Bispecific fusion polypeptides and uses thereof

Abstract

The present invention relates to bispecific fusion polypeptides that bind to vascular endothelial growth factor (VEGF) A and VEGFC, comprising a first domain that specifically recognizes VEGFA and a second domain that specifically recognizes VEGFC. The present invention also relates to methods of using the bispecific fusion polypeptides for treating or preventing diseases associated with VEGFA or VEGFC.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fusion polypeptides, and more particularly to bispecific fusion polypeptides that bind VEGFA and VEGFC and uses thereof. BACKGROUND

[0002] Angiogenesis refers to the process of small blood vessels formation from endothelial cells that already exist in the tissue. Angiogenesis is a process of proliferation and migration of endothelial cells from existing blood vessels in the tissue. Angiogenesis and vasculogenesis are essential in development and subsequent homeostasis, but can be pathogenic in cancer and various ophthalmic diseases.

[0003] The vascular endothelial growth factor (VEGF) family has been identified as being very important for angiogenesis and vasculogenesis. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, VEGFE and placenta growth factor (PLGF). The VEGF receptors (VEGFR) mainly include three subtypes of VEGFR1, VEGFR2 and VEGFR3. Different VEGFs bind to respective VEGFR subtypes, resulting in VEGFR phosphorylation and thus exerting corresponding biological effects. Therefore, blocking the binding of VEGF to its receptor VEGFR can effectively inhibit the proliferation of endothelial cells, vascular proliferation and leakage, and thus achieve the purpose of treating diseases related to endothelial cell proliferation and angiogenesis. SUMMARY

[0004] To solve one of the technical problems in the prior art, the present application provides a new fusion polypeptide which can effectively block the binding of multiple vascular endothelial growth factors (VEGF) to their receptors VEGFR, thereby inhibiting the proliferation of endothelial cells, vascular proliferation and leakage.

[0005] One aspect of the present application provides a bispecific fusion polypeptide that binds vascular endothelial growth factor (VEGF) A and VEGFC, the bispecific fusion polypeptide comprising: a first domain that specifically recognizes VEGFA, and a second domain that specifically recognizes VEGFC.

[0006] In some embodiments of the present application, the first domain can be a vascular endothelial growth factor receptor (VEGFR) or a functional fragment thereof that specifically recognizes VEGFA. In some embodiments of the present application, the first domain can be an antibody or a functional fragment thereof that specifically recognizes VEGFA.

[0007] In some embodiments of the application, the second domain can be a VEGFR that specifically recognizes VEGFC, or a functional fragment thereof. In some embodiments of the application, the second domain can be an antibody or a functional fragment thereof that specifically recognizes VEGFC.

[0008] In some embodiments of the application, the first domain can comprise immunoglobulin (Ig) like domain 2 of VEGFR1 and Ig like domain 3 of VEGFR2. In some embodiments of the application, the first domain can comprise an antibody Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a scFv fragment, a nanobody, a heavy chain variable region (VH) fragment, or a light chain variable region (VL) fragment that specifically binds to VEGFA. In some embodiments of the application, the first domain can comprise Ig like domain 2 of VEGFR1, Ig like domain 3 of VEGFR2, and Ig like domain 4 of VEGFR2.

[0009] In some embodiments of the application, the second domain comprises Ig like domain 1, Ig like domain 2, and Ig like domain 3 of VEGFR3. In some embodiments of the application, the second domain comprises an antibody Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a scFv fragment, a nanobody fragment, a VH fragment, or a VL fragment that specifically binds to VEGFC.

[0010] In some embodiments of the application, the bispecific fusion polypeptide further comprises a third domain, wherein the third domain comprises an Fc region of an immunoglobulin. In some embodiments of the application, the immunoglobulin is selected from the group consisting of IgA, IgG, IgM, IgD, and IgE. In some embodiments of the application, the immunoglobulin is IgG. In some embodiments of the application, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4.

[0011] In some embodiments of the application, the first domain, the second domain, and / or the third domain are directly linked or linked via a linker.

[0012] In some embodiments of the application, the bispecific fusion polypeptide comprises the first domain, the second domain, and the third domain. In some embodiments of the application, the bispecific fusion polypeptide comprises the first domain, the second domain, and the third domain connected in any manner. In some embodiments of the application, the bispecific fusion polypeptide comprises the first domain-second domain-third domain from N-terminus to C-terminus. In some embodiments of the application, the bispecific fusion polypeptide comprises the first domain-third domain-second domain from N-terminus to C-terminus. In some embodiments of the application, the bispecific fusion polypeptide comprises the second domain-first domain-third domain from N-terminus to C-terminus. In some embodiments of the application, the bispecific fusion polypeptide comprises the second domain-third domain-first domain from N-terminus to C-terminus. In some embodiments of the application, the bispecific fusion polypeptide comprises the third domain-first domain-second domain from N-terminus to C-terminus. In some embodiments of the application, the bispecific fusion polypeptide comprises the third domain-second domain-first domain from N-terminus to C-terminus.

[0013] In some embodiments of the application, the bispecific fusion polypeptide comprises the first domain and the third domain. In some embodiments of the application, the first domain is directly connected to the N-terminus of the third domain, or connected to the N-terminus of the third domain through a linker, and vice versa.

[0014] In some embodiments of the application, the bispecific fusion polypeptide comprises the first domain and the second domain. In some embodiments of the application, the first domain is directly connected to the N-terminus of the second domain, or connected to the N-terminus of the second domain through a linker, and vice versa.

[0015] In some embodiments of the application, the bispecific fusion polypeptide comprises the second domain and the third domain. In some embodiments of the application, the second domain is directly connected to the N-terminus of the third domain, or connected to the N-terminus of the third domain through a linker, and vice versa.

[0016] In some embodiments of the application, the Fc region of the immunoglobulin has an amino acid sequence as set forth in any one of SEQ ID NOs: 12-15, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 12-15.

[0017] In some embodiments of the application, the linker comprises at least 6 amino acids. In some embodiments of the application, the linker has an amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 8, or an amino acid sequence having 1, 2, or 3 insertions, substitutions, or deletions compared to the amino acid sequence set forth in SEQ ID NO: 7 or 8.

[0018] In some embodiments of the application, the first domain can comprise an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, and an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments of the application, the first domain can comprise an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments of the application, the first domain comprises an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9.

[0019] In some embodiments of the application, the second domain comprises: an amino acid sequence as set forth in SEQ ID NO: 3, an amino acid sequence in which the amino acid corresponding to amino acid position 75 of SEQ ID NO: 3 is not asparagine, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3; an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4; and an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5.

[0020] In some embodiments of the application, the amino acid sequence corresponding to amino acid position 75 of SEQ ID NO: 3, which is not asparagine, is an amino acid sequence in which amino acid position 75 is substituted for glutamine, aspartic acid, glutamic acid, arginine, or lysine. In some embodiments of the application, the amino acid sequence corresponding to amino acid position 75 of SEQ ID NO: 3, which is not asparagine, is an amino acid sequence as set forth in SEQ ID NO: 6.

[0021] In some embodiments of the application, the second domain comprises an amino acid sequence as set forth in SEQ ID NO: 10, an amino acid sequence corresponding to amino acid position 80 of SEQ ID NO: 10, which is not asparagine, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments of the application, the amino acid sequence corresponding to amino acid position 80 of SEQ ID NO: 10, which is not asparagine, is an amino acid sequence in which amino acid position 80 is substituted for glutamine, aspartic acid, glutamic acid, arginine, or lysine. In some embodiments of the application, the amino acid sequence corresponding to amino acid position 80 of SEQ ID NO: 10, which is not asparagine, is an amino acid sequence as set forth in SEQ ID NO: 11.

[0022] In some embodiments of the application, the bispecific fusion polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17. In some embodiments of the application, the bispecific fusion polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments of the application, the bispecific fusion polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 19 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments of the application, the bispecific fusion polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 20 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments of the application, the bispecific fusion polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 21 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21. In some embodiments of the application, the bispecific fusion polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 22 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22. In some embodiments of the application, the bispecific fusion polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 23 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23. In some embodiments of the application, the bispecific fusion polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24.

[0023] Another aspect of the application provides an isolated nucleic acid molecule encoding a bispecific fusion polypeptide of the application.

[0024] Yet another aspect of the application provides a nucleic acid delivery vehicle comprising an isolated nucleic acid molecule of the application. According to some embodiments of the application, the nucleic acid delivery vehicle can be derived from an adenovirus, an adeno-associated virus, a lentivirus, or other acceptable nucleic acid delivery vehicle.

[0025] A further aspect of the present application provides a bispecific binding molecule of vascular endothelial growth factor (VEGF) A and VEGFC, which bispecific binding molecule comprises a dimer of the bispecific fusion polypeptides of the present application. In some embodiments of the present application, the fusion polypeptides of the present application form homodimers. In some embodiments of the present application, the fusion polypeptides of the present application form heterodimers.

[0026] In some embodiments of the present application, the bispecific fusion polypeptides of the present application comprise a first domain, a second domain, and a third domain, which third domain comprises an Fc region of an immunoglobulin. In some embodiments of the present application, a bispecific fusion polypeptide comprising a first domain-second domain-third domain from N-terminus to C-terminus forms a dimer. In some embodiments of the present application, a bispecific fusion polypeptide comprising a first domain-third domain-second domain from N-terminus to C-terminus forms a dimer. In some embodiments of the present application, a bispecific fusion polypeptide comprising a second domain-first domain-third domain from N-terminus to C-terminus forms a dimer. In some embodiments of the present application, a bispecific fusion polypeptide comprising a second domain-third domain-first domain from N-terminus to C-terminus forms a dimer. In some embodiments of the present application, a bispecific fusion polypeptide comprising a third domain-first domain-second domain from N-terminus to C-terminus forms a dimer. In some embodiments of the present application, a bispecific fusion polypeptide comprising a third domain-second domain-first domain from N-terminus to C-terminus forms a dimer.

[0027] A further aspect of the present application provides a host cell comprising the isolated nucleic acid molecule of the present application.

[0028] A further aspect of the present application provides a pharmaceutical composition. In some embodiments of the present application, the pharmaceutical composition comprises the bispecific fusion polypeptide of the present application, and a pharmaceutically acceptable carrier. In some embodiments of the present application, the pharmaceutical composition comprises the isolated nucleic acid molecule of the present application, and a pharmaceutically acceptable carrier. In some embodiments of the present application, the pharmaceutical composition comprises the nucleic acid delivery vector of the present application, and a pharmaceutically acceptable carrier. In some embodiments of the present application, the pharmaceutical composition comprises the bispecific binding molecule of the present application, and a pharmaceutically acceptable carrier.

[0029] In some embodiments of the application, the pharmaceutical composition is in the form of a tablet, a powder, a granule, a pill, an injection, a suspension, a powder, an emulsion, an aerosol, a gel, an eye drop, a sustained release, or a sustained release implant. In some embodiments, the pharmaceutical composition can be formulated into an injectable formulation. In some embodiments, the formulation is suitable for intravitreal injection, subcutaneous, intradermal, intramuscular, intravenous, intrathecal, or intraspinal administration.

[0030] Yet another aspect of the present application provides a kit comprising the pharmaceutical composition of the present application, which is packaged in a container. In some embodiments of the present application, the container is a glass ampoule, a glass bottle, a plastic ampoule, a plastic bottle, a plastic bag, or a prefilled syringe. In some embodiments, the present application relates to a pharmaceutical unit dosage form suitable for parenteral administration to humans, which comprises a pharmaceutical composition as described herein in a suitable container. In some embodiments, the suitable container is a prefilled syringe. In some embodiments, the prefilled syringe comprises a needle.

[0031] Yet another aspect of the present application provides a method of treating or preventing a disease associated with VEGFA or VEGFC. The method comprises administering to a subject a therapeutically effective amount of the bispecific fusion polypeptide of the present application, the isolated nucleic acid molecule of the present application, the nucleic acid delivery vector comprising the isolated nucleic acid molecule of the present application, the bispecific binding molecule of the present application, or the pharmaceutical composition of the present application.

[0032] In some embodiments of the present application, the disease is selected from the group consisting of neovascular eye diseases and cancer-related indications. In some embodiments of the present application, the disease is selected from the group consisting of age-related macular degeneration, diabetic macular edema, diabetic retinopathy, neovascular glaucoma, retinal vein occlusion, corneal neovascularization, corneal neovascularization after corneal transplantation, breast cancer, kidney cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, bladder cancer, pancreatic cancer, liver cancer, cervical cancer, and glioblastoma.

[0033] DETAILED DESCRIPTION

[0034] Definitions

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied, for the purposes of interpretation of this specification, and, as appropriate, to the singular form as well as to the plural form of each term herein, and to each combination of terms herein unless in specific contexts a different grouping is explicitly indicated.

[0036] The expressions "a," "an," and "the" as used herein include the plural reference unless the context clearly indicates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

[0037] The term "about" as used herein means ±20% of the number that follows. In some embodiments, the term "about" means ±10% of the number that follows. In some embodiments, the term "about" means ±5% of the number that follows.

[0038] The term "vascular endothelial growth factor" or "VEGF" as used herein refers to the 165-amino acid vascular endothelial growth factor, and related 121-, 189-, and 206-amino acid vascular endothelial growth factors, and the like (as described in Leung et al., Science 246: 1306 (1989) and Houck et al., Mol. Endocrin. 5: 1806 (1991)), as well as naturally occurring allelic and processed forms of these growth factors. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, and placenta growth factor (PLGF). VEGF is a highly specific mitogen for vascular endothelial cells, with effects on increasing vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and vascular formation.

[0039] The term "vascular endothelial growth factor receptor," "VEGF receptor," or "VEGFR" as used herein refers to a cellular receptor for VEGF, typically a cell surface receptor found on vascular endothelial cells, and variants thereof that retain the ability to bind VEGF. The VEGFR family includes primarily VEGFRl, VEGFR2, and VEGFR3. The three members of the VEGFR family include an extracellular domain, a transmembrane domain, and an intracellular tyrosine kinase domain, with seven immunoglobulin (Ig)-like domains in the extracellular domain (as described in, e.g., Figure 1(As shown). The seven immunoglobulin domains from the N-terminus of the protein are called Ig-like domain 1, Ig-like domain 2, Ig-like domain 3, Ig-like domain 4, Ig-like domain 5, Ig-like domain 6, and Ig-like domain 7, respectively. VEGFR binds to VEGF through its extracellular domains, and the Ig-like domains of VEGFR have a high affinity for VEGF. VEGFR1 and VEGFR2 are expressed on most vascular endothelial cells, while VEGFR3 is mainly expressed on lymphatic endothelial cells. VEGFR can initiate related intracellular signaling pathways by binding to VEGFR1 and VEGFR2, participating in physiological and pathological processes such as cell proliferation and angiogenesis. It can not only promote angiogenesis and increase vascular permeability, but also prevent apoptosis, thereby maintaining blood vessels. It has been reported that the second Ig-like domain of VEGFR1 (i.e., Ig-like domain 2) determines the binding of this receptor to VEGF (Terri Davis-Smyth et al., The EMBO Journal, vol. 15 no. 18, pp. 4919-4927, 1996). VEGFC can bind to VEGFR2 and VEGFR3, primarily regulating lymphangiogenesis. VEGFB may play a role in non-angiogenic tumors, while VEGFC and VEGFD can play roles in angiogenesis and neovascularization in cancerous tissues. VEGFE is also a potential angiogenesis factor. VEGFR3 has also been reported to bind to VEGFD. PLGF activates signaling pathways by specifically binding to its receptor VEGFR1 / Flt-1, mediating the interaction between endothelial cells and stromal cells, and also influencing endothelial cell differentiation and maturation.

[0040] As used herein, the term "fusion polypeptide" refers to two or more proteins or fragments thereof that are collinearly linked by their respective peptide backbones using genetic expression or protein synthesis methods that encode protein polynucleotides. Preferably, the polypeptide or fragments thereof are from different sources. In some embodiments, the fusion polypeptide includes peptide fragments from different sources, such as fragments from VEGFR1, VEGFR2, VEGFR3, immunoglobulins, or antibodies.

[0041] As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a bispecific fusion peptide or a bispecific binding molecule) and its binding ligand (e.g., an antigen). Binding affinity is typically expressed as the dissociation constant (K0). D ) represents the dissociation constant (K) D ) is the dissociation rate (k d ) and binding rate (k a The ratio of ), i.e., K D =kd / k a Affinity can be measured by routine methods known in the art, e.g., surface plasmon resonance (SPR).

[0042] The term "specifically recognizes" or "specifically binds" as used herein means that the fusion polypeptide or specific binding molecule has recognition and binding selectivity for the ligand and can be distinguished from non-specific or nonspecific binding. In one embodiment, the bispecific fusion polypeptide or bispecific binding molecule of the application binds to an unrelated protein to a degree that is less than about 10% of the binding to VEGFA and VEGFC, as measured, for example, by SPR. In certain embodiments, the bispecific fusion polypeptide or bispecific binding molecule provided herein binds to VEGFA and VEGFC with a Kd of 10 D M or less, for example, 10 -7 M or less, for example, 10 -10 M to 10 -11 M.

[0043] The term "substitution" as used herein in reference to an amino acid means that at least one amino acid residue in an amino acid sequence is replaced with another, different "replacement" amino acid residue. The term "insertion" as used herein in reference to an amino acid means the incorporation of at least one additional amino acid into an amino acid sequence. While an insertion typically consists of the insertion of 1 or 2 amino acid residues, larger "peptide insertions" can also be made, e.g., insertions of about 3 to 5 or even up to about 10, 15 or 20 amino acid residues. As disclosed above, the inserted residues can be naturally occurring or non-naturally occurring. The term "deletion" as used herein in reference to an amino acid means the removal of at least one amino acid residue from an amino acid sequence.

[0044] A fusion polypeptide of the present disclosure, or a fragment thereof, can comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A "conservative amino acid substitution" is where an amino acid residue is replaced with an amino acid residue having a side chain of similar charge, size, and / or shape. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in the present context, an essential or non-essential amino acid residue in a fusion polypeptide or linker is preferably replaced with another amino acid residue from the same side chain family. In certain embodiments, an amino acid stretch can be replaced with a stretch of different order and / or composition of structural similarity and side chain family membership. Alternatively, in certain embodiments, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ability of the fusion polypeptides of the invention to bind to a desired target.

[0045] The term "antibody" as used herein includes intact antibodies and any antigen binding fragment (i.e., "antigen binding portion," "antigen binding polypeptide," or "immunobinder"), or single chains thereof. An "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable (VH) region and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable (VL) region and a light chain constant region (CL). Each VH and VL region contains three regions of high sequence variability, referred to as hypervariable regions or complementarity determining regions (CDRs), CDR1, CDR2, and CDR3. The sequence variability of the regions outside of the CDR regions in the VH and VL regions is relatively conservative and is referred to as the framework region (FR). Each of VH or VL has four framework regions, designated FR1, FR2, FR3, and FR4.

[0046] The five major classes of immunoglobulins are IgA, IgD, IgE, IgG, and IgM, and these major classes can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The CL of different types (kappa or lambda) of immunoglobulins are essentially identical in length, but the CH of different classes of immunoglobulins differ in length, e.g., IgG, IgA and IgD include CHI, CH2 and CH3, while IgM and IgE include CHI, CH2, CH3 and CH4. The hinge region, which is located between CHI and CH2, is rich in proline and is flexible, allowing for changes in the distance between antigen binding sites, which is advantageous for antibodies that bind to epitopes located at different positions. The hinge region is susceptible to hydrolysis by papain, pepsin, and the like, resulting in different hydrolysis fragments. The site of papain hydrolysis of Ig is near the N-terminus of the two heavy chains connected by a disulfide bond in the hinge region, which can cleave Ig into two identical Fab fragments and one Fc fragment. The Fab fragment is a fragment antigen binding (Fab), which is composed of a complete light chain and the VH and CHI domains of the heavy chain.

[0047] The term "Fc region" of an "antibody," "immunoglobulin," or "Ig" as used herein refers to the C-terminal constant domains or constant region (CH) of an immunoglobulin heavy chain or fragment thereof that is a fragment crystallizable (Fc), which is composed of the CH2 and CH3 domains of an Ig. In the present context, the term "Fc region" includes wild-type Fc regions and variant Fc regions. A wild-type Fc region denotes an amino acid sequence that is identical to the amino acid sequence of a naturally occurring Fc region. SEQ ID NO: 9 shows the Fc region of wild-type human IgGl. The term "variant (human) Fc region" denotes an amino acid sequence that differs from that of a wild-type (human) Fc region by virtue of at least one amino acid mutation. In some embodiments, the variant Fc region has at least one amino acid mutation, e.g., 1 to 10 amino acid mutations, compared to the wild-type Fc region. In some embodiments, the variant Fc region has 1 to 3 amino acid mutations compared to the wild-type Fc region.

[0048] The term "heterodimer" as used herein refers to a fusion polypeptide of the present application formed by two different fusion polypeptides of the present application.

[0049] The term "individual" or "subject" as used herein refers to a mammal, including, but not limited to, humans and non-human mammals, for example, mammals include, but are not limited to, domesticated animals (such as cows, horses, dogs, sheep, goats, cats, and dogs), primates (such as humans and monkeys), and rodents (such as rabbits, mice, and rats).

[0050] As used herein, numerical ranges are understood to be inclusive of the numbers from the lower and upper ends of the ranges. For example, a range from 1 to 20 should be understood to include any number from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0051] The term "linker" as used herein refers to a (peptide) linker of natural and / or synthetic origin, consisting of linear amino acids. Each domain in the bispecific fusion polypeptides of the present application can be linked by a linker, wherein each linker is fused and / or otherwise linked (e.g., via a peptide bond) to at least two polypeptides or domains. In some embodiments, the amino acid sequences of all linkers present in the bispecific fusion polypeptides of the present application are identical. In other embodiments, the amino acid sequences of at least two linkers present in the bispecific fusion polypeptides of the present application are different. The linker should have a length suitable to link two or more monomeric domains in this manner, the linker being able to ensure that the different domains to which it is linked fold correctly and are suitably presented to functionally exert their biological activity. In different embodiments, the linker has a flexible conformation. Suitable flexible linkers include, for example, having glycine, glutamine, and / or serine residues. In some embodiments, the amino acid residues in the linker can be arranged in small repeating units of up to 5 amino acids, for example having the amino acid sequence set forth in GGGSGG (SEQ ID NO: 6) or GGGSGGG (SEQ ID NO: 7).

[0052] "Percent (%) sequence identity" with respect to a reference amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that have the same amino acid residues as in the reference amino acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, exclusive of any conservative substitutions as part of the sequence identity. To determine percent amino acid sequence identity, sequences can be aligned using a variety of methods within the skill in the art, for example, using BLAST, ALIGN or Megalign (DNASTAR) software. Those of skill in the art will be able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0053] The term "pharmaceutically acceptable carrier" as used herein refers to a nontoxic component of a pharmaceutical formulation other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0054] The term "treatment" as used herein refers to reducing and / or ameliorating a disorder and / or symptoms associated therewith, and preventing the worsening of the symptoms of the disorder. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing disease progression, ameliorating or palliating one or more symptoms of the disease, and remission or improved prognosis. It will be understood, however, that the effects of treatment are not required to be total or complete.

[0055] The term "effective amount" as used herein refers to the amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

[0056] Examples and figures are provided below to help understand the present application. It should be understood, however, that these examples and figures are only intended to illustrate the present application, but do not constitute any limitation. The actual scope of protection of the present application is set forth in the claims. It should be understood that any modification and change can be made without departing from the spirit of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 The structural schematic diagram of three subtypes of VEGFR family, VEGFR1, VEGFR2 and VEGFR3 is shown.

[0058] Figure 2 The structural schematic diagram of the constructed fusion polypeptide chain is shown. The same domains as in Figure 1 The same domains as in the same pattern are filled, and "*" indicates the mutation in Ig-like domain 1 of VEGFR3.

[0059] Figure 3 The schematic diagram of the expressed fusion polypeptide dimer is shown.

[0060] Figure 4 The binding of the bispecific protein of the present application to VEGFA and VEGFC is shown. (A) shows the ELISA results of the binding of the bispecific proteins BiFpC1, BiFpC2, BiFpC3, BiFpC4 and BiFpC5 of the present application to VEGFA; (B) shows the ELISA results of the binding of the bispecific proteins of the present application to VEGFC; (C) shows the ELISA results of the binding of the bispecific proteins BiFpC2 and BiFpC6 of the present application to VEGFA (VA) or VEGFC (VC).

[0061] Figure 5The bi-specific proteins of the present application, BiFpC2, BiFpC6, were shown to bind to VEGFA and VEGFC simultaneously. (A) BiFpC2 was shown to bind to VEGFA and VEGFC simultaneously. (B) BiFpC6 was shown to bind to VEGFA and VEGFC simultaneously.

[0062] Figure 6 The bi-specific proteins of the present application were shown to inhibit VEGFA or VEGFC induced HUVEC cell proliferation. (A) BiFpC2 was shown to inhibit VEGFA induced HUVEC cell proliferation. (B) BiFpC6 was shown to inhibit VEGFA induced HUVEC cell proliferation. (C) BiFpC2 was shown to inhibit VEGFC induced HUVEC cell proliferation. (D) BiFpC6 was shown to inhibit VEGFC induced HUVEC cell proliferation.

[0063] Figure 7 The bi-specific proteins of the present application were shown to compete for binding to VEGFA and VEGFC. (A) BiFpC2 was shown to compete with Eylea for binding to VEGFA. (B) BiFpC6 was shown to compete with VEGFR2 for binding to VEGFA. (C) BiFpC2 was shown to compete with VEGFR3-Fc for binding to VEGFC. (D) BiFpC6 was shown to compete with VEGFR3 for binding to VEGFC. DETAILED DESCRIPTION

[0064] In order to make the objectives, technical solutions, and advantages of the present application clearer, the following further describes the present application in conjunction with examples. The specific examples described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessarily obscuring the concepts of the present disclosure. Such structures and technologies are also described in many publications, for example, Sambrook, J., Fritsch, E.F., and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

[0065] Example 1: Construction of expression plasmids

[0066] In this example, the Ig-like domain 2 of VEGFR1, the Ig-like domain 3 of VEGFR2, the Ig-like domains 1 to 3 of VEGFR3, and a single chain antibody fragment (scFv) against VEGFA as shown in SEQ ID NO: 16 were combined with human IgG1-Fc as shown in Table 1 below.

[0067] Table 1: Sequence constitution of fusion polypeptides.

[0068]

[0069]

[0070] Note: "R1D2" means Ig-like domain 2 of VEGFR1. "R2D3" means Ig-like domain 3 of VEGFR2. "R3D1D2D3" means Ig-like domain 1, Ig-like domain 2 and Ig-like domain 3 of VEGFR3. "Fc" means Fc region of human immunoglobulin IgG1. "Fc(FL)" means Fc (full-length), which is Fc region of human IgG1, with 5 amino acids "EPKSC" and one more disulfide bond in hinge region than "Fc". The mutation site shown is numbered by amino acid from N-terminus of the fusion polypeptide chain.

[0071] According to the structure shown in the above table, eight fusion polypeptide chains were obtained, and their amino acid sequences are as follows, respectively:

[0072] BiFpC1: SEQ ID NO: 17;

[0073] BiFpC2: SEQ ID NO: 18;

[0074] BiFpC3 chain 1: SEQ ID NO: 19;

[0075] BiFpC3 chain 2: SEQ ID NO: 20;

[0076] BiFpC4: SEQ ID NO: 21;

[0077] BiFpC5 chain 1: SEQ ID NO: 22;

[0078] BiFpC5 chain 2: SEQ ID NO: 23;

[0079] BiFpC6: SEQ ID NO: 24.

[0080] Figure 2 The structural schematic diagram of the above fusion polypeptide chains is shown, and "*" in the figure indicates that there is a mutation in the amino acid sequence.

[0081] The nucleic acid sequences encoding the above eight fusion polypeptide chains were inserted into the transient expression vector ptt5 or the stable expression vector FTP002, respectively, using standard molecular biology techniques (see, e.g., Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press). Specifically, BiFpC1, BiFpC2, BiFpC4 and BiFpC6 were inserted into the multiple cloning site downstream of the EF-1 alpha promoter of the vector FTP002; BiFpC3 chain 1, BiFpC3 chain 2, BiFpC5 chain 1 and BiFpC5 chain 2 were inserted into the multiple cloning site of the vector ptt5. Eight plasmid constructs FTP002-BiFpC1, FTP002-BiFpC2, ptt5-BiFpC3 chain 1, ptt5-BiFpC3 chain 2, FTP002-BiFpC4, ptt5-BiFpC5 chain 1, ptt5-BiFpC5 chain 2, FTP002-BiFpC6 were obtained. The correctness of the inserted sequences was verified by sequencing.

[0082] Example 2: Expression of bispecific proteins

[0083] The stable expression vectors FTP002-BiFpC1, FTP002-BiFpC2, FTP002-BiFpC4, FTP002-BiFpC6 constructed in Example 1 were transfected into the Chinese hamster cell line CHO HD-BIOP3 (Horizon Discovery Ltd., USA) using the cationic polymer transfection reagent (ATG fect solution, Cat. No. ATO00017, Wuhan Pu Jian Bio-technology) according to the manufacturer's instructions. FectoCHO medium (Cat. No. ATO00018, Wuhan Pu Jian Bio-technology) containing 4 mM L-glutamine (Cat. No. A3704, Applichem) was used for the transfection. The cells were selected in the presence of 1 mg / ml Geneticin (Cat. No. 10131-017, InvivoGen) for 2-3 weeks. The cell lines were expanded and frozen in liquid nitrogen. TMCHO HD-BIOP3 cells were cultured in CD medium (Cat# 716-06L, Polyplus). The expression vector encodes the EF-1 alpha promoter and the gene for glutamine synthetase (GS). The expression of GS allows the biochemical synthesis of glutamine, an amino acid required for the growth of CHO HD-BIOP3 cells. After transfection, cells were subjected to mixed selection with 25 μΜ L-methionine sulfoximine (MSX, Cat# M111096, Aladdin, China). The inhibition of GS by MSX was used to increase the stringency of selection. Cells with the integration of the expression vector cDNA into the transcriptionally active region of the host cell genome were selected against CHO HD-BIOP3 wild type cells, which do not express endogenous levels of GS. Transfected cells were seeded in 96-well plates at low density (about 0.8 cells / well) to allow the stable expression of cells to approach exponential growth. Master wells were screened for bispecific protein expression and then expanded in serum-free Dynamis medium (Cat# A26175-02, Gibco, USA) in suspension culture for the expression of bispecific proteins.

[0084] The transient expression vectors ptt5-BiFpC3 chain 1 and ptt5-BiFpC3 chain 2 were co-transfected into HEK-293 cell line using cationic polymer transfection reagent (ATG fect solution, Cat# ATO00017, Wuhan Pk Biotech) according to the manufacturer’s instructions. The transiently expressed bispecific proteins were expressed in HEK-293 cells, which were cultured in an incubator at 37 °C, 95% relative humidity, 5% CO2 until the cells were harvested at a certain degree of cell viability (without the selection and culture of monoclonal cells). The transient expression vectors ptt5-BiFpC5 chain 1 and ptt5-BiFpC5 chain 2 were used to express bispecific proteins in a similar way.

[0085] Figure 3 A schematic diagram of the expressed bispecific protein is shown.

[0086] Example 3: Purification of bispecific protein by affinity chromatography and detection of its expression level and aggregation properties

[0087] The bispecific protein expressed in Example 2 was secreted into the culture medium. The culture medium containing the bispecific protein was applied to a protein A affinity column Unimab 50 (Cat. No. 17010-050100, Nanjing Nucleon Biotech Co., Ltd., Suzhou, China) which had been equilibrated with a phosphate buffer solution (pH 7.4). Then, the bound bispecific protein was eluted by a 0.1 M sodium citrate buffer solution (pH 3.4). The eluted bispecific protein was adjusted to a stable state by adjusting the solution pH with an acid (such as 0.1 M citric acid) or a base (0.1 M Tris), and was placed at 4°C for detection or was placed at -80°C for storage after being aliquoted.

[0088] Take 1 mL of the bispecific protein, and use ultraviolet spectrophotometry according to the “Chinese Pharmacopoeia 2020 Edition” to record the absorption light at 280 nm, divide by the absorption coefficient of the bispecific protein, and obtain the concentration of the bispecific protein. The expression amount of the bispecific protein can be calculated using the formula: concentration (mg / mL) x affinity elution volume (mL) / CHO cell supernatant volume (L) = bispecific protein expression amount (mg / L). The expression amount can also be expressed by determining the cell supernatant titer, and the experimental method is provided by Nonsame (Chengdu) Biotechnology Co., Ltd.

[0089] In addition, the aggregation characteristics of the expressed bispecific protein were detected. The expressed candidate bispecific protein was detected by size exclusion chromatography (SEC) HPLC. The sample to be tested was diluted to 1.0 mg / mL, and 10 μL (10 μg) was injected for analysis on a size exclusion column (Sepax Zenix-C SEC-300). The flow rate was 0.65 mL / min, the column temperature was 35°C, the detection was at 214 nm, and the mobile phase was 200 mM phosphate buffer + 300 mM NaCl, pH 7.0. The bispecific protein multimer, main peak, and fragments were determined by integrating the appropriate peaks.

[0090] Table 2: Expression amount and aggregation characteristics of the candidate bispecific protein after affinity chromatography.

[0091]

[0092]

[0093] According to the data in Table 2, it can be seen that the expression levels of the bispecific proteins with different structures differ greatly, wherein the expression levels of BiFpC2, BiFpC5 and BiFpC6 are relatively high, more than 25 mg / L, while the expression levels of other bispecific proteins are relatively low, only about 1 / 5 of the expression levels of the aforementioned three bispecific proteins. In addition, the purities of the protein samples obtained after one-step affinity purification of different structures also differ greatly, for example, BiFpC5, although the expression level is high, but the purity of the main peak (the purity of the main peak in the detection result of SEC-HPLC can reflect the target product) is obviously low, and the main impurity is a fragmented molecule; for another example, BiFpC3, the expression level is low, and at the same time contains more (more than 20%) multimer impurities. The above all shows that different molecular structure designs have great differences in expression level and purity.

[0094] Example 4: Detection of the binding of the bispecific protein of the application to VEGFA and VEGFC

[0095] This example detects the binding of the bispecific protein of the application to VEGFA and VEGFC by ELISA, respectively.

[0096] 4.1 Detection of the binding of the bispecific protein to VEGFA

[0097] A 96-well plate was coated with 50 pL / well of 0.2 pg / mL human VEGFA (Cat. No. Z03073-10 pg, Genscript, China) in phosphate buffered saline (1 x PBS), the plate was sealed and coated overnight at 4°C. After washing each well twice with wash buffer 1 x PBST (8.01 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4, 0.05% Tween-20, pH 7.4), 200 pL / well of PBST containing 1% bovine serum albumin BSA (BIOFROXX) was added for blocking. After sealing the 96-well plate and incubating for 2 hours at 37°C, each well was washed twice with 1 x PBST to remove the blocking reagent. The bispecific proteins of the application BiFpC1, BiFpC2, BiFpC3, BiFpC4 and BiFpC5, and the positive control Eylea were added to each well (50 pL / well) in a 4-fold serial dilution (diluted in 1 x PBS) and incubated for 1 hour at 37°C. Then, each well was washed three times with 1 x PBST to remove unbound bispecific proteins. Subsequently, 100 pL / well of 1 :8000 diluted HRP-conjugated goat anti-human IgG (H+L) (Cat. No. Ab6858, Abeam, USA) was added and incubated for 0.5 hours at 37°C. After washing the plate three times with 1 x PBST, TMB substrate (Solarbio, 100 pL / well) was added. Incubation was performed for 10 minutes at 37°C in the dark. Finally, 100 pL of 2 M sulfuric acid was added to each well to stop the reaction. The optical density at 450 nm was measured using a microplate reader Synergy Lx (BioTek Instruments, USA).EC 50 The concentration of the protein sample needed to represent 50% binding of VEGFA by the bispecific protein, the positive control or the negative control protein. The tests were performed in duplicate.

[0098] The results are shown in Figure 4 A. As shown in Figure 4 A, the bispecific proteins of the application BiFpC1, BiFpC2, BiFpC3, BiFpC4 and BiFpC5 could bind to VEGFA in a concentration-dependent manner, with binding effects comparable to the positive control Eylea.

[0099] 4.2 Detection of the binding of the bispecific proteins to VEGFC

[0100] A 96-well plate was coated with 50 pL / well of 2.0 pg / mL VEGFC (Cat. No. Z03286-10 pg, Genscript, China) in phosphate buffered saline (1 x PBS), the plate was sealed and coated overnight at 4°C. After washing each well twice with wash buffer 1 x PBST (8.01 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4, 0.05% Tween-20, pH 7.4), 200 pL / well of PBST containing 1% BSA (BIOFROXX) was added for blocking. After sealing the plate and incubation at 37°C for 2 hours, each well was washed twice with 1 x PBST to remove blocking reagent. Four-fold serial dilutions of the bispecific proteins BiFpC1, BiFpC2, BiFpC3, BiFpC4 and BiFpC5 (diluted in phosphate buffered saline 1 x PBS), as well as the positive control OPT-302 (0.04-40 nM, diluted in phosphate buffered saline 1 x PBS) were added to each well (100 pL / well) and incubated at 37°C for 1 hour. Each well was washed three times with 1 x PBST to remove unbound bispecific proteins, followed by the addition of 100 pL / well of 1 :8000 diluted HRP-conjugated goat anti-human IgG (H+L) (Cat. No. Ab6858, Abeam, USA) and incubation at 37°C for 0.5 hours. The plate was washed three times with 1 x PBST and TMB substrate (Solarbio, 100 pL / well) was added. Incubation was performed for 10 minutes in the dark at 37°C. The reaction was finally stopped by adding 100 pL 2M sulfuric acid to each well. The optical density was immediately measured using a colorimetric microplate reader Synergy Lx (BioTek Instruments, USA) set to 450 nm. 50 The concentration of the protein sample needed to represent 50% binding of VEGFC by the bispecific protein, the positive control or the negative control protein. The tests were performed in duplicate.

[0101] The results are shown in Figure 4 B. It can be seen from Figure 4 B that the BiFpC1, BiFpC2, BiFpC3 and BiFpC4 of the present application bind to VEGFC in a concentration-dependent manner with a binding strength comparable to the positive control OPT-302. The bispecific protein BiFpC5 does not substantially differ from VEGFC binding.

[0102] Furthermore it can be seen from Figure 4As can be seen from A-B, different structural forms have a great difference in the binding force to the two target targets: for the VEGFA target, BiFpC1, BiFpC2 and BiFpC3 use the same binding domain, and are consistent with Eylea, but the binding force of each structure to VEGFA is different; at the same time, BiFpC4 and BiFpC5 use another same binding domain, but the binding force to VEGFA is also different. And the existing literature shows that the binding domain used by BiFpC1, BiFpC2 and BiFpC3 has a much higher binding force to VEGFA than the binding domain used by BiFpC4 and BiFpC5, but from the data obtained from the experiment of the present embodiment, it can be seen that BiFpC4 and BiFpC5 have higher affinity; for the VEGFC target, BiFpC1, BiFpC2, BiFpC3, BiFpC4, BiFpC5 and the control molecule OPT302 use the same VEGFC binding domain, but the binding force to the target is still very different, especially BiFpC5, which completely loses the binding ability to VEGFC. The above shows that even if the same target binding domain is used, due to the difference in the overall structure, different molecules will exhibit completely different target binding abilities, which may increase, decrease, or even be lost.

[0103] 4.3 Detection of the binding of BiFpC2 and BiFpC6 to VEGFA or VEGFC

[0104] This embodiment compares the binding of BiFpC2 and BiFpC6 to the antigens VEGFA or VEGFC.

[0105] Coat a 96-well plate with 100 μL / well of 0.5 μg / mL VEGFA (catalog number: Z03073-10 μg, Genscript, China) in carbonate buffer (1×CBS). Seal the plate and coat overnight at 4°C. Wash each well twice with 1×PBST washing buffer (8.01 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4, 0.05% Tween-20, pH 7.4), then block with 200 μL / well of PBST containing 1% BSA (BIOFROXX). Seal the plate and incubate at 37°C for 2 hours, then wash each well twice with 1×PBST to remove the blocking reagent. Serial dilutions of BiFpC2 or BiFpC6 (all diluted in 1×PBS phosphate buffer) were added to each well (100 μL / well) and incubated at 37°C for 1 hour. Each well was washed three times with 1×PBST to remove unbound bispecific proteins, followed by the addition of 100 μL / well of a 1:10000 dilution of HRP-conjugated goat anti-human IgG (H+L) (catalog number Ab97225, Abcam, USA), and incubated at 37°C for 0.5 hours. The plate was washed three times with 1×PBST, and then TMB substrate (Tiangen, 100 μL / well) was added. Incubation was performed in the dark at 37°C for 10 minutes. Finally, 50 μL of 2M sulfuric acid was added to each well. The optical density was immediately measured using a Synergy Lx microplate reader set to 450 nm (BioTek Instruments, USA). 50 This indicates the protein sample concentration required for the bispecific protein to bind 50% of the VEGFA amount. All tests were performed in duplicate.

[0106] Coat a 96-well plate with 100 μL / well of VEGFC (catalog number: Z03286-10 μg, Genscript, China) at 2.0 μg / mL in carbonate buffer (1×CBS), seal the plate, and incubate overnight at 4°C. Wash each well twice with 1×PBST washing buffer (8.01 g NaCl, 0.2 g KCl, 1.42 g Na₂HPO₄, 0.27 g KH₂PO₄, 0.05% Tween-20, pH 7.4), then block with 200 μL / well of PBST containing 1% BSA (BIOFROXX). Seal the plate and incubate at 37°C for 2 hours, then wash each well twice with 1×PBST to remove the blocking reagent. Serial dilutions (4-fold) of bispecific proteins BiFpC2 and BiFpC6 (diluted in 1×PBS phosphate buffer) were added to each well (100 μL / well) and incubated at 37°C for 1 hour. Each well was washed three times with 1×PBST to remove unbound bispecific proteins, followed by the addition of 100 μL / well of 1:10000 dilution of HRP-conjugated goat anti-human IgG (H+L) (catalog number Ab97225, Abcam, USA), and incubated at 37°C for 0.5 hours. The plate was washed three times with 1×PBST, and TMB substrate (Tiangen, 100 μL / well) was added. The plate was incubated in the dark at 37°C for 10 minutes. Finally, 50 μL of 2M sulfuric acid was added to each well to stop the reaction. The optical density was immediately measured using a Synergy Lx microplate reader set to 450 nm (BioTek Instruments, USA). EC 50 This indicates the protein sample concentration required for the bispecific protein to bind 50% of the VEGFC amount. All tests were performed in duplicate.

[0107] like Figure 4 As shown in C, the bispecific proteins BiFpC2 and BiFpC6 of the present invention can bind to VEGFA or VEGFC in a concentration-dependent manner, and the two bispecific proteins have considerable affinity for VEGFA and VEGFC.

[0108] Example 5: Detection of the binding affinity of bispecific proteins to VEGFA and VEGFC

[0109] As described above, VEGFA can bind to VEGFR1 and VEGFR2, and VEGFC can bind to VEGFR2 and VEGFR3. To demonstrate that the candidate bispecific proteins constructed in this invention can specifically bind to VEGFA and VEGFC, this embodiment further tested the binding affinity of the candidate bispecific proteins using surface plasmon resonance (SPR). Taking into account the aforementioned data, the structures BiFpC1, BiFpC2, BiFpC4, and BiFpC6, with relatively ideal data, were further selected, and their target affinity was evaluated using Biacore.

[0110] 5.1 Detection of the binding affinity between bispecific proteins and VEGFA

[0111] On a Biacore S200 or Biacore 8000 instrument (GE Healthcare), surface plasmon resonance assays were performed at an analysis temperature of 25°C using HEPES-EP+ (GE Healthcare, 10 mM Hepes pH 7.4 + 150 mM NaCl + 3 mM EDTA + 0.05% (v / v) surfactant P20) as the run buffer. The binding affinity of candidate bispecific proteins BiFpC1, BiFpC2, BiFpC4, and BiFpC6 to human VEGFA (catalog number: Z03073-10 μg, Genscript, China) was determined. 10 μg / mL of the four candidate proteins, along with the VEGFA-specific positive control Eylea (Bayer, Germany), were captured onto a protein A chip (catalog number: 29157555, GE Healthcare) via protein A conjugation. In addition, human VEGFA was diluted in running buffer to prepare human VEGFA solutions with concentrations of 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 and 0 (blank) nM.

[0112] Each analysis cycle consisted of: (1) capturing the candidate proteins in flow cell 2 (Fc2); (2) injecting VEGFA solution into flow cells 1 and 2 (Fc1 and Fc2) at 30 μL / min (120 s); (3) monitoring the dissociation phase by flowing 1×HBS-EP+ buffer through the chip at 30 μL / min for 360 s; and (4) regenerating the protein A chip surface by injecting 10 mM glycine hydrochloride buffer, pH 1.5. (Biacore was used.) TM The Insight Evaluation software uses a standard dual reference and fit with a 1:1 binding model to process data in order to determine the binding rate (k). a ) and dissociation rate (k d According to equation K)D =k d / k a Calculate the equilibrium dissociation constant (K) D The results are shown in Table 3 below.

[0113] Table 3: Binding affinity of candidate bispecific proteins to VEGFA.

[0114]

[0115] As can be seen from the results in Table 3, BiFpC1, BiFpC2, BiFpC4, and BiFpC6 of the present invention all exhibit high binding affinity for VEGFA. D Similar to Eylea, it can reach 10. -11 M level.

[0116] 5.2 Detection of the binding affinity between bispecific proteins and VEGFCs

[0117] The binding affinity of the bispecific protein of the present invention to human VEGFCs was determined by surface plasmon resonance assay using HEPES-EP+ (GE Healthcare, 10 mM Hepes pH 7.4 + 150 mM NaCl + 3 mM EDTA + 0.05% (v / v) surfactant P20) as the run buffer and an analysis temperature set to 25 °C on a Biacore S200 or Biacore 8000 instrument (GE Healthcare).

[0118] Bispecific proteins BiFpC1, BiFpC2, BiFpC4, and BiFpC6, along with the positive control OPT-302 which specifically recognizes VEGFC, were captured onto a Protein A chip (catalog number: 29157555, GE Healthcare) at a fixed concentration of 10 μg / mL via conjugation with Protein A. Human VEGFC concentrations of 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, and 0 (blank) nM were prepared by dilution in running buffer (catalog number: Z03286-10 μg, Genscript, China). Each analysis cycle consisted of the following steps: (1) capturing the bispecific protein in flow cell 2 (Fc2); (2) injecting VEGFC into flow cells 1 and 2 (Fc1 and Fc2) at 30 μL / min (120 s); (3) flowing 1×HBS-EP+ buffer through the chip at 30 μL / min for 360 s to monitor the dissociation phase; and (4) regenerating the chip surface with 10 mM glycine hydrochloride buffer, pH 1.5. Biacore was used. TMThe Insight Evaluation software uses a standard dual reference and fit with a 1:1 binding model to process data in order to determine the binding rate (k). a ) and dissociation rate (k d ). For example, from relation K D =k d / k a Calculate the equilibrium dissociation constant (K) D The results are shown in Table 4 below.

[0119] Table 4: Binding affinity of candidate bispecific proteins to VEGFC.

[0120]

[0121]

[0122] As can be seen from the results in Table 4, the BiFpC1, BiFpC2, BiFpC4, and BiFpC6 of the present invention all exhibit high binding affinity to VEGFC. D It can reach 10 -10 The M-level, with BiFpC2 and BiFpC6 even reaching 10. -11 Grade M, far exceeding the positive control OPT-302 (10 -10 The M level (higher M level) demonstrates a higher affinity for VEGFC.

[0123] The results show that all four tested structures exhibit strong VEGFA affinity, but their affinity for VEGFC varies significantly. BiFpC2 and BiFpC6 demonstrate extremely strong VEGFC affinity, approximately ten times that of the control molecule OPT-302. This further illustrates that even using the same structural domains, differences in molecular structure can greatly influence a molecule's binding ability to its target molecule.

[0124] Example 6: Verification of the simultaneous binding of the bispecific protein of the present invention to VEGFA and VEGFC

[0125] This embodiment tested whether the bispecific proteins BiFpC2 and BiFpC6 of the present invention could bind to VEGFA and VEGFC simultaneously.

[0126] On a Biacore 8000 instrument, surface plasmon resonance assays were performed using HEPES-EP+ (GE Healthcare, 10 mM Hepes pH 7.4 + 150 mM NaCl + 3 mM EDTA + 0.05% (v / v) surfactant P20) as the run buffer at an analysis temperature of 25 °C to determine the ability of the bispecific proteins BiFpC2 and BiFpC6 of the present invention to bind to human VEGFA and / or VEGFC.

[0127] The bispecific proteins BiFpC2 and BiFpC6 expressed in Example 3 were captured at a fixed concentration of 10 μg / mL onto the Protein A chip (catalog number: 29157555, GE Healthcare) via conjugation with Protein A. Then, using the ABA continuous injection mode, the specific operation is as follows: capture the bispecific protein in flow cell 2 (Fc2) for 30-40 seconds, then inject 20 nM VEGFA or 500 nM VEGFC into flow cells 1 and 2 (Fc1 and Fc2) at a rate of 30 μL / min (120 seconds). Next, inject 500 nM VEGFC or 20 nM VEGFA into flow cells 1 and 2 (Fc1 and Fc2) at a rate of 30 μL / min (120 seconds). Finally, regenerate the chip surface with 10 mM glycine hydrochloride buffer (pH 1.5) at a rate of 30 μL / min (60 seconds). (Biacore was used.) TM The Insight Evaluation software analyzes the data using a standard dual-reference and fit model with a 1:1 combination, and the results are shown below. Figure 5 middle.

[0128] like Figure 5 As shown in Figure A, the increase in resonance units from the two ligands bound to the bispecific protein (initial 22.3 RU from VEGFA, followed by an additional 8.8 RU from human VEGFC), and the increase in resonance units through another injection sequence (initial 17.3 RU from VEGFC, followed by an additional 12.8 RU from human VEGFA), confirm that the bispecific protein BiFpC2 of the present invention can simultaneously bind to human VEGFA and human VEGFC. Furthermore, after the bispecific protein BiFpC2 binds to the two ligands in different injection sequences, the cumulative sum of resonance units is 31.1 RU and 30.1 RU, respectively, with no significant difference between the two.

[0129] like Figure 5As shown in Figure B, the increase in resonance units from the two ligands bound to the bispecific protein (initial 30.6 RU from VEGFA, followed by an additional 9.8 RU from human VEGFC), and the increase in resonance units through another injection sequence (initial 17.6 RU from VEGFC, followed by an additional 22.0 RU from human VEGFA), confirms that the other bispecific protein BiFpC6 of the present invention can simultaneously bind to human VEGFA and human VEGFC. Furthermore, the cumulative sum of resonance units after BiFpC6 binds to the two ligands in different injection sequences is 40.3 RU and 39.9 RU, respectively, with no significant difference between the two.

[0130] Example 7: Stability determination of the bispecific protein of the present invention at different temperatures

[0131] The bispecific proteins BiFpC2 and BiFpC6 obtained in Example 3 were subjected to affinity chromatography and then placed in a 20 mM citrate-sodium citrate buffer system at pH 4.8. The samples were then stored and collected according to the stability protocol described below.

[0132] Samples were stored at low temperature (4°C) for 1, 3, 6, 15, 30, 60, and 90 days; at room temperature (25°C) for 3, 6, 15, and 30 days; and at high temperature (40°C) for 1, 3, and 6 days. After sampling at time zero and at each sampling point, bispecific protein samples were analyzed by size exclusion chromatography (SEC) HPLC based on the percentage of the target protein's molecular weight (main peak %). Specifically, the sample was diluted to 1.0 mg / mL and 10 μL (10 μg) was injected into a molecular sieve column (Sepax Zenix-C SEC-300) for analysis. The flow rate was 0.65 mL / min, the column temperature was 35°C, and detection was performed at 214 nm. The mobile phase was 200 mM phosphate buffer + 300 mM NaCl, pH 7.0. The bispecific protein peak (main peak), polymorphic peaks, and fragment peaks were determined by integrating appropriate peaks. The results were obtained by analyzing the chromatography using ChemStation, and the percentage of the main peak was calculated using the ratio of the AUC of the peaks eluted between polymeric and fragmentary peaks to the total AUC. The results are shown in Table 5 below.

[0133] Table 5: Stability of bispecific proteins BiFpC2 and BiFpC6 at different temperatures.

[0134]

[0135]

[0136] The results in the table above show that the bispecific proteins BiFpC2 and BiFpC6 are stable under both room temperature and low temperature conditions. Without the addition of other excipients, after 30 days at room temperature (25°C), the peak percentage of BiFpC2 changed from 84.2% to 81.1%, and that of BiFpC6 changed from 87.0% to 84.6%. After 90 days at a low temperature (4°C), the peak percentage of BiFpC2 changed from 84.2% to 84.9%, and that of BiFpC6 changed from 87.0% to 85.8%. In contrast, both BiFpC2 and BiFpC6 are less stable at high temperatures. After 6 days at 40°C, the peak percentage of BiFpC2 changed from 84.2% to 35.3%, and that of BiFpC6 changed from 87.0% to 49.7%. Therefore, it is important to avoid exposing the bispecific proteins to high temperatures during the process. BiFpC6 exhibits better stability than BiFpC2.

[0137] Example 8: Inhibition of VEGFA or VEGFC-induced HUVEC cell proliferation by the bispecific protein of the present invention.

[0138] HUVEC cells are human umbilical vein endothelial cells and are the most commonly used cell line for in vitro detection of drugs related to the VEGF / VEGFR signaling pathway. The HUVEC cells used in this example were purchased from Sciencell (catalog number #8000).

[0139] 8.1 Effects of VEGFA on HUVEC cell proliferation

[0140] VEGFA is an endothelial growth factor that initiates intracellular signaling pathways by binding to cell membrane surface receptors VEGFR1 and VEGFR2, and participates in physiological and pathological processes such as cell proliferation and angiogenesis.

[0141] The dosage range of the bispecific protein, from 0.1 ng / mL to 1 μg / mL, was evaluated. 50 μL of each test concentration of the bispecific protein BiFpC2 was added to wells containing 50 μL of 10 ng / mL (final concentration) VEGFA. The test was performed in duplicate. Eylea, a commercially available neutralizing protein of VEGFA, was used as a positive control, and wells containing only the assay medium served as solvent blank controls. Eylea was tested at the same concentration range as the bispecific protein BiFpC2. 96-well plates containing the VEGFA / protein mixture were incubated at 37°C, 95% relative humidity, and 5% CO2 for 60 minutes.

[0142] HUVEC cells were cultured in ECM complete medium (containing 5% FBS, penicillin G (1×), streptomycin (1×), and endothelial cell growth additive ECGS (1×)) at 37°C in a 5% CO2 incubator. After 24 hours, the cells were incubated with 1× DPBS (calcium-free) 2+ Mg 2+ Wash the cells and digest them with 1 mL of 0.6% trypsin / EDTA to detach them from the bottom of the culture flask. Then, add 9 mL of ECM complete medium to stop the digestion reaction. Subsequently, centrifuge the cells at 1000×g at room temperature for 5 minutes. Resuspend the cell clumps in assay medium (ECM assay medium containing 0.5% FBS). Measure the cell density using an automated cell counter (Countstar, IC1000, Macody). After adjusting the density, add 5000 cells (in 100 μL) / well to the center well of a 96-well plate, and add DPBS to the surrounding wells to prevent medium evaporation during culture. Incubate the 96-well plate overnight in a tissue culture incubator (37°C, 95% relative humidity, 5% CO2). After 24 hours, add 100 μL of protein / VEGFA mixture to the HUVEC cells cultured in the 96-well plate and continue incubation for another 72 hours.

[0143] After the reaction is complete, use The cell viability assay kit (Promega, USA) was used to test HUVEC cell viability according to the product instructions. Simply put, the assay kit was brought to room temperature... Buffer solution is injected into the syringe Substrate, mix well, and add 100 μL / well to the cells in a 96-well plate from which the 100 μL / well culture medium has been discarded. Incubate at room temperature for 10 minutes, then measure the chemiluminescence using a Synergy Lx microplate reader (BioTek Instruments, USA). Results are shown below. Figure 6 In A. Figure 6 The relative luminescence values ​​in A were calculated using a four-parameter inhibition curve fitting (GraphPad Prism, version 5.0) of the data, where the concentration of either the bispecific protein BiFpC2 or Eylea was used as the IC50 when 50% of the VEGFA-induced response was inhibited. 50 .like Figure 6 As shown in Figure A, the bispecific protein BiFpC2 of this invention inhibits VEGFA-induced HUVEC cell proliferation in a concentration-dependent manner. This demonstrates that the bispecific protein BiFpC2 of this invention can effectively neutralize VEGFA.

[0144] Using a similar method to that used for BiFpC2, the inhibitory effect of BiFpC6 on VEGFA-induced HUVEC cell proliferation was determined and verified. The detection method is described below:

[0145] The dosage range of the bispecific protein, from 0.46 ng / mL to 3 μg / mL (final concentration), was evaluated. 45 μL of the bispecific protein BiFpC6 at each test concentration was added to wells containing 45 μL of 45 ng / mL VEGFA. The test was performed in duplicate. Wells containing only the test medium served as solvent blank controls. 96-well plates containing the VEGFA / protein mixture were incubated at 37 °C, 95% RHU, and 5% CO2 for 60 min.

[0146] HUVEC cells were cultured in T75 cell culture flasks containing ECM complete medium (containing 5% FBS, penicillin and streptomycin (1×), and endothelial cell growth additive ECGS (1×)) and incubated at 37°C in a 5% CO2 incubator. For proliferation assays, 24 hours before adding the test samples, cells were washed with 1×PBS and digested with 3 mL of 0.25% trypsin / EDTA to detach the cells from the bottom of the culture flask. The digestion reaction was then terminated by adding 7 mL of ECM complete medium. Subsequently, the cells were centrifuged at 150×g at room temperature for 5 minutes. The cell clumps were resuspended in assay medium (ECM medium containing 0.5% FBS). Cell density was measured using an automated cell counter (Countstar, IC1000, Macody). After density adjustment, 5000 cells (in 100 μL) were added to the center wells of a 96-well plate, with sterile water added to the surrounding wells to prevent medium evaporation during culture. The 96-well plates were placed in a tissue culture incubator (37°C, 95% relative humidity, 5% CO2) overnight. After 24 hours, a protein / VEGFA mixture (80 μL) was added to the HUVEC cells cultured in the 96-well plates, and incubation continued for another 72 hours, with a final VEGFA concentration of 10 ng / mL.

[0147] After the reaction, HUVEC cell viability was assessed using the CellTiterGlo cell viability assay kit. In short, the assay kit was equilibrated to room temperature, 90 μL of the assay reagent was added to each well, and the cells were shaken on a shaker for 2 minutes. After reacting at room temperature for 10 minutes, the cells were then analyzed using Fluoroskan. TM Chemiluminescence was measured using a ThermoFisher microplate reader. Results are shown below. Figure 6 B in. Figure 6 In inhibition curves were generated using four-parameter curve fitting (GraphPad Prism, version 8.0) in B, where the concentration of the bispecific protein BiFpC6 was used as the IC50 when the 50% VEGFA-induced response was inhibited. 50.like Figure 6 As shown in Figure B, the bispecific protein BiFpC6 of this invention inhibits VEGFA-induced HUVEC cell proliferation in a concentration-dependent manner. This demonstrates that the bispecific protein BiFpC6 of this invention can effectively neutralize VEGFA.

[0148] 8.2 Regarding VEGFC-induced HUVEC cell proliferation

[0149] VEGFC is an endothelial growth factor that initiates intracellular signaling pathways by binding to cell membrane surface receptors VEGFR2 and VEGFR3, and participates in physiological and pathological processes such as cell proliferation and angiogenesis.

[0150] The dosage range of the bispecific protein, from 0.01 μg / mL to 200 μg / mL, was evaluated. 50 μL of each test concentration of the bispecific protein BiFpC2 was added to wells containing 50 μL of 100 ng / mL (final concentration) VEGFC. The assay was performed in duplicate. Monoclonal antibody FTL001 (Sound Biopharma, China) was used as a negative control in the assay, and wells containing only the assay medium served as solvent blank controls. The control monoclonal antibody was tested at the same mass concentration range as the bispecific protein. 96-well plates containing the VEGFC / protein mixture were incubated at 37 °C, 95% relative humidity, and 5% CO2 for 60 minutes.

[0151] HUVEC cells were cultured as usual in complete medium (endothelial cell culture medium ECM containing 5% FBS, penicillin G (1×), streptomycin (1×), and endothelial cell growth additive ECGS (1×)). After 24 hours, the cells were cultured in 1× DPBS (calcium-free). 2+ Mg 2+ The cells were washed and detached from the bottom of the culture flask by digestion with 1 mL of 0.6% trypsin / EDTA, and 9 mL of complete culture medium was added to terminate the digestion reaction. The cells were then centrifuged at 1000×g at room temperature for 5 minutes. The cell clumps were resuspended in assay medium (ECM assay medium containing 0.5% FBS). The cell density was measured using an automated cell counter (Countstar, IC1000, Macody). After adjusting the density, 5000 cells (in 100 μL) were added to the center wells of a 96-well plate, and DPBS was added to the surrounding wells to prevent culture medium evaporation during culture. The 96-well plate was placed in a tissue culture incubator (37°C, 95% relative humidity, 5% CO2) overnight. After 24 hours, a bispecific protein (or monoclonal antibody) / VEGFC mixture (100 μL) was added to the HUVEC cells and incubated for 72 hours.

[0152] After the reaction is complete, according to (Promega, USA) Instructions: Equilibrate the test kit to room temperature before use. Before use, […]. Buffer is injected into the syringe. Substrate and mix well before use. Discard 100 μL of culture medium, then add 100 μL of the prepared assay solution. Incubate at room temperature for 10 minutes, then measure the chemiluminescence using a Synergy Lx microplate reader (BioTek Instruments, USA). Results are shown below. Figure 6 C.

[0153] Figure 6 The relative luminescence value in C was calculated using a four-parameter inhibition curve fitting (GraphPad Prism, version 5.0) of the data, where the concentration of the bispecific protein BiFpC2 or the negative control was used as the IC50 when 50% of the VEGFC-induced response was inhibited. 50 .from Figure 6 As shown in Figure C, the bispecific protein BiFpC2 inhibited VEGFC-induced HUVEC cell proliferation in a concentration-dependent manner. This demonstrates that the bispecific protein of this invention can effectively neutralize VEGFC.

[0154] Using a similar method to BiFpC2, the inhibitory effect of BiFpC6 on VEGFC-induced HUVEC cell proliferation was also verified. The detection method is described below:

[0155] The dosage range of the bispecific protein, from 1.5 ng / mL to 10 μg / mL (final concentration), was evaluated. 45 μL of the bispecific protein BiFpC6 at each test concentration was added to wells containing 45 μL of 225 ng / mL VEGFC. The test was performed in duplicate. Wells containing only the test medium served as solvent blank controls. 96-well plates containing the VEGFC / protein mixture were incubated at 37 °C, 95% ROH, and 5% CO2 for 60 min.

[0156] HUVEC cells were cultured in T75 cell culture flasks containing ECM complete medium (containing 5% FBS, penicillin and streptomycin (1×), and endothelial cell growth additive ECGS (1×)) and incubated at 37°C in a 5% CO2 incubator. For proliferation assays, 24 hours before adding the test samples, cells were washed with 1×PBS and digested with 3 mL of 0.25% trypsin / EDTA to detach the cells from the bottom of the culture flask. The digestion reaction was then terminated by adding 7 mL of ECM complete medium. Subsequently, the cells were centrifuged at 150×g at room temperature for 5 minutes. The cell clumps were resuspended in assay medium (ECM medium containing 0.5% FBS). Cell density was measured using an automated cell counter (Countstar, IC1000, Macody). After density adjustment, 5000 cells (in 100 μL) were added to the center wells of a 96-well plate, with sterile water added to the surrounding wells to prevent medium evaporation during culture. The 96-well plates were placed in a tissue culture incubator (37°C, 95% relative humidity, 5% CO2) overnight. After 24 hours, 80 μL of the protein / VEGFC mixture was added to the HUVEC cells cultured in the 96-well plates, and incubation continued for another 72 hours, with the final VEGFC concentration being 50 ng / mL.

[0157] After the reaction, HUVEC cell viability was assessed using the CellTiterGlo cell viability assay kit. In short, the assay kit was equilibrated to room temperature, 90 μL of the assay reagent was added to each well, and the cells were shaken on a shaker for 2 minutes. After reacting at room temperature for 10 minutes, the cells were then analyzed using Fluoroskan. TM Chemiluminescence was measured using a ThermoFisher microplate reader. Results are shown below. Figure 6 D. Figure 6 Inhibition curves were generated using four-parameter curve fitting (GraphPad Prism, version 8.0) in D, where the concentration of the bispecific protein BiFpC6 was used as the IC50 when the 50% VEGFC-induced response was inhibited. 50 .like Figure 7 As shown in Figure D, the bispecific protein BiFpC6 of this invention inhibits VEGFC-induced HUVEC cell proliferation in a concentration-dependent manner. This demonstrates that the bispecific protein BiFpC6 of this invention can effectively neutralize VEGFCs.

[0158] Example 9: Determination of the competitive binding of the bispecific protein of the present invention to VEGFA and VEGFC

[0159] 9.1 Competitive Combinations of VEGFA

[0160] 1.0 μg / mL Eylea (Bayer, Germany) in 1×PBS (8.01 g NaCl, 0.2 g KCl, 1.42 g Na₂HPO₄, 0.27 g KH₂PO₄, 0.05% Tween-20, pH 7.4) was used to coat 96-well plates at a rate of 100 μL / well. The plates were sealed and incubated overnight at 4°C. After washing each well twice with 1×PBST (1×PBS, 0.05% Tween-20, pH 7.4), 200 μL / well of PBST containing 1% BSA (BIOFROXX) was added for blocking. The plates were sealed and incubated at 37°C for 2 hours, and then each well was washed twice with 1×PBST to remove the blocking reagent. The bispecific protein BiFpC2 of this invention, the positive control Conbercept ophthalmic injection (Kanghong, China), and the negative control human IgG1, kappa Isotype Control (Catalog No. HG1K, Sino Biological, China) were serially diluted 3-fold (in 1×PBS) and pre-incubated with 5 nM VEGFA (Catalog No. Z03073, Genscript, China) dimer at 37°C for 1 hour. After pre-incubation, 100 μL of the co-incubator was added to each well and incubated at 37°C for 1 hour. Each well was washed three times with 1×PBST, and then 100 μL of 1:75000 diluted VEGFA rabbit monoclonal antibody (Catalog No. 11066-R105, Sino Biological, China) was added to each well and incubated at 37°C for 0.5 hours. Each well was washed three times with 1×PBST, followed by the addition of 100 μL / well of a 1:75000 dilution of goat anti-rabbit IgG (H+L) polyclonal antibody (catalog number 511203, ZEN BIO, China). The plate was washed three times with 1×PBST, and 100 μL / well of TMB substrate (catalog number PR1200, Solarbio) was added. The plate was incubated in the dark at 37°C for 10 minutes. Finally, 50 μL of 2M sulfuric acid was added to each well to stop the reaction. The optical density at 450 nm was measured using a Synergy Lx microplate reader (BioTek Instruments, USA). Tests were performed in duplicate. Results are shown below. Figure 7 In A, IC 50 This indicates the concentration of the bispecific protein or positive control when 50% of VEGFA is competitively bound. Figure 7 As shown in Figure A, the bispecific protein BiFpC2 of this invention competitively binds to VEGFA in a concentration-dependent manner with Eylea, and its competitive binding activity is comparable to that of the positive control conbercept. The IC50 of the bispecific protein BiFpC2 is... 50 The concentration was 10.6 nM, and the IC50 of the positive control was...50 The concentration was 16.9 nM. The negative control showed no competitive activity.

[0161] The competitive binding ability of BiFpC6 to VEGFA was also verified using a method similar to that used for BiFpC2. The detection method is described below:

[0162] 0.5 μg / mL VEGFA165 (catalog number Z03073-50, GenScript, China) in carbonate coating buffer (sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, ultrapure water 1000 mL, pH 9.6) was used to coat a 96-well plate at a rate of 100 μL / well. The plate was sealed and coated overnight at 4°C. After washing each well twice with 1×PBST (1×PBS, 0.05% Tween-20, pH 7.4), 260 μL / well of PBST containing 1% BSA (catalog number A8020, Solarbio, China) was added for blocking. The plate was sealed and incubated at 37°C for 1.5 h, and then each well was washed twice with 1×PBST to remove the blocking reagent. The bispecific protein BiFpC6 of this invention was serially diluted 3-fold (in 1×PBST + 0.1% BSA) and mixed thoroughly with 0.5 μg / mL Human VEGFR2 (catalog number KDR-H5227, AcroBiosystems, China) at a 1:1 ratio. 100 μL of this mixture was added to each well of the corresponding microplate and incubated at 37°C for 1 hour. Each well was washed three times with 1×PBST, followed by the addition of 100 μL / well of a 1:8000 dilution of HRP-Anti 6*His antibody (catalog number HRP-66005, Proteintech, China), and incubated at 37°C for 45 minutes. The microplate was then washed three times with 1×PBST, and 100 μL / well of TMB substrate (catalog number PA107-01, Tiangen Biotech (Beijing) Co., Ltd., China) was added, and incubated at 37°C in the dark for 10 minutes. Finally, 100 μL of oxalic acid stop solution (63.0 g anhydrous oxalic acid, 1000 mL ultrapure water) was added to each well to terminate the reaction. The optical density at 450 nm was measured using a microplate reader (Beijing Pulang New Technology Co., Ltd., China). Results are as follows: Figure 7 As shown in Figure B, the bispecific protein BiFpC6 of this invention competitively binds to VEGFA165 with Human VEGFR2 in a concentration-dependent manner, wherein the IC50 of the bispecific protein BiFpC6 is... 50 It was 996.3 ng / mL.

[0163] 9.2 Competitive Combinations for VEGFC

[0164] 96-well plates were coated with 1.25 μg / mL VEGFR3-Fc (catalog number FL4-H5251, ACRO Biosystems, China) in 1×PBS (8.01 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4, 0.05% Tween-20, pH 7.4) at 50 μL / well. The plates were sealed and coated overnight at 4°C. Each well was washed twice with 1×PBST (1×PBS, 0.05% Tween-20, pH 7.4), followed by blocking with 200 μL / well of PBST containing 1% BSA (BIOFROXX). The plates were sealed and incubated at 37°C for 2 hours, followed by washing twice with 1×PBST. Bispecific protein BiFpC2 and negative control human IgG1, kappa Isotype Control (catalog number HG1K, Sino Biological, China) were serially diluted 3-fold (in 1×PBS) and pre-incubated with 2.5 nM VEGFC-his (catalog number VEC-H4225, AcroBiosystems, China) dimer at 37°C for 1 hour. After pre-incubation, the co-incubator was added to each well (100 μL / well) and incubated at 37°C for 1 hour. Each well was washed three times with 1×PBST, followed by the addition of 100 μL / well of 1:5000 diluted HRP-conjugated 6*His, His-Tag monoclonal antibody (catalog number 66005, Proteintech, USA) at 37°C for 0.5 hours. Wash the plate three times with 1×PBST, then add TMB substrate (catalog number PR1200, Solarbio, 100 μL / well). Incubate in the dark at 37°C for 10 minutes. Finally, stop the reaction by adding 50 μL of 2M sulfuric acid to each well. Measure the optical density at 450 nm using a Synergy Lx microplate reader (BioTek Instruments, USA). Perform the test in duplicate. Results are shown below. Figure 7 In C. For example... Figure 7 As shown in Figure C, the bispecific protein BiFpC2 competitively binds to the natural receptor VEGFR3 in a concentration-dependent manner to VEGFC. The IC50 of the bispecific protein BiFpC2 is... 50 The activity was 4.8 nM, while the negative control showed no competitive activity.

[0165] Using a method similar to that used for BiFpC2, the competitive binding ability of BiFpC6 to VEGFC was also verified. The detection method is described below:

[0166] Coat a 96-well plate with 100 μL / well of 1 μg / mL Human VEGFC Protein (His Tag) (catalog number 10542-H08H, Beijing Yiqiao Shenzhou Technology Co., Ltd., China) in carbonate coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, 1000 mL ultrapure water, pH 9.6). Seal the plate and incubate overnight at 4°C. Wash each well twice with 1×PBST (1×PBS, 0.05% Tween-20, pH 7.4), then block with 260 μL / well of PBST containing 1% BSA (catalog number A8020, Solarbio, China). Seal the plate and incubate at 37°C for 1.5 hours, then wash each well twice with 1×PBST. BiFpC6, a bispecific protein, was serially diluted 3-fold (in 1×PBST + 0.1% BSA) with 0.1 μg / mL of Biotinylated Human VEGFR3 / FLT4 Protein, His, Avitag. TM (Catalog No. FL4-H82E1, AcroBiosystems, China) After thorough mixing at a 1:1 ratio, add 100 μL / well to the corresponding well of the ELISA plate and incubate at 37°C for 1 hour. Wash each well three times with 1×PBST, then add 100 μL / well of 1:8000 diluted Streptavidin-HRP(SA) (Catalog No. YXXO5701-HRP, Pujian Biotechnology (Wuhan) Co., Ltd., China) and incubate at 37°C for 45 minutes. Wash the plate three times with 1×PBST, then add 100 μL / well of TMB substrate (Catalog No. PA107-01, Tiangen Biotech (Beijing) Co., Ltd., China) and incubate at 37°C in the dark for 10 minutes. Finally, add 100 μL of oxalic acid stop solution (63.0 g anhydrous oxalic acid, 1000 mL ultrapure water) to each well to stop the reaction. The optical density at 450 nm was measured using an ELISA reader (Beijing Pulang New Technology Co., Ltd., China). The results are as follows: ​ As shown in Figure D, the bispecific protein BiFpC6 competitively binds to the natural receptor VEGFR3 in a concentration-dependent manner to VEGFC. The IC50 of the bispecific protein BiFpC6 is... 50 It was 5405 ng / mL.

[0167] Example 10: In vivo testing of the bispecific protein of the present invention in a cynomolgus monkey (Macaca fascicularis) nCNV model.

[0168] Eight cynomolgus monkeys (Guangxi Xiongsen Primate Experimental Animal Breeding and Development Co., Ltd.) were used as a model of wet age-related macular degeneration (neoAMD). The day of model establishment was designated as Day 1. Choroidal neovascularization (CNV) was induced in both eyes of the cynomolgus monkeys using fundus laser photocoagulation. On Day 15, fundus fluorescein angiography (FFA) was performed to assess fundus fluorescein leakage, and CNV leakage was graded according to the following criteria:

[0169] Level 1, no high fluorescence was observed in the light spot;

[0170] Level 2, high fluorescence in the light spot but no fluorescence leakage;

[0171] Level 3, high fluorescence in the light spot, slight fluorescence leakage, leakage does not exceed the edge of the light spot;

[0172] Level 4, high fluorescence in the light spot, severe fluorescence leakage, leakage extends beyond the edge of the light spot.

[0173] On day 16, five animals with grade 4 fluorescent leakage spots were selected and divided into two groups (grouping was based on the average leakage area and grade 4 spot rate to ensure no significant difference between the two groups). One group of three animals was administered the bispecific protein BiFpC2 of this invention; the other group of two animals was administered the positive control Eylea. On the day of grouping, the animals were injected intravitreally, unilaterally, with a single injection of 100 μL of 20 μM bispecific protein BiFpC2 or 43 μM positive control Eylea. During the experiment, the animals underwent routine clinical observation daily, and were weighed and subjected to routine ophthalmic examinations on days 1, 15, 18, 22, and 29. Fundus photography (FP), FFA, and optical coherence tomography (OCT) were performed on days 1, 15, 22, and 29. The effectiveness was evaluated based on four indicators: the fluorescent leakage area of ​​grade 4 spots and the thickness of subretinal high reflectance signal material (SHRM). The results are as follows:

[0174] Grade 4 fluorescence spot rate: On D22 (1 week after administration) and D29 (2 weeks after administration), compared with before treatment, animals treated with bispecific protein BiFpC2 and positive control Eylea showed a significantly reduced grade 4 fluorescence spot rate, and no statistical difference was observed between the bispecific protein BiFpC2 group and the positive control Eylea group (p>0.05).

[0175] Average leakage area of ​​grade 4 fluorescent spots: On D15 (before drug administration), there was no statistically significant difference in the average leakage area of ​​grade 4 fluorescent spots between the bispecific protein BiFpC2 group and the positive control Eylea group (p>0.05); on D29, both bispecific protein BiFpC2 and positive control Eylea reduced the fluorescent leakage area, and there was no statistically significant difference in the average fluorescent leakage area between the two groups (p>0.05).

[0176] Reduction in average fluorescence leakage area and improvement rate of grade 4 fluorescent spots: On days 22 and 29, there was no statistically significant difference in the reduction in average fluorescence leakage area between the bispecific protein BiFpC2 group and the positive control group (p>0.05). On day 29, there was no statistically significant difference in the improvement rate of average fluorescence leakage area between the bispecific protein BiFpC2 group and the positive control group (p>0.05).

[0177] Mean SHRM thickness: At D15, D22 and D29, there was no statistically significant difference in the mean SHRM thickness between the bispecific protein BiFpC2 group and the positive control Eylea group (p>0.05).

[0178] This embodiment uses a cynomolgus monkey choroidal neovascularization model, administering a single intravitreal injection of 100 μL of 20 μM bispecific protein BiFpC2 or 43 μM of the positive control Eylea per eye. At 1 week (D22) and 2 weeks (D29) post-administration, it was observed that the bispecific protein BiFpC2, at only half the molar concentration of the positive control Eylea, reduced the fluorescent leakage area and decreased the thickness of highly reflective subretinal material, with effects comparable to Eylea.

[0179] Example 11. In vivo testing of the bispecific protein of the present invention in a rat nCNV model.

[0180] In a rat nCNV model, the percentage of grade 4 fluorescent spots, the area of ​​grade 4 fluorescent spots, and retinal thickness were measured at 7, 14, and 21 days after administration of the bispecific protein. The results showed that, under the experimental conditions of this embodiment, the high-dose BiFpC2 group (15 μg / eye, once intravitreal injection) had a certain inhibitory effect on angiogenesis and leakage in the rat model of choroidal neovascularization.

[0181] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A bispecific fusion polypeptide that binds vascular endothelial growth factor (VEGF) A and VEGFC, characterized in that, the polypeptide consists of a first domain specifically recognizing VEGFA, a second domain specifically recognizing VEGFC and a third domain, which are directly connected or connected by a linker; the first domain comprises immunoglobulin (Ig) like domain 2 of VEGFR1 and Ig like domain 3 of VEGFR2, the second domain comprises Ig like domain 1, Ig like domain 2 and Ig like domain 3 of VEGFR3, the third domain comprises Fc region of immunoglobulin; the bispecific fusion polypeptide consists of the first domain-second domain-third domain, which are directly connected or connected by a linker, from N-terminus to C-terminus; the first domain is in order from N-terminus to C-terminus the amino acid sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 2; the second domain is the amino acid sequence as shown in SEQ ID NO: 10; the third domain is the amino acid sequence as shown in SEQ ID NO:

13.

2. The bispecific fusion polypeptide of claim 1, wherein, the linker comprises at least 6 amino acids.

3. The bispecific fusion polypeptide of claim 2, wherein, the amino acid sequence of the linker is as shown in SEQ ID NO:

7.

4. The bispecific fusion polypeptide of claim 3, wherein, the amino acid sequence of the bispecific fusion polypeptide is as shown in SEQ ID NO:

24.

5. An isolated nucleic acid molecule encoding the bispecific fusion polypeptide of any one of claims 1 to 4.

6. A nucleic acid delivery vector comprising the isolated nucleic acid molecule of claim 5.

7. The nucleic acid delivery vector of claim 6, wherein the nucleic acid delivery vector comprises adenovirus, adeno-associated virus, lentivirus or other acceptable nucleic acid delivery vector.

8. A bispecific binding molecule for vascular endothelial growth factor (VEGF) A and VEGFC, characterized in that, the bispecific binding molecule comprises a dimer of the bispecific fusion polypeptide of any one of claims 1 to 4.

9. The bispecific binding molecule of claim 8, wherein, the dimer is a homodimer or a heterodimer.

10. A host cell comprising the isolated nucleic acid molecule of claim 5.

11. A pharmaceutical composition comprising the bispecific fusion polypeptide of any one of claims 1 to 4, the isolated nucleic acid molecule of claim 5, the nucleic acid delivery vector of claim 6 or 7, or the bispecific binding molecule of claim 8 or 9, and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 11, wherein, the pharmaceutical composition is in the form of a tablet, a powder, a granule, a pill, an injection, a suspension, a powder, an emulsion, an aerosol, a gel, an eye drop, a sustained release or a sustained release implant.

13. Use of the bispecific fusion polypeptide of any one of claims 1 to 4, the isolated nucleic acid molecule of claim 5, the nucleic acid delivery vector of claim 6 or 7, the bispecific binding molecule of claim 8 or 9, or the pharmaceutical composition of claim 11 or 12 in the manufacture of a medicament for treating or preventing a disease associated with VEGFA or VEGFC, which disease is selected from the group consisting of a neovascular eye disease or a cancer, which cancer is selected from the group consisting of renal cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, colorectal cancer, non-small cell lung cancer, liver cancer, cervical cancer or glioblastoma.

14. Use according to claim 13, characterized in that, The neovascular eye disease is selected from age-related macular degeneration, diabetic retinopathy, neovascular glaucoma, retinal vein occlusion or corneal neovascularization.

15. Use according to claim 14, characterized in that, The diabetic retinopathy is diabetic macular edema and the corneal neovascularization is post corneal transplant neovascularization.

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