences; The method can be obtained by a method comprising the steps of:

[0063] In some embodiments, the present invention also provides functional variants of the single domain antibodies of the present invention. Such functional variants can be obtained by introducing mutations into the coding nucleic acid sequences of the exemplary single domain antibodies of the present invention, e.g., CDR and / or FR sequences, by methods well known in the art, e.g., random or site-directed mutagenesis, and then screening for variants that retain the desired properties (e.g., screening by phage display libraries). Typically, functional variants retain significant sequence identity with the parent single domain antibody (or VHH domain). Preferably, functional variants retain the desired biological properties of the parent single domain antibody (or VHH domain), e.g., the variant has comparable (e.g., at least 50%, 60%, 70%, 80%, preferably 90% or more) biological activity, or improved biological activity (e.g., 110% to 150% or more) compared to the biological activity of the parent. The desired biological properties include, but are not limited to, for example, target antigen-binding affinity (e.g., measured by KD value), activity that inhibits the binding of a target antigen to a receptor (e.g., measured by IC50 value), inhibitory activity that inhibits activation of a target antigen-induced signaling pathway (e.g., measured by IC50 value), inhibition of angiogenesis and / or vascular leakage in in vitro or in vivo experiments, and inhibition of tumor growth / survival in in vitro or in vivo experiments.

[0064] In some embodiments, the invention provides affinity variants of the single domain antibody polypeptides of the invention. Preferably, said affinity variants exhibit one or more amino acid changes in the amino acid sequence relative to the parent single domain antibody from which they are derived, whereby the affinity variants have an altered binding affinity to the target antigen compared to the parent antibody. Typically, the affinity variants exhibit improved antigen binding affinity over the parent. The improvement may be, but is not limited to, a lower KD value, a faster off-rate, or an increased (or decreased) cross-reactivity of the target antigen with homologous proteins of non-human species. Often, the affinity variants have one or more amino acid residue substitutions in the CDRs compared to the parent. The substitutions may be conservative or non-conservative. In one embodiment, the affinity variants of the invention have a total of 10 or less, 6 or less, or 1-5, e.g. 1, 2, 3, 4 or 5 amino acid substitutions in CDRs 1-3 compared to the parent. In some embodiments, the affinity variants are mutated in CDR2 compared to the parent. In another embodiment, the affinity variants are mutated in CDRs 2 and CDR3 compared to the parent. Affinity variants can be obtained by various affinity maturation methods known in the art, including mutating CDRs, using E. coli mutator strains, DNA shuffling, phage display, and the like.

[0065] "Complementarity determining region", "CDR region" or "CDR" (which may be used interchangeably herein with hypervariable region "HVR") is an amino acid region in an antibody variable region that is primarily responsible for binding to an antigen epitope. Herein, the CDRs of a single domain antibody or VHH domain are generally referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. In some embodiments, the present invention provides an anti-VEGFC single domain antibody, wherein the antibody has the following CDR sequences: (i) the three CDR sequences of the VHH sequence of SEQ ID NO: 4, (ii) the three CDR sequences of the VHH sequence of SEQ ID NO: 8; (iii) the three CDR sequences of the VHH sequence of SEQ ID NO: 12; (iv) the three CDR sequences of the VHH sequence of SEQ ID NO: 16; (v) the three CDR sequences of the VHH sequence of SEQ ID NO: 20, and (vi) the three CDR sequences of the VHH sequence of SEQ ID NO: 24; The VHH domain comprises the

[0066] Preferably, the CDR sequences are defined according to Kabat or according to Chothia, or CDR1 is defined according to a combination of Kabat and Chothia, and CDR2 and CDR3 are defined according to Kabatc; or,

[0067] Here, the antibody comprises a mutant of any one of the CDR sequences selected from (i) to (vi) above, and for example, with respect to any one of the CDR sequences (i) to (vi) above, the mutant has 1 to 8, 1 to 5, or 1, 2, 3, 4, 5, or 6 amino acid changes, preferably conservative amino acid substitutions.

[0068] In some embodiments, the present invention provides an anti-VEGF C single domain antibody comprising a VHH domain consisting of three CDRs and four FRs, represented by the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein: (i) CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:1, CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, and CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:3; (ii) CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10, and CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11; (iii) CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18, and CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19, or (iv) CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21, CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22, and CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; or, Here, the antibody comprises a mutant of any one of the CDR sequences selected from (i) to (vi) above, and for example, with respect to any one of the CDR sequences (i) to (vi) above, the mutant has 1 to 8, 1 to 5, or 1, 2, 3, 4, 5, or 6 amino acid changes, preferably conservative amino acid substitutions.

[0069] Preferably, the anti-VEGFC single domain antibody of the invention comprises a VHH domain having the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, and CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19.

[0070] More preferably, the anti-VEGFC single domain antibody of the invention comprises a VHH domain having the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:21, CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:22, and CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:23.

[0071] The "variable region" or "variable domain" of a single domain antibody is the region that is involved in antigen binding in a single domain antibody. The heavy chain variable domain in a single domain antibody is also referred to herein as a VHH domain. As is known in the art, one or more residues in a VHH variable region can be modified, for example, modified residues, particularly conservative residue replacements, in one or more CDR regions and / or in one or more framework regions, but the modified antibody still essentially retains at least one biological property (for example, antigen binding ability) of the antibody molecule before the change.

[0072] For example, residues in the CDR regions can be mutated to improve one or more binding properties (e.g., affinity) of the antibody. The antigen-binding properties or other functional properties of the mutated antibody can be evaluated in in vitro or in vivo assays. Preferably, conservative substitutions are introduced. Preferably, no more than one, two, three, four or five residue changes are introduced in the CDR regions. Additionally, residues in the framework regions can be mutated, for example, to improve the properties of the antibody. For example, one or more framework residues can be "backmutated" to the corresponding germline sequence residues.

[0073] CDR grafting is another antibody variable region modification method known in the art. Since CDR sequences are responsible for most antibody-antigen interactions, recombinant antibody variants can be constructed that simulate the properties of known antibodies. In the antibody variants, CDR sequences from a known antibody are grafted into the framework regions of a different antibody with different properties. Thus, in one embodiment, the invention relates to an anti-VEGFC single domain antibody that comprises CDR sequences from one heavy chain variable region of the exemplary VHH single domain antibody in FIG. 19, but with different framework region sequences. Framework region sequences for replacement can be obtained from public DNA databases that contain germline antibody gene sequences or from VEGFC antibody sequences reported in the public literature. Germline DNA encoding human heavy chain variable region genes can be obtained, for example, from the GenBank database. The antibody protein sequences and the protein sequences in the databases can be compared by sequence similarity search tools such as Gapped BLAST. Preferably, the framework sequences for substitution have structural similarity to the framework sequences of the antibodies of the invention selected for alteration, e.g., framework sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity. In some embodiments, antibody humanization can be performed according to the methods of Example 7.

[0074] Accordingly, in one embodiment, the invention provides an anti-VEGFC single domain antibody comprising, or alternatively consisting of, an amino acid sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20 and 24, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0075] In another embodiment, the invention provides an anti-VEGFC single domain antibody, the antibody comprising: (i) comprising a VHH sequence having the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof; or (ii) comprising a VHH sequence having the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof; or (iii) comprising a VHH sequence having the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof; or (iv) comprising a VHH sequence having the amino acid sequence shown in SEQ ID NO: 16 or a variant thereof; or (v) comprising a VHH sequence having the amino acid sequence shown in SEQ ID NO: 20 or a variant thereof; or (vi) Contains a VHH sequence having the amino acid sequence shown in SEQ ID NO: 24 or a variant thereof.

[0076] Wherein, the variant has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity in amino acid sequence with reference VHH sequence (preferably over the entire length or in the three regions of CDR1, CDR2 and CDR3). In one embodiment, the variant has at least 1 and no more than 30, 10, or 5, 4, 3, 2, 1 or 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in amino acid sequence with reference VHH sequence (preferably over the entire length or in the three regions of CDR1, CDR2 and CDR3). Preferably, the difference between the variant and reference VHH sequence is not in the CDR region.

[0077] Preferably, the anti-VEGFC single domain antibody of the invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 20. More preferably, the anti-VEGFC single domain antibody of the invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 24.

[0078] II. Fusion Proteins / Chimeric Polypeptides In a further aspect, the present invention provides a polypeptide comprising at least one single domain antibody or VHH domain of the invention, wherein said polypeptide comprises a single domain monoclonal antibody or VHH domain of the invention and another peptide / polypeptide sequence linked to its N-terminus or C-terminus or N-terminus and C-terminus.

[0079] In some embodiments, a single domain antibody or VHH domain of the invention (e.g. a camelid VHH domain or a humanized form thereof) can be linked to the constant region, e.g. the Fc region, of a human antibody to produce a VHH-Fc polypeptide, which in one embodiment comprises a single domain antibody of the invention and its C-terminal Fc portion.

[0080] In a further embodiment, the VHH-Fc polypeptide comprises an Fc portion from a camelid. In one embodiment, the VHH-Fc polypeptide is produced and isolated by immunization of the camelid, such as an alpaca. Various methods are known in the art for immunizing camelids and isolating the VHH antibodies produced against the target antigen.

[0081] In some further embodiments, the VHH-Fc polypeptide comprises an Fc portion derived from a human or non-human primate. In further embodiments, the VHH-Fc polypeptide comprises a human IgG Fc region, e.g., a human IgG1 Fc region, e.g., an Fc region of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0082] In one embodiment, a VHH-Fc polypeptide according to the invention comprises a connecting peptide, eg an Fc portion, fused to the C-terminus of a single domain monoclonal antibody via the hinge region.

[0083] In a further embodiment, the VHH-Fc polypeptide according to the present invention can dimerize via the Fc portion with another polypeptide chain comprising an Fc portion (e.g. another VHH-Fc polypeptide, the same or different). Thus, in a further embodiment, the present invention also provides a homo- or heteromultimeric protein comprising the VHH-Fc polypeptide of the present invention. In one preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing of two identical VHH-Fc polypeptide chains.

[0084] In addition to the VHH-Fc polypeptides and heavy chain antibodies described above, the present invention also provides other fusion proteins / chimeric polypeptides comprising at least one single domain antibody polypeptide according to the invention.

[0085] In some embodiments, the fusion protein / chimeric polypeptide of the invention comprises one single domain antibody or VHH domain according to the invention. In another embodiment, the fusion protein / chimeric polypeptide of the invention comprises multiple single domain antibodies or VHH domains according to the invention. The fusion protein according to the invention may be monospecific or multispecific. In some embodiments, the fusion protein / chimeric polypeptide of the invention comprises only specificity for VEGFC. In a further embodiment, the fusion protein / chimeric polypeptide of the invention, in addition to comprising a single domain antibody or VHH domain(s) according to the invention against VEGFC, also comprises specificity for another antigen, for example specificity for VEGFA.

[0086] As will be understood by those skilled in the art, each component in the fusion protein is operably linked so that it can perform its desired function. In one embodiment, the components of the fusion protein may be directly linked or linked via a linking peptide or linker. In some embodiments, the linker comprises an amino acid sequence derived from an immunoglobulin hinge region, or comprises a flexible amino acid sequence, such as a sequence consisting of glycine and serine, such as (G4S)n or G(G4S)n, where n=1, 2, 3, 4, 5, 6 or 7, preferably 2, 3 or 4.

[0087] III. Bispecific binding molecules In a further aspect, the present invention provides a method for producing a method for the treatment of a cancer (i) a first antigen-binding moiety that specifically binds VEGF-C; (ii) a second antigen-binding moiety that specifically binds VEGF-A; and The present invention provides an anti-VEGFC / VEGFA bispecific binding molecule comprising: The bispecific molecules of the invention provide dual antagonistic activity against VEGF-A and VEGF-C.

[0088] In some embodiments, a bispecific binding molecule of the invention comprises: (i) inhibiting activation of the VEGFR2 signaling pathway induced by hVEGF-A alone, which can be determined by measuring the IC50 value of the inhibitory activity, preferably by a KDR reporter assay, for example, the method described in Example 10, wherein the IC50 value is, for example, 0.1 nM to 10 nM, for example, about 0.2 nM to 1 nM, for example, 0.6 nM, or about 1 nM to 3 nM, for example, about 1 nM; (ii) inhibiting activation of the VEGFR2 signaling pathway induced by hVEGF-C alone, which can be determined by measuring the IC50 value of the inhibitory activity, preferably by a KDR reporter assay, for example, the method described in Example 10, wherein the IC50 value is 0.1 nM to 1 nM, for example, about 0.2 nM to 0.6 nM; (iii) inhibiting hVEGF-C-induced lymphatic cell proliferation, which can be determined preferably by measuring the IC50 value of the inhibitory activity by a cell proliferation assay, for example, the method described in Example 11, wherein the IC50 value is 0.01 nM to 0.5 nM, for example, about 0.1 nM to 0.5 nM; (iv) inhibiting endothelial cell survival and proliferation co-induced by hVEGF-C and hVEGF-A, with the level of inhibition reaching at least 80%, 85%, 90%, 95% or about 100%, preferably determined by measuring the IC50 value of the inhibitory activity by a cell proliferation assay, for example, the method described in Example 12, wherein the IC50 value is 0.1 nM to 0.5 nM, for example, about 0.2 nM; (v) inhibiting angiogenesis co-induced by hVEGF-C and hVEGF-A, with an inhibition level of at least 80%, 85%, 90%, 95% or about 100%, preferably as determined by measuring the inhibitory activity by an endothelial cell tube formation assay, e.g., the method described in Example 13; (vi) inhibiting angiogenesis in tumors (e.g., solid tumors, e.g., melanoma); (vii) inhibiting the growth of a tumor (e.g., a solid tumor, e.g., melanoma), e.g., in a tumor-bearing animal model, e.g., the tumor inhibition rate by single-agent administration reaches 50% or more in a tumor-bearing animal model described in Example 14; and (viii) inhibiting the onset and / or progression of angiogenesis-related eye diseases, for example, by reducing the level of angiogenesis, reducing vascular leakage, and inhibiting edema and / or thickening of the fundus retina due to angiogenesis; The compound has one or more biological activities selected from the following:

[0089] Thus, in some embodiments, the invention provides anti-VEGFA / VEGFC bispecific binding molecules that can be used to treat angiogenesis-related diseases, such as cancer (e.g., solid tumors) and ocular diseases (e.g., macular degeneration AMD).

[0090] The anti-VEGFC component of the anti-VEGFA / VEGFC bispecific binding molecule according to the invention may comprise any of the above-mentioned anti-VEGFC single domain antibody polypeptides of the invention, or any of the anti-VEGFC VHH domains according to the invention. Preferably, said anti-VEGFC component comprises or consists of an anti-VEGFC VHH domain according to the invention, more preferably said VHH domain is a humanized VHH domain. In one embodiment, said VHH domain comprises the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, in one embodiment, said VHH domain comprises the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 9, 10 and 11, in one preferred embodiment, said VHH domain comprises the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 17, 18 and 19, and in a further preferred embodiment, said VHH domain comprises the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 21, 22 and 23. In a further embodiment, the VHH domain comprises an amino acid sequence selected from (i) SEQ ID NO: 4, 8, 12, 16, 20 or 24, or (ii) an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of (i), or (iii) an amino acid sequence having at least 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 amino acid changes (e.g., substitutions, deletions and / or insertions, preferably substitutions, more preferably conservative substitutions) to the amino acid sequence of (i). In a preferred embodiment, the VHH domain comprises or consists of the amino acid sequence of SEQ ID NO: 20 or 24. In one embodiment, an anti-VEGFA / VEGFC bispecific binding molecule according to the invention comprises at least one, such as two or more (preferably two) anti-VEGFC single domain antibodies or VHH domains of the invention.

[0091] The anti-VEGFA component of the anti-VEGFA / VEGFC bispecific binding molecule according to the present invention can be provided by any molecule that contains a VEGFA binding domain, including, but not limited to, an anti-VEGFA antibody, such as a single chain Fv antibody, a Fab antibody, a Fab' antibody, a (Fab)2 antibody, a single domain antibody and a nanobody; an anti-VEGFA trap molecule, such as a trap molecule that contains the extracellular domain of the VEGFA receptor VEGFR1, a trap molecule that contains the extracellular domain of the VEGFA receptor VEGFR2, or a trap molecule that contains the extracellular domains of VEGFR1 and VEGFR2; and a fusion protein or chimeric polypeptide of a VEGFA binding domain and the Fc portion of an immunoglobulin. In the bispecific antibody molecule according to the present invention, the anti-VEGFA component is linked or fused to the C-terminus or N-terminus of the anti-VEGFA component. In some embodiments, the anti-VEGFA component can be covalently linked to the N-terminus or C-terminus, preferably the C-terminus, of the anti-VEGFA component, directly or preferably via a linker sequence. In some embodiments, anti-VEGFA moieties according to the invention bind to human VEGFA and inhibit the binding of VEGFA to its receptors VEGFR1 and / or VEGFR2 and the signal transduction induced thereby.

[0092] In some preferred embodiments, the anti-VEGFA moiety according to the present invention is provided by an Fc chimeric polypeptide comprising a VEGFA binding domain, whereby the anti-VEGFA moiety comprises a VEGFA binding domain in the form of an Fc fusion protein. In another preferred embodiment, the anti-VEGFA moiety is provided by an anti-VEGFA Fab antibody, whereby the anti-VEGFA moiety comprises a VEGFA binding domain in the form of an Fab antibody. In embodiments where the anti-VEGFA moiety is an Fc fusion protein, preferably the VEGFA binding domain may be at the N-terminus or C-terminus of the Fc moiety, and the anti-VEGFA Fc moiety is linked to the opposite end of the Fc moiety. In embodiments where the anti-VEGFA moiety is an Fab antibody, preferably the anti-VEGFA Fc moiety is linked to the C-terminus of the Fab antibody.

[0093] In some embodiments, the bispecific binding proteins of the invention according to the invention may include a linker linking the anti-VEGFA component and the anti-VEGFC component. In one embodiment, the linker is a peptide about 6 to about 30 amino acids in length, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. Preferably, the linker comprises a G(G4S)n or G(G4S)n amino acid sequence, where n=an integer of 1, 2, 3, 4 or 5, preferably n=2, 3 or 4.

[0094] Bispecific binding molecules in the form of Fc fusion proteins In some embodiments, the invention accordingly provides an anti-VEGFA / VEGFC bispecific binding molecule, wherein said binding molecule comprises an anti-VEGFA component and an anti-VEGFC component in the form of an Fc fusion protein, wherein said anti-VEGFA component comprises a VEGF-A binding domain fused to an immunoglobulin Fc region, and said anti-VEGF-C component comprises any anti-VEGF-C single domain antibody polypeptide described in the present invention, in particular a VHH single domain antibody polypeptide consisting of any anti-VEGF-C VHH domain described in the present invention. In one embodiment, said anti-VEGFFC component is linked to said anti-VEGFA component via its Fc region, preferably covalently linked, more preferably linked via a linker. In further embodiments, said VEGF-A binding domain and said anti-VEGF-C VHH domain are located at both ends of the Fc region, respectively. In some preferred embodiments, said VEGF-A binding domain is located at the N-terminus of the Fc region, and said anti-VEGF-C VHH domain is located at the C-terminus of the Fc region.

[0095] In one embodiment, the VEGF-A binding domain is a VEGF-A binding polypeptide. In a further embodiment, the VEGF-A binding polypeptide comprises an extracellular VEGFA binding domain derived from a VEGFA receptor, such as VEGFR1 and / or VEGFR2. In one embodiment, the VEGFA binding polypeptide comprises a third Ig domain of VEGFR2 genetically fused to a second Ig domain of VEGFR1. In one embodiment, the VEGF-A binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:26, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. Preferably, the VEGF-A binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:26.

[0096] In one embodiment, the Fc region is a human IgG Fc region, e.g., a human IgG1 Fc region, and preferably comprises, or alternatively consists of, the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto.

[0097] In one embodiment, the anti-VEGFA component comprising a VEGFA binding polypeptide and an Fc region comprises or consists of the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0098] In some embodiments, the anti-VEGFA / VEGFC bispecific binding molecules of the invention form dimers through the dimerization effect of the Fc region.

[0099] In one preferred embodiment, in the anti-VEGFA / VEGFC bispecific binding molecule of the invention, the anti-VEGFA binding polypeptide according to the invention and the anti-VEGFC VHH domain according to the invention are fused to the N-terminus and C-terminus of an immunoglobulin Fc region, respectively, and the fusion polypeptide chain formed therewith forms a dimer by dimerization of the Fc region. Thus, in one preferred embodiment, the invention provides an anti-VEGFA / VEGFC bispecific binding molecule comprising a first polypeptide chain and a second polypeptide chain, wherein said first polypeptide chain and said second polypeptide chain comprise a fusion polypeptide formed by fusing the anti-VEGFA binding polypeptide according to the invention and the anti-VEGFC VHH domain according to the invention to the N-terminus and C-terminus of an immunoglobulin Fc region, respectively, and wherein said first polypeptide chain and said second polypeptide chain may be the same or different. Preferably, the first polypeptide chain and the second polypeptide chain are the same, and the anti-VEGFA / VEGFC bispecific binding molecule of the invention is a homodimeric protein comprising a first polypeptide chain and a second polypeptide chain. In one embodiment, the first and second polypeptide chains are the same and each comprise a VEGF-A binding polypeptide consisting of the amino acid sequence of SEQ ID NO:26, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.In a further embodiment, the first and second polypeptide chains are the same and each comprises an anti-VEGF-C VHH domain comprising a CDR1, CDR2 and CDR3 sequence selected from the amino acid sequence of SEQ ID NO: 4, 8, 12, 16, 20 or 24, preferably the VHH domain comprises a CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 1 to 3, SEQ ID NO: 9 to 11, SEQ ID NO: 17 to 19 or SEQ ID NO: 21 to 23, more preferably the VHH domain comprises or consists of an amino acid sequence selected from SEQ ID NO: 4, 8, 12, 16, 20 or 24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. In a further embodiment, the first and second polypeptide chains are the same and each comprises an Fc region derived from human IgG1. In a further embodiment, the first and second polypeptide chains are the same, and wherein the anti-VEGFC VHH domain according to the invention is fused to the C-terminus of the immunoglobulin Fc region via a linker. In one embodiment, the linker comprises a G(G4S)n or G(G4S)n amino acid sequence, where n=an integer of 1, 2, 3, 4 or 5, preferably n=2, 3 or 4. In a further embodiment, the first and second polypeptide chains are the same and comprise an amino acid sequence selected from SEQ ID NOs: 40-41 and 44-47, respectively, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the first and second polypeptide chains are the same and comprise an amino acid sequence as shown in SEQ ID NO: 47, respectively.

[0100] Bispecific binding molecules in the form of Fab antibodies In some further embodiments, the present invention provides an anti-VEGFA / VEGFC bispecific binding molecule, wherein the binding molecule comprises an anti-VEGFA component and an anti-VEGFC component in the form of a Fab antibody, wherein the anti-VEGFA component comprises a heavy chain variable region VH and a light chain variable region VL that pair to form a VEGF-A binding domain, and which are fused to an immunoglobulin heavy chain constant region CH1 and a light chain constant region CL, respectively, to form an anti-VEGFA Fab antibody. In some further embodiments, the anti-VEGFC component is linked, preferably covalently linked, more preferably linked via a linker, to the anti-VEGFA component in the form of a Fab antibody by the C-terminus of the CH1 and / or CL constant regions of the anti-VEGFA Fab antibody. In a further embodiment, the anti-VEGFA Fab antibody is linked to one anti-VEGF-C component each at the C-terminus of the CH1 constant region and the CL constant region, respectively. Preferably, said anti-VEGF-C component comprises any anti-VEGF-C single domain antibody polypeptide according to the invention, in particular a VHH single domain antibody polypeptide consisting of any anti-VEGF-C VHH domain according to the invention.

[0101] In some embodiments of the bispecific binding molecules according to the invention, the anti-VEGFA Fab antibody according to the invention comprises a Fab heavy chain formed by linking the heavy chain variable region VH and the immunoglobulin heavy chain constant region CH1, and a Fab light chain formed by the light chain variable region VL and the immunoglobulin constant region VL. In another embodiment, the anti-VEGFA Fab antibody according to the invention comprises a Fab light chain formed by linking the heavy chain variable region VH and the immunoglobulin constant region VL, and a Fab heavy chain formed by linking the light chain variable region VL and the immunoglobulin heavy chain constant region CH1.

[0102] In some embodiments, the anti-VEGFA Fab antibody according to the present invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises the amino acid sequences of HCDR1 to 3 of SEQ ID NOs: 34 to 36, and the VL comprises the amino acid sequences of LCDR1 to 3 of SEQ ID NOs: 37 to 39. In further embodiments, the anti-VEGFA Fab antibody is a chimeric or humanized antibody, preferably a humanized antibody. In a further embodiment, the VEGF Fab antibody comprises a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH comprises the amino acid sequence of SEQ ID NO:29, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO:32, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably, the VH comprises the amino acid sequence of SEQ ID NO:29, and the VL comprises the amino acid sequence of SEQ ID NO:32.

[0103] In some further embodiments, the anti-VEGFA Fab antibody according to the invention comprises a CH1 constant region of human immunoglobulin IgG1. In another embodiment, the anti-VEGFA Fab antibody according to the invention comprises a human kappa light chain constant region CL. In one embodiment, the anti-VEGFA Fab antibody comprises a heavy chain CH1 constant region having an amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. In a further embodiment, the anti-VEGFA Fab antibody comprises a light chain CL constant region having an amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0104] In some embodiments, preferably, the anti-VEGFA Fab antibody according to the invention comprises a Fab heavy chain consisting of a VH and a CH1 according to the invention (VH-CH1 chain) and a Fab light chain consisting of a VL and a CL according to the invention (VL-CL chain). More preferably, the anti-VEGFA Fab antibody according to the invention comprises a Fab heavy chain having an amino acid sequence of SEQ ID NO:28 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and a Fab light chain having an amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. More preferably, the anti-VEGFA Fab antibody according to the invention comprises an amino acid sequence of SEQ ID NO:28 and an amino acid sequence of SEQ ID NO:31.

[0105] In some further embodiments of the bispecific binding molecule according to the invention, the anti-Fab antibody according to the invention is linked to the anti-VEGFC single domain antibody of the invention or the anti-VEGFC VHH domain of the invention at the C-terminus of CH1 and CL, respectively, directly or preferably via a linker. Thus, in one embodiment, the invention provides an anti-VEGFA / VEGFC bispecific binding molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a Fab heavy chain of an anti-VEGFA Fab antibody according to the invention and an anti-VEGFC VHH domain linked to the C-terminus of its CH1, and the second polypeptide chain comprises a Fab light chain of an anti-VEGFA Fab antibody according to the invention and an anti-VEGFC VHH domain linked to the C-terminus of its CL. Preferably, the Fab heavy chain consists of VH and CH1 (VH-CH1) and the Fab light chain consists of VL and CL (VL-CL).

[0106] In one preferred embodiment, the present invention provides an anti-VEGFA / VEGFC bispecific binding molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises from N-terminus to C-terminus a VH-CH1-linker-VHH and the second polypeptide chain comprises from N-terminus to C-terminus a VL-CL-linker-VHH, wherein said VHH is an anti-VEGFA VHH domain of the present invention, and said VH-CH1 and VL-CL pair to form an anti-VEGF-A Fab antibody of the present invention, optionally wherein CH1 and CL may be directly linked to the VHH domain without the use of a linker. In some embodiments, the VHH domain comprises CDR1, CDR2 and CDR3 sequences selected from the amino acid sequence of SEQ ID NO: 4, 8, 12, 16, 20 or 24; preferably, the VHH domain comprises CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1 to 3, SEQ ID NO: 9 to 11, SEQ ID NO: 17 to 19 or SEQ ID NO: 21 to 23; more preferably, the VHH domain comprises or consists of an amino acid sequence selected from SEQ ID NO: 4, 8, 12, 16, 20 or 24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and even more preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 20 or 24.In some embodiments, the VH comprises the amino acid sequence of HCDR1 to 3 of SEQ ID NOs: 34 to 36, and the VL comprises the amino acid sequence of LCDR1 to 3 of SEQ ID NOs: 37 to 39. Preferably, the VH and VL are humanized. More preferably, the VH comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. Even more preferably, the VH comprises the amino acid sequence of SEQ ID NO: 29, and the VL comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CH1 comprises the CH1 constant region of human immunoglobulin IgG1. In another embodiment, the CL comprises the human kappa light chain constant region CL. In one embodiment, the CH1 comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. In a further embodiment, the CL comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. In one embodiment, the linker comprises a G(G4S)n or G(G4S)n amino acid sequence, where n=1, 2, 3, 4 or 5 integers, preferably n=2, 3 or 4.

[0107] In a further preferred embodiment, the present invention provides an anti-VEGFA / VEGFC bispecific binding molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are - a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, - a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 49 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, - a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 51 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, Selected from In a preferred embodiment, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 48 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 49, or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43, or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 48 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 49, or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 50 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 51. More preferably, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 48 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 49.

[0108] The invention also provides immunoconjugates (e.g., conjugated to toxins or small chemical molecules) comprising the single domain antibodies, fusion proteins and bispecific binding molecules of the invention, as well as pharmaceutical compositions and drug combinations, in which the single domain antibodies, fusion proteins and bispecific binding molecules of the invention may comprise additional therapeutic agents, such as other therapeutic agents available for the anticipated use of the single domain antibodies, fusion proteins and bispecific binding molecules of the invention, such as chemotherapeutic agents, radiotherapeutic agents, anti-angiogenic molecules, immunosuppressants, antifibrotic agents, neuroprotective agents and tumor suppressor molecules.

[0109] IV. Polynucleotides, Vectors and Hosts The present invention provides a nucleic acid encoding any of the above molecules of the invention (single domain antibodies, fusion proteins and bispecific binding molecules). Further provided is a vector comprising said nucleic acid. In one embodiment, the vector is an expression vector. Further provided is a host cell comprising said nucleic acid or said vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell or a 293 cell). In another embodiment, the host cell is prokaryotic.

[0110] In one aspect, the invention provides a nucleic acid encoding any of the above anti-VEGFC single domain antibodies or VHH domains. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human VEGFC antigen binding ability. In some embodiments, the nucleic acid is operably linked in frame to a nucleic acid encoding another peptide / polypeptide, so that when expressed from a suitable expression vector, it produces a fusion protein or chimeric polypeptide comprising the single domain antibody or VHH domain and another peptide / polypeptide. To facilitate production and purification, the single domain antibody or VHH domain may be fused at the N-terminus to a secretion signal peptide and / or a tag peptide that contributes to purification, such as a hexahistidine tag. The single domain antibody or VHH domain may be fused at the C-terminus to the Fc portion of an immunoglobulin to form a VHH-Fc antibody.

[0111] In a further aspect, the invention provides a nucleic acid encoding any of the above anti-VEGFA / VEGFC bispecific binding molecules. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human VEGFA and human VEGFC antigen binding ability. In one embodiment, the nucleic acid encoding the first and second polypeptide chains of the bispecific antibody molecule can be in the same vector or in different vectors. In a further embodiment, the nucleic acid encoding the first and second polypeptide chains of the bispecific antibody molecule can be expressed by introducing them into the same or different host cells. Thus, in some embodiments, a method of producing a bispecific binding molecule of the invention comprises culturing a host cell comprising a nucleic acid encoding the first and second polypeptide chains under conditions suitable for expression of the first and second polypeptide chains of the molecule to produce a bispecific binding molecule of the invention.

[0112] As will be apparent to those skilled in the art, due to the degenerate nature of the genetic code, each antibody or polypeptide amino acid sequence can be encoded by more than one nucleic acid sequence.

[0113] Nucleic acid sequences encoding the molecules of the invention can be produced using methods well known in the art, for example, by de novo solid phase DNA synthesis or by PCR amplification.

[0114] In one embodiment, provide one or more vectors that contain the nucleic acid of the present invention.In one embodiment, vector is expression vector, for example, prokaryotic expression vector or eukaryotic expression vector.Vector includes but is not limited to virus, plasmid, cosmid, lambda phage or yeast artificial chromosome (YAC).

[0115] In one embodiment, a host cell is provided that comprises one or more polynucleotides of the invention. In some embodiments, a host cell is provided that comprises an expression vector of the invention. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce an antibody molecule of the invention. Suitable host cells for replicating and supporting the expression of the antibody molecule of the invention are well known in the art. If necessary, such cells can be transfected or transduced with a particular expression vector, and the vector-containing cells can be cultured in large quantities to inoculate large-scale fermenters so that sufficient quantities of the molecule of the invention can be obtained for clinical application. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be utilized. Examples of mammalian host cell lines that can be used include SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), dog kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki & Wu, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, Humana Press, Totowa, NJ), p. 255-268 (2003). In a preferred embodiment, the host cell is a CHO, HEK293 or NSO cell.

[0116] V. Production and Purification of Molecules of the Invention In a further aspect, the invention provides a method for producing a molecule of the invention (single domain antibodies, fusion proteins and bispecific binding molecules), said method comprising culturing a host cell comprising a polynucleotide encoding a polypeptide chain of said molecule under conditions suitable for expressing said polypeptide chain, and assembling said polypeptide chains to produce said molecule under conditions suitable for assembling said polypeptide chains into said molecule.

[0117] For recombinant production, the polynucleotides encoding the polypeptide chains of the molecules of the invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed by methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages or yeast artificial chromosomes (YACs). Once an expression vector for expression, including one or more polynucleotides of the invention, has been prepared, the expression vector can be transfected or introduced into a suitable host cell. To this end, various techniques can be utilized, such as, for example, protoplast fusion, calcium phosphate co-precipitation, electroporation, retroviral transduction, viral transfection, particle gun, liposome-based transfection or other common techniques.

[0118] The antibody molecules prepared as described herein can be purified by known conventional techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill in the art.

[0119] In one embodiment, the single domain antibodies or VHH domains of the invention can be produced in bacteria, such as E. coli. In one embodiment, the single domain antibodies can be expressed in the periplasm of the bacteria, and then isolated and optionally further purified by methods known in the art. Furthermore, the single domain antibodies, VHH domains, fusion polypeptides and bispecific molecules can be expressed in eukaryotic cells. In one embodiment, the cells are 293 cells, and after expression, isolated and purified, for example, by protein A column.

[0120] The purity of the antibody molecules of the present invention can be determined by any one of a variety of well-known analytical methods, including size-exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. A variety of assays known in the art can be used to identify, screen or characterize the physical / chemical properties and / or biological activity of the antibody molecules provided herein.

[0121] VI.Measurement method The molecules provided herein (single domain antibodies, fusion proteins and bispecific molecules) can be identified, screened or phenotypically characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0122] Binding of the molecules of the invention to human VEGFA and / or human VEGFC can be measured by methods known in the art, such as, for example, ELISA, Western blot, or exemplary methods disclosed in the Examples herein. For example, recombinant VEGFA and / or human VEGFC proteins can be used to measure the binding dynamics of the molecules (e.g., K DIn some embodiments, the binding equilibrium dissociation constant, e.g., monovalent or bivalent binding affinity, of a molecule to human VEGFA and / or human VEGFC can be measured at a temperature of 30° C. by a light interference bioassay (e.g., Fortebio affinity assay), such as the method set forth in Example 3.

[0123] The VEGFA and / or VEGFC antagonistic / inhibitory activity of the molecules of the invention can be measured by methods known in the art, such as ELISA inhibition tests, receptor fluorescent reporter molecule activation tests, cell proliferation tests, or exemplary methods disclosed in the Examples herein. For example, the inhibitory activity of molecules that inhibit the binding of human VEGFA and / or human VEGFC to their associated receptors can be measured by ELISA inhibition tests, such as the methods shown in Example 4. Cell-based receptor reporter assays, such as the methods described in Examples 5 or 10, can be used to measure the inhibition of the activation of the VEGFR2 signaling pathway induced by hVEGF-A alone or hVEGF-C alone or in combination, for example, in NFAT-RE-luc2P / KDR HEK293 cells. Cell proliferation assays, such as the CCK-8 test, such as the methods described in Examples 6 or 11, can be used to measure the in vitro inhibition of cell survival and / or proliferation induced by hVEGF-A alone or hVEGF-C alone or in combination, in lymphocytes or endothelial cells.

[0124] The antiangiogenic effect of the molecule of the present invention can be measured by methods known in the art, for example, in vitro test and / or in vivo animal experiment. For example, VEGFA and VEGFC can be used to induce human umbilical vein endothelial cell (HUVEC) lumen formation in an in vitro endothelial cell lumen formation experiment, for example, the method shown in Example 13, and the inhibitory effect of the molecule on VEGFA and VEGFC-induced primary cell lumen formation can be detected. For example, in a tumor-bearing mouse model, the antiangiogenic and / or antitumor effect of the molecule can be detected, for example, according to the method shown in Example 14. Alternatively, alternatively, for example, a laser-induced choroidal neovascularization animal model can be used, and the inhibitory state of the molecule on choroidal neovascularization can be observed after molecule administration, for example, by fundus color photography, fluorescein fundus angiography, optical coherence tomography.

[0125] VII. Pharmaceutical Compositions, Drug Combinations, and Reagent Kits In one aspect, the present invention provides a composition, such as a pharmaceutical composition, comprising a molecule of the present invention (single domain antibody, fusion protein, or bispecific molecule) formulated with a pharma- ceutically acceptable vector. As used herein, a "pharmaceutically acceptable vector" includes any or all of physiologically compatible solvents, dispersion media, isotonicity agents, and absorption delaying agents, etc. The pharmaceutical composition of the present invention is suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., injection or infusion). In one embodiment, the molecule of the present invention is formulated as a pharmaceutical composition suitable for injection administration. In some embodiments, the molecule of the present invention is formulated as a pharmaceutical composition suitable for intraperitoneal injection. In further embodiments, the molecule of the present invention is formulated as a pharmaceutical composition suitable for ocular injection administration (e.g., intravitreal injection administration). In some embodiments, the molecule of the present invention is the only active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise an antibody molecule as described herein and one or more therapeutic agents.

[0126] In another embodiment, the present invention also provides a pharmaceutical combination comprising a molecule of the invention (single domain antibody, fusion protein or bispecific molecule) and one or more therapeutic agents.

[0127] The therapeutic agent applied in the pharmaceutical compositions and drug combinations of the present invention may be any one of the therapeutic agents selected from the following classes: (i) antiangiogenic agents, (ii) immunosuppressants, (iii) antifibrotic agents, (iv) neuroprotective agents, and (v) drugs having tumor-suppressing activity.

[0128] The compositions of the present invention may be in various forms. These forms include liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form is determined by the desired mode of administration and therapeutic use. A typical preferred composition is in the form of an injectable or infusible solution. A preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (ip), intramuscular) injection. In one preferred embodiment, the antibody molecule is administered by intravenous infusion or injection. In another preferred embodiment, the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.

[0129] The phrases "parenteral administration" and "parenteral mode of administration" as used herein refer to modes of administration other than enteral and topical administration, which are generally given by injection and include, but are not limited to, intravenous, intramuscular, intraarterial, intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.

[0130] Therapeutic compositions should generally be sterile and stable under preparation and storage conditions. The compositions can be prepared in solution, microemulsion, dispersion, liposome or lyophilized form. Sterile injectable solutions can be prepared by adding the active compound (i.e., antibody molecule) in a given amount to a suitable solvent, followed by filtration and sterilization. Dispersions are generally prepared by adding the active compound to a sterile solvent, which contains a basic dispersion medium and other ingredients. A coating agent such as lecithin can be used. In the case of dispersions, surfactants can be used to maintain the proper fluidity of the solution. Absorption of injectable compositions can be prolonged by including in the composition an agent that delays absorption, for example, monostearate and gelatin.

[0131] The pharmaceutical compositions of the invention may contain a "therapeutically effective amount" or a "prophylactically effective amount" of the molecules of the invention. A "therapeutically effective amount" refers to an amount that effectively achieves a desired therapeutic result at a required dosage for a required period of time. A therapeutically effective amount can vary depending on many factors, such as the disease state, the age, sex and weight of the individual. A therapeutically effective amount is any amount in which toxic or adverse effects do not outweigh the beneficial effects of the treatment. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to untreated subjects. The inhibitory ability of the molecules of the invention on a measurable parameter (e.g., tumor volume) can be evaluated in an animal model system that shows efficacy in human tumors.

[0132] A "prophylactically effective amount" refers to an amount to effectively achieve a desired prophylactic result at a necessary dosage for a necessary period of time. Generally, since a prophylactic dose is used prior to or at an early stage of disease in a subject, the prophylactically effective amount will be less than the therapeutically effective amount.

[0133] A reagent kit comprising the antibody molecule described herein is also within the scope of the present invention.The reagent kit may also comprise one or more other elements, for example, instructions, other reagents such as markers or coupling reagents, pharma-ceutically acceptable vectors, and devices or other materials for administration to subjects.

[0134] VIII. Uses and Methods of the Molecules of the Invention In one aspect, the present invention provides in vivo and in vitro uses and applications of the molecules of the invention.

[0135] In some embodiments, the uses and methods of the present invention involve administering the molecules of the present invention in vivo and / or in vitro: - binding to VEGFA antigen and / or VEGFC antigen, - inhibition of binding of VEGFA and / or VEGFC to their associated receptors, such as VEGFR2 and / or VEGFR3; - Inhibition of VEGFA and / or VEGFC-induced activation of VEGFR2 and / or VEGFR3 cell signaling, - VEGFA and / or VEGFC-induced inhibition of endothelial cell survival, proliferation and / or migration; - VEGFC-induced inhibition of lymphocyte survival and / or proliferation, -Inhibition of VEGFC-induced lymphangiogenesis and proliferation and migration of lymphatic endothelial cells - Inhibition of VEGFA and / or VEGFC-induced angiogenesis and / or vascular leakage, - Inhibition of tumor angiogenesis and / or growth and / or metastasis, It relates to the application of

[0136] In some embodiments, the antibody molecule of the invention or a pharmaceutical composition comprising the antibody molecule of the invention is used as a medicament to treat and / or prevent a disease or as a diagnostic tool for a disease in an individual, preferably said individual is a mammal, more preferably a human.

[0137] In some embodiments, the present invention provides a method of treating angiogenesis-related diseases, comprising administering to a subject a molecule of the present invention (single domain antibody polypeptide or a fusion protein thereof, or a bispecific binding protein) or a pharmaceutical composition thereof. In some embodiments, the disease is a solid tumor, preferably melanoma, wherein administration of the bispecific binding protein inhibits angiogenesis in the tumor and / or tumor growth. In other embodiments, the disease is an ocular disease, preferably age-related macular degeneration, diabetic retinopathy, retinal vascular occlusion and corneal neovascularization.

[0138] In some embodiments the invention provides the use of a single domain antibody polypeptide of the invention or a fusion protein thereof or a bispecific binding molecule of the invention for preparing a medicament for treating and / or preventing a disease in a subject and / or for preparing a diagnostic tool for diagnosing a disease, wherein said disease is preferably an angiogenesis-associated disease, such as a solid tumor and an eye disease.

[0139] In any of the above embodiments, one or more additional active agents, such as chemotherapeutic and / or cancer therapeutic agents or anti-angiogenic therapies, may further be co-administered with the single domain antibody polypeptide or bispecific binding protein of the invention. The co-administration may be in the form of simultaneous, parallel or sequential administration in any order.

[0140] In a further aspect, the present invention also provides a diagnostic method for detecting the presence of a relevant antigen in a biological sample, such as serum, semen, urine or tissue biopsy sample (e.g. from a hyperproliferative or cancerous lesion), in vitro or in vivo. The diagnostic method comprises (i) contacting the sample (and optionally a control sample) with a molecule of the invention (single domain antibody, fusion protein or bispecific binding molecule) (labeled or unlabeled) or administering said antibody molecule to a subject under conditions allowing the interaction to occur, and (ii) detecting the formation of a complex between said molecule and the sample (and optionally the control sample). The formation of a complex indicates the presence of the relevant antigen and may, in some cases, indicate suitability or need for a treatment and / or prophylaxis as described herein. Example 1

[0141] Example 1. Preparation of a phage immunization library Construction of alpaca immune or synthetic libraries 1.1 Two healthy adult alpacas (Chengdu NBBIOLAB Co., Ltd.) were selected. 0.5 mg of recombinant protein antigen human VEGFC (Beijing Sino Biological Co., Ltd.) was mixed uniformly with Freund's adjuvant in a 1:1 ratio, and the alpacas were immunized a total of four times by multiple subcutaneous injections on the back, with an immunization interval of two weeks. 1.2 Collect 50 mL of alpaca peripheral blood, isolate lymphocytes, and obtain 2.5 x 10 viable cells. 7 1 mL of Trizol reagent (THERMO) was added per cell, and total RNA was extracted by chloroform / isopropyl alcohol precipitation. 10 μg of RNA was used as a template, and reverse transcription was performed using PrimeScript Reverse Transcription Reagent Kit (Takara). Using cDNA as a template, nucleic acids encoding VHH were produced by PCR amplification, and then a phage display vector that expresses and displays a fusion polypeptide of a Flag-tagged and hexahistidine-tagged VHH fragment and a phage gpIII protein was inserted, and E. coli TG1 competent cells were transformed with the display vector to construct and produce a VHH antibody library. In brief, the first and second PCR reactions were performed using cDNA as a template (for detailed methods, see J Immunol Methods. 2007 July 31; 324(1-2): 13-25). The pC3-HF vector and the product of the second PCR were double-cleaved with SacI and SalI (Thermo), respectively, and the enzyme-cleaved products were reacted with T4 ligase (Thermo). TG1 competent cells were electrically transformed to construct a VHH antibody library, and the bacterial solution was frozen and stored at -80°C. The resuscitated bacterial solution was inoculated into 100 mL of YT-AG medium (Shanghai Sangon Co., Ltd.), and helper phages were added to infect the bacteria. The bacteria were then resuspended in 2xYT-AK medium (Shanghai Sangon Co., Ltd.) and cultured overnight at 37°C and 200 rpm. The culture supernatant was collected, and recombinant phages were prepared using the PEG / NaCl precipitation method. 1.3 The recombinant phages were subjected to panning experiments with biotin-labeled antigen VEGFC (Beijing Sino Biological Co., Ltd.) in triplicates. 50 μL of Dynabeads® M280 magnetic beads (Thermo Co., Ltd.) and an appropriate amount of biotin-labeled antigen were added to each tube, incubated at room temperature for 30 min, and then 1 × 10 12 cfu recombinant phages were added and incubated at room temperature for 1 h. The resulting mixture was added to 1 mL of PBST and washed 10 times, with each wash time being 5 min. Finally, 0.5 mL of glycine buffer at pH 2.5 was added to elute the antigen-binding recombinant phages, which were then infected into TG1 and cultured overnight, infected with helper phages, and recombinant phages were prepared for the next panning experiment, and TG1 bacterial clones containing positive VHHs were identified. 1.4 Binding The binding activity was detected by ELISA, and the clones were sequenced. VEGFC antigen (Beijing Sino Biological Co., Ltd.) was taken in advance, diluted to 0.5 μg / mL with PBS buffer, coated on a 96-well ELISA plate, and left in a refrigerator at 4 °C overnight. The antigen-coated plate was washed three times with PBST, and blocking agent was added to 300 μL / well, and the plate was left to stand at room temperature for 1 h for blocking. The plate was washed three times with PBST, and 80 μL of blocking agent + 20 μL of expression supernatant of TG1 bacteria of the positive VHH identified in 1.3 above was added, and the plate was shaken at room temperature for 1 h. After washing three times with PBST, 100 μL / well of Anti-Flag / HRP secondary antibody (Sigma) diluted with blocking agent was added and shaken at room temperature for 40 min. After washing six times with PBST, 100 μL / well of TMB color development solution was added and color development was allowed to continue for 5 min to 15 min away from light. 100 μL / well of stop solution was then added. The values ​​were read using a microplate reader and the OD450nm absorbance was measured. Bacterial clones with a reading greater than 0.5 were selected and sent to GENEWIZ for sequencing, and TG1 bacterial monoclones containing each corresponding VHH sequence were selected, glycerol was added, and the clones were frozen in a -80℃ refrigerator.

[0142] Example 2. Production and purification of prokaryotic antibodies The positive VHH-containing TG1 monoclone obtained above was used for expression and purification to obtain a VHH antibody protein. TG1 bacteria containing the VHH expression plasmid identified in Example 1 was inoculated into 800 mL of LB-Amp medium and cultured at 37 ° C. and 200 rpm until the OD600 value reached 0.5 to 0.6. 1 mM IPTG was added to the bacterial solution to induce expression of the VHH fragment, and cultured overnight at 28 ° C. and 200 cpm. The culture supernatant was collected and centrifuged, and then 15 mL of PB + 1 mg / mL of polymyxin was added and resuspended to dissolve the bacterial periplasm, centrifuged again, and filtered through a 0.22 μm filter membrane. The periplasm lysate was passed through a 1 mL Ni Sepharose precolumn, washed twice with PBS, and 0.5 M imidazole was added to elute the target protein, and the protein concentration was measured by ultraviolet light. The protein concentration of the eluted target protein was measured by ultraviolet light, dispensed into multiple tubes, and stored in a refrigerator at -40 ° C. An alpaca VHH library was screened to identify the following HCDR motifs: HCDR1:GSXFSXYAMG, HCDR2:ATTSGGSTLYADSVKG, HCDR3:XWRGSDPENY, We obtained two anti-VEGFC VHH antibodies (LA49G9 and LA63G12) that share the same VHH domain. Based on the two obtained VHH antibodies, humanized VHH antibodies (LA49G9.2 and LA63G12.1) and affinity matured humanized VHH antibodies (am63G12-14B11 and am63G12-5G8-18B9) were produced. Please refer to the sequence listing and Figure 19 for the CDR amino acid sequences and VHH amino acid sequences of the VHH antibodies of the present invention, as well as the sequence numbers. In the VHH antibody biological activity detection experiments in the following Examples, humanized LA49G9.2 and LA63G12.1 molecule-related experiments were performed using dimerized VHH-Fc antibodies, and the rest were performed using VHH single domain antibodies.

[0143] Example 3. Measurement of binding dynamics between the antibody of the present invention and antigen using biolayer interference technology The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human VEGFC was measured using biolayer interferometry (ForteBio). ForteBio affinity measurements were performed according to conventional methods (Estep, P et al., High throughput solution based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):p270-8). Thirty minutes before the start of the experiment, an appropriate number of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors were taken depending on the number of samples and immersed in SD buffer (PBS 1x, BSA 0.1%, Tween®-20 0.05%). 100 μL of SD buffer, antibody and antigen (human VEGFC (Beijing Sino Biological)) were taken and added to a 96-well black polystyrene half-area microplate (Greiner, 675076). The plate was arranged according to the sample position, and the sensor position was selected. The instrument parameters were set as follows: baseline, loading ~ 1 nm, baseline, association and dissociation steps were performed, the execution time of each step was determined by the binding and dissociation rate of the sample, the rotation speed was 400 rpm, and the temperature was 30 °C. K was measured using ForteBio analysis software. D The values ​​were analyzed. In the experiments described in the above assay, the affinities of the antibodies are as shown in Table 1. [Table 2] [Table 3] In the above studies, the antibodies LA49G9 and LA63G12 had monovalent KD values ​​with human VEGFC of 1.00E-08M and 1.64E-08M, respectively, while the affinity-matured antibodies Am63G12-14B11 and Am63G2-5G8-18B9 had further improved binding affinity with monovalent KD values ​​of 6.21E-09M and 5.70E-09M, respectively. The antibodies LA49G9, LA49G9.2, LA63G12, and LA63G12.1 had bivalent affinity KD values ​​with human VEGFC of 6.13E-10M, 6.45E-10M, 6.56E-10M, and 6.22E-10M, respectively.

[0144] Example 4. ELISA inhibition experiment of anti-VEGFC VHH antibodies The inhibitory effect of the antibody of the present invention on the binding of hVEGF C and its receptor KDR was measured. SA (streptavidin) was diluted to 1 μg / mL, and 100 μL / well was spread on a microplate and placed at 4°C overnight. The plate was washed three times with PBST, and 3% BSA was added for 1.5 h blocking. The plate was washed three times with PBST, and 50 ng / mL VEGFC-biotin (human VEGFC labeled with biotin) was added and incubated for 1.5 h. 50 μL of antibody and VEGFR2-Fc (final concentration 0.2 μg / mL, Beijing Sino Biological) or VEGFR3-Fc (final concentration 0.2 μg / mL, Beijing Sino Biological) were added after pre-incubation for 20 min. The plate was washed three times with PBST, and anti-human FcHRP antibody (diluted 1:10000, BETHYL) was added and incubated for 30 min. After washing six times with PBST, the plate was incubated with TMB (SOLARBIO) for 5 minutes for color development, and the OD450nm was read after stopping the development. The inhibition results of the anti-VEGF VHH antibodies obtained in the present invention are shown in Figure 1. The candidate molecules LA49G9, LA63G12 and the humanized antibody LA63G12.1, and the affinity matured molecules Am63G12-14B11 and Am63G2-5G8-18B9 antibodies can all inhibit the binding of VEGFC to VEGFR2 and VEGFR3, and the two affinity matured molecules have essentially comparable inhibition IC50 values ​​to the positive control molecule OPT-302 (VEGF-C-trap, SEQ ID NO: 52).

[0145] Example 5. Inhibition experiment of VEGFC-induced activation of HEK293 KDR reporter by anti-VEGFCVHH antibody VEGFC can bind to the related receptor VEGFR2 (KDR), activate the VEGFR2 signaling pathway, and induce the survival, proliferation, and migration of vascular endothelial cells. In this study, we used the KDR reporter experimental system and NFAT-RE-luc2P / KDR HEK293 cells (Promega Cat CS181401) to detect the inhibitory effect of gradient-diluted antibodies on the VEGFC activation-related receptor signaling pathway. The experimental methodology was as described by the supplier: Take out NFAT-RE-luc2P / KDR HEK293 cells that were replaced with experimental medium (DMEM medium containing 10% FBS) 3 days ago, aspirate off the old medium, wash once with PBS, digest the cells with 1mL of Accutase solution (Sigma) until the cells become round and come off the wall, stop the reaction with 5mL of dilution medium (DMEM medium containing 10% FBS), aspirate the cells into a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the medium, add 10mL of dilution medium to resuspend the cells, mix uniformly, and count. The cell viability should be 90% or more. The cell density in the dilution medium should be 0.8 x 10 6 The solution was adjusted to cells / mL and added at 50 μL / well to a 96-well white cell culture plate according to the experimental layout. A mixture of hVEGFC (R&D) with a concentration of 200ng / mL and gradient-diluted test antibodies (including the VHH antibody of the present invention, a positive control molecule (OPT-302), and a negative control antibody (IgG isotype control antibody)) was prepared and left to stand for 30 minutes, after which 50μL / well was added to a 96-well white cell culture plate containing cells, and incubated for 6 hours in a 37℃, 5% carbon dioxide incubator. A blank control group to which no antibody or VEGFC was added, and a VEGF-C experimental group to which only VEGFC was added but no antibody were added were also set up. The 96-well white cell culture plate that had been incubated for 6 h was removed from the carbon dioxide incubator and equilibrated to room temperature for 10 to 15 min. Bio-Glo Luciferase Assay System (Promega), which had been removed in advance and equilibrated to room temperature, was added to the 96-well white cell culture plate at 100 μL / well according to the experimental layout, and incubated at room temperature for 5 min away from light. The fluorescence values ​​were read by a multifunction microplate reader, the chemiluminescence mode was selected as the plate reading mode, the end point method was selected as the plate reading type, the wavelength was set to full wavelength, the fluorescence was collected row by row, and the collection time for each row was 1000 ms. The results are shown in FIG. 2, in which LA63G12 and LA49G9 can completely suppress VEGFC-induced activation of the KDR signaling pathway.

[0146] Example 6. Inhibition experiment of VEGFC-induced BaF3-FLT4 cell proliferation by anti-VEGFC VHH antibody In this study, antibodies and recombinant human VEGFC protein were co-incubated with BaF3-FLT4, BaF3 cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) that overexpress FLT4 (VEGFR3). The viable cell count was detected by CCK-8 reagent kit (Dojindo Chemical Industries) to reflect the inhibitory effect of different antibodies on VEGFC-induced BaF3-FLT4 proliferation. BaF3 cells were infected with a lentivirus carrying the FLT4 gene to obtain BaF3-FLT4 cells that overexpress FLT4. The cell proliferation inhibition test was carried out according to the instruction manual of the CCK-8 reagent kit. The experimental medium was prepared using 1640 medium containing 10% FBS. The test antibodies (including the VHH antibody of the present invention, the positive control molecule (OPT-302) and the negative control antibody (IgG isotype control antibody)) had a maximum final concentration of 20 μg / mL and were diluted 1:3 in sequence. A blank control group (blank) to which no antibody or VEGFC was added, and a VEGF-C experimental group to which only VEGFC but no antibody was added were also set up. In the test system, hVEGFC (R&D) had a final concentration of 20 ng / mL and BaF3-FLT4 cells were cultured at 2 × 10 5 The final concentration was 100 μL of the system per well in a 96-well plate, and the plate was incubated at 37° C. in a CO2 incubator for 72 h. Then, 15 μL of CCK-8 was added to each well and incubated for 4 h in a 37 °C, CO2 incubator. The absorbance value was measured by dual wavelengths, the detection wavelength was 450 nm, and the reference wavelength was 620 nm. The OD450 to OD620 values ​​were measured. The experimental results are shown in Figure 3. Both of the antibodies LA63G12 and LA49G9 of the present invention can effectively inhibit hVEGF-induced BaF3-FLT4 survival and proliferation in vitro.

[0147] Example 7. Humanization of anti-VEGFC VHH antibody, activity detection, and protein expression and purification The LA49G9 and LA63G12 antibodies were humanized by the following steps: (1) determining the CDR loop structure; (2) find the closest homologous sequence for each V / J region in the human germline sequence database; (3) screening for the closest human germline heavy chain match and the lowest amount of reversion mutations; (4) constructing the CDR regions of the chimeric antibody onto a human framework region; (5) using the sequence and structural features to determine amino acid positions in the framework region that function to maintain the CDRs; (6) making back mutations (reverting to the input amino acid type) at sequence positions determined to be important; (7) The amino acids at risk sites were optimized. Humanized antibodies LA49G9.2 and LA63G12.1 were obtained and the antibody sequences were sequenced. For transfection and expression of VHH single domain antibodies or VHH-Fc antibodies in 293 cells, plasmids containing nucleic acids encoding anti-VEGF C antibodies were prepared. Depending on the required transfection volume, expi-293 cells (Invitrogen) were passaged to a cell density of 1.5 × 10 6 The cell density was adjusted to approximately 3 × 10 6 The transfection volume was 1 / 10 of the final volume of F17 medium (Gibco, A13835-01) was used as the transfection buffer, and the appropriate plasmid was added and mixed uniformly. The appropriate polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid (the ratio of plasmid to PEI is 1:3 for 293F cells), mixed uniformly, and then incubated at room temperature for 10 min to obtain the DNA / PEI mixture. After resuspending the cells in the DNA / PEI mixture, they were incubated at 36.5°C, 8% CO2 for 24 h, and then supplemented with 2% of the transfection volume of FEED (Sigma) and cultured under conditions of 36.5°C, 120 rpm, and 8% CO2. On the 6th day of continuous culture or when the cell activity was ≦60%, the cell supernatant was collected and purified. Prior to purification, the collected medium was centrifuged at 4500 rpm for 30 min and the cells were discarded. The supernatant was then filtered through a 0.22 μL filter. A Protein A column (Hitrap Mabselect Sure 5 × 5 mL, GE, 11-0034-95) was equilibrated with 10 mL of binding buffer (sodium phosphate 20 mM, NaCl 150 mM, pH 7.0). The filtered supernatant was added to the purification column and re-equilibrated with 15 mL of binding buffer. The eluate was collected by adding 5 mL of elution buffer (citric acid + sodium citrate 0.1 M, pH 3.5) and 80 μL of Tris-HCl per mL of eluate was added. The collected antibody was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044) for concentration detection. The activity of the humanized antibody was measured according to the methods described in Examples 3 to 6. The results are shown in Tables 1 and 2 above, and in Figures 1, 4, and 5, respectively. As is clear from the results, the humanized antibody had antigen hVEGFC binding activity and VEGFR2 / VEGFR3 receptor inhibitory activity comparable to those of the parent antibody, inhibited VEGFC-induced activation of the VEGFR2 signaling pathway in a cell-based assay (Figure 4), and inhibited the proliferation of Baf3-FLT4 cells induced by the binding of VEGFC to VEGFR3 (Figure 5).

[0148] Example 8. Anti-VEGFC VHH affinity maturation The LA63G12.1 humanized single domain antibody gene was selected as the template, and random amino acid mutations were introduced into the antigen binding region (CDR), and degenerate primers (Genewiz) containing the NNK genetic code and framework region-specific primers were designed and synthesized. The antibody mutant gene library was amplified by overlap extension PCR (OE-PCR). The PCR fragments and vectors were enzymatically cleaved and ligated in the same manner as in Example 1, and TG1 bacteria were transformed to prepare a recombinant phage library, and phage panning was performed three times. Prokaryotic protein expression was performed according to Example 2, and the clone binding activity was detected by ELISA and sequenced and analyzed. Finally, two mutants with obviously improved affinity were selected, with clone numbers am63G12-14B11 and am63G12-5G8-18B9. Eukaryotic VHH single domain antibody expression was performed according to Example 7. The affinity matured VHH detection methods were performed as described in Example 4, Example 5 and Example 6. The results are shown in Figure 1, Figure 6 and Figure 7, respectively. As can be seen from the results, the affinity matured antibodies have further improved anti-VEGFC biological activity compared to the parent antibody.

[0149] Example 9. Construction of anti-VEGFA / VEGFC bispecific antibodies Anti-VEGFA / VEGFC bispecific antibodies were constructed using LA63G12, LA63G12.1, am63G12-14B11 and am63G12-5G8-18B9 and expressed as eukaryotic proteins in 293 cells according to Example 7. Briefly, The VHH single domain antibody was linked to the C-terminus of the Fc region of VEGF-trap (Aflibercept, Eylea) by a linker to construct a bispecific antibody comprising a first polypeptide chain and a second polypeptide chain as shown in Figure 18A. VEGF-trap is a recombinant fusion protein (SEQ ID NO: 25) consisting of a VEGF-A binding domain portion (SEQ ID NO: 26) from human VEGF receptors 1 and 2 fused at the C-terminus to a human IgG1 Fc region (SEQ ID NO: 27), which, in a dimeric form, provides high affinity VEGF_A binding and inhibits VEGF_A-induced activation of the VEGFR signaling pathway. The VHH single domain antibody was linked by a linker to the C-terminus of the Fab portion of the anti-VEGF_A antibody Lucentis (ranibizumab) to construct a bispecific antibody comprising a first polypeptide chain and a second polypeptide chain as shown in Figure 18B. Lucentis is a recombinant humanized IgG1 kappa isotype monoclonal antibody, in which a VH-CH1 polypeptide chain having the amino acid sequence shown in SEQ ID NO:28 pairs with a VL-CL polypeptide chain having the amino acid sequence shown in SEQ ID NO:29 to form a Fab that binds to human VEGF-A and inhibits its biological activity. Table 3 below lists the bispecific antibodies that were constructed and their compositions. [Table 4]

[0150] Example 10. Inhibition of VEGFA or C-induced activation of HEK293 KDR reporter by anti-VEGFA / VEGFC bispecific antibody VEGFA or VEGFC can bind to the related receptor VEGFR2 (KDR), activate the VEGFR2 signaling pathway, and induce effects such as survival, proliferation, and migration of vascular endothelial cells. This study utilized the KDR reporter experimental system and NFAT-RE-luc2P / KDR HEK293 cells (Promega Cat CS181401) to detect the inhibitory effect of gradient-diluted antibodies on VEGFA and VEGFC activation-related receptor signaling pathways. The experimental method was basically as described in Example 5. Briefly, NFAT-RE-luc2P / KDR HEK293 cells (0.8 × 10 6 A 96-well white cell culture plate containing hVEGFA (R&D) at a concentration of 100 ng / mL or hVEGFC (R&D) at a concentration of 200 ng / mL and a gradient dilution of the test antibody was prepared and left to stand for 30 min. 50 μL / well of the mixture was added to a 96-well white cell culture plate containing NFAT-RE-luc2P / KDR HEK293 cells and incubated for 6 h in a 37°C, 5% CO2 incubator. A blank control group (blank) without any antibody or VEGFC was prepared, an experimental group with only VEGFC but no antibody, and an experimental group with VEGFC and an IgG isotype control antibody were prepared at the same time. For comparison, we also detected the inhibitory activity of the anti-VEGF A molecule IBI304 (sequence number 53) and the bispecific antibody Faricimab (anti-Ang-2 / anti-VEGF-A) on the VEGF-A signaling pathway, and the inhibitory activity of the anti-VEGF-C molecule OPT-302 on the VEGF-C signaling pathway. The 96-well white cell culture plate that had been incubated for 6 h was removed from the carbon dioxide incubator and equilibrated to room temperature for 10 to 15 min. Bio-Glo Luciferase Assay System (Promega), which had been removed in advance and equilibrated to room temperature, was added to the 96-well white cell culture plate at 100 μL / well according to the experimental layout, and incubated at room temperature for 5 min away from light. The fluorescence values ​​were read by a multifunction microplate reader, the chemiluminescence mode was selected as the plate reading mode, the end point method was selected as the plate reading type, the wavelength was set to full wavelength, the fluorescence was collected row by row, and the collection time for each row was 1000 ms. The detection results are shown in Figures 8 and 9. Both the tested anti-VEGFA and anti-VEGFC bispecific antibodies can inhibit VEGFA (Figure 8) or VEGFC (Figure 9)-induced activation of the KDR signaling pathway.

[0151] Example 11. Inhibition of VEGFC-induced proliferation of BaF3-FLT4 cells by anti-VEGFA / VEGFC bispecific antibody The bispecific antibody described in the present invention was used in a BaF3-FLT4 proliferation experiment system to detect the effect of the antibody on VEGFC-induced BaF3-FLT4 proliferation. The experimental method was essentially as described in Example 6. Experimental media were prepared using 1640 medium containing 10% FBS. Test antibodies had a maximum final concentration of 10 nM and were serially diluted 1:3. In the test system, hVEGFC (R&D) had a final concentration of 20 ng / mL, and BaF3-FLT4 cells were diluted at 2 × 10 5 with a final concentration of cells / mL. In the measurement, a blank control group (blank) in which no antibody or VEGFC was added, an experimental group in which only VEGFC but no antibody was added, and an experimental group in which VEGFC and an IgG isotype control antibody were added were set up. At the same time, for comparison, the inhibitory activity of the anti-VEGF-C molecule OPT-302 on VEGFC-induced BaF3-FLT4 cell proliferation was also detected. The detection results are shown in FIG. 10, which show that all of the tested anti-VEGFA / VEGFC bispecific antibodies can inhibit VEGFC-induced proliferation of BaF3-FLT4 cells.

[0152] Example 12. Inhibition of VEGFA+VEGFC-induced HUVEC proliferation by anti-VEGFA / VEGFC bispecific antibody VEGFA and VEGFC act on related receptors such as VEGFR in vascular endothelial cells, promoting the survival, proliferation and migration of vascular endothelial cells, and further inducing angiogenesis. In this experiment, VEGFA and VEGFC were used in combination to induce the survival and proliferation of human umbilical vein endothelial cells (HUVECs), and the inhibitory effect of antibodies on the survival and proliferation of primary cells induced by VEGFA and VEGFC was detected. In this example, the survival and proliferation of HUVECs were measured by CCK-8. Specifically, the cells were treated one day before, seeded on a 96-well culture plate at 2000 cells / well, and incubated in a 37°C, 5% carbon dioxide incubator for 24 hours. After the cells attached to the wall, experimental medium (DMEM medium) containing or not containing VEGFA with a final concentration of 5 ng / mL and VEGFC with a final concentration of 50 ng / mL and / or gradient diluted antibodies was prepared, replaced the endothelial cell medium in the 96-well plate, and incubated in an incubator at 37°C with 5% carbon dioxide for 72 hours. In the experiment, the cells were divided into groups as follows: Blank group: DMEM medium VEGFA group: DMEM medium + 5ng / mL VEGFA VEGFC group: DMEM medium + 50ng / mL VEGFC VEGFA+VEGFC group: DMEM medium + 5ng / mL VEGFA + 50ng / mL VEGFC IgG group: DMEM medium + 5ng / mL VEGFA + 50ng / mL VEGFC + isotype control IgG IBI304+OPT-302 group: DMEM medium + 5ng / mL VEGFA + 50ng / mL VEGFC + IBI304 + OPT-302 (IBI304 and OPT-302 were mixed at a molar concentration ratio of 1:1), Bispecific antibody group: EGM-2 medium + 5 ng / mL VEGFA + 50 ng / mL VEGFC + bispecific antibody to be tested. After incubating the cells in the experimental medium, 10 μL / well of CCK-8 detection solution (Dojindo Chemical) was added and incubated in a 37°C, 5% carbon dioxide incubator for 12 to 24 hours. The absorbance OD was measured using a multi-function microplate reader. 450 ~OD620 The value of was read. In the experiment described above, the detection results are shown in Figure 11. The tested anti-VEGFA and anti-VEGFC bispecific antibodies can completely inhibit VEGFA+VEGFC-induced HUVEC cell proliferation and survival.

[0153] Example 13. Inhibition of VEGFA+VEGFC-induced HUVEC tube formation by anti-VEGFA / VEGFC bispecific antibodies VEGFA and VEGFC act on vascular endothelial cells to promote the formation of tube-like structures in vascular endothelial cells and further induce the formation of angiogenic structures. In this experiment, VEGFA and VEGFC were used to induce tube formation in human umbilical vein endothelial cells (HUVECs), and the inhibitory effect of antibodies on VEGFA- and VEGFC-induced tube formation in primary cells was detected. In this example, the inhibition of HUVEC tube formation by anti-VEGFA / VEGFC bispecific antibody was detected by HUVEC tube formation experiment. Specifically, Matrigel (BD) was placed on ice and melted one day before, added to a 96-well plate at 100 μL / well, and hardened in a 37°C, CO2 incubator for 30 min. Cells were treated with Accutase solution, seeded in a 96-well culture plate at 20,000 cells / well, and incubated in a 37°C, 5% carbon dioxide incubator for 24 h. Experimental media (EGM2) were prepared with or without VEGFA having a final concentration of 10 ng / mL and VEGFC having a final concentration of 50 ng / mL and / or gradient diluted antibodies. HUVEC cells were resuspended in different experimental media, seeded in a 96-well culture plate at 20,000 cells / well, and incubated in a 37°C, 5% carbon dioxide incubator for 24 h. In the experiment, the cells were divided into groups as follows: Blank group: EGM-2 medium VEGFA group: EGM-2 medium + 5ng / mL VEGFA VEGFC group: EGM-2 medium + 50ng / mL VEGFC VEGFA+VEGFC group: EGM-2 medium + 5ng / mL VEGFA + 50ng / mL VEGFC IgG group: EGM-2 medium + 5ng / mL VEGFA + 50ng / mL VEGFC + 20nM isotype control IgG IBI304+OPT-302 group: EGM-2 medium + 5ng / mL VEGFA + 50ng / mL VEGFC + 20nM IBI304 + 20nM OPT-302 IEX04-056 group: EGM-2 medium + 5ng / mL VEGFA + 50ng / mL VEGFC + 20nM IEX04-056 IEX04-067 group: EGM-2 medium + 5ng / mL VEGFA + 50ng / mL VEGFC + 20nM IEX04-067. The number of tubes was calculated from the microscopic images. The detection results are shown in Figure 12. Figure 12A shows the tube formation image, and Figure 12B shows the statistical results of tube formation. Anti-VEGFA and anti-VEGFC bispecific antibodies can completely inhibit VEGFA+VEGFC-induced HUVEC cell tube formation.

[0154] Example 14. Inhibition of A375 tumor angiogenesis by anti-VEGFA / VEGFC bispecific antibody Overexpression of VEGFA and VEGFC in tumor cells can induce angiogenesis in vivo and promote tumor growth. In this example, A375 human malignant melanoma cells were cultured at 3 × 10 6 The anti-angiogenic and anti-tumor effects of the anti-VEGFA / VEGFC antibody of the present invention were measured in nude mice by inoculating each mouse with 1000 ng / mL of the VEGFA / VEGFC antibody. Human nude mouse: Female nude mice with BALB / c background were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., with SPF grade. After receiving the mice, they were kept for 7 days to acclimate before the start of the experiment. cell: Human A375 cells were purchased from ATCC (CAT#: CRL-1619) and routinely subcultured for subsequent in vivo experiments in strict accordance with the manufacturer's instructions. Cells were harvested by centrifugation and resuspended in sterile PBS to a cell density of 1.5 × 10 7 On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the armpit of mice to establish A375 tumor-bearing mouse models. Dosage: Mice were randomly divided into groups (6 mice per group). 7 and 21 days after tumor cell inoculation, the tumor volume of each mouse was detected. The dosage and method are shown in Table 4. PBS (purchased from Gibco) was used as a negative control and administered on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, 17th, and 19th days after inoculation, respectively, and the tumor volume and body weight of the mice were monitored twice a week. Body weight and tumor volume were measured before each administration, and the relative tumor inhibition rate (TGI%) was calculated on the 21st day after inoculation, with the calculation formula: TGI%=100%×(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-tumor volume before administration of control group). Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured with a caliper, and the tumor volume was calculated as V=L×W. 2 The weight was measured using an electronic balance. [Table 5] As shown in Figures 13A and 13B and Table 5, the results of tumor inhibition rate showed that on the 21st day after inoculation, IEX04-056 single agent showed tumor inhibition effect at different doses. On the 21st day after inoculation, the tumor inhibition rate was 54% at 1 mg / kg, 71% at 5 mg / kg, and 74% at 25 mg / kg. Therefore, the bispecific binding molecules against VEGFA and VEGFC of the present invention have obvious tumor inhibition effect. [Table 6] Sectioning and CD31 (vascular endothelial marker) staining of tumor tissues on days 7 and 21 after inoculation demonstrated microvascularization in the tumor tissue. Wax blocks embedded in OCT embedding medium were placed on a freezing microtome and cut into sections with a thickness of 6 μm. 1. After leaving the sections at room temperature for 30 minutes, they were fixed with pre-cooled acetone at 4°C and then dried at room temperature. 2. Rinse by immersing in PBST for 5 min each time (PBST: 1X PBS + 0.05% Tween (registered trademark) 20) three times. 3. After circling with a pap pen, block with 10% goat serum (diluted with PBST), drop 100 μL onto each tissue, and block at room temperature. 4. Add 100μL of primary antibody PE-anti-mouse CD31 directly to the plate and incubate overnight at 4℃. 5. The next day, rinse with PBST for 5 minutes three times. 6. Stain cell nuclei with DAPI, drop 100μL onto each tissue, and incubate for 5min away from light. 7. Discard the DAPI staining solution and rinse with PBST for 5 min three times. The specimens were mounted with a prolong diamond antifade mountant (Invitrogen) and then scanned with an automated quantitative analysis scanner. The results are shown in Figure 13C. After treatment with IEX04-056, the angiogenesis of A375 tumors was significantly reduced.

[0155] Example 15. Efficacy test of laser-induced choroidal neovascularization This experiment utilized a laser-induced choroidal neovascularization model in cynomolgus monkeys to measure the anti-angiogenic effect of the anti-VEGFA / VEGFC bispecific antibody of the invention. Species: Macaque, Grade: Normal grade, Age: 2.5-5 years, Weight: 2.45kg-5.55kg, Average 4.02kg, Weight at time of modeling: 3.35kg-4.35kg. In this study, photocoagulation was performed around the fovea of ​​the fundus macula of cynomolgus monkeys using a laser to induce choroidal neovascularization in the fundus, and an animal model similar to human choroidal neovascularization was established. Fluorescein fundus angiography was performed before and on the 20th day after photocoagulation to determine the modeling status. Sixteen cynomolgus monkeys (half male and half female) that were successfully modeled were selected and divided into four groups, namely the model control group, the IEX04-056 group, the IEX04-067 group, and the Eylea+OPT-302 group, with four monkeys per group. On the 21st day after photocoagulation, IEX04-056, IEX04-067, or Eylea+OPT-302 were injected into the vitreous of both eyes according to the doses in Table 6 below, and the model control group was administered the same volume of 0.9% sodium chloride injection. The animals in each group were subjected to color fundus photography, fluorescein fundus angiography, and optical coherence tomography on days 7, 14, 21, and 28 after administration, respectively, to observe the inhibition of choroidal neovascularization by the test product. After euthanasia on day 29 after administration, both eyes were removed and subjected to histological examination by HE staining. [Table 7] Color fundus photography and fluorescein angiography Evaluation metrics: (1) Grading of fluorescent spots Grading criteria for spots photographed by fluorescent angiography after modeling: Grade 1: no strong fluorescence in the spot; Grade 2: strong fluorescence in the spot but no leakage of fluorescein; Grade 3: strong spot fluorescence, mild leakage of fluorescein, leakage not beyond the edge of the spot; Grade 4: Spot has strong fluorescence, significant leakage of fluorescein, leakage beyond the edge of the spot; Spots of grades 1 to 4 should be counted and the grade of fundus laser spots should be recorded at each examination. (2) Fluorescein leakage area improvement rate (%) Fluorescein leakage area improvement rate (%) = (fluorescein leakage area before administration - fluorescein leakage area after administration) / fluorescein leakage area before administration × 100% (3) Reduction in fluorescein leakage area Reduction in fluorescein leakage area = fluorescein leakage area before administration - fluorescein leakage area after administration Optical Coherence Tomography (OCT) Evaluation metrics: (1) Fundus retinal thickening improvement rate Fundus retinal thickening improvement rate (%) = retinal thickness before administration - retinal thickness after administration x 100 Pre-treatment retinal thickness - Pre-modeling retinal thickness (2) Reduction in fundus retinal thickness Reduction in fundus retinal thickness = retinal thickness before administration - retinal thickness after administration The results of color fundus photography and fluorescence angiography are shown in Figures 14 and 15. As can be seen from the results, the antibody of the present invention exhibited a significant antiangiogenic effect 28 days after administration, proving that the antibody of the present invention has a significant inhibitory effect on laser-induced fundus neovascularization (P<0.001) and has the function of protecting vascular integrity. The results of OCT are shown in Figure 16. As can be seen from the results, the antibody of the present invention significantly inhibited retinal thickening 14 to 28 days after administration (P<0.05), proving that the antibody of the present invention has the function of inhibiting retinal edema and thickening caused by neovascularization. Histopathological examination Twenty-nine days after administration to the cynomolgus monkeys, the animals were anesthetized with sodium pentobarbital according to their body weight (intravenously injected at approximately 30 mg / kg, the dose can be adjusted according to the animal's physical condition), euthanized by exsanguination from the abdominal aorta or femoral artery, gross observation was performed, and both eyeballs were removed. Both eyes of some animals were fixed in modified Davidson's fixative, embedded in paraffin, sectioned, and laser modeling areas were selected for histopathological examination, including routine HE staining. In pathological sections, the antibody of the present invention was able to better improve pathological changes such as edema, hyperplasia, and fibrosis at the laser injury site compared to the combination of the anti-VEGFA molecule Eylea and the anti-VEGFC molecule OPT-032, and showed better retinal morphological improvement (Figures 17A and 17B).

[0156] Sequence Listing LA49G9 antibody CDR1 sequence SEQ ID NO:1 GSMFSMYAMG LA49G9 antibody CDR2 sequence SEQ ID NO:2 ATTSGGSTLYADSVKG LA49G9 antibody CDR3 sequence SEQ ID NO:3 YWRGSDPENY LA49G9 antibody VHH sequence SEQ ID NO:4 QLQLVESGGGLVQPGGSLRLSCAASGSMFSMYAMGWYRQAPGKQRELVAATTSGGSTLYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCYTYWRGSDPENYWGRGTQVTVSS LA49G9.2 antibody CDR1 sequence SEQ ID NO:5 GSMFSMYAMG LA49G9.2 antibody CDR2 sequence SEQ ID NO:6 ATTSGGSTLYADSVKG LA49G9.2 antibody CDR3 sequence SEQ ID NO:7 YWRGSDPENY LA49G9.2 antibody VHH sequence SEQ ID NO:8 EVQLVESGGGLVQPGGSLRLSCAASGSMFSMYAMGWYRQAPGKQRELVAATTSGGSTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCYTYWRGSDPENYWGQGTQVTVSS LA63G12 antibody CDR1 sequence SEQ ID NO:9 GSSFSPYAMG LA63G12 antibody CDR2 sequence SEQ ID NO:10 ATTSGGSTLYADSVKG LA63G12 antibody CDR3 sequence SEQ ID NO:11 HWRGSDPENY LA63G12 antibody VHH sequence SEQ ID NO: 12 QLQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVAATTSGGSTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS LA63G12.1 antibody CDR1 sequence SEQ ID NO:13 GSSFSPYAMG LA63G12.1 antibody CDR2 sequence SEQ ID NO:14 ATTSGGSTLYADSVKG LA63G12.1 antibody CDR3 sequence SEQ ID NO:15 HWRGSDPENY LA63G12.1 antibody VHH sequence SEQ ID NO: 16 EVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATTSGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS am63G12.1-14B11 antibody CDR1 sequence SEQ ID NO:17 GSSFSPYAMG am63G12.1-14B11 antibody CDR1 sequence SEQ ID NO:18 ATSEGGFTLYADSVKG am63G12.1-14B11 antibody CDR1 sequence SEQ ID NO:19 HWRGSDPENY am63G12.1-14B11 antibody VHH sequence SEQ ID NO:20 EVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATSEGGFTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS am63G12.1-5G8-18B9 antibody CDR1 sequence SEQ ID NO:21 GSSFSPYAMG am63G12.1-5G8-18B9 antibody CDR2 sequence SEQ ID NO:22 ATSSGGFTLYADSVKG am63G12.1-5G8-18B9 antibody CDR3 sequence SEQ ID NO:23 YYREYDPEMY am63G12.1-5G8-18B9 antibody VHH sequence SEQ ID NO:24 EVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATSSGGFTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYTYYREYDPEMYWGQGTQVTVSS Aflibercept full sequence SEQ ID NO:25 SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLS PSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Aflibercept VEGF-A binding domain: SEQ ID NO: 26 SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK Aflibercept human IgG1 Fc region: SEQ ID NO: 27 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Ranibizumab Fab VH-CH1: SEQ ID NO: 28 EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL Ranibizumab Fab VH: SEQ ID NO: 29 EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSS Ranibizumab Fab CH1: SEQ ID NO: 30 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL Ranibizumab Fab VL-CL: SEQ ID NO: 31 DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ranibizumab Fab VL: SEQ ID NO: 32 DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK Ranibizumab Fab CL: SEQ ID NO: 33 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ranibizumab Fab: HCDR1 SEQ ID NO: 34 GYDFTHY Ranibizumab Fab: HCDR2 SEQ ID NO: 35 NTYTGE Ranibizumab Fab: HCDR3 SEQ ID NO: 36 YPYYYGTSHWYFDV Ranibizumab Fab: LCDR1 SEQ ID NO: 37 QDISNYLN Ranibizumab Fab: LCDR2 SEQ ID NO: 38 FTSSLHS Ranibizumab Fab: LCDR3 SEQ ID NO: 39 QQYSTVPWT IEX04-026 SEQ ID NO:40 SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYP SSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGGGGGSGGGGSGGGGSQLQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVAATTSGGSTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS IEX04-037 SEQ ID NO:41 SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATTSGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS IEX04-039 VH-CH1-Linker-VHH Sequence No. 42 EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATTSGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS IEX04-039 VL-CL-linker-VHH SEQ ID NO: 43 DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATTSGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS IEX04-041 SEQ ID NO:44 SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWE YPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATTSGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS IEX04-042 SEQ ID NO:45 SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPS SKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATTSGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS IEX04-046 SEQ ID NO:46 SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYP SSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATSEGGFTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS IEX04-056 SEQ ID NO:47 SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYP SSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHPCPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATSSGGFTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYTYYREYDPEMYWGQGTQVTVSS IEX04-067 VH-CH1-リンカー-VHH SEQ ID NO:48 EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLASSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATSEGGFTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYCYMHWRGSDPENYWGQGTQVTVSS IEX04-067 VL-CL-Linker-VHH SEQ ID NO: 49 DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATSEGGFTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYMHWRGSDPENYWGQGTQVTVSS IEX04-069 VH-CH1-linker-VHH SEQ ID NO:50 EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATSSGGFTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYTYYREYDPEMYWGQGTQVTVSS IEX04-069 VL-CL-Linker-VHH SEQ ID NO:51 DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSSFSPYAMGWYRQAPGKQRELVSATSSGGFTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCYTYYREYDPEMYWGQGTQVTVSS Positive control antibody, anti-VEGF-C (VEGFR3-trap molecule) OPT-302, SEQ ID NO: 52 YSMTPPTLNITEESHVIDTGDSLSISCRGQHPLEWAWPGAQEAPATGDKDSEDTGVVRDCEGTDARPYCKVLLLHEVHAQDTGSYVCYYKYIKARIEGTTAASSYVFVRDFEQPFINKPDTLLVNRKDAMWVPCLVSIPGLNVTLRSQSSVLWPDGQEVVWDDRRGMLVSTPLLHDALYLQCETTWGDQDFLSNPFLVHITGNELYDIQLLPRKSLELLVGEKLVLNCTVWAEFNSGVTFDWDYPGKQAERGKWVPERRSQQTHTELSSILTIHNVSQHDLGSYVCKANNGIQRFRESTEVIVHEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IBI304 molecular sequence SEQ ID NO:53 PFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTVVLSPSHGIELSVGE KLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGGGGGGGGGDKTHTCPLCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A single domain antibody (sdAb) polypeptide that specifically binds to human VEGFC, comprising a VHH domain having the following formula: comprising or consisting of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the VHH domain is (i) CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21, CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22, and CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; (ii) CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18, and CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; (iii) a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11; or (iv) CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1, CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3; The polypeptide comprising:

2. The VHH domain is (i) an amino acid sequence selected from SEQ ID NOs: 24, 20, 16, 12, 8, and 4, preferably the amino acid sequence of SEQ ID NO: 20, more preferably the amino acid sequence of SEQ ID NO: 24; (ii) an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of (i); (iii) an amino acid sequence having at least 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 amino acid changes (e.g., substitutions, deletions, and / or insertions, preferably substitutions, more preferably conservative substitutions) relative to the amino acid sequence of (i); or (iv) is humanized; The polypeptide of claim 1.

3. A protein comprising at least one single domain antibody polypeptide according to claim 1, said protein being a fusion protein or a chimeric polypeptide, preferably said protein being a VHH-Fc antibody.

4. 1. A bispecific binding protein comprising: (i) a first antigen-binding moiety that specifically binds to human VEGFC; (ii) a second antigen-binding moiety that specifically binds human VEGFA; and Including, wherein the first antigen-binding moiety comprises the single domain antibody polypeptide of claim 1; and The bispecific binding protein inhibits the binding of VEGFA to its VEGF receptor and inhibits the binding of VEGFC to its VEGF receptor. The bispecific binding protein.

5. 5. The bispecific binding protein of claim 4, wherein the first antigen binding moiety is linked to the second antigen binding moiety via a linker, preferably the linker comprises a G(GS)n or (GS)n amino acid sequence, where n=1, 2, 3, 4 or 5, preferably n=2, 3 or 4.

6. The second antigen-binding component may be an anti-VEGFA antibody (e.g., a single chain Fv antibody, a Fab antibody, a Fab′ antibody, a (Fab) ２ 5. The bispecific binding protein of claim 4, selected from the group consisting of antibodies, single domain antibodies and nanobodies, VEGF-A trap molecules, or Fc fusion proteins comprising a VEGFA binding domain.

7. the second antigen-binding component is an Fc fusion protein comprising a VEGF-A binding domain from a VEGFR1 and / or VEGFR2 receptor; Preferably, the VEGF-A binding domain comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; More preferably, the second antigen-binding moiety comprises the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

8. 8. The bispecific binding protein of claim 7, wherein the single domain antibody polypeptide that binds to VEGFC is linked to the C-terminus of the Fc fusion polypeptide.

9. The bispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain and the second polypeptide chain are the same and each comprise an amino acid sequence selected from SEQ ID NOs: 47, 40-41, and 44-46, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; 9. The bispecific binding protein of claim 8, wherein the first polypeptide chain and the second polypeptide chain are preferably the same and each comprise the amino acid sequence set forth in SEQ ID NO:

47.

10. the second antigen-binding component comprises an anti-VEGF-A Fab antibody consisting of VH-CH1 and VL-CL; Preferably, the VH comprises the amino acid sequences of HCDR1 to HCDR3 of SEQ ID NOs: 34 to 36, respectively, and the VL comprises the amino acid sequences of LCDR1 to LCDR3 of SEQ ID NOs: 37 to 39, respectively; More preferably, the VH comprises the amino acid sequence of SEQ ID NO: 29 and the VL comprises the amino acid sequence of SEQ ID NO: 32; More preferably, the Fab fragment comprises the amino acid sequences of SEQ ID NO:28 and SEQ ID NO:

31.

11. 11. The bispecific binding protein of claim 10, wherein the single domain antibody polypeptide is linked to the C-terminus of the VH-CH1 and / or VL-CL of the Fab antibody, preferably via a linker.

12. The bispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises the single domain antibody polypeptide fused to the C-terminus of the VH-CH1 polypeptide of the Fab antibody; and 12. The bispecific binding protein of claim 11 , wherein the second polypeptide chain comprises the single domain antibody polypeptide fused to the C-terminus of the VL-CL polypeptide of the Fab antibody.

13. The first polypeptide chain and the second polypeptide chain a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; - a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 51 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. selected from the group consisting of 13. The bispecific binding protein of claim 12, wherein preferably the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 48 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:

49.

14. A polynucleotide encoding the single domain antibody polypeptide of claim 1, a protein comprising said single domain antibody polypeptide, or a bispecific binding protein comprising a first antigen-binding moiety comprising said single domain antibody polypeptide and a second antigen-binding moiety that specifically binds to human VEGF A.

15. An expression vector comprising the polynucleotide of claim 14.

16. A host cell transfected with the vector of claim 15.

17. 17. A method for producing a single domain antibody polypeptide, a protein comprising said single domain antibody polypeptide, or a bispecific binding protein comprising said single domain antibody polypeptide, said method comprising culturing the host cell of claim 16 and recovering the single domain antibody polypeptide, said protein, or said bispecific binding protein produced from said host cell.

18. A pharmaceutical composition comprising the single domain antibody polypeptide of claim 1 or a protein comprising said single domain antibody polypeptide, and a pharmaceutically acceptable vector.

19. A pharmaceutical composition comprising the bispecific binding protein of claim 4 and a pharmaceutically acceptable vector.

20. 20. The pharmaceutical composition of claim 19 for use in treating an angiogenesis-related disease.

21. 21. The pharmaceutical composition for use according to claim 20, wherein the disease is a solid tumor, preferably the disease is melanoma.

22. 21. The pharmaceutical composition for use according to claim 20, wherein the disease is an ocular disease, preferably the disease is age-related macular degeneration, diabetic retinopathy, retinal vascular occlusion or corneal neovascularization.

23. 10. Use of a single domain antibody polypeptide according to claim 1, or a protein comprising said single domain antibody polypeptide, for preparing a drug for treating and / or preventing a disease in a subject and / or for preparing a diagnostic tool for diagnosing a disease, wherein said disease is preferably an angiogenesis-associated disease, such as a solid tumor or an eye disease.

24. Use of the bispecific binding protein of claim 4 for preparing a medicament for treating and / or preventing a disease in a subject and / or for preparing a diagnostic tool for diagnosing a disease, wherein the disease is preferably an angiogenesis-related disease, such as a solid tumor or an eye disease.

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