VEGF-C muteins for selective lymphatic stimulation

Abstract

The present invention provides a VEGF-C mutant protein that selectively binds to VEGFR-3 relative to VEGFR-2. The invention also provides methods of inducing lymphangiogenesis in a subject in need thereof by administering to the subject an effective amount of the disclosed VEGF-C mutein. The invention also provides methods of treating a disease or condition (e.g., cancer) in a subject in need thereof by administering to the subject an effective amount of the disclosed VEGF-C mutein.

Description

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 317,614, filed on March 8, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with U.S. Government support under CA239444 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0005] Sequence Listing

[0006] This application contains a sequence listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on March 8, 2023, is named 251609_000089_SL.xml, and is 347,984 bytes in size. Field of the Invention

[0007] The present invention generally relates to vascular endothelial growth factor C (VEGF-C) mutant proteins, which have selective binding to vascular endothelial growth factor receptor 3 (VEGFR-3) relative to vascular endothelial growth factor receptor 2 (VEGFR-2). The present invention also relates to a method of inducing lymphangiogenesis in a subject in need thereof by administering an effective amount of a VEGF-C mutant protein or a functional fragment thereof to the subject. The present invention also relates to a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of a VEGF-C mutant protein or a functional fragment thereof to the subject.

[0008] background

[0009] Immune surveillance against pathogens and tumors in the central nervous system is thought to be limited due to the lack of lymphatic drainage. The meningeal lymphatic vasculature has been shown to be manipulated to generate a better immune response against brain tumors. When tumors are confined to the central nervous system, immunity to glioblastoma antigens mediated by CD8 T cells is very limited, resulting in uncontrolled tumor growth. However, ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in draining deep cervical lymph nodes, migration of CD8 T cells to tumors, rapid clearance of glioblastoma, and durable anti-tumor memory responses. Furthermore, transfection of mRNA constructs expressing VEGF-C synergizes with checkpoint blockade therapy to eradicate existing glioblastomas.

[0010] Vascular endothelial growth factor C (VEGF-C) binds to vascular endothelial growth factor receptor-2 (VEGFR-2), promoting blood vessel growth (angiogenesis) and regulating vascular permeability, and binds to vascular endothelial growth factor receptor-3 (VEGFR-3), promoting lymphatic vessel growth (lymphangiogenesis). VEGF-C acts primarily on lymphatic endothelial cells (LECs) through VEGFR-3, thereby promoting their survival, growth, and migration. Angiogenesis is essential for the growth of primary and metastatic tumors. There is a need to reduce unwanted angiogenesis while promoting lymphangiogenesis. SUMMARY OF THE INVENTION

[0011] Various non-limiting aspects and embodiments of the invention are described below.

[0012] In one aspect, the present invention provides an isolated vascular endothelial growth factor C (VEGF-C) mutant protein or a functional fragment thereof, wherein compared with the wild-type VEGF-C protein from the same species, the VEGF-C mutant protein or a functional fragment thereof has reduced or no ability to stimulate the proliferation of blood endothelial cells, but retains the ability to stimulate the proliferation of lymphatic endothelial cells.

[0013] In one aspect, the present invention provides an isolated vascular endothelial growth factor C (VEGF-C) mutant protein or a functional fragment thereof, wherein the VEGF-C mutant protein or a functional fragment thereof (i) has a reduced binding affinity to vascular endothelial growth factor receptor-2 (VEGFR-2) compared to the wild-type VEGF-C protein from the same species, (ii) has the ability to bind to vascular endothelial growth factor receptor-3 (VEGFR-3) and generate signal transduction through it, and (iii) comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the wild-type VEGF-C protein from the same species.

[0014] In some embodiments of any of the above methods, compared to the wild-type VEGF-C protein from the same species, the VEGF-C mutant protein or its functional fragment produces reduced or no signal transduction through VEGFR-2. In some embodiments of any of the above methods, the VEGF-C mutant protein or its functional fragment does not produce signal transduction through VEGFR-2. In some embodiments of any of the above methods, the signal through VEGFR-2 is determined by measuring the AKT phosphorylation and / or ERK phosphorylation levels that VEGFR-2 depends on in blood endothelial cells, by wound healing assays, by proliferation assays, or by angiogenesis assays. In some embodiments of any of the above methods, the signal transduction through VEGFR-3 is determined by measuring the AKT-phosphorylation and / or ERK-phosphorylation levels that VEGFR-3 depends on in lymphatic endothelial cells, by wound healing assays (scratch assays), by proliferation assays, or by angiogenesis assays.

[0015] In some embodiments of any of the methods described above, the VEGF-C mutant protein or functional fragment thereof does not induce angiogenesis.

[0016] In some embodiments of any of the above methods, the VEGF-C mutant protein is a mutant protein of a wild-type VEGF-C protein comprising amino acids 111-211 of SEQ ID NO: 4, or a mutant protein of a polypeptide defined by corresponding positions within a wild-type VEGF-C protein of another species. In some embodiments of any of the above methods, the wild-type VEGF-C protein comprises amino acids 111-211 of SEQ ID NO: 4.

[0017] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises one or more mutations selected from the group consisting of mutations at residues T112, L115, D119, Q126, T144, N145, K149, N163, S164, E165, I184, V186, L188 and P192, wherein the positions of the residues are defined relative to SEQ ID NO:4, or one or more mutations selected from the group consisting of mutations at corresponding residues within a wild-type VEGF-C protein of another species, or one or more mutations at residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, I188, V190, L192 and P196, wherein the positions of the residues are defined relative to SEQ ID NO:1.

[0018] In some embodiments of any of the above methods, the VEGF-C mutant protein or functional fragment thereof further comprises a mutation at residue C133, wherein the position of the residue is defined relative to SEQ ID NO: 4, or comprises a mutation at a corresponding residue in a wild-type VEGF-C protein of another species. In some embodiments, the mutation at residue C133 is a C133A mutation.

[0019] In some embodiments of any of the above methods, the VEGF-C mutant protein is a mutant protein of a wild-type VEGF-C protein comprising amino acids 115-215 of SEQ ID NO: 1, or a mutant protein of a polypeptide defined by corresponding positions within a wild-type VEGF-C protein of another species. In some embodiments, the wild-type VEGF-C protein comprises amino acids 115-215 of SEQ ID NO: 1.

[0020] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises one or more mutations selected from mutations at residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, I188, V190, L192 and P196, wherein the positions of the residues are defined relative to SEQ ID NO: 1.

[0021] In some embodiments of any of the above methods, the VEGF-C mutant protein or functional fragment thereof further comprises a mutation at residue C137, wherein the position of the residue is defined relative to SEQ ID NO: 1, or comprises a mutation at a corresponding residue in a wild-type VEGF-C protein of another species. In some embodiments, the mutation at residue C137 is a C137A mutation.

[0022] In some embodiments of any of the above methods, 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.

[0023] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises one or more mutations selected from mutations at residues N167, S168 and / or L192.

[0024] In some embodiments of any of the above methods, 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.

[0025] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation.

[0026] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:160 or SEQ ID NO:56.

[0027] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:160 or SEQ ID NO:56.

[0028] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation and an S168G mutation.

[0029] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:161 or SEQ ID NO:57.

[0030] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:161 or SEQ ID NO:57.

[0031] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation and an L192I mutation.

[0032] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:162 or SEQ ID NO:58.

[0033] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:162 or SEQ ID NO:58.

[0034] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation, an S168G mutation, and an L192I mutation.

[0035] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:163 or SEQ ID NO:59.

[0036] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:163 or SEQ ID NO:59.

[0037] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation.

[0038] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:166 or SEQ ID NO:62.

[0039] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:166 or SEQ ID NO:62.

[0040] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation and an S168G mutation.

[0041] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:167 or SEQ ID NO:63.

[0042] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:167 or SEQ ID NO:63.

[0043] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation and an L192I mutation.

[0044] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:168 or SEQ ID NO:64.

[0045] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:168 or SEQ ID NO:64.

[0046] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation, an S168G mutation, and an L192I mutation.

[0047] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:169 or SEQ ID NO:65.

[0048] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:169 or SEQ ID NO:65.

[0049] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an S168G mutation.

[0050] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:172 or SEQ ID NO:68.

[0051] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:172 or SEQ ID NO:68.

[0052] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an S168G mutation and an L192I mutation.

[0053] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:174 or SEQ ID NO:70.

[0054] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:174 or SEQ ID NO:70.

[0055] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an N167H mutation.

[0056] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:184 or SEQ ID NO:80.

[0057] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:184 or SEQ ID NO:80.

[0058] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation and an S168R mutation.

[0059] In some embodiments of any of the methods described above, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO:192 or SEQ ID NO:88.

[0060] In some embodiments of any of the methods described above, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO:192 or SEQ ID NO:88.

[0061] In one aspect, provided herein is a fusion protein or conjugate comprising the VEGF-C mutant protein or a functional fragment thereof disclosed herein, wherein the mutant protein or the functional fragment thereof is fused and / or conjugated to one or more heterologous parts.

[0062] In some embodiments, the one or more heterologous moieties are selected from an immunoglobulin or a functional fragment thereof, albumin or a functional fragment thereof, an albumin binding antibody or a functional fragment thereof, and a polyethylene glycol (PEG) polymer.

[0063] 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 Fc effector functions. 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.

[0064] In one aspect, provided herein is an isolated polynucleotide molecule encoding a VEGF-C mutant protein or a functional fragment thereof or a fusion protein of the present disclosure.

[0065] In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 210 or SEQ ID NO: 108. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 211 or SEQ ID NO: 109. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 212 or SEQ ID NO: 110. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 213 or SEQ ID NO: 111. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 216 or SEQ ID NO: 114. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 217 or SEQ ID NO: 115. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 218 or SEQ ID NO: 116. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 219 or SEQ ID NO: 117. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 222 or SEQ ID NO: 120. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 224 or SEQ ID NO: 122. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 234 or SEQ ID NO: 132. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of SEQ ID NO: 242 or SEQ ID NO: 140. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence encoding a VEGF-C mutant protein or a functional fragment thereof, which is operably linked to a promoter.

[0066] In some embodiments, the polynucleotide molecule is mRNA.

[0067] In some embodiments, the polynucleotide molecule comprises one or more nucleotide modifications. In some embodiments, the one or more nucleotide modifications are a 5' cap, 5-methylcytosine, or pseudouridine.

[0068] In one aspect, provided herein are vectors comprising a polynucleotide molecule of the present disclosure.

[0069] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is derived from herpes virus, cytomegalovirus, poliovirus, alphavirus, vaccinia virus, rabies virus, adeno-associated virus (AAV), retrovirus, lentivirus or adenovirus.

[0070] In one aspect, provided herein are particles comprising a polynucleotide molecule of the disclosure. In some embodiments, the particle is a nanoparticle, a lipid particle, a microparticle, a lipid nanoparticle, a polymer particle, or a virus-like particle (VLP).

[0071] In one aspect, provided herein is a host cell comprising a polynucleotide of the present disclosure or a vector of the present disclosure.

[0072] In one aspect, the present invention provides a method for producing a VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof, comprising culturing a host cell of the present disclosure under conditions of expressing the VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof.

[0073] In one aspect, provided herein is a VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof produced by the method of the present disclosure.

[0074] In one aspect, provided herein is a kit comprising a VEGF-C mutant protein or a functional fragment thereof of the present disclosure or a fusion protein or conjugate of the present disclosure, and optionally instructions for use.

[0075] In one aspect, provided herein is a kit comprising a polynucleotide of the disclosure or a vector of the disclosure, or a particle of the disclosure, and optionally instructions for use.

[0076] In one aspect, provided herein is a pharmaceutical composition comprising a VEGF-C mutant protein of the present disclosure 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.

[0077] In some embodiments, the composition includes mRNA encoding a VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof as an mRNA-nanoparticle formulation.

[0078] In some embodiments, the pharmaceutical composition further comprises an immunotherapeutic agent. In some embodiments, the immunotherapeutic 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-BLTA antibody, an anti-GITR antibody, an anti-4-IBB antibody or an anti-Ox-40 antibody.

[0079] 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 intracisternal administration. In some embodiments, the pharmaceutical composition is formulated for eye drop administration. In some embodiments, the pharmaceutical composition is formulated for intraocular administration.

[0080] In one aspect, provided herein is a method of inducing lymphangiogenesis in a subject in need thereof, the method comprising administering to the subject an effective amount of a VEGF-C mutant protein of the present disclosure 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, or a pharmaceutical composition of the present disclosure.

[0081] In some embodiments of any of the above methods, the administration of the VEGF-C mutant protein or its functional fragment, fusion protein, conjugate, polynucleotide molecule, carrier, particle or pharmaceutical composition does not cause one or more side effects associated with the administration of wild-type VEGF-C protein or corresponding fusion protein, conjugate, polynucleotide molecule, carrier, particle or pharmaceutical composition. In some embodiments, the one or more side effects are angiogenesis and / or increased intraocular pressure (IOP).

[0082] In some embodiments of any of the above methods, the VEGF-C mutant protein or a functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisternalis, intravitreally, by eye drops, subcutaneously, intradermally, by inhalation, by long-dwelling catheter, orally, topically or systemically.

[0083] In some embodiments of any of the methods described above, the VEGF-C mutein or a functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle or pharmaceutical composition thereof is administered to the cisterna magna or directly to the lymphatic system.

[0084] In some embodiments of any of the methods described above, the subject has a disease or condition selected from the group consisting of cancer, coronary vascular function, osmoregulation, cardiac ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, a disease or condition associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennicham syndrome, Milroy disease, Turner syndrome, age-related macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, and retinal edema.

[0085] In some embodiments of any of the methods described above, the disease or condition associated with lymph node transplantation is breast cancer-related lymphedema, idiopathic lymphedema, and / or heart failure-related lymphedema.

[0086] In one aspect, provided herein is a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a VEGF-C mutant protein of the present disclosure 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, or a pharmaceutical composition of the present disclosure.

[0087] In some embodiments, wherein the methods provided herein are methods of treating a disease or condition in a subject in need thereof, the disease or condition is cancer, coronary vascular function, osmoregulation, cardiac ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, a disease or condition associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennican syndrome, Milroy 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, liquid cancer, kidney cancer, bladder urothelial cancer, prostate cancer, pheochromocytoma, bile duct cancer, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, 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 cell tumor, craniopharyngioma, meningioma, CNS lymphoma, pineal tumor, retinoblastoma, uveal melanoma and rhabdoid tumor.

[0088] In some embodiments, the method for treating a disease or condition of a subject in need also includes administering an immunotherapeutic agent. In some embodiments, the immunotherapeutic 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-BLTA antibody, an anti-GITR antibody, an anti-4-IBB antibody or an anti-Ox-40 antibody. In some embodiments, VEGF-C mutant protein or its functional fragment, fusion protein, conjugate, polynucleotide molecule, carrier, particle or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisternal, intravitreally, by eye drops, subcutaneously, intradermally, by inhalation, by long indwelling catheter, orally, topically or systemically. In some embodiments, the VEGF-C mutant protein or a functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle or pharmaceutical composition thereof is administered to the cisterna magna or directly to the lymphatic system.

[0089] In some embodiments, wherein the method provided herein is a method of treating a disease or condition in a subject in need thereof, and wherein the disease is cancer, the method further comprises administering to the subject an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is selected from surgery, radiation therapy, administration of a chemotherapeutic agent, immunotherapy, and any combination thereof.

[0090] In one aspect, the present invention provides a method for regulating intraocular pressure in a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutant protein of the present disclosure or a functional fragment thereof, a fusion protein or conjugate, a polynucleotide molecule, a carrier, a particle or a pharmaceutical composition, 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 carrier or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above. In some embodiments, a VEGF-C mutant protein or a corresponding wild-type VEGF-C protein or a functional fragment thereof, a fusion protein, a conjugate, a polynucleotide molecule, a carrier, a particle or a pharmaceutical composition, 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 carrier or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above is administered to the posterior portion of the eye. In some embodiments, administration is intraocular administration. In some embodiments, intraocular administration is intravitreal, by eye drops, or subretinal administration.

[0091] In one aspect, the present invention provides a method for removing unwanted fluid in the eye of a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutant protein of the present disclosure or a functional fragment thereof, a fusion protein or a conjugate, a polynucleotide molecule, a carrier, a particle or a pharmaceutical composition or a corresponding wild-type VEGF-C protein or a functional fragment thereof or a fusion protein or a conjugate thereof or a polynucleotide molecule encoding the wild-type VEGF-C protein or a functional fragment thereof or a carrier or particle comprising the polynucleotide molecule or a pharmaceutical composition comprising any of the above. In some embodiments, the unwanted fluid is optic nerve, retina, subretinal, choroidal or suprachoroidal fluid. In some embodiments, the subject suffers from glaucoma, macular edema, central serous chorioretinopathy, retinal edema, papilledema, macular degeneration or diabetic retinopathy. In some embodiments, administration is intraocular administration. In some embodiments, intraocular administration is intravitreal, by eye drops or subretinal administration.

[0092] In one aspect, provided herein is a method for providing neuroprotection in a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutant protein or a functional fragment thereof, a fusion protein or conjugate, a polynucleotide molecule or a vector, a particle or a pharmaceutical composition of the present disclosure, or a corresponding wild-type VEGF-C protein or a functional fragment thereof, a fusion protein or a conjugate, or a polynucleotide molecule encoding the wild-type VEGF-C protein or a functional fragment thereof, or a vector or a particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

[0093] In one aspect, provided herein is a vaccine comprising the VEGF-C mutant protein or a functional fragment, fusion protein or conjugate thereof, polynucleotide molecule, vector, particle, or pharmaceutical composition of the present disclosure.

[0094] In one aspect, provided herein is a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a vaccine of the present disclosure.

[0095] In some embodiments of any of the methods described above, the subject is a human.

[0096] In another aspect, provided herein is a method for generating a library of VEGF-C mutant proteins that selectively bind to VEGFR-3, wherein the amino acid residues comprising the common binding interface on the VEGF-C protein that binds to both VEGFR-3 and VEGFR-2 are diversified to other amino acids by mutagenesis of the corresponding nucleic acid sequence.

[0097] In another aspect, the present invention provides a yeast cell library for selecting VEGF-C mutant proteins, which comprises a plurality of yeast cells, wherein the yeast cells comprise a cell wall peptide anchor sequence, a linker peptide and a VEGF-C mutant protein sequence.

[0098] These and other aspects described herein will become apparent to those of ordinary skill in the art from the accompanying description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1A-1B An overview of the pleiotropic effects of VEGF-C is described. Figure 1A is a schematic diagram showing that VEGF-C binds to both VEGFR-2 and VEGFR-3, promoting angiogenesis and lymphangiogenesis, respectively. Figure 1B Schematic representation of a designed VEGF-C mutant protein showing specific binding to VEGFR-3 and loss of binding to VEGFR-2.

[0100] Figure 2A-2B The design of VEGF-C display constructs for dimerization and screening is described. Figure 2AMultiple methods for displaying VEGF-C on yeast surfaces were shown, which were used to optimize VEGF-C dimerization on the surface. The resulting combination showed that wild-type VEGF-C had high binding affinity for VEGFR-3 and lower affinity for VEGFR-2. Figure 2B is a schematic diagram showing the selection of a yeast display library for finding VEGFR-3 specific VEGF-C mutant proteins.

[0101] Figure 3 Depicted is the design of a VEGF-C mutant protein library. A structure-guided primer library was designed to generate approximately 10 3 The structures of VEGF-C binding to VEGFR-3 and VEGFR-2 were studied to identify key residues on the binding site surface. Amino acids that can undergo significant polarization changes in these residues were selected to design primers. SEQ ID NOs: 27-34 and 34-39 are disclosed in the figure in order of appearance.

[0102] Figure 4 Describe VEGF-C mutant protein for VEGFR-3 by positive selection, and for VEGFR-2 combination by negative selection.Wild-type VEGF-C shows the combination with yeast cells expressing VEGFR-2 and VEGFR-3 (upper row, WT).The first two rounds are selected for the combination with VEGFR-3.In subsequent rounds, the VEGFR-3 of reduced amount is used to select the mutant protein with high binding affinity to VEGFR-3.Meanwhile, VEGFR-2 is included and negatively selected to enrich the mutant protein that loses binding affinity to VEGFR-2 but has high binding power with VEGFR-3.

[0103] Figure 5 Depicted is the confirmation of receptor affinity preferences after the final rounds of selection. Mutant proteins that were positively selected for VEGFR-3 and negatively selected for VEGFR-2 showed high binding to VEGFR-3 but no binding to VEGFR-2, even at receptor concentrations 100-fold higher than wild-type binding.

[0104] Figure 6 Depicted is the confirmation of receptor affinity preferences after the final rounds of selection. Clonal mutant proteins after rounds 5 and 6 of selection were sequenced to identify unique clones enriched in the mutant protein population. Unique mutant protein residues were identified to create specific mutations in wild-type VEGF-C for validation.

[0105] Figure 7A summary of the sequence, mutation rate, and affinity of the selected mouse clones and the final LS-VEGF-C is depicted. The positions and identities of the key residues are listed in the first two rows. The shaded columns represent mutations at contact positions, all of which inform the final mutations in WT-VEGF-C that produced LS-VEGF-C. The mutation rates of clones and specific residue positions are also indicated. The affinity of WT- and LS-VEGF-C for VEGFR-2 and VEGFR-3 was determined by SPR.

[0106] Figure 8 WT VEGF-C and VEGFR-2 and VEGFR-3 affinity after the 6th round of selection are depicted.

[0107] Fig. 9 Describe the VEGF-C mutant protein of separation and show the specific signal transduction by VEGFR-3 in vitro.Use human umbilical vein endothelial cells (HUVEC) and human dermal lymphatic endothelial cells (juvenile HUVEC-j and adult HUVEC-a) to assess VEGF-A, wild-type VEGF-C and VEGF-C mutant protein (163I and RTI) signal transduction in vitro.HUVEC specifically expresses VEGFR-2, and HDLEC expresses VEGFR-2 and VEGFR-3, thus allowing to assess every kind of receptor signal transduction.With every kind of protein stimulation cell, collect protein lysate and carry out western blot analysis to detect ERK phosphorylation (downstream of VEGFR-2 and VEGFR-3).

[0108] Fig.10 Depicted is the use of VEGF-C mutant proteins in vivo. A single intravitreal administration of VEGF-C mutant proteins resulted in a sustained decrease in intraocular pressure (IOP) in wild-type mice compared to their counterparts.

[0109] Fig.11 Describe the use of VEGF-C mutant proteins in vivo. Wild-type VEGF-C and VEGF-C mutant proteins were evaluated in vivo for the treatment of brain tumors. In combination with anti-PD-1 antibodies, VEGF-C mutant proteins showed significant therapeutic benefits in the treatment of brain tumors.

[0110] Fig.12 VEGFR-2 and VEGFR-3 affinities of WT VEGF-C and the previously established VEGFR-3 specific ligand VEGF-C152S / C-156S are depicted.

[0111] Fig.13Describe the isolated VEGF-C mutant protein showing specific signal transduction through VEGFR-3 in vivo. Wild-type VEGF-C and VEGF-C mutant protein (RTI) signal transduction were evaluated in vivo. These vectors were injected into the eyes of mice. After 24 hours, the eyeballs were removed and made into single cell suspensions to examine AKT phosphorylation. Wild-type VEGF-C retains signal transduction through VEGFR-2 in blood endothelial cells, while mutant VEGF-C (RTI) no longer has signal transduction. Both wild-type protein and RTI mutant protein send signals through VEGFR-3 in lymphatic endothelial cells.

[0112] Fig.14 The administration route was shown to result in a differential decrease in IOP in vivo. VEGF-C or VEGF-C mutant proteins (RTI) were administered via eye drops, injection into the anterior chamber (AC), or intravitreal. In the AC, both wild-type and mutant proteins had similar activity, whereas the mutant proteins exhibited the ability to selectively lower IOP when administered via eye drops or intravitreal.

[0113] Figures 15A-15D Directed evolution of VEGF-C variants for lymphatic vessel-specific VEGFR-3 binding is shown. Fig.15A The mVEGFR2 affinity of WT VEGF-C and mutant proteins after the 6th selection iteration is shown. Fig. 15B The mVEGFR3 affinity of WT VEGF-C and mutant proteins after the 6th selection iteration is shown. Fig. 15C Shown are downstream ERK phosphorylation in HUVECs (R2+) after administration of control (n=3), VEGF-A (n=3), WTVEGF-C (n=3), and LS-VEGF-C (n=3). Fig.15D 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) is shown. Fig. 15C and 15D Data are expressed as mean ± sem, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, ns not significant.

[0114] Figures 16A-16B It was shown that administration of LS-VEGF-C resulted in a reduction in lymphedema in a mouse lymphedema model. LS-VEGF-C was administered subcutaneously near the lymph node ligation site in mice and resulted in a significant reduction in ankle size compared to controls. Mice received two treatments to demonstrate the repeat administration capability of LS-VEGF-C. Figures 16C-16DTumor-bearing mice were treated with tumor vaccines and PD-1 and either LS-VEGF-C or WT-VEGF-C. Fig. 16C Tumor growth curves are shown. Fig.16D The survival rate of mice is shown.

[0115] Figures 17A-17E showed that LS-VEGF-C caused changes in intraocular lymphangiogenesis without increasing vascular permeability. Fig.17A Shown are intraocular pressure measurements following topical (eye drops), intracameral, and intravitreal administration of WT-VEGF-C in WT mice. Fig. 17B Shown are intraocular pressure measurements following topical (eye drops), intracameral, and intravitreal administration of LS-VEGF-C in WT mice. Fig. 17C Representative fundus (left column), fluorescent angiogenesis (middle column), and OCT (right column) images following intravitreal administration of VEGF-A, WT-VEGF-C, and LS-VEGF-C are shown. Fig.17D A normalized measure of fluorescence intensity in fluorescein angiography is shown in 2 and indicates the permeability of blood vessels to the extravascular space; a sharp spike above the baseline (normalized value approximately 20) indicates the presence of non-leaky vessels, and a persistently elevated value indicates increased permeability. Fig.17E Shown is a comparison of repeated measurements of absorbance of systemically delivered Evans Blue in the eye following IVT administration of PBS (control), VEGF-A, WT VEGF-C, and LS-VEGF-C, indicating permeability of retinal vessels to the extravascular space. Fig.17E , ***P<0.005, ****P<0.001, *P<0.05, ns not significant.

[0116] Figures 18A-18H LS-VEGF-C was shown to provide neuroprotection in a glaucoma model. Fig.18A is a schematic representation of the microbead mouse model of glaucoma and its evaluation by IOP measurement. Fig.18BShown are summary (mean ± sem, left) and individual IOP measurements (right) over 28 days for WT mice (control, n = 12), glaucoma microbead mouse model (induced glaucoma, n = 12), glaucoma microbead mouse model with IVTLS-VEGF-C (induced glaucoma + LS-VEGF-C (IVT), n = 12), glaucoma microbead mouse model with intravitreally administered LS-VEGF-C (induced glaucoma + LS-VEGF-C (IVT), n = 12), glaucoma microbead mouse model with topically applied LS-VEGF-C (induced glaucoma + LS-VEGF-C (eye drops)), n = 12), and WT mice with intravitreally administered LS-VEGF-C (LS-VEGF-C (IVT), n = 12). Fig.18C Confocal images showing Brn3a and DAPI labeling of the retina after intravitreal injection of NMDA (excitotoxicity studies) were compared between WT mice (control), microbead glaucoma model (induced glaucoma), and microbead glaucoma model with intravitreal (+LS-VEGF-CIVT) and topical (+LS-VEGF-C eye drops) LS-VEGF-C. Fig.18D Shown are the Brn3a cell counts observed in the image fields (control, n=7; microbead glaucoma model, n=6; microbead glaucoma model+IVTLS-VEGF-C, n=7; microbead glaucoma model+LS-VEGF-C eye drops, n=8). Fig.18E is a schematic representation of the DBA2J pigment dispersion mouse model of glaucoma. Fig.18F Shown are summary (mean ± sem, left) and individual IOP measurements (right) over 28 days for WT mice (control, n = 12), glaucoma microbead mouse model (induced glaucoma, n = 12, glaucoma microbead mouse model with IVTLS-VEGF-C (induced glaucoma + LS-VEGF-C (IVT), n = 12), glaucoma microbead mouse model with intravitreally administered LS-VEGF-C (induced glaucoma + LS-VEGF-C (IVT), n = 12), glaucoma microbead mouse model with topically applied LS-VEGF-C (induced glaucoma + LS-VEGF-C (eye drops)), n = 12), and glaucoma microbead mouse model with intravitreally administered LS-VEGF-C (LS-VEGF-C (IVT), n = 12). Figure 18G Images of RGC axons in the optic nerves of control D2-Gpnmb+ mice and DBA2J mice receiving different treatments (untreated (spontaneous glaucoma), +LS-VEGF-C IVT, +LS-VEGF-C eye drops) are shown. Fig.18HShown are the RGC axon cell counts observed in the image field (control, n=3; DBA2J glaucoma, n=4; LS-VEGF-C IVT, n=4; LS-VEGF-C eye drops, n=4).

[0117] Fig.19 Data from VEGFR2 and VEGFR3 binding assays of WT VEGF-C to VEGFR2 and VEGFR3 are shown.

[0118] Figures 20A-20F It was shown that in a lymphedema mouse model, administration of LS-VEGF-C resulted in a reduction in lymphedema. LS-VEGF-C was injected subcutaneously in mice near the lymph node ligation site and resulted in a significant reduction in ankle size compared to controls. Mice received two treatments to demonstrate the repeat administration capability of LS-VEGF-C.

[0119] Figures 21A-21D These results suggest that LS-VEGF-C provides a unique mechanism of action and that sustained effects were observed. Fig.21A Shown are IOP measurements (mean ± sem) over 10 days following topical (left), intracameral (middle), and intravitreal (right) administration of WT-VEGF-C and LS-VEGF-C. Fig. 21B is a graphic representation with arrows showing the flow of fluid into and out of the eye. Fig. 21C IOP measurements following topical administration of currently FDA-approved glaucoma medications are shown. Data points for the first hour were excluded due to confounding with the IOP-lowering effects of photoanesthesia. Fig.21D Comparison of IOP measurements (mean ± sem) of control mice, mice treated with FDA-approved topical glaucoma drugs at the time point at which the maximum IOP reduction was observed (dorzolamide, Dz, at 2 hours; Rhopressa, Rho, at 2 hours; Brimonidine, Bri, at 2 hours; Latanoprost, Lat, at 3 hours; Timolol, at 2 hours), 2 days after IV injection of LS-VEGF-C, and mice administered LS-VEGF-C intravitreally two days earlier at the time point at which the maximum IOP reduction was observed for topical administration of FDA-approved drugs. Data are shown as mean ± sem, with *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, ns not significant.

[0120] Figures 22A-22D Images of the anterior side of the eye are shown, showing similar signs to its posterior counterpart. Fig.22A Images of control WT mice focusing on the cornea and iris are shown (n=3). Fig. 22BImages of WT mice after IVT administration of VEGF-A are shown with focus on the cornea and iris (n=3). Neovascularization and corneal opacity were observed. Fig. 22C Images of WT mice after IVT administration of WT-VEGF-C are shown with focus on the cornea and iris (n=3). Corneal opacity was observed. Fig.22D Images of WT mice after IVT injection of LS-VEGF-C are shown with focus on the cornea and iris (n=3).

[0121] Figures 23A-23B LS-VEGF-C was shown to provide neuroprotection in a glaucoma model. Fig.23A Shown are confocal images of Brn3a and DAPI labeling of the retina after intravitreal injection of NMDA (excitotoxicity studies). Fig. 23B The number of RGC cells observed in the image field for NMDA-facilitated excitability studies is shown.

[0122] Fig.24 An overview of VEGFR2 binding of the hLS-VEGF-C variants is shown and summarizes the binding of the hVEGF-C variants to hVEGFR2 compared to WT VEGF-C, expressed as a percentage of the yeast population that stains positive. Lower values ​​are expected, indicating lower VEGFR2 engagement.

[0123] Fig.25 The relative VEGFR3 versus (vs) VEGFR2 bias of LS-hVEGF-C clones is shown. Fig.25 Figures containing hVEGFR3 binding (expressed as mean fluorescence intensity MFI) relative to hVEGFR2 binding (expressed as percentage of yeast population that stains positive). For the upper figure, 100 nM of VEGFR3 was used. For the lower figure, 10 nM of hVEGFR3 was used. Selected points are marked with the number of the LS-hVEGF-C mutant protein. Clones in the lower right quadrant of these figures are preferred because they show a biased binding to VEGFR3 relative to VEGFR2.

[0124] Fig.26 VEGFR2 and VEGFR3 binding of LS-hVEGF-C is shown; Fig.24 and 25 Specific values ​​for hVEGFR2 and hVEGFR3 binding for each clone are depicted in .

[0125] Fig. 27 Binding isotherms of selected LS-hVEGF-C muteins against hVEGFR3 are depicted. Fig. 27 Specific EC50 values ​​and R2 values ​​used for curve fitting are provided at the bottom.

[0126] Figures 28A-28M The mass of human VEGF-C mutein fused to human IgG1 Fc is shown. Fig.28A The quality of the human C137A clone fusion is shown. Fig.28B The quality of the human clone 7 fusion is shown. Fig.28C The mass of the human clone 8 fusion is shown. Fig.28D The quality of the human clone 9 fusion is shown. Fig.28E The quality of the human clone 10 fusion is shown. Fig.28F The quality of the human clone 13 fusion is shown. Figure 28G The quality of the human clone 14 fusion is shown. Fig.28H The quality of the human clone 15 fusion is shown. Fig.28I The quality of the human clone 19 fusion is shown. Figure 28K The quality of the human clone 21 fusion is shown. Figure 28L The quality of the human clone 31 fusion is shown. Figure 28M The quality of the human clone 39 fusion is shown.

[0127] Fig.29 Shown are the effects and intraocular pressure of mice treated intravitreally with RTI-Fc muteins.

[0128] Fig.30 Shown is the reduction in IOP in mice treated intravitreally with LS-VEGF-C having either albumin-conjugated or Fc-conjugated monomeric form.

[0129] Detailed Description

[0130] The present invention provides a VEGF-C mutant protein and a method of inducing lymphangiogenesis in a subject in need thereof, the method comprising administering an effective amount of the VEGF-C mutant protein to the subject.

[0131] The terms “a,” “an,” and “the” do not denote a limitation of quantity, but rather denote the presence of “at least one” of the referenced item.

[0132] The term "about" when used before a numerical value indicates that the value may vary within a reasonable range, for example, within ±10%, ±5% or ±1% of the stated value, including the stated value.

[0133] The terms "patient", "individual", "subject", "mammal" and "animal" are used interchangeably herein to refer to mammals, including but not limited to humans and veterinary animals (e.g., cats, dogs, rabbits, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.

[0134] The terms "treat" or "treatment" of a state, disorder or condition include: (1) preventing, delaying or reducing the incidence and / or likelihood of at least one clinical or subclinical symptom of the state, disorder or condition in a subject who may have or is susceptible to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) ameliorating the disease, i.e., causing regression of the state, disorder or condition or at least one clinical or subclinical symptom thereof. The benefit to the subject treated is statistically significant or at least perceptible to the patient or physician.

[0135] As used herein, the terms "mutein protein", "mutein polypeptide" and "mutein" are used interchangeably to refer to a protein with an altered amino acid sequence compared to its wild-type counterpart. Amino acid sequence changes may include amino acid substitutions, deletions or additions.

[0136] As used herein, the term "lymphangiogenesis" refers to the process of lymphatic vessel formation and / or stimulation of lymphatic vasculature function.

[0137] In one aspect, provided herein is an isolated vascular endothelial growth factor C (VEGF-C) mutant protein or a functional fragment thereof that selectively binds to VEGFR-3 relative to VEGFR-2.

[0138] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof has reduced or no ability to stimulate blood endothelial cell proliferation, but retains the ability to stimulate lymphatic endothelial cell proliferation, compared to a wild-type VEGF-C protein from the same species.

[0139] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof (i) has reduced binding affinity for VEGFR-2 compared to the wild-type VEGF-C protein from the same species, (ii) has the ability to bind to VEGFR-3 and generate signal transduction through VEGFR-3, and (iii) comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the wild-type VEGF-C protein from the same species.

[0140] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof has a binding affinity for VEGFR-2 that is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more compared to the wild-type VEGF-C protein from the same species.

[0141] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof has the ability to bind to VEGFR-3 and generate signaling through VEGFR-3, which is approximately the same, increased, or slightly decreased compared to the wild-type VEGF-C protein from the same species.

[0142] In cells, VEGF-C is produced in the form of an inactive propeptide. Converting enzymes (such as furin, PC5 or PC7) cleave between the VEGF homology domain and the C-terminal filament homology domain to produce pro-VEGF-C. Pro-VEGF-C is able to bind to VEGFR-3, but does not activate it. The second proteolytic cleavage of the dissociation protein and metalloproteinase 3 (ADAMTS3) with thrombospondin motif removes the two terminal domains, thereby producing 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 the case of 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 No. P49767). In the case of wild-type murine VEGF-C, the mature VEGF-C protein corresponds to amino acids 111-211 (SEQ ID NO: 292) of the murine VEGF-C propeptide (SEQ ID NO: 4).

[0143] In some embodiments, the VEGF-C mutant protein can be a mutant protein of a human VEGF-C protein. The human VEGF-C mutant protein can comprise 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% or 99% identical to amino acids 115-215 of the following polypeptide sequence:

[0144] MHLLGFFSVACSLLAAAALLPGPREAPAAAAAFESGLDLSDAEPDAGEATAYASKDLEEQLRSVSSVDELMTVLYPEYWKMYKCQLRKGGWQHNREQANLNSRTEETIKFAAA HYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLDVYRQVHSIIR RSLPATLPQCQAANKTCPTNYMWNNHICRCLAQEDFMFSSDAGDDSTDGFHDICGPNKELDEETCQCVCRAGLRPASCGPHKELDRNSCQCVCKNKLFPSQCGANREFDENTCQ CVCKRTCPRNQPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFSQPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFS(SEQ ID NO: 1; wild-type VEGF-C propeptide; UniProt database accession number P49767).

[0145] The polynucleotide molecule encoding the human VEGF-C mutant protein may comprise 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% or 99% identical to nucleotides 343-645 of the following VEGF-C coding sequence:

[0146]

[0147] The human VEGF-C mutant protein may comprise 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% or 99% identical to the following polypeptide sequence:

[0148] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (wild-type human mature VEGF-C protein; SEQ ID NO: 101).

[0149] The polynucleotide molecule encoding the human VEGF-C mutant protein may comprise 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% or 99% identical to the following VEGF-C coding sequence:

[0150] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTT GCAATTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG(SEQ ID NO: 153; nucleotide sequence encoding human mature VEGF-C protein).

[0151] In certain embodiments, the VEGF-C mutant protein comprises one or more mutations at residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, I188, V190, L192, P196, and combinations thereof, wherein the positions of the mutations are defined relative to SEQ ID NO: 1. In certain embodiments, the mutation at L119 is an L119E mutation. In certain embodiments, the mutation at L119 is an L119M mutation. In certain embodiments, the mutation at D123 is a D123N mutation. In certain embodiments, the mutation at Q130 is a Q130K mutation. In certain embodiments, the mutation at N167 is an N167R mutation. In certain embodiments, the mutation at N167 is an N167I mutation. In certain embodiments, the mutation at N167 is an N167Q mutation. In certain embodiments, the mutation at N167 is an N167H mutation. In certain embodiments, the mutation at S168 is a S168G mutation. In certain embodiments, the mutation at S168 is a S168R mutation. In certain embodiments, the mutation at V190 is a V190T mutation. In certain embodiments, the mutation at L192L is a L192I mutation.

[0152] In some embodiments, the VEGF-C mutant protein can be a mutant protein of a mouse VEGF-C protein. In certain embodiments, the mouse VEGF-C mutant protein can comprise 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% or 99% identical to amino acids 111-211 of the following polypeptide sequence:

[0153] MHLLCFLSLACSLLAAALIPSPREAPATVAAFESGLGFSEAEPDGGEVKAFEGKDLEEQLRSVSSVDELMSVLYPDYWKMYKCQLRKGGWQQPTLNTRTGDSVKFAAA HYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGGCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLDVYRQ VHSIIRRSLPATLPQCQAANKTCPTNYVWNNYMCRCLAQQDFIFYSNVEDDSTNGFHDVCGPNKELDEDTCQCVCKGGLRPSSCGPHKELDRDSCQCVCKNKLFPNSC GANREFDENTCQCVCKRTCPRNQPLNPGKCACECTENTQKCFLKGKKFHHQTCSCYRRPCANRLKHCDPGLSFSEEVCRCVPSYWKRPHLN (mouse wild-type VEGF-C propeptide; SEQ ID NO: 4).

[0154] The polynucleotide molecule encoding the murine VEGF-C mutant protein may comprise 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% identical to nucleotides 331-631 of the following VEGF-C coding sequence:

[0155]

[0156] In some embodiments, the VEGF-C mutant protein can be a mutant protein of a mouse VEGF-C protein. In certain embodiments, the mouse VEGF-C mutant protein can comprise 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% identical to the following polypeptide sequence:

[0157] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (wild-type murine mature VEGF-C protein; SEQ ID NO: 292).

[0158] The polynucleotide molecule encoding the murine VEGF-C mutant protein may comprise 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% identical to the following VEGF-C coding sequence:

[0159] AACACAGAGATCCTGAAAAGTATTGATAATGAGTGGAGAAAGACTCAATGCATGCCACGTGAGGTGTGTATAGATGTGGGGAAGGAGTTTGGAGCAGCCACAAACACCTTCTTTAAACCTCCATGTGTGTCCGTCTACAGATGTGGGGTTGCTGCAACAGCGAGGGGCTGCAGTGCATGAACACCAGCACAGGTTACCTCAGCAAGACGTTGTTTGAAATTACAGTGCCTCTCTCACAAGGCCCCAAACCAGTCACAATCAGTTTTGCCAATCACACTTCCTGCCGGTGCATGTCTAAACTG (nucleotide sequence encoding wild-type murine mature VEGF-C protein; SEQ ID NO: 293).

[0160] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0161] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167QVEGF-C mutein; SEQ ID NO: 160).

[0162] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0163] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGTCTGAGG GGCTCCAATGTATGAACACGAGTACGtctTACTTGAGTAAGACCTTGTTCGAAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167QVEGF-C mutant protein; SEQ ID NO: 210).

[0164] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0165] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167Q and S168G VEGF-C mutant protein; SEQ ID NO: 161).

[0166] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0167] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGCGAGGGGC TCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167Q and S168GVEGF-C mutant proteins; SEQ ID NO: 211).

[0168] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0169] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human N167Q and L192 IVEGF-C mutant protein; SEQ ID NO: 162).

[0170] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0171] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGTCTGAGGGGC TCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167Q and L192 IVEGF-C mutant proteins; SEQ ID NO: 212).

[0172] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0173] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human N167Q, S168G and L192I VEGF-C mutant protein; SEQ ID NO: 163).

[0174] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0175] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGCGAGGGGCTCC AATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167Q, S168G and L192 IVEGF-C mutant proteins; SEQ ID NO: 213).

[0176] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0177] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167IVEGF-C mutein; SEQ ID NO: 166).

[0178] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0179] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCTCTGAGG GGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167 IVEGF-C mutant protein; SEQ ID NO: 216).

[0180] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0181] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167I and S168G VEGF-C mutant protein; SEQ ID NO: 167).

[0182] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0183] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCGGCGAGGGGGC TCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167I and S168GVEGF-C mutant proteins; SEQ ID NO: 217).

[0184] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0185] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human N167I and L192I VEGF-C mutant protein; SEQ ID NO: 168).

[0186] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0187] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCTCTGAGGGGG CTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167I and L192 IVEGF-C mutant proteins; SEQ ID NO: 218).

[0188] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0189] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human N167I, S168G and L192I VEGF-C mutant protein; SEQ ID NO: 169).

[0190] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0191] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCGGCGAGGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167I, S168G and L192I VEGF-C mutant proteins; SEQ ID NO: 219).

[0192] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0193] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human S168G VEGF-C mutant protein; SEQ ID NO: 172).

[0194] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0195] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGC AATGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (Coder S168G Nucleotide sequence of VEGF-C mutant protein; SEQ ID NO: 222).

[0196] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0197] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human S168G and L192I VEGF-C mutant protein; SEQ ID NO: 174).

[0198] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0199] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human S168G and L192I VEGF-C mutant proteins; SEQ ID NO: 224).

[0200] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0201] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCHSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167H VEGF-C mutant protein; SEQ ID NO: 184).

[0202] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0203] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGC CACTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (encoder N167H Nucleotide sequence of VEGF-C mutant protein; SEQ ID NO: 234).

[0204] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0205] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCIREGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167I and S168R VEGF-C mutant protein; SEQ ID NO: 192).

[0206] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0207] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCAGAGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167I and S168R VEGF-C mutant proteins; SEQ ID NO: 242).

[0208] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises one or more mutations selected from mutations at residues N167, S168 and / or L192, wherein the positions of the mutations are defined relative to SEQ ID NO: 1.

[0209] 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, wherein the positions of the mutations are defined relative to SEQ ID NO: 1.

[0210] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation, wherein the position of the mutation is defined relative to SEQ ID NO:1.

[0211] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation and an S168G mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0212] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation and an L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0213] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation, an S168G mutation, and an L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0214] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation, wherein the position of the mutation is defined relative to SEQ ID NO:1.

[0215] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation and an S168G mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0216] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation and an L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0217] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation, an S168G mutation, and an L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0218] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an S168G mutation, wherein the position of the mutation is defined relative to SEQ ID NO:1.

[0219] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an S168G mutation and an L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0220] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an N167H mutation, wherein the position of the mutation is defined relative to SEQ ID NO:1.

[0221] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation and an S168R mutation, wherein the positions of the mutations are relative to SEQ ID NO: 1.

[0222] In certain embodiments, the VEGF-C mutant protein may comprise a functional fragment of human VEGF-C.

[0223] In certain embodiments, the VEGF-C mutant protein can comprise 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 polypeptide sequence of any one of SEQ ID NOs: 154-159, SEQ ID NOs: 164-165, SEQ ID NOs: 170-171, SEQ ID NO: 173, SEQ ID NOs: 175-183, SEQ ID NOs: 185-191, or SEQ ID NOs: 193-203.

[0224] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein can comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to any one of SEQ ID NOs: 204-209, SEQ ID NOs: 214-215, SEQ ID NOs: 220-221, SEQ ID NOs: 223, SEQ ID NOs: 225-233, SEQ ID NOs: 235-241, or SEQ ID NOs: 243-253.

[0225] In certain embodiments, the VEGF-C mutant protein may comprise a functional fragment of mouse VEGF-C. In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0226] NTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (murine VEGF-C mutant protein, SEQ ID NO: 23).

[0227] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0228] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGCGGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATTCTG AGGGGCTCCAATGTATGAACACGAGTACGGGTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding mouse VEGF-C mutant protein, SEQ ID NO: 24).

[0229] In certain embodiments, the VEGF-C mutant protein comprises one or more mutations at residues T112, L115, D119, Q126, T144, N145, K149, N163, S164, E165, I184, V186, L188, P192, and combinations thereof, wherein the positions of the mutations are defined relative to SEQ ID NO: 4. In certain embodiments, the mutation at L115 is an L115E mutation. In certain embodiments, the mutation at L115 is an L115M mutation. In certain embodiments, the mutation at D119 is a D119N mutation. In certain embodiments, the mutation at Q126 is a Q126K mutation. In certain embodiments, the mutation at N163 is an N163R mutation. In certain embodiments, the mutation at N163 is an N163I mutation. In certain embodiments, the mutation at N163 is an N163Q mutation. In certain embodiments, the mutation at S164 is an S164G mutation. In certain embodiments, the mutation at V186 is a V184T mutation. In certain embodiments, the mutation at L188 is a L188I mutation. In some embodiments, the VEGF-C mutant protein comprises the mutation N163I.

[0230] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0231] MHLLCFLSLACSLLAAALIPSPREAPATVAAFESGLGFSEAEPDGGEVKAFEGKDLEEQLRSVSSVDELMSVLYPDYWKMYKCQLRKGGWQQPTLNTRTGDSVKF AAAHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL DVYRQVHSIIRRSLPATLPQCQAANKTCPTNYVWNNYMCRCLAQQDFIFYSNVEDDSTNGFHDVCGPNKELDEDTCQCVCKGGLRPSSCGPHKELDRDSCQCVCKN KLFNPNSCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTENTQKCFLKGKKFHHQTCSCYRRPCANRLKHCDPGLSFSEEVCRCVPSYWKRPHLN (mouse N163I VEGF-C mutein; SEQ ID NO: 6).

[0232] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise 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 nucleotide sequence:

[0233]

[0234] In some embodiments, the VEGF-C mutant protein comprises mutations N163R, V186T, and L188I, wherein the positions of the mutations are defined relative to SEQ ID NO:4.

[0235] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0236] MHLLCFLSLACSLLAAALIPSPREAPATVAAFESGLGFSEAEPDGGEVKAFEGKDLEEQLRSVSSVDELMSVLYPDYWKMYKCQLRKGGWQQPTLNTRTGDSVKF AAAHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSK LDVYRQVHSIIRRSLPATLPQCQAANKTCPTNYVWNNYMCRCLAQQDFIFYSNVEDDSTNGFHDVCGPNKELDEDTCQCVCKGGLRPSSCGPHKELDRDSCQCVC KNKLFFPNSCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTENTQKCFLKGKKFHHQTCSCYRRPCANRLKHCDPGLSFSEEVCRCVPSYWKRPHLN(ratRTI VEGF-C mutein, SEQ ID NO: 8).

[0237] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0238]

[0239] In certain embodiments, the VEGF-C mutein comprises a mutation at residue C133, wherein the position of the mutation is defined relative to SEQ ID NO: 4. In certain embodiments, the mutation at residue C133 is a C133A mutation.

[0240] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0241] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (murine VEGF-C mutant protein with C133A mutation, SEQ ID NO: 9).

[0242] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0243] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGCGGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATTCTGAGGGGCTCCAATGTATGAACACGAGTACGGGTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding a murine VEGF-C mutant protein having a C133A mutation, SEQ ID NO: 10).

[0244] In certain embodiments, the VEGF-C mutein comprises mutations C133A and N163I, wherein the positions of the mutations are defined relative to SEQ ID NO:4.

[0245] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0246] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (having C133A and N163I mutations, SEQ ID NO: 17).

[0247] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0248] AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGCAAAACTCAATGTATGCCACGGGAGGTTGCAATAGACGTGGGAAAGGAATTTGGCGCCGCCACGAATACCTTTTTCAAGCCTCCCTGTGTGAGTGTTTACAGATGTGGTGGTTGTTGCATATCAGAGGGATTGCAGTGCATGAACACAAAGTACAGGTTACTTGAGTAAAACATTGTTTGAAATCACAGTACCATTGTCCCAAGGCCCTAAGCCGGTTACGATCTCTTTCGCCAATCATACGTCATGCCGCTGTATGAGTAAGTTG (nucleotide sequence encoding a murine VEGF-C mutant protein having C133A and N163I mutations, SEQ ID NO: 18).

[0249] In some embodiments, the VEGF-C mutant protein comprises mutations C133A, N163R, V186T, and L188I, wherein the positions of the mutations are defined relative to SEQ ID NO:4.

[0250] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0251] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKL (murine VEGF-C mutant protein with C133A, N163R, V186T and L188I mutations, SEQ ID NO: 13).

[0252] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0253] AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGAAAAACCCAATGTATGCCACGCGAGGTAGCGATAGATGTGGGCAAAGAATTCGGCGCCGCGACGAACACCTTTTTCAAGCCCCCTTGCGTCTCCGTATATAGATGCGGTGGATGTTGCCGATCCGAGGGCCTTCAGTGTATGAACACATCTACTGGCTATTTGAGCAAGACGCTCTTTGAGATTACAACACCAATTAGTCAAGGTCCCAAGCCTGTTACCATCTCTTTCGCTAACCACACTTCATGCCGCTGTATGAGTAAGTTG (nucleotide sequence encoding a murine VEGF-C mutant protein having C133A, N163R, V186T and L188I mutations, SEQ ID NO: 14).

[0254] In certain embodiments, the VEGF-C mutein comprises a mutation at residue C137, wherein the position of the mutation is defined relative to SEQ ID NO: 1. In certain embodiments, the mutation at residue C137 is a C1337A mutation.

[0255] In certain embodiments, the human VEGF-C mutant protein may comprise 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:

[0256] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human VEGF-C mutant protein with C133A mutation; SEQ ID NO: 100).

[0257] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0258] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human VEGF-C mutant protein having C133A mutation, SEQ ID NO: 152).

[0259] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0260] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A and N167Q VEGF-C muteins; SEQ ID NO: 56).

[0261] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0262] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A and N167Q VEGF-C mutant proteins; SEQ ID NO: 108).

[0263] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0264] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A, N167Q and S168G VEGF-C mutant protein; SEQ ID NO: 57).

[0265] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0266] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167Q and S168G VEGF-C mutant proteins; SEQ ID NO: 109).

[0267] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0268] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human C137A, N167Q and L192I VEGF-C mutant protein; SEQ ID NO: 58).

[0269] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the following nucleotide sequence:

[0270] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167Q and L192I VEGF-C mutant proteins; SEQ ID NO: 110).

[0271] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0272] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human C137A, N167Q, S168G and L192I VEGF-C mutant protein; SEQ ID NO: 59).

[0273] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0274] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167Q, S168G and L192I VEGF-C mutant proteins; SEQ ID NO: 111).

[0275] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0276] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A and N167I VEGF-C mutant proteins; SEQ ID NO: 62).

[0277] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0278] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCTCTGAGGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A and N167I VEGF-C mutant proteins; SEQ ID NO: 114).

[0279] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0280] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A, N167I and S168G VEGF-C mutant protein; SEQ ID NO: 63).

[0281] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0282] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCGGCGAGGGGTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167I and S168G VEGF-C mutant proteins; SEQ ID NO: 115).

[0283] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0284] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human C137A, N167I and L192I VEGF-C mutant protein; SEQ ID NO: 64).

[0285] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0286] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCTCTGAGGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167I and L192I VEGF-C mutant proteins; SEQ ID NO: 116).

[0287] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0288] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human C137A, N167I, S168G and L192I VEGF-C mutant protein; SEQ ID NO: 65).

[0289] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0290] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCGGCGAGGGGTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167I, S168G and L192I VEGF-C mutant proteins; SEQ ID NO: 117).

[0291] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0292] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A and S168G VEGF-C mutant protein; SEQ ID NO: 68).

[0293] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0294] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A and S168G VEGF-C mutant proteins; SEQ ID NO: 120).

[0295] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0296] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human C137A, S168G and L192I VEGF-C mutant protein; SEQ ID NO: 70).

[0297] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0298] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, S168G and L192I VEGF-C mutant proteins; SEQ ID NO: 122).

[0299] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0300] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCHSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A and N167H VEGF-C muteins; SEQ ID NO: 80).

[0301] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0302] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCACTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A and N167H VEGF-C mutant proteins; SEQ ID NO: 132).

[0303] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0304] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIREGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A, N167I and S168R VEGF-C mutant proteins; SEQ ID NO: 88).

[0305] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0306] AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCAGAGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167I and S168R VEGF-C mutant proteins; SEQ ID NO: 140).

[0307] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises one or more mutations selected from mutations at residues C137, N167, S168 and / or L192, wherein the positions of the mutations are defined relative to SEQ ID NO: 1.

[0308] In some embodiments, the mutation at C137 is C137A, the mutation at N167 is N167I mutation, N167Q mutation, or N167H mutation; the mutation at S168 is S168G mutation or S168R mutation; and / or the mutation at L192 is L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO: 1.

[0309] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation and a N167Q mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0310] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation, a N167Q mutation, and a S168G mutation, wherein the positions of the mutations are defined relative to SEQ ID NO: 1.

[0311] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation, a N167Q mutation, and a L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO: 1.

[0312] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation, a N167Q mutation, a S168G mutation, and a L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO: 1.

[0313] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation and a N167I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0314] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation, a N167I mutation, and a S168G mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0315] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation, a N167I mutation, and a L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0316] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation, a N167I mutation, a S168G mutation, and a L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0317] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation and a S168G mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0318] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation, a S168G mutation, and a L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0319] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation and a N167H mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0320] In some embodiments, the VEGF-C mutant protein or a functional fragment thereof comprises a C137A mutation, a N167I mutation, and a S168R mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0321] In certain embodiments, the VEGF-C mutant protein can comprise 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 SEQ ID NO: 50-55, SEQ ID NO: 60-61, SEQ ID NO: 66-67, SEQ ID NO: 69, SEQ ID NO: 71-79, SEQ ID NO: 81-87 or SEQ ID NO: 89-99.

[0322] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein can comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to any one of SEQ ID NOs: 102-107, SEQ ID NOs: 112-113, SEQ ID NOs: 118-119, SEQ ID NOs: 121, SEQ ID NOs: 123-131, SEQ ID NOs: 133-139, or SEQ ID NOs: 141-151.

[0323] The VEGF-C mutant proteins or fragments thereof described herein include variants having single or multiple amino acid substitutions, deletions or additions, which retain the biological properties (eg, binding affinity or immune effector activity) of the VEGF-C mutant proteins or fragments thereof.

[0324] These variants may include: (i) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (ii) variants in which one or more amino acids are added to or deleted from a polypeptide, (iii) variants in which one or more amino acids include a substitution, and (iv) variants in which the VEGF-C mutant protein or its fragment is fused or conjugated with another peptide or polypeptide (e.g., a fusion partner, a protein tag) or other chemical moiety (which can confer useful properties to the VEGF-C mutant protein or its fragment, e.g., an epitope of an antibody, a polyhistidine sequence, a biotin moiety, etc.). The VEGF-C mutant protein or its fragment described herein may include variants in which an amino acid residue from one species is substituted with a corresponding residue in another species, whether in a conservative position or a non-conservative position. In other embodiments, an amino acid residue in a non-conservative position is substituted with a conservative or non-conservative residue. Amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc.

[0325] Amino acid substitutions can be conservative, meaning that the substituted amino acid has chemical properties similar to the original amino acid. One skilled in the art will understand which amino acids have 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).

[0326] In some embodiments, the VEGF-C mutant protein is modified to extend its circulation half-life. Strategies for extending the half-life of recombinant proteins include, but are not limited to, fusion with immunoglobulins or fragments thereof (e.g., the Fc domain of IgG); fusion with albumin or its albumin fragment; fusion with albumin-binding antibodies or their antibody fragments (e.g., scFv, Fab, or single domain antibodies (VHH)); or chemical modification with polyethylene glycol (PEG).

[0327] In some embodiments, the VEGF-C mutant protein or fragment or variant thereof is fused and / or conjugated to one or more heterologous moieties (e.g., but not limited to, peptides, polypeptides, small molecules, polymers, nucleic acids, lipids, carbohydrates, etc.).

[0328] In certain embodiments, the VEGF-C mutant protein is fused and / or conjugated with a portion that provides a longer half-life for the VEGF-C mutant protein. In some embodiments, the VEGF-C mutant protein is fused and / or conjugated with a portion that specifically binds to albumin (including but not limited to small molecules, peptides, polypeptides, or lipids that bind to albumin). In some embodiments, the VEGF-C mutant protein is fused and / or conjugated with an immunoglobulin constant region (Fc domain), scFv, Fab, single domain antibody (VHH), immunoglobulin, or its heavy or light chain.

[0329] In certain embodiments, the VEGF-C mutant protein is fused and / or conjugated to a polymer, including but not limited to lipopolymers, polyethylene glycol (PEG), biodegradable polymers such as PLA (poly(lactic acid)) and PLGA (poly(lactic-glycolic acid)), polysaccharides, polysaccharides, poly(propylene glycol), polyoxyethylated polyols, polyethylene ethers, copolymers of ethylene glycol and propylene glycol polyvinyl alcohol, dextran, hyaluronic acid, chitin, and the like.

[0330] In certain embodiments, the VEGF-C mutant protein is fused and / or conjugated to an Fc domain. In certain embodiments, the Fc sequence fused or conjugated to the VEGF-C mutant protein of the present invention may comprise 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:

[0331] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 21).

[0332] In certain embodiments, the polynucleotide molecule encoding the Fc sequence may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the following nucleotide sequence:

[0333] ACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTGgcggccgcTGAACCCAAAAGTTGTGACAAAACGCACACATGCCCGCCTTGCCCCGCCCCGGAGCTACTGGGCGGACCCTCTGTGTTTCTGTTTCCACCAAAGCCGAAAGATACTCTTATGATTTCCAGGACACCTGAAGTCACCTGCGTTGTTGTGGATGTTAGCCATGAAGATCCCGAAGTGAAGTTTAACTGGTACGTAGACGGCGTGGAAGTACACAATGCAAAAACGAAACCTAGAGAAGAACAATATCAATCAACCTATAGGGTAGTGTCAGTCCTTACAGTCCTACACCAGGACTGGCTTAATGGGAAAGAGTATAAATGCAAGGTCAGTAACAAGGCGCTACCCGCTCCAATAGAGAAAACTATCTCTCTAAGGCTAAAGGCCAGCCGCGTGAGCCCCAGGTATATACACTGCCACCATCCAGGGAGGAAATGACAAAGAACCAGGTGTCCCTGACTTGCTTAGTAAAAGGGTTTTATCCATCCGACATCGCCGTGGAATGGGAATCTAATGGTCAGCCTGAAAACAATTACAAGACAACACCTCCCGTTCTTGATAGCGACGGATCTTTTTTCCTTTACTCTAAATTGACTGTAGATAAAAGTAGGTGGCAGCAAGGTAACGTCTTTTCATGCAGCGTGATGCACGAGGCATTACACAACCATTACACACAGAAATCATTGTCATTAAGTCCAGGGAAG(SEQ ID NO:22).

[0334] In certain embodiments, the VEGF-C mutant protein can comprise 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:

[0335] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (murine VEGF-C mutein with C133A mutation and Fc conjugation, SEQ ID NO:11)

[0336] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0337]

[0338] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0339] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (murine VEGF-C mutein with C133A, N163R, V186T and L188I mutations and with Fc conjugation, SEQ ID NO: 15).

[0340] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0341]

[0342] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0343] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (murine VEGF-C mutein with C133A and N163I mutations and Fc conjugation, SEQ ID NO: 19).

[0344] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0345]

[0346] In some embodiments, the IgG Fc domain comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the VEGF-C mutein comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the VEGF-C mutein consists of the amino acid sequence of SEQ ID NO: 15.

[0347] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0348] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (human C137A with Fc conjugation VEGF-C protein; SEQ ID NO: 266).

[0349] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0350]

[0351] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0352] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (Human C137A and N167QVEGF-C muteins with Fc conjugation; SEQ ID NO: 267).

[0353] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0354]

[0355] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0356] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (with Fc-conjugated human C137A, N167Q and S168G VEGF-C mutant protein; SEQ ID NO: 268).

[0357] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0358]

[0359] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0360] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (with Fc-conjugated human C137A, N167Q and L192I VEGF-C mutant protein; SEQ ID NO: 269).

[0361] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0362]

[0363] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0364] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (Human C137A, N167Q, S168G and L192I with Fc conjugation VEGF-C mutant protein; SEQ ID NO: ).

[0365] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0366]

[0367] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0368] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGCCISEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (Human C137A and N167IVEGF-C mutant protein with Fc conjugation; SEQ ID NO: 271).

[0369] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0370]

[0371] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0372] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (with Fc-conjugated human C137A, N167I and S168G VEGF-C mutant protein; SEQ ID NO: 272).

[0373] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0374]

[0375] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0376] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (with Fc-conjugated human C137A, N167I and L192I VEGF-C mutant protein; SEQ ID NO: 273).

[0377] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0378]

[0379] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0380] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (Human C137A, N167I, S168G and L192I with Fc conjugation VEGF-C mutant protein; SEQ ID NO: 274).

[0381] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0382]

[0383] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0384] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (human C137A and S168GVEGF-C mutant protein with Fc conjugation; SEQ ID NO:275)

[0385] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0386]

[0387] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0388] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (with Fc-conjugated human C137A, S168G and L192I VEGF-C mutant protein; SEQ ID NO: 276).

[0389] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the following nucleotide sequence:

[0390]

[0391] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0392] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCHSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (Human C137A and N167HVEGF-C mutant protein with Fc conjugation; SEQ ID NO: 277).

[0393] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0394]

[0395] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0396] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIREGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (with Fc-conjugated human C137A, N167I and S168R VEGF-C mutant protein; SEQ ID NO: 278).

[0397] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the following nucleotide sequence:

[0398]

[0399] In some embodiments, the present invention relates to modifying IgG Fc domains to reduce Fc effector functions. This modification can be achieved by various techniques, including but not limited to amino acid substitution, deletion or addition of Fc domains. For example, modification may involve replacing one or more amino acid residues in the Fc domain with non-natural amino acids, changing the glycosylation pattern or introducing steric hindrance to the Fc domain. In some embodiments, the modified Fc domains show reduced binding affinity to Fc receptors or complement proteins, thereby resulting in reduced Fc effector functions. Examples of modified Fc domains showing reduced Fc effector functions include but are not limited to Fc variants with point mutations (e.g., 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 produce therapeutic antibodies with improved safety and reduced adverse immune response risks. The modified Fc domain can also be used to design antibody-based therapies with desired effector functions (e.g., reduced ADCC, complement activation, or immune complex formation). In some embodiments, the IgG Fc domain is modified to reduce Fc effector functions. 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. In some embodiments, the IgG Fc domain is an IgG4 variant comprising one or more mutations at residues F234 or L235. In some embodiments, the IgG Fc domain is an IgG4 variant comprising one or more mutations at F234A or L235A. In some embodiments, the IgG Fc domain is an IgG1 variant comprising one or more mutations at residues L234 or L235. In some embodiments, the IgG Fc domain is an IgG1 variant comprising one or more mutations at L234A or L235A. In some embodiments, the IgG Fc domain can be a cross-subclass domain. For example, but not limited to, the IgG Fc domain can be an IgG2 variant with point mutations from IgG4 (e.g., H268Q / V309L / A330S / P331S).

[0400] In some embodiments, the Fc domain comprises one or more mutations.In some embodiments, the Fc domain is mutated, glycoengineered, or otherwise modified to reduce Fc effector function.

[0401] In some embodiments, the Fc domain comprises one or more mutations that eliminate critical glycosylation sites required for effector function.

[0402] In some embodiments, the Fc domain comprises a mutation at residue N82, wherein the position of the residue is defined relative to SEQ ID NO: 21. In some embodiments, the mutation at N82 defined relative to SEQ ID NO: 21 is an N82Q mutation, an N82A mutation, or an N82G mutation.

[0403] In certain embodiments, the VEGF-C mutein is fused and / or conjugated to albumin. In certain embodiments, the albumin is human albumin.

[0404] In some embodiments, the VEGF-C mutant protein is fused and / or conjugated to the albumin or Fc domain via a linker. 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-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 a protease (e.g., trypsin, chymotrypsin, or thrombin); or (Leu-Val-Pro-Arg) (SEQ ID NO: 305), which can be cleaved by the 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, which can form a triazole bond with an alkyne-containing molecule; or (cyclohexyl-Ala-Pro-Pro), which contains the unnatural amino acid cyclohexyl-alanine, which can form a rigid cyclohexane structure. Examples of such linkers include, but are not limited to, linkers having the following amino acid sequence: GGGGGSGGGGSGGGGS (SEQ ID NO: 294).

[0405] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0406] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLHHHHHHHH (murine VEGF-C mutant protein with C133A and N163I mutations having 8xHis (SEQ ID NO: 306) aa sequence; SEQ ID NO: 254).

[0407] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0408] AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGCAAAACTCAATGTATGCCACGGGAGGTTGCAATAGACGTGGGAAAGGAATTTGGCGCCGCCACGAATACCTTTTTCAAGCCTCCCTGTGTGAGTGTTTACAGATGTGGTGGTTGTTGCATATCAGAGGGATTGCAGTGCATGAACACAAGTACAGTTACTTGAGTAAAACATTGTTTGAAATCACAGTACCATTGTCCCAAGGCCCTAAGCCGGTTACGATCTCTTTCGCCAATCATACGTCATGCCGCTGTATGAGTAAGTTGCATCACCATCACCATCATCACCAT (nucleotide sequence encoding murine VEGF-C mutant protein with C133A and N163I mutations having 8xHis (SEQ ID NO: 306) aa sequence; SEQ ID NO: 260).

[0409] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0410] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKLHHHHHHHH (murine VEGF-C mutant protein with C133A, N163R, V186T and L188I mutations having 8xHis (SEQ ID NO: 306) aa sequence; SEQ ID NO: 255).

[0411] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0412] AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGAAAAACCCAATGTATGCCACGCGAGGTAGCGATAGATGTGGGCAAAGAATTCGGCGCCGCGACGAACACCTTTTTCAAGCCCCCTTGCGTCTCCGTATATAGATGCGGTGGATGTTGCCGATCCGAGGGCCTTCAGTGTATGAACACATCTACTGGCTATTTGAGCAAGACGCTCTTTGAGATTACAACACCAATTAGTCAAGGTCCCAAGCCTGTTACCATCTCTTTCGCTAACCACACTTCATGCCGCTGTATGAGTAAGTTGCATCACCATCACCATCATCACCAT (nucleotide sequence encoding murine VEGF-C mutant protein with C133A, N163R, V186T and L188I mutations having 8xHis (SEQ ID NO: 306) aa sequence; SEQ ID NO: 261).

[0413] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0414] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (with C133A and N163I mutations AAA clone scar Fc(N297Q) fused to 6xHis(SEQ ID NO: 25) aa sequence of mouse VEGF-C mutant protein; SEQ ID NO: 256).

[0415] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0416]

[0417] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0418] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQ STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (with C133A, N163R, V186T and L188I mutations AAA clone scar Fc (N297Q) fused to the murine VEGF-C mutant protein of the 6xHisaa sequence; SEQ ID NO: 257).

[0419] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0420]

[0421] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0422] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (with C133A and N163I mutations AAA clone scar and linker mouse serum albumin fusion GS8xHis (SEQ ID NO:306) aa sequence of mouse VEGF-C mutant protein; SEQ ID NO:258).

[0423] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0424]

[0425] In certain embodiments, the VEGF-C mutant protein may comprise 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:

[0426] NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (murine VEGF-C mutant protein with C133A, N163R, V186T and L188I mutations AAA clone scar and linker mouse serum albumin fusion GS8xHis (SEQ ID NO: 306) aa sequence; SEQ ID NO: 259).

[0427] In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein may comprise a sequence having at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following nucleotide sequence:

[0428]

[0429] In some embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof is mRNA.

[0430] mRNA may offer several advantages to AAV and other gene delivery systems, which include, but are not limited to, one or more of the following: mRNA may be highly customizable, mRNA may prevent recognition by pattern recognition receptors and nucleases to allow for sustained expression, mRNA may offer well-controlled expression kinetics with repeat dosing options, and mRNA may offer a low risk of integration into the genome due to its localization in the cytosol. mRNA may also be cost-effective.

[0431] In some embodiments, the polynucleotide molecules encoding VEGF-C mutant proteins or functional fragments thereof contain modified nucleotides, such as 5-methylcytosine and pseudouridine substitutions, which can increase stability, reduce deamination, reduce nuclease activity, reduce innate recognition, or increase translation efficiency of the polynucleotide molecules. In some embodiments, the modified nucleotides are 5-methylcytosine or pseudouridine. In some embodiments, the polynucleotide molecules encoding VEGF-C mutant proteins contain a 5' cap.

[0432] The mRNA may contain modified nucleotides. In some embodiments, the modified nucleotide is 5-methylcytosine or pseudouridine. In some embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment comprises a 5' cap. In some embodiments, the 5' cap is added using CleanCapReagentAG. CleanCap is composed of C32H43N15O24P4 and allows high capping efficiency, thereby producing more active mRNA. Cap1 does not activate pattern recognition receptors and is important for the proficiency of in vivo expression. Without wishing to be bound by theory, any one or more of 5-methylcytosine, pseudouridine and 5' cap can improve the stability of mRNA, thereby prolonging the expression of VEGF-C mutant protein or its functional fragment.

[0433] In some embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment is contained in a viral vector. Exemplary viral vectors include, but are not limited to, herpes virus, cytomegalovirus, poliovirus, alphavirus, vaccinia virus, rabies virus, adeno-associated virus (AAV), retrovirus, slow virus and adenovirus. The retrovirus can be a slow virus. The recombinant virus particle can be derived from an adeno-associated virus (AAV). In some embodiments, AAV is AAV2. In some embodiments, AAV is AAV5. In some embodiments, AAV is AAV9.

[0434] In some embodiments, VEGF-C mutant protein or its functional fragment can be administered in a dosage regimen involving a combination of mRNA and AAV. One or more mRNA administrations can be performed to quickly obtain high expression, for example within 2 hours after delivery of mRNA. The expression of VEGF-C mutant protein or its functional fragment provided by AAV may take 7-14 days, or even up to four weeks, depending on the serotype. The administration of protein or its functional fragment can be performed to obtain transient expression. The co-administration of both mRNA and AAV or the continuous administration for a short period of time can provide the sustained expression of VEGF-C mutant protein or its functional fragment. Without being bound by theory, the administration of protein can provide transient expression and controlled expression dynamics, and multiple doses can be used. Without being bound by theory, the administration of mRNA can provide transient expression, controlled expression dynamics and high expression, and multiple doses can be used. Without being bound by theory, the administration of AAV can provide delayed expression and high-level expression. Expression dynamics can be effectively and sensitively measured using ELISA and Western blotting.

[0435] In some embodiments, the polynucleotide molecules encoding VEGF-C mutant proteins or their functional fragments are contained in liposomes. VEGF-C mutant proteins or their functional fragments can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, attached to liposomes by connecting molecules associated with liposomes and polynucleotides, encapsulated in liposomes, complexed with liposomes, dispersed in solutions containing lipids, mixed with lipids, combined with lipids, contained in lipids as suspensions, containing micelles or complexed with micelles, or otherwise associated with lipids. Liposomes containing VEGF-C mutant proteins or their functional fragments can exist in a bilayer structure, as micelles, or have a "collapsed" structure. Liposomes can also be simply dispersed in a solution, possibly forming aggregates of uneven size or shape. For example, nucleotides (e.g., siRNA) can be encapsulated in neutral liposomes using methods involving ethanol and calcium. The shape can be that of a spherical vesicle. In various embodiments, liposomes can include one or more concentric layers of lipid bilayer molecules. In some embodiments, the lipid component includes a combination 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.

[0436] In some embodiments, the polynucleotide molecules encoding VEGF-C mutant proteins or their functional fragments are attached to nanoparticles or polymers. In certain embodiments, the nanoparticles of the present invention also include at least one agent that specifically binds to a specific type or class of cells and / or other specific types of compounds (e.g., a portion targeting a specific cell or cell type). In some embodiments, the nanoparticle is a nanosphere. In some embodiments, the polymer is dextran, poly (amine-to-ester), poly (β-amino-ester), polyethylene imine, poly-L-lysine, polyethylene glycol or dendrimer.

[0437] In some embodiments, the polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof is contained in a recombinant viral particle or in a virus-like particle (VLP).

[0438] In some embodiments, the VEGF-C mutant protein is produced and administered in the form of a "masked" prodrug that is activated after administration to a patient. In some cases, this may confer the desired tissue or tumor-specific activity to the mutant protein, for example by limiting the expression of endogenous proteases that cleave the prodrug and release active "mature" fragments. In some embodiments, the VEGF-C mutant protein is a variant of the full-length VEGF-C propeptide and is activated by a protease (e.g., ADAMTS2, plasmin, furin, cathepsin D, thrombin, and / or KLK3 / PSA). In other embodiments, the VEGF-C mutant protein is fused to an inhibitory peptide that is proteolytically released by an endogenous protease. Inhibitory peptides can take many forms, including but not limited to antibodies or antibody fragments that bind to VEGF-C mutant proteins to mask their interactions with VEGFR-3, VEGFR-3 extracellular domains or fragments thereof that competitively inhibit VEGF-C from interacting with VEGFR-3 on cells, and any other binding agents (e.g., VHH, fibronectin domains, knottins, lipocalin, leucine-rich repeats, etc.). Non-limiting example sets of protease cleavage sites are described below. Inhibitory peptides can be separated from biologically active VEGF-C mutant proteins by enterokinase cleavage site (EKCS) peptides (which are recognized and cleaved by endogenous protease enterokinase present in the small intestine). In this way, prodrugs can be designed to be in an inactive state before reaching the small intestine, where prodrugs can be activated by enterokinase cleavage. Alternatively, inhibitory peptides can be separated from biologically active VEGF-C mutant proteins by furin cleavage site (FCS) peptides that can be recognized and cleaved by furin, and furin is an endogenous protease that is overexpressed in many tumor cells. By designing a prodrug so that it is specifically cleaved by furin, the activation of the prodrug can be localized to the tumor microenvironment, thereby achieving targeted therapy. In addition, 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 is overexpressed in many tumors). In this way, the activation of the prodrug can be localized to the tumor microenvironment, thereby achieving targeted VEGF-C mutant protein therapy.

[0439] In one aspect, a method for inducing lymphangiogenesis in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a VEGF-C mutant protein or a functional fragment thereof or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the foregoing.

[0440] In one aspect, provided herein is a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a VEGF-C mutant protein of the present disclosure or a functional fragment thereof, or a fusion protein 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, or a pharmaceutical composition of the present disclosure.

[0441] In some embodiments, the disease or condition is cancer, coronary vascular function, osmoregulation, cardiac ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, a disease or condition associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennicham syndrome, Milroy disease, Turner syndrome, age-related macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, and retinal edema.

[0442] 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 cell tumor, craniopharyngioma, meningioma, CNS lymphoma, pineal tumor, retinoblastoma, uveal melanoma and rhabdoid tumor.

[0443] In some embodiments, the VEGF-C mutant protein or its functional fragment, fusion protein, conjugate, polynucleotide molecule, carrier, particle or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisternal, intravitreally, by eye drops, subcutaneously, intradermally, by inhalation, by long indwelling catheter, orally, topically or systemically. 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 intracisternal administration. In some embodiments, the pharmaceutical composition is formulated for eye drop administration. 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 administration by inhalation. In some embodiments, the pharmaceutical composition is formulated for administration by long indwelling catheter. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for topical administration (eg, as a cream or gel).

[0444] In one aspect, a method for treating cancer in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a VEGF-C mutant protein or a functional fragment thereof or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the foregoing.

[0445] In one aspect, a pharmaceutical composition is provided, comprising a VEGF-C mutant protein or a functional fragment thereof and optionally an immunotherapeutic agent.

[0446] In one aspect, a method for generating a library of VEGF-C mutant proteins or functional fragments thereof is provided, wherein the VEGF-C mutant proteins or functional fragments thereof have (i) specific binding to VEGF; and (ii) reduced binding to VEGFR-2 compared to wild-type VEGF-C.

[0447] In certain embodiments, the cancer is a 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., astrocytomas, glioblastomas, oligodendrogliomas, brain stem gliomas, juvenile pilocytic astrocytomas, and optic gliomas), ependymomas, subependymomas, primitive neuroectodermal tumors, gangliogliomas, schwannomas, germ cell tumors, craniopharyngiomas, meningiomas, CNS lymphomas, pineal tumors, retinoblastomas, uveal melanomas, and rhabdoid tumors.

[0448] Gliomas can be any tumor derived from brain glial tissue. In some embodiments, gliomas can be mixed gliomas. Gliomas can be low-grade gliomas or high-grade gliomas. Gliomas can be supratentorial, infratentorial or pontine. Examples of gliomas include, but are not limited to, glioblastomas.

[0449] In certain embodiments, cancer is glioblastoma. In certain embodiments, cancer is glioblastoma multiforme (GBM). The initial diagnosis of GBM is usually performed using CT or MRI, wherein glioblastoma usually shows as annular enhanced lesions. Diagnosis can be confirmed based on biopsy (e.g., stereotactic biopsy or craniotomy to remove the tumor).

[0450] In certain embodiments, the cancer is a metastatic cancer. In certain embodiments, the cancer is a metastatic cancer that has spread to the brain or central nervous system of the subject. In certain embodiments, the cancer is a metastatic brain cancer.

[0451] In certain embodiments, 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, liquid cancer (e.g., acute myeloid leukemia), kidney cancer, bladder urothelial carcinoma, prostate cancer, pheochromocytoma, bile duct cancer, liver: hepatocellular carcinoma, pancreatic ductal adenocarcinoma, thymoma, sarcoma, mesothelioma, testicular cancer, and colorectal cancer.

[0452] In certain embodiments, the VEGF-C mutant protein or its functional fragment of the present disclosure (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be used to treat or regulate cardiac function or disease. Examples of such cardiac function or disease include, but are not limited to, coronary vascular function, osmotic regulation, cardiac ischemia, and restenosis.

[0453] In certain embodiments, the VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the foregoing) of the present disclosure can be used in pulmonology.

[0454] In certain embodiments, the VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the foregoing) of the present disclosure can be used to treat fibrosis.

[0455] In certain embodiments, the VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) of the present disclosure can be used in immunology.

[0456] In certain non-limiting embodiments, the VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the foregoing) of the present disclosure can be used for lymph node transplantation.

[0457] In certain embodiments, the VEGF-C mutant protein or its functional fragment of the present disclosure (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be used for intestinal health. In certain non-limiting embodiments, the VEGF-C mutant protein or its functional fragment can be used to treat colitis and chronic liver disease.

[0458] In certain embodiments, the VEGF-C mutant protein or its functional fragment (or its fusion protein or conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the foregoing) of the present disclosure can be used in nephrology.

[0459] In certain non-limiting embodiments, the VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the foregoing) of the present disclosure can be used to treat polycystic kidney disease.

[0460] In certain non-limiting embodiments, the VEGF-C mutant protein or its functional fragment of the present disclosure (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be used to treat Alzheimer's disease, Parkinson's disease, stroke, and cerebral ischemia with lung injury. In certain embodiments, the VEGF-C mutant protein or its functional fragment has neuroregenerative properties.

[0461] In certain non-limiting embodiments, the VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the foregoing) of the present disclosure can be used for wound healing.

[0462] In certain non-limiting embodiments, the VEGF-C mutant protein or its functional fragment of the present disclosure (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be used to treat lymphedema. Non-limiting examples of lymphedema include, but are not limited to, primary lymphedema, secondary lymphedema, and hereditary lymphedema.

[0463] In certain embodiments, the VEGF-C mutant protein or its functional fragment of the present disclosure (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be used to treat genetic diseases. Non-limiting examples of such genetic diseases include, but are not limited to, Milroy disease, Hennicum syndrome, and Turner syndrome.

[0464] In certain embodiments, the VEGF-C mutant protein or its functional fragment of the present disclosure (or its fusion protein or conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be used to treat eye diseases. Non-limiting examples of such eye diseases include, but are not limited to, age-related macular degeneration, glaucoma, diabetic retinopathy, central serous chorioretinopathy, macular edema, and retinal edema.

[0465] In certain embodiments, the VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) of the present disclosure can be used in combination with chimeric antigen receptor (CAR) T cells.

[0466] In certain embodiments, the VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) of the present disclosure can be used in combination with a cancer vaccine.

[0467] In some embodiments, the method does not comprise administering a tumor-specific antigen to the subject.

[0468] In certain embodiments, the VEGF-C mutant protein or its functional fragment of the present disclosure (or its fusion protein or conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be used in combination with an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. The immune checkpoint inhibitor can target PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, BTLA, GITR, 4-1BB or Ox-40. The immune checkpoint inhibitor can 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.

[0469] VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate or polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein or vector or particle containing the polynucleotide molecule or pharmaceutical composition containing any of the above) and immunotherapeutic agent can be administered in combination. For example, VEGF-C mutant protein and immunotherapeutic agent are administered in the same composition.

[0470] Alternatively, the VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the foregoing) and the immunotherapeutic agent can be administered sequentially.

[0471] In various embodiments, the VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) is administered before the administration of the immunotherapeutic agent. The VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be administered locally to the brain or central nervous system (e.g., cerebellomedullary cisterna magna), and then the immunotherapeutic agent can be administered systemically (e.g., intravenously). The VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be administered intratumorally, and then the immunotherapeutic agent can be administered systemically (e.g., intravenously). VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be administered intrathecally, and then the immunotherapeutic agent can be administered systemically (e.g., intravenously). VEGF-C mutant protein or its functional fragment (or its fusion protein or its conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be administered directly into the lymphatic system, and then the immunotherapeutic agent can be administered systemically (e.g., intravenously).

[0472] In various embodiments, the method for treating cancer further comprises administering an additional anti-cancer treatment to the subject. Examples of additional anti-cancer treatments include, but are not limited to, surgery, radiotherapy, administration of chemotherapeutic agents, and any combination thereof. These additional anti-cancer treatments may be administered before, simultaneously with, or after the administration of a VEGF-C mutant protein or a functional fragment thereof (or a fusion protein or a conjugate thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the foregoing).

[0473] In various embodiments, the subject is a human patient. The human patient can be a child or an adult.

[0474] In various embodiments, the method can effectively treat cancer in a subject. In some embodiments, the method can effectively induce lymphangiogenesis of tumors in the brain or central nervous system of a subject. In various embodiments, lymphangiogenesis can be confirmed by MRI imaging, for example, where contrast agents can be used to calculate the diameter of the lymphatic vasculature. In various embodiments, lymphangiogenesis can be confirmed by continuously collecting CSF to measure VEGFA, VEGFB, VEGFC or VEGFD concentrations. In various embodiments, lymphangiogenesis can be confirmed by continuously collecting CSF to measure VEGFC concentrations. The method can effectively reduce tumor volume. In some embodiments, the method can effectively reduce tumor volume in the brain or central nervous system of a subject. In various embodiments, the method can effectively provide immune memory against tumors. Without wishing to be bound by theory, the low clinical efficacy of immunotherapy in GBM patients may be due to low antigens collected from the CNS in a stable state and in the early stages of tumor development. Administration of VEGF-C mutant proteins or functional fragments thereof can increase the amount of antigen sampling occurring in the brain, thereby improving the efficacy and results of any other immunotherapy (e.g., anti-CTLA-4 antibodies) administered. Although the role of VEGFC in cancer is thought to be to promote metastasis through (lymph) angiogenesis, the inventors unexpectedly found that in the brain, VEGF-C can reduce tumor size by increasing immune surveillance. VEGF-C can stimulate lymphatic endothelial cell proliferation and enhance lymphatic function through VEGFR-3.

[0475] In one aspect, the present invention provides a method for regulating intraocular pressure in a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutant protein or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof, or a fusion protein thereof, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above, or the 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 comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

[0476] In one aspect, the present invention provides a method for removing unwanted fluid from the eye of a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutant protein of the present disclosure or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof, or a fusion protein thereof, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above, or the 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 comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

[0477] In some embodiments, the unwanted fluid is optic nerve, retinal, subretinal, choroidal, or suprachoroidal fluid.

[0478] In some embodiments, wherein the method of the present disclosure is a method of regulating intraocular pressure in a subject in need thereof or a method of removing unwanted fluid in the eye of a subject in need thereof, the subject having glaucoma, macular edema, central serous chorioretinopathy, retinal edema, papilledema, macular degeneration, or diabetic retinopathy.

[0479] In some embodiments, wherein the method of the present disclosure is a method of regulating intraocular pressure in a subject in need thereof or a method of removing unwanted fluid from the eye of a subject in need thereof, the VEGF-C mutant protein or a functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, carrier, particle or pharmaceutical composition is administered to the back of the eye. In some embodiments, administration is intraocular administration. In some embodiments, intraocular administration is intravitreal, by eye drops or subretinal administration.

[0480] In one aspect, the present invention provides a method for providing neuroprotection in a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutant protein of the present disclosure or its functional fragment or its fusion protein or conjugate, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above, or a 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 comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above. In some embodiments, neuroprotection is provided, but not limited to, for macular degeneration, glaucoma, stroke, Alzheimer's disease or Parkinson's disease. In some embodiments, administration is intraocular administration. In some embodiments, intraocular administration is intravitreal, by eye drops or subretinal administration.

[0481] In one aspect, the present invention provides a vaccine comprising the VEGF-C mutant protein or its functional fragment or its fusion protein or conjugate of the present disclosure, or a polynucleotide molecule encoding the VEGF-C mutant protein or its functional fragment or its fusion protein, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

[0482] In one aspect, the present invention provides a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a vaccine of the present disclosure. In some embodiments, the immune response is, but is not limited to, an anti-cancer mediated immune response, a vaccine mediated immune response, an anti-viral immune response, an anti-bacterial immune response, or any other anti-pathogen immune response. Example

[0483] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, but not to limit, the disclosed embodiments.

[0484] Example 1. Generation of VEGF-C mutant proteins

[0485] Therapeutic stimulation of lymphatic vessel development (lymphangiogenesis) holds promise as an emerging therapeutic paradigm for a wide range of indications, from cardiovascular disease to cancer immunotherapy. However, specific pharmacological agents that promote lymphangiogenesis without stimulating potentially deleterious angiogenesis are currently unavailable. Notably, vascular endothelial growth factor C (VEGF-C), a major physiological driver of lymphangiogenesis, agonizes pro-lymphangiogenic signaling through VEGFR-3 but also signals through pro-angiogenic VEGFR-2. Here, directed evolution using yeast surface display was used to generate a lymphatic vessel-specific VEGF-C variant that binds exclusively to VEGFR-3 but does not signal through VEGFR-2 (LS-VEGF-C). LS-VEGF-C exhibited superior preclinical efficacy in models of lymphangiogenesis and anti-PD-1 cancer immunotherapy compared with wild-type VEGF-C. The biased effects of LS-VEGF-C on lymphangiogenesis versus angiogenesis were further determined by investigating its effects on enhancing retroocular lymphatic drainage. LS-VEGF-C significantly reduced IOP in normotensive mice and two different mouse models of ocular hypertension without increasing angiogenesis and vascular permeability as seen with WT-VEGF-C treatment. LS-VEGF-C also exhibited IOP-independent neuroprotective properties in a model of retinal ganglion cell death caused by excitotoxic injury. Collectively, these results highlight the feasibility and preclinical efficacy of treating lymphangiogenesis with a VEGFR-3-specific agonist and reveal an unexpected role for lymphangiogenesis in controlling IOP.

[0486] Protein expression, purification, and biotinylation

[0487] The extracellular domains of murine 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 pEZT vector with N-terminal H7 signal peptide, C-terminal AviTag and hexa-histidine tag (SEQ ID NO: 25) and expressed by transient transfection of Expi293 cells (ThermoFisher) according to the manufacturer's instructions. Proteins were enriched from cell supernatants by Ni-NTA chelating resin and further purified by size exclusion chromatography (Column SEC650, Bio-rad) into a final buffer of HEPES-buffered saline (HBS; 10 mM HEPES, pH 7.5, 150 mM NaCl). Protein biotinylation was performed at room temperature for 2 hours using in-house purified BirA ligase in 0.1 mM bicine (pH 8.3), 10 mM ATP, 10 mM magnesium acetate and 0.5 mM biotin (Avidity, #BIO500). The biotinylated protein was then purified by gel filtration as described above. The biotinylation efficiency was assessed using an SDS / PAGE streptavidin shift assay.

[0488] Yeast display of VEGF-C

[0489] Murine VEGF-C (amino acids 112-215) was cloned into the C-terminal display vector pCT-GCN42 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. Yeast was maintained and expanded in liquid synthetic glucose medium (SDCAA) with casamino acids at 30°C, and then expression was induced in liquid synthetic glucose medium (SDCAA) with casamino acids at 20°C for 24-48 hours. The displayed protein levels were verified by staining with a C-terminal Myc tag antibody (Cell Signaling Technology, #3739S). Biotinylated VEGFR-2 and VEGFR-3 binding was detected using fluorescent streptavidin-phycoerythrin secondary antibodies and quantified by flow cytometry using a Sony SA3800 flow cytometer.

[0490] Figure 2AMultiple methods for displaying VEGF-C on yeast surfaces were shown, which were used to optimize VEGF-C dimerization on the surface. The resulting combination showed that wild-type VEGF-C had high binding affinity for VEGFR-3 and lower affinity for VEGFR-2. Figure 2B is a schematic diagram showing the selection of a yeast display library for finding VEGFR-3 specific VEGF-C mutant proteins.

[0491] Fourteen residues were identified from the three-dimensional structural analysis of the VEGF-C:VEGFR3 and VEGF-C:VEGFR2 complexes. A library randomizing these residues was constructed using assembly PCR ( Figure 3 The PCR product was further amplified with primers containing homology to the main chain of the yeast display vector to achieve homologous recombination. The transformed yeast was recovered and amplified in SDCAA medium.

[0492] Mutant protein selection

[0493] Experiment was carried out using BiacoreT100 and carried out at 25°C. Protein concentration was quantitatively measured by 280nm absorbance using Nanodrop2000 spectrometer (ThermoScientific). Biotinylated protein VEGFR-2 or VEGFR-3 was fixed on Biacore biotin CAPture sensor chip (Cytiva LifeSciences). Unrelated biotinylated protein was immobilized to serve as a reference surface for nonspecific binding. Single cycle kinetics was used to measure the serial dilutions of WT-VEGF-C, LS-VEGF-C and C125S in Hepes buffer saline-P+ buffer (GE Healthcare). According to the manufacturer's instructions, the reagent provided in the CAPture test kit was used to regenerate VEGFR-2 and VEGFR-3 surfaces. All data were analyzed using BiacoreT100 evaluation software 2.0 with 1:1 Langmuir binding model.

[0494] Murine VEGF-C library construction and selection

[0495] Crystal structures of VEGF-C / VEGFR-2 complex (PDBID2XIW) and VEGF-C / VEGFR-3 (PDBID4BSK) were aligned to analyze VEGF-C and its receptor interface. 14 positions in mVEGF-C that contact mVEGFR-2 and mVEGFR-3 were identified to create a mutant library (Table 1). The library was assembled using synthetic degenerate primers covering these mutant residues (Table 2). The PCR product was further amplified with primers containing homology to the vector and co-electroporated into EBY100 competent yeast with the linearized pCT-GCN42 vector. The resulting library was then measured to contain 10 8 A transformant. Figure 3 Depicted is the design of a VEGF-C mutant protein library. A structure-guided primer library was designed to generate approximately 10 3 The structures of VEGF-C binding to VEGFR-3 and VEGFR-2 were studied to identify key residues on the surface of the binding site. Amino acids that could undergo significant polarization changes among these residues were selected to design primers.

[0496] Table 1. VEGF-C library design.

[0497] Table 2. VEGF-C library primers

[0498] The transformed yeast were recovered and amplified in SDCAA medium at 30°C and induced in SGCAA medium at 20°C for 24-48 hours. The naive library was selected with 2 rounds of 1 μM mVEGFR-3 to enrich for mVEGFR-3 positive binders using LS columns (Miltenyi, #130-042-401) and magnetic selection. Starting from round 3, flow cytometric sorting was performed using a Sony SH800 cell sorter, and the yeast population was counter-selected with 1 μM mVEGFR-2 monomer and selected with 100 nM mVEGFR-3. For rounds 4-6, the counter-selection reagent mVEGFR-2 concentration was gradually increased to 0.25 μM (round 4) and 0.5 μM (rounds 5 and 6) tetramers, while the mVEGFR-3 concentration was reduced to 10 nM (round 4), 2 nM (round 5) or 1 nM (round 6). Yeast after each round of selection is retained and strengthened at the same time after all selections are completed to check their binding affinity to VEGFR-2 and VEGFR-3. After the last round of selection, VEGF-C plasmids were extracted from the amplified SDCAA cultured yeast and transformed into Escherichia coli for cloning and sequencing (Table 3). The polymerized unique clones were re-transformed into yeast to titrate their binding preferences for VEGFR-2 and VEGFR-3. The VEGF-C mutants finally selected were expressed by the insect cell pFastbac expression system and purified in FPLC with a SEC column. Finally, the monodispersed protein was clarified by removing endotoxins for in vivo animal treatment. Figure 4 As shown, wild-type VEGF-C shows binding to yeast expressing VEGFR-2 and VEGFR-3 (upper row, WT). The first two rounds of selection were for binding to VEGFR-3. In subsequent rounds, reduced amounts of VEGFR-3 were used to select for mutant proteins with high binding affinity to VEGFR-3. At the same time, VEGFR-2 was included and negatively selected to enrich for mutant proteins that lost binding affinity to VEGFR-2 but had high binding to VEGFR-3. Figure 5 Depicted is the confirmation of receptor affinity preferences after the final rounds of selection. Mutant proteins selected as positive for VEGFR-3 and negative for VEGFR-2 showed high binding to VEGFR-3 and no binding to VEGFR-2, even at receptor concentrations 100-fold higher than wild-type binding. Figure 6 Depicted is the confirmation of receptor affinity preferences after the last few rounds of selection. Clonal mutant proteins after rounds 5 and 6 of selection were sequenced to identify unique clones enriched in the mutant protein population. Unique mutant protein residues were identified to generate specific mutations in wild-type VEGF-C for validation.

[0499] Figure 7Depicted is the confirmation of receptor affinity preferences after the last few rounds of selection. Clonal mutant proteins after rounds 5 and 6 of selection were sequenced to identify unique clones enriched in the mutant protein population. Unique mutant protein residues were identified to generate specific mutations in wild-type VEGF-C for validation.

[0500] Figure 8 Depicted is the confirmation of receptor affinity preference after the final round of selection against the human VEGF receptor. A mutant protein selected positively for VEGFR-3 and negatively for VEGFR-2 showed high binding to VEGFR-3 and no binding to VEGFR-2, even at receptor concentrations 100-fold higher than wild-type binding.

[0501] Table 3. Results of the last round of sequencing of VEGF-C.

[0502] Engineered Lymphatic Specific VEGF-C (LS-VEGF-C)

[0503] Increased lymphatic drainage demonstrates therapeutic benefit in pathologies such as lymphedema, where fluid accumulation due to disruption of the lymphatic infrastructure is directly addressed. 2 From a more immunological perspective, exploitation of meningeal lymphatics could increase brain antigen drainage via lymphangiogenesis and promote an enhanced immune response to glioblastoma. 3 These applications demonstrate not only the therapeutic potential of harnessing lymphangiogenesis but also its diversity of application, even within the immune-privileged space.

[0504] Previous studies have shown that VEGFR-2 mainly promotes angiogenesis 4 , while VEGFR-3 promotes lymphangiogenesis 5-8 Vascular endothelial growth factor C (VEGF-C) is a major growth factor that promotes lymphatic vessel growth and pumping; however, its pleiotropic effects (binding to VEGFR-2 and VEGFR-3) Fig.19 ) limits its use as a therapy and as a molecular tool to study in vivo signaling. In addition, the ability of VEGF-C to promote adequate lymphangiogenesis is variable, and high doses are often required to produce significant effects. 9 ; Altogether, these shortcomings hinder the full realization of the therapeutic potential of VEGF-C-driven lymphangiogenesis.

[0505] To overcome these limitations, attempts were made to engineer a more selective but more potent VEGF-C mutant protein. To develop a mutant VEGF-C that only binds to VEGFR-3 and promotes lymphangiogenesis, 14 residues on the receptor binding interface were identified by structural analysis and randomized ( Figure 3 ). Using the yeast display directed evolution platform ( Figure 2B ), a library was created that was subjected to six rounds of selection with selectivity for VEGFR-3 and counter-selection for VEGFR-2 ( Figure 4 After this final selection, a panel of variants showed conservative, exclusive binding to VEGFR-3, but not to humans ( Figure 8 ) and the mouse version of the receptor ( Fig.15A and 15B ) showed no binding to VEGFR-2. Notably, the VEGFR-3 binding of these variants was superior to the VEGFR-3 binding of the previously established VEGFR-3 specific ligand VEGF-C152S / C-156S ( Fig.12 After inspection of the clones, seven key residues 115, 119, 126, 163, 164, 186, and 188 were identified, leading to our final variant (LS-VEGF-C); biophysical characterization by surface plasmon resonance showed that LS-VEGF-C retained picomolar affinity for VEGFR-3 while completely inhibiting VEGFR-2 affinity compared to WT-VEGF-C ( Figure 6 and 7 ).

[0506] To assess whether functionality was preserved, HUVEC and HDLEC cells, which are vascular endothelial cells and lymphatic endothelial cells, respectively, were used. Figure 1B )) After that, downstream signaling was examined by measuring ERK activation in cells. HUVECs with VEGFR-2 expression showed similar enhanced ERK activation by VEGF-A and WT-VEGF-C but not LS-VEGF-C ( Fig. 15C ). In contrast, HDLECs showed similar ERK phosphorylation between WT-VEGF-C and LS-VEGF-C ( Fig. 15C), which reflects the conserved VEGFR-3 binding and signaling in the mutant proteins. Investigation of cell proliferation after VEGF administration confirmed this trend; vascular endothelial cells had increased proliferation in response to VEGF-A and WT-VEGF-C, while lymphatic endothelial cells had increased proliferation in response to any VEGF, but was most statistically significant in response to LS-VEGF-C ( Fig.15D Since lymphatic endothelial cells display both VEGFR-2 and VEGFR-3, it is not surprising that VEGF-A and WT-VEGF-C also lead to increased proliferation of lymphatic endothelial cells. 5 Together, these data suggest that LS-VEGF-C is a novel mutant form of VEGF-C that is highly specific for VEGFR-3 binding and lymphangiogenesis.

[0507] mRNA / Nanoparticles

[0508] VEGF-C wild-type and mutant protein encoding mRNAs were synthesized by TriLink BioTechnologies with complete substitution of pseudouridine and 5-methylcytosine bases, capped using CleanCapreagent AG and polyadenylated (120A). mRNA was mixed at a ratio of 1 ug per 0.1 μL in vivo JETPEI (Polyplus Transfection), vortexed for 30 seconds, and incubated at room temperature for 15 minutes before use.

[0509] cell

[0510] Human umbilical vascular endothelial cells (HUVEC) and human dermal lymphatic endothelial cells (HDLEC) were obtained from (Promocell). They were cultured in MV medium (Promocell) containing supplements (0.05 mL fetal bovine serum / mL, 0.004 mL endothelial cell growth supplement / mL, 10 ng recombinant human EGF / mL, 90 ug heparin / mL, 1 ug hydrocortisone / mL).

[0511] Western blotting

[0512] HEK293T cells were transfected with VEGFCmRNA constructs combined with liposomes. Supernatants were removed from these cells and incubated with HUVEC, HDLEC-j, or HDLEC-a cells. In other experiments, VEGF-C or VEGF-A proteins were added directly to the culture medium and placed on top of the cells. The samples were dissolved in RIPA buffer and boiled with sample buffer for 5 minutes. In other experiments, HUVEC and HDLEC cells were treated with 100ng / mL VEGF-A, WT-VEGF-C, or LS-VEGF-C in RPMI medium containing 1% FBS supplement. The samples were trypsinized (0.05%) and quenched with RPMI medium (1% FBS supplement). The supernatant was aspirated, the cell pellet was resuspended, then dissolved in RIPA buffer, and then boiled with sample buffer for 5 minutes.

[0513] Western blotting was performed in a similar manner as previously reported 3 . Briefly, 10% gels were used, each gel was run at 10 mA for 30 minutes, and each gel was run at 40 mA until the molecular weight standards were properly separated. Wet transfer was performed at 120 mA for 90 minutes on ice for each gel. After blocking with milk-TBST and washing 3 times with TBST, anti-pERK was used at a concentration of 1:1000 and incubated overnight in the cold room. After washing, HRP-conjugated anti-rabbit secondary antibodies were used at a concentration of 1:500 for 2 hours at room temperature and imaged using the ChemiDocMP imaging system (Bio-Rad).

[0514] Mouse

[0515] Six to ten-week-old mixed-sex C57BL / 6J (WT), DBA2J, and DBA / 2J-Gpnmb+ / SjJ mice were purchased from the Jackson Laboratory and subsequently bred and maintained at Yale University. All procedures used in this study (sex-matched, age-matched) were in accordance with U.S. federal guidelines and the institutional policies of the Yale School of Medicine Animal Care and Use Committee.

[0516] MTT assay

[0517] A 96-well plate was used and 10,000 cells were inoculated per well. The cells were first starved for 12 hours in RPMI medium without supplements, then washed with PBS and then subjected to experimental conditions. The negative control was treated with RPMI medium containing 1% FBS supplement. The experimental conditions included adding 100ng / mL VEGF-A, WT-VEGF-C or LS-VEGF-C to the negative control medium (RPMI medium containing 1% FBS supplement). A standard curve was made by cell titration. Then 10uL 12mMMTT stock solution was added to each sample. After homogenizing each well by pipetting up and down, the absorbance was measured at 570nm, and the cell count was determined by matching with the standard curve.

[0518] Endothelial cell isolation

[0519] The cornea, retina, choroid, and optic nerve were isolated for single cell dissociation. These tissues were mixed with 1 mg / ml −1 Collagenase D (Roche) and 30 μg ml −1 DnaseI (Sigma-Aldrich) was digested at 37°C for 45 minutes. The samples were then pipetted up and down to mechanically dissociate the tissue and filtered through a 70 micron filter. The samples were then spun down at 500xg for 5 minutes. The cell pellets were then resuspended in FACS buffer (PBS containing 2% FBS and 1 mM EDTA) for staining.

[0520] Flow cytometry

[0521] Prior to immunostaining, nonspecific binding was blocked using Fc receptor blocking solution (TruStain FcX™, 101320, BioLegend) at 4°C for 10 minutes. Subsequently, cells were stained with the corresponding antibodies at 4°C for 30 minutes. The cells were then washed to remove excess antibodies and resuspended in FACS buffer. Samples were run on an Attune NxT flow cytometer and then analyzed using FlowJo software (10.8.1, TreeStar).

[0522] For AKT phosphorylation staining, surface markers were first stained on ice for 30 minutes. Cells were then fixed and stained according to the instructions of the BD Phosflow kit. Samples were run on an Attune NxT flow cytometer and analyzed using FlowJo software (10.8.1, TreeStar).

[0523] Intraocular pressure measurement

[0524] IOP measurements followed consensus recommendations (https: / / iovs.arvojournals.org / article.aspx?articleid=2778419). Mice were briefly exposed to isoflurane for sedation, after which IOP was measured with an iCare tonometer. IOP measurements were the average of 6 measurements during one sedation session. Mice were consistently positioned so that the probe was perpendicular to the surface of the eyeball during measurement.

[0525] Apply to eyes

[0526] Intraperitoneal injection of ketamine (50 mg / kg −1 ) and xylazine (5mgkg −1 ) mixture, the mice then received topical, intravitreal or intracameral administration. Topical administration involves placing 5uL of the 1ug / uL solution on the eye. Intravitreal and intracameral administration involve a small puncture along the edge of the cornea for entry of the Hamilton syringe. For intracameral administration, the needle enters the anterior chamber. For intravitreal administration, the needle enters the vitreous humor space. Both intravitreal and intracameral administration involve 1ug / uL solution. After application, the eye is covered with artificial tear ointment. Any eye not evaluated is also covered with artificial tear ointment to avoid dryness.

[0527] Combination therapy

[0528] After IVT administration of LS-VEGF-C, two days later, ketamine (25 mg kg −1 ) and xylazine (2.5 mg / kg −1 The mice were anesthetized with a mixture of 1% dapoxetine and 1% dapoxetine. Subsequently, the eyes received a topical application of a US FDA-approved drug to evaluate the combination effect.

[0529] Microbead Model

[0530] Adapted from Sappington et al. 18 Ketamine (50 mg / kg) was injected intraperitoneally −1 ) and xylazine (5mgkg −1 ) were anesthetized and received intracameral administration of polystyrene beads. Artificial tear ointment was then applied topically to avoid corneal drying.

[0531] Intravitreal AAV injection and imaging

[0532] WT mice were intravitreally injected with AAV (dose) containing PBS, VEGF-A (concentration), WT-VEGF-C (concentration), or LS-VEGF-C (concentration). One day later, ketamine (50 mg kg −1 ) and xylazine (5 mg kg−1 ) mixture to anesthetize mice. 100uL of dye at a concentration of 20mg / mL was also injected intraperitoneally. Eyes were dilated with 1% tropicamide. After incubation for 5 minutes, mice were placed on a scaffold for fundus examination, fluorescein angiography, and OCT imaging on Phoenix Micron IV.

[0533] Absorbance measurement of Evans blue

[0534] For Evans blue readout, Evans blue was injected intraperitoneally instead of the dye. After 4 hours, the mice were euthanized and perfused with PBS. The eyes were then isolated and homogenized with beads and then run on a microplate reader (Abs_max at 620 nm).

[0535] Tissue processing and microscopy

[0536] For retinal wholemount, the mouse eyeballs were first removed and then fixed in 1% formaldehyde. After carefully removing the optic nerve, cornea, and sclera, the isolated retina was cut into four parts, with the cut located halfway along the optic nerve. After staining with Brn3a and DAPI, confocal imaging was performed on a Leica SP8 microscope.

[0537] For cross sections of the optic nerve, the optic nerve was fixed in 4% and then processed and embedded in a resin / OTC / paraffin mixture. The block was then sectioned on a microtome / cryostat. The cross sections were then visualized on a transmission electron microscope.

[0538] NMDA stimulation studies

[0539] Adapted from Schlüter et al. 31 , 10 nmol NMDA was delivered intravitreally, and RGCs were assessed by confocal microscopy of retinal whole mounts on day 1 after injection, as described above.

[0540] Statistical analysis

[0541] All statistical analyses were performed using GraphPad Prism software. Data were analyzed using two-tailed unpaired Student's t-test or paired Student's t-test using Prism software. Statistical significance was defined as *P<0.05, **P<0.01, and ***P<0.001.

[0542] Intravitreal injection

[0543] Mice were anesthetized using ketamine and xylazine. One drop of 0.5% tropicamide was applied to the eye. The mouse was positioned to expose the superior nasal region of the eye, and a 33 g needle was used to puncture the superior nasal sclera at the pars plana. The mouse head was fixed and the needle was placed at a 45 degree angle. After the needle tip was inserted, 2 μL of the mRNA-nanoparticle formulation was injected and the needle was left in for 5 seconds to prevent reflux. Antimicrobial eye ointment was then applied to prevent infection, and the mouse was placed in a heated cage until fully recovered. Fig.10 Depicted is the use of VEGF-C mutant proteins in vivo. A single intravitreal administration of VEGF-C mutant proteins resulted in a sustained decrease in intraocular pressure (IOP) in wild-type mice compared to counterpart mice.

[0544] Tumor inoculation

[0545] Ketamine (50 mg / kg) was injected intraperitoneally −1 ) and xylazine (5mgkg −1 ) mixture to anesthetize the mice. The mice were shaved and then placed in a stereotaxic frame. After disinfecting the scalp with alcohol and betadine, an incision was made in the midline of the scalp to expose the coronal and sagittal sutures, and a skull burr hole was made 2 mm lateral to the sagittal suture and 0.5 mm posterior to the bregma. A 10 μl Hamilton syringe containing tumor cells was inserted into the skull burr hole 2.5 mm deep from the brain surface and kept balanced for 1 min before infusion. A microinfusion pump (World Precision Instruments) was used to infuse the cells at a rate of 1 μl min −1 3 μl of tumor cells were infused. After the infusion was completed, the syringe was left in place for another minute and then removed. The skull burr hole was filled with bone wax and fixed with nails and the skin was cleaned. Analgesics (meloxicam and buprenorphine, 1 mg / kg) were administered intramuscularly. −1 ), the mice were placed in a heated cage until fully recovered. Fig.11 Describes the use of VEGF-C mutant proteins in vivo. Wild-type VEGF-C and VEGF-C mutant proteins were evaluated in vivo for the treatment of brain tumors. In combination with anti-PD-1 antibodies, VEGF-C mutant proteins showed significant therapeutic benefit in the treatment of brain tumors. In addition, as Fig.12 As shown in the literature, the VEGF-C mutants reported in the literature lose their binding affinity to both VEGFR-2 and VEGFR-3. Mutants such as C152S (previously reported in the literature) lose their activity to both VEGFR-2 and VEGFR-3, thereby losing their powerful lymphangiogenic activity. Fig.13As shown, the isolated VEGF-C mutant proteins of the present disclosure show specific signaling through VEGFR-3 in vivo. Wild-type VEGF-C and VEGF-C mutant protein (RTI) signaling were evaluated in vivo. These vectors were injected into the eyes of mice. After 24 hours, the eyeballs were removed and single cell suspensions were made to examine AKT phosphorylation. Wild-type VEGF-C retained signaling through VEGFR-2 in blood endothelial cells, while mutant VEGF-C (RTI) no longer had signaling. Both the wild-type protein and the RTI mutant protein signaled through VEGFR-3 in lymphatic endothelial cells. In addition, as Fig.14 As shown, the route of administration results in a differential decrease in IOP in vivo. VEGF-C or VEGF-C mutant proteins (RTI) can be administered by eye drops, injection into the anterior chamber (AC), or intravitreal administration. In the AC, both wild-type and mutant proteins have similar activities, while when administered by eye drops or intravitreal administration, the mutant proteins exhibit the ability to selectively reduce IOP.

[0546] Intracisternal injection

[0547] For intracisternal injection, mice were anesthetized with ketamine and xylazine, and the nape of the neck was shaved and washed with alcohol. A 2 cm incision was made at the base of the skull, and the nape of the neck muscles were separated using forceps. After visualization of the cisterna magna, the dura mater was punctured using a Hamilton syringe with a 15-gauge, 33-gauge needle. The injection was performed at 1 μl / min. −1 Each mouse was injected with 3 microliters of mRNA vector (4-5 μg) at a rate of 100 μg / cm2. After the injection was completed, the needle was left in the body for another 3 minutes to prevent backflow. The skin was fixed and cleaned with nails, and the same postoperative procedures as tumor inoculation were performed.

[0548] Example 2. LS-VEGF-C exhibits potent therapeutic effects in a variety of settings.

[0549] To confirm that LS-VEGF-C acts through VEGFR-3 signaling in vivo, LS-VEGF-C and WT-VEGF-C were first administered to wild-type mice, and then endothelial cells (CD45 - CD31 + ) activation of downstream signaling. Regardless of VEGFR-3 expression, WT-VEGF-C showed a significant increase in AKT phosphorylation in endothelial cells ( Fig.13 In contrast, LS-VEGF-C was not expressed in cells expressing VEGFR-3 (CD45 - CD31 + VEGFR3 +) induced significantly higher AKT phosphorylation, whereas in cells not expressing VEGFR-3 (CD45 - CD31 + VEGFR3 - ), indicating that the in vivo activity of LS-VEGF-C reflects the activity observed in vitro.

[0550] Since its discovery, the lymphatic regulatory potential of VEGF-C has been demonstrated in vivo in a number of pathological processes. Here, we tested the ability of LS-VEGF-C to provide therapeutic lymphangiogenesis in several of these models. First, we induced a mouse hindlimb lymphedema model using local lymph node ablation performed by surgical ablation. 10 LS-VEGF-C administration was more effective in reducing swelling than WT-VEGF-C, and the degree of swelling reduction was comparable to that in normal conditions ( Fig.16A , 16B , 20A, and 20B). Not only was it effective as a single agent in this setting after a single dose, but the molecule also showed sustained activity after multiple administrations, highlighting its potential as a chronic disease therapeutic. Second, LS-VEGF-C was evaluated as an adjuvant to anti-PD-1 cancer immunotherapy in a mouse melanoma model. LS-VEGF-C showed superior preclinical efficacy to WT-VEGF-C in terms of both tumor volume reduction and survival ( Fig. 16C , 16D , 20C, and 20D). Finally, LS-VEGF-C was used as an adjunct to checkpoint inhibitor therapy for the treatment of glioblastoma. LS-VEGF-C exhibited activity comparable to WT-VEGF-C, resulting in survival in the majority of treated animals, which otherwise resulted in lethal outcomes ( Fig.20E and 20F ). These experimental groups confirm the conservation of the biological function of LS-VEGF-C in controlling biophysical properties and immunological outcomes, while demonstrating its superiority as an in vivo pharmacological agent over the wild-type growth factor.

[0551] Example 3. Biophysical modulation using ocular lymphatic vessels.

[0552] Previous studies have shown that the eye has a lymphatic vasculature that drains the anterior chamber; the conjunctiva has been described previously. 11 and ciliary body 12 The lymphatic vessels in the , and the post-glial lymphatic clearance system. 13 Our group recently discovered that ocular lymphatics are compartmentalized and well-established, and that the anterior and posterior parts of the eye have distinct lymphatic drainage systems. By expanding these lymphatic structures through lymphangiogenesis, we hypothesized that ocular administration of LS-VEGF-C would lead to increased drainage and reduced intraocular pressure in the eye.

[0553] WT-VEGF-C was injected intracamerally (similar to previous reports 14 ) and resulted in a decrease in intraocular pressure (IOP) measurements after intravitreal injection ( Fig.17A In contrast, LS-VEGF-C resulted in a sustained and enhanced reduction in IOP following any method of administration, whether topical, intracameral, or intravitreal ( Fig. 17B The greater impact and versatility of LS-VEGF-C may stem from its VEGFR-3 specificity, whereas WT-VEGF-C has two possible receptors to bind, namely VEGFR-2, which acts as a VEGF-C sink. 15 This may also contribute to the lack of phenotypic effects of WT-VEGF-C in eye drops, as well as the hindrance to rapid clearance of many topical applications and the multiple ocular barriers that need to be overcome. 16 Interestingly, in mRNA form, WT-VEGF-C failed to reduce IOP when administered topically or intravitreally, but performed similarly to the more potent LS-VEGF-C when administered intracamerally ( Fig.21A , highlighting the importance of delivery method and vehicle). Importantly, LS-VEGF-C demonstrated a durable IOP-lowering effect, with IOP reductions observed within hours and lasting for days, compared to commonly used glaucoma eye drops which only showed activity for a few hours ( Fig. 21B and 21C LS-VEGF-C is significantly more active as a single agent than other glaucoma drugs and does not require combination therapy to achieve a stable IOP reduction ( Fig.21D The efficacy of LS-VEGF-C in the normotensive IOP model demonstrates the translation of our in vitro findings into a physiological phenomenon.

[0554] Example 4. LS-VEGF-C reduces vascular side effects in vivo.

[0555] To evaluate the off-target effects of VEGF growth factors and vascular changes, the retinal vasculature was imaged after intravitreal injection of VEGF-A, WT-VEGF-C, and LS-VEGF-C. Fundus photographs showed hyperreflective spots and increased vascular tortuosity ( Fig. 17C Left column). Fluorescein angiography showed enhanced visibility of retinal parenchymal permeability similar to the anatomical vascular findings. VEGF-A and WT-VEGF-C resulted in increased dye presence in the extravascular space ( Fig. 17C middle column), which can be quantified by measuring the fluorescence intensity of the entire image ( Fig.17DIn the control and LS-VEGF-C administered retinas, no intensities exceeding 40 were observed except overlying major blood vessels, whereas in the VEGF-A and WT-VEGF-C administered retinas, large patches showed intensities exceeding 40. Optical coherence tomography (OCT) showed findings consistent with angiography, with pockets of fluid adjacent to the choroid / retinal pigment epithelium layer in eyes administered with VEGF-A or WT-VEGF-C, but not in eyes administered with LS-VEGF-C ( Fig. 17C Right column). As a parallel approach to verify these findings, retinal vascular permeability was assessed by measuring Evans blue absorbance after systemic injection of Evans blue and perfusion of mice with PBS. Similar to the angiographic data, these data indicate that LS-VEGF-C is able to mitigate increased vascular permeability, revealing an intact, healthy retinal vasculature ( Fig.17E On the front side of the eye, VEGF-A causes uncontrolled angiogenesis in the iris and cornea as well as corneal opacities, indicative of edema or fluid leakage into the anterior chamber ( Fig. 22B WT-VEGF-C resulted in similar corneal opacity but without significant neovascularization, whereas LS-VEGF-C showed physical findings similar to controls ( Fig. 22C and 22D LS-VEGF-C not only demonstrates its utility by being shown to mitigate unwanted vascularity, its characterization as a novel molecular tool also elucidates the contrasting effects of VEGFR-2 and VEGFR-3 signaling in vivo.

[0556] Example 5. LS-VEGF-C provides neuroprotection in ocular hypertension.

[0557] After demonstrating IOP-lowering efficacy in wild-type mice, we examined whether LS-VEGF-C could provide therapeutic benefit in a glaucoma model. Glaucoma is characterized as a neurodegenerative disease caused by the death of retinal ganglion cells (RGCs), often associated with elevated intraocular pressure. 17 First, a microbead model was used, which blocks the intraocular outflow pathway and induces an IOP increase in a short period of time. 18 , reminiscent of acute angle-closure glaucoma ( Fig.18A). After the beads were applied to the anterior chamber, the mice experienced an average increase of 20 mmHg in IOP. LS-VEGF-C was administered via two different routes to assess its efficacy. Intravitreal (IVT) administration of LS-VEGF-C was able to restore elevated IOP to baseline levels on average. A closer look revealed that the effect was bimodal, with mice experiencing either a 10 mmHg decrease in IOP, similar to values ​​seen in wild-type mice treated with LS-VEGF-C, or mice not responding well and continued elevated IOP. Of note, topical application of LS-VEGF-C had limited benefit. A decrease in IOP was observed in the mice, but this decrease was not sustained and never fell below their baseline measurements ( Fig.18B ). RGC imaging (indicated by Brn3a staining) showed that LS-VEGF-C provided neuroprotection to RGCs. Under induced glaucoma conditions, RGCs were significantly reduced, which was improved after intravitreal administration of LS-VEGF-C ( Fig.18C Although eye drop administration as a group did not appear to have a statistically significant effect, a bimodal effect was observed in the retina, with Brn3a cell numbers similar to controls and other cell numbers similar to eyes with glaucomatous ( Fig.18D ).

[0558] To further evaluate the effect of LS-VEGF-C on glaucoma, we utilized the spontaneously glaucomatous mouse, the DBA2J pigment dispersion model. 19 As a negative control to establish a baseline, the D2-Gpnmb+ model was used, which has a normally functioning Gpnmb gene and therefore no iris pigment dispersion that leads to spontaneous glaucoma. Although the mechanical injury to the anterior chamber is similar to that of microbead injection, this model presents a more chronic disease state, requiring a lead time of 6-8 months to show significantly elevated IOP ( Fig.18E After screening for 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 ( Fig.18F ). RGC axons in the optic nerves of these mice were evaluated and the results showed that LS-VEGF-C was able to maintain axonal integrity in this glaucoma model. Interestingly, while IVT administration showed a significant decrease in pressure and corresponding optic nerve axonal health, topical administration did not show significant preservation of axonal integrity despite a similar decrease in IOP ( Figure 18G and 18H ).

[0559] Although the exact mechanism of neuronal death in ocular neurodegenerative diseases remains unclear, unifying theories include the accumulation of neurotoxic metabolites or excitatory neuronal signaling. 20,21Given that VEGF-C is associated with lymphatic drainage, which allows clearance of metabolites and macromolecules from organs 22,23 We hypothesize that, in addition to its ability to lower IOP, LS-VEGF-C may have neuroprotective properties in glaucoma. Intravitreal injection of NMDA resulted in widespread RGC death, whereas simultaneous intravitreal administration of LS-VEGF-C prevented this ( Fig.23A and 23B Together, these results suggest that LS-VEGF-C is able to provide neuroprotection in several glaucoma models through multimodal mechanisms that target IOP and neurotoxin clearance.

[0560] Example 6. Human LS-VEGF-C screening.

[0561] Yeast display of LS-hVEGF-C variants: Candidate LS-VEGF-C sequences (SEQ ID: 1-50 and 100) and WThVEGF-C were synthesized by TwistBiosciences and inserted into the yeast display system as described above. All clones contained the C137A mutation to enhance biochemical stability (independent of VEGFR3 bias). After induction of LS-hVEGF-C expression, yeast strains were stained with 500nM biotinylated hVEGFR2, 100nM biotinylated hVEGFR3, or 10nM VEGFR3 for one hour at 4˚C. The yeast were then washed with FACS buffer (PBS + 0.5% BSA + 0.5mM EDTA) and stained with a fluorescent streptavidin secondary antibody. Receptor binding was then quantified by flow cytometry. For Fig. 27 In the binding titration study in , yeast were incubated with a range of 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 was detected as described above. Table 4 shows the human clones tested. Table 5 shows the selected human clones.

[0562] Table 4. Human VEGF-C clones tested.

[0563] Table 5. Selected human VEGF-C clones

[0564] Example 7. Characterization of human LS-VEGF-C-Fc.

[0565] Human VEGFC mutant proteins fused to human IgG1Fc (N297Q) were transiently expressed in Expi293 cells using the Expifectamine293 transfection kit (Thermo-Fisher Scientific) according to the manufacturer's instructions. The expression supernatant was then subjected to Ni-NTA chromatography and protein yield was quantified in mg / L (Table 6). The quality was then assessed by size exclusion chromatography using a Superose 6 column (GE Healthcare) ( Figures 28A-28M ).

[0566] Table 6. Expression of human VEGFC muteins fused to human IgG1 Fc.

[0567] Example 8. VEGFR3 expression in lymphatic vessels may be a source of mediating the reduction of intraocular pressure.

[0568] VEGFR3fl / fl mice were bred with Cdh5aCRE, NESTINCRE and PROX1CRE (all ERT2, tamoxifen-induced) mice to generate tamoxifen-induced conditional knockout mice. In the control group, mice received corn oil only, and in the other groups, mice received tamoxifen every day for 7 days. Then, all mice were injected with beads into the anterior chamber to produce ocular hypertension. After confirming the increase in intraocular pressure (baseline), all mice received intravitreal RTI-Fc treatment and the intraocular pressure was measured 7 days later.

[0569] Fig.29 The data presented in show that VEGFR3 expression in prox1- and cdh5a-positive cells leads to a decrease in IOP, but not in nestin-positive 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 suggests that VEGFR3 expression in lymphatic vessels may be the source of the IOP-mediating decrease, indicating that RTI activity is particularly important in lymphatic endothelial cells.

[0570] Example 9. Evaluation of VEGF-C mutein conjugates for intraocular pressure reduction.

[0571] Wild-type mice were treated intravitreally with LS-VEGF-C as monomer, albumin conjugate, or Fc conjugate, and the reduction in IOP was assessed.

[0572] The monomer had little effect, while the albumin conjugate and the Fc conjugate had similar peak reductions in IOP, with the Fc conjugate showing an advantage over time ( Fig.30 ).

[0573] discuss

[0574] This article provides the creation of a lymphatic vessel-specific VEGF-C that provides a new molecular tool for studying lymphatic biology. Although dysregulation of the lymphatic vasculature has been implicated in many disease processes, the translation of these findings into the clinic has been limited by the lack of methods to specifically stimulate the lymphatic system. Previous efforts identified mutant proteins that lost VEGFR-2 binding, but also significantly lost binding to VEGFR-3. 24-26 To address these clinical hurdles, we created a library of VEGF-C mutant proteins to generate LS-VEGF-C proteins that have picomolar binding to VEGFR-3 but not to VEGFR-2. By exploiting this novel property, we highlight for the first time the potential of this novel molecule to treat a variety of diseases known to harbor lymphatic dysfunction. By completely abrogating the angiogenic activity, the activity of LS-VEGF-C could be further extended to new disease spaces. We demonstrate the superior efficacy of LS-VEGF-C as a therapy for glaucoma, a disease with clearly identifiable biophysical dysfunction that could benefit from lymphatic therapy, excluding WT-VEGF-C as a possible candidate due to the risk of ocular angiogenesis. Not only does LS-VEGF-C exhibit robust in vivo activity consistent with its binding properties, it also does so without causing angiogenesis or vascular permeability.

[0575] This is not the first time that a lymphatic-based therapy has been proposed for glaucoma. The trabecular meshwork and Schlemm's canals display features of lymphatic vasculature and have been identified as areas that can be stimulated by lymphatic signaling. 14,27,28 This concept has also been applied to mapping aqueous humor in NHPs and humans. 29 In addition, the uveoscleral lymphatic pathway is also believed to be involved in the drainage of aqueous humor. 12 . As mentioned above, the focus of IOP reduction has been specifically on the anterior chamber, with all medications and surgical procedures targeting components of the anterior eye. However, glaucoma pathology occurs in the posterior eye, with optic nerve damage being the primary sign of progression. Here we decided that focusing on IOP reduction in this compartment would provide a unique approach to complement current therapeutic strategies. As a therapeutic molecule, LS-VEGF-C demonstrated a potent IOP-lowering effect in two models of ocular hypertension, resulting in significant preservation of neuronal health. In addition to this, we demonstrated that stimulation of the optic nerve glymphatic system can drain neurotoxic molecules out of the eye, thereby providing neuroprotection.

[0576] Most importantly, LS-VEGF-C is not only a new agent, but also a tool to answer questions about lymphoid biology. Previous reports have attempted to stimulate VEGFR-3 specifically and concluded that without VEGFR-2 signaling, the effects were not as strong. 30 . In contrast, LS-VEGF-C exhibited stronger phenotypic differences compared to WT-VEGF-C in vivo. This is likely due to a combination of factors including 1) without VEGFR-2 binding, VEGF-C is not sequestered from its lymphatic binding partner VEGFR-3, and 2) LS-VEGF-C retains picomolar binding affinity for VEGFR-3. This makes our mutant protein not only an excellent candidate for clinical relevance requiring lymphatic stimulation, but also a unique tool to separate VEGFR-2 (angiogenesis) and VEGFR-3 (lymphangiogenesis) signaling. It is conceivable that it will be useful in diseases with distinct indications such as lymphedema 2 The latest discoveries on neurodegenerative diseases 22,23 ) has the potential to have a significant impact on human health and disease. In summary, the results to date from the development of VEGFR-3-specific ligands establish the therapeutic potential and versatility of lymphangiogenesis, even in immune-privileged spaces.

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[0609] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention other than those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. These modifications are intended to fall within the scope of the appended claims. It should also be understood that all values ​​are approximate and are for description only.

[0610] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. An isolated vascular endothelial growth factor C (VEGF-C) mutant protein or a functional fragment thereof, wherein compared with the wild-type VEGF-C protein from the same species, the VEGF-C mutant protein or the functional fragment thereof has reduced or no ability to stimulate the proliferation of blood endothelial cells, but retains the ability to stimulate the proliferation of lymphatic endothelial cells.

2. An isolated vascular endothelial growth factor C (VEGF-C) mutant protein or a functional fragment thereof, wherein the VEGF-C mutant protein or a functional fragment thereof (i) has a reduced binding affinity to vascular endothelial growth factor receptor-2 (VEGFR-2) compared to a wild-type VEGF-C protein from the same species, (ii) has the ability to bind to vascular endothelial growth factor receptor-3 (VEGFR-3) and generate signal transduction through it, and (iii) comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of a wild-type VEGF-C protein from the same species.

3. The VEGF-C mutant protein or a functional fragment thereof according to claim 1 or claim 2, wherein the VEGF-C mutant protein or a functional fragment thereof produces reduced or no signal transduction through VEGFR-2 compared to the wild-type VEGF-C protein from the same species.

4. The VEGF-C mutant protein or a functional fragment thereof according to claim 3, wherein the VEGF-C mutant protein or a functional fragment thereof does not produce signal transduction through VEGFR-2.

5. The VEGF-C mutant protein or functional fragment thereof according to claim 3 or claim 4, wherein the signal transduction through VEGFR-2 is determined by measuring the VEGFR-2-dependent AKT-phosphorylation and / or ERK-phosphorylation levels in blood endothelial cells, by a wound healing assay (scratch assay), by a proliferation assay or by an angiogenesis assay.

6. The VEGF-C mutant protein or functional fragment thereof according to any one of claims 2 to 5, wherein signaling through VEGFR-3 is determined by measuring VEGFR-3-dependent AKT-phosphorylation and / or ERK-phosphorylation levels in lymphatic endothelial cells, by a wound healing assay, by a proliferation assay or by an angiogenesis assay. 7 . The VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 6 , wherein the VEGF-C mutant protein or a functional fragment thereof does not induce angiogenesis.

8. The VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 7, wherein the VEGF-C mutant protein is a mutant protein of the wild-type VEGF-C protein comprising amino acids 111-211 of SEQ ID NO: 4, or a mutant protein of a polypeptide defined by corresponding positions within the wild-type VEGF-C protein of another species.

9. The VEGF-C mutant protein or a functional fragment thereof according to claim 8, wherein the wild-type VEGF-C protein comprises amino acids 111-211 of SEQ ID NO:

4.

10. The VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 9, wherein the VEGF-C mutant protein or a functional fragment thereof comprises one or more mutations selected from mutations at residues T112, L115, D119, Q126, T144, N145, K149, N163, S164, E165, I184, V186, L188 and P192, wherein the positions of the residues are defined relative to SEQ ID NO: 4, or comprises one or more mutations selected from mutations at corresponding residues in a wild-type VEGF-C protein of another species.

11. The VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 10, wherein the VEGF-C mutant protein or a functional fragment thereof further comprises a mutation at residue C133, wherein the position of the residue is defined relative to SEQ ID NO: 4, or comprises a mutation at the corresponding residue in a wild-type VEGF-C protein of another species.

12. The VEGF-C mutant protein or a functional fragment thereof according to claim 11, wherein the mutation at residue C133 is a C133A mutation.

13. The VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 7, wherein the VEGF-C mutant protein is a mutant protein of the wild-type VEGF-C protein comprising amino acids 115-215 of SEQ ID NO: 1, or a mutant protein of a polypeptide defined at corresponding positions in a wild-type VEGF-C protein of another species.

14. The VEGF-C mutant protein or a functional fragment thereof according to claim 13, wherein the wild-type VEGF-C protein comprises amino acids 115-215 of SEQ ID NO:

1.

15. The VEGF-C mutant protein or functional fragment thereof according to any one of claims 1 to 7 and 13 to 14, wherein the VEGF-C mutant protein or functional fragment thereof comprises one or more mutations selected from mutations at residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, I188, V190, L192 and P196, wherein the positions of the residues are defined relative to SEQ ID NO:

1.

16. The VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 7 and 13 to 15, wherein the VEGF-C mutant protein or a functional fragment thereof further comprises a mutation at residue C137, wherein the position of the residue is defined relative to SEQ ID NO: 1, or comprises a mutation at the corresponding residue in the wild-type VEGF-C protein of another species.

17. The VEGF-C mutant protein or a functional fragment thereof according to claim 16, wherein the mutation at residue C137 is a C137A mutation.

18. The VEGF-C mutant protein or functional fragment thereof according to any one of claims 15 to 17, wherein the mutation at residue L119 is L119E mutation or L119M mutation; the mutation at residue D123 is D123N mutation; the mutation at residue Q130 is Q130K mutation; the mutation at residue N167 is N167R mutation, N167I mutation, N167Q mutation or N167H mutation; the mutation at residue S168 is S168G mutation or S168R mutation; the mutation at residue V190 is V190T mutation; and / or the mutation at residue L192 is L192I mutation.

19. The VEGF-C mutant protein or a functional fragment thereof according to any one of claims 15 to 18, wherein the VEGF-C mutant protein or a functional fragment thereof comprises one or more mutations selected from mutations at residues N167, S168 and / or L192.

20. The VEGF-C mutant protein or a functional fragment thereof according to claim 19, wherein the mutation at residue N167 is N167I mutation, N167Q mutation or N167H mutation; the mutation at residue S168 is S168G mutation or S168R mutation; and / or the mutation at residue L192 is L192I mutation.

21. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation.

22. The VEGF-C mutant protein of claim 21, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 160 or SEQ ID NO:

56.

23. The VEGF-C mutant protein of claim 22, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 160 or SEQ ID NO:

56.

24. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation and an S168G mutation.

25. The VEGF-C mutant protein of claim 24, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO:

57.

26. The VEGF-C mutant protein of claim 25, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO:

57.

27. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation and an L192I mutation.

28. The VEGF-C mutant protein of claim 27, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO:

58.

29. The VEGF-C mutant protein of claim 28, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO:

58.

30. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an N167Q mutation, an S168G mutation and an L192I mutation.

31. The VEGF-C mutant protein of claim 30, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 163 or SEQ ID NO:

59.

32. The VEGF-C mutant protein of claim 31, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 163 or SEQ ID NO:

59.

33. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation.

34. The VEGF-C mutant protein of claim 33, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 166 or SEQ ID NO:

62.

35. The VEGF-C mutant protein of claim 34, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 166 or SEQ ID NO:

62.

36. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation and an S168G mutation.

37. The VEGF-C mutant protein of claim 36, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 167 or SEQ ID NO:

63.

38. The VEGF-C mutant protein of claim 37, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 167 or SEQ ID NO:

63.

39. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation and an L192I mutation.

40. The VEGF-C mutant protein of claim 39, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 168 or SEQ ID NO:

64.

41. The VEGF-C mutant protein of claim 40, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 168 or SEQ ID NO:

64.

42. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation, an S168G mutation and an L192I mutation.

43. The VEGF-C mutant protein of claim 42, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 169 or SEQ ID NO:

65.

44. The VEGF-C mutant protein of claim 43, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 169 or SEQ ID NO:

65. The VEGF-C mutant protein or the functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or the functional fragment thereof comprises an S168G mutation.

46. ​​The VEGF-C mutant protein of claim 45, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO:

68.

47. The VEGF-C mutant protein of claim 46, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO:

68. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an S168G mutation and an L192I mutation.

49. The VEGF-C mutant protein of claim 48, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO:

70.

50. The VEGF-C mutant protein of claim 49, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO:

70.

51. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an N167H mutation.

52. The VEGF-C mutant protein of claim 51, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO:

80.

53. The VEGF-C mutant protein of claim 52, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO:

80.

54. The VEGF-C mutant protein or a functional fragment thereof according to claim 20, wherein the VEGF-C mutant protein or a functional fragment thereof comprises an N167I mutation and an S168R mutation.

55. The VEGF-C mutant protein of claim 54, wherein the VEGF-C mutant protein comprises the amino acid sequence of SEQ ID NO: 192 or SEQ ID NO:

88.

56. The VEGF-C mutant protein of claim 55, wherein the VEGF-C mutant protein consists of the amino acid sequence of SEQ ID NO: 192 or SEQ ID NO:

88.

57. A fusion protein or conjugate comprising the VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 56, wherein the mutant protein or the functional fragment thereof is fused and / or conjugated to one or more heterologous parts.

58. The fusion protein or conjugate of claim 57, wherein the one or more heterologous moieties are selected from immunoglobulins or functional fragments thereof, albumin or functional fragments thereof, albumin binding antibodies or functional fragments thereof, and polyethylene glycol (PEG) polymers.

59. The fusion protein or conjugate of claim 58, wherein the immunoglobulin or functional fragment thereof comprises an IgG Fc domain.

60. The fusion protein of claim 59, wherein the IgG Fc domain is modified to reduce Fc effector function.

61. The fusion protein of claim 60, wherein the IgG Fc domain comprises a mutation at residue N297.

62. The fusion protein of claim 61, wherein the mutation at residue N297 is selected from the group consisting of N297Q, N297A and N297G.

63. An isolated polynucleotide molecule encoding the VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 56 or the fusion protein or conjugate according to any one of claims 57 to 62.

64. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 210 or SEQ ID NO:

108.

65. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 211 or SEQ ID NO:

109.

66. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 212 or SEQ ID NO:

110.

67. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 213 or SEQ ID NO:

111.

68. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 216 or SEQ ID NO:

114.

69. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 217 or SEQ ID NO:

115.

70. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 218 or SEQ ID NO:

116.

71. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 219 or SEQ ID NO:

117.

72. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 222 or SEQ ID NO:

120.

73. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 224 or SEQ ID NO:

122.

74. The polynucleotide molecule 63 of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 234 or SEQ ID NO:

132.

75. The polynucleotide molecule of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 242 or SEQ ID NO:

140.

76. The polynucleotide molecule of any one of claims 63-75, wherein the polynucleotide molecule comprises a nucleotide sequence encoding the VEGF-C mutant protein or a functional fragment thereof, and the nucleotide sequence is operably linked to a promoter.

77. The polynucleotide molecule of any one of claims 63-75, wherein the polynucleotide molecule is mRNA.

78. The polynucleotide molecule of any one of claims 63-77, wherein the polynucleotide molecule comprises one or more nucleotide modifications.

79. The polynucleotide molecule of claim 78, wherein the one or more nucleotide modifications are a 5' cap, 5-methylcytosine or pseudouridine.

80. A vector comprising the polynucleotide molecule of any one of claims 63-79.

81. The vector of claim 80, wherein the vector is a viral vector.

82. The vector of claim 81, wherein the viral vector is derived from a herpes virus, a cytomegalovirus, a poliovirus, an alphavirus, a vaccinia virus, a rabies virus, an adeno-associated virus (AAV), a retrovirus, a lentivirus, or an adenovirus.

83. A particle comprising the polynucleotide molecule of any one of claims 63-79.

84. The particle of claim 83, wherein the particle is a nanoparticle, a lipid particle, a microparticle, a lipid nanoparticle, a polymer particle, or a virus-like particle (VLP).

85. A host cell comprising the polynucleotide of any one of claims 63-79 or the vector of any one of claims 80-82.

86. A method for producing a VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof, comprising culturing the host cell of claim 85 under conditions for expressing the VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof.

87. A VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof, which is produced by the method of claim 86.

88. A kit comprising the VEGF-C mutant protein or functional fragment thereof according to any one of claims 1 to 56 or the fusion protein or conjugate according to any one of claims 57 to 62, and optionally instructions for use.

89. A kit comprising the polynucleotide of any one of claims 63-79 or the vector of any one of claims 80-82 or the particle of claim 83 or claim 84, and optionally instructions for use.

90. A pharmaceutical composition comprising a VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 56, a fusion protein or a conjugate according to any one of claims 57 to 62, a polynucleotide molecule according to any one of claims 63 to 79, a vector according to any one of claims 80 to 82, or particles according to claim 83 or claim 84, and a pharmaceutically acceptable carrier or diluent.

91. The pharmaceutical composition of claim 90, wherein the composition comprises mRNA encoding a VEGF-C mutant protein or a functional fragment thereof or a fusion protein thereof as an mRNA-nanoparticle formulation.

92. The pharmaceutical composition of claim 90 or 91, further comprising an immunotherapeutic agent.

93. The pharmaceutical composition of claim 92, wherein the immunotherapeutic agent is an immune checkpoint inhibitor.

94. The pharmaceutical composition of claim 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.

95. The pharmaceutical composition of claim 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-BLTA antibody, an anti-GITR antibody, an anti-4-IBB antibody or an anti-Ox-40 antibody.

96. The pharmaceutical composition of any one of claims 90-95, wherein the composition is formulated for intrathecal administration.

97. The pharmaceutical composition of any one of claims 90-95, wherein the composition is formulated for intratumoral administration.

98. The pharmaceutical composition of any one of claims 90-95, wherein the composition is formulated for systemic administration.

99. The pharmaceutical composition of any one of claims 90-95, wherein the composition is formulated for intracisternal administration.

100. The pharmaceutical composition of any one of claims 90-95, wherein the composition is formulated for eye drop administration.

101. The pharmaceutical composition of any one of claims 90-95, wherein the composition is formulated for intraocular administration.

102. A method for inducing lymphangiogenesis in a subject in need thereof, the method comprising administering to the subject an effective amount of a VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 56, or a fusion protein or conjugate according to any one of claims 57 to 62, or a polynucleotide molecule according to any one of claims 63 to 79, or a vector according to any one of claims 80 to 82, or a particle according to claim 83 or claim 84, or a pharmaceutical composition according to any one of claims 90 to 101.

103. The method of claim 102, wherein administration of the VEGF-C mutant protein or a functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle or pharmaceutical composition does not cause one or more side effects associated with administration of a wild-type VEGF-C protein or a corresponding fusion protein, conjugate, polynucleotide molecule, vector, particle or pharmaceutical composition.

104. The method of claim 103, wherein the one or more side effects are angiogenesis and / or increased intraocular pressure (IOP).

105. The method of any one of claims 102-104, wherein the VEGF-C mutant protein or a functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisternalis, intravitreally, by eye drops, subcutaneously, intradermally, by inhalation, by a long-dwelling catheter, orally, topically or systemically.

106. The method of any one of claims 102-104, wherein the VEGF-C mutant protein or a functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle or pharmaceutical composition thereof is administered to the cisterna magna or directly to the lymphatic system.

107. The method of any one of claims 102-107, wherein the subject has a disease or condition selected from the group consisting of cancer, coronary vascular function, osmotic regulation, cardiac ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, a disease or condition associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennicham syndrome, Milroy disease, Turner syndrome, age-related macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, and retinal edema.

108. The method of claim 107, wherein the disease or condition associated with lymph node transplantation is breast cancer-related lymphedema, idiopathic lymphedema, and / or heart failure-related lymphedema.

109. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a VEGF-C mutant protein or functional fragment thereof according to any one of claims 1 to 56, or a fusion protein or conjugate according to any one of claims 57 to 62, or a polynucleotide molecule according to any one of claims 63 to 79, or a vector according to any one of claims 80 to 82, or a particle according to claim 83 or claim 84, or a pharmaceutical composition according to any one of claims 90 to 101.

110. The method of claim 109, wherein the disease or condition is cancer, coronary vascular function, osmotic regulation, cardiac ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, a disease or condition associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennicham syndrome, Milroy disease, Turner syndrome, age-related macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, and retinal edema.

111. The method of claim 110, wherein 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, liquid cancer, kidney cancer, bladder urothelial carcinoma, prostate cancer, pheochromocytoma, bile duct cancer, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, thymoma, sarcoma, mesothelioma, testicular cancer, or colorectal cancer.

112. The method of claim 110, wherein the cancer is located in the brain or central nervous system of the subject.

113. The method of claim 110, wherein the cancer is selected from the group consisting of glioma, ependymoma, subependymoma, primitive neuroectodermal tumor, ganglioglioma, schwannoma, germ cell tumor, craniopharyngioma, meningioma, CNS lymphoma, pineal tumor, retinoblastoma, uveal melanoma, and rhabdoid tumor.

114. The method of any one of claims 109-113, further comprising administering an immunotherapeutic agent.

115. The method of claim 114, wherein the immunotherapeutic agent is an immune checkpoint inhibitor.

116. The method of claim 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.

117. The method of claim 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-BLTA antibody, an anti-GITR antibody, an anti-4-IBB antibody, or an anti-Ox-40 antibody.

118. The method of any one of claims 109-117, wherein the VEGF-C mutant protein or a functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisternalis, intravitreally, by eye drops, subcutaneously, intradermally, by inhalation, by a long-dwelling catheter, orally, topically or systemically.

119. The method of any one of claims 109-117, wherein the VEGF-C mutant protein or a functional fragment, fusion protein, conjugate, polynucleotide molecule, vector, particle or pharmaceutical composition thereof is administered to the cisterna magna or directly to the lymphatic system.

120. The method of any one of claims 109-119, wherein the disease is cancer, and wherein the method further comprises administering to the subject an additional anti-cancer therapy.

121. The method of claim 120, wherein the additional anti-cancer treatment is selected from the group consisting of surgery, radiation therapy, administration of a chemotherapeutic agent, immunotherapy, and any combination thereof.

122. A method for regulating intraocular pressure in a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 56, or a fusion protein or conjugate according to any one of claims 57 to 62, or a polynucleotide molecule according to any one of claims 63 to 79, or a vector according to any one of claims 80 to 82, or a particle according to claim 83 or claim 84, or a pharmaceutical composition according to any one of claims 90 to 101, 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 comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

123. The method of claim 122, wherein the VEGF-C mutant 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 the polynucleotide molecule encoding the wild-type VEGF-C protein or its functional fragment or the vector or particle comprising the polynucleotide molecule or the pharmaceutical composition comprising any of the above is administered to the posterior part of the eye.

124. The method of any one of claims 122-123, wherein the administration is intraocular administration.

125. The method of claim 124, wherein the intraocular administration is intravitreal, by eye drops, or subretinal.

126. A method for removing unwanted fluid from the eye of a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 56, or a fusion protein or conjugate according to any one of claims 57 to 62, or a polynucleotide molecule according to any one of claims 63 to 79, or a vector according to any one of claims 80 to 82, or a particle according to claim 83 or claim 84, or a pharmaceutical composition according to any one of claims 90 to 101, or a corresponding wild-type VEGF-C protein or a functional fragment thereof, or a fusion protein or conjugate, or a polynucleotide molecule encoding the wild-type VEGF-C protein or a functional fragment thereof, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

127. The method of claim 126, wherein the unwanted fluid is optic nerve, retinal, subretinal, choroidal, or suprachoroidal fluid.

128. The method of any one of claims 122-127, wherein the subject suffers from glaucoma, macular edema, central serous chorioretinopathy, retinal edema, papilledema, macular degeneration, or diabetic retinopathy.

129. The method of any one of claims 124-126, wherein the administration is intraocular administration.

130. The method of claim 127, wherein the intraocular administration is intravitreal, by eye drops, or subretinal.

131. A method for providing neuroprotection to a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutant protein or a functional fragment thereof according to any one of claims 1 to 56, or a fusion protein or conjugate according to any one of claims 57 to 62, or a polynucleotide molecule according to any one of claims 63 to 79, or a vector according to any one of claims 80 to 82, or a particle according to claim 83 or claim 84, or a pharmaceutical composition according to any one of claims 90 to 101, or a corresponding wild-type VEGF-C protein or a functional fragment thereof, or a fusion protein or a conjugate thereof, or a polynucleotide molecule encoding the wild-type VEGF-C protein or a functional fragment thereof, or a vector or particle comprising the polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

132. A vaccine comprising the VEGF-C mutant protein or functional fragment thereof according to any one of claims 1 to 56, or the fusion protein or conjugate according to any one of claims 57 to 62, or the polynucleotide molecule according to any one of claims 63 to 79, or the vector according to any one of claims 80 to 82, or the particles according to claim 83 or claim 84, or the pharmaceutical composition according to any one of claims 90 to 101.

133. A method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of the vaccine of claim 132.

134. The method of any one of claims 102-133, wherein the subject is a human.

135. A method for generating a library of VEGF-C mutant proteins that selectively bind to VEGFR-3, wherein the amino acid residues comprising the common binding interface on the VEGF-C protein that binds to both VEGFR-3 and VEGFR-2 are diversified to other amino acids by mutagenesis of the corresponding nucleic acid sequence.

136. A yeast cell library for selecting VEGF-C mutant proteins, comprising a plurality of yeast cells, wherein the yeast cells comprise a cell wall peptide anchor sequence, a connecting peptide and a VEGF-C mutant protein sequence.

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