VEGF-C muteins for selective lymphatic stimulation

Abstract

The present invention provides VEGF-C muteins that selectively bind to VEGFR-3 over VEGFR-2. The present invention also provides methods of inducing lymphangiogenesis in a subject in need of such induction by administering to the subject an effective amount of a VEGF-C mutein of the present disclosure. The present invention also provides methods of treating a disease or condition (e.g., cancer) in a subject in need of such treatment by administering to the subject an effective amount of a VEGF-C mutein of the present disclosure.

Description

Cross-reference of related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 317,614, filed on 8 March 2022, which is incorporated herein by reference in its entirety for all purposes. Description of federally funded research and development.

[0002] This invention was made with government support under grant number CA239444, awarded by the National Institutes of Health, USA. The U.S. Government has certain rights in this invention. Sequence Listing

[0003] This application includes a sequence list, which has been electronically submitted in XML file format and is incorporated herein by reference in its entirety. The XML copy created on March 8, 2023, has the filename 251609_000089_SL.xml and a size of 347,984 bytes. [Technical Field]

[0004] The present invention generally relates to vascular endothelium. Cell proliferation Vascular endothelium is more important than factor receptor-2 (VEGFR-2). Cell proliferation Vascular endothelium that selectively binds to factor receptor-3 (VEGFR-3). Cell proliferation Factor C (VEGF-C) Mutant protein The present invention also relates to the induction of lymphangiogenesis in subjects requiring an effective dose of VEGF-C Mutant protein The present invention also relates to a method for inducing lymphangiogenesis by administering a functional fragment thereof to a target. Mutant protein The procedure includes the step of administering the functional fragment thereof to the target. [Background technology]

[0005] Immune surveillance against pathogens and tumors in the central nervous system is thought to be limited by insufficient lymphatic drainage. Manipulation of the meningeal lymphatic system has been shown to elicit a stronger immune response against brain tumors. Immunity against glioblastoma antigens mediated by CD8 T cells is greatly restricted when the tumor is confined to the central nervous system, and tumor growth cannot be controlled. However, the Cell proliferation ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes the priming of CD8 T cells in the deep cervical lymph nodes of the drainage region, the migration of CD8 T cells into the tumor, the rapid removal of glioblastoma, and the promotion of a long-lasting anti-tumor memory response. Furthermore, transfection of an mRNA construct expressing VEGF-C acts synergistically with checkpoint blockade therapy to eradicate existing glioblastoma.

Summary of the Invention

Problems to be Solved by the Invention

[0006] vascular endothelial Cell proliferation growth factor C (VEGF-C) promotes blood vessel growth (angiogenesis) and regulates blood vessel permeability, and binds to vascular endothelial growth factor receptor-2 (VEGFR-2) and vascular endothelial growth factor receptor-3 (VEGFR-3) that promotes lymphatic vessel growth (lymphangiogenesis). VEGF-C acts mainly on lymphatic endothelial cells (LECs) via VEGFR-3 to promote survival, Cell proliferation , and migration. Angiogenesis is essential for the growth of both primary tumors and metastases. It is necessary to reduce unwanted angiogenesis while promoting lymphangiogenesis. Cell proliferation

Means for Solving the Problems

Means for Solving the Problems

[0007] In one aspect, provided herein is an isolated vascular endothelial

[0008] growth factor C (VEGF-C) Cell proliferation ​ mutation A protein or a functional fragment thereof, where VEGF-C mutation The protein or its functional fragment has reduced or no ability to stimulate vascular endothelial cell proliferation compared to the wild-type VEGF-C protein, which is of the same origin but retains the ability to stimulate lymphatic endothelial cell proliferation.

[0009] In one embodiment, the herein provides isolated vascular endothelium. Cell proliferation Factor C (VEGF-C) mutation A protein or a functional fragment thereof, where VEGF-C mutation The protein or its functional fragment is (i) compared to the wild-type VEGF-C protein of the same species, Cell proliferation (ii) Vascular endothelium has reduced binding affinity to factor receptor-2 (VEGFR-2) Cell proliferation (iii) It has the ability to bind to factor receptor-3 (VEGFR-3) and generate VEGFR-3-mediated signaling, and (iii) contains an amino acid sequence that is at least 70% identical to the amino acid sequence of the same species' wild-type VEGF-C protein.

[0010] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment generates VEGFR-2-mediated signaling in reduced amounts or at all compared to the wild-type VEGF-C protein of the same species. In some embodiments of any of the methods described above, VEGF-C mutationThe protein or its functional fragment does not generate VEGFR-2-mediated signaling. In some embodiments of any of the methods described above, VEGFR-2-mediated signaling is determined by measuring VEGFR-2-dependent AKT phosphorylation and / or ERK phosphorylation levels in vascular endothelial cells by a wound healing assay, a proliferation assay, or angiogenesis assay. In some embodiments of any of the methods described above, VEGFR-3-mediated signaling is determined by measuring VEGFR-3-dependent AKT phosphorylation and / or ERK phosphorylation levels in lymphatic endothelial cells by a wound healing assay (scratch assay), a proliferation assay, or angiogenesis assay.

[0011] In some embodiments of any of the methods described above, VEGF-C mutation Proteins or their functional fragments do not induce angiogenesis.

[0012] In some embodiments of any of the methods described above, VEGF-C mutation The protein is a polypeptide defined by amino acids 111-211 of SEQ ID NO: 4 or the corresponding position of another wild-type VEGF-C protein. Mutant protein In some embodiments of any of the methods described above, the wild-type VEGF-C protein contains amino acids 111-211 of SEQ ID NO: 4.

[0013] In some embodiments of any of the methods described above, VEGF-C mutationThe protein or its functional fragment includes: one or more mutations selected from mutations in residues T112, L115, D119, Q126, T144, N145, K149, N163, S164, E165, I184, V186, L188, and P192, the residue position defined in relation to SEQ ID NO: 4; or mutations in the corresponding residues in another wild-type VEGF-C protein; or one or more mutations in residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, I188, V190, L192, and P196, the residue position defined in relation to SEQ ID NO: 1.

[0014] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment further includes a mutation at residue C133, where the position of the residue is defined in relation to SEQ ID NO: 4, or a mutation at the corresponding residue in another wild-type VEGF-C protein. In some embodiments, the mutation at residue C133 is a C133A mutation.

[0015] In some embodiments of any of the methods described above, VEGF-C mutation The protein is a polypeptide defined by amino acids 115-215 of SEQ ID NO: 1 or the corresponding position in another wild-type VEGF-C protein. Mutant protein In some embodiments, the wild-type VEGF-C protein contains amino acids 115-215 of SEQ ID NO: 1.

[0016] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains one or more mutations selected from mutations in residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, I188, V190, L192, and P196, the residue positions being defined in relation to SEQ ID NO: 1.

[0017] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment further includes a mutation at residue C137, where the position of the residue is defined in relation to SEQ ID NO: 1, or a mutation at the corresponding residue in the wild-type VEGF-C protein of another species. In some embodiments, the mutation at residue C137 is a C137A mutation.

[0018] In some embodiments of any of the methods described above: a mutation in residue L119 is an L119E mutation or an L119M mutation; a mutation in residue D123 is a D123N mutation; a mutation in residue Q130 is a Q130K mutation; a mutation in residue N167 is an N167R mutation, an N167I mutation, an N167Q mutation or an N167H mutation; a mutation in residue S168 is an S168G mutation or an S168R mutation; a mutation in residue V190 is a V190T mutation; and / or a mutation in residue L192 is an L192I mutation.

[0019] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains one or more mutations selected from the mutations at residues N167, S168, and / or L192.

[0020] In some embodiments of any of the methods described above: the mutation at residue N167 is an N167I mutation, an N167Q mutation, or an N167H mutation; the mutation at residue S168 is an S168G mutation or an S168R mutation; and / or the mutation at residue L192 is an L192I mutation.

[0021] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the N167Q mutation.

[0022] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 160 or SEQ ID NO: 56.

[0023] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 160 or SEQ ID NO: 56.

[0024] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the N167Q mutation and the S168G mutation.

[0025] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO: 57.

[0026] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO: 57.

[0027] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the N167Q mutation and the L192I mutation.

[0028] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 58.

[0029] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 58.

[0030] In some embodiments of any of the methods described above, VEGF-C mutationThe protein or its functional fragment contains the N167Q mutation, the S168G mutation, and the L192I mutation.

[0031] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 163 or SEQ ID NO: 59.

[0032] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 163 or SEQ ID NO: 59.

[0033] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the N167I mutation.

[0034] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 166 or SEQ ID NO: 62.

[0035] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 166 or SEQ ID NO: 62.

[0036] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the N167I and S168G mutations.

[0037] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 167 or SEQ ID NO: 63.

[0038] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 167 or SEQ ID NO: 63.

[0039] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the N167I mutation and the L192I mutation.

[0040] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 168 or SEQ ID NO: 64.

[0041] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 168 or SEQ ID NO: 64.

[0042] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the N167I mutation, the S168G mutation, and the L192I mutation.

[0043] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 169 or SEQ ID NO: 65.

[0044] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 169 or SEQ ID NO: 65.

[0045] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the S168G mutation.

[0046] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 68.

[0047] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 68.

[0048] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the S168G mutation and the L192I mutation.

[0049] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 70.

[0050] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 70.

[0051] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the N167H mutation.

[0052] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO: 80.

[0053] In some embodiments of any of the methods described above, VEGF-C mutation The protein consists of the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO: 80.

[0054] In some embodiments of any of the methods described above, VEGF-C mutation The protein or its functional fragment contains the N167I mutation and the S168R mutation.

[0055] In some embodiments of any of the methods described above, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 192 or SEQ ID NO: 88.

[0056] In some embodiments of any of the methods described above, VEGF-C mutationThe protein consists of the amino acid sequence of SEQ ID NO: 192 or SEQ ID NO: 88.

[0057] In one embodiment, the VEGF-C disclosed herein is provided herein. mutation A fusion protein or conjugate comprising a protein or a functional fragment thereof, where mutation The protein or its functional fragment is fused and / or conjugated with one or more heterologous parts.

[0058] In some embodiments, one or more heterogeneous parts are selected from immunoglobulin or functional fragments thereof, albumin or functional fragments thereof, albumin-conjugated antibodies or functional fragments thereof, and polyethylene glycol (PEG) polymers.

[0059] In some embodiments, the immunoglobulin or its functional fragment comprises an IgG Fc domain. In some embodiments, the IgG Fc domain is modified to reduce its Fc effector function. In some embodiments, the IgG Fc domain comprises a mutation at residue N297. In some embodiments, the mutation at residue N297 is selected from N297Q, N297A, and N297G.

[0060] In one embodiment, the VEGF-C provided herein is provided in this specification. mutation An isolated polynucleotide molecule encoding a protein or a functional fragment thereof, or a fusion protein of the present disclosure.

[0061] In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 210 or SEQ ID NO: 108. In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 211 or SEQ ID NO: 109. In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 212 or SEQ ID NO: 110. In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 213 or SEQ ID NO: 111. In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 216 or SEQ ID NO: 114. In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 217 or SEQ ID NO: 115. In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 218 or SEQ ID NO: 116. In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 219 or SEQ ID NO: 117. In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 222 or SEQ ID NO: 120. In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 224 or SEQ ID NO: 122. In some embodiments, the polynucleotide molecule includes the nucleotide sequence of SEQ ID NO: 234 or SEQ ID NO: 132. In some embodiments, the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 242 or SEQ ID NO: 140. In some embodiments, the polynucleotide molecule comprises VEGF-C operably linked to a promoter. mutation It contains a nucleotide sequence that codes for a protein or a functional fragment thereof.

[0062] In some embodiments, the polynucleotide molecule is mRNA.

[0063] In some embodiments, the polynucleotide molecule includes one or more nucleotide modifications. In some embodiments, one or more nucleotide modifications are a 5' cap, 5-methylcytosine, or pseudouridine.

[0064] In one embodiment, what is provided herein is a vector comprising the polynucleotide molecule of the present disclosure.

[0065] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is derived from a herpesvirus, cytomegalovirus, poliovirus, alphavirus, vacciniavirus, rabies virus, adeno-associated virus (AAV), retrovirus, lentivirus, or adenovirus.

[0066] In one embodiment, what is provided herein are particles comprising the polynucleotide molecules of the present disclosure. In some embodiments, the particles are nanoparticles, lipid particles, fine particles, lipid nanoparticles, polymer particles, or virus-like particles (VLPs).

[0067] In one embodiment, what is provided herein is a host cell comprising the polynucleotide or vector of the disclosure.

[0068] In one embodiment, what is provided herein is VEGF-C mutation A method for producing a protein or a functional fragment thereof, or a fusion protein thereof, wherein the host cells of the present disclosure are VEGF-C mutation A method comprising the step of culturing a protein or a functional fragment thereof, or a fusion protein thereof, under conditions in which it is expressed.

[0069] In one embodiment, provided herein is VEGF-C manufactured by the method of the present disclosure. mutation It is a protein, a functional fragment thereof, or a fusion protein thereof.

[0070] In one embodiment, the VEGF-C provided herein is provided in this specification. mutation A kit comprising a protein or a functional fragment thereof, or a fusion protein or conjugate of the present disclosure, and optionally, instructions for use.

[0071] In one embodiment, what is provided herein is a kit comprising the polynucleotides or vectors of the disclosure, or particles of the disclosure, and optionally, instructions for use.

[0072] In one embodiment, the VEGF-C provided herein is provided in this specification. mutation A pharmaceutical composition comprising a protein or a functional fragment thereof, or a fusion protein or conjugate of the present disclosure, or a polynucleotide molecule of the present disclosure, or a vector of the present disclosure, or a particle of the present disclosure, and a pharmaceutically acceptable carrier or diluent.

[0073] In some embodiments, the compositions, VEGF-C mutation The product contains mRNA encoding a protein or a functional fragment thereof, or a fusion protein thereof, as an mRNA nanoparticle formulation.

[0074] In some embodiments, the pharmaceutical composition further comprises an immunotherapy agent. In some embodiments, the immunotherapy agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, BTLA, GITR, 4-1BB, or Ox-40. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-BTLA antibody, an anti-GITR antibody, an anti-4-1BB antibody, or an anti-Ox-40 antibody.

[0075] In some embodiments, the pharmaceutical composition is formulated for intrathecal administration. In some embodiments, the pharmaceutical composition is formulated for intratumoral administration. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for intracisional administration. In some embodiments, the pharmaceutical composition is formulated for ophthalmic administration. In some embodiments, the pharmaceutical composition is formulated for intraocular administration.

[0076] In one embodiment, the foregoing provides a method for inducing lymphangiogenesis in a subject requiring induction of lymphangiogenesis, the method comprising an effective amount of VEGF-C of the foregoing disclosure. mutation The process includes administering a protein or a functional fragment thereof, or a fusion protein or conjugate of the Disclosure, or a polynucleotide molecule of the Disclosure, or a vector of the Disclosure, or a particle of the Disclosure, or a pharmaceutical composition of the Disclosure to a subject.

[0077] In some embodiments of any of the methods described above, VEGF-C mutation Administration of a protein or its functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition does not cause one or more side effects associated with the administration of wild-type VEGF-C protein or the corresponding fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition. In some embodiments, one or more side effects are angiogenesis and / or increased intraocular pressure (IOP).

[0078] In some embodiments of any of the methods described above, VEGF-C mutation Proteins or their functional fragments, fusion proteins, conjugates, polynucleotide molecules, vectors, particles, or pharmaceutical compositions are administered intrathecally, intraocularly, intratumorally, intracisionally, intravitreously, by eye drops, subcutaneously, intradermally, by inhalation, by long-term indwelling catheter, orally, topically, or systemically.

[0079] In some embodiments of any of the methods described above, VEGF-C mutation Proteins or their functional fragments, fusion proteins, conjugates, polynucleotide molecules, vectors, particles, or pharmaceutical compositions are administered directly into the cisterna macrophylaxis or into the lymphatic system.

[0080] In some embodiments of any of the methods described above, the subject has a disease or condition selected from cancer, coronary vascular function, osmolality regulation, myocardial ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, diseases or conditions associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennecum syndrome, Milroy's disease, Turner syndrome, age-related macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, and retinal edema.

[0081] In some embodiments of any of the methods described above, the diseases or conditions associated with lymph node transplantation are breast cancer-related lymphedema, idiopathic lymphedema, and / or heart failure-related lymphedema.

[0082] In one embodiment, the foregoing provides a method for treating a disease or condition requiring treatment, the method comprising an effective amount of VEGF-C of the foregoing disclosure. mutation The process includes administering a protein or a functional fragment thereof, or a fusion protein or conjugate of the Disclosure, or a polynucleotide molecule of the Disclosure, or a vector of the Disclosure, or a particle of the Disclosure, or a pharmaceutical composition of the Disclosure to a subject.

[0083] The methods provided herein are methods for treating a disease or condition requiring treatment of that disease or condition, in some embodiments the disease or condition is cancer, coronary vascular function, osmolality, myocardial ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, disease or condition associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennecum syndrome, Milroy's disease, Turner syndrome, age-related macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, and retinal edema. In some embodiments, the cancer is melanoma, lung cancer, breast cancer, gastric cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, head and neck squamous cell carcinoma, thyroid cancer, humoral cancer, kidney cancer, urothelial carcinoma, prostate cancer, pheochromocytoma, cholangiocarcinoma, hepatocellular carcinoma, invasive pancreatic ductal carcinoma, thymoma, sarcoma, mesothelioma, testicular cancer, or colorectal cancer. In some embodiments, the cancer is located in the brain or central nervous system of the subject. In some embodiments, the cancer is selected from glioma, ependymoma, subependymoma, primitive neuroectodermal tumor, ganglioglioma, schwannoma, germ tumor, craniopharyngioma, meningioma, CNS lymphoma, pineal gland tumor, retinoblastoma, uveal melanoma, and rhabdoid tumor.

[0084] In some embodiments, a method for treating a target disease or condition requiring treatment of the disease or condition further includes the step of administering an immunotherapy agent. In some embodiments, the immunotherapy agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, BTLA, GITR, 4-1BB, or Ox-40. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-LAG-3 antibody, anti-BTLA antibody, anti-GITR antibody, anti-4-1BB antibody, or anti-Ox-40 antibody. In some embodiments, VEGF-C mutationProteins or their functional fragments, fusion proteins, conjugates, polynucleotide molecules, vectors, particles, or pharmaceutical compositions are administered intrathecally, intraocularly, intratumorally, intracisionally, intravitreously, by eye drops, subcutaneously, intradermally, by inhalation, by long-term indwelling catheter, orally, topically, or systemically. In some embodiments, VEGF-C mutation Proteins or their functional fragments, fusion proteins, conjugates, polynucleotide molecules, vectors, particles, or pharmaceutical compositions are administered directly into the cisterna macrophylaxis or into the lymphatic system.

[0085] The methods provided herein are methods for treating a disease or condition requiring treatment, and the disease is cancer. In some embodiments, the methods further include a step of administering additional anti-cancer treatment to the target. In some embodiments, the additional anti-cancer treatment is selected from surgery, radiotherapy, administration of chemotherapeutic agents, immunotherapy, and any combination thereof.

[0086] In one embodiment, the foregoing provides a method for regulating intraocular pressure in an object requiring regulation of intraocular pressure, the method comprising an effective amount of VEGF-C of the foregoing disclosure. mutation The process includes administering to a subject a protein or functional fragment thereof, a fusion protein or conjugate, a polynucleotide molecule, a vector, particles, or a pharmaceutical composition, or a corresponding wild-type VEGF-C protein or functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding a wild-type VEGF-C protein or functional fragment thereof, or a vector or particles containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above. In some embodiments, VEGF-C mutationA protein or the corresponding wild-type VEGF-C protein, or its functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition, or the corresponding wild-type VEGF-C protein or its functional fragment, or its fusion protein or conjugate, or a polynucleotide molecule encoding the wild-type VEGF-C protein or its functional fragment, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, is administered to the posterior segment of the eye. In some embodiments, administration is intraocular. In some embodiments, intraocular administration is intravitreous, ophthalmic, or subretinal.

[0087] In one embodiment, the foregoing provides a method for removing unwanted intraocular fluid from an eye of a subject requiring removal of unwanted intraocular fluid, the method comprising an effective amount of VEGF-C of the foregoing disclosure. mutation The process includes administering to a subject a protein or its functional fragment, a fusion protein or conjugate, a polynucleotide molecule, a vector, particles, or a pharmaceutical composition, or a corresponding wild-type VEGF-C protein or its functional fragment, or its fusion protein or conjugate, or a polynucleotide molecule encoding a wild-type VEGF-C protein or its functional fragment, or a vector or particles containing the polynucleotide molecule, or a pharmaceutical composition containing any of the above. In some embodiments, the unwanted body fluid is optic nerve fluid, retinal fluid, subretinal fluid, choroidal fluid, or epichoroidal fluid. In some embodiments, the subject has glaucoma, macular edema, central serous chorioretinopathy, retinal edema, papilledema, or macular degeneration. disease or suffering from diabetic retinopathy. In some embodiments, administration is intraocular. In some embodiments, intraocular administration is intravitreous, ophthalmic, or subretinal.

[0088] In one embodiment, the foregoing provides a method for providing neuroprotection to an object requiring neuroprotection, the method comprising an effective amount of VEGF-C of the foregoing disclosure. mutationThe procedure includes administering to a subject a protein or its functional fragment, a fusion protein or conjugate, a polynucleotide molecule, or a vector, particle, or pharmaceutical composition, or a corresponding wild-type VEGF-C protein or its functional fragment, or its fusion protein or conjugate, or a polynucleotide molecule encoding a wild-type VEGF-C protein or its functional fragment, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above.

[0089] In one embodiment, the VEGF-C provided herein is provided in this specification. mutation A vaccine comprising a protein or its functional fragment, fusion protein or conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition.

[0090] In one embodiment, the foregoing provides a method for inducing an immune response in a subject requiring induction of an immune response, the method comprising the step of administering an effective amount of the vaccine of the foregoing disclosure to the subject.

[0091] In some embodiments of any of the methods described above, the subject is a human.

[0092] In a further embodiment, the herein provides VEGF-C that selectively binds to VEGFR-3. Mutant protein This method generates a library in which the amino acid residues constituting the common binding interface on VEGF-C that binds to both VEGFR-3 and VEGFR-2 are diversified to other amino acids by introducing mutations into the corresponding nucleic acid sequences.

[0093] In a further embodiment, what is provided herein is VEGF-C mutation This is a yeast cell library for protein selection, and the yeast cell library contains cell wall peptide anchor sequences, linker peptides, and VEGF-C Mutant protein It contains multiple yeast cells that include the sequence.

[0094] These and other embodiments described herein will be apparent to those skilled in the art from the following description, claims, and drawings. [Brief explanation of the drawing]

[0095] [Figure 1A] Figures 1A and 1B illustrate the pleiotropism of VEGF-C. Figure 1A is a schematic diagram showing that VEGF-C binds to both VEGFR-2 and VEGFR-3, which promote angiogenesis and lymphangiogenesis, respectively. [Figure 1B] Figure 1B is a schematic diagram of a designed VEGF-C mutant protein that loses its binding to VEGFR-2 and exhibits specific binding to VEGFR-3. [Figure 2A] Figures 2A and 2B show VEGF-C display components for dimerization and screening. Figure 2A shows several methods for displaying VEGF-C on the yeast surface, which were used to optimize surface dimerization of VEGF-C. The final combination displayed wild-type VEGF-C with high binding affinity to VEGFR-3 and low affinity to VEGFR-2. [Figure 2B] Figure 2B is a schematic diagram showing the selection of yeast display libraries aimed at discovering VEGFR-3-specific VEGF-C mutant proteins. [Figure 3] Figure 3 shows the design of the VEGF-C mutant protein library. A structure-based primer library was designed to generate approximately 103 combinations of VEGF-C mutant proteins. The structures of VEGF-C binding to VEGFR-3 and VEGFR-2 were studied, and key residues on the surface of the binding site were identified. Amino acids that could potentially cause significant polarity changes at these residues were selected for primer design. The figure shows sequence numbers 27-34 and 34-39, respectively, in order of appearance. [Figure 4]Figure 4 shows that the VEGF-C mutant protein was positively selected for VEGFR-3 binding and negatively selected for VEGFR-2 binding. Wild-type VEGF-C showed binding to yeast cells expressing both VEGFR-2 and VEGFR-3 (top row, WT). The first two rounds selected for binding to VEGFR-3. Subsequent rounds utilized the decrease in VEGFR-3 levels to select mutant proteins with high binding affinity to VEGFR-3. Simultaneously, VEGFR-2 was included, and its negative selection enriched mutant proteins that had high binding affinity to VEGFR-3 but lost binding affinity to VEGFR-2. [Figure 5] Figure 5 shows confirmation of receptor affinity priority after the final selection round. Mutant proteins that were positively selected for VEGFR-3 and negatively selected for VEGFR-2 showed high binding affinity to VEGFR-3 and no binding to VEGFR-2, even at receptor concentrations 100 times higher than wild-type binding. [Figure 6-1] Figure 6 shows confirmation of receptor affinity priority after the final selection round. Clonal mutant proteins were sequenced after selection rounds 5 and 6 to identify unique clones enriched within the mutant protein population. Unique mutant protein residues were identified, and specific mutations were created in wild-type VEGF-C for validation. [Figure 6-2] Continuation of Figure 6-1. [Figure 7] Figure 7 shows an overview of the sequence, mutation rate, and affinity of the selected mouse clones and the final LS-VEGF-C. The positions and IDs of important residues are listed in the first two rows. Shaded columns indicate mutations at contact sites, all of which promote the final mutations in WT-VEGF-C that create LS-VEGF-C. Affinity for VEGFR-2 and VEGFR-3 was determined by SPR for both WT-VEGF-C and LS-VEGF-C. [Figure 8]Figure 8 shows the affinities of VEGFR-2 and VEGFR-3 between WT VEGF-C and after selection round 6. [Figure 9] Figure 9 shows that isolated VEGF-C mutant proteins exhibited specific signaling via VEGFR-3 in vitro. Signaling of VEGF-A, wild-type VEGF-C, and VEGF-C mutant proteins (163I and RTI) was evaluated in vitro using human umbilical vein endothelial cells (HUVECs) and human dermal lymphatic endothelial cells (young HDLEC-j and adult HDLEC-a). HUVECs specifically express VEGFR-2, while HDLECs express both VEGFR-2 and VEGFR-3, allowing for evaluation of signaling at each receptor. Cells were stimulated with each protein, and protein lysates were collected and subjected to Western blotting to detect ERK phosphorylation (downstream of VEGFR-2 and VEGFR-3). [Figure 10] Figure 10 shows the use of the VEGF-C mutant protein in vivo. A single intravitreal administration of the VEGF-C mutant protein resulted in a sustained reduction in intraocular pressure (IOP) in wild-type mice compared to their counterparts. [Figure 11] Figure 11 shows the use of VEGF-C mutant protein in vivo. Wild-type VEGF-C and VEGF-C mutant protein were evaluated in vivo for the treatment of brain tumors. When combined with an anti-PD-1 antibody, VEGF-C mutant protein showed a significant therapeutic advantage in treating brain tumors. [Figure 12] Figure 12 shows the VEGFR-2 and VEGFR-3 affinities of WT VEGF-C and the previously established VEGFR-3 specific ligands, VEGF-C152S / C-156S. [Figure 13]Figure 13 shows that isolated VEGF-C mutant proteins exhibited specific signaling via VEGFR-3 in vivo. Signaling of wild-type VEGF-C and VEGF-C mutant protein (RTI) was evaluated in vivo. These vectors were injected into the eyes of mice. After 24 hours, the eyeballs were extracted and single-cell suspensions were prepared to check for AKT phosphorylation. Wild-type VEGF-C retained VEGFR-2-mediated signaling in vascular endothelial cells, while mutant VEGF-C (RTI) no longer exhibited signaling. Both wild-type and RTI mutant proteins exhibited VEGFR-3-mediated signaling in lymphatic endothelial cells. [Figure 14] Figure 14 shows that the in vivo reduction of IOP differed depending on the route of administration. VEGF-C (wt) or VEGF-C mutant protein (rti) was administered by eye drops, injection into the anterior chamber (AC), or intravitreous injection. In the AC, both wild-type (wt) and mutant protein (rti) exhibited similar activity, but the mutant protein showed a selective ability to lower intraocular pressure when administered by eye drops or intravitreous injection. [Figure 15A] Figures 15A–15D show the directional evolution of lymphocyte-specific VEGFR-3 binding. Figure 15A shows the mVEGFR2 affinity of WT VEGF-C and mutant proteins after repeated selection round 6. [Figure 15B] Figure 15B shows the mVEGFR3 affinity of WT VEGF-C and the mutant protein after repeated selection round 6. [Figure 15C] Figure 15C shows the ERK phosphorylation of downstream HUVEC(R2+) after administration of control (n=3), VEGF-A (n=3), WT VEGF-C (n=3), and LS-VEGF-C (n=3). [Figure 15D]Figure 15D shows the proliferation of HUVEC (R2+) and HDLEC (R2+R3+) after administration of control (n=3), VEGF-A (n=3), WT VEGF-C (n=3), and LS-VEGF-C (n=3). In Figures 15C and 15D, data are shown as mean ± sem, with *P<0.05, **P<0.01, ***P<0.005, and ****P<0.001, where ns means "nonsignificant". [Figure 16A] Figures 16A–16B show that administration of LS-VEGF-C reduced lymphedema in a mouse model of lymphedema. LS-VEGF-C was administered subcutaneously near the lymphatic ligation site in mice, resulting in a significant reduction in ankle size compared to controls. Mice were treated twice to demonstrate the potential for repeat administration of LS-VEGF-C. [Figure 16B] Continuation of Figure 16A. [Figure 16C] Figures 16C-16D show mice with tumors treated with a tumor vaccine containing LS-VEGF-C or WT-VEGF-C and PD-1. Figure 16C shows the tumor growth curve. [Figure 16D] Figure 16D shows the survival rate of mice. [Figure 17A] Figures 17A-17E show that LS-VEGF-C induced changes in lymphangiogenesis in the eyeball without increasing vascular permeability. Figure 17A shows intraocular pressure measurements after topical (eye drop) administration, anterior chamber administration, and intravitreous administration of WT-VEGF-C in WT mice. [Figure 17B] Figure 17B shows intraocular pressure measurements after topical (eye drop) administration, anterior chamber administration, and intravitreous administration of LS-VEGF-C in WT mice. [Figure 17C] Figure 17C shows representative fundus images (left column), fluorescence angiography images (center column), and OCT images (right column) after intravitreal administration of VEGF-A, WT-VEGF-C, and LS-VEGF-C. [Figure 17D]Figure 17D shows normalized measurements of fluorescence intensity across two multiple fluorescein angiography images, which indicate vascular permeability to extravascular areas; a steep peak above baseline (approximately 20 in normalized values) indicates the presence of leak-free vessels, while a sustained increase indicates increased permeability. [Figure 17E] Figure 17E shows repeated-measures comparisons of systemically delivered Evans Blue absorbance in the eyeball after IVT administration of PBS (control), VEGF-A, WT VEGF-C, and LS-VEGF-C, which indicates the permeability of retinal vessels to the extravascular region. For Figure 17E, ***P<0.005, ****P<0.001, and *P<0.05, where ns means "nonsignificant". [Figure 18A] Figures 18A–18H show that LS-VEGF-C provides neuroprotection in a glaucoma model. Figure 18A is a schematic diagram of a microbead mouse model of glaucoma and its evaluation by IOP measurement. [Figure 18B] Figure 18B shows a 28-day summary (mean ± sem, left) and individual IOP measurements (right) for WT mice (control, n=12), a microbead mouse model of glaucoma (induced glaucoma, n=12), a microbead mouse model of glaucoma treated with IVT of LS-VEGF-C (induced glaucoma + LS-VEGF-C (IVT), n=12), a microbead mouse model of glaucoma treated with intravitreal administration of LS-VEGF-C (induced glaucoma + LS-VEGF-C (IVT), n=12), a microbead mouse model of glaucoma treated with topical administration of LS-VEGF-C (induced glaucoma + LS-VEGF-C (eye drops), n=12), and WT mice treated with intravitreal administration of LS-VEGF-C (LS-VEGF-C (IVT), n=12). [Figure 18C]Figure 18C shows Brn3a and DAPI-labeled confocal images of the retina after excitotoxicity studies of intravitreal-injected NMDA. We compared WT mice (control), a microbead glaucoma model (induced glaucoma), and a microbead glaucoma model treated with intravitreal administration (+LS-VEGF-C IVT) and topical administration (+LS-VEGF-C eye drops). [Figure 18D] Figure 18D shows the number of Brn3a cells observed within the image field (control, n=7; microbead glaucoma model, n=6; microbead glaucoma model + IVT LS-VEGF-C, n=7; microbead glaucoma model + LS-VEGF-C eye drops, n=8). [Figure 18E] Figure 18E is a schematic diagram of a DBA2J pigment-dispersed mouse model of glaucoma. [Figure 18F] Figure 18F shows a 28-day summary (mean ± sem, left) and individual IOP measurements (right) for WT mice (control, n=12), a microbead mouse model of glaucoma (induced glaucoma, n=12), a microbead mouse model of glaucoma treated with IVT of LS-VEGF-C (induced glaucoma + LS-VEGF-C (IVT), n=12), a microbead mouse model of glaucoma treated with intravitreal administration of LS-VEGF-C (induced glaucoma + LS-VEGF-C (IVT), n=12), a microbead mouse model of glaucoma treated with topical administration of LS-VEGF-C (induced glaucoma + LS-VEGF-C (eye drops), n=12), and WT mice treated with intravitreal administration of LS-VEGF-C (LS-VEGF-C (IVT), n=12). [Figure 18G] Figure 18G shows images of RGC axons in the optic nerve of control D2-Gpnmb+ mice and DBA2J mice that received the following different treatments: untreated (spontaneous glaucoma), +LS-VEGF-C IVT, and +LS-VEGF-C eye drops. [Figure 18H] Figure 18H shows the number of RGC axonal cells observed within the image field (control, n=3; DBA2J glaucoma, n=4; LS-VEGF-C IVT, n=4; LS-VEGF-C eye drops, n=4). [Figure 19] Figure 19 shows data from the VEGFR2 and VEGFR3 binding assay of WT VEGF-C to VEGFR2 and VEGFR3. [Figure 20A] Figures 20A–20B show that administration of LS-VEGF-C reduced lymphedema in a mouse model of lymphedema. LS-VEGF-C was administered subcutaneously near the lymphatic ligation site in mice, resulting in a significant reduction in ankle size compared to controls. Mice were treated twice to demonstrate the potential for repeat administration of LS-VEGF-C. [Figure 20B] Continuation of Figure 20A. [Figure 20C] In a mouse model of melanoma, LS-VEGF-C was evaluated as an adjuvant for anti-PD-1 cancer immunotherapy. LS-VEGF-C demonstrated superior preclinical efficacy compared to WT-VEGF-C in both tumor volume reduction and survival. [Figure 20D] Continuation of Figure 20C. [Figure 20E] LS-VEGF-C was used as an adjuvant therapy for checkpoint inhibitor therapy in the treatment of glioblastoma. LS-VEGF-C showed activity comparable to WT-VEGF-C and resulted in the survival of the majority of treated animals, which would otherwise have had a fatal outcome. [Figure 20F] Continuation of Figure 20E. [Figure 21A] Figures 21A-21D demonstrate that LS-VEGF-C provides a unique mechanism of action that allows for sustained effects. Figure 21A shows IOP measurements (mean ± sem) over 10 days after local (left), anterior chamber (center), and intravitreal (right) administration of WT-VEGF-C and LS-VEGF-C. [Figure 21B] Figure 21B is a schematic diagram that includes arrows showing the flow of bodily fluids into and out of the eyeball. [Figure 21C] Figure 21C shows IOP measurements after topical administration of currently FDA-approved medications for glaucoma. Data points from the first hour have been excluded due to the ambiguous IOP-reducing effect of mild anesthesia. [Figure 21D]Figure 21D compares IOP measurements (mean ± sem) of control mice, mice treated with FDA-approved topical glaucoma drugs at the time of maximum IOP reduction (dorzolamide, Dz, 2 hours; Lopressa, Rho, 2 hours; brimonidine, Bri, 2 hours; latanoprost, Lat, 3 hours; timolol, 2 hours), mice 2 days after IV injection of LS-VEGF-C, and mice treated with topical administration of FDA-approved drugs at the time of maximum IOP reduction in mice that had received intravitreal LS-VEGF-C 2 days prior. Data are shown as mean ± sem, with *P<0.05, **P<0.01, ***P<0.005, and ****P<0.001, where ns means "nonsignificant". [Figure 22A] Figures 22A–22D show images of the anterior side of the eyeball, which exhibit similar signs to the posterior side. Figure 22A shows images of control WT mice focused on the cornea and iris (n=3). [Figure 22B] Figure 22B shows images of WT mice after in vitro VEGF-A administration, with focus on the cornea and iris (n=3). Neovascularization and corneal opacity were observed. [Figure 22C] Figure 22C shows images of WT mice after IVT administration of WT-VEGF-C, with focus on the cornea and iris (n=3). Corneal opacity was observed. [Figure 22D] Figure 22D shows images of WT mice after IVT administration of LS-VEGF-C, with focus on the cornea and iris (n=3). [Figure 23A] Figures 23A–23B demonstrate that LS-VEGF-C provides neuroprotection in a glaucoma model. Figure 23A shows Brn3a and DAPI-labeled confocal images of the retina after an excitotoxicity study of intravitreal-injected NMDA. [Figure 23B] Figure 23B shows the number of RGC cells observed within the image field from an NMDA-promoted excitability study. [Figure 24]Figure 24 shows an overview of VEGFR2 binding for hLS-VEGF-C variants, summarizing the binding of hVEGF-C variants to hVEGFR2 compared to WT VEGF-C, expressed as a percentage of the yeast population that was stain-positive. Lower values ​​indicate less involvement of VEGFR2, which is desirable. [Figure 25] Figure 25 shows the relative bias between VEGFR3 and VEGFR2 for LS-hVEGF-C clones. Figure 25 includes plots of hVEGFR3 binding (represented by mean fluorescence intensity MFI) and hVEGFR2 binding (represented by the percentage of stain-positive yeast population). The upper graph uses 100 nM hVEGFR3. The lower graph uses 10 nM hVEGFR3. The selected points are labeled with the number of LS-hVEGF-C mutant proteins. Clones in the lower right quadrant of these plots are preferred because they show a bias towards binding to VEGFR3 rather than VEGFR2. [Figure 26] Figure 26 shows the VEGFR2 and VEGFR3 binding for LS-hVEGF-C; the specific values ​​of hVEGFR2 and hVEGFR3 for each clone are shown in Figures 24 and 25. [Figure 27] Figure 27 shows the binding isotherms of the selected LS-hVEGF-C mutant proteins to hVEGFR3. Specific EC50 and R2 values ​​for curve fitting are provided at the bottom of Figure 27. [Figure 28A] Figures 28A–28M show the quality of human VEGF-C mutant proteins fused to human IgG1 Fc. Figure 28A shows the quality of the human C137A clone fusion. [Figure 28B] Figure 28B shows the quality of the human clone 7 fusion. [Figure 28C] Figure 28C shows the quality of the human clone 8 fusion. [Figure 28D] Figure 28D shows the quality of the human clone 9 fusion. [Figure 28E] Figure 28E shows the quality of the 10-human clone fusion. [Figure 28F]Figure 28F shows the quality of the human clone 13 fusion. [Figure 28G] Figure 28G shows the quality of the 14-human clone fusion. [Figure 28H] Figure 28H shows the quality of the 15-human clone fusion. [Figure 28I] Figure 28I shows the quality of the human clone 16 fusion. [Figure 28J] Figure 28J shows the quality of the human clone 19 fusion. [Figure 28K] Figure 28K shows the quality of the human clone 21 fusion. [Figure 28L] Figure 28L shows the quality of the human clone 31 fusion. [Figure 28M] Figure 28M shows the quality of the human clone 39 fusion. [Figure 29] Figure 29 shows the effects on mice treated intravitreously with the RTI-Fc mutant protein and their intraocular pressure. [Figure 30] Figure 30 shows the decrease in intraocular pressure in mice treated with LS-VEGF-C in monomer form, albumin conjugate, or Fc conjugate. [Modes for carrying out the invention]

[0096] This invention provides VEGF-C to induce lymphangiogenesis in the body of subjects who require the induction of lymphangiogenesis. Mutant protein The present invention provides a method for obtaining an effective amount of VEGF-C mutation This includes a step of administering a protein as the target.

[0097] The terms "a, an" and "the" do not indicate a quantitative limitation, but rather indicate that there is "at least one" item being referred to.

[0098] When used before a number, the term "about" indicates that the number may vary within a reasonable range, for example, within ±10%, ±5%, or ±1% of the stated value, and may also include the stated value.

[0099] The terms “patient,” “individual,” “subject,” “mammal,” and “animal” are used herein interchangeably and are not limited to humans, as well as mammals, including domestic animals (e.g., cats, dogs, rabbits, cattle, horses, sheep, pigs, etc.) and experimental animal models. In one preferred embodiment, the subject is human.

[0100] The terms “treat” a state, disorder, or condition, or “treatment,” include: (1) preventing, delaying, or reducing the occurrence and / or likelihood of the occurrence of at least one clinical or subclinical symptom of a state, disorder, or condition in a subject who is suffering from or predisposed to a state, disorder, or condition but has not yet experienced or presented any clinical or subclinical symptom of the state, disorder, or condition; or (2) inhibiting a state, disorder, or condition, i.e., preventing, reducing, or delaying the progression or (in the case of maintenance therapy) relapse of the disease, or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, i.e., causing regression of the state, disorder, or condition, or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated must be statistically significant or at least perceptible to the patient or physician.

[0101] As used herein, the term " mutation "protein (mutein protein)", mutation "Potential polypeptide (mutein polypeptide)", and " Mutant protein"(mutein)" refers to a protein used as an interchangeable protein, characterized by a modified amino acid sequence compared to its wild-type counterpart. Amino acid sequence changes may include amino acid substitutions, deletions, or additions.

[0102] As used herein, the term "lymphangiogenesis" refers to the process of lymphatic formation and / or stimulation of lymphatic system function.

[0103] In one embodiment, the herein provides isolated vascular endothelium that selectively binds to VEGFR-3 rather than VEGFR-2. Cell proliferation Factor C (VEGF-C) mutation It is a protein or a functional fragment thereof.

[0104] In some embodiments, VEGF-C mutation The protein or its functional fragment has reduced or no ability to stimulate vascular endothelial cell proliferation compared to the wild-type VEGF-C protein, which is of the same origin but retains the ability to stimulate lymphatic endothelial cell proliferation.

[0105] In some embodiments, VEGF-C mutation The protein or functional fragment thereof (i) has reduced binding affinity to VEGFR-2 compared to the wild-type VEGF-C protein of the same species, (ii) has the ability to bind to VEGFR-3 and generate VEGFR-3-mediated signaling, and (iii) contains an amino acid sequence that is at least 70% identical to the amino acid sequence of the wild-type VEGF-C protein of the same species.

[0106] In some embodiments, VEGF-C mutation The protein or its functional fragment has a binding affinity to VEGFR-2 that is reduced by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or higher percentages compared to the wild-type VEGF-C protein of the same species.

[0107] In some embodiments, VEGF-C mutation The protein or its functional fragment has the ability to bind to VEGFR-3 and generate VEGFR-3-mediated signaling that is generally identical, increased, or slightly decreased compared to the wild-type VEGF-C protein of the same species.

[0108] In cells, VEGF-C is produced as an inactive propeptide. Convertases such as furin, PC5, or PC7 cleave the VEGF homology domain and the C-terminal silk homology domain to generate pro-VEGF-C. Pro-VEGF-C can bind to VEGFR-3 but does not activate it. A second proteolytic cleavage by A-disintegrin with thrombospondin motif 3 and metalloproteinase (ADAMTS3) removes both terminal domains, yielding a mature, active VEGF-C protein. See, for example, Rauniyar et al., Front. Bioeng. Biotechnol., 2018, Vol. 6, Art. 7, doi.org / 10.3389 / fbioe.2018.00007. In wild-type human VEGF-C, the mature VEGF-C protein (SEQ ID NO: 101) corresponds to amino acids 115-215 of the wild-type human VEGF-C propeptide (SEQ ID NO: 1; UniProt Accession #P49767). In wild-type mouse VEGF-C, the mature VEGF-C protein corresponds to amino acids 111-211 (SEQ ID NO: 292) of the mouse VEGF-C propeptide (SEQ ID NO: 4).

[0109] In some embodiments, VEGF-C mutation The protein is human VEGF-C protein. mutation It can be a protein. Human VEGF-C mutation The protein may contain sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 115–215 of the following polypeptide sequences: MHLLGFFSVACSLLAAAALLPGPREAPAAAAAFESGLDLSDAEPDAGEATAYASKDLEEQLRSVSSVDELMTVLYPEYWKMYKCQLRKGGWQHNREQANLNSRTEETIKFAAAHYNTEILKSIDNE WRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLDVYRQVHSIIRRSLPATLPQCQAANKTCPTNYMWNN HICRCLAQEDFMFSSDAGDDSTDGFHDICGPNKELDEETCQCVCRAGLRPASCGPHKELDRNSCQCVCKNKLFPSQCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFSQPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFS (Sequence ID 1; Wild-type VEGF-C propeptide; UniProt database accession number P49767).

[0110] Human VEGF-C mutation A protein-coding polynucleotide molecule may contain sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 343–645 of the sequence encoding VEGF-C below:

[0111] Human VEGF-C mutation The protein may contain sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Wild-type human mature VEGF-C protein; SEQ ID NO: 101).

[0112] Human VEGF-C mutation Protein-coding polynucleotide molecules may contain sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following VEGF-C encoding sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (Sequence ID 153; nucleotide sequence encoding human mature VEGF-C protein).

[0113] In a specific embodiment, VEGF-C Mutant proteinThis includes one or more mutations in residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, I188, V190, L192, P196 and combinations thereof, where the location of the mutation is defined in relation to SEQ ID NO: 1. In certain embodiments, the mutation at L119 is the L119E mutation. In certain embodiments, the mutation at L119 is the L119M mutation. In certain embodiments, the mutation at D123 is the D123N mutation. In certain embodiments, the mutation at Q130 is the Q130K mutation. In certain embodiments, the mutation at N167 is the N167R mutation. In certain embodiments, the mutation at N167 is the N167I mutation. In certain embodiments, the mutation at N167 is the N167Q mutation. In certain embodiments, the mutation at N167 is the N167H mutation. In certain embodiments, the mutation at S168 is the S168G mutation. In certain embodiments, the mutation at S168 is the S168R mutation. In certain embodiments, the mutation at V190 is the V190T mutation. In certain embodiments, the mutation at L192L is the L192I mutation.

[0114] In some embodiments, VEGF-C mutation The protein is the mouse VEGF-C protein. mutation It may be a protein. In certain embodiments, mouse VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 111-211 of the following polypeptide sequences: MHLLCFLSLACSLLAAALIPSPREAPATVAAFESGLGFSEAEPDGGEVKAFEGKDLEEQLRSVSSVDELMSVLYPDYWKMYKCQLRKGGWQQPTLNTRTGDSVKFAAAHY NTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLDVYRQVHSI IRRSLPATLPQCQAANKTCPTNYVWNNYMCRCLAQQDFIFYSNVEDDSTNGFHDVCGPNKELDEDTCQCVCKGGLRPSSCGPHKELDRDSCQCVCKNKLFPNSCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTENTQKCFLKGKKFHHQTCSCYRRPCANRLKHCDPGLSFSEEVCRCVPSYWKRPHLN (Mouse wild-type VEGFC propeptide; SEQ ID NO: 4).

[0115] Mouse VEGF-C Mutant protein Polynucleotide molecules encoding VEGF-C may contain sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to nucleotides 331-631 of the following VEGF-C encoding sequence:

[0116] In some embodiments, VEGF-C mutation The protein is the mouse VEGF-C protein. mutation It may be a protein. In certain embodiments, mouse VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Wild-type mouse mature VEGF-C protein; SEQ ID NO: 292).

[0117] Mouse VEGF-C mutation Protein-coding polynucleotide molecules may contain sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to the following VEGF-C-coding sequences: AACACAGAGATCCTGAAAAGTATTGATAATGAGTGGAGAAAGACTCAATGCATGCCACGTGAGGTGTGTATAGATGTGGGGAAGGAGTTTGGAGCAGCCACAAACACCTTCTTTAAACCTCCATGTGTGTCCGTCTACAGATGTGGGGGTTGCTGCAACAGCGAGGGGCTGCAGTGCATGAACACCAGCACAGGTTACCTCAGCAAGACGTTGTTTGAAATTACAGTGCCTCTCTCACAAGGCCCCAAACCAGTCACAATCAGTTTTGCCAATCACACTTCCTGCCGGTGCATGTCTAAACTG (nucleotide sequence encoding mature VEGF-C in wild-type mouse; SEQ ID NO: 293).

[0118] In a specific embodiment, VEGF-C mutation The protein may contain sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL(Human N167Q VEGF-C mutation Protein; sequence number 160).

[0119] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTG CCAGTCTGAGGGGCTCCAATGTATGAACACGAGTACGtctTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG(Human N167Q VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 210).

[0120] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Human N167Q and S168G VEGF-C) mutation Protein; SEQ ID NO: 161).

[0121] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAG GGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human N167Q and S168G VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 211).

[0122] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (Human N167Q and L192I VEGF-C) mutation Protein; SEQ ID NO: 162).

[0123] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAG TCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human N167Q and L192I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 212).

[0124] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (Human N167Q, S168G, and L192I VEGF-C) mutation Protein; SEQ ID NO: 163).

[0125] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGC GAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human N167Q, S168G, and L192I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 213).

[0126] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL(Human N167I VEGF-C mutation Protein; (Sequence ID 166).

[0127] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTG CATCTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG(Human N167I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 216).

[0128] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Human N167I and S168G VEGF-C) mutation Protein; SEQ ID NO: 167).

[0129] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATC GGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human N167I and S168G VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 217).

[0130] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (Human N167I and L192I VEGF-C) mutation Protein; SEQ ID NO: 168).

[0131] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATC TCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human N167I and L192I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 218).

[0132] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (Human N167I, S168G and L192I VEGF-C) mutation Protein; SEQ ID NO: 169).

[0133] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCGGC GAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human N167I, S168G and L192I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 219).

[0134] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL(Human S168G VEGF-C mutation Protein; SEQ ID NO: 172).

[0135] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTG CAATGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG(Human S168G VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 222).

[0136] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (Human S168G and L192I VEGF-C) mutation Protein; SEQ ID NO: 174).

[0137] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAAT GGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human S168G and L192I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 224).

[0138] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCHSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL(Human N167H VEGF-C mutation Protein; SEQ ID NO: 184).

[0139] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTG CCACTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG(Human N167H VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 234).

[0140] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCIREGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Human N167I and S168R VEGF-C) mutation Protein; SEQ ID NO: 192).

[0141] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATC AGAGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (Human N167I and S168R VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 242).

[0142] In some embodiments, VEGF-C mutation The protein or its functional fragment contains one or more mutations selected from mutations at residues N167, S168, and / or L192, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0143] In some embodiments, the mutation at N167 is an N167I mutation, an N167Q mutation, or an N167H mutation; the mutation at S168 is an S168G mutation, or an S168R mutation; and / or the mutation at L192 is an L192I mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0144] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the N167Q mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0145] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the N167Q mutation and the S168G mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0146] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the N167Q mutation and the L192I mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0147] In some embodiments, VEGF-C mutation The protein or its functional fragment includes the N167Q mutation, the S168G mutation, and the L192I mutation, where the location of the mutation is defined in relation to Sequence ID No. 1.

[0148] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the N167I mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0149] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the N167I mutation and the S168G mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0150] In some embodiments, VEGF-C mutation The protein or a functional fragment thereof comprises an N167I mutation and an L192I mutation, where the positions of the mutations are defined in relation to SEQ ID NO: 1.

[0151] In some embodiments, VEGF-C mutation The protein or a functional fragment thereof comprises an N167I mutation, an S168G mutation, and an L192I mutation, where the positions of the mutations are defined in relation to SEQ ID NO: 1.

[0152] In some embodiments, VEGF-C mutation The protein or a functional fragment thereof comprises an S168G mutation, where the position of the mutation is defined in relation to SEQ ID NO: 1.

[0153] In some embodiments, VEGF-C mutation The protein or a functional fragment thereof comprises an S168G mutation and an L192I mutation, where the positions of the mutations are defined in relation to SEQ ID NO: 1.

[0154] In some embodiments, VEGF-C mutation The protein or a functional fragment thereof comprises an N167H mutation, where the position of the mutation is defined in relation to SEQ ID NO: 1.

[0155] In some embodiments, VEGF-C mutation The protein or a functional fragment thereof comprises an N167I mutation and an S168R mutation, where the positions of the mutations are defined in relation to SEQ ID NO: 1.

[0156] In certain embodiments, VEGF-C Mutant protein may comprise a functional fragment of human VEGF-C.

[0157] In certain embodiments, VEGF-C Mutant proteinIt may include a sequence that is at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the polypeptide sequences of SEQ ID NO: 154-159, SEQ ID NO: 164-165, SEQ ID NO: 170-171, SEQ ID NO: 173, SEQ ID NO: 175-183, SEQ ID NO: 185-191, or SEQ ID NO: 193-203.

[0158] In certain embodiments, VEGF-C Mutant protein The polynucleotide molecule encoding it may include a sequence that has at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any of the sequences of SEQ ID NO: 204-209, SEQ ID NO: 214-215, SEQ ID NO: 220-221, SEQ ID NO: 223, SEQ ID NO: 225-233, SEQ ID NO: 235-241, or SEQ ID NO: 243-253.

[0159] In certain embodiments, VEGF-C Mutant protein may include a functional fragment of mouse VEGF-C. In certain embodiments, VEGF-C Mutant protein may include a sequence that is at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequence: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (mouse VEGF-C mutation protein, SEQ ID NO: 23).

[0160] In certain embodiments, VEGF-C Mutant proteinPolynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGCGGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGC AATTCTGAGGGGCTCCAATGTATGAACACGAGTACGGGTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG(Mouse VEGF-C mutation Protein-coding nucleotide sequence (SEQ ID NO: 24).

[0161] In a specific embodiment, VEGF-C Mutant proteinThis includes one or more mutations in residues T112, L115, D119, Q126, T144, N145, K149, N163, S164, E165, I184, V186, L188, P192 and combinations thereof, where the location of the mutation is defined in relation to SEQ ID NO: 4. In certain embodiments, the mutation at L115 is the L115E mutation. In certain embodiments, the mutation at L115 is the L115M mutation. In certain embodiments, the mutation at D119 is the D119N mutation. In certain embodiments, the mutation at Q126 is the Q126K mutation. In certain embodiments, the mutation at N163 is the N163R mutation. In certain embodiments, the mutation at N163 is the N163I mutation. In certain embodiments, the mutation at N163 is the N163Q mutation. In certain embodiments, the mutation at S164 is the S164G mutation. In certain embodiments, the mutation at V186 is the V184T mutation. In certain embodiments, the mutation at L188 is the L188I mutation. In some embodiments, VEGF-C mutation The protein contains the N163I mutation.

[0162] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: MHLLCFLSLACSLLAAALIPSPREAPATVAAFESGLGFSEAEPDGGEVKAFEGKDLEEQLRSVSSVDELMSVLYPDYWKMYKCQLRKGGWQQPTLNTRTGDSVKFAAAHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLD VYRQVHSIIRRSLPATLPQCQAANKTCPTNYVWNNYMCRCLAQQDFIFYSNVEDDSTNGFHDVCGPNKELDEDTCQCVCKGGLRPSSCGPHKELDRDSCQCVCKNK LFPNSCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTENTQKCFLKGKKFHHQTCSCYRRPCANRLKHCDPGLSFSEEVCRCVPSYWKRPHLN(Mouse N163I VEGF-C mutation Protein; (SEQ ID NO: 6).

[0163] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein (SEQ ID NO: 5).

[0164] In some embodiments, VEGF-C mutation The protein contains mutations N163R, V186T, and L188I, where the mutation locations are defined in relation to SEQ ID NO: 4.

[0165] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: MHLLCFLSLACSLLAAALIPSPREAPATVAAFESGLGFSEAEPDGGEVKAFEGKDLEEQLRSVSSVDELMSVLYPDYWKMYKCQLRKGGWQQPTLNTRTGDSVKF AAAHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKL DVYRQVHSIIRRSLPATLPQCQAANKTCPTNYVWNNYMCRCLAQQDFIFYSNVEDDSTNGFHDVCGPNKELDEDTCQCVCKGGLRPSSCGPHKELDRDSCQCVCK NKLFPNSCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTENTQKCFLKGKKFHHQTCSCYRRPCANRLKHCDPGLSFSEEVCRCVPSYWKRPHLN(Mouse RTI VEGFC mutation Protein (SEQ ID NO: 8).

[0166] In a specific embodiment, VEGF-C Mutant proteinPolynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein (SEQ ID NO: 7).

[0167] In a specific embodiment, VEGF-C Mutant protein This involves a mutation at residue C133, where the location of the mutation is defined in relation to SEQ ID NO: 4. In certain embodiments, the mutation at residue C133 is a C133A mutation.

[0168] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL(C133A mutant mouse VEGF-C mutation Protein, SEQ ID NO: 9)

[0169] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGCGGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATTCTGAGGGGCTCCAATGTATGAACACGAGTACGGGTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (VEGF-C having the C133A mutation mutation A nucleotide sequence encoding the protein, SEQ ID NO: 10).

[0170] In certain embodiments, VEGF-C mutation ​​​​​​​​​​​​​​​​Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGCAAAACTCAATGTATGCCACGGGAGGTTGCAATAGACGTGGGAAAGGAATTTGGCGCCGCCACGAATACCTTTTCAAGCCTCCCTGTGTGAGTGTTTACAGATGTGGTGGTTGTTGCATATCAGAG GGATTGCAGTGCATGAACACAAGTACAGGTTACTTGAGTAAAACATTGTTTGAAATCACAGTACCATTGTCCCAAGGCCCTAAGCCGGTTACGATCTCTTTCGCCAATCATACGTCATGCCGCTGTATGAGTAAGTTG (Mouse VEGF-C with C133A and N163I mutations mutation Nucleotide sequence encoding a protein (SEQ ID NO: 18).

[0173] In some embodiments, VEGF-C mutation The protein contains mutations C133A, N163R, V186T, and L188I, where the mutation locations are defined in relation to SEQ ID NO: 4.

[0174] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKL(Mouse VEGF-C with C133A, N163R, V186T, and L188I mutations) mutationProtein (SEQ ID NO: 13).

[0175] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGAAAAACCCAATGTATGCCACGCGAGGTAGCGATAGATGTGGGCAAAGAATTCGGCGCCGCGACGAACACCTTTTTCAAGCCCCCTTGCGTCTCCGTATATAGATGCGGTGGATGTTGCCGATCCGAGGGCCTTC AGTGTATGAACACATCTACTGGCTATTTGAGCAAGACGCTCTTTGAGATTACAACACCAATTAGTCAAGGTCCCAAGCCTGTTACCATCTCTTTCGCTAACCACACTTCATGCCGCTGTATGAGTAAGTTG(Mouse VEGF-C with C133A, N163R, V186T, and L188I mutations mutation Protein-coding nucleotide sequence, SEQ ID NO: 14).

[0176] In a specific embodiment, VEGF-C Mutant protein This involves a mutation at residue C137, where the location of the mutation is defined in relation to Sequence ID No. 1. In a particular embodiment, the mutation at residue C137 is a C137A mutation.

[0177] In a specific embodiment, human VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL(Human VEGF-C with C133A mutation) mutation Protein; (SEQ ID NO: 100).

[0178] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATTC TGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (Human VEGF-C with C133A mutation mutation A nucleotide sequence that codes for a protein (SEQ ID NO: 152).

[0179] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Human C137A and N167Q VEGF-C) mutation Protein; SEQ ID NO: 56).

[0180] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAG TCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A and N167Q VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 108).

[0181] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Human C137A, N167Q and S168G VEGF-C) mutation Protein; SEQ ID NO: 57).

[0182] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGC GAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A, N167Q and S168G VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 109).

[0183] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (Human C137A, N167Q and L192I VEGF-C) mutation Protein; SEQ ID NO: 58).

[0184] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGTCT GAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A, N167Q and L192I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 110).

[0185] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (Human C137A, N167Q, S168G and L192I VEGF-C) mutation Protein; SEQ ID NO: 59).

[0186] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGCGAG GGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A, N167Q, S168G and L192I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 111).

[0187] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Human C137A and N167I VEGF-C) mutation Protein; (SEQ ID NO: 62).

[0188] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATC TCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A and N167I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 114).

[0189] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Human C137A, N167I and S168G VEGF-C) mutation Protein; SEQ ID NO: 63).

[0190] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCGGC GAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A, N167I and S168G VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 115).

[0191] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (Human C137A, N167I and L192I VEGF-C) mutation Protein; (SEQ ID NO: 64).

[0192] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCTCT GAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A, N167I and L192I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 116).

[0193] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (Human C137A, N167I, S168G and L192I VEGF-C) mutation Protein; SEQ ID NO: 65).

[0194] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCGGCGAG GGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A, N167I, S168G and L192I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 117).

[0195] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Human C137A and S168G VEGF-C) mutation Protein; SEQ ID NO: 68).

[0196] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAAT GGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A and S168G VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 120).

[0197] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (Human C137A, S168G and L192I VEGF-C) mutation Protein; SEQ ID NO: 70).

[0198] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATGGC GAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A, S168G and L192I VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 122).

[0199] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCHSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Human C137A and N167H VEGF-C) mutation Protein; SEQ ID NO: 80).

[0200] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAC TCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A and N167H VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 132).

[0201] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIREGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Human C137A, N167I and S168R VEGF-C) mutation Protein; SEQ ID NO: 88).

[0202] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCAGA GAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (human C137A, N167I and S168R VEGF-C mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 140).

[0203] In some embodiments, VEGF-C mutation The protein or its functional fragment contains one or more mutations selected from mutations at residues C137, N167, S168, and / or L192, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0204] In some embodiments, the mutation at C137 is C137A, the mutation at N167 is N167I, N167Q, or N167H; the mutation at S168 is S168G or S168R; and / or the mutation at L192 is L192I, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0205] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the C137A mutation and the N167Q mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0206] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the C137A mutation, the N167Q mutation, and the S168G mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0207] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the C137A mutation, the N167Q mutation, and the L192I mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0208] In some embodiments, VEGF-C mutation The protein or its functional fragment includes the C137A mutation, N167Q mutation, S168G mutation, and L192I mutation, where the location of the mutation is defined in relation to Sequence ID No. 1.

[0209] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the C137A mutation and the N167I mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0210] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the C137A mutation, the N167I mutation, and the S168G mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0211] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the C137A mutation, the N167I mutation, and the L192I mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0212] In some embodiments, VEGF-C mutation The protein or its functional fragment includes the C137A mutation, N167I mutation, S168G mutation, and L192I mutation, where the location of the mutation is defined in relation to Sequence ID No. 1.

[0213] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the C137A mutation and the S168G mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0214] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the C137A mutation, the S168G mutation, and the L192I mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0215] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the C137A mutation and the N167H mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0216] In some embodiments, VEGF-C mutation The protein or its functional fragment contains the C137A mutation, the N167I mutation, and the S168R mutation, where the location of the mutation is defined in relation to SEQ ID NO: 1.

[0217] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the polypeptide sequences of sequence numbers 50-55, 60-61, 66-67, 69, 71-79, 81-87, or 89-99.

[0218] In a specific embodiment, VEGF-C Mutant protein The polynucleotides encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any of the sequence numbers 102-107, 112-113, 118-119, 121, 123-131, 133-139, or 141-151.

[0219] VEGF-C as described herein Mutant protein Or a fragment thereof contains the VEGF-C described above. Mutant protein Or, variants having one or more amino acid substitutions, deletions, or additions that preserve the biological properties of the fragment (e.g., binding affinity or immunoeffector activity).

[0220] These variants include: (i) variants in which one or more amino acid residues are substituted with conserved or non-conserved amino acids; (ii) variants in which one or more amino acids are added to or deleted from the polypeptide; (iii) variants in which one or more amino acids contain substituents; and (iv) the VEGF-C described above. Mutant protein Or the fragments may include, for example, an antibody epitope, a polyhistidine sequence, a biotin moiety, etc., of VEGF-C Mutant protein Or variants fused or conjugated with another peptide or polypeptide (e.g., fusion partner, protein tag) or other chemical moiety that can impart useful properties to the fragment. VEGF-C as described herein Mutant proteinAlternatively, the fragment may include variants in which certain amino acid residues are substituted with corresponding residues of other species at either a conserved or non-conserved position. In other embodiments, the amino acid residue at a non-conserved position is substituted with a conserved or non-conserved residue. Amino acid substitutions are generally based on the relative similarity of substituents on the amino acid side chains, such as hydrophobicity, hydrophilicity, charge, size, etc.

[0221] Amino acid substitutions may be conservative, meaning that the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art will understand which amino acids share similar chemical properties. For example, the following groups of amino acids have similar chemical properties such as size, charge, and polarity: Group I (Ala, Ser, Thr, Pro, Gly); Group II (Asp, Asn, Glu, Gln); Group III (His, Arg, Lys); Group IV (Met, Leu, Ile, Val, Cys); Group V (Phe, Thy, Trp).

[0222] In some embodiments, VEGF-C mutation Proteins are modified to extend their circulating half-life. Strategies for extending the half-life of recombinant proteins include, but are not limited to, fusion to immunoglobulins or immunoglobulin fragments such as the Fc domain of IgG; fusion to albumin or albumin fragments; fusion to albumin-binding antibodies or their antibody fragments such as scFv, Fab, or single-domain antibodies (VHH); or chemical modification with polyethylene glycol (PEG).

[0223] In some embodiments, VEGF-C Mutant protein Or fragments thereof or variants thereof may be fused and / or conjugated to one or more heterologous parts of peptides, polypeptides, small molecules, polymers, nucleic acids, lipids, sugars, etc., but are not limited to these.

[0224] In a specific embodiment, VEGF-C Mutant protein VEGF-C Mutant proteinIt is fused and / or conjugated to a portion that provides a long half-life. In some embodiments, VEGF-C Mutant protein This includes, but is not limited to, small molecules, peptides, polypeptides, or lipids that bind to albumin, and is fused and / or conjugated to a portion that specifically binds to albumin. In some embodiments, VEGF-C Mutant protein It is fused and / or conjugated to the constant region (Fc domain) of an immunoglobulin, scFv, Fab, single-domain antibody (VHH), immunoglobulin, or their heavy or light chain.

[0225] In a specific embodiment, VEGF-C Mutant protein These polymers are fused and / or conjugated to polymers including, but not limited to, lipid polymers, polyethylene glycol (PEG), biodegradable polymers such as PLA (poly(lactic acid)) and PLGA (poly(lactic acid-glycolic acid)), polysaccharides, poly(propylene glycol), polyoxyethylated polyols, polyvinyl ethers, copolymers of ethylene glycol and propylene glycol, polyvinyl alcohol, dextran, hyaluronic acid, and chitin.

[0226] In a specific embodiment, VEGF-C Mutant protein It is fused to and / or conjugated to the Fc domain. In certain embodiments, the VEGF-C of the present invention Mutant protein The Fc sequence fused or conjugated to may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 21).

[0227] In certain embodiments, a polynucleotide molecule encoding an Fc sequence may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: (Sequence ID 22).

[0228] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Mouse VEGF-C with C133A mutation and Fc conjugation) mutation Protein (SEQ ID NO: 11).

[0229] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: mutation Protein-coding nucleotide sequence, SEQ ID NO: 12).

[0230] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(C133A, N163R, V186T, and L188I mutations, and mouse VEGF-C with Fc conjugation) mutation Protein (SEQ ID NO: 15).

[0231] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: mutation Protein-coding nucleotide sequence (SEQ ID NO: 16).

[0232] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Mouse VEGF-C with C133A and N163I mutations and Fc conjugation) mutation Protein (SEQ ID NO: 19).

[0233] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: mutation Protein-coding nucleotide sequence (SEQ ID NO: 20).

[0234] In some embodiments, the IgG Fc domain includes the amino acid sequence of SEQ ID NO: 21. In some embodiments, VEGF-C mutation The protein contains the amino acid sequence of SEQ ID NO: 11. In some embodiments, VEGF-C Mutant protein This includes the amino acid sequence of SEQ ID NO: 19. In some embodiments, VEGF-C Mutant protein This consists of the amino acid sequence of SEQ ID NO: 19. In some embodiments, VEGF-C Mutant protein This includes the amino acid sequence of SEQ ID NO: 15. In some embodiments, VEGF-C Mutant protein This consists of the amino acid sequence of SEQ ID NO: 15.

[0235] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (Human C137A VEGF-C protein with Fc conjugation; SEQ ID NO: 266).

[0236] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences:

[0237] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A and N167Q with Fc conjugation VEGF-C mutation Protein; SEQ ID NO: 267).

[0238] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 280).

[0239] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A, N167Q and S168G VEGF-C with Fc conjugation) mutation Protein; SEQ ID NO: 268).

[0240] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 281).

[0241] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A, N167Q and L192I VEGF-C with Fc conjugation) mutation Protein; SEQ ID NO: 269).

[0242] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 282).

[0243] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A, N167Q, S168G and L192I VEGF-C with Fc conjugation) mutation Protein; sequence number).

[0244] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 283).

[0245] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A and N167I VEGF-C with Fc conjugation) mutation Protein; SEQ ID NO: 271).

[0246] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 284).

[0247] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A, N167I and S168G VEGF-C with Fc conjugation) mutation Protein; SEQ ID NO: 272).

[0248] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 285).

[0249] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A, N167I and L192I VEGF-C with Fc conjugation) mutation Protein; SEQ ID NO: 273).

[0250] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 286).

[0251] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A, N167I, S168G and L192I VEGF-C with Fc conjugation) mutation Protein; SEQ ID NO: 274).

[0252] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 287).

[0253] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A and S168G VEGF-C with Fc conjugation) mutation Protein; SEQ ID NO: 275).

[0254] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 288).

[0255] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A, S168G and L192I VEGF-C with Fc conjugation) mutation Protein; SEQ ID NO: 276).

[0256] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 289).

[0257] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCHSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A and N167H with Fc conjugation VEGF- mutation Protein; SEQ ID NO: 277).

[0258] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 290).

[0259] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIREGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(Human C137A, N167I and S168R VEGF-C with Fc conjugation) mutation Protein; SEQ ID NO: 278).

[0260] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 291).

[0261] In some embodiments, the present invention relates to modifying the IgG Fc domain to reduce Fc effector function. Such modifications can be achieved by a variety of techniques, including but not limited to amino acid substitutions, deletions, or additions to the Fc domain. For example, modifications may include substituting one or more amino acid residues in the Fc domain with non-native amino acids, altering the glycosylation pattern, or introducing steric hindrance to the Fc domain. In some embodiments, the modified Fc domain exhibits reduced binding affinity to Fc receptors or complement proteins, resulting in reduced Fc effector function. Examples of modified Fc domains exhibiting reduced Fc effector function include, but are not limited to, Fc variants with point mutations such as N297A, L234A / L235A, S239D / I332E, S298A / IgG1 / IgG3, L309D / I332E, M428L, and N434S, or Fc variants with additional glycosylation sites. These modifications can be used to create therapeutic antibodies with improved safety profiles and reduced risk of adverse immune responses. Modified Fc domains can also be used to design antibody-based therapeutics with desired effector functions, such as reduced ADCC, complement activation, or immune complex formation. In some embodiments, the IgG Fc domain is modified to reduce Fc effector function. In some embodiments, the IgG Fc domain contains a mutation at residue N297. In some embodiments, the mutation at residue N297 is selected from N297Q, N297A, and N297G. In some embodiments, the IgG Fc domain is an IgG4 variant containing one or more mutations at residue F234 or L235. In some embodiments, the IgG Fc domain is an IgG4 variant containing one or more mutations F234A or L235A. In some embodiments, the IgG Fc domain is an IgG1 variant containing one or more mutations at residue L234 or L235. In some embodiments, the IgG Fc domain is an IgG1 variant containing one or more L234A or L235A mutations.In some embodiments, the IgG Fc domain may be a cross-subclass domain. For example, but not limited to, the IgG Fc domain may be an IgG2 variant having a point mutation from IgG4 (e.g., H268Q / V309L / A330S / P331S).

[0262] In some embodiments, the Fc domain contains one or more mutations. In some embodiments, the Fc domain is mutated to reduce Fc effector function, modified by glycoengineering, or otherwise modified.

[0263] In some embodiments, the Fc domain contains one or more mutations that excise important glycosylation sites necessary for effector function.

[0264] In some embodiments, the Fc domain includes a mutation at residue N82, where the position of the residue is defined in relation to Sequence ID No. 21. In some embodiments, the mutation at N82 defined in relation to Sequence ID No. 21 is an N82Q mutation, an N82A mutation, or an N82G mutation.

[0265] In a specific embodiment, VEGF-C Mutant protein It is fused to and / or conjugated to albumin. In certain embodiments, the albumin is human albumin.

[0266] In some embodiments, VEGF-C Mutant proteinThese are fused to and / or conjugated to albumin or Fc domains via linkers. Examples of linkers include, but are not limited to, proline-rich linkers, acidic linkers, basic linkers, cleavable linkers, or rigid linkers. Examples of proline-rich linkers include, but are not limited to, (Pro-Pro-Gly)3 (SEQ ID NO: 295), (Pro-Gly)5 (SEQ ID NO: 296), or (Pro-Pro-Pro-Gly)3 (SEQ ID NO: 297). Examples of acidic linkers include, but are not limited to, (Glu-Ser-Glu-Ser)3 (SEQ ID NO: 298), (Asp-Glu)5 (SEQ ID NO: 299), or (Glu-Asp-Glu-Asp-Glu)3 (SEQ ID NO: 300). Examples of basic linkers include, but are not limited to, (Lys-Ser-Lys-Ser)3 (SEQ ID NO: 301), (Arg-Lys-Arg-Lys)3 (SEQ ID NO: 302), or (Lys-Arg-Lys-Arg-Lys)3 (SEQ ID NO: 303). Examples of cleavable linkers include, but are not limited to, (Gly-Gly-Ser)3 (SEQ ID NO: 304), which can be cleaved by proteases such as trypsin, chymotrypsin, or thrombin, or (Leu-Val-Pro-Arg) (SEQ ID NO: 305), which can be cleaved by protease factor Xa. Examples of rigid linkers include, but are not limited to, (azido-Lys-Arg-Pro)m, which contains the unnatural amino acid azido-lysine and can form triazole bonds with alkyne-containing molecules, or (cyclohexyl-Ala-Pro-Pro), which contains the unnatural amino acid cyclohexyl-alanine and can form a rigid cyclohexane structure. Examples of such linkers include, but are not limited to, the linker having the amino acid sequence: GGGGGSGGGGSGGGGS (SEQ ID NO: 294).

[0267] In a specific embodiment, VEGF-C Mutant proteinThis may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLHHHHHHHH(8xHis(SEQ ID NO: 306)aa sequence and mouse VEGF-C with C133A and N163I mutations) mutation Protein; SEQ ID NO: 254).

[0268] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGCAAAACTCAATGTATGCCACGGGAGGTTGCAATAGACGTGGGAAAGGAATTTGGCGCCGCCACGAATACCTTTTTCAAGCCTCCCTGTGTGAGTGTTTACAGATGTGGTGGTTGTTGCATATCAGAGGGATTGCAGTGCATGAACACAAGTACAGGTTACTTGAGTAAAACATTGTTTGAAATCACAGTACCATTGTCCCAAGGCCCTAAGCCGGTTACGATCTCTTTCGCCAATCATACGTCATGCCGCTGTATGAGTAAGTTGCATCACCATCACCATCATCACCAT(8xHis (SEQ ID NO. 306) aa sequence and C133A and N163I mutations) mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 260).

[0269] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKLHHHHHHHH(8xHis(SEQ ID NO: 306)aa sequence and mouse VEGF-C with C133A, N163R, V186T and L188I mutations) mutation Protein; SEQ ID NO: 255).

[0270] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGAAAAACCCAATGTATGCCACGCGAGGTAGCGATAGATGTGGGCAAAGAATTCGGCGCCGCGACGAACACCTTTTTCAAGCCCCCTTGCGTCTCCGTATATAGATGCGGTGGATGTTGCCGATCCGAGGGCCTTCAGTGTATGAACACATCTACTGGCTATTTGAGCAAGACGCTCTTTGAGATTACAACACCAATTAGTCAAGGTCCCAAGCCTGTTACCATCTCTTTCGCTAACCACACTTCATGCCGCTGTATGAGTAAGTTGCATCACCATCACCATCATCACCAT(8xHis(SEQ ID NO: 306)aa sequence and mouse VEGF-C with C133A, N163R, V186T and L188I mutations) mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 261).

[0271] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (C133A and N163I variant AAA cloning scar) Mouse VEGF-C containing Fc(N297Q) fused 6xHis(SEQ ID NO: 25)aa sequence mutation Protein; SEQ ID NO: 256).

[0272] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 262).

[0273] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(C133A, N163R, V186T and L188I mutant AAA cloning scar Fc(N297Q) fusion 6xHis aa-arranged mouse VEGF-C mutation Protein; SEQ ID NO: 257).

[0274] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 263).

[0275] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (C133A and N163I mutant AAA cloning scar and Linker mouse serum albumin fusion GS Mouse VEGF-C with 8xHis (sequence number 306)aa sequence mutation Protein; SEQ ID NO: 258).

[0276] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: mutation Protein; SEQ ID NO: 264).

[0277] In a specific embodiment, VEGF-C Mutant protein This may include sequences that are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following polypeptide sequences: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (C133A, N163R, V186T and L188I mutant AAA cloning scar and linker mouse serum albumin fusion GS Mouse VEGF-C with 8xHis (sequence number 306)aa sequence mutation Protein; SEQ ID NO: 259).

[0278] In a specific embodiment, VEGF-C Mutant protein Polynucleotide molecules encoding may contain sequences having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the following nucleotide sequences: mutation A nucleotide sequence that codes for a protein; (SEQ ID NO: 265).

[0279] In some embodiments, VEGF-C Mutant protein Alternatively, the polynucleotide molecule encoding its functional fragment is mRNA.

[0280] mRNA can offer several advantages to AAV and other gene delivery systems, including, but not limited to, one or more of the following: mRNA is highly customizable; mRNA can prevent recognition by pattern recognition receptors and nucleases, enabling sustained expression; mRNA can provide well-controlled expression dynamics through repeated dosing options; and mRNA localizes in the cytoplasm, reducing the risk of integration into the genome. mRNA can also be more cost-effective.

[0281] In some embodiments, VEGF-C mutation Polynucleotide molecules encoding proteins or functional fragments thereof may contain modified nucleotides, such as 5-methylcytosine and pseudouridine substitutions, which can improve the stability of the polynucleotide molecule, reduce deamination, decrease nuclease activity, reduce innate recognition, or improve translation efficiency. In some embodiments, the modified nucleotide is 5-methylcytosine or pseudouridine. In some embodiments, VEGF-C Mutant protein The polynucleotide molecule encoding this includes a 5' cap.

[0282] The mRNA may contain modified nucleotides. In some embodiments, the modified nucleotides are 5-methylcytosine or pseudouridine. In some embodiments, VEGF-C Mutant proteinThe polynucleotide molecule or functional fragment thereof encoding contains a 5' cap. In some embodiments, the 5' cap is added using CleanCap Reagent AG. CleanCap is composed of C32H43N15O24P4, enabling high capping efficiency and resulting in more active mRNA. Cap1 does not activate the pattern recognition receptor and is important for good in vivo expression. While we do not wish to be bound by theory, one or more of 5-methylcytosine, pseudouridine, and the 5' cap may improve mRNA stability, thereby promoting VEGF-C Mutant protein Alternatively, the expression of the functional fragment can be extended.

[0283] In some embodiments, VEGF-C Mutant protein Alternatively, polynucleotide molecules encoding the functional fragment thereof are contained within the viral vector. Exemplary viral vectors include, but are not limited to, herpesviruses, cytomegaloviruses, polioviruses, alphaviruses, vaccinia viruses, rabies viruses, adenoviruses (AAVs), retroviruses, lentiviruses, and adenoviruses. Retroviruses may be lentiviruses. Recombinant viral particles may be derived from adenoviruses (AAVs). In some embodiments, the AAV is AAV2. In some embodiments, the AAV is AAV5. In some embodiments, the AAV is AAV9.

[0284] In some embodiments, VEGF-C Mutant protein Alternatively, the functional fragment can be administered in a drug regimen requiring a combination of mRNA and AAV. High expression can be rapidly achieved, for example, within 2 hours after mRNA delivery, with one or more mRNA doses. VEGF-C provided by AAV Mutant proteinThe expression of the functional fragment may take 7 to 14 days, or up to 4 weeks, depending on the serotype. Immediate expression can be obtained by administering the protein or its functional fragment. By administering both mRNA and AAV together or in succession over a short period, VEGF-C can be expressed. Mutant protein Alternatively, it can provide sustained expression of its functional fragment. While we do not wish to be bound by theory, protein administration can provide immediate expression and controlled expression dynamics, and multiple doses are possible. While we do not wish to be bound by theory, mRNA administration can provide immediate expression, controlled expression dynamics, and high expression, and multiple doses are possible. While we do not wish to be bound by theory, AAV administration can provide delayed expression and high levels of expression. Expression dynamics can be measured effectively and sensitively using ELISA and Western blotting.

[0285] In some embodiments, VEGF-C Mutant protein Alternatively, the polynucleotide molecule encoding its functional fragment is contained within the liposome. VEGF-C Mutant protein Alternatively, the functional fragment may be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the polynucleotide, enclosed within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in a micelle or complexed with a micelle, or otherwise associated with a lipid. VEGF-C Mutant proteinLiposomes containing the functional fragment may exist within a bilayer structure, as micelles, or in a "collapsed" structure. Liposomes may also simply be dispersed in solution and may form aggregates that are not uniform in size or shape. For example, nucleotides (e.g., siRNA) can be encapsulated in neutral liposomes using a method requiring ethanol and calcium. The shape may be that of a spherical vesicle. In various embodiments, liposomes may contain one or more concentric layers of lipid bilayer molecules. In some embodiments, the lipid components include combinations of Cl2-200, XTC, MC3, NC98-5, DLinDMA, HGT5001cis, HGT5001trans, HGT5000, HGT4003, DLinKC2DMA, ALNY100, ICE, DLinKC2DMA, CHOL, DOPE, DMG-PEG-2000, Cl2-200, DOPE, CHOL, and DMGPEG2K.

[0286] In some embodiments, VEGF-C Mutant protein Alternatively, a polynucleotide molecule encoding the functional fragment is attached to the nanoparticle or polymer. In certain embodiments, the nanoparticles of the present invention further include at least one activator (e.g., a portion targeting a specific cell or cell type) that specifically binds to a particular type or category of cells and / or other particular types of compounds. In some embodiments, the nanoparticles are nanospheres. In some embodiments, the polymer is dextran, poly(amine coester), poly(β-aminoester), polyethyleneimine, poly-L-lysine, polyethylene glycol, or a dendrimer.

[0287] In some embodiments, VEGF-C Mutant protein Alternatively, polynucleotide molecules encoding such functional fragments are contained within recombinant virus particles or virus-like particles (VLPs).

[0288] In some embodiments, VEGF-C Mutant proteinIt is produced and administered as a “masked” prodrug that is activated after administration to the patient. In some cases, this is achieved by the restricted expression of an endogenous protease that, for example, cleaves the prodrug and releases an active “mature” fragment. Mutant protein It can provide the desired tissue or tumor-specific activity. In some embodiments, VEGF-C Mutant protein This is a variant of the full-length VEGF-C propeptide, which is activated by proteases such as ADAMTS2, plasmin, furin, cathepsin D, thrombin, and / or KLK3 / PSA. In other embodiments, VEGF-C Mutant protein It is fused to an inhibitory peptide released by proteolysis by endogenous proteases. The inhibitory peptide can take many forms, including but not limited to: VEGF-C Mutant protein Antibodies or antibody fragments that bind to and weaken the interaction with VEGFR-3; VEGFR-3 extracellular domains or fragments thereof that competitively inhibit the interaction between VEGF-C and VEGFR-3 on cells; and any other binding factors (e.g., VHH, fibronectin domain, Nottin, Lipocalin, leucine-rich repeat, etc.). A non-limiting set of examples of protease cleavage sites is described below. Inhibitory peptides are enterokinase cleavage site (EKCS) peptides that are recognized and cleaved by enterokinase, an endogenous protease present in the small intestine, thereby inhibiting the biological activity of VEGF-C. Mutant protein This allows for the separation of the prodrug from the enterokinase. This enables the prodrug to be designed to be inactive until it reaches the small intestine, where it can be activated by enterokinase cleavage. Alternatively, the inhibitory peptide can be activated by a furin cleavage site (FCS) peptide, which is recognized and cleaved by furin, an endogenous protease overexpressed in many tumor cells, thereby enabling the biological activity of VEGF-C. Mutant proteinIt can be isolated from furin. By designing the prodrug to be specifically cleaved by furin, the activation of the prodrug can be localized to the tumor microenvironment, leading to targeted therapy. Furthermore, the prodrug can be designed to be cleaved by other endogenous proteases that are overexpressed in specific tumors. For example, the prodrug can be designed to be cleaved by matrix metalloproteinases MMP or urokinase-type plasminogen activator uPA, which are overexpressed in many tumors. This allows the activation of the prodrug to be localized to the tumor microenvironment, leading to targeted VEGF-C Mutant protein It can provide therapy.

[0289] In one embodiment, a method is provided for inducing lymphangiogenesis in the body of a subject requiring induction of lymphangiogenesis, the method comprising an effective amount of VEGF-C mutation Proteins or functional fragments thereof, or fusion proteins or conjugates thereof, or VEGF-C mutation The procedure includes administering to a subject a polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particles containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above.

[0290] In one embodiment, the foregoing provides a method for treating a disease or condition requiring treatment, the method comprising an effective amount of VEGF-C of the foregoing disclosure. mutation The process includes administering a protein or a functional fragment thereof, or a fusion protein of the Disclosure, or a polynucleotide molecule of the Disclosure, or a vector of the Disclosure, or a particle of the Disclosure, or a pharmaceutical composition of the Disclosure, to a subject.

[0291] In some embodiments, the disease or condition is cancer, coronary vascular function, osmolality, myocardial ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, disease or condition associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennecum syndrome, Milroy's disease, Turner syndrome, age-related macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, and retinal edema.

[0292] In some embodiments, the cancer is located in the brain or central nervous system of the subject. In some embodiments, it is selected from glioma, ependymoma, subependymoma, primitive neuroectodermal tumor, ganglioglioma, schwannoma, germ tumor, craniopharyngioma, meningioma, CNS lymphoma, pineal tumor, retinoblastoma, uveal melanoma, and rhabdoid tumor.

[0293] In some embodiments, VEGF-C mutation Proteins or their functional fragments, fusion proteins, conjugates, polynucleotide molecules, vectors, particles, or pharmaceutical compositions are administered intrathecally, intraocularly, intratumorally, intracisor magnum, intravitreously, by eye drops, subcutaneously, intradermally, by inhalation, by indwelling catheters, orally, topically, or systemically. In some embodiments, the pharmaceutical composition is formulated for intrathecal administration. In some embodiments, the pharmaceutical composition is formulated for intratumor administration. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for intracisor magnum administration. In some embodiments, the pharmaceutical composition is formulated for eye drops. In some embodiments, the pharmaceutical composition is formulated for intraocular administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intradermal administration. In some embodiments, the pharmaceutical composition is formulated for inhalation administration. In some embodiments, the pharmaceutical composition is formulated for administration via indwelling catheters. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for topical administration (e.g., as a cream or gel).

[0294] In one embodiment, a method is provided for treating a target cancer requiring treatment, the method comprising an effective amount of VEGF-C mutation Proteins or functional fragments thereof, or fusion proteins or conjugates thereof, or VEGF-C mutation The procedure includes administering to a subject a polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particles containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above.

[0295] In one embodiment, VEGF-C Mutant protein Alternatively, a pharmaceutical composition is provided comprising a functional fragment thereof and, optionally, an immunotherapy agent.

[0296] In one embodiment, VEGF-C Mutant protein Alternatively, a method for generating a library of its functional fragments is provided, and VEGF-C Mutant protein Alternatively, the functional fragment may have: (i) specific binding affinity to VEGF; and (ii) reduced binding affinity to VEGFR-2 compared to wild-type VEGF-C.

[0297] In certain embodiments, the cancer is melanoma. In certain embodiments, the cancer is located in the brain or central nervous system of the subject. Examples of such cancers include, but are not limited to, gliomas (e.g., astrocytoma, glioblastoma, oligodendroglioma, brainstem glioma, juvenile pilocytic astrocytoma, optic glioma), ependymoma, subependymoma, primitive neuroectodermal tumor, ganglioglioma, schwannoma, germoma, craniopharyngioma, meningioma, CNS lymphoma, pineal tumor, retinoblastoma, uveal melanoma, and rhabdoid tumor.

[0298] A glioma may be any tumor arising from the glial tissue of the brain. In some embodiments, the glioma may be a mixed glioma. The glioma may be a low-grade or high-grade glioma. The glioma may be located in the supratentorial, infratentorial, or pontine regions. Examples of gliomas include, but are not limited to, glioblastomas.

[0299] In certain embodiments, the cancer is glioblastoma. In certain embodiments, the cancer is glioblastoma pleomorphicum (GBM). Initial diagnosis of GBM is generally made using CT or MRI, where glioblastoma usually presents as a lesion exhibiting ring-enhancement. Confirmation of the diagnosis can be made based on biopsy, such as stereotactic biopsy, or craniotomy with tumor resection.

[0300] In certain embodiments, the cancer is metastatic cancer. In certain embodiments, the cancer is metastatic cancer that has spread to the brain or central nervous system of the subject. In certain embodiments, the cancer is metastatic brain cancer.

[0301] In certain embodiments, cancers include melanoma, lung cancer, breast cancer, stomach cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, head and neck squamous cell carcinoma, thyroid cancer, humoral cancer (e.g., acute myeloid leukemia), kidney cancer, urothelial carcinoma, prostate cancer, pheochromocytoma, cholangiocarcinoma, hepatocellular carcinoma, invasive pancreatic ductal carcinoma, thymoma, sarcoma, mesothelioma, testicular cancer, and colorectal cancer.

[0302] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing such polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used to treat or modulate cardiac function or cardiac disease. Examples of such cardiac function or cardiac disease include, but are not limited to, coronary vascular function, osmolality, myocardial ischemia, and restenosis.

[0303] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutationA polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used in respiratory medicine.

[0304] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used for the treatment of fibrosis.

[0305] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used in immunology.

[0306] In certain non-limiting embodiments, VEGF-C of the Disclosure Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used for lymph node transplantation.

[0307] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutationA polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used for intestinal health. In certain non-limiting embodiments, VEGF-C Mutant protein Alternatively, its functional fragments can be used for the treatment of colitis and chronic liver disease.

[0308] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used in nephrology.

[0309] In certain non-limiting embodiments, VEGF-C of the Disclosure Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used for the treatment of polycystic kidney disease.

[0310] In certain non-limiting embodiments, VEGF-C of the Disclosure Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used for the treatment of Alzheimer's disease, Parkinson's disease, stroke, and cerebral ischemia with lung injury. In certain embodiments, VEGF-C Mutant protein Alternatively, its functional fragments possess nerve regeneration properties.

[0311] In certain non-limiting embodiments, VEGF-C of the Disclosure Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used for wound healing.

[0312] In certain non-limiting embodiments, VEGF-C of the Disclosure Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used for the treatment of lymphedema. Non-exclusive examples of lymphedema include primary lymphedema, secondary lymphedema, and hereditary lymphedema.

[0313] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing such polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used for the treatment of hereditary disorders. Non-exclusive examples of such hereditary disorders include Milroy's disease, Henecum syndrome, and Turner syndrome.

[0314] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutationA polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used for the treatment of eye diseases. Non-limiting examples of such eye diseases include age-related macular degeneration, glaucoma, diabetic retinopathy, central serous chorioretinopathy, macular edema, and retinal edema.

[0315] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used in combination with chimeric antigen receptor (CAR) T cells.

[0316] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used in combination with a cancer vaccine.

[0317] In some embodiments, the above method does not include the step of administering a tumor-specific antigen.

[0318] In certain embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutationA polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be used in combination with an immunotherapy agent. In some embodiments, the immune checkpoint inhibitor may target PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, BTLA, GITR, 4-1BB, or Ox-40. The immune checkpoint inhibitor may be an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-BTLA antibody, an anti-GITR antibody, an anti-4-IBB antibody, or an anti-Ox-40 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0319] VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, may be administered together with an immunotherapy agent. For example, VEGF-C Mutant protein The immunotherapy agent is administered in the same composition.

[0320] Alternatively, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particles containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, and an immunotherapy agent may be administered sequentially.

[0321] In various embodiments, VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutationA polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, is administered before the administration of the immunotherapy agent. VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be administered locally to the brain or central nervous system (e.g., to the cisterna magna), after which the immunotherapy agent can be administered systemically (e.g., intravenously). VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be administered intratumorally, after which an immunotherapy agent can be administered systemically (e.g., intravenously). VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be administered intrathecally, after which the immunotherapy agent can be administered systemically (e.g., intravenously). VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation A polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, can be directly administered to the lymphatic system, after which the immunotherapy agent can be administered systemically (e.g., intravenously).

[0322] In various embodiments, methods for treating cancer further include a step of administering additional anti-cancer treatments. Examples of additional anti-cancer treatments include, but are not limited to, surgery, radiation therapy, administration of chemotherapeutic agents, and any combination thereof. These additional anti-cancer treatments include VEGF-C Mutant protein or its functional fragment (or its fusion protein or conjugate, or VEGF-C) mutation It may be administered before, simultaneously with, or after the administration of a polynucleotide molecule encoding a protein, a functional fragment thereof, or a fusion protein thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above.

[0323] In various embodiments, the subjects are human patients. These human patients may be children or adults.

[0324] In various embodiments, the above method is effective for treating the target cancer. In some embodiments, the above method is effective for inducing lymphangiogenesis in the target brain or central nervous system tumor. In various embodiments, lymphangiogenesis can be confirmed by MRI imaging, where, for example, the diameter of the lymphatic system can be calculated using a contrast agent. In various embodiments, lymphangiogenesis can be confirmed by serial CSF collection to measure the concentrations of VEGFA, VEGFB, VEGFC, or VEGFD. In various embodiments, lymphangiogenesis can be confirmed by serial CSF collection to measure the concentration of VEGFC. The above method may be effective for reducing tumor volume. In some embodiments, the above method is effective for reducing the volume of the target brain or central nervous system tumor. In various embodiments, the above method is effective for providing immunological memory against the tumor. While we do not wish to be bound by theory, the low clinical efficacy of immunotherapy in GBM patients may be due to low antigen sampling from the CNS between the steady state and the early stages of tumor development. VEGF-C Mutant proteinAlternatively, administration of its functional fragment can increase the amount of antigen sampling occurring in the brain, thereby improving the efficacy and outcomes of any other immunotherapy administered (e.g., anti-CTLA-4 antibody). While the role of VEGFCs in cancer has been thought to be to promote metastasis via (lymphoid) angiogenesis, we have surprisingly shown that in the brain, VEGF-C can reduce tumor size by increasing immune surveillance. VEGF-C can stimulate the proliferation of lymphatic endothelial cells via VEGFR-3, thereby enhancing lymphatic function.

[0325] In one embodiment, the foregoing provides a method for regulating intraocular pressure in an individual requiring intraocular pressure regulation, the method comprising an effective amount of VEGF-C mutation Proteins or functional fragments thereof, or fusion proteins or conjugates thereof, or VEGF-C mutation The procedure includes administering to a subject a polynucleotide molecule encoding a protein or a functional fragment thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, or a corresponding wild-type VEGF-C protein or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding a wild-type VEGF-C protein or a functional fragment thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above.

[0326] In one embodiment, the foregoing provides a method for removing unwanted intraocular fluid from an eye of a subject requiring removal of unwanted intraocular fluid, the method comprising an effective amount of VEGF-C of the foregoing disclosure. mutation Proteins or functional fragments thereof, or fusion proteins or conjugates thereof, or VEGF-C mutationThe procedure includes administering to a subject a polynucleotide molecule encoding a protein or a functional fragment thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above, or a corresponding wild-type VEGF-C protein or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding a wild-type VEGF-C protein or a functional fragment thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above.

[0327] In some embodiments, the unwanted bodily fluids are optic nerve fluid, retinal fluid, subretinal fluid, choroidal fluid, or epichoroidal fluid.

[0328] The methods disclosed herein are methods for adjusting intraocular pressure in subjects requiring adjustment of intraocular pressure, or for removing excess intraocular fluid in subjects requiring removal of excess intraocular fluid. In some embodiments, subjects include glaucoma, macular edema, central serous chorioretinopathy, retinal edema, papilledema, and macular degeneration. disease or suffers from diabetic retinopathy.

[0329] In some embodiments, the method disclosed herein is a method for adjusting intraocular pressure in a subject requiring adjustment of intraocular pressure, or a method for removing excess intraocular fluid in a subject requiring removal of excess intraocular fluid, and involves VEGF-C mutation Proteins or functional fragments thereof, fusion proteins, conjugates, polynucleotide molecules, vectors, particles, or pharmaceutical compositions are administered to the posterior segment of the eye. In some embodiments, administration is intraocular. In some embodiments, intraocular administration is intravitreous, ophthalmic, or subretinal.

[0330] In one embodiment, the foregoing provides a method for providing neuroprotection to an object requiring neuroprotection, the method comprising an effective amount of VEGF-C of the foregoing disclosure. mutation Proteins or functional fragments thereof, or fusion proteins or conjugates thereof, or VEGF-C mutationThe procedure includes administering to a subject a polynucleotide molecule encoding a protein or a functional fragment thereof, or a vector or particle containing the polynucleotide molecule, or a pharmaceutical composition containing any of the above, or a corresponding wild-type VEGF-C protein or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding the wild-type VEGF-C protein or a functional fragment thereof, or a vector or particle containing the polynucleotide molecule, or a pharmaceutical composition containing any of the above. In some embodiments, neuroprotection is, but is not limited to, macular degeneration. disease It is provided for glaucoma, stroke, Alzheimer's disease, or Parkinson's disease. In some embodiments, administration is intraocular. In some embodiments, intraocular administration is intravitreous, ophthalmic, or subretinal.

[0331] In one embodiment, the VEGF-C provided herein is provided in this specification. mutation Proteins or functional fragments thereof, or fusion proteins or conjugates thereof, or VEGF-C mutation A vaccine comprising a polynucleotide molecule encoding a protein or a functional fragment thereof, or a vector or particle containing the above polynucleotide molecule, or a pharmaceutical composition containing any of the above.

[0332] In one embodiment, the foregoing provides a method for inducing an immune response in a subject requiring induction of an immune response, the method comprising the step of administering an effective amount of the vaccine of the foregoing to the subject. In some embodiments, the immune response is, but is not limited to, an anti-cancer mediated immune response, a vaccine mediated immune response, an antiviral immune response, an antibacterial immune response, or another anti-pathogen immune response. [Examples]

[0333] To further illustrate some of the embodiments disclosed herein, the following examples are provided. These examples are intended to illustrate the embodiments of the disclosure and are not intended to limit them.

[0334] Example 1. VEGF-C Mutant protein Generation Stimulation of lymphangiogenesis (new lymphatic vessel formation) for therapeutic purposes is expected to be a new therapeutic paradigm for a wide range of indications, from cardiovascular disease to cancer immunotherapy. However, there are currently no specific pharmacological agents that promote lymphangiogenesis without stimulating potentially harmful angiogenesis. In particular, vascular endothelium is a major physiological driver of lymphangiogenesis. Cell proliferation Factor C (VEGF-C) stimulates not only pro-lymphangiogenic signaling via phosphovector VEGFR-3 but also pro-angiogenic signaling via VEGFR-2. Here, using directed evolution via yeast surface display, we generated a lymphocyte-specific VEGF-C variant (LS-VEGF-C) that binds only to VEGFR-3 and does not transmit signals via VEGFR-2. Compared to wild-type VEGF-C, LS-VEGF-C showed superior preclinical efficacy in models of lymphangiogenesis and anti-PD-1 cancer immunotherapy. The lymphangiogenic-biased effect of LS-VEGF-C was further determined here by studying its effect on enhancing lymphatic drainage in the posterior segment. LS-VEGF-C significantly reduced intraocular pressure in normotensive mice and two separate ocular-tension mouse models without the increased angiogenesis and vascular permeability seen with WT-VEGF-C treatment. LS-VEGF-C also exhibited neuroprotective properties in a model of retinal ganglion cell death induced by excitotoxic injury, independently of intraocular pressure. Taken together, these results highlight the feasibility and preclinical efficacy of treatment-oriented lymphangiogenesis by VEGFR-3-specific agonism and reveal the unexpected impact of lymphangiogenesis on intraocular pressure control.

[0335] Protein expression, purification, and biotinylation The extracellular domains of mouse VGFR2 (first three Ig domains, amino acids 20-326), VGFR3 (amino acids 25-329), human VGFR2 (amino acids 19-325), and VGFR3 (amino acids 25-329) were cloned into a pEZT vector along with the N-terminal H7 signal peptide, C-terminal AviTag, and hexahistidine tag (SEQ ID NO: 25). These were expressed by transient transfection of Expi293 cells (Thermo Fisher) according to the manufacturer's instructions. The proteins were enriched from the cell supernatant with Ni-NTA chelate resin and further purified in HEPES-buffered saline (HBS; 10 mM HEPES, pH 7.5, 150 mM NaCl), the final buffer, by size exclusion chromatography (Column SEC650, Bio-rad). Biotinylation of proteins was performed at room temperature for 2 hours using in-house purified BirA ligase enzyme in 0.1 mM bicin (pH 8.3), 10 mM ATP, 10 mM magnesium acetate, and 0.5 mM biotin (Avidity, #BIO500). The biotinylated proteins were then purified by gel filtration as described above. Biotinylation efficiency was evaluated using an SDS / PAGE streptavidin shift assay.

[0336] Yeast display of VEGF-C Mouse VEGF-C (amino acids 112-215) was cloned into the C-terminal display vector pCT-GCN42 along with the yeast GCN4 sequence RMKQLEDKIEELLSKIYHLENEIARLKKLIGER (SEQ ID NO: 26) to promote in situ dimerization, and displayed on the surface of the yeast strain Saccharomyces cerevisiae EBY100. The yeast was grown in liquid synthetic dextrose (SDCAA) medium containing casamino acids at 30°C, and then expression was induced in liquid synthetic glucose (SGCAA) medium containing casamino acids at 20°C for 24-48 hours. The level of the displayed protein was verified by staining with a C-terminal Myc-tagged antibody (Cell Signaling Technology, #3739S). The binding of biotinylated VEGFR-2 and VEGFR-3 was detected using a fluorescent streptavidin-phycoerythrin secondary antibody and quantified using a Sony SA3800 flow cytometer.

[0337] Figure 2A shows several methods for displaying VEGF-C on the yeast surface, which were used to optimize surface dimerization of VEGF-C. The final combination displayed wild-type VEGF-C with high binding affinity to VEGFR-3 and low affinity to VEGFR-2. Figure 2B shows VEGFR-3-specific VEGF-C. Mutant protein This is a schematic diagram showing the selection of yeast display libraries aimed at the discovery of [specific feature].

[0338] Fourteen residues were identified from the three-dimensional structural analysis of the VEGF-C:VEGFR3 and VEGF-C:VEGFR2 complexes. A randomized library of these residues (Figure 3) was constructed using assembly PCR. To enable homologous recombination, the PCR product was further amplified using primers homologous to the yeast display vector backbone. Transformed yeast was harvested and grown in SDCAA medium.

[0339] Mutant protein Selection The experiment was conducted at 25°C using a Biacore T100. Protein concentration was quantified by absorbance at 280 nm using a Nanodrop2000 spectrometer (Thermo Scientific). Biotinylated proteins VEGFR-2 or VEGFR-3 were immobilized on a Biacore Biotin CAPture sensor chip (Cytiva Life Sciences). Unrelated biotinylated proteins were immobilized to function as reference surfaces for nonspecific binding. Measurements were performed using single-cycle kinetics with serial dilutions of WT-VEGF-C, LS-VEGF-C, and C125S in HEPES-buffered saline-P+ buffer (GE Healthcare). The VEGFR-2 and VEGFR-3 surfaces were regenerated using reagents provided in the CAPture kit according to the manufacturer's instructions. All data were analyzed using Biacore T100 evaluation software 2.0 with a 1:1 Langmuir binding model.

[0340] Building and selecting a mouse VEGF-C library The crystal structures of the VEGF-C / VEGFR-2 complex (PDB ID 2XIW) and VEGF-C / VEGFR-3 (PDB ID 4BSK) were aligned, and VEGF-C and its receptor interface were analyzed. Fourteen positions within mVEGF-C that contact both mVEGFR-2 and mVEGFR-3 were identified, and a mutant library was created (Table 1). The library was assembled using synthetic denaturing primers that cover these mutated residues (Table 2). The PCR products were further amplified using primers homologous to the vector and co-electroperforated in EBY100 competent yeast with a linearized pCT-GCN42 vector. The resulting library was later measured and found to be 10 8 It contained 1 transformant. Figure 3 shows VEGF-C Mutant protein This shows the library design. 3 Individual VEGF-C Mutant proteinTo generate combinations, we designed a structure-based primer library. We studied the structures of VEGF-C that bind to VEGFR-3 and VEGFR-2 and identified key residues on the surface of the binding site. Amino acids that could potentially cause significant polarity changes at these residues were selected for primer design.

[0341] [Table 1]

[0342] [Table 2]

[0343] Transformed yeast cells were harvested and grown in SDCAA medium at 30°C, followed by induction in SGCAA medium at 20°C for 24–48 hours. Naive libraries were selected using two rounds of 1 μM mVEGFR-3, and mVEGFR-3-positive binding factors were enriched using an LS column (Miltenyi, #130-042-401) and magnetic selection. From round 3 onwards, the yeast population was counter-selected using 1 μM mVEGFR-2 monomer and selected using 100 nM mVEGFR-3 by flow cytometry sorting on a Sony SH800 cell sorter. For rounds 4-6, the concentration of the counter-selection reagent mVEGFR-2 was gradually increased to 0.25 μM (round 4) and 0.5 μM (rounds 5 and 6) tetramers, while the mVEGFR-3 concentration was decreased to 10 nM (round 4), 2 nM (round 5), or 1 nM (round 6). Yeast was retained after each selection round and simultaneously boosted to check binding affinity to VEGFR-2 and VEGFR-3 once all selections were complete. After the final selection round, the VEGF-C plasmid was extracted from the grown SDCAA cultured yeast and transformed into E. coli for colony sequencing (Table 3). The collected unique clones were retransformed into yeast and titrated for binding preference to VEGFR-2 and VEGFR-3. The finally selected VEGF-C mutants were expressed by an insect cell pFastbac expression system and purified by FPLC using an SEC column. The monodisperse protein was ultimately removed by endotoxin removal for in vivo animal treatment. As shown in Figure 4, wild-type VEGF-C showed binding to yeast expressing both VEGFR-2 and VEGFR-3 (top row, WT). The first two rounds selected binding to VEGFR-3. Subsequent rounds utilized the decrease in VEGFR-3 levels to select high binding affinity to VEGFR-3. Mutant protein This was selected. At the same time, VEGFR-2 was also included, and by negative selection, it lost its binding affinity to VEGFR-2 while retaining high binding affinity to VEGFR-3. Mutant proteinThe receptor was enriched. Figure 5 shows confirmation of receptor affinity preference after the final selection round. It was positively selected for VEGFR-3 and negatively selected for VEGFR-2. Mutant protein Even at receptor concentrations 100 times higher than wild-type binding, it showed high binding affinity to VEGFR-3 and no binding to VEGFR-2. Figure 6 shows confirmation of receptor affinity priority after the final selection round. Clones after selection rounds 5 and 6. Mutant protein Sequencing, Mutant protein We identified unique clones that were enriched within the population. Mutant protein We identified specific residues and created specific mutations in wild-type VEGF-C for verification.

[0344] Figure 7 shows confirmation of receptor affinity priority after the final selection round. Clones after selection rounds 5 and 6. Mutant protein Sequencing, Mutant protein Unique clones that enriched the population were identified. Mutant protein We identified the residues and purified mutations specific to wild-type VEGF-C for validation.

[0345] Figure 8 shows confirmation of receptor affinity preference for human VEGF receptors after the final round of selection. Positive selection was observed for VEGFR-3, and negative selection was observed for VEGFR-2. Mutant protein Even at receptor concentrations 100 times higher than wild-type binding, it exhibits high binding affinity to VEGFR-3 and no binding to VEGFR-2.

[0346] [Table 3]

[0347] Production of lymphocyte-specific VEGF-C (LS-VEGF-C) Increased lymphatic drainage offers therapeutic benefits for conditions such as lymphedema, directly addressing fluid accumulation caused by the disruption of the lymphatic system infrastructure. 2From a more immunological perspective, utilizing meningeal lymphatic vessels increases the efflux of brain antigens through lymphangiogenesis, thereby promoting the enhancement of the immune response against glioblastoma. 3 These applications demonstrate not only the therapeutic potential of utilizing lymphangiogenesis but also its versatility, allowing it to be applied even in areas with immune privileges.

[0348] Previous studies have shown that VEGFR-2 primarily promotes angiogenesis. 4 VEGFR-3 promotes lymphangiogenesis. 5~8 This has been shown. Cell proliferation Factor C (VEGF-C) is a major growth factor that can promote lymphatic vessel growth and pumping, but its multidirectional nature (binding to both VEGFR-2 and VEGFR-3, Figure 19) limits its use as a therapeutic agent and as a molecular tool for in vivo signaling research. Furthermore, the ability of VEGF-C to promote sufficient lymphangiogenesis varies, and high doses are often required for a significant effect. 9 Overall, these drawbacks prevent the full realization of the therapeutic potential of VEGF-C-driven lymphangiogenesis.

[0349] To overcome these limitations, we are developing VEGF-C, which is more selective yet still potent. Mutant proteinThe creation of such a mutant VEGF-C was desired. To develop a mutant VEGF-C that binds exclusively to VEGFR-3 and promotes lymphangiogenesis, 14 residues of the receptor binding interface were identified by structural analysis for randomization (Figure 3). Using a yeast display-directed evolution platform (Figure 2B), a library was created that underwent six selection rounds for selectivity to VEGFR-3 and reverse selectivity to VEGFR-2 (Figure 4). After this final selection, the population of variants showed conserved exclusive binding to VEGFR-3 in both the human (Figure 8) and mouse versions of the receptor, while failing to bind to VEGFR-2 (Figures 15A and 15B). Notably, the VEGFR-3 binding of these variants surpassed that of the previously established VEGFR-3 specific ligand VEGF-C152S / C-156S (Figure 12). Clonal analysis identified seven key residues, 115, 119, 126, 163, 164, 186, and 188, to obtain the final variant of the present invention (LS-VEGF-C); biophysical characterization by surface plasmon resonance showed that LS-VEGF-C retains picomolar affinity for VEGFR-3 while completely suppressing VEGFR-2 affinity compared to WT-VEGF-C (Figures 6 and 7).

[0350] To assess whether functionality was conserved, HUVEC cells and HDLEC cells, which are vascular endothelial cells and lymphatic endothelial cells, respectively, were used. Administration of VEGF-A, LS-VEGF-C, and WT-VEGF-C, which show binding to VEGFR-2, VEGFR-3, or both (Figure 1B), resulted in downstream signaling being detected by measuring intracellular ERK activation. HUVEC cells expressing VEGFR-2 showed similarly enhanced ERK activation with VEGF-A and WT-VEGF-C, but not with LS-VEGF-C (Figure 15C). In contrast, HDLEC cells showed similar levels of ERK phosphorylation with WT-VEGF-C and LS-VEGF-C (Figure 15C), reflecting conserved VEGFR-3 binding and signaling in the mutant proteins. This trend was confirmed by studies on cell proliferation during VEGF administration; vascular endothelial cells proliferated more with VEGF-A and WT-VEGF-C, while lymphatic endothelial cells proliferated more with all VEGFs, although LS-VEGF-C showed the most statistically significant increase (Figure 15D). Since lymphatic endothelial cells present both VEGFR-2 and VEGFR-3, it is not necessarily surprising that VEGF-A and WT-VEGF-C also increase the proliferation of lymphatic endothelial cells. 5 These data, when combined, suggest that LS-VEGF-C is a novel variant of VEGF-C with high specificity for VEGFR-3 binding and lymphangiogenesis.

[0351] mRNA / nanoparticles VEGF-C wild type and Mutant protein The mRNA encoding the substance was synthesized by TriLink Bio Technologies by complete substitution of pseudouridine and 5-methylcytosine bases, capped using CleanCap reagent AG, and polyadenylated (120A). The mRNA was mixed with 0.1 μL of in vivo-jetPEI (Polyplus Transfection) per 1 ug, stirred in a vortex mixer for 30 seconds, incubated at room temperature for 15 minutes, and then used.

[0352] Cell Human umbilical vein endothelial cells (HUVEC) and human dermal lymphatic endothelial cells (HDLEC) were obtained from (Promocell). These were cultured in MV medium (Promocell) containing supplements (0.05 mL / mL fetal bovine serum, 0.004 mL / mL endothelial cell growth supplement, 10 ng / mL recombinant human EGF, 90 μg / mL heparin, 1 μg / mL hydrocortisone).

[0353] Western blot HEK293T cells were transfected with a VEGFC mRNA construct combined with lipofectamine. Supernatants were collected from these cells and incubated with HUVEC, HDLEC-j, or HDLEC-a cells. In other experiments, VEGF-C or VEGF-A protein was added directly to the medium and placed on top of the cells. Samples were lysed in RIPA buffer and boiled in sample buffer for 5 minutes. In other experiments, HUVEC and HDLEC cells were treated with 100 ng / mL VEGF-A, WT-VEGF-C, or LS-VEGF-C in RPMI medium containing 1% FBS supplement. Samples were trypsinized (0.05%) and quenched with RPMI medium (1% FBS supplement). The supernatant was aspirated, and the cell pellet was resuspended and then lysed in RIPA buffer, and then boiled in sample buffer for 5 minutes.

[0354] Western blotting was as previously reported 3The procedure was carried out in a similar manner. Briefly, a 10% gel was used and processed at 10 mA per gel for 30 minutes, and then at 40 mA per gel until the ladder was properly separated. Wet transfer was performed on ice at 120 mA per gel for 90 minutes. After blocking with skim milk powder-TBST and three washes with TBST, the gels were incubated overnight in a cold room with anti-pERK at a concentration of 1:1000. After washing, HRP-conjugated anti-rabbit secondary antibody was used at a concentration of 1:500 at room temperature for 2 hours, and imaging was performed using a ChemiDoc MP imaging system (Bio-Rad).

[0355] mouse Mixed-sex C57BL / 6J(WT), DBA2J, and DBA / 2J-Gpnmb+ / SjJ mice, aged 6–10 weeks, were purchased from Jackson Laboratory and bred and reared at Yale University. All procedures used in this study (sex matching, age matching) complied with federal guidelines and the institutional policies of the Yale School of Medicine Animal Care and Use Committee.

[0356] MTT assay Using a 96-well plate, 10,000 cells were seeded per well. First, the cells were starved for 12 hours in RPMI medium without supplements, then washed with PBS and processed to meet experimental conditions. A negative control was prepared by processing RPMI medium containing 1% FBS supplement. The experimental conditions consisted of adding 100 ng / mL of VEGF-A, WT-VEGF-C, or LS-VEGF-C to the negative control medium (RPMI medium containing 1% FBS supplement). A standard curve was created by cell titration. Subsequently, 10 μL of 12 mM MTT stock solution was added to each sample. After homogenizing each well by pipetting, absorbance was measured at 570 nm, and the cell number was determined by comparison with the standard curve.

[0357] Isolation of endothelial cells For single-cell dissociation, the cornea, retina, choroid, and optic nerve were isolated. These tissues were then treated with 1 mg ml of RPMI at 37°C. -1 Collagenase D (Roche) and 30 μg ml -1 The cells were digested with DNase I (Sigma-Aldrich) for 45 minutes. The samples were then pipetted to mechanically dissociate the tissue and filtered through a 70 μm filter. The samples were then centrifuged at 500 x g for 5 minutes. Finally, the cell pipettes were resuspended in FACS buffer (PBS containing 2% FBS and 1 mM EDTA) for staining.

[0358] Flow cytometry Prior to immunohistochemistry, nonspecific binding was blocked using an Fc receptor blockade solution (TruStain FcX®, 101320, BioLegend) for 10 minutes at 4°C. The cells were then stained with the corresponding antibody for 30 minutes at 4°C. The cells were then washed to remove excess antibody and resuspended in FACS buffer. Samples were processed using an Attune NxT flow cytometer and analyzed using FlowJo software (10.8.1, Tree Star).

[0359] For AKT phosphorylation staining, the surface marker was first stained on ice for 30 minutes. Then, the cells were fixed and stained according to the instructions on the BD Phosflow kit. The samples were processed using an Attune NxT flow cytometer and analyzed using FlowJo software (10.8.1, Tree Star).

[0360] Measurement of intraocular pressure Intraocular pressure (IOP) was measured according to consensus-based recommendations (https: / / iovs.arvojournals.org / article.aspx?articleid=2778419). Mice were briefly exposed to isoflurane for sedation, after which IOP was measured using an iCare tonometer. IOP values ​​are the average of six measurements taken during a single sedation session. The mice were kept in a constant position so that the probe was perpendicular to the eye surface during measurement.

[0361] Intraocular administration Ketamine (50 mg kg) -1 ) and xylazine (5 mg kg -1 After intraperitoneal injection of a mixture with ), mice were administered locally, intravitreously, or intrachorally. Local administration involved placing only 5 μL of a 1 μg / μL solution on the eyeball. Intravitreous and intrachorally administered mice were given small holes along the edge of the cornea to allow access for a Hamilton syringe. In intrachorally administered mice, the needle entered the anterior chamber. In intravitreous administered mice, the needle entered the vitreous fluid space. Both intravitreous and intrachorally administered mice used a 1 μg / μL solution. After administration, the eyeballs were covered with artificial tear ointment. Eyeballs not being evaluated were also covered with artificial tear ointment to prevent drying.

[0362] Combination therapy Two days after IVT administration of LS-VEGF-C, ketamine (25 mg kg) was administered. -1 ) and xylazine (2.5 mg kg -1 Mice were anesthetized by intraperitoneal injection of a mixture of ) and ). Subsequently, the FDA-approved drug was administered topically into the eyeball to evaluate the combined effect.

[0363] Microbead Model Sappington et al. 18 Based on this, ketamine (50mg kg -1 ) and xylazine (5 mg kg -1 Mice were anesthetized by intraperitoneal injection of a mixture of ) and then administered polystyrene beads into the anterior chamber. Artificial tears ointment was then applied topically to prevent drying.

[0364] Intravitreous AAV injection and imaging WT mice were intravitreal-administered PBS, VEGF-A (concentration), WT-VEGF-C (concentration), or LS-VEGF-C (concentration) in an AAV (dose). One day later, ketamine (50 mg kg) was administered. -1 ) and xylazine (5 mg kg -1Mice were anesthetized by intraperitoneal injection of a mixture of ( ). 100 μL of a 20 mg / mL dye was also injected intraperitoneally. The pupils were dilated with 1% tropicamide. After a 5-minute incubation, the mice were mounted for fundus imaging, fluorescein angiography, and OCT imaging using Phoenix Micron IV.

[0365] Absorbance measurement of Evans Blue For Evans Blue reading, Evans Blue was injected intraperitoneally instead of the dye. After 4 hours, the mice were euthanized and perfused with PBS. The eyeballs were isolated, homogenized with beads, and then processed with a plate reader (Abs_max at 620 nm).

[0366] Tissue processing and microscopic examination For retinal whole-mount specimens, mouse eyeballs were first extracted and then fixed with 1% formaldehyde. After carefully removing the optic nerve, cornea, and sclera, the isolated retina was divided into quarters by cutting the optic nerve halfway through. After staining with Brn3a and DAPI, confocal imaging was performed using a Leica SP8 microscope.

[0367] For cross-sections of the optic nerve, the optic nerve was fixed with 4% resin, then treated and embedded in a resin / OTC / paraffin mixture. Next, the block was thinned using a microtome / cryostat. The cross-section was then visualized using a transmission electron microscope.

[0368] Research on excitation by NMDA Schluter et al. 31 Based on the above, 10 nmol of NMDA was delivered intravitreously, and one day after injection, RGC was evaluated by confocal microscopy of retinal whole-mount specimens as described above.

[0369] statistical analysis All statistical analyses were performed using GraphPad Prism software. Data were analyzed using either an independent two-tailed Student t-test or a paired Student t-test with Prism software. Statistical significance was defined as *P<0.05, **P<0.01, and ***P<0.001.

[0370] Intravitreal injection Mice were anesthetized with ketamine and xylazine. One drop of 0.5% tropicamide was applied to the eyeball. The mouse was positioned so that the superior nasal region of the eyeball was exposed, and the sclera of the superior nasal region was punctured at the level of the ciliary body splenium using a 33g needle. The mouse's head was fixed, and the needle was positioned at a 45° angle. After inserting the tip, 2 μL of mRNA-nanoparticle formulation was injected, and the needle was left in place for 5 seconds to prevent reflux. Then, an ophthalmic antibacterial ointment was applied to prevent infection, and the mouse was placed in a heated cage until it had fully recovered. Figure 10 shows in vivo VEGF-C Mutant protein This indicates the use of VEGF-C. Mutant protein A single intravitreal administration of the drug resulted in a sustained reduction in intraocular pressure (IOP) in wild-type mice compared to its counterpart.

[0371] Inoculation of tumors Ketamine (50 mg kg) administered by intraperitoneal injection -1 ) and xylazine (5 mg kg -1 Mice were anesthetized with a mixture of ( ). The mice's heads were shaved and placed in a stereotactic frame. After sterilizing the scalp with alcohol and betadine, a midline scalp incision was made to expose the coronal and sagittal sutures, and a burr hole was made 2 mm laterally to the sagittal suture and 0.5 mm posterior to Buglemer's. A 10 μl Hamilton syringe filled with tumor cells was inserted into the burr hole to a depth of 2.5 mm from the surface of the brain and allowed to settle for 1 minute before injection. Using a microinfusion pump (World Precision Instruments), 3 μl of tumor cells were injected at a rate of 1 μl min -1The injection was administered. After the injection was complete, the syringe was left in place for another minute before being removed. The burr hole was filled with bone wax, the skin was secured with staples, and the area was cleaned. Analgesics (meloxicam and buprenorphine, 1 mg kg) were administered. -1 After intramuscular administration of VEGF-C, the mice were placed in heated cages until they had fully recovered. Figure 11 shows the in vivo effects of VEGF-C. Mutant protein This indicates the use of wild-type VEGF-C and VEGF-C. Mutant protein This was evaluated in vivo in relation to the treatment of brain tumors. When combined with an anti-PD-1 antibody, VEGF-C Mutant protein It demonstrated significant therapeutic benefits in treating brain tumors. Furthermore, as shown in Figure 12, the VEGF-C variants reported in the literature have lost their binding affinity to both VEGFR-2 and VEGFR-3. Variants such as C152S, previously reported in the literature, have lost their activity to both VEGFR-2 and VEGFR-3 and have lost their potent lymphangiogenic activity. As shown in Figure 13, the isolated VEGF-C of this disclosure Mutant protein This showed specific signaling via VEGFR-3 in vivo. Wild-type VEGF-C and VEGF-C Mutant protein The signaling pathway of (RTI) was evaluated in vivo. These vectors were injected into the eyes of mice. After 24 hours, the eyeballs were extracted and single-cell suspensions were prepared to check for AKT phosphorylation. Wild-type VEGF-C retained VEGFR-2-mediated signaling in vascular endothelial cells, while mutant VEGF-C (RTI) no longer exhibited signaling. Mutant protein In both cases, signal transduction was mediated via VEGFR-3 within lymphatic endothelial cells.

[0372] Intracisternal injection For intracisional injection, mice were anesthetized with ketamine and xylazine, and their dorsal necks were shaved and washed with alcohol. A 2 cm incision was made at the base of the skull, and the muscles of the dorsal neck were separated using forceps. After visualizing the cisterna magna, the dura mater was punctured using a Hamilton syringe equipped with a 15° 33 gauge needle. 3 microliters of mRNA vector (4-5 μg) per mouse were administered at a rate of 1 μl per minute. -1 The drug was administered at the specified rate. After the injection was complete, the needle was left in place for an additional 3 minutes to prevent reflux. The skin was stapled and cleaned, and the same postoperative procedures as for tumor inoculation were performed.

[0373] Example 2. LS-VEGF-C exhibits potent therapeutic effects under multiple conditions. To confirm that LS-VEGF-C acts via VEGFR-3 signaling in vivo, LS-VEGF-C and WT-VEGF-C were first administered to wild-type mice, and then Phosflow was used to analyze the activation of downstream signaling in endothelial cells (CD45). - CD31 + ) was evaluated. WT-VEGF-C showed a significant increase in AKT phosphorylation in endothelial cells, regardless of VEGFR-3 expression (Figure 13). In contrast, LS-VEGF-C showed an increase in VEGFR-3-expressing cells (CD45 - CD31 + VEGFR3 + ) induced significantly higher AKT phosphorylation, but cells that do not express VEGFR-3 (CD45 - CD31 + VEGFR3 - This was not the case in the other study. This suggests that the in vivo activity of LS-VEGF-C reflects what was observed in vitro.

[0374] The potential of VEGF-C to modulate lymphatic vessels has been demonstrated in vivo in many pathological processes since its discovery. Here, we tested the ability of LS-VEGF-C to provide therapeutic lymphangiogenesis in some of these models. First, a mouse model of hindlimb lymphedema. 10This was induced by local lymphangiectomy via surgical resection. Administration of LS-VEGF-C effectively reduced swelling to a near-normal state compared to WT-VEGF-C (Figures 16A, 16B, 20A, and 20B). The molecule was not only effective as a monotherapy under these conditions after a single dose, but also showed sustained activity after multiple doses, highlighting its potential as a therapeutic agent for chronic diseases. Next, LS-VEGF-C was evaluated as an adjuvant for anti-PD-1 cancer immunotherapy in a mouse model of melanoma. LS-VEGF-C showed superior preclinical efficacy compared to WT-VEGF-C in both tumor volume reduction and survival (Figures 16C, 16D, 20C, and 20D). Finally, LS-VEGF-C was used as an adjuvant therapy for checkpoint inhibitor therapy for the treatment of glioblastoma. LS-VEGF-C exhibited activity comparable to WT-VEGF-C, resulting in the survival of the majority of treated animals, which would otherwise have had a fatal outcome (Figures 20E and 20F). These experiments established the conserved biological function of LS-VEGF-C in controlling both biophysical properties and immunological outcomes, while simultaneously demonstrating its superiority over wild-type growth factor as an in vivo pharmacological agent.

[0375] Example 3. Utilization of ocular lymphatic vessels for biophysical regulation. Previous studies have shown that the eye has a lymphatic system that drains from the anterior compartment; conjunctiva 11 and ciliary body 12 The lymphatic vessels and the posterior glymphatic clearance system. 13 However, this has been explained in the past. In recent years, a team of inventors has identified that the ocular lymphatic vessels are compartmentalized, and it has been established that the anterior and posterior parts of the eye have separate lymphatic drainage systems. It has been hypothesized that intraocular administration of LS-VEGF-C leads to increased drainage and a decrease in intraocular pressure by expanding these lymphatic structures through lymphangiogenesis.

[0376] WT-VEGF-C has been reported in the past. 14 Similarly, intrachoritus injection and intravitreal injection resulted in low intraocular pressure (IOP) measurements (Figure 17A). In contrast, LS-VEGF-C resulted in a sustained and enhanced decrease in IOP after any method of administration, whether local, intrachoritus, or intravitreal (Figure 17B). The effectiveness and versatility of LS-VEGF-C may stem from its VEGFR-3 specificity, while WT-VEGF-C has two receptors it can bind to, namely VEGFR-2 acting as a VEGF-C sink. 15 This presents a problem. This may be due to the lack of expressive effect of WT-VEGF-C in the case of eye drops, as well as the need to overcome rapid clearance and multiple intraocular barriers, which hinder multiple topical applications. 16 Interestingly, in mRNA form, WT-VEGF-C did not exhibit an IOP-reducing effect when administered topically or intravitreally, but when administered anterior chamber, it functioned similarly to the more effective LS-VEGF-C (Figure 21A), highlighting the importance of both delivery method and vehicle. Importantly, LS-VEGF-C showed a long-term effect in lowering intraocular pressure compared to commonly used glaucoma eye drops that exhibit activity for only a few hours, with IOP reduction observed within a few hours and lasting up to several days (Figures 21B and 21C). The activity of LS-VEGF-C as a monotherapy was significantly higher than other glaucoma drugs, and combination therapy was not necessary to achieve a flat range in IOP reduction (Figure 21D). The efficacy of LS-VEGF-C in a normotensive IOP model demonstrates that our in vitro findings translate into physiological phenomena.

[0377] Example 4. LS-VEGF-C eliminates vascular side effects in vivo. To evaluate off-target effects and vascular changes induced by VEGF growth factor, the retinal vascular system was imaged after intravitreous administration of VEGF-A, WT-VEGF-C, and LS-VEGF-C. Fundus photographs showed that, compared to the PBS control, VEGF-A and WT-VEGF-C exhibited hyperreflective spots along with increased vascular tortuosity (Figure 17C, left column). Fluorescein angiography revealed similar anatomical vascular findings, but with improved visibility of penetration into the retinal stroma. VEGF-A and WT-VEGF-C increased the presence of the dye in the extravascular region (Figure 17C, center column), which could be quantified by measuring the fluorescence intensity across the entire image (Figure 17D). No intensities above 40 were observed in the control and LS-VEGF-C administered retinas, except in areas overlapping major vessels, while multiple large localized patches showed intensities above 40 in the VEGF-A and WT-VEGF-C administered retinas. Optical coherence tomography (OCT) showed consistent findings with angiography, with fluid pockets observed near the choroid / retinal pigment epithelium after administration of VEGF-A or WT-VEGF-C to the eye, but not after administration of LS-VEGF-C (Figure 17C, right column). As a parallel method to verify these findings, retinal vascular permeability was evaluated by measuring the absorbance of Evans blue after systemic injection of Evans blue and perfusion of mice with PBS. Similar to the angiographic data, these data revealed that LS-VEGF-C could mitigate the increase in vascular permeability and show an unchanged, healthy retinal vascular system (Figure 17E). Anteriorly to the eye, VEGF-A resulted in uncontrolled neovascularization in the iris and cornea, along with corneal opacity indicating edema or leakage of fluid into the anterior chamber (Figure 22B). WT-VEGF-C produced similar corneal opacity but without apparent neovascularization, while LS-VEGF-C showed physical findings similar to the control (Figures 22C and 22D). LS-VEGF-C not only demonstrates its usefulness by alleviating undesirable vascular distribution, but its properties as a novel molecular tool also elucidate the contrasting in vivo effects of VEGFR-2 and VEGFR-3 signaling.

[0378] Example 5. LS-VEGF-C provides neuroprotection in ocular hypertension. Having demonstrated an IOP-reducing effect in wild-type mice, we investigated whether LS-VEGF-C could provide therapeutic benefits in a glaucoma model. Glaucoma is characterized as a neurodegenerative disease caused by the death of retinal ganglion cells (RGCs) and is often associated with elevated intraocular pressure. 17 First, a microbead model was used. This model blocks the outflow pathway within the eye, thereby inducing a rapid increase in IOP. 18 This is reminiscent of acute angle-closure glaucoma (Figure 18A). Mice showed an average increase of 20 mmHg in IOP after bead administration into the anterior chamber. LS-VEGF-C was administered via two different routes to evaluate its efficacy. Intravitreal (IVT) administration of LS-VEGF-C was able to return the elevated IOP to baseline on average. In more detail, this effect was observed to be bimodal, with a 10 mmHg decrease in IOP in mice, which was comparable to wild-type mice treated with LS-VEGF-C or mice with a poor response and persistently elevated IOP. In particular, the benefit provided by locally administered LS-VEGF-C was limited. While the intraocular pressure of mice decreased, this was not sustained and never fell below baseline levels (Figure 18B). Imaging of RGCs indicated by Brn3a staining demonstrated that LS-VEGF-C provides neuroprotection to RGCs. In induced glaucoma, a clear decrease in RGCs was evident, which improved with intravitreal administration of LS-VEGF-C (Figure 18C). In one group, ophthalmic administration did not appear to have a statistically significant effect, but a bimodal effect was observed in retinas with a similar number of Brn3a cells as the control and a similar number of other cells as the glaucoma eye (Figure 18D).

[0379] To further evaluate the effects of LS-VEGF-C in glaucoma, we used a DBA2J dye dispersion model in mice with spontaneously occurring glaucoma. 19A D2-Gpnmb+ model was used as a negative control to establish a baseline. Because it possesses a normally functioning Gpnmb gene, it lacks the iris pigment dispersion that leads to spontaneously occurring glaucoma. Mechanical damage to the anterior chamber was similar to that with microbead injection, but this model exhibited a more chronic pathology, with a lead time of 6–8 months to show a significant increase in IOP (Figure 18E). After screening mice with elevated IOP, PBS or LS-VEGF-C was administered topically or intravitreally. In contrast to the microbead model, both administration methods resulted in a decrease in IOP within the range of control mice (Figure 18F). RGC axons in the optic nerves of these mice were evaluated, demonstrating that LS-VEGF-C could, to some extent, preserve axonal integrity in this glaucoma model. Interestingly, IVT administration showed a significant pressure reduction and corresponding optic nerve axonal health, while topical administration, despite similarly decreasing IOP, did not show significant preservation of axonal integrity (Figures 18G and 18H).

[0380] The precise mechanisms of neuronal cell death in ophthalmic neurodegenerative diseases remain unclear, but a unified theory includes the accumulation of neurotoxic metabolites or excitatory nerve signals. 20,21 VEGF-C is involved in lymphatic drainage, which enables the clearance of metabolites and macromolecules within organs. 22,23 Therefore, it was theorized that LS-VEGF-C may possess neuroprotective properties in glaucoma beyond its ability to reduce IOP. Intravitreal injection of NMDA resulted in widespread RGC death, which was prevented by simultaneous intravitreal administration of LS-VEGF-C (Figures 23A and 23B). Taken together, these results demonstrate that LS-VEGF-C can provide neuroprotection in multiple models of glaucoma through a multimodal mechanism targeting IOP and neurotoxin clearance.

[0381] Example 6. Screening of human LS-VEGF-C. Yeast display of LS-hVEGF-C variants: Candidate human LS-VEGF-C sequences (SEQ ID NOs. 1-50 and 100) and WT hVEGF-C were synthesized by Twist Biosciences and inserted into the yeast display system as described above. All clones contained the C137A mutation to enhance biochemical stability (unrelated to bias towards VEGFR3). After induction of LS-hVEGF-C expression, yeast strains were stained with 500 nM biotinylated hVEGFR2, 100 nM biotinylated hVEGFR3, or 10 nM VEGFR3 at 4°C for 1 hour. Subsequently, the yeast was washed with FACS buffer (PBS + 0.5% BSA + 0.5 mM EDTA) and stained with fluorescent streptavidin secondary antibody. Receptor binding was then quantified by flow cytometry. For the binding titration study shown in Figure 27, yeast was incubated at various hVEGFR3 concentrations: 1.00 μM, 316 nM, 100 nM, 31.6 nM, 10.0 nM, 3.16 nM, 1.00 nM, and 316 pM. hVEGFR3 binding detection was performed as described above. Table 4 shows the human clones tested. Table 5 shows the selected human clones.

[0382] [Table 4] TIFF2023172964000005.tif174170

[0383] [Table 5]

[0384] Example 7. Characterization of human LS-VEGF-C-Fc. Using the Expifectamine 293 transfection kit (Thermo-Fisher Scientific) according to the manufacturer's instructions, human VEGFCs were transfected with human IgG1 Fc (N297Q). Mutant proteinThe protein was transiently expressed in Expi293 cells. Next, the expression supernatant was subjected to Ni-NTA chromatography, and the protein yield was quantified in milligrams / liter (Table 6). Subsequently, the quality was evaluated by size exclusion chromatography using a Superose 6 column (GE Healthcare) (Figures 28A-28M).

[0385] [Table 6]

[0386] Example 8. VEGFR3 expression in lymphatic vessels is likely to be a mediating factor in the reduction of intraocular pressure. Tamoxifen-inducible conditional knockout mice were created by crossing VEGFR3fl / fl mice with Cdh5aCRE, NESTINCRE, and PROX1CRE (all ERT2, tamoxifen-inducible) mice. Under control conditions, mice were given only corn oil, while under other conditions, mice were given tamoxifen daily for 7 days. Subsequently, all mice were induced to have ocular hypertension by bead injection into the anterior chamber. After confirmation of elevated intraocular pressure (baseline), all mice were treated intravitreally with RTI-Fc, and intraocular pressure was measured after 7 days.

[0387] The data shown in Figure 29 demonstrates that VEGFR3 expression in prox1 and cdh5a-positive cells leads to a decrease in intraocular pressure, but not in nestin cells. Since prox1 is expressed in neurons and lymphatic vessels, cdh5a is expressed in lymphatic vessels and blood vessels, and nestin is expressed in neurons, this demonstrates that VEGFR expression in lymphatic vessels is likely the cause of the decrease in intraocular pressure. RTI activity has been shown to be particularly important in lymphatic endothelial cells.

[0388] Example 9. VEGF-C in relation to the reduction of intraocular pressure Mutant protein Conjugate's evaluation. Wild-type mice were treated with monomeric intravitreous LS-VEGF-C, albumin conjugation, or Fc conjugation, and the reduction in intraocular pressure was evaluated.

[0389] Although the above monomers had little effect, albumin conjugation and Fc conjugation showed similar peak reductions in IOP, with Fc conjugation demonstrating superiority over time (Figure 30).

[0390] Consider The creation of lymphospecific VEGF-C, which provides a novel molecular tool for the study of lymphobiology, is provided herein. While dysregulation of the lymphatic system is involved in many disease processes, the clinical application of these findings is limited due to the lack of methods to specifically stimulate lymphatic vessels. Previous efforts have resulted in not only the loss of VEGFR-2 binding, but also a significant loss of binding to VEGFR-3. Mutant protein It has been identified 24~26 To address these clinical barriers, this specification uses VEGF-C Mutant protein We created a library to generate the LS-VEGF-C protein, which exhibits picomolar binding to VEGFR-3 but not to VEGFR-2. This novel property highlights, for the first time herein, the potential of this new molecule to treat various diseases known to involve lymphatic dysfunction. By completely eliminating angiogenesis, the activity of LS-VEGF-C could be further extended to new disease areas. The excellent efficacy of LS-VEGF-C as a treatment for glaucoma is demonstrated herein. Glaucoma is a disease characterized by clearly identifiable biophysical dysfunction and can benefit from lymphatic therapy. WT-VEGF-C has been ruled out as a candidate due to the risk of angiogenesis in the eye. LS-VEGF-C not only exhibited potent in vivo activity consistent with its binding properties, but also did not induce angiogenesis or vascular permeability.

[0391] This is not the first time lymphatic-based therapies have been proposed for glaucoma. The trabecular meshwork and Schlemm's canal exhibit signature characteristics of the lymphatic system and have been identified as regions that can be stimulated by lymphatic signaling. 14,27,28 This concept has also been applied to NHP and human aqueous humor mapping. 29 Furthermore, the uveoscleral outflow pathway is also thought to be involved in aqueous humor drainage. 12 As mentioned above, the focus in reducing intraocular pressure is particularly narrowed to the anterior compartment, and all drugs and surgical procedures target the components of the anterior segment. However, the pathogenesis of glaucoma occurs in the posterior segment, and optic nerve damage is the main sign of progression. In this specification, we decided to provide a unique approach that complements current treatment strategies by reducing IOP focused on this compartment. As a therapeutic molecule, LS-VEGF-C showed a potent effect in reducing IOP in two ocular hypertension models described herein, resulting in the maintenance of nerve cell health. Furthermore, this specification demonstrates that stimulating the optic lymphatic vessels can remove neurotoxic molecules from the eye, enabling neuroprotection.

[0392] Most importantly, LS-VEGF-C is not only a novel pharmacological agent, but also a tool for solving questions in lymphobiology. Previous reports had concluded that it only specifically stimulated VEGFR-3 and was not very potent without VEGFR-2 signaling. 30 In contrast, LS-VEGF-C showed a stronger phenotypic difference compared to WT-VEGF-C. This is thought to be due to a combination of factors, including 1) the absence of VEGF-C that is sequestered from its lymphoid binding partner VEGFR-3 in the absence of VEGFR-2 binding, and 2) LS-VEGF-C retains binding affinity to VEGFR-3 at picomolar concentrations. This leads us to believe that Mutant proteinThis not only presents an excellent candidate for clinical significance requiring lymphatic stimulation, but also offers a unique tool for decoupling VEGFR-2 (angiogenic) signaling from VEGFR-3 (lymphangiogenic) signaling. Its potential applications include lymphedema. 2 From diseases with clear signs such as these, to recent discoveries in neurodegeneration 22,23 This extends to the extent of having a significant impact on human health and disease. In summary, the results obtained herein from the development of VEGFR-3 specific ligands establish therapeutic potential and versatility, even in areas with immunological privileges. References

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[0394] The present invention is not limited in scope by the specific embodiments described herein. In practice, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the above description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Furthermore, it should be understood that all values ​​are approximations and are provided for illustrative purposes only.

[0395] Patents, patent applications, publications, product descriptions, and protocols are referenced throughout this application, and their disclosures are incorporated by reference in their entirety for any purpose. Finally, preferred embodiments of the present invention are described in separate sections. [Embodiment 1] Isolated vascular endothelial growth factor C (VEGF-C) mutant protein or functional fragment thereof, wherein the VEGF-C mutant protein or functional fragment thereof has reduced or no ability to stimulate vascular endothelial cell proliferation compared to a wild-type VEGF-C protein of the same origin that retains the ability to stimulate lymphatic endothelial cell proliferation. [Embodiment 2] An isolated vascular endothelial growth factor C (VEGF-C) mutant protein or functional fragment thereof, wherein the VEGF-C mutant protein or functional fragment thereof (i) has reduced binding affinity to vascular endothelial growth factor receptor-2 (VEGFR-2) compared to a similarly derived wild-type VEGF-C protein, (ii) has the ability to bind to vascular endothelial growth factor receptor-3 (VEGFR-3) and generate VEGFR-3-mediated signaling, and (iii) contains an amino acid sequence that is at least 70% identical to the amino acid sequence of a similarly derived wild-type VEGF-C protein. [Embodiment 3] The VEGF-C mutant protein or functional fragment thereof according to Embodiment 1 or 2 generates a reduced amount or no VEGFR-2-mediated signaling compared to the wild-type VEGF-C protein of the same species. [Embodiment 4] The VEGF-C mutant protein or functional fragment thereof according to Embodiment 3, wherein the VEGF-C mutant protein or functional fragment thereof does not generate signal transduction via VEGFR-2. [Embodiment 5] The VEGFR-2-mediated signal transduction is determined by measuring VEGFR-2-dependent AKT phosphorylation and / or ERK phosphorylation levels in vascular endothelial cells by a wound healing assay (scratch assay), a proliferation assay, or angiogenesis assay, in the VEGF-C mutant protein or functional fragment thereof according to Embodiment 3 or 4. [Embodiment 6] The VEGFR-3-mediated signaling is determined by measuring VEGFR-3-dependent AKT phosphorylation and / or ERK phosphorylation levels in lymphatic endothelial cells by a wound healing assay, a proliferation assay, or angiogenesis assay, in the VEGF-C mutant protein or functional fragment thereof according to any one of Embodiments 2 to 5. [Embodiment 7] The VEGF-C mutant protein or functional fragment thereof described above does not induce angiogenesis, as described in any of Embodiments 1 to 6. [Embodiment 8] The VEGF-C mutant protein or functional fragment thereof according to any one of Embodiments 1 to 7 is a mutant protein of the wild-type VEGF-C protein comprising a polypeptide defined by amino acids 111 to 211 of SEQ ID NO: 4 or the corresponding position of another wild-type VEGF-C protein. [Embodiment 9] The wild-type VEGF-C protein is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 8, comprising amino acids 111-211 of SEQ ID NO: 4. [Embodiment 10] The VEGF-C mutant protein or functional fragment thereof is one or more mutations selected from mutations in residues T112, L115, D119, Q126, T144, N145, K149, N163, S164, E165, I184, V186, L188, and P192, wherein the position of the residue is defined in relation to SEQ ID NO: 4; or a VEGF-C mutant protein or functional fragment thereof according to any of Embodiments 1 to 9, comprising a mutation in a corresponding residue in another type of wild-type VEGF-C protein. [Embodiment 11] The VEGF-C mutant protein or functional fragment thereof further comprises a mutation at residue C133, wherein the position of the residue is defined in relation to SEQ ID NO: 4, or a mutation at a corresponding residue in another type of wild-type VEGF-C protein, as described in any of Embodiments 1 to 10. [Embodiment 12] The mutation at residue C133 is a C133A mutation, as described in Embodiment 11, for the VEGF-C mutant protein or its functional fragment. [Embodiment 13] The VEGF-C mutant protein or functional fragment thereof according to any one of Embodiments 1 to 7 is a mutant protein of the wild-type VEGF-C protein comprising a polypeptide defined by amino acids 115 to 215 of SEQ ID NO: 1 or the corresponding position in another wild-type VEGF-C protein. [Embodiment 14] The wild-type VEGF-C protein is the VEGF-C mutant protein or functional fragment thereof described in Embodiment 13, comprising amino acids 115-215 of SEQ ID NO: 1. [Embodiment 15] The VEGF-C mutant protein or functional fragment thereof, as described in any of embodiments 1 to 7 and 13 to 14, comprises one or more mutations selected from mutations in residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, I188, V190, L192, and P196, wherein the position of the residue is defined in relation to SEQ ID NO: 1. [Embodiment 16] The VEGF-C mutant protein or functional fragment thereof further comprises a mutation at residue C137, wherein the position of the residue is defined in relation to SEQ ID NO: 1, or a mutation at a corresponding residue in another type of wild-type VEGF-C protein, as described in any of Embodiments 1 to 7 and 13 to 15. [Embodiment 17] The mutation at residue C137 is a C137A mutation, as described in Embodiment 16, for the VEGF-C mutant protein or its functional fragment. [Embodiment 18] The mutation at residue L119 is an L119E mutation or an L119M mutation; the mutation at residue D123 is a D123N mutation; the mutation at residue Q130 is a Q130K mutation; the mutation at residue N167 is an N167R mutation, an N167I mutation, an N167Q mutation or an N167H mutation; the mutation at residue S168 is an S168G mutation or an S168R mutation; the mutation at residue V190 is a V190T mutation; and / or the mutation at residue L192 is an L192I mutation, a VEGF-C mutant protein or a functional fragment thereof according to any of embodiments 15 to 17. [Embodiment 19] The VEGF-C mutant protein or functional fragment thereof according to any one of embodiments 15 to 18 comprises one or more mutations selected from mutations in residues N167, S168, and / or L192. [Embodiment 20] The VEGF-C mutant protein or functional fragment thereof according to Embodiment 19, wherein the mutation at residue N167 is an N167I mutation, an N167Q mutation, or an N167H mutation; the mutation at residue S168 is an S168G mutation, or an S168R mutation; and / or the mutation at residue L192 is an L192I mutation. [Embodiment 21] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the N167Q mutation. [Embodiment 22] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 21, comprising the amino acid sequence of SEQ ID NO: 160 or SEQ ID NO: 56. [Embodiment 23] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 22, comprising the amino acid sequence of SEQ ID NO: 160 or SEQ ID NO: 56. [Embodiment 24] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the N167Q mutation and the S168G mutation. [Embodiment 25] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 24, comprising the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO: 57. [Embodiment 26] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 25, comprising the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO: 57. [Embodiment 27] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the N167Q mutation and the L192I mutation. [Embodiment 28] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 27, comprising the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 58. [Embodiment 29] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 28, comprising the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 58. [Embodiment 30] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the N167Q mutation, the S168G mutation, and the L192I mutation. [Embodiment 31] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 30, comprising the amino acid sequence of SEQ ID NO: 163 or SEQ ID NO: 59. [Embodiment 32] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 31, comprising the amino acid sequence of SEQ ID NO: 163 or SEQ ID NO: 59. [Embodiment 33] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the N167I mutation. [Embodiment 34] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 33, comprising the amino acid sequence of SEQ ID NO: 166 or SEQ ID NO: 62. [Embodiment 35] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 34, comprising the amino acid sequence of SEQ ID NO: 166 or SEQ ID NO: 62. [Embodiment 36] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the N167I mutation and the S168G mutation. [Embodiment 37] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 36, comprising the amino acid sequence of SEQ ID NO: 167 or SEQ ID NO: 63. [Embodiment 38] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 37, comprising the amino acid sequence of SEQ ID NO: 167 or SEQ ID NO: 63. [Embodiment 39] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the N167I mutation and the L192I mutation. [Embodiment 40] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 39, comprising the amino acid sequence of SEQ ID NO: 168 or SEQ ID NO: 64. [Embodiment 41] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 40, comprising the amino acid sequence of SEQ ID NO: 168 or SEQ ID NO: 64. [Embodiment 42] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the N167I mutation, the S168G mutation, and the L192I mutation. [Embodiment 43] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 42, comprising the amino acid sequence of SEQ ID NO: 169 or SEQ ID NO: 65. [Embodiment 44] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 43, comprising the amino acid sequence of SEQ ID NO: 169 or SEQ ID NO: 65. [Embodiment 45] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, which includes the S168G mutation. [Embodiment 46] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 45, comprising the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 68. [Embodiment 47] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 46, comprising the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 68. [Embodiment 48] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the S168G mutation and the L192I mutation. [Embodiment 49] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 48, comprising the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 70. [Embodiment 50] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 49, comprising the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 70. [Embodiment 51] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the N167H mutation. [Embodiment 52] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 51, comprising the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO: 80. [Embodiment 53] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 52, comprising the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO: 80. [Embodiment 54] The VEGF-C mutant protein or functional fragment thereof is the VEGF-C mutant protein or functional fragment thereof according to Embodiment 20, comprising the N167I mutation and the S168R mutation. [Embodiment 55] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 54, comprising the amino acid sequence of SEQ ID NO: 192 or SEQ ID NO: 88. [Embodiment 56] The VEGF-C mutant protein is the VEGF-C mutant protein according to Embodiment 55, comprising the amino acid sequence of SEQ ID NO: 192 or SEQ ID NO: 88. [Embodiment 57] A fusion protein or conjugate comprising a VEGF-C mutant protein or a functional fragment thereof as described in any of Embodiments 1 to 56, wherein the mutant protein or functional fragment thereof is fused and / or conjugated with one or more heterologous moieties. [Embodiment 58] The fusion protein or conjugate according to Embodiment 57, wherein the one or more heterogeneous portions are selected from immunoglobulin or a functional fragment thereof, albumin or a functional fragment thereof, an albumin-conjugated antibody or a functional fragment thereof, and a polyethylene glycol (PEG) polymer. [Embodiment 59] The immunoglobulin or functional fragment thereof comprises an IgG Fc domain, and is a fusion protein or conjugate according to Embodiment 58. [Embodiment 60] The fusion protein according to embodiment 59, wherein the IgG Fc domain is modified to reduce Fc effector function. [Embodiment 61] The IgG Fc domain is the fusion protein according to Embodiment 60, comprising a mutation at residue N297. [Embodiment 62] The fusion protein according to embodiment 61, wherein the mutation at residue N297 is selected from N297Q, N297A, and N297G. [Embodiment 63] An isolated polynucleotide molecule encoding a VEGF-C mutant protein or a functional fragment thereof as described in any of Embodiments 1 to 56, or a fusion protein or conjugate as described in any of Embodiments 57 to 62. [Embodiment 64] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 210 or SEQ ID NO: 108. [Embodiment 65] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 211 or SEQ ID NO: 109. [Embodiment 66] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 212 or SEQ ID NO: 110. [Embodiment 67] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 213 or SEQ ID NO: 111. [Embodiment 68] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 216 or SEQ ID NO: 114. [Embodiment 69] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 217 or SEQ ID NO: 115. [Embodiment 70] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 218 or SEQ ID NO: 116. [Embodiment 71] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 219 or SEQ ID NO: 117. [Embodiment 72] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 222 or SEQ ID NO: 120. [Embodiment 73] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 224 or SEQ ID NO: 122. [Embodiment 74] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 234 or SEQ ID NO: 132. [Embodiment 75] The polynucleotide molecule according to Embodiment 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 242 or SEQ ID NO: 140. [Embodiment 76] The polynucleotide molecule according to any one of embodiments 63 to 75, wherein the polynucleotide molecule comprises a nucleotide sequence encoding the VEGF-C mutant protein or a functional fragment thereof operably linked to a promoter. [Embodiment 77] The polynucleotide molecule according to any one of embodiments 63 to 75, wherein the polynucleotide molecule is mRNA. [Embodiment 78] The polynucleotide molecule is a polynucleotide molecule according to any one of embodiments 63 to 77, comprising one or more nucleotide modifications. [Embodiment 79] The polynucleotide molecule according to Embodiment 78, wherein the one or more nucleotide modifications are a 5' cap, 5-methylcytosine, or pseudouridine. [Embodiment 80] A vector comprising a polynucleotide molecule as described in any of embodiments 63 to 79. [Embodiment 81] The vector is a viral vector, as described in Embodiment 80. [Embodiment 82] The viral vector is derived from a herpesvirus, cytomegalovirus, poliovirus, alphavirus, vaccinia virus, rabies virus, adenovirus (AAV), retrovirus, lentivirus, or adenovirus, as described in Embodiment 81. [Embodiment 83] Particles comprising a polynucleotide molecule as described in any of embodiments 63 to 79. [Embodiment 84] The particles according to Embodiment 83, wherein the particles are nanoparticles, lipid particles, fine particles, lipid nanoparticles, polymer particles, or virus-like particles (VLPs). [Embodiment 85] A host cell comprising a polynucleotide according to any of embodiments 63 to 79 or a vector according to any of embodiments 80 to 82. [Embodiment 86] A method for producing a VEGF-C mutant protein or a functional fragment thereof, or a fusion protein thereof, comprising the step of culturing the host cells described in Embodiment 85 under conditions in which the VEGF-C mutant protein or a functional fragment thereof, or a fusion protein thereof, is expressed. [Embodiment 87] A VEGF-C mutant protein or a functional fragment thereof, or a fusion protein thereof, produced by the method described in Embodiment 86. [Embodiment 88] A kit comprising a VEGF-C mutant protein or a functional fragment thereof as described in any of Embodiments 1 to 56, or a fusion protein or conjugate as described in any of Embodiments 57 to 62, and optionally, instructions for use. [Embodiment 89] A kit comprising a polynucleotide according to any of Embodiments 63 to 79, a vector according to any of Embodiments 80 to 82, or particles according to Embodiment 83 or 84, and optionally, instructions for use. [Embodiment 90] A pharmaceutical composition comprising a VEGF-C mutant protein or a functional fragment thereof as described in any of Embodiments 1 to 56, or a fusion protein or conjugate as described in any of Embodiments 57 to 62, or a polynucleotide molecule as described in any of Embodiments 63 to 79, or a vector as described in any of Embodiments 80 to 82, or particles as described in Embodiment 83 or 84, and a pharmaceutically acceptable carrier or diluent. [Embodiment 91] The pharmaceutical composition according to Embodiment 90, wherein the composition comprises mRNA encoding the VEGF-C mutant protein or a functional fragment thereof, or a fusion protein thereof, as an mRNA nanoparticle formulation. [Embodiment 92] A pharmaceutical composition according to embodiment 90 or 91, further comprising an immunotherapy agent. [Embodiment 93] The pharmaceutical composition according to Embodiment 92, wherein the immunotherapy agent is an immune checkpoint inhibitor. [Embodiment 94] The pharmaceutical composition according to Embodiment 93, wherein the immune checkpoint inhibitor targets PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, BTLA, GITR, 4-1BB, or Ox-40. [Embodiment 95] The pharmaceutical composition according to Embodiment 94, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-BTLA antibody, an anti-GITR antibody, an anti-4-IBB antibody, or an anti-Ox-40 antibody. [Embodiment 96] The pharmaceutical composition described above is a pharmaceutical composition according to any one of embodiments 90 to 95, which is formulated for intrathecal administration. [Embodiment 97] The pharmaceutical composition described above is formulated for intratumor administration, as described in any of embodiments 90 to 95. [Embodiment 98] The pharmaceutical composition described above is a pharmaceutical composition according to any one of embodiments 90 to 95, which is formulated for systemic administration. [Embodiment 99] The pharmaceutical composition described above is a pharmaceutical composition according to any one of embodiments 90 to 95, which is formulated for intracisional administration. [Embodiment 100] The pharmaceutical composition described above is a pharmaceutical composition according to any one of embodiments 90 to 95, which is formulated for ophthalmic administration. [Embodiment 101] The pharmaceutical composition described above is a pharmaceutical composition according to any one of embodiments 90 to 95, which is formulated for intraocular administration. [Embodiment 102] A method for inducing lymphangiogenesis in a subject requiring induction of lymphangiogenesis, the method comprising the step of administering to the subject an effective amount of a VEGF-C mutant protein or functional fragment thereof according to any of Embodiments 1 to 56, or a fusion protein or conjugate according to any of Embodiments 57 to 62, or a polynucleotide molecule according to any of Embodiments 63 to 79, or a vector according to any of Embodiments 80 to 82, or particles according to Embodiment 83 or 84, or a pharmaceutical composition according to any of Embodiments 90 to 101. [Embodiment 103] The method according to Embodiment 102, wherein the administration of the VEGF-C mutant protein or its functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition does not cause one or more side effects associated with the administration of wild-type VEGF-C protein or the corresponding fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition. [Embodiment 104] The method according to embodiment 103, wherein one or more of the aforementioned side effects are angiogenesis and / or increased intraocular pressure (IOP). [Embodiment 105] The method according to any one of Embodiments 102 to 104, wherein the VEGF-C mutant protein or its functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisionally, intravitreously, by eye drops, subcutaneously, intradermally, by inhalation, by a long-term indwelling catheter, orally, topically, or systemically. [Embodiment 106] The method according to any one of Embodiments 102 to 104, wherein the VEGF-C mutant protein or its functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered directly into the cisterna macrocystoides or into the lymphatic system. [Embodiment 107] The method according to any one of embodiments 102 to 107, wherein the subject has a disease or condition selected from cancer, coronary vascular function, osmolality regulation, myocardial ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, disease or condition related to lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennecum syndrome, Milroy's disease, Turner syndrome, age-related macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, and retinal edema. [Embodiment 108] The method according to Embodiment 107, wherein the disease or condition associated with lymph node transplantation is breast cancer-related lymphedema, idiopathic lymphedema, and / or heart failure-related lymphedema. [Embodiment 109] A method for treating a disease or condition in a subject requiring treatment, the method comprising the step of administering to the subject an effective amount of a VEGF-C mutant protein or functional fragment thereof according to any of Embodiments 1 to 56, or a fusion protein or conjugate according to any of Embodiments 57 to 62, or a polynucleotide molecule according to any of Embodiments 63 to 79, or a vector according to any of Embodiments 80 to 82, or particles according to Embodiment 83 or 84, or a pharmaceutical composition according to any of Embodiments 90 to 101. [Embodiment 110] The method according to Embodiment 109, wherein the disease or condition is cancer, coronary vascular function, osmolality regulation, myocardial ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, disease or condition associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennecum syndrome, Milroy's disease, Turner syndrome, age-related macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, and retinal edema. [Embodiment 111] The method according to Embodiment 110, wherein the cancer is melanoma, lung cancer, breast cancer, stomach cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, head and neck squamous cell carcinoma, thyroid cancer, humoral cancer, kidney cancer, urothelial carcinoma, prostate cancer, pheochromocytoma, bile duct cancer, hepatocellular carcinoma, invasive pancreatic ductal carcinoma, thymoma, sarcoma, mesothelioma, testicular cancer, or colorectal cancer. [Embodiment 112] The method according to Embodiment 110, wherein the cancer is located in the brain or central nervous system of the subject. [Embodiment 113] The method according to Embodiment 110, wherein the cancer is selected from glioma, ependymoma, subependymoma, primitive neuroectodermal tumor, ganglioglioma, schwannoma, germ tumor, craniopharyngioma, meningioma, CNS lymphoma, pineal tumor, retinoblastoma, uveal melanoma, and rhabdoid tumor. [Embodiment 114] The method according to any one of embodiments 109 to 113, further comprising the step of administering an immunotherapy agent. [Embodiment 115] The method according to Embodiment 114, wherein the immunotherapy agent is an immune checkpoint inhibitor. [Embodiment 116] The method according to Embodiment 115, wherein the immune checkpoint inhibitor targets PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, BTLA, GITR, 4-1BB, or Ox-40. [Embodiment 117] The method according to Embodiment 116, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-BTLA antibody, an anti-GITR antibody, an anti-4-IBB antibody, or an anti-Ox-40 antibody. [Embodiment 118] The method according to any one of Embodiments 109 to 117, wherein the VEGF-C mutant protein or its functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisionally, intravitreously, by eye drops, subcutaneously, intradermally, by inhalation, by a long-term indwelling catheter, orally, topically, or systemically. [Embodiment 119] The VEGF-C mutant protein or its functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered directly into the cisterna macrocystoides or into the lymphatic system, according to any one of Embodiments 109 to 117. [Embodiment 120] The disease is cancer, and the method is the method according to any one of embodiments 109 to 119, comprising the step of administering an additional anti-cancer treatment to the subject. [Embodiment 121] The method according to Embodiment 120, wherein the additional anti-cancer treatment is selected from surgery, radiotherapy, administration of chemotherapeutic agents, immunotherapy, and any combination thereof. [Embodiment 122] A method for regulating intraocular pressure in a subject requiring regulation of intraocular pressure, the method comprising the step of administering to the subject an effective amount of a VEGF-C mutant protein or functional fragment thereof according to any of Embodiments 1 to 56, or a fusion protein or conjugate according to any of Embodiments 57 to 62, or a polynucleotide molecule according to any of Embodiments 63 to 79, or a vector according to any of Embodiments 80 to 82, or particles according to Embodiment 83 or 84, or a pharmaceutical composition according to any of Embodiments 90 to 101; or a corresponding wild-type VEGF-C protein or functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding the wild-type VEGF-C protein or functional fragment thereof, or a vector or particles containing the polynucleotide molecule, or a pharmaceutical composition containing any of the above. [Embodiment 123] The method according to Embodiment 122, wherein the VEGF-C mutant protein or the corresponding wild-type VEGF-C protein, or a functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition thereof, or the corresponding wild-type VEGF-C protein or its functional fragment, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding the wild-type VEGF-C protein or its functional fragment, or a vector or particle containing the polynucleotide molecule, or a pharmaceutical composition containing any of the above, is administered to the posterior eye. [Embodiment 124] The method according to embodiment 122 or 123, wherein the administration is intraocular. [Embodiment 125] The method according to Embodiment 124, wherein the intraocular administration is intravitreous administration, ophthalmic administration, or subretinal administration. [Embodiment 126] A method for removing unwanted intraocular fluid from a subject requiring removal of unwanted intraocular fluid, the method comprising the step of administering to the subject an effective amount of a VEGF-C mutant protein or functional fragment thereof according to any of Embodiments 1 to 56, or a fusion protein or conjugate according to any of Embodiments 57 to 62, or a polynucleotide molecule according to any of Embodiments 63 to 79, or a vector according to any of Embodiments 80 to 82, or particles according to Embodiment 83 or 84, or a pharmaceutical composition according to any of Embodiments 90 to 101; or a corresponding wild-type VEGF-C protein or functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding the wild-type VEGF-C protein or functional fragment thereof, or a vector or particles containing the polynucleotide molecule, or a pharmaceutical composition containing any of the above. [Embodiment 127] The method according to embodiment 126, wherein the aforementioned unwanted bodily fluid is optic nerve fluid, retinal fluid, subretinal fluid, choroidal fluid, or epichoroidal fluid. [Embodiment 128] The method according to any one of embodiments 122 to 127, wherein the subject is suffering from glaucoma, macular edema, central serous chorioretinopathy, retinal edema, papilledema, macular degeneration, or diabetic retinopathy. [Embodiment 129] The method according to any one of embodiments 124 to 126, wherein the administration is intraocular. [Embodiment 130] The method according to Embodiment 129, wherein the intraocular administration is intravitreous administration, ophthalmic administration, or subretinal administration. [Embodiment 131] A method for providing neuroprotection to a subject requiring neuroprotection, the method comprising the step of administering to the subject an effective amount of a VEGF-C mutant protein or functional fragment thereof according to any of Embodiments 1 to 56, or a fusion protein or conjugate according to any of Embodiments 57 to 62, or a polynucleotide molecule according to any of Embodiments 63 to 79, or a vector according to any of Embodiments 80 to 82, or particles according to Embodiment 83 or 84, or a pharmaceutical composition according to any of Embodiments 90 to 101; or a corresponding wild-type VEGF-C protein or functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding the wild-type VEGF-C protein or functional fragment thereof, or a vector or particles containing the polynucleotide molecule, or a pharmaceutical composition containing any of the above. [Embodiment 132] A vaccine comprising a VEGF-C mutant protein or a functional fragment thereof as described in any of Embodiments 1 to 56, or a fusion protein or conjugate as described in any of Embodiments 57 to 62, or a polynucleotide molecule as described in any of Embodiments 63 to 79, or a vector as described in any of Embodiments 80 to 82, or particles as described in Embodiment 83 or 84, or a pharmaceutical composition as described in any of Embodiments 90 to 101. [Embodiment 133] A method for inducing an immune response in a subject requiring induction of an immune response, the method comprising the step of administering an effective amount of the vaccine described in Embodiment 132 to the subject. [Embodiment 134] The method according to any one of embodiments 102 to 133, wherein the subject is a human. [Embodiment 135] A method for generating a library of VEGF-C mutant proteins that selectively bind to VEGFR-3, wherein the amino acid residues constituting a common binding interface on VEGF-C that binds to both VEGFR-3 and VEGFR-2 are diversified to other amino acids by introducing mutations into the corresponding nucleic acid sequences. [Embodiment 136] A yeast cell library for the selection of VEGF-C mutant proteins, comprising a plurality of yeast cells containing a cell wall peptide anchor sequence, a linker peptide, and the sequence of the VEGF-C mutant protein.

Claims

1. Isolated vascular endothelial growth factor C (VEGF-C) mutant protein or functional fragment thereof, wherein (i) its binding affinity to vascular endothelial growth factor receptor-2 (VEGFR-2) is reduced compared to the same-origin wild-type VEGF-C protein, (ii) it has the ability to bind to vascular endothelial growth factor receptor-3 (VEGFR-3) and generate VEGFR-3-mediated signaling, and (iii) it contains an amino acid sequence that is at least 70% identical to the amino acid sequence of the same-origin wild-type VEGF-C protein.

2. A VEGF-C mutant protein or a functional fragment thereof according to claim 1, which does not induce angiogenesis.

3. The wild-type VEGF-C protein comprises amino acids 115 to 215 of SEQ ID NO: 1, as described in claim 1, for the VEGF-C mutant protein or functional fragment thereof.

4. A VEGF-C mutant protein or functional fragment thereof according to claim 1, comprising one or more mutations selected from mutations in residues N167, S168, L192, T116, L119, D123, Q130, T148, N149, K153, E169, I188, V190, and P196 of the wild-type VEGF-C protein comprising amino acids 115 to 215 of SEQ ID NO: 1 (the positions of the residues are defined in relation to SEQ ID NO: 1), or one or more mutations in the corresponding residues of another wild-type VEGF-C protein.

5. A VEGF-C mutant protein or functional fragment thereof according to claim 4, further comprising a mutation at residue C137 (the position of the residue is defined in relation to SEQ ID NO: 1), or a mutation at a corresponding residue in another wild-type VEGF-C protein.

6. The VEGF-C mutant protein or functional fragment thereof according to claim 5, wherein the mutation at residue C137 is a C137A mutation.

7. The VEGF-C mutant protein or functional fragment thereof according to claim 4, wherein the mutation at residue N167 is an N167R mutation, an N167I mutation, an N167Q mutation, or an N167H mutation; the mutation at residue S168 is an S168G mutation or an S168R mutation; the mutation at residue L192 is an L192I mutation; the mutation at residue L119 is an L119E mutation or an L119M mutation; the mutation at residue D123 is a D123N mutation; the mutation at residue Q130 is a Q130K mutation; and / or the mutation at residue V190 is a V190T mutation.

8. A VEGF-C mutant protein or a functional fragment thereof according to claim 4, comprising one or more mutations selected from mutations in residues N167, S168, and / or L192.

9. The VEGF-C mutant protein or functional fragment thereof according to claim 8, wherein the mutation at residue N167 is an N167I mutation, an N167Q mutation, or an N167H mutation; the mutation at residue S168 is an S168G mutation, or an S168R mutation; and / or the mutation at residue L192 is an L192I mutation.

10. (a) comprising the N167Q mutation, or (b) containing the N167Q mutation and the S168G mutation, or (c) containing the N167Q mutation and the L192I mutation, or (d) including the N167Q mutation, the S168G mutation, and the L192I mutation, or (e) containing the N167I mutation, or (f) containing the N167I mutation and the S168G mutation, or (g) containing the N167I mutation and the L192I mutation, or (h) Including the N167I mutation, the S168G mutation, and the L192I mutation, or (i) containing the S168G mutation, or (j) containing the S168G mutation and the L192I mutation, or (k) containing the N167H mutation, or (l) Including the N167I mutation and the S168R mutation, The VEGF-C mutant protein or a functional fragment thereof according to claim 9.

11. (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 160 or SEQ ID NO: 56, (b) containing or consisting of the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO: 57, (c) containing or consisting of the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 58, (d) containing or consisting of the amino acid sequence of SEQ ID NO: 163 or SEQ ID NO: 59, (e) containing or consisting of the amino acid sequence of SEQ ID NO: 166 or SEQ ID NO: 62, (f) containing or consisting of the amino acid sequence of SEQ ID NO: 167 or SEQ ID NO: 63, (g) containing or consisting of the amino acid sequence of SEQ ID NO: 168 or SEQ ID NO: 64, (h) containing or consisting of the amino acid sequence of SEQ ID NO: 169 or SEQ ID NO: 65, (i) containing or consisting of the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 68, (j) containing or consisting of the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 70, (k) containing or consisting of the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO: 80, (l) Consists of or includes the amino acid sequence of SEQ ID NO: 192 or SEQ ID NO: 88 The VEGF-C mutant protein according to claim 10.

12. A fusion protein or conjugate comprising a VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 11, wherein the mutant protein or functional fragment thereof is fused and / or conjugated with one or more heterologous moieties.

13. The fusion protein or conjugate according to claim 12, wherein the one or more heterogeneous portions are selected from immunoglobulin or a functional fragment thereof, albumin or a functional fragment thereof, an albumin-conjugated antibody or a functional fragment thereof, and a polyethylene glycol (PEG) polymer.

14. An isolated polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 11.

15. (a) comprising the nucleotide sequence of SEQ ID NO: 210 or SEQ ID NO: 108, or (b) containing the nucleotide sequence of SEQ ID NO: 211 or SEQ ID NO: 109, or (c) containing the nucleotide sequence of SEQ ID NO: 212 or SEQ ID NO: 110, or (d) containing the nucleotide sequence of SEQ ID NO: 213 or SEQ ID NO: 111, or (e) containing the nucleotide sequence of SEQ ID NO: 216 or SEQ ID NO: 114, or (f) containing the nucleotide sequence of SEQ ID NO: 217 or SEQ ID NO: 115, or (g) containing the nucleotide sequence of SEQ ID NO: 218 or SEQ ID NO: 116, or (h) containing the nucleotide sequence of SEQ ID NO: 219 or SEQ ID NO: 117, or (i) containing the nucleotide sequence of SEQ ID NO: 222 or SEQ ID NO: 120, or (j) containing the nucleotide sequence of SEQ ID NO: 224 or SEQ ID NO: 122, or (k) containing the nucleotide sequence of SEQ ID NO: 234 or SEQ ID NO: 132, or (l) Containing the nucleotide sequence of SEQ ID NO: 242 or SEQ ID NO: 140, The polynucleotide molecule according to claim 14.

16. A vector comprising the polynucleotide molecule described in claim 14.

17. A particle comprising the polynucleotide molecule described in claim 14.

18. The particles according to claim 17, wherein the particles are nanoparticles, lipid particles, fine particles, lipid nanoparticles, polymer particles, or virus-like particles (VLPs).

19. A host cell comprising the polynucleotide molecule described in claim 14.

20. A method for producing a VEGF-C mutant protein or a functional fragment thereof, or a fusion protein thereof, comprising culturing the host cells described in claim 19 under conditions in which the VEGF-C mutant protein or a functional fragment thereof, or a fusion protein thereof, is expressed.

21. A kit comprising a VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 11, or a fusion protein or conjugate containing the VEGF-C mutant protein or a functional fragment thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof, or a vector containing the polynucleotide molecule, or particles containing the polynucleotide molecule, and optionally, instructions for use.

22. A pharmaceutical composition comprising a VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 11, or a fusion protein or conjugate containing the VEGF-C mutant protein or a functional fragment thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof, or a vector containing the polynucleotide molecule, or particles containing the polynucleotide molecule, and a pharmaceutically acceptable carrier or diluent.

23. The pharmaceutical composition according to claim 22, comprising mRNA encoding the VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof as an mRNA nanoparticle formulation.

24. The pharmaceutical composition according to claim 22, formulated for intrathecal administration, intratumoral administration, systemic administration, intracisional administration, ophthalmic administration, or intraocular administration.

25. A composition for inducing lymphangiogenesis in subjects requiring the induction of lymphangiogenesis, comprising a VEGF-C mutant protein or a functional fragment thereof as described in any one of claims 1 to 11, or a fusion protein or conjugate containing the VEGF-C mutant protein or a functional fragment thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof, or a vector containing the polynucleotide molecule, or particles containing the polynucleotide molecule.

26. The composition according to claim 25, which is administered intrathecally, intraocularly, intratumorally, intracisterna magnum, intraanterior chamber, intravitreously, by eye drops, subretinal, subcutaneously, intradermally, by inhalation, by a long-term indwelling catheter, orally, topically, or systemically, or by administration to the cisterna magnum or directly into the lymphatic system.

27. A composition for treating a disease or condition in a subject requiring treatment of a disease or condition, comprising a VEGF-C mutant protein or a functional fragment thereof as described in any one of claims 1 to 11, or a fusion protein or conjugate containing the VEGF-C mutant protein or a functional fragment thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof, or a vector containing the polynucleotide molecule, or particles containing the polynucleotide molecule.

28. The composition according to claim 27, wherein the disease or condition is cancer, coronary vascular function, osmolality regulation, myocardial ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, disease or condition related to lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, excess intraocular fluid, Hennecum syndrome, Milroy's disease, Turner syndrome, ocular hypertension, macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, retinal edema, or papilledema.

29. The composition according to claim 27, which is administered intrathecally, intraocularly, intratumorally, intracisterna magnum, intraanterior chamber, intravitreously, by eye drops, subretinal, subcutaneously, intradermally, by inhalation, by a long-term indwelling catheter, orally, topically, or systemically, or by administration to the cisterna magnum or directly into the lymphatic system.

30. A composition for regulating intraocular pressure in subjects requiring regulation of intraocular pressure, or for removing unwanted intraocular fluid in subjects requiring removal of unwanted intraocular fluid, comprising a VEGF-C mutant protein or a functional fragment thereof as described in any one of claims 1 to 11, or a fusion protein or conjugate containing the VEGF-C mutant protein or a functional fragment thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof, or a vector containing the polynucleotide molecule, or particles containing the polynucleotide molecule.

31. The composition according to claim 30, which is administered intravitreously, as eye drops, or subretinally.

32. The composition according to claim 30, wherein the subject is suffering from glaucoma, macular edema, central serous chorioretinopathy, retinal edema, papilledema, macular degeneration, or diabetic retinopathy.

33. A composition for providing neuroprotection in subjects requiring neuroprotection, comprising a VEGF-C mutant protein or a functional fragment thereof as described in any one of claims 1 to 11, or a fusion protein or conjugate containing the VEGF-C mutant protein or a functional fragment thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof, or a vector containing the polynucleotide molecule, or particles containing the polynucleotide molecule.

34. A method for generating a library of VEGF-C mutant proteins that selectively bind to VEGF-3, comprising diversifying the amino acid residues constituting the common binding interface on VEGF-C that binds to both VEGF-3 and VEGF-2 by introducing mutations into the corresponding nucleic acid sequences to other amino acids.

35. A yeast cell library for the selection of VEGF-C mutant proteins, comprising multiple yeast cells containing a cell wall peptide anchor sequence, a linker peptide, and a VEGF-C mutant protein sequence.

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