Tri-specific fusion proteins and uses thereof

Abstract

The present invention relates to multispecific fusion proteins, and more particularly to tri-specific fusion proteins that specifically bind to VEGF A, VEGF C and Ang2. The invention also relates to polynucleotides encoding the proteins, expression vectors and host cells, pharmaceutical compositions thereof, and methods and uses for treating neovascularization-related diseases.

Description

Tri-specific fusion protein and its use Technical Field

[0001] The present invention relates to the field of multispecific fusion proteins, and more specifically to trispecific fusion proteins that specifically bind to VEGF A, VEGF C, and Ang2. The present invention also relates to polynucleotides encoding the proteins, expression vectors, and host cells, as well as pharmaceutical compositions thereof, and methods and uses for treating neovascularization-related diseases. Background Art

[0002] Vascular endothelial growth factor (VEGF) is a major regulator of vascular development and blood and lymphatic vessel function in adults during health and disease. The VEGF family is currently known to consist of five structurally related factors: VEGFA, VEGFB, VEGFC, VEGFD, and placental growth factor (PlGF). VEGF family members primarily exist as homodimeric polypeptides that bind to their respective VEGF receptors to induce signaling and trigger corresponding biological effects.

[0003] Vascular endothelial growth factor A (VEGF-A) has multiple isoforms, including VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206. These isoforms are generated by alternative splicing, with VEGF165 being the predominant isoform. VEGF-A participates in the angiogenesis process by interacting with the tyrosine kinase receptors VEGFR-1 and VEGFR-2. Blocking the VEGF-A pathway has been proposed to improve diseases associated with neovascularization. Such VEGF-A blockers / antagonists include neutralizing antibodies targeting VEGF-A, and soluble decoy receptors and Trap molecules that prevent VEGF-A from binding to its normal receptors. Aflibercept (also known as Aflibercept, VEGFA-Trap, trade name Elyea) is a recombinant fusion protein formed by the ligand-binding extracellular domains of human VEGF receptors 1 and 2 fused to the Fc region of human IgG1. It has been approved for the treatment of neovascularization-related 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) shares approximately 30% sequence homology with VEGF-165. VEGF-C exerts its effects by binding to the tyrosine kinase receptors VEGFR2 and VEGFR3, participating in the neovascularization process and lymphangiogenesis. VEGFR2 is expressed on vascular endothelial cells and also binds to VEGF-A. VEGFR3 is expressed on vascular endothelial cells and lymphatic cells but does not bind to VEGF-A. In a variety of metastatic tumor models, VEGF-C / VEGFR3 signaling pathway blockers have been used to inhibit tumor lymphangiogenesis and metastasis. In addition, VEGF-C Trap molecules, such as OPT-032 (a VEGFC / D inhibitor constructed from the ligand-binding extracellular domain of VEGFR3) developed by Opthea Ltd., have been proposed for the treatment of neovascularization-related retinal diseases.

[0005] In addition to the VEGF family, human angiopoietin (ANG) is also thought to be involved in vascular development and postnatal angiogenesis. Angiopoietin-2 (also known as Angiopoietin-2, ANGPT2, or Ang2) is a member of the angiopoietin family. This protein is a secreted glycoprotein composed of three parts: a secretory signal peptide, an N-terminal coiled-coil domain (CCD), and a C-terminal fibrinogen-like domain (FLD). The FLD domain of the angiopoietin protein is highly conserved and is responsible for binding to the Tie2 receptor. Preclinical studies using acute ocular inflammation models and choroidal neovascularization models (CNV models) have shown that ANG-2 and VEGF are synergistically involved in pathological neovascularization and vascular permeability. Therefore, in addition to anti-VEGF therapy, the Tie2 / angiopoietin pathway has also been proposed as a therapeutic target for vascular-related retinopathy. For example, the combination of the anti-Ang2 antibody nesvacumab (Regeneron, Tarrytown, NY, USA) and aflibercept (anti-VEGF-A) is being used to treat neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME). In addition, the anti-VEGF-A / Ang2 bispecific antibody faricimab (co-developed by Roche, Basel, Switzerland and Genentech, South San Francisco, CA, USA) has also been proposed for the treatment of nAMD and DME.

[0006] Abnormal vascular permeability and angiogenesis are known to be involved in a variety of disease processes, including cancer, autoimmunity, and retinopathy. The development of existing drugs that block angiogenic signaling pathways has brought therapeutic benefits to patients with some diseases. However, the treatment of such diseases still faces challenges in terms of drug accessibility, efficacy, patient compliance, and treatment burden. Therefore, there is an urgent need for new drug molecules that can effectively target and ameliorate abnormal angiogenesis to meet these therapeutic needs.

[0007] Summary of the Invention

[0008] To address these needs, the present inventors designed and developed a trispecific fusion protein molecule capable of specifically targeting and binding VEGF-A, VEGF-C, and ANG2. This trispecific molecule exhibits strong binding and blocking effects on each target antigen and demonstrates excellent anti-angiogenesis activity in animal models. Based on this foundation, the present inventors developed the present invention.

[0009] Therefore, in a first aspect, the present invention provides a tri-specific fusion protein comprising:

[0010] a polypeptide chain comprising (i) a VEGF-C binding domain; (ii) an ANG2 binding domain and (iii) a VEGF-A binding domain,

[0011] wherein the VEGF-C binding domain and the ANG2 binding domain comprise or consist of a VHH domain that specifically binds to VEGF-C or ANG2, respectively, and

[0012] wherein the VEGF-A binding domain comprises or consists of a ligand capture domain that specifically binds to VEGF-A, wherein the ligand capture domain comprises an extracellular domain from VEGFR,

[0013] Wherein, preferably, the polypeptide further comprises (iv) a dimerization domain, in particular a dimerization domain selected from the hinge region, CH3 region and Fc region.

[0014] In some embodiments, the polypeptide comprises a hinge region. In other embodiments, the polypeptide comprises a CH3 region, and preferably the polypeptide comprises a CH3 region with a hinge region at the N-terminus. In further embodiments, the polypeptide comprises an Fc region, and preferably the polypeptide comprises an Fc region with a hinge region at the N-terminus. In some embodiments, the hinge region comprises a CPPC or CPPCPPC amino acid sequence, and preferably the hinge region comprises an amino acid sequence selected from SEQ ID NOs: 12-19 and 45-46, more preferably an amino acid sequence of SEQ ID NO: 12 or 13. In some embodiments, the Fc region is an Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the CH3 region is a CH3 region of human IgG1, IgG2, IgG3, or IgG4.

[0015] In yet another aspect, the present invention provides polynucleotides, vectors, host cells encoding the tri-specific fusion proteins of the present invention; and pharmaceutical compositions, pharmaceutical combinations, and kits comprising the molecules of the present invention.

[0016] In another aspect, the present invention provides the use of the trispecific fusion protein of the present invention for treatment or as a drug, or the use of the trispecific fusion protein of the present invention for preventing and / or treating diseases or for preparing a drug for preventing and / or treating diseases, such as neovascularization-related diseases, such as tumors and eye diseases.

[0017] In yet another aspect, the present invention also provides diagnostic uses of the trispecific fusion proteins of the present invention.

[0018] In yet another aspect, the present invention also provides a multi-specific fusion protein with a unique configuration, wherein the fusion protein comprises:

[0019] A polypeptide chain comprising a first VHH domain that specifically binds to a first antigen, a second VHH domain that specifically binds to a second antigen, and a ligand capture domain that specifically binds to a third antigen, wherein the polypeptide further comprises a dimerization domain selected from a hinge region, a CH3 region, or an Fc region. In some preferred embodiments, the dimerization domain is a hinge region, and preferably, the hinge region is connected to the C-terminus of the ligand capture domain.

[0020] In yet another aspect, the present invention also provides a hinge region comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 12-19 and 45-46, and preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 12 or 13. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A shows the SDS-PAGE purity of the anti-VEGF A / VEGF C / Ang2 multispecific fusion protein after one-step purification with Protein A. NR indicates protein samples treated in non-reducing loading buffer; R indicates protein samples treated in reducing loading buffer.

[0022] FIG1B shows the SEC-HPLC purity of the anti-VEGF A / VEGF C / Ang2 multispecific fusion protein after one-step purification with Protein A.

[0023] FIG2 shows an experiment in which anti-VEGF A / VEGF C / Ang2 multispecific fusion protein blocks VEGF A-induced HEK 293KDR reporter molecule activation.

[0024] FIG3 shows an experiment in which anti-VEGF A / VEGF C / Ang2 multispecific fusion protein blocks VEGF C-induced HEK 293KDR reporter molecule activation.

[0025] FIG4 shows that anti-VEGF A / VEGF C / Ang2 multispecific fusion protein blocks VEGF C-induced Baf3-FLT4 proliferation experiment.

[0026] FIG5 shows an ELISA blocking experiment in which the anti-VEGF A / VEGF C / Ang2 multispecific fusion protein blocked the binding of Ang2 to Tie2.

[0027] FIG6 shows a blocking experiment in which an anti-VEGF A / VEGF C / Ang2 multispecific fusion protein blocks the binding of Ang2 to Tie2-expressing cells.

[0028] FIG7 shows that the anti-VEGF A / VEGF C / Ang2 multispecific fusion protein inhibits the Ang2-induced Tie2 phosphorylation experiment.

[0029] FIG8 shows that the anti-VEGF A / VEGF C / Ang2 multi-specific fusion protein inhibits the HUVEC proliferation induced by VEGF A+C.

[0030] FIG9 shows the changes in A375 tumor volume over time under different administration methods in the A375 subcutaneous tumor model neovascularization experiment.

[0031] FIG10 shows fundus angiography images showing that anti-VEGF A / VEGF C / Ang2 multispecific fusion protein inhibits laser-induced choroidal neovascularization.

[0032] FIG. 11 shows the improvement rate (%) of leakage area measured by FFA on day 5 of administration in the laser-induced CNV model.

[0033] FIG12 shows that anti-VEGF A / VEGF C / Ang2 multispecific fusion protein inhibits DL-AAA-induced retinal neovascularization (RNV).

[0034] FIG13 shows that anti-VEGF A / VEGF C / Ang2 multispecific fusion protein inhibits vascular leakage.

[0035] Figure 14 shows an exemplary configuration of an anti-VEGF A / VEGF C / Ang2 multispecific fusion protein according to the present invention, wherein VHH1 and VHH2 independently represent a VEGFC binding domain or an ANG2 binding domain, respectively, and the ligand capture domain represents a VEGA ligand capture domain from a VEGF receptor.

[0036] Detailed Description of the Invention

[0037] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0038] definition

[0039] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0040] As used herein, the term "comprising" or "including" is intended to include the stated elements, integers, or steps, but does not exclude any other elements, integers, or steps. For example, when reference is made to an antigen-binding domain "comprising" a particular sequence, it is intended to encompass an antigen-binding domain consisting of that particular sequence.

[0041] As used herein, the term "fusion protein" refers to a protein comprising at least one polypeptide chain formed by the fusion of at least two heterologous polypeptide sequences, optionally via a connecting peptide. Fusion proteins can be produced by recombinant expression and can be in the form of monomers or polymers (e.g., dimers) comprising one or more of the polypeptide chains. In one embodiment, the fusion protein of the present invention is a dimeric protein formed by two identical polypeptide chains.

[0042] The term "heterologous" when referring to a polypeptide chain comprising a fusion protein means that the polypeptide chain comprises two or more subsequences that do not occur in the same protein or polypeptide in nature.

[0043] The term "recombinant" as used herein refers to the production of a polypeptide or protein by a biological host. The host can be selected from, but is not limited to, mammalian expression systems, insect cell expression systems, yeast expression systems, and bacterial expression systems. In one embodiment, the polypeptide / protein of the present invention is a recombinant polypeptide / protein expressed in a prokaryotic (e.g., E. coli) or eukaryotic host cell (e.g., mammalian host cell).

[0044] As used herein, the term "monospecific" refers to a polypeptide / protein molecule that has one or more antigen binding sites, each of which binds to the same epitope of the same antigen.

[0045] As used herein, the term "multispecific" refers to a polypeptide / protein molecule that has 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 present invention provides a trispecific fusion protein comprising at least one polypeptide chain comprising antigen-binding sites with binding specificities for three different antigens: VEGF-A, VEGF-C, and ANG2.

[0046] As used herein, the terms "antigen binding site" and "antigen binding domain" are used interchangeably to refer to the region of a molecule that actually binds to the target antigen. Examples of antigen binding sites include, but are not limited to, the variable domains of antibodies and the extracellular ligand-binding domains of receptors. Preferably, the VEGFC antigen-binding site used in the trispecific fusion proteins of the present invention is provided by the variable domain of an anti-VEGFC heavy chain antibody (i.e., "VHH"); the ANG2 antigen-binding site used in the trispecific fusion proteins of the present invention is provided by the variable domain of an anti-ANG2 heavy chain antibody (i.e., "VHH"); and the VEGFA antigen-binding site used in the trispecific binding proteins of the present invention is provided by a VEGFA ligand-trap domain (i.e., a VEGF receptor extracellular domain polypeptide fragment that specifically binds to VEGFA) or by an artificial ligand "mini-Trap" domain.

[0047] The term "VHH domain" is used herein to refer to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain, also referred to as a single variable domain fragment or a nanobody fragment. The VHH domain is different from the conventional VH domain of a four-chain immunoglobulin in that it does not need to be paired with a light chain variable domain to form an antigen binding site. Such a VHH domain can be derived from a heavy chain antibody produced in a Camelidae species (e.g., camel, alpaca, dromedary, llama, and guanaco). Other species besides Camelidae can also produce heavy chain antibodies that naturally lack light chains, and the VHH domains contained in such heavy chain antibodies are also within the scope of the present invention. The variable domain of a heavy chain antibody (i.e., VHH domain) is the same as the heavy chain variable region (VH) in a conventional four-chain IgG antibody. It is the domain responsible for binding the antibody to the antigen and is structurally composed of three complementarity determining regions (CDRs) and four conserved framework regions (FRs). They are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some cases, for therapeutic applications of VHH domains, it is desirable to reduce their immunogenicity. Therefore, 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).

[0048] As used herein, the term "ligand trap" molecule refers to a fusion protein comprising the extracellular domain of a receptor protein that interacts with the ligand, fused to the Fc region of an immunoglobulin. Currently, a variety of trap molecules have been developed that "capture" VEGF ligands, including VEGFA and VEGFC, from the VEGF receptor. These trap molecules can be used to bind to the corresponding ligand in the extracellular environment and reduce its concentration.

[0049] As used herein, the terms "ligand capture domain" and "mini-Trap" domain are used interchangeably to refer to polypeptide fragments that, unlike Trap molecules, do not contain an immunoglobulin Fc region and that consist primarily of a ligand-interacting extracellular domain from (at least one) receptor protein. It should be understood that the receptor extracellular domain that interacts with the ligand contained in the ligand capture domain and the mini-Trap domain can be in the form of a native sequence or a modified sequence, such as a native extracellular domain sequence from one receptor, or a hybrid extracellular domain sequence composed of extracellular domains from different receptors, or a variant sequence in which amino acid mutations are introduced into the native extracellular domain or hybrid extracellular binding domain. In one embodiment, the VEGFA binding domain of the present invention comprises or consists of a mini-trap domain that "captures" VEGFA. The mini-trap domain is capable of competing with the native VEGFA cell receptor for binding to VEGFA, thereby inhibiting VEGFA-induced signaling. In one embodiment, the VEGFA-binding mini-trap domain has a length of no more than 350 amino acids, preferably no more than 250 amino acids. In one embodiment, the VEGFA-binding mini-trap domain is formed by fusing the extracellular ligand-binding domains from VEGF receptors 1 and 2, and preferably has amino acids of human origin to minimize the immunogenicity of the molecule in humans.

[0050] As used herein, the term "connecting peptide", "flexible connecting peptide" or "linker" refers to a short amino acid sequence of no more than 50 amino acid residues consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T), used alone or in combination, and / or hinge region sequences from the natural hinge region of an immunoglobulin or its truncated and / or modified form. In one embodiment, the connecting peptide has a length of 5-50 amino acids, for example, 10, 15, 20, 25, or 30 amino acids. In one embodiment, the connecting peptide comprises the amino acid sequence (G4S)n (SEQ ID NO: 55), wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6, or 7. In one embodiment, the connecting peptide comprises the amino acid sequence TS (G4S) n(SEQ ID NO: 56), wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In one embodiment, the connecting peptide comprises the amino acid sequence G(G4S)n (SEQ ID NO: 57), wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In one embodiment, the connecting peptide comprises the amino acid sequence (GGGGG)n (SEQ ID NO: 58) or (GRPGS)n (SEQ ID NO: 59), wherein n is an integer of 2, 3, 4, 5, 6 or 7, for example, n is an integer of 2 or 3. In yet another embodiment, the connecting peptide comprises a hinge region, for example, a hinge region having the amino acid sequence of CPPC (SEQ ID NO: 60) or CPPCPPC (SEQ ID NO: 61). In some further embodiments, the connecting peptide further comprises a short linker of 5-15, for example 10 amino acids in length, located at the C-terminus of the hinge region, in particular a short linker composed of glycine (G) and / or serine (S) and / or threonine residues (T), thereby conferring the ability of the polypeptide comprising the connecting peptide to pair and dimerize in the hinge region, and conferring the segment flexibility required for the normal function of the antigen-binding domains at both ends of the connecting peptide.

[0051] Herein, the term "hinge region" refers to a native hinge region from an immunoglobulin IgG, or a truncated form thereof that at least retains the core hinge segment, or a variant sequence thereof, provided that the hinge region comprises at least 2, for example 2-4 or preferably 3, cysteine ​​residues capable of forming interchain disulfide bonds. These cysteine ​​residues in the hinge region may be residues originally present in the native hinge region, or residues introduced by amino acid substitution and / or addition. The hinge region of a natural immunoglobulin heavy chain is well known in the art and is composed of a segment between the Fab and Fc portions. According to crystallographic data, the natural IgG hinge region can be divided into an upper hinge region, an intermediate (core) hinge region, and a lower hinge region. The upper hinge region is the segment from the end of the Fab to the first inter-heavy chain disulfide bridge; the core hinge region is the polyproline core segment from the first inter-heavy chain disulfide bridge to the last inter-heavy chain disulfide bridge; and the lower hinge region is the segment from the last inter-heavy chain disulfide bridge to the starting residue of Fc. According to the EU numbering system, the hinge region of the human heavy chain IgG1 extends from Glu216 to Pro230 or to Gly236 of the heavy chain, with the polyproline segment from Cys226 to Cys229 forming the core segment, which divides the entire hinge region into the upper hinge region and the lower hinge region. For a review of immunoglobulin hinge regions, see General Characteristics of Immunoglobulin Molecules, ROALD NEZLIN, in The Immunoglobulins, 1998, https: / / doi.org / 10.1016 / B978-012517970-6 / 50001-1, which is incorporated herein by reference. Hinge region sequences of human immunoglobulin IgG isotypes IgG1, IgG2, IgG3, and IgG4 are published at https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. Those skilled in the art can easily determine the hinge region sequence corresponding to IgG1 residues 216-230 or residues 216-236 in other immunoglobulins by comparison with the IgG1 sequence. In this article, the term "hinge region" does not include immunoglobulin Fc region sequences or variant sequences thereof. In some embodiments according to the present invention, the hinge region has a length of 4 to 25 amino acids, such as 8-22, especially 10-20 amino acids in length, such as 11, 12, 13, 14, 15, 16, 17 or 18 amino acids in length. In some preferred embodiments, the hinge region for the fusion protein polypeptide chain of the present invention comprises CPPC or preferably CPPCPPC amino acid sequence, whereby the two polypeptide chains can pair through the hinge region and form a stable dimer.In some further preferred embodiments, the hinge region comprises an amino acid sequence selected from SEQ ID NOs: 12-19 and 45-46.

[0052] As used herein, the terms "Fc region" and "Fc domain" are used interchangeably to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc region" comprises two or three constant domains, i.e., a CH2 domain, a CH3 domain, and an optional CH4 domain. The Fc region that can be used in the fusion protein of the present invention includes, but is not limited to, an Fc region of IgG1, IgG2, IgG3, or IgG4 having a native sequence or variant sequence. Unless otherwise indicated herein, the amino acid residues in the hinge region and Fc region and each constant region of the heavy chain are numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the term "Fc region" or "Fc domain" does not include the heavy chain variable region VH and light chain variable region VL, as well as the heavy chain constant region CH1 and light chain constant region CL of an immunoglobulin, but may or may not include all or part of the hinge region. In the case where the Fc region does not have a natural N-terminal hinge region sequence; in some embodiments of the present invention, the Fc region may be artificially connected to a hinge region as defined herein at its N-terminus, for example, a hinge region comprising an amino acid sequence selected from SEQ ID NOs: 12-19 and 45-46.

[0053] In this article, the term "native sequence Fc region" encompasses naturally occurring various immunoglobulin Fc region sequences, such as various Ig subtypes and the Fc region sequences of their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes:from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In some embodiments, human IgG heavy chain Fc region comprises an amino acid sequence extending from Cys226 or from Pro230 to the heavy chain carboxyl terminus. However, the C-terminal terminal lysine (Lys447) in the Fc region may be present or absent. In further embodiments, human IgG heavy chain Fc region carries a hinge sequence or a partial hinge sequence of a natural immunoglobulin at the N-terminus, such as, according to EU numbering, a sequence from E216 to T225 or a sequence from D221 to T225. As used herein, the term "variant sequence Fc region" refers to an Fc region polypeptide comprising modifications relative to a native sequence Fc region polypeptide. The modifications may be additions, deletions, or substitutions of amino acid residues. Substitutions may include naturally occurring amino acids and non-naturally occurring amino acids. The purpose of the modifications may be to alter the binding of the Fc region to its receptor and the effector functions thereby elicited.

[0054] As used herein, the terms "CH3 region" and "CH3 domain" are used interchangeably to define the CH3 constant region of an immunoglobulin heavy chain. The term encompasses both native sequence CH3 regions and variant CH3 regions. Fc regions useful in the fusion proteins of the present invention include, but are not limited to, CH3 regions of IgG1, IgG2, IgG3, or IgG4 having native or variant sequences.

[0055] As used herein, the term "native sequence CH3 region" encompasses naturally occurring sequences of various immunoglobulin Fc regions, such as the CH3 region sequences of various Ig subclasses and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520.). In some embodiments, the human IgG heavy chain CH3 region comprises an amino acid sequence extending from Gly341 to the carboxyl terminus of the heavy chain, wherein the C-terminal lysine (Lys447) of the CH3 region may or may not be present. As used herein, the term "variant sequence CH3 region" refers to a CH3 region polypeptide comprising modifications relative to a native sequence CH3 region polypeptide. The modifications may be additions, deletions, or substitutions of amino acid residues. Substitutions may include naturally occurring amino acids and non-naturally occurring amino acids. The purpose of the modifications may be to alter the binding of the CH3 region to its receptor and the effector functions thereby induced.

[0056] As used herein, the term "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" refers to a region in an antibody variable domain (VH / VL or VHH) that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. CDRs are numbered sequentially from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. CDRs located within the heavy chain antibody variable region (VHH domain) are sometimes also referred to as HCDR1, HCDR2, and HCDR3. In a given heavy chain antibody variable region amino acid sequence, its CDR sequence can be determined using a variety of schemes 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 antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department 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 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 Components", Journal of Molecular Biology, 406, 228-256 (2011)).

[0057] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0058] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0059] In addition, CDRs can also be identified based on having the same Kabat numbering position as a reference CDR sequence. In the present invention, unless otherwise indicated, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0060] As used herein, an "isolated" protein or polypeptide (e.g., a fusion protein of the invention) is one that has been separated from the components of its natural environment. In some embodiments, the protein or polypeptide is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0061] As used herein, the term "VEGFA" or "VEGF-A" refers to vascular endothelial growth factor A (e.g., human VEGFA protein under 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 binding domain in a molecule that specifically binds to human VEGFA.

[0062] As used herein, the term "VEGFC" or "VEGF-C" refers to vascular endothelial growth factor C (e.g., human VEGFC protein under accession number UniProt P49767). VEGFC binds to the receptors VEGFR2 (also known as KDR) and VEGFR3 (also known as FLT4). As used herein, "antigen binding specificity for VEGFC" refers to a binding site or domain in a molecule that specifically binds to human VEGFC.

[0063] As used herein, the term "ANG2" (also referred to as ANGPT2 or ANGP2) refers to angiopoietin 2 (e.g., the human ANG2 protein under accession number UniProt O15123). ANG2 binds to the receptor Tie2 (also known as TEK), inducing tyrosine phosphorylation of TEK / TIE2. ANG2 can synergize with VEGF to promote endothelial cell migration and proliferation. As used herein, "antigen binding specificity for ANG2" refers to a binding site or domain in a molecule that specifically binds to human ANG2.

[0064] As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0065] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the 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 described herein.

[0066] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. Such variants may comprise conservative modifications.

[0067] For polypeptide sequences, "conservative modifications" include replacements, 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 a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative replacements: 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 modification" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody or binding protein.

[0068] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and the progeny derived therefrom. Host cells are any type of cell system that can be used to produce the tri-specific fusion proteins of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0069] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other 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., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which the recombinant polynucleotide is incorporated.

[0070] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., 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, the individual is a human.

[0071] The term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the present invention are used to delay the development of the disease or to slow the progression of the disease.

[0072] The term "anti-tumor effect" or "tumor inhibition effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival. The terms "tumor" and "cancer" are used interchangeably herein to encompass various solid tumors.

[0073] As used herein, the term "neovascularization-related disease" or "neovascularization-related disease" refers to a disease, disorder, and / or condition in which the onset, development, and / or progression of the disease involve neovascularization (including angiogenesis, lymphangiogenesis, and / or both). Such diseases, disorders, or conditions would benefit from blocking the biological activity of VEGF-C, VEGF-A, ANG2, or any two or three thereof.

[0074] The fusion protein of the present invention

[0075] A variety of IgG and IgG-like antibodies targeting VEGF and angiopoietin signaling pathways have been proposed, including anti-VEGF-A, anti-VEGF-C, and anti-ANG2 antibodies, as well as bispecific antibodies against VEGF and Ang2. However, there have been no reports of trispecific molecules that simultaneously block VEGF A, VEGF C, and Ang2.

[0076] The inventors have constructed a trispecific fusion protein molecule with high binding affinity and specificity for three target antigens simultaneously. They have demonstrated that targeted blockade of the three major signaling pathways involved in abnormal angiogenesis—the VEGF-A, VEGF-C, and Angiopoietin 2 pathways—through a single molecule can help curb the progression of neovascularization-related diseases, thereby providing greater clinical benefits to patients. Furthermore, compared to combination therapy requiring multiple injections, a single administration of the trispecific molecule can simultaneously achieve anti-VEGF A, anti-VEGF C, and anti-Ang2 effects, advantageously reducing dosing frequency. Furthermore, the strong blocking activity of the trispecific molecule against the three target antigens also helps extend the dosing cycle.

[0077] Furthermore, in some preferred embodiments, the trispecific molecules of the present invention exhibit good drugability and a relatively low molecular weight through their specially designed molecular form, which is advantageous for production and therapeutic administration. In particular, in the treatment of ocular diseases, drug molecules are often administered intravitreally as injectable solutions. At the same mass concentration, molecules with a smaller molecular weight can have a relatively higher molar concentration, thereby reducing dosing frequency, improving patient compliance, and reducing the treatment burden.

[0078] Thus, in one aspect, the present invention provides a trispecific fusion protein comprising (i) a VEGF-A binding domain, (ii) a VEGF-C binding domain, and (iii) an ANG2 binding domain fused into a single polypeptide chain, wherein: the VEGF-C binding domain and the ANG2 binding domain comprise or consist of a VHH domain; and the VEGF-A binding domain comprises, consists of, or consists essentially of an extracellular binding domain from a VEGF receptor that specifically binds to VEGFA (also referred to herein as a VEGFA ligand capture domain or mini-Trap domain). In some preferred embodiments, the polypeptide chain further comprises (iv) a dimerization domain, particularly a dimerization domain selected from a hinge region, a CH3 region, or an Fc region. In some embodiments of the fusion proteins of the present invention, the dimerization domain is a hinge region of 10-20 amino acids in length. Preferably, the hinge region comprises the core hinge region sequence CPPC (SEQ ID NO: 60) from immunoglobulin IgG, or its modified sequence CPPCPPC (SEQ ID NO: 61), such that the two polypeptide chains can pair through the hinge region and form a stable dimeric fusion protein. In other embodiments, as an alternative to the hinge region, the polypeptide chain comprises an Fc region or a CH3 region, or in other embodiments, the polypeptide chain comprises an Fc region or a CH3 region with a hinge region at the N-terminus. In some embodiments, the hinge region, CH3 region, or Fc region is present at the C-terminus of the polypeptide chain; in other embodiments, the hinge region, CH3 region, or Fc region is located between the two binding domains of the polypeptide chain. For example, in some embodiments, the VEGF-A binding domain is connected to the VEGF-C binding domain or the ANG2 binding domain, or both, at the C-terminus via the hinge region, the CH3 region, or the Fc region. In some preferred embodiments, the polypeptide chain of the fusion protein of the present invention does not contain an immunoglobulin Fc region or a modified form thereof. In some further preferred embodiments, the fusion protein of the present invention is formed by connecting a connecting peptide of 5-30 amino acids in length and a hinge region to connect the binding domains of (i) to (iii).

[0079] In yet another aspect, the present invention also provides a multi-specific fusion protein with a unique configuration, wherein the fusion protein comprises:

[0080] A polypeptide chain comprising a first VHH domain that specifically binds to a first antigen, a second VHH domain that specifically binds to a second antigen, and a ligand capture domain that specifically binds to a third antigen, wherein the polypeptide further comprises a dimerization domain selected from a hinge region, a CH3 region, or an Fc region. Preferably, the dimerization domain is a hinge region, and preferably, the hinge region is connected to the C-terminus of the ligand capture domain.

[0081] The hinge region used in the multi-specific fusion protein can be any hinge region described herein. In some preferred aspects, the hinge region comprises or consists of an amino acid sequence selected from SEQ ID NOs: 12-19 and 45-46, more preferably comprises or consists of an amino acid sequence of SEQ ID NO: 12 or 13.

[0082] In addition to the hinge region, CH3 region or Fc region, in some aspects, the multi-specific fusion protein of the present invention also preferably comprises one or two additional connecting peptides to ensure that each domain contained in the polypeptide chain can perform its normal function. The sequence of the connecting peptide is not particularly limited, for example, the connecting peptide can be any connecting peptide sequence described herein. In some embodiments, the connecting peptide can be 5-12 amino acids in length, preferably comprising the amino acid sequence (GRPGS)n (SEQ ID NO: 59) or (GGGGG)n (SEQ ID NO: 58), wherein n=2 or 3.

[0083] Therefore, in some embodiments, the present invention also provides such a multi-specific fusion protein, which comprises a polypeptide chain having the following structure from N-terminus to C-terminus:

[0084] (i) a first VHH domain, a first connecting peptide, a ligand capture domain, a hinge region, a second connecting peptide, and a second VHH domain,

[0085] (ii) ligand capture domain, hinge region, first connecting peptide, first VHH domain, second connecting peptide, second VHH domain, or (iii) first VHH domain, first connecting peptide, second VHH domain, second connecting peptide, ligand capture domain, hinge region.

[0086] In some embodiments, the present invention also provides such a multi-specific fusion protein, wherein the fusion protein comprises a polypeptide chain having the following structure from N-terminus to C-terminus:

[0087] (i) a first VHH domain, a first connecting peptide, a ligand capture domain, an Fc region or a CH3 region, a second connecting peptide, and a second VHH domain,

[0088] (ii) a ligand capture domain, an Fc region or a CH3 region, a first connecting peptide, a first VHH domain, a second connecting peptide, a second VHH domain, or

[0089] (iii) a first VHH domain, a first connecting peptide, a second VHH domain, a second connecting peptide, a ligand capture domain, an Fc region or a CH3 region,

[0090] Optionally, the Fc region or CH3 region has a hinge region at the N-terminus.

[0091] It will be understood that in the aforementioned embodiment, the first, second and third antigens may be different from each other, and thus the multi-specific fusion protein is a tri-specific fusion protein; but in some cases, the first antigen and the second antigen may be the same, and thus the multi-specific fusion protein is a bi-specific fusion protein.

[0092] The first and second VHH domains used in the multi-specific fusion protein of the present invention can be the same or different and can be directed against the same or different target antigens. Those skilled in the art can select the first and second VHH domains to be used in combination according to specific application requirements.

[0093] The ligand-capture domain used in the multi-specific fusion proteins of the present invention can be a ligand-interaction domain from a natural or artificial receptor that binds the third antigen. For example, in some preferred aspects, the ligand-capture domain is the VEGF-A binding domain from the extracellular region of VEGFR1 / 2. In some more preferred aspects, the multi-specific fusion protein is a tri-specific fusion protein against VEGFA / ANG2 / VEGFC according to the present invention.

[0094] The following describes in detail the components comprising the fusion protein and an exemplary fusion protein molecule, using the anti-VEGFA / ANG2 / VEGFC trispecific fusion protein of the present invention as an example. Those skilled in the art will appreciate that, unless the context clearly indicates otherwise, any combination of the technical features of these components is contemplated by the present invention. Furthermore, those skilled in the art will appreciate that, unless the context clearly indicates otherwise, the fusion proteins of the present invention may include any such combination of features.

[0095] VEGF-A binding domain

[0096] In the present invention, the VEGF-A binding domain used in the fusion protein of the present invention is provided by a ligand capture domain or mini-Trap domain from a VEGF receptor. In some embodiments, the capture domain or mini-Trap domain comprises a natural sequence or artificial sequence extracellular domain from a VEGF receptor (particularly, VEGFR1 and / or VEGFR2) that specifically binds to VEGFA.

[0097] VEGF receptor (VEGFR) is a tyrosine kinase receptor with an extracellular region consisting of 7 immunoglobulin (Ig)-like domains. In this article, the Ig-like domains are numbered sequentially from the N-terminal to the C-terminal of the VEGFR protein. For example, human VEGFR-1 comprises seven Ig-like domains numbered 1, 2, 3, 4, 5, 6 and 7, with Ig-like domain 1 at the N-terminal of the extracellular domain and Ig-like domain 7 at the C-terminal of the extracellular domain. VEGFR interacts with ligands through the Ig-like domains. VEGFR-1 (Flt-1) binds to VEGF-A, VEGF-B and PIGF, and can function as a decoy receptor for VEGF or the regulator of VEGFR-2. VEGFR-2 (KDR / Flk-1) binds to all VEGF isoforms and is the main mediator of VEGF-induced angiogenesis signaling. VEGFR-3 (Flt-4) binds to VEGF-C and VEGF-D, but not to VEGF-A, and acts as a mediator of lymphangiogenesis. It has been demonstrated that partial Ig-like domains from the extracellular regions of the receptors VEGFR1 or VEGFR2, or their hybrid forms, are sufficient to capture VEGFA ligands and compete with native VEGFRs for binding to VEGFA. For a more detailed description of such Ig-like domains, see, for example, U.S. Patent No. 7,531,173, Yu, D. et al. (2012). Mol. Ther. 20(3):938-947, and Holash, J. et al. (2002). PNAS. 99(17):11393-11398, which are hereby incorporated by reference in their entirety.

[0098] In one embodiment, therefore, according to the invention, the VEGF-A binding domain comprises at least one Ig-like domain, for example at least two or three Ig-like domains, from VEGFR1 and / or VEGFR2. The VEGFRs that can be used to provide the VEGFA binding domain of the invention can be from mammals, such as humans, baboons, chimpanzees, mice or rats, preferably from humans. In one embodiment, the VEGF-A binding domain according to the present invention comprises Ig-like domains 1-3 of VEGFR-1 (e.g., human VEGFR-1), Ig-like domains 2-3 of VEGFR-1 (e.g., human VEGFR-1), Ig-like domains 1-3 of VEGFR-2 (e.g., human VEGFR-2), or Ig-like domain 2 of VEGFR-1 (e.g., human VEGFR-1) and Ig-like domain 3 of VEGFR-2 (e.g., human VEGFR-2), or Ig-like domain 2 of VEGFR-1 (e.g., human VEGFR-1) and Ig-like domain 3-4 of VEGFR-2 (e.g., human VEGFR-2). In a preferred embodiment, the VEGF-A binding domain according to the present invention comprises Ig-like domain 2 of VEGFR-1 (e.g., human VEGFR-1) and Ig-like domain 3 of VEGFR-2 (e.g., human VEGFR-2). In addition, the present invention also contemplates VEGF-A binding domains having mutations in the above-mentioned native sequence Ig-like domains from VEGFR, including chemically modified forms, or variant forms having 1-10 amino acid substitutions, deletions, or additions.

[0099] In a preferred embodiment, the VEGFA binding domain comprises the Ig-like domain 3 of VEGFR2 (e.g., human VEGFR2) genetically fused to the C-terminus of the Ig-like domain 2 of VEGFR1 (e.g., human VEGFR1). Preferably, the VEGFA binding domain comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity thereto, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 9. Most preferably, the VEGF-A binding domain comprises the amino acid sequence of SEQ ID NO: 9, or consists of the amino acid sequence shown in SEQ ID NO: 9.

[0100] VEGF-C binding domain

[0101] In the trispecific fusion protein according to the present invention, the VEGF-C binding domain is provided by a VHH domain. Thus, in some embodiments, the VEGF-C binding domain according to the present invention comprises a VHH domain that specifically binds to VEGF-C. Preferably, the VHH comprises a CDR1, CDR2, and CDR3 sequence having a variable region as shown in SEQ ID NO:4. The CDR sequence range of the amino acid sequence of the variable region SEQ ID NO:4 can be defined according to the Kabat, AbM, Chothia, Contact, or IMGT schemes, or can be defined according to any two, more, or all of these definition schemes. In one embodiment, the VEGF-C binding domain according to the present invention comprises, in the variable region having SEQ ID NO:4, a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence defined according to Kabat or Chothia; or a CDR1 sequence defined according to a combination of Kabat and Chothia, and CDR2 and CDR3 sequences defined according to Kabat.

[0102] In one embodiment, the VEGF-C binding domain according to the present invention comprises

[0103] (i) a CDR1 comprising or consisting of SEQ ID NO: 1;

[0104] (ii) a CDR2 comprising or consisting of SEQ ID NO: 2; and

[0105] (iii) a CDR3 comprising or consisting of SEQ ID NO: 3.

[0106] In one embodiment, the VEGF-C binding domain according to the present invention comprises the amino acid sequence set forth in SEQ ID NO:4. In another embodiment, the VEGF-C binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:4 and retains the ability to specifically bind to VEGF-C. In another preferred embodiment, the VEGF-C binding domain comprises an amino acid sequence that has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions (e.g., conservative substitutions) compared to SEQ ID NO:4 and retains the ability to specifically bind to VEGF-C. Preferably, the amino acid additions, deletions, and / or substitutions do not occur in the CDR regions. Most preferably, in the tri-specific fusion protein according to the present invention, the VEGF-C binding domain according to the present invention comprises the amino acid sequence set forth in SEQ ID NO:4, or consists of the amino acid sequence set forth in SEQ ID NO:4.

[0107] ANG-2 binding domain

[0108] In trispecific fusion proteins according to the present invention, the ANG-2 binding domain is provided by a VHH domain. Thus, in some embodiments, the ANG-2 binding domain according to the present invention comprises a VHH domain that specifically binds to ANG-2. Preferably, the VHH comprises the CDR1, CDR2, and CDR3 sequences of the variable region represented by SEQ ID NO:8. The CDR sequence ranges of the amino acid sequence of the variable region represented by SEQ ID NO:8 can be defined according to the Kabat, AbM, Chothia, Contact, or IMGT schemes, or can be defined according to any two, more, or all of these schemes. In one embodiment, the ANG-2 binding domain according to the present invention comprises, in the variable region represented by SEQ ID NO:8, a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence defined according to Kabat or Chothia; or a CDR1 sequence defined according to a combination of Kabat and Chothia, and CDR2 and CDR3 sequences defined according to Kabat.

[0109] In one embodiment, the ANG-2 binding domain according to the invention comprises

[0110] (i) a CDR1 comprising or consisting of SEQ ID NO: 5;

[0111] (ii) a CDR2 comprising or consisting of SEQ ID NO: 6; and

[0112] (iii) a CDR3 comprising or consisting of SEQ ID NO: 7.

[0113] In one embodiment, the ANG-2 binding domain according to the present invention comprises the amino acid sequence set forth in SEQ ID NO: 8. In another embodiment, the ANG-2 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 8 and that retains the ability to specifically bind to ANG-2. In yet another preferred embodiment, the ANG-2 binding domain comprises an amino acid sequence that has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 8 and that retains the ability to specifically bind to ANG-2. Preferably, the amino acid additions, deletions, and / or substitutions do not occur in the CDR regions. Most preferably, in a tri-specific fusion protein according to the present invention, the ANG-2 binding domain according to the present invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8.

[0114] Dimerization domain

[0115] Hinge area

[0116] In addition to the three antigen-binding domains, in some embodiments, the polypeptide chain of the fusion protein according to the present invention preferably further comprises a hinge region capable of driving polypeptide chain dimerization. In some embodiments, the hinge region comprises at least two, or preferably three, cysteine ​​residues. In other embodiments, the hinge region is included at the C-terminus of the VEGF-A binding domain or is present at the C-terminus of the polypeptide chain.

[0117] In one embodiment, the hinge region comprises CPPC (SEQ ID NO: 60) or CPPCPPC (SEQ ID NO: 61) amino acid sequence. In one embodiment, the hinge region comprises an amino acid sequence from a hinge region or a portion of a hinge region of a (e.g., human) IgG isotype (IgG1, IgG2, IgG3, or IgG4) fused to the N-terminus of CPPC (SEQ ID NO: 60) or CPPCPPC (SEQ ID NO: 61), or a mutant sequence thereof, or consists of said sequence. In one embodiment, the hinge region has a length of no more than 20 amino acid sequences, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In another embodiment, the hinge region has a length of 10-15 amino acid sequences.

[0118] In one embodiment, the hinge region comprises or consists of an amino acid sequence selected from SEQ ID NOs: 12-19 and 45-46.

[0119] In some more preferred embodiments, the hinge region comprises, or consists of, the amino acid sequence of SEQ ID NO: 12; or comprises, or consists of the amino acid sequence of SEQ ID NO: 13.

[0120] Fc region

[0121] In addition to the three antigen-binding domains, the polypeptide chain of the fusion protein according to the present invention, in some embodiments, further comprises an Fc region capable of driving dimerization of the polypeptide chain.

[0122] In the trispecific fusion proteins of the present invention, the applicable Fc region can be an Fc region or CH3 region from any natural immunoglobulin molecule, or a variant thereof. For example, the Fc region can comprise two or three constant region domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. Preferably, the Fc region comprises from N-terminus to C-terminus: CH2-CH3, more preferably from N-terminus to C-terminus: hinge region-CH2-CH3. In some embodiments, the Fc region used in the molecules of the present invention is an Fc region from IgG (especially human IgG), for example, an Fc region of IgG1, IgG2 or IgG4, preferably an Fc region from human IgG1. Preferably, the Fc region comprises SEQ ID NO: 35 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0123] CH3 area

[0124] In addition to the three antigen-binding domains, the polypeptide chain of the fusion protein according to the present invention, in some embodiments, further comprises a CH3 region capable of driving dimerization of the polypeptide chain.

[0125] In the trispecific fusion proteins of the present invention, the applicable CH3 region can be a CH3 region from any natural immunoglobulin molecule, or a variant thereof. Preferably, the CH3 region has a hinge region or a flexible connecting peptide at the N-terminus. In some embodiments, the CH3 region used in the molecules of the present invention is a CH3 region from IgG (especially human IgG), for example, a CH3 region of IgG1, IgG2 or IgG4, preferably a CH3 region from human IgG1. Preferably, the CH3 region comprises SEQ ID NO: 47 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0126] Connector peptide

[0127] In the trispecific fusion protein according to the present invention, the antigen-binding domains can be connected by a connecting peptide. There is no specific limitation on the connecting peptide that can be used in the fusion protein of the present invention. After reading this specification, those skilled in the art can easily determine the applicable connecting peptide sequence based on the components to be connected and the connection position.

[0128] In some embodiments, each antigen binding domain of the present invention, and optionally dimerization domain, is connected by a connecting peptide. In one embodiment, the connecting peptide is a flexible connecting peptide of 5-50 amino acids, preferably a connecting peptide comprising glycine (G) and / or serine (S) and / or threonine residues (T). In one embodiment, the connecting peptide has a length of 5-50 amino acids, for example, 8, 10, 15, 20, 25 or 30 amino acids in length, or has an amino acid length falling between any two integers. In one embodiment, the connecting peptide comprises an amino acid sequence (GRPGS) n (SEQ ID NO: 59) or (GGGGG) n (SEQ ID NO: 58), wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7, preferably, n=2 or 3. In some embodiments, the connecting peptide comprises a hinge region. In some embodiments, the connecting peptide does not comprise a hinge region.

[0129] In some embodiments, the fusion protein polypeptide chain of the present invention includes at least one (and preferably one) connecting peptide comprising a hinge region. The connecting peptide can include 20-50 amino acid lengths, especially 20-30 amino acid lengths. The hinge region can have a length of no more than 20 amino acid sequences, for example, with a 10-15 amino acid sequence length. In some embodiments, the connecting peptide includes or consists of a short linker of 5-20 amino acids of the hinge region and its C-terminus. In some embodiments, the short linker is a short linker comprising or consisting of an amino acid residue selected from glycine (G) and / or serine (S) and / or threonine residue (T), for example, with 5, 8, 10, 12, 15, 20 amino acid lengths, or with an amino acid length falling between any two integers. In other embodiments, the short linker is a short linker comprising or consisting of an amino acid sequence of SEQ ID NO: 10 or 11. Preferably, the connecting peptide comprises a sequence of SEQ ID NO: 20 or 21. In some preferred embodiments, the connecting peptide comprising the hinge region is linked to the C-terminus of the VEGFA binding domain, and the VEGFA binding domain is linked to the ANG2 binding domain or the VEGFC binding domain or both at the C-terminus through the connecting peptide.

[0130] In other embodiments, the fusion protein polypeptide chain of the present invention is connected together by a connecting peptide that does not contain a hinge region. When the polypeptide chain includes a hinge region dimerization domain, the hinge region dimerization domain is preferably located at the C-terminus of the polypeptide chain and is directly connected to the binding domain located at the C-terminus of the polypeptide chain. When the polypeptide chain includes an Fc region or a CH3 region dimerization domain, the Fc region or CH3 region can be directly (for example, when the Fc region or CH3 region has a hinge region at the N-terminus), or through a connecting peptide, connected to the C-terminus of any antigen-binding domain. In these embodiments, the connecting peptide can include 5-20 amino acids in length, more preferably 5-15 amino acids in length. In some embodiments, the connecting peptide is a short linker comprising or consisting of an amino acid residue selected from glycine (G) and / or serine (S) and / or threonine residue (T), for example, having 5, 8, 10, 12, 15, 20 amino acids in length, or having an amino acid length falling between any two integers. In other embodiments, the connecting peptide comprises the amino acid sequence of SEQ ID NO: 10 or 11.

[0131] Exemplary tri-specific fusion proteins

[0132] In the trispecific fusion protein according to the present invention, the connection order between each antigen binding domain and optionally the dimerization domain is not particularly limited. Those skilled in the art can easily determine the applicable connection order as needed after reading this specification.

[0133] In some embodiments, the present invention provides trispecific fusion proteins in which a VEGF-C binding domain and an ANG2 binding domain are linked to a VEGF-A binding domain, either separately or in tandem.

[0134] In some embodiments, the present invention provides a tri-specific fusion protein, wherein the VEGF-C binding domain and the ANG2 binding domain are respectively linked to the N-terminus or C-terminus of the VEGF-A binding domain, preferably the ANG2 binding domain is bound to the N-terminus of the VEGF-A binding domain and the VEGF-C binding domain is bound to the C-terminus of the VEGF-A binding domain.

[0135] In other embodiments, the present invention provides trispecific fusion proteins in which a VEGF-C binding domain and an ANG2 binding domain are linked in tandem and further linked to the C-terminus of a VEGF-A binding domain. In some embodiments, the VEGF-C binding domain is linked to the N-terminus of the ANG2 binding domain. In other embodiments, the VEGF-C binding domain is linked to the C-terminus of the ANG2 binding domain.

[0136] In some embodiments, the fusion protein further comprises a hinge region, and preferably the hinge region is located at the C-terminus of the VEGF-A binding domain, or at the C-terminus of the polypeptide chain.

[0137] In other embodiments, the fusion protein further comprises an Fc region, and preferably the Fc region is located at the C-terminus of the VEGF-A binding domain, or at the C-terminus of the polypeptide chain.

[0138] In other embodiments, the fusion protein further comprises a CH3 region, and preferably the CH3 region is located at the C-terminus of the VEGF-A binding domain, or at the C-terminus of the polypeptide chain.

[0139] In any of the above embodiments, the linkage may be direct, or via a linker peptide as described herein.

[0140] In some preferred embodiments, the trispecific fusion protein according to the present invention comprises a hinge region. Preferably, the trispecific fusion protein comprises at least one polypeptide chain comprising, from N-terminus to C-terminus:

[0141] (i) ANG2 binding domain, first connecting peptide, VEGFA binding domain, hinge region, second connecting peptide, VEGFC binding domain,

[0142] (ii) a VEGFC binding domain, a first connecting peptide, a VEGFA binding domain, a hinge region, a second connecting peptide, and an ANG2 binding domain,

[0143] (iii) a VEGFA binding domain, a hinge region, a first connecting peptide, an ANG2 binding domain, a second connecting peptide, a VEGFC binding domain, or

[0144] (iv) a VEGFA binding domain, a hinge region, a first connecting peptide, a VEGFC binding domain, a second connecting peptide, and an ANG2 binding domain,

[0145] (v) an ANG2 binding domain, a first connecting peptide, a VEGFC binding domain, a second connecting peptide, a VEGFA binding domain, a hinge region, or

[0146] (vi) VEGFC binding domain, first connecting peptide, ANG2 binding domain, second connecting peptide, VEGFA binding domain, hinge region.

[0147] In one embodiment, the first connecting peptide and the second connecting peptide can be identical or different. In some embodiments, the first and second connecting peptides are short linkers comprising or consisting of an amino acid residue selected from glycine (G) and / or serine (S) and / or threonine residues (T), for example, having 5, 8, 10, 12, 15, 20 amino acid lengths, or having an amino acid length falling between any two integers. In some embodiments, the first connecting peptide and / or the second connecting peptide comprise an amino acid sequence (G4S)n (SEQ ID NO: 55), wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, or 4. In one embodiment, the first connecting peptide and / or the second connecting peptide are composed of an amino acid sequence (G4S)2 (SEQ ID NO: 62) or (G4S)3 (SEQ ID NO: 63). In another embodiment, the first connecting peptide and / or the second connecting peptide comprise an amino acid sequence TS (G4S)n (SEQ ID NO: 56), wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, or 4. In another embodiment, the first connecting peptide and / or the second connecting peptide comprise the amino acid sequence (GGGGG)n (SEQ ID NO: 58), wherein n is an integer equal to or greater than 2, for example, n is an integer of 2 or 3. In another embodiment, the first connecting peptide and / or the second connecting peptide comprise the amino acid sequence (GGGGG)n (SEQ ID NO: 58) or (GRPGS)n (SEQ ID NO: 59), wherein n is an integer equal to or greater than 2, for example, n is an integer of 2 or 3. In a preferred embodiment, the first and second connecting peptides each comprise the amino acid sequence of SEQ ID NO: 10. In another preferred embodiment, the first and second connecting peptides each comprise the amino acid sequence of SEQ ID NO: 11.

[0148] In some embodiments, the hinge region comprises the CPPC (SEQ ID NO: 60) or CPPCPPC (SEQ ID NO: 61) amino acid sequence. In one embodiment, the hinge region comprises, or consists of, an amino acid sequence selected from SEQ ID NOs: 12-19 and 45-46. In a preferred embodiment, the hinge region comprises, or consists of, the amino acid sequence of SEQ ID NO: 12. In another preferred embodiment, the hinge region comprises, or consists of, the amino acid sequence of SEQ ID NO: 13. In a preferred embodiment, the hinge region comprises, or consists of, the amino acid sequence of SEQ ID NO: 15. In another preferred embodiment, the hinge region comprises, or consists of, the amino acid sequence of SEQ ID NO: 16. In a preferred embodiment, the hinge region comprises, or consists of, the amino acid sequence of SEQ ID NO: 17. In another preferred embodiment, the hinge region comprises, or consists of, the amino acid sequence of SEQ ID NO: 18. In a preferred embodiment, the hinge region comprises, or consists of, the amino acid sequence of SEQ ID NO: 45. It has been found that in the fusion protein of the present invention comprising a hinge region as a dimerization domain, a hinge region comprising a sequence such as SEQ ID NO: 12, 13, 15, 16, 17, 18 or 45 is more conducive to improving the stability of the fusion protein dimer and thereby improving its production performance and drugability.

[0149] In some preferred embodiments, the present invention provides a trispecific fusion protein comprising at least one polypeptide chain, wherein the polypeptide chain comprises, from N-terminus to C-terminus: an ANG2 binding domain, a first connecting peptide, a VEGFA binding domain, a hinge region, a second connecting peptide, and a VEGFC binding domain. Preferably, the polypeptide chain comprises

[0150] (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28, 48 and 50; or

[0151] (ii) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of (i); or

[0152] (iii) an amino acid sequence having at least 1-30, or 1-20, or 1-15, or 1-10, or 1-5 amino acid changes (e.g., substitutions, deletions and / or insertions, preferably substitutions, more preferably conservative substitutions) relative to the amino acid sequence of (i).

[0153] In a preferred embodiment, the present invention provides a trispecific fusion protein comprising the following polypeptide chain, wherein the polypeptide chain comprises:

[0154] (i) the amino acid sequence of SEQ ID NO: 24; or

[0155] (ii) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of (i); or

[0156] (iii) an amino acid sequence having at least 1-30, or 1-20, or 1-15, or 1-10, or 1-5 amino acid changes (e.g., substitutions, deletions and / or insertions, preferably substitutions, more preferably conservative substitutions) relative to the amino acid sequence of (i).

[0157] Most preferably, the present invention provides a trispecific fusion protein comprising the following polypeptide chain, wherein the polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 24. Preferably, the fusion protein is a dimer consisting of two of the polypeptide chains.

[0158] In a preferred embodiment, the present invention provides a trispecific fusion protein comprising the following polypeptide chain, wherein the polypeptide chain comprises:

[0159] (i) the amino acid sequence of SEQ ID NO: 25; or

[0160] (ii) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of (i); or

[0161] (iii) an amino acid sequence having at least 1-30, or 1-20, or 1-15, or 1-10, or 1-5 amino acid changes (e.g., substitutions, deletions and / or insertions, preferably substitutions, more preferably conservative substitutions) relative to the amino acid sequence of (i).

[0162] Most preferably, the present invention provides a trispecific fusion protein comprising the following polypeptide chain, wherein the polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 25. Preferably, the fusion protein is a dimer consisting of two of the polypeptide chains.

[0163] In other preferred embodiments, the present invention provides a trispecific fusion protein comprising at least one polypeptide chain, wherein the polypeptide chain comprises, from N-terminus to C-terminus: a VEGFA binding domain, a hinge region, a first connecting peptide, an ANG2 binding domain, a second connecting peptide, and a VEGFC binding domain. Preferably, the polypeptide chain comprises

[0164] (i) an amino acid sequence selected from SEQ ID NOs: 29-32; or

[0165] (ii) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of (i); or

[0166] (iii) an amino acid sequence having at least 1-30, or 1-20, or 1-15, or 1-10, or 1-5 amino acid changes (e.g., substitutions, deletions and / or insertions, preferably substitutions, more preferably conservative substitutions) relative to the amino acid sequence of (i).

[0167] Preferably, the present invention provides a trispecific fusion protein comprising the following polypeptide chains, wherein the polypeptide chains comprise or consist of the amino acid sequence of SEQ ID NO: 29, 30, or 31. Preferably, the fusion protein is a dimer consisting of two of the polypeptide chains.

[0168] In other preferred embodiments, the present invention provides a trispecific fusion protein comprising at least one polypeptide chain, wherein the polypeptide chain comprises, from N-terminus to C-terminus: an ANG2 binding domain, a first connecting peptide, a VEGFA binding domain, a second connecting peptide, a VEGFC binding domain, and a hinge region. Preferably, the polypeptide chain comprises

[0169] (i) an amino acid sequence selected from SEQ ID NOs: 33-34; or

[0170] (ii) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of (i); or

[0171] (iii) an amino acid sequence having at least 1-30, or 1-20, or 1-15, or 1-10, or 1-5 amino acid changes (e.g., substitutions, deletions and / or insertions, preferably substitutions, more preferably conservative substitutions) relative to the amino acid sequence of (i).

[0172] Preferably, the present invention provides a trispecific fusion protein comprising the following polypeptide chain, wherein the polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 33 or 34. Preferably, the fusion protein is a dimer consisting of two of the polypeptide chains.

[0173] In some further embodiments, the present invention also provides a fusion protein comprising the following polypeptide chain, wherein the polypeptide chain comprises from N-terminus to C-terminus:

[0174] (i) an ANG2 binding domain, a first connecting peptide, a VEGFA binding domain, an Fc region or a CH3 region, a second connecting peptide, and a VEGFC binding domain,

[0175] (ii) a VEGFC binding domain, a first connecting peptide, a VEGFA binding domain, an Fc region or a CH3 region, a second connecting peptide, and an ANG2 binding domain,

[0176] (iii) a VEGFA binding domain, an Fc region or a CH3 region, a first connecting peptide, an ANG2 binding domain, a second connecting peptide, a VEGFC binding domain, or

[0177] (iv) a VEGFA binding domain, an Fc region or a CH3 region, a first connecting peptide, a VEGFC binding domain, a second connecting peptide, and an ANG2 binding domain,

[0178] (v) an ANG2 binding domain, a first connecting peptide, a VEGFC binding domain, a second connecting peptide, a VEGFA binding domain, an Fc region, or a CH3 region, or

[0179] (vi) VEGFC binding domain, a first connecting peptide, an ANG2 binding domain, a second connecting peptide, a VEGFA binding domain, an Fc region or a CH3 region.

[0180] In one embodiment, the first connecting peptide and the second connecting peptide can be identical or different. In some embodiments, the first and second connecting peptides are short linkers comprising or consisting of an amino acid residue selected from glycine (G) and / or serine (S) and / or threonine residues (T), for example, having 5, 8, 10, 12, 15, 20 amino acid lengths, or having an amino acid length falling between any two integers. In some embodiments, the first connecting peptide and / or the second connecting peptide comprise an amino acid sequence (G4S)n (SEQ ID NO: 55), wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, or 4. In one embodiment, the first connecting peptide and / or the second connecting peptide are composed of an amino acid sequence (G4S)2 (SEQ ID NO: 62) or (G4S)3 (SEQ ID NO: 63). In another embodiment, the first connecting peptide and / or the second connecting peptide comprise an amino acid sequence TS (G4S)n (SEQ ID NO: 56), wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, or 4. In another embodiment, the first connecting peptide and / or the second connecting peptide comprise the amino acid sequence (GGGGG)n (SEQ ID NO: 58), wherein n is an integer equal to or greater than 2, for example, n is an integer of 2 or 3. In another embodiment, the first connecting peptide and / or the second connecting peptide comprise the amino acid sequence (GGGGG)n (SEQ ID NO: 58) or (GRPGS)n (SEQ ID NO: 59), wherein n is an integer equal to or greater than 2, for example, n is an integer of 2 or 3. In a preferred embodiment, the first and second connecting peptides each comprise the amino acid sequence of SEQ ID NO: 10. In another preferred embodiment, the first and second connecting peptides each comprise the amino acid sequence of SEQ ID NO: 11.

[0181] In some embodiments, the Fc region comprises a native hinge region sequence or a variant hinge region sequence, preferably the Fc region is an Fc region from human IgG1, more preferably the Fc region comprises the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 85%, 90%, 95% or 99% identity thereto.

[0182] In some embodiments, the CH3 region is an Fc region from human IgG1, more preferably comprising the amino acid sequence of SEQ ID NO: 47 or an amino acid sequence having at least 85%, 90%, 95% or 99% identity thereto. Preferably, the CH3 region has a hinge region at the N-terminus.

[0183] In some embodiments, the present invention provides a tri-specific fusion protein comprising the following polypeptide chain, wherein the polypeptide chain comprises:

[0184] (i) the amino acid sequence of SEQ ID NO: 22 or 49; or

[0185] (ii) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of (i); or

[0186] (iii) an amino acid sequence having at least 1-30, or 1-20, or 1-15, or 1-10, or 1-5 amino acid changes (e.g., substitutions, deletions and / or insertions, preferably substitutions, more preferably conservative substitutions) relative to the amino acid sequence of (i).

[0187] Most preferably, the present invention provides a trispecific fusion protein comprising the following polypeptide chains, wherein the polypeptide chains comprise or consist of the amino acid sequence of SEQ ID NO: 22 or 49. Preferably, the fusion protein is a dimer consisting of two of the polypeptide chains.

[0188] In some embodiments, the trispecific fusion protein of the present invention has a biological activity selected from any one or more of the following:

[0189] (i) High affinity binding to VEGFA, VEGFC, and ANG2, preferably, as determined using thin-layer biofilm interferometry, such as the assay described in Example 3, with a bivalent binding affinity constant KD value of 1-10 nM or less for VEGF-A; and a bivalent binding affinity constant KD value of 0.1-1 nM for VEGFC and ANG2;

[0190] (ii) blocking VEGF-A and / or VEGF-C-induced VEGFR2 signaling pathway activation;

[0191] (iii) blocking VEGF-C-induced VEGFR3 signaling pathway activation;

[0192] (iv) blocking ANG2 binding to Tie2-expressing cells;

[0193] (v) blocking ANG2-induced activation of the Tie2 signaling pathway;

[0194] (vi) inhibiting endothelial cell survival and proliferation induced by both VEGF-C and VEGF-A;

[0195] (vii) inhibiting neovascularization of tumors (e.g., solid tumors such as melanoma);

[0196] (viii) inhibiting the growth of tumors (e.g., solid tumors such as melanoma);

[0197] (ix) inhibiting the occurrence and / or development of neovascularization-related eye diseases, such as reducing the level of neovascularization, reducing vascular leakage, and inhibiting retinal edema and / or thickening caused by neovascularization.

[0198] Thus, in some embodiments, the present invention provides tri-specific fusion proteins against VEGFA / VEGFC / ANG2 that can be used to treat neovascularization-related diseases, such as cancer (eg, solid tumors) and ocular diseases.

[0199] Polynucleotides, vectors and hosts

[0200] The present invention provides nucleic acids encoding any of the above tri-specific fusion proteins of the present invention. Also provided are vectors comprising the nucleic acids. In one embodiment, the vector is an expression vector. Also provided are host cells comprising the nucleic acids or the vectors. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells). In another embodiment, the host cell is prokaryotic.

[0201] Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification. As will be appreciated by those skilled in the art, due to codon degeneracy, each polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. In addition, to facilitate production and purification, the nucleic acid encoding the tri-specific fusion protein of the present invention may also include a signal peptide encoding a secretory signal fused to the N-terminus of the fusion protein and / or a tag peptide fused to the C-terminus of the fusion protein to facilitate purification, such as a hexahistidine tag.

[0202] The nucleic acid encoding the tri-specific fusion protein of the present invention can be introduced into a vector for amplification or expression. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0203] The present invention also provides host cells comprising expression vectors of the present invention. Host cells suitable for replication and support expression of the fusion protein of the present invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors as needed, and large quantities of cells containing the vectors can be cultivated for inoculating large-scale fermenters to obtain sufficient amounts of the present molecules for clinical applications. 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 used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine 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, NSO, P3X63 and Sp2 / 0, etc. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO, HEK293 or NSO cell.

[0204] Production and purification of the fusion protein of the present invention

[0205] In another aspect, the present invention provides a method for producing a tri-specific fusion protein of the present invention, the method comprising: culturing a host cell comprising a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the fusion protein, thereby producing the fusion protein. For the purpose of this recombinant production, methods well known to those skilled in the art can be used to construct an expression vector comprising a polynucleotide encoding the polypeptide chain of the fusion protein of the present invention. A variety of techniques can be used to transfect or introduce the expression vector into suitable host cells, including, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques. After the fusion protein is expressed from the host cell, it can be recovered and / or purified using techniques known in the art.

[0206] Methods that can be used to purify the fusion proteins of the present invention include, but are not limited to, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be apparent to those skilled in the art. In one embodiment, after expression, the trispecific fusion protein of the present invention is isolated and purified by Protein A column.

[0207] The purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activities of the fusion proteins provided herein can be identified, screened or characterized by a variety of assays known in the art.

[0208] Assay

[0209] The fusion proteins provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0210] The binding of the fusion protein of the present invention to human VEGFA and / or human VEGFC and / or human ANG2 can be determined by methods known in the art, such as ELISA, Western blotting, flow cytometry, etc., or the exemplary methods disclosed in the Examples herein. For example, the binding kinetics (e.g., K) of the molecules can be determined using recombinant VEGFA and / or human VEGFC and / or human ANG2 proteins in a biointerferometry assay. D In some embodiments, the equilibrium dissociation constant, e.g., bivalent binding affinity, of a molecule binding to human VEGFA and / or human VEGFC and / or human ANG2 is determined using a biointerferometry assay (e.g., Fortebio affinity measurement), such as the method described in Example 3, at 30°C. The binding activity of the fusion proteins of the present invention to cells naturally or recombinantly expressing VEGFA and / or human VEGFC and / or human ANG2 protein can also be tested by flow cytometry. For example, see the ANG2 cell binding assay described in Example 7.

[0211] The VEGFA and / or VEGFC and / or ANG2 antagonist / inhibitory activity of the fusion proteins of the present invention can be determined by methods known in the art, such as ELISA blocking assays, receptor fluorescent reporter activation assays, cell-based receptor phosphorylation assays, cell proliferation assays, or the exemplary methods disclosed in the Examples herein. For example, a cell-based receptor reporter assay, such as the method described in Example 4, using, for example, NFAT-RE-luc2P / KDR HEK293 cells, can be used to determine whether the molecule blocks activation of the VEGFR2 signaling pathway induced by hVEGF-A alone, hVEGF-C alone, or a combination thereof. Alternatively, a cell proliferation assay, such as the CCK-8 assay, such as the method described in Examples 5 or 9, using lymphocytes or endothelial cells, can be used to determine whether the molecule inhibits cell survival and / or proliferation induced by hVEGF-A alone, hVEGF-C alone, or a combination thereof in vitro.

[0212] The anti-neovascularization effects of the molecules of the present invention can be measured by methods known in the art, such as in vitro assays and / or in vivo animal studies. For example, an in vitro endothelial cell tube formation assay, such as the method described in Example 9, can be used to induce tube formation in human umbilical vein endothelial cells (HUVECs), using VEGFA and VEGF C, and then measuring the inhibitory effect of the molecules on VEGF A- and VEGF C-induced tube formation in primary cells. The anti-neovascularization and / or anti-tumor effects of the molecules can also be measured, for example, in tumor-bearing mouse models, such as the method described in Example 10. Alternatively, an animal model of laser-induced choroidal neovascularization or a DL-AA-induced retinal neovascularization can be used. After administration of the molecules, the inhibition of choroidal neovascularization or retinal neovascularization by the molecules can be observed, for example, by fundus color photography, fluorescein angiography, or optical coherence tomography.

[0213] Pharmaceutical compositions, drug combinations, and kits

[0214] In one aspect, the present invention provides compositions, for example, pharmaceutical compositions comprising the tri-specific fusion proteins of the present invention formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible. The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion). In one embodiment, the fusion protein of the present invention is formulated as a pharmaceutical composition suitable for injection. In some embodiments, the fusion protein of the present invention is formulated as a pharmaceutical composition suitable for intraperitoneal injection. In further embodiments, the fusion protein of the present invention is formulated as a pharmaceutical composition suitable for ocular injection (e.g., intravitreal injection). In some embodiments, the fusion protein of the present invention is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise a fusion protein molecule as described herein and one or more other therapeutic agents.

[0215] In another aspect, the present invention also provides a pharmaceutical combination comprising a fusion protein of the present invention and one or more therapeutic agents.

[0216] The therapeutic agents suitable for use in the pharmaceutical compositions and drug combinations of the present invention can be selected from any of the following categories: (i) anti-angiogenic drugs; (ii) immunosuppressive drugs; (iii) anti-fibrotic drugs; (iv) neuroprotective agents; and (v) drugs with tumor inhibitory effects.

[0217] The compositions of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Common preferred compositions are in the form of injectable solutions or infusible solutions. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (ip), intramuscular) injection. In a 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.

[0218] As used herein, the phrases "parenteral administration" and "parenteral administration" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intradermal, intraperitoneal, transtracheal, subcutaneous injection, and infusion.

[0219] Therapeutic compositions should generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, dispersion, liposome or lyophilized form. Sterile injectable solutions can be prepared by adding the active compound to a suitable solvent in the required amount and then filtering and sterilizing. Typically, dispersions are prepared by incorporating the active compound into a sterile solvent containing a basic dispersion medium and other ingredients. Coating agents such as lecithin can be used. In the case of dispersions, the appropriate fluidity of the solution can be maintained by using a surfactant. Prolonged absorption of the injectable composition can be caused by including substances that delay absorption, such as monostearate and gelatin, in the composition.

[0220] The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a molecule of the present invention. A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount may vary depending on a variety of factors such as the disease state, age, sex, and weight of the individual. A therapeutically effective amount is an amount in which any toxic or deleterious effects are outweighed by the therapeutically beneficial effects. 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 still more preferably at least about 80% relative to an untreated subject. The ability of a molecule of the present invention to inhibit a measurable parameter (e.g., tumor volume) can be evaluated in an animal model system that is predictive of efficacy in human tumors.

[0221] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, a prophylactic dose is less than a therapeutically effective amount because a prophylactic dose is used in a subject before or at an earlier stage of the disease.

[0222] Kits comprising the antibody molecules described herein are also within the scope of the present invention. The kit may comprise one or more other elements, such as instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0223] Uses and methods

[0224] In one aspect, the present invention provides in vivo and in vitro uses and application methods of the fusion protein of the present invention.

[0225] In some embodiments, the uses and methods of the present invention involve applying the fusion proteins of the present invention in vivo and / or in vitro to:

[0226] - binds to VEGFA antigen and / or VEGFC antigen and / or ANG2 antigen,

[0227] - blocking the binding of VEGFA and / or VEGFC and / or ANG2 to their cognate receptors, such as VEGFR2 and / or VEGFR3 and / or Tie2;

[0228] - Inhibition of VEGFA and / or VEGFC and / or ANG2-induced activation of VEGFR2 and / or VEGFR3 and / or Tie2 cell signaling;

[0229] - Inhibition of VEGFA and / or VEGFC and / or ANG2-induced vascular endothelial cell survival, proliferation and / or migration;

[0230] - Inhibition of VEGFC and / or ANG2-induced lymphocyte survival and / or proliferation;

[0231] - Inhibits VEGFC and / or ANG2-induced lymphangiogenesis and lymphatic endothelial cell growth and migration;

[0232] - Inhibition of VEGFA and / or VEGFC and / or ANG2-induced neovascularization and / or vascular leakage;

[0233] -Inhibit tumor neovascularization and / or growth and / or metastasis.

[0234] Therapeutic applications

[0235] In one aspect, the present invention provides uses of the trispecific molecules of the present invention for preventing / treating neovascularization-related diseases and corresponding treatment methods. In some embodiments, the disease or condition is a disease or condition that would benefit from inhibition of neovascularization in diseased tissue, including, but not limited to, solid tumors and ophthalmic diseases. In some embodiments, the disease is a solid tumor, preferably melanoma, wherein administration of the bispecific binding protein inhibits neovascularization and / or tumor growth within the tumor. In other embodiments, the disease is an ocular disease.

[0236] In the treatment methods according to the present invention, the antibodies of the present invention may be used alone or in combination with other treatment modalities or therapeutic agents, as appropriate. Other treatment modalities / therapeutic agents that can be used in combination with the antibodies of the present invention include, but are not limited to, other anti-angiogenic drugs, such as integrins, plasma kallikrein, etc., immunosuppressive drugs, such as anti-complement HTRA1, anti-fibrotic drugs, such as anti-PDGF and anti-CTGF, etc., and neuroprotective drugs, such as NGF, etc.

[0237] In some embodiments, the fusion protein of the present invention or a pharmaceutical composition comprising the fusion protein of the present invention is used as a drug for treating and / or preventing a disease in an individual, preferably a mammal, more preferably a human.

[0238] In some embodiments, the present invention provides a method for treating a neovascularization-related disease, comprising administering a fusion protein of the present invention or a pharmaceutical composition thereof to a subject. In some embodiments, the disease is a solid tumor, wherein administration of the trispecific fusion protein inhibits neovascularization and / or tumor growth within the tumor. In other embodiments, the disease is an ocular disease.

[0239] In some embodiments, the present invention provides the use of the fusion protein of the present invention in the preparation of a medicament for treating and / or preventing a disease in a subject, wherein the disease is preferably a neovascularization-related disease, such as a solid tumor and an ocular disease. In some embodiments, the ocular disease is choroidal neovascularization or retinal neovascularization.

[0240] In any of the above embodiments, one or more additional active agents, such as chemotherapeutic agents and / or cancer therapeutic agents or anti-angiogenic therapies, can be further administered in combination with the trispecific fusion proteins of the present invention. The combined administration can be administered simultaneously, concurrently, or sequentially in any order.

[0241] Detection and diagnostic applications

[0242] In yet another aspect, the present invention also provides a diagnostic method for detecting the presence of relevant antigens in a biological sample, such as serum, semen or urine or a 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 (labeled or unlabeled) fusion protein of the present invention or administering the fusion protein to a subject under conditions that allow interaction to occur, and (ii) detecting the formation of a complex between the fusion protein and the sample (and optionally a control sample). The formation of a complex indicates the presence of relevant antigens, and in some cases, can show the suitability or demand for treatment and / or prevention as described herein. In some embodiments, the present invention also provides the use of its fusion protein of the present invention in the preparation of a diagnostic tool for preparing a disease, wherein the disease is preferably a neovascularization-related disease, such as a solid tumor and an eye disease. Example

[0243] Example 1. Production and purification of multi-specific fusion proteins

[0244] Expi293F cells (purchased from Thermo Fisher Scientific) were subcultured in Expi293F cell culture medium (purchased from Thermo Fisher Scientific). The cell density was checked one day before transfection and adjusted to 2×10 6The cell density was adjusted to 3 × 10 cells / ml on the day of transfection. 6 cells / ml.

[0245] Opti-MEM medium (purchased from Gibco) with 1 / 10 of the final volume of the transfected Expi293F cells was used as the transfection buffer. 10 μg of the corresponding recombinant plasmid was added to each mL of transfection buffer and mixed. 30 μg of polyethylenimine (PEI) (Polysciences, catalog number: 23966) was added to each mL of transfection buffer and mixed. The mixture was incubated at room temperature for 20 minutes. The PEI / DNA mixture was then gently poured into the Expi293F cell suspension and mixed. The cells were then cultured on a shaker at 8% CO2, 36.5°C, and 120 rpm.

[0246] After 16-18 hours of culture, the culture flask was supplemented with 200 g / L FEED (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone) at 1 / 50 the volume of the post-transfection culture, a glucose solution at a final concentration of 4 g / L, and VPA (valproic acid) at a final concentration of 2 mM / L. The cells were gently mixed and incubated on a shaker at 8% CO₂, 36.5°C, and 120 rpm. Culture was continued for 6 days, and the culture was harvested and centrifuged at 4000 rpm for 30 minutes. The cell supernatant was filtered through a 0.45 μM filter and purified by affinity chromatography and gel chromatography.

[0247] The specific affinity chromatography purification steps are as follows: use Amsphere TM A3 (JSR Life Sciences, catalog number: 10000327-C05) Protein A affinity chromatography column was used. Prior to purification, the column and tubing were detoxified with 0.1 M NaOH for 2 h. The tubing and column were then rinsed with distilled water. The column was equilibrated with 5 column volumes of 1× PBS (Gibco, catalog number: 10010023). The collected supernatant was passed through the column and then rinsed with 10 column volumes of 1× PBS to remove nonspecifically bound proteins. The column was then rinsed with 5 column volumes of elution buffer (100 mM glycine, pH 2.7). The eluate was collected and the pH of the protein was adjusted to 6.0 with 2 M Tris for further gel chromatography.

[0248] The specific gel exchange chromatography purification procedure is as follows: a Superdex 200 Increase 10 / 300GL (GE Healthcare, catalog number: 28-9909-44) gel chromatography column is selected and placed in the AKTApure system. The AKTApure system equipped with the Superdex 200 Increase 10 / 300GL gel chromatography column is detoxified with 0.1M NaOH for 2 hours, then the system and column are rinsed with distilled water; the column is equilibrated with 2-5 column volumes of 1× PBS until the conductivity and pH are stable; the protein obtained by affinity chromatography is loaded and eluted with 1× PBS. Impurities such as aggregates are removed based on the UV absorption peak, and a high-purity sample is collected. The sample is filtered through a 0.22μm membrane, and the protein concentration is determined.

[0249] The amino acid sequence and sequence number of the IAR044 multi-specific fusion protein of the present invention expressed and purified based on the above-mentioned procedure are shown in the summary section of the attached sequence listing.

[0250] Example 2. Purity Identification of Multispecific Fusion Protein Dimers

[0251] SDS-PAGE electrophoresis analysis: Take protein samples, add reducing loading buffer (Yisheng Biotechnology, catalog number: 20315ES05) or non-reducing loading buffer (Beijing Biolabs, catalog number: WE0289), heat at 70 degrees Celsius for 10 minutes, take an appropriate amount of sample and add precast gel 4–20% TGX TM Precast Gel (Bio-rad, catalog number: 4561095) was added, and a protein marker (Biorad, catalog number: 1610375) was added as a control. Electrophoresis was performed at 150V for 50 minutes, and the gel was stained with a protein stainer (GenScript, L00760C). The gel was then photographed with a gel imager (Shanghai Tianneng, Tanon-1600).

[0252] SEC-HPLC analysis: Take a protein sample and use a size exclusion chromatography column to test the purity. The chromatographic column is a TSKgel G3000Swxl (7.8×300mm, 5μm) analytical column, the guard column is a TSKgel guardcolumn Swxl (6.0×40mm, 7μm), the mobile phase is 20mmol / L phosphate buffer + 400mmol / L NaClO4, pH 6.8, the injection plate temperature is 10℃, the column temperature is 25℃, the injection volume is 50μl, the flow rate is 0.5ml / min, the acquisition time is 30 minutes, and the FLD fluorescence detector is used with a PMT value of 7, an excitation wavelength of 285nm, and an emission wavelength of 340nm. Take the sample to be tested and dilute it to 1.0mg / ml with the mobile phase as the test solution. Take the formulation buffer and dilute it with the same treatment method as above as the blank solution. Take 50μl of the blank solution and the test solution and inject it into the liquid chromatograph to start the detection.

[0253] CE-SDS (Capillary Gel Electrophoresis) Analysis: Protein samples were collected and tested for purity using capillary gel electrophoresis. Uncoated capillaries were used with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20.2 cm. Before electrophoresis, the capillary column was flushed with 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and a running gel at 70 psi. Dilute the sample to be tested to 1.0 mg / ml with an appropriate amount of ultrapure water. Place 50 μl of this diluted sample in a 1.5 ml centrifuge tube and add 45 μl of pH 6.5 sample buffer (weigh 0.32 g of citric acid monohydrate and 2.45 g of disodium hydrogen phosphate dodecahydrate in 45 ml of ultrapure water, dilute to 50 ml to prepare citric acid-phosphate buffer. Accurately measure 200 μl of this buffer, add 80 μl of 10% (w / v) sodium lauryl sulfate solution, add water to 1 ml, and mix thoroughly) and 5 μl of 250 mmol / L NEM solution (weigh 62 mg of N-ethylmaleimide and dissolve in 2 ml of ultrapure water). Mix thoroughly, heat at 70 ± 2°C for 10 ± 2 minutes, cool to room temperature, and transfer to a sample vial as the test sample solution. Prepare a blank solution by taking the same volume of formulation buffer as the test sample and following the same procedure as above. Sample injection conditions: -5 kV for 20 seconds; separation voltage: -10 kV for 35 minutes. The capillary column temperature was controlled at 25°C, and the detection wavelength was 220 nm.

[0254] Figure 1A shows SDS-PAGE images of representative multispecific fusion proteins after one-step purification with Protein A. As shown, all tested multispecific fusion proteins formed dimers of the correct size; IAR044-028, 030, 032, and 038, 039, and 040 all formed higher proportions of dimers compared to the IAR044-022 molecule with an unmodified hinge region.

[0255] Figure 1B shows the results of SEC-HPLC purity analysis of IAR044-034, 035, and 036 molecules.

[0256] Table 1 below shows the expression yield, SEC-HPLC purity and CE-SDS purity of a variety of different fusion proteins using different first and second connecting peptides and optionally a C-terminal hinge region.

[0257] Table 1. Fusion protein expression yield and purity

[0258] Example 3. Determination of the binding kinetics of the multi-specific fusion protein of the present invention with antigens by biofilm thin layer interferometry (BLI) and surface plasmon resonance (SPR)

[0259] The equilibrium dissociation constant (KD) of the multispecific fusion protein of the present invention binding to the target antigen was determined using thin-layer biofilm interferometry (ForteBio). ForteBio affinity determination was performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0260] Half an hour before the start of the experiment, according to the number of samples, take an appropriate number of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors and soak them in SD buffer (PBS1×, BSA 0.1%, Tween-20 0.05%). Take 100μl of SD buffer, multi-specific fusion protein, antigen (including human Ang2 (Beijing Yiqiao), and human VEGF165 (R&D) and human VEGF C (R&D)), and add them to a 96-well black polystyrene half-volume microplate (Greiner, 675076). Arrange the plate according to the sample position and select the sensor position. The instrument setting parameters are as follows: running steps: Baseline, Loading~1nm, Baseline, Association and Dissociation; the running time of each step depends on the sample binding and dissociation rate, the rotation speed is 400rpm, and the temperature is 30℃. Use ForteBio analysis software to analyze K D value.

[0261] In the experiments described in the above assays, the affinities of representative multi-specific fusion proteins are shown in Tables 2-4:

[0262] Table 2. Affinity constants (equilibrium dissociation constants) of the ForteBio assay for bivalent binding to VEGFA antigen

[0263] Table 3. Affinity constants (equilibrium dissociation constants) of the ForteBio assay for bivalent binding to the antigen VEGFC

[0264] Table 4. Affinity constants (equilibrium dissociation constants) of the ForteBio assay for bivalent binding to the antigen ANG2

[0265] * Indicates that the dissociation constant exceeds the detection limit of ForteBio

[0266] In the above experiments, the bivalent binding affinity KD values ​​of antibodies IAR044-030 and IAR044-032 to human VEGF A were 5.15E-10M and 5.17E-10M, respectively; the bivalent binding affinity KD values ​​of antibodies IAR044-030 and IAR044-032 to human VEGF C were 1.04E-9M and 1.01E-9M, respectively; and the bivalent binding affinity KD values ​​of antibodies IAR044-030 and IAR044-032 to human Ang2 were 2.06E-10M and 2.20E-10M, respectively.

[0267] The equilibrium dissociation constants (KD) of the antibodies expressed and purified in Example 1 binding to human VEGF A, VEGF C and Ang2 were determined using surface plasmon resonance (SPR).

[0268] The KD of the antibody was determined by Biacore (GE Healthcare, T200). After the antibody was captured on the chip with an anti-human Fc antibody, the affinity and kinetic constants were obtained by detecting the binding and dissociation between the antigen human TIGIT and the captured antibody. The specific method is as follows: Using an amino coupling kit (BR-1006-33, GE Healthcare), the anti-human Fc antibody (R&D product number MAB110) or antigen was coupled to the surface of a CM5 chip (29-1496-03, GE Healthcare) with a coupling height of approximately 6000 RU. 1M ethanolamine was injected to block the remaining active sites. The diluted antibody was captured in the fourth channel of the CM5 chip at a flow rate of 10 μl / min for 60 seconds. The third channel was used as a blank control channel. Then, gradient dilutions of antigens (including human Ang2 (Beijing Sino-Qiao), human VEGF165 (R&D), and human VEGF C (R&D)) were injected into both channels of the chip in ascending concentration order, with an association time of 180 seconds and a dissociation time of 600 seconds. Finally, the chip was regenerated using Glycine pH 1.5 (BR-1003-54, GE Healthcare). Kinetic analysis of the data was performed using Biacore T200 analysis software using a 1:1 binding model. The results are shown in Tables 5-7 below.

[0269] Table 5. Affinity constants (equilibrium dissociation constants) of antigen-antibody binding detected by SPR

[0270] Table 6. Affinity constants (equilibrium dissociation constants) of antigen-antibody bivalent binding detected by SPR

[0271] Table 7. Affinity constants (equilibrium dissociation constants) of antigen-antibody bivalent binding detected by SPR

[0272] In the above experiments, the bivalent binding affinity KD values ​​of antibodies IAR044-030 and IAR044-032 to human VEGF A were 1.51E-10M and 2.03E-10M, respectively; the bivalent binding affinity KD values ​​of antibodies IAR044-030 and IAR044-032 to human VEGF C were <2.34E-10M and <2.76E-10M, respectively; and the bivalent binding affinity KD values ​​of antibodies IAR044-030 and IAR044-032 to human Ang2 were 9.32E-12M and 1.20E-11M, respectively.

[0273] Example 4 Anti-VEGF A / VEGF C / Ang2 multispecific fusion protein blocks VEGF A or C-induced HEK293KDR reporter activation

[0274] VEGF A or VEGF C can bind to the related receptor VEGFR2 (also known as KDR), activating the VEGFR2 signaling pathway and inducing endothelial cell survival, proliferation, and migration. This study utilized the KDR reporter assay system and NFAT-RE-luc2P / KDR HEK293 cells (Promega Cat CS181401) to examine the blocking effect of serially diluted fusion proteins on VEGFA or VEGFC-activated receptor signaling pathways. The experimental methods were performed according to the supplier's instructions.

[0275] Remove the NFAT-RE-luc2P / KDR HEK293 cells that had been replaced with experimental culture medium (DMEM culture medium containing 10% FBS) 3 days in advance, remove the old culture medium, and wash once with PBS. Then digest the cells with 1 ml of Accutase solution until the cells become round and detach from the wall, and terminate the reaction with 5 ml of dilution culture medium (DMEM culture medium containing 10% FBS). Pipette the cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the culture medium. Add 10 ml of dilution culture medium to resuspend the cells, mix well, and count. The cell viability should be above 90%. Adjust the cell density to 0.8×10 with dilution culture medium. 6 cells / ml, 50 μl / well was added into a 96-well white cell culture plate according to the experimental layout.

[0276] Prepare a mixture containing 120 ng / ml VEGF A (R&D) or 200 ng / ml VEGF C (R&D) and serially diluted test fusion proteins. After the mixture is allowed to rest for 30 minutes, 50 μl / well is added to a 96-well white cell culture plate containing cells and incubated in a 37°C, 5% CO2 incubator for 6 hours. Simultaneously, set up blank controls, controls containing only VEGFA or VEGFC, and controls containing an IgG isotype control antibody, the anti-VEGF A molecule Elylea, or the anti-VEGF C molecule OPT302 in place of the test fusion protein.

[0277] Remove the 96-well white cell culture plate from the CO2 incubator after incubation for 6 hours and equilibrate to room temperature for 10-15 minutes. Add 100 μl / well of the Bio-Glo Luciferase Assay System (Promega), which had been previously equilibrated to room temperature, to the 96-well white cell culture plate according to the experimental layout. Incubate at room temperature in the dark for 5 minutes.

[0278] Use a multifunctional microplate reader to read the fluorescence value. Select the chemiluminescence mode for plate reading mode, the endpoint method for plate reading type, and the wavelength as full wavelength. Collect fluorescence column by column, and the collection time for each column is 1000ms.

[0279] In the experiments described in the above assays, the test results are shown in Figures 2 and 3 , indicating that the tested trispecific fusion proteins of the present invention can block the activation of the KDR signaling pathway induced by VEGF A or VEGF C.

[0280] Example 5 Anti-VEGF A / VEGF C / Ang2 multispecific fusion protein blocks VEGF C-induced BaF3-FLT4 proliferation

[0281] In this study, the multispecific fusion proteins of the present invention and recombinant human VEGFC protein were co-incubated with BaF3-FLT4 cells (BaF3 cells overexpressing FLT4 (VEGFR3), Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). The number of viable cells was determined using a CCK-8 kit (Tongren Chemical), demonstrating the inhibitory effects of the different fusion proteins on VEGFC-induced BaF3-FLT4 proliferation.

[0282] BaF3 cells were infected with a lentivirus carrying the FLT4 gene to obtain FLT4-overexpressing BaF3 cells, BaF3-FLT4.

[0283] The cell proliferation inhibition test was performed according to the CCK-8 kit instructions. The experimental culture medium was prepared using 1640 medium containing 10% FBS. The highest final concentration of the test antibody was 10 nM, and the antibody was diluted in a 1:3 ratio. A blank control group and a VEGF-C experimental group with only VEGFC added were also set up. In the test system, the final concentration of hVEGFC (R&D) was 20 ng / ml, and the final concentration of BaF3-FLT4 cells was 2x10 5 Cells were plated in a 96-well plate at 100 μl per well and incubated at 37°C in a CO2 incubator for 72 hours. Subsequently, 15 μl of CCK-8 was added to each well and incubated at 37°C in a CO2 incubator for 4 hours. Absorbance was measured using dual-wavelength spectrophotometry with a detection wavelength of 450 nm and a reference wavelength of 620 nm. OD450 - OD620 values ​​were calculated.

[0284] The experimental results are shown in Figure 4. The tri-specific fusion protein of the present invention can effectively inhibit the survival and proliferation of BaF3-FLT4 induced by hVEGFC in vitro.

[0285] Example 6 Anti-VEGF A / VEGF C / Ang2 multispecific fusion protein blocks the binding of Ang2 to Tie2

[0286] The ability of the multi-specific fusion protein of the present invention and the control antibody Faricimab to block the binding between human Ang2 and hTie2 was detected by ELISA.

[0287] hTie2 protein (Beijing Yiqiao) was resuspended in PBS and prepared at a concentration of 2ug / ml for coating the ELISA plate overnight. The plate was blocked with 5% BSA for 1 hour. The biotin antigen Recombinant Biotinylated hAngiopoietin-2 protein (R&D) was diluted to 600ng / ml and added to the ELISA plate at 50μl / well. The multi-specific fusion protein prepared as described above was serially diluted starting from the highest concentration, with a total of 8 or 12 dilution gradients. The serially diluted fusion protein was added to the ELISA plate at 50μl / well and incubated on ice with PBS for 30min, with the final antigen concentration being 300ng / ml. The ELISA plate containing the mixture of antigen and fusion protein was incubated for 90min, washed three times with PBS, the supernatant was discarded, and 100μl Avidin-HRP (Invitrogen) diluted 1:10000 was added to each well, incubated at room temperature for 30min, and washed six times with PBS. Use 100ul / well TMB colorimetric solution (solarbio) for 1 min, and stop with 100ul / well stop solution (solarbio). Read OD using a microplate reader. 450 and OD 620 Absorbance value.

[0288] The experimental results showed that the multispecific fusion protein of the present invention and the control antibody Faricimab achieved a complete blocking effect, and in terms of IC50 values, the multispecific fusion protein of the present invention was significantly superior to the control antibody Faricimab. (See Figure 5)

[0289] Example 7 Anti-VEGF A / VEGF C / Ang2 multispecific fusion protein blocks the binding of human Ang2-Fc protein to 293 cells overexpressing Tie2

[0290] The ability of the multispecific fusion proteins of the present invention (IAR044-030 and IAR044-032) and control antibodies to block the binding of human Ang2-hFc to Tie2 on the cell surface was detected by flow cytometry (FACS).

[0291] Expi-293 cells overexpressing human Tie2 (293-tie2 cells) were generated by transfection with a pCHO1.0 vector (Invitrogen) carrying the human Tie2 gene cloned into MCS.

[0292] The antigen hAng2-Fc protein (Beijing Yiqiao) was diluted to 4 μg / ml and added to the plate at 50 μl / well. Anti-ANG2 molecules (IAR044-030, IAR044-032, and control antibody Faricimab) were diluted 2-fold starting from a maximum concentration of 800 nM, for a total of 12 dilutions. 50 μl / well was added to the plate and incubated on ice with PBS for 30 minutes. The final antigen concentration was 2 μg / ml, and the highest final concentration of anti-ANG2 molecules was 400 nM. 293-tie2 cells were adjusted to 2×10 5 Cells were centrifuged at 300 g for 5 min, the supernatant discarded, and resuspended in the antigen / anti-ANG2 mixture. Incubated on ice for 30 min. Add 100 μl of PBS per well, centrifuge at 300 g for 5 min, and wash once with PBS. Add 100 μl of goat anti-human IgG-PE (SouthernBiotech) at a 1:200 dilution per well and incubate on ice for 20 min. Add 100 μl of PBS per well, centrifuge at 300 g for 5 min, and wash once with PBS. Resuspend cells in 100 μl of PBS and measure cell fluorescence using a flow cytometer (BD Biosciences). Concentration-dependent curves were fitted using GraphPad based on the MFI. The results are shown in Figure 6.

[0293] The experimental results showed that IAR044-030, IAR044-032, and the control antibody Faricimab achieved complete blocking effect. In terms of IC50, IAR044-030 and IAR044-032 were significantly superior to the control antibody. (See Figure 6)

[0294] Example 8 Anti-VEGF A / VEGF C / Ang2 Multispecific Fusion Protein Inhibits Tie2 Phosphorylation Induced by Human Ang2-Fc Protein in 293-tie2 Cells

[0295] To detect the inhibitory effect of the fusion protein of the present invention on hAng2-Fc-induced tie2 phosphorylation, an hAng2-induced phosphorylation assay was used.

[0296] In this experiment, the fusion protein of the present invention and recombinant hAng2-fc protein were co-incubated with expi293 cells overexpressing Tie2 (293-tie2 cells). The content of phosphorylated Tie2 in the system was detected to reflect the inhibitory effect of different fusion proteins on hAng2-fc-induced Tie2 phosphorylation.

[0297] Take 293-tie2 cells expressing Tie2 and dilute to 2x10 6 cell / ml, 100ul per well was added to a 96-well plate, centrifuged at 400g for 5min, and the supernatant was removed.

[0298] Experimental culture medium was prepared using Expi293 medium. The test fusion protein was diluted in a 1:2 ratio to a maximum final concentration of 60 μg / ml. The final concentration of hAng2-fc was 2.5 μg / ml. The anti-VEGFA / Ang2 bispecific antibody Farcimab, as well as a bispecific fusion protein (IEX04-56) with a similar anti-VEGFC VHH domain as the test fusion protein, or a bispecific antibody (IEX04-012) with the same anti-Ang2 VHH domain, were used as controls.

[0299] Resuspend cells in 100 μl of experimental culture medium per well and incubate at 37°C for 15 minutes. Remove the culture medium by centrifugation and add 100 μl of NP-40 lysis buffer containing 1% protease and phosphatase inhibitors. Incubate on ice for 30 minutes. Centrifuge at 2000 g, collect the supernatant, and store at -80°C.

[0300] The pTie2 concentration was detected according to the phosphorylation tie2 ELISA kit (R&D, catalog number: DYC2720E). Briefly, the capture antibody was coated onto the enzyme labeling plate at a concentration of 4ug / ml at 4°C overnight. Washed three times with PBST and blocked with 5% BSA for 1h. Add 100ul of the sample to be tested (can be diluted 2-3 times) and the control pTie2 (for making a standard curve) and incubated at room temperature for 2h. Washed three times with PBST, added 100ul of HRP-conjugated anti-pTyr antibody and incubated at room temperature for 2h. Washed 6 times with PBST, added 100ul of TMB for color development, and after 15min, added 100ul of stop buffer to terminate the reaction. Determine OD 450 -OD 620 value.

[0301] The experimental results are shown in Figure 7. The multi-specific fusion protein of the present invention can effectively inhibit hAng2-Fc-induced Tie2 phosphorylation in 293 cells in vitro, and its inhibitory activity is superior to that of the positive control antibody Farcimab.

[0302] Example 9 Anti-VEGF A / VEGF C / Ang2 multispecific fusion protein inhibits VEGF A+VEGF C-induced HUVEC proliferation

[0303] VEGF A and VEGF C act on related receptors such as VEGFR in vascular endothelial cells, promoting endothelial cell survival, proliferation, and migration, thereby inducing neovascularization. This experiment used VEGF A and VEGF C to co-induce the survival and proliferation of human umbilical vein endothelial cells (HUVECs) to examine the inhibitory effect of the multi-specific fusion protein of the present invention on VEGF A- and VEGF C-induced primary cell survival and proliferation.

[0304] In this example, HUVEC survival and proliferation were measured using CCK-8 assays. The specific method was as follows: cells were treated one day in advance, plated at 2,000 cells / well in a 96-well culture plate, and incubated in a 37°C, 5% CO2 incubator for 24 hours. After the cells attached, the endothelial cell culture medium in the 96-well plate was replaced with experimental culture medium containing a final concentration of 5 ng / ml VEGF A and 60 ng / ml VEGF C, along with serially diluted fusion proteins. The culture medium was then replaced with the endothelial cell culture medium in the 96-well plate, and the plates were incubated in a 37°C, 5% CO2 incubator for 72 hours. The anti-VEGFA molecule Aflibercept and the anti-VEGFC molecule OPT302, as well as a 1:1 molar mixture thereof, as well as an anti-VEGF C / VEGF A bispecific fusion protein (IEX04-056) and an anti-ANG2 / VEGF A bispecific antibody (IEX04-012) were used as controls.

[0305] After the cells were incubated with the experimental medium, CCK-8 detection solution (Tongren Chemical) was added at 10 μl / well and placed in a 37°C, 5% carbon dioxide incubator for 12 to 24 hours. The absorbance OD was measured using a multifunctional microplate reader. 450 -OD 620 Read the value.

[0306] In the experiment described in the above assay, the test results are shown in Figure 8. The tri-specific fusion protein of the present invention can completely inhibit the proliferation and survival of HUVEC cells induced by VEGF A + VEGF C.

[0307] Example 10 Anti-VEGF A / VEGF C / Ang2 multispecific fusion protein inhibits A375 tumor growth and neovascularization experiment

[0308] In this example, A375 human malignant melanoma cells were cultured at 3×10 6 cell inoculated into each mouse, and the anti-angiogenesis and anti-tumor effects of the multi-specific fusion protein of the present invention were determined in nude mice.

[0309] Human nude mice:

[0310] Female nude mice of BALB / c background were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (SPF grade). Mice were acclimated for 7 days after arrival before the study began.

[0311] cell:

[0312] Human A375 cells were purchased from ATCC (CAT#:CRL-1619) and routine subculture was performed in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed with PBS and Matrigel at a ratio of 1:1 to prepare a cell concentration of 1.5x10 7 On day 0, mice were subcutaneously injected with A375 cell suspension, 0.2 mL / mouse, i.e., the inoculation volume was 3x10 6 cells / mouse.

[0313] Dosage:

[0314] Mice were randomly divided into groups (6 mice per group). The dosage and method of administration are shown in Table 8. PBS (purchased from Gibco) was used as a negative control and was administered on days 6, 8, 10, 12, 14, 16, 18, and 20 after inoculation. The tumor volume and body weight of the mice were monitored twice a week. Body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 21st day after inoculation, and the calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: A vernier caliper was used to measure the maximum long axis (L) and maximum wide axis (W) of the tumor, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0315] Table 8. Experimental design

[0316] *: The structures of the control molecules IEX04-010 and IEX04-056 are shown in SEQ ID NOs: 43 and 44.

[0317] The tumor inhibition rate results (as shown in FIG9 and Table 9) show that the trispecific molecule of the present invention targeting VEGF A, VEGF C and Ang2 has a significant inhibitory effect on tumors.

[0318] Table 9. Tumor inhibition rate on day 21

[0319] Example 11. Laser-induced choroidal neovascularization efficacy test

[0320] This experiment used a mouse laser-induced choroidal neovascularization model to determine the anti-neovascularization effect of the trispecific fusion protein of the present invention. The experimental animals were SPF-grade C57 mice weighing 18-30 g.

[0321] This study used laser photocoagulation around the fovea of ​​the mouse fundus to induce choroidal neovascularization, establishing an animal model similar to human choroidal neovascularization. Twenty-four hours before photocoagulation, mice were divided into six groups: model control, Aflibercept, IEX04-010, IEX04-056, IAR044-030, and IAR044-032, with five mice in each group. Within 24 hours after photocoagulation, the test article was administered intravitreally at the doses listed in Table 10 below. The model control group received an equal volume of 0.9% sodium chloride solution. On days 3, 5, and 10 after photocoagulation, fundus color photography and fluorescein angiography were performed to assess the inhibitory effect of the test article on choroidal neovascularization. Ten days after administration, mice were euthanized, and both eyes were examined for histological examination with hematoxylin and eosin staining.

[0322] Table 10: Experimental design table

[0323] Fundus color photography and fluorescein angiography

[0324] Evaluation indicators:

[0325] (1) Fluorescent spot rating

[0326] Grading standards for the light spots photographed by fluorescence angiography after modeling:

[0327] Level 1: The light spot does not show high fluorescence;

[0328] Grade 2: The spot has high fluorescence but no fluorescein leakage;

[0329] Grade 3: The spot has high fluorescence and slight fluorescein leakage, which does not exceed the edge of the spot;

[0330] Level 4: The spot has high fluorescence and significant fluorescein leakage, which exceeds the edge of the spot.

[0331] Count the laser spots of levels 1-4, and record the level of fundus laser spots in each examination.

[0332] (2) Improvement rate of fluorescein leakage area

[0333] Fluorescein leakage area improvement rate (%) = (fluorescein leakage area before administration - fluorescein leakage area after administration) / fluorescein leakage area before administration * 100%

[0334] The results are shown in Figures 10 and 11. Although the number of test samples was limited, it can be seen that the multi-specific fusion protein IAR044-032 of the present invention showed a significant anti-angiogenesis trend after 5 days of administration.

[0335] Example 12. DL-AAA-induced retinal neovascularization assay

[0336] This experiment used the Dutch rabbit retinal neovascularization model to detect the inhibitory effect of molecules on retinal neovascularization.

[0337] Description of experimental animals

[0338] Species and strain: Dutch rabbit

[0339] Animal level: Ordinary

[0340] Source of experimental animals: Pizhou Dongfang Breeding Co., Ltd., Experimental Animal Production License No.: SCXK(Su)2017-0002, Experimental Animal Quality Certificate No.: NO.202227644, NO.202259452.

[0341] Age of start of medication (D1): 8-15 months

[0342] Body weight before the start of drug administration (D1): 2.46-3.44 kg.

[0343] Number of animals: 29 qualified animals (all female) were used for modeling, and 36 eyes were enrolled in the study by intravitreal injection of DL-α-AAA (DL-α-aminoadipic acid).

[0344] Experimental methods

[0345] Animal grouping and experimental design

[0346] Before drug administration, the animals were divided into groups according to the fluorescence leakage area of ​​the RNV of the model eye, as shown in Table 11. Drug administration began on D1.

[0347] Table 11 Animal grouping and experimental design

[0348] #: Indicates that the model is successfully established and the eye gender of the animals included in the group is not limited.

[0349] Red-free image

[0350] All animals had red-free fundus images collected at screening (12 weeks after modeling), before enrollment, D8, D15, D29, D43, D47 (only animal #2318791), D57, and D71. The examination areas were the temporal, central, and nasal nerve fiber layers, and the RNV morphology was observed.

[0351] Fundus fluorescein angiography (FFA)

[0352] All animals underwent FFA examinations at screening (12 weeks after modeling), before enrollment, and on days 8, 15, 29, 43, 47 (animal #2318791 only), 57, and 71. Prior to FFA, animals were intravenously administered 10% sodium fluorescein injection (10 mg / kg, 100 mg / mL). Early and late FFA images were collected, with images within approximately 1.5 minutes considered early and images between approximately 1.5 and 3 minutes considered late. The presence of fluorescence leakage in the fundus was used to determine RNV generation and leakage. The area of ​​fluorescence leakage was measured, and the reduction in fluorescence leakage area and the improvement rate of fluorescence leakage area were calculated: Fluorescence leakage area reduction = fluorescence leakage area before administration - fluorescence leakage area after administration; Fluorescence leakage area improvement rate = fluorescence leakage area reduction / fluorescence leakage area before administration × 100%.

[0353] The results showed (Figure 12) that during the test period, the IEX04-012, IEX04-056, IAR044-030, and IAR044-030 administered at a dose of 0.03 mg / eye were all able to effectively inhibit fluorescence leakage. The inhibitory effect of IEX04-056 can last until D57, while the inhibitory effect of IEX04-012 and low-dose and high-dose IAR044-030 can last until D71. The efficacy of IEX04-012 and low-dose IAR044-030 is better than that of Moreover, the efficacy of low-dose IAR044-030 is stronger than that of IEX04-012.

[0354] Example 13. Evans blue staining experiment of mouse vascular leakage

[0355] Subcutaneous injection of VEGF A, Ang2, and VEGF C can induce neovascularization in vivo and promote leakage. In this example, mice were given an intravenous injection of Evans Blue in advance. 30 minutes later, 50 ng of VEGF A, 500 ng of Ang2, and 500 ng of VEGF C were subcutaneously injected into the mice to induce the model. Each localized point was injected at one site, 3 sites on each side, for a total of 6 sites per mouse. Simultaneously, the following drugs were injected according to different groups as shown in Table 12 below. Photos were taken 20 minutes after administration, and the leakage area was counted.

[0356] Table 12 Animal groups and experimental design

[0357] The results are shown in Figure 13. As shown in the figure, the multi-specific fusion protein molecules (IAR044-030, IAR044-032, IAR044-019) can significantly inhibit vascular leakage, and the effect is better than that of clinical drugs and bispecific drugs.

[0358] Sequence Listing Overview

[0359] This application is accompanied by a sequence listing comprising a number of nucleic acid and amino acid sequences. The following table provides an overview of the sequences included.

Claims

1. A trispecific fusion protein comprising: a polypeptide chain comprising (i) a VEGF-C binding domain; (ii) an ANG2 binding domain and (iii) a VEGF-A binding domain, in, The VEGF-C binding domain and the ANG2 binding domain comprise or consist of a VHH domain that specifically binds to VEGF-C or ANG2, respectively, and wherein the VEGF-A binding domain comprises or consists of a ligand capture domain that specifically binds to VEGF-A, wherein the ligand capture domain comprises an extracellular domain from a VEGF receptor (VEGFR), Preferably, the polypeptide further comprises (iv) a dimerization domain selected from the group consisting of a hinge region, a CH3 region and an Fc region.

2. The fusion protein of claim 1, wherein The polypeptide chain of the fusion protein comprises a hinge region, wherein the hinge region comprises a CPPC (SEQ ID NO: 60) or CPPCPPC (SEQ ID NO: 61) amino acid sequence, preferably the hinge region comprises an amino acid sequence selected from SEQ ID NOs: 12-19 and SEQ ID NOs: 45-46, more preferably an amino acid sequence of SEQ ID NO: 12 or 13.

3. The fusion protein of claim 1, wherein The VEGF-C binding domain comprises the CDR1-3 sequences contained in SEQ ID NO: 4, preferably, comprises the CDR1 sequence of SEQ ID NO: 1 or consisting thereof; comprises the CDR2 sequence of SEQ ID NO: 2 or consisting thereof; and comprises the CDR3 sequence of SEQ ID NO: 3 or consisting thereof; Still more preferably, the VEGF-C binding domain comprises SEQ ID NO: 4, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity thereto, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO:

4. Most preferably, the VEGF-C binding domain comprises the amino acid sequence of SEQ ID NO: 4, or consists of the amino acid sequence shown in SEQ ID NO:

4.

4. The fusion protein of claims 1-3, wherein the ANG2 binding domain comprises the CDR1-3 sequences contained in SEQ ID NO: 8, preferably, a CDR1 sequence comprising SEQ ID NO: 5 or consisting thereof; a CDR2 sequence comprising SEQ ID NO: 6 or consisting thereof; and a CDR3 sequence comprising SEQ ID NO: 7 or consisting thereof; Still more preferably, the ANG2-binding domain comprises SEQ ID NO: 8, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity thereto, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO:

8. Most preferably, the ANG2 binding domain comprises the amino acid sequence of SEQ ID NO: 8, or consists of the amino acid sequence shown in SEQ ID NO:

8.

5. The fusion protein of claims 1 to 4, wherein the VEGF-A binding domain comprises the immunoglobulin-like (Ig) domain 2 of VEGFR-1 and the Ig-like domain 3 of VEGFR-2, and preferably comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity thereto, or an amino acid sequence having one or more (preferably 1 to 10, more preferably 1 to 5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 9, Most preferably, the VEGF-A binding domain comprises the amino acid sequence of SEQ ID NO: 9, or consists of the amino acid sequence shown in SEQ ID NO:

9.

6. The fusion protein of claims 1-5, wherein the polypeptide chain of the fusion protein comprises from N-terminus to C-terminus: (i) ANG2 binding domain, first connecting peptide, VEGFA binding domain, hinge region, second connecting peptide, VEGFC binding domain, (ii) a VEGFC binding domain, a first connecting peptide, a VEGFA binding domain, a hinge region, a second connecting peptide, and an ANG2 binding domain, (iii) VEGFA binding domain, hinge region, first connecting peptide, ANG2 binding domain, second connecting peptide, VEGFC binding domain, (iv) a VEGFA binding domain, a hinge region, a first connecting peptide, a VEGFC binding domain, a second connecting peptide, and an ANG2 binding domain, (v) an ANG2 binding domain, a first connecting peptide, a VEGFC binding domain, a second connecting peptide, a VEGFA binding domain, a hinge region, or (vi) VEGFC binding domain, first connecting peptide, ANG2 binding domain, second connecting peptide, VEGFA binding domain, hinge region; Alternatively, the polypeptide chain of the fusion protein comprises from N-terminus to C-terminus: (i) an ANG2 binding domain, a first connecting peptide, a VEGFA binding domain, an Fc region or a CH3 region, a second connecting peptide, and a VEGFC binding domain, (ii) a VEGFC binding domain, a first connecting peptide, a VEGFA binding domain, an Fc region or a CH3 region, a second connecting peptide, and an ANG2 binding domain, (iii) a VEGFA binding domain, an Fc region or a CH3 region, a first connecting peptide, an ANG2 binding domain, a second connecting peptide, and a VEGFC binding domain, (iv) a VEGFA binding domain, an Fc region or a CH3 region, a first connecting peptide, a VEGFC binding domain, a second connecting peptide, and an ANG2 binding domain, (v) an ANG2 binding domain, a first connecting peptide, a VEGFC binding domain, a second connecting peptide, a VEGFA binding domain, an Fc region, or a CH3 region, or (vi) a VEGFC binding domain, a first connecting peptide, an ANG2 binding domain, a second connecting peptide, a VEGFA binding domain, an Fc region or a CH3 region, Optionally, the polypeptide chain of the fusion protein further comprises a hinge region connected to the N-terminus of the Fc region or the CH3 region.

7. The fusion protein of claim 6, wherein: (i) the hinge region comprises an amino acid sequence selected from SEQ ID NOs: 12-19 and 45-46, more preferably an amino acid sequence of SEQ ID NO: 12 or 13; (ii) the first and second connecting peptides are 5-12 amino acids in length, preferably comprising the amino acid sequence (GRPGS)n (SEQ ID NO: 59) or (GGGGG)n (SEQ ID NO: 58), wherein n=2 or 3; (iii) the Fc region is an Fc region from human IgG, more preferably the Fc region comprises the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence that is at least 85%, 90%, 95% or 99% identical thereto, (iv) The CH3 region is a CH3 region from human IgG, more preferably the CH3 region comprises the amino acid sequence of SEQ ID NO: 47 or an amino acid sequence having at least 85%, 90%, 95% or 99% identity thereto.

8. The fusion protein of claims 1-7, wherein the polypeptide chain comprises: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-34 and 48-50; or (ii) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of (i); or (iii) an amino acid sequence having at least 1-30, or 1-20, or 1-15, or 1-10, or 1-5 amino acid changes (e.g., substitutions, deletions and / or insertions, preferably substitutions, more preferably conservative substitutions) relative to the amino acid sequence of (i).

9. The fusion protein of claims 1-8, wherein the polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 24 or 25.

10. The fusion protein of claims 1-9, which is a dimeric protein comprising two identical polypeptide chains.

11. A polynucleotide encoding the fusion protein of claims 1-10.

12. An expression vector comprising the polynucleotide of claim 11.

13. A host cell transfected with the vector of claim 12.

14. A method for producing the fusion protein of claims 1 to 10, comprising culturing the host cell of claim 13, and recovering the produced fusion protein.

15. A pharmaceutical composition comprising the fusion protein of claims 1 to 10 and a pharmaceutically acceptable carrier.

16. A method for treating a disease related to neovascularization, comprising administering the fusion protein of claims 1 to 10 or a pharmaceutical composition thereof to a subject.

17. The method of claim 16, wherein the disease is a solid tumor, and wherein administration of the fusion protein inhibits neovascularization within the tumor and / or tumor growth.

18. The method of claim 16, wherein the disease is an ocular disease, such as choroidal neovascularization or retinal neovascularization.

19. Use of the fusion protein of claims 1-10 as a medicine, as a medicine for treating a disease, or as a diagnostic tool for diagnosing a disease, or in the preparation of a medicine for treating and / or preventing a disease in a subject and / or for the preparation of a diagnostic tool for diagnosing a disease, wherein the disease is preferably a neovascularization-related disease, such as solid tumors and eye diseases.

20. A multi-specific fusion protein comprising: A polypeptide chain comprising a first VHH domain that specifically binds to a first antigen, a second VHH domain that specifically binds to a second antigen, and a ligand capture domain that specifically binds to a third antigen, wherein the polypeptide further comprises a dimerization domain selected from a hinge region, a CH3 region, or an Fc region, preferably, wherein the hinge region, the CH3 region, or the Fc region is connected to the C-terminus of the ligand capture domain.

21. The fusion protein of claim 20, wherein the polypeptide chain comprises: (i) a first VHH domain, a first connecting peptide, a ligand capture domain, a hinge region, a second connecting peptide, and a second VHH domain, (ii) a ligand capture domain, a hinge region, a first connecting peptide, a first VHH domain, a second connecting peptide, a second VHH domain, or (iii) a first VHH domain, a first connecting peptide, a second VHH domain, a second connecting peptide, a ligand capture domain, and a hinge region, Preferably, wherein the hinge region comprises an amino acid sequence selected from SEQ ID NOs: 12-19 and 45-46, more preferably an amino acid sequence of SEQ ID NO: 12 or 13, Preferably, the first and second connecting peptides comprise the amino acid sequence (GRPGS)n (SEQ ID NO: 59) or (GGGGG)n (SEQ ID NO: 58), wherein n=2 or 3, Still more preferably, the first and second antigens are different from each other and are independently selected from VEGF-C and ANG2, respectively, and the third antigen is VEGF-A.

22. A hinge region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-19 and 45-46.

23. The hinge region of claim 22, comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13.

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