VEGF-C muteins for selective lymphatic stimulation

Abstract

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

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 317,614, filed March 8, 2022, which is incorporated by reference in its entirety for all purposes. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. CA239444 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention. SEQUENCE LISTING

[0003] This application contains a Sequence Listing, which has been submitted electronically in XML file format and is incorporated by reference in its entirety into this application. The XML copy created on March 8, 2023 has the filename 251609_000089_SL.xml and is 347,984 bytes in size. [Technical field]

[0004] The present invention generally relates to vascular endothelial growth factor C (VEGF-C) muteins that selectively bind to vascular endothelial growth factor receptor-3 (VEGFR-3) over 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 to the subject an effective amount of a VEGF-C mutein or a functional fragment thereof. The present invention also relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a VEGF-C mutein or a functional fragment thereof. [Background technology]

[0005] Immune surveillance against pathogens and tumors in the central nervous system is thought to be limited by a lack of lymphatic drainage. It has been shown that by manipulating the meningeal lymphatic system, a stronger immune response can be generated against brain tumors. Immunity to glioblastoma antigens mediated by CD8 T cells is severely limited when tumors are confined to the central nervous system, leading to failure to control tumor growth. However, ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining deep cervical lymph nodes, migration of CD8 T cells into tumors, rapid clearance of glioblastomas, and long-lasting antitumor memory responses. Furthermore, transfection of mRNA constructs expressing VEGF-C acts synergistically with checkpoint blockade therapy to eradicate existing glioblastomas. Summary of the Invention [Problem to be solved by the invention]

[0006] Vascular endothelial growth factor C (VEGF-C) binds to vascular endothelial growth factor receptor-2 (VEGFR-2), which promotes blood vessel growth (angiogenesis) and regulates vascular permeability, and to vascular endothelial growth factor receptor-3 (VEGFR-3), which promotes lymphatic vessel growth (lymphangiogenesis). VEGF-C acts primarily through VEGFR-3 on lymphatic endothelial cells (LECs) to promote their survival, growth, and migration. Angiogenesis is essential for the growth of both primary tumors and metastases. There is a need to reduce undesirable angiogenesis while promoting lymphangiogenesis. [Means for solving the problem]

[0007] Various non-limiting aspects and embodiments of the present invention will now be described.

[0008] In one aspect, provided herein is an isolated vascular endothelial growth factor C (VEGF-C) mutein protein or functional fragment thereof, wherein said VEGF-C mutein protein or functional fragment thereof has reduced or no ability to stimulate vascular endothelial cell proliferation compared to a wild-type VEGF-C protein of the same species but which retains the ability to stimulate lymphatic endothelial cell proliferation.

[0009] In one aspect, provided herein is an isolated vascular endothelial growth factor C (VEGF-C) mutein protein or functional fragment thereof, wherein said VEGF-C mutein protein or functional fragment thereof (i) has 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 signaling through VEGFR-3, and (iii) comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of said wild-type VEGF-C protein from the same species.

[0010] In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof produces reduced or no signaling through VEGFR-2 compared to the wild-type VEGF-C protein from the same species. In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof does not produce signaling through VEGFR-2. In some embodiments of any of the above methods, the signaling through VEGFR-2 is determined by measuring VEGFR-2-dependent AKT phosphorylation and / or ERK phosphorylation levels in vascular endothelial cells by a wound healing assay, a proliferation assay, or an angiogenesis assay. In some embodiments of any of the above methods, the 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 (scratch assay), a proliferation assay, or an angiogenesis assay.

[0011] In some embodiments of any of the above-described methods, the VEGF-C mutein protein or functional fragment thereof does not induce angiogenesis.

[0012] In some embodiments of any of the above methods, the VEGF-C mutein protein is a mutein of a wild-type VEGF-C protein that includes amino acids 111-211 of SEQ ID NO:4, or a polypeptide defined by the corresponding positions in another wild-type VEGF-C protein. In some embodiments of any of the above methods, the wild-type VEGF-C protein includes amino acids 111-211 of SEQ ID NO:4.

[0013] In some embodiments of any of the above-mentioned methods, the VEGF-C mutein protein or functional fragment thereof comprises one or more mutations selected from: a mutation at residues T112, L115, D119, Q126, T144, N145, K149, N163, S164, E165, 1184, V186, L188, and P192, wherein said positions of said residues are defined with reference to SEQ ID NO:4; or a mutation at a corresponding residue in another wild-type VEGF-C protein; or one or more mutations at residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, 1188, V190, L192, and P196, wherein said positions of said residues are defined with reference to SEQ ID NO:1.

[0014] In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof further comprises a mutation at residue C133, said position of said residue being defined with reference to SEQ ID NO: 4, or a mutation at the corresponding residue in another wild-type VEGF-C protein. In some embodiments, the mutation at residue C133 is a C133A mutation.

[0015] In some embodiments of any of the above-described methods, the VEGF-C mutein protein is a mutein of a wild-type VEGF-C protein that includes amino acids 115-215 of SEQ ID NO:1, or a polypeptide defined by the corresponding positions in another species of the wild-type VEGF-C protein. In some embodiments, the wild-type VEGF-C protein includes amino acids 115-215 of SEQ ID NO:1.

[0016] In some embodiments of any of the above-described methods, the VEGF-C mutein 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, the above positions of the above residues being defined in relation to SEQ ID NO:1.

[0017] In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof further comprises a mutation at residue C137, said position of said residue being defined with reference to SEQ ID NO: 1, or a mutation at the corresponding residue in said wild-type VEGF-C protein of another species. In some embodiments, said mutation at residue C137 is a C137A mutation.

[0018] In some embodiments of any of the above-mentioned 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.

[0019] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises one or more mutations selected from mutations at residues N167, S168, and / or L192.

[0020] In some embodiments of any of the aforementioned 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.

[0021] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises an N167Q mutation.

[0022] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:160 or SEQ ID NO:56.

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

[0024] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises an N167Q mutation and an S168G mutation.

[0025] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:161 or SEQ ID NO:57.

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

[0027] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises an N167Q mutation and an L192I mutation.

[0028] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:162 or SEQ ID NO:58.

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

[0030] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises an N167Q mutation, an S168G mutation, and an L192I mutation.

[0031] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:163 or SEQ ID NO:59.

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

[0033] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises an N167I mutation.

[0034] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:166 or SEQ ID NO:62.

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

[0036] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises an N167I mutation and an S168G mutation.

[0037] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:167 or SEQ ID NO:63.

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

[0039] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises an N167I mutation and an L192I mutation.

[0040] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:168 or SEQ ID NO:64.

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

[0042] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises an N167I mutation, an S168G mutation, and an L192I mutation.

[0043] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:169 or SEQ ID NO:65.

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

[0045] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises a S168G mutation.

[0046] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:172 or SEQ ID NO:68.

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

[0048] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises a S168G mutation and a L192I mutation.

[0049] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:174 or SEQ ID NO:70.

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

[0051] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises an N167H mutation.

[0052] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:184 or SEQ ID NO:80.

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

[0054] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein or functional fragment thereof comprises an N167I mutation and an S168R mutation.

[0055] In some embodiments of any of the aforementioned methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO:192 or SEQ ID NO:88.

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

[0057] In one aspect, provided herein is a fusion protein or conjugate comprising a VEGF-C mutein protein or functional fragment thereof disclosed herein, wherein the mutein protein or functional fragment thereof is fused and / or conjugated to one or more heterologous moieties.

[0058] In some embodiments, the one or more heterologous moieties are selected from an immunoglobulin or a functional fragment thereof, an albumin or a functional fragment thereof, an albumin-binding antibody or a functional fragment thereof, and a polyethylene glycol (PEG) polymer.

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

[0060] In one aspect, provided herein is an isolated polynucleotide molecule encoding a VEGF-C mutein protein or functional fragment thereof of the present disclosure, or a fusion protein of the present disclosure.

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

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

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

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

[0065] In some embodiments, the vector is a viral vector, hi some embodiments, 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.

[0066] In one aspect, provided herein is a particle comprising a polynucleotide molecule of the present disclosure. In some embodiments, the particle is a nanoparticle, a lipid particle, a microparticle, a lipid nanoparticle, a polymeric particle, or a virus-like particle (VLP).

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

[0068] In one aspect, provided herein is a method for producing a VEGF-C mutein protein or a functional fragment thereof, or a fusion protein thereof, comprising culturing a host cell of the present disclosure under conditions in which the VEGF-C mutein protein or a functional fragment thereof, or a fusion protein thereof is expressed.

[0069] In one aspect, provided herein is a VEGF-C mutein protein, or a functional fragment thereof, or a fusion protein thereof, produced by the methods of the disclosure.

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

[0071] In one aspect, provided herein is a kit comprising a polynucleotide of the present disclosure, or a vector of the present disclosure, or a particle of the present disclosure, and optionally, instructions for use.

[0072] In one aspect, provided herein is a pharmaceutical composition comprising a VEGF-C mutein protein or functional fragment thereof of the present disclosure, 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 pharma- ceutically acceptable carrier or diluent.

[0073] In some embodiments, the composition comprises an mRNA encoding the VEGF-C mutein protein or a functional fragment thereof, or a fusion protein thereof, as an mRNA nanoparticle formulation.

[0074] In some embodiments, the pharmaceutical composition further comprises an 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-BTLA antibody, an anti-GITR antibody, an anti-4-IBB antibody, or an anti-Ox-40 antibody.

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

[0076] In one 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 mutein protein or functional fragment thereof of the present disclosure, 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.

[0077] In some embodiments of any of the above methods, the administration of the VEGF-C mutein protein or 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, hi some embodiments, the one or more side effects are neovascularization and / or elevated intraocular pressure (IOP).

[0078] In some embodiments of any of the above-described methods, the VEGF-C mutein protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisternally, intravitreally, ophthalmically, subcutaneously, intradermally, by inhalation, via a chronic indwelling catheter, orally, topically, or systemically.

[0079] In some embodiments of any of the above-described methods, the VEGF-C mutein protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered into the cisterna magna or directly into the lymphatic system.

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

[0081] In some embodiments of any of the aforementioned methods, the disease or condition associated with lymph node transplantation is breast cancer-associated lymphedema, idiopathic lymphedema, and / or heart failure-associated lymphedema.

[0082] In one aspect, provided herein is a method of treating a disease or condition in a subject in need of such treatment, comprising administering to the subject an effective amount of a VEGF-C mutein protein or functional fragment thereof of the present disclosure, 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.

[0083] In some embodiments, where the methods provided herein are methods of treating a disease or condition in a subject in need of such treatment, the disease or condition is cancer, coronary vascular function, osmoregulation, cardiac ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, diseases or conditions associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennekam's syndrome, Milroy's disease, Turner's 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, squamous cell carcinoma of the head and neck, thyroid cancer, liquid cancer, kidney cancer, urothelial cancer, prostate cancer, pheochromocytoma, cholangiocarcinoma, hepatocellular carcinoma, invasive pancreatic ductal carcinoma, thymoma, sarcoma, mesothelioma, testicular cancer, or colorectal cancer. In some embodiments, the cancer is 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, germinoma, craniopharyngioma, meningioma, CNS lymphoma, pineal tumor, retinoblastoma, uveal malignant melanoma, and rhabdoid tumor.

[0084] In some embodiments, the method of treating a disease or condition in a subject in need thereof further comprises 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-BTLA antibody, an anti-GITR antibody, an anti-4-IBB antibody, or an anti-Ox-40 antibody. In some embodiments, the VEGF-C mutein protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisternally, intravitreally, ophthalmically, subcutaneously, intradermally, by inhalation, via a chronic indwelling catheter, orally, topically, or systemically. In some embodiments, the VEGF-C mutein protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered into the cisterna magna or directly into the lymphatic system.

[0085] In some embodiments where the methods provided herein are methods of treating a disease or condition in a subject in need thereof and the disease is cancer, the method further comprises administering to the subject an additional anti-cancer treatment, in some embodiments, the additional anti-cancer treatment is selected from surgery, radiation therapy, administration of a chemotherapeutic agent, immunotherapy, and any combination thereof.

[0086] In one aspect, provided herein is a method of modulating intraocular pressure in a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutein protein or functional fragment thereof, fusion protein or conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition of the present disclosure, or a corresponding wild-type VEGF-C protein or functional fragment thereof, fusion protein or conjugate thereof, or a polynucleotide molecule encoding said wild-type VEGF-C protein or functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above. In some embodiments, the VEGF-C mutein protein or the corresponding wild-type VEGF-C protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition, or the corresponding wild-type VEGF-C protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition, or the corresponding wild-type VEGF-C protein or functional fragment thereof, fusion protein or conjugate thereof, or a polynucleotide molecule encoding said wild-type VEGF-C protein or functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above, is administered to the posterior segment of the eye. In some embodiments, the administration is intraocular administration, hi some embodiments, the intraocular administration is intravitreal administration, eye drop administration, or subretinal administration.

[0087] In one aspect, provided herein is a method of removing unwanted fluid in the eye of a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutein protein or functional fragment thereof, a fusion protein or conjugate, a polynucleotide molecule, a vector, a particle, or a pharmaceutical composition of the present disclosure, or a corresponding wild-type VEGF-C protein or functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said wild-type VEGF-C protein or functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above. In some embodiments, the unwanted fluid is optic nerve fluid, retinal fluid, subretinal fluid, choroidal fluid, 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, the administration is intraocular. In some embodiments, the intraocular administration is intravitreal administration, eye drop administration, or subretinal administration.

[0088] In one aspect, provided herein is a method of providing neuroprotection to a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutein protein or functional fragment thereof, fusion protein or conjugate, polynucleotide molecule, or vector, particle, or pharmaceutical composition of the present disclosure, or a corresponding wild-type VEGF-C protein or functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said wild-type VEGF-C protein or functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

[0089] In one aspect, provided herein is a vaccine comprising a VEGF-C mutein protein or a functional fragment thereof, a fusion protein or conjugate, a polynucleotide molecule, a vector, a particle, or a pharmaceutical composition of the present disclosure.

[0090] 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.

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

[0092] In a further aspect, provided herein is a method for generating a library of VEGF-C muteins that selectively bind to VEGFR-3, in which amino acid residues that constitute the covalent binding interface on VEGF-C that binds to both VEGFR-3 and VEGFR-2 are diversified to other amino acids by mutagenesis of the corresponding nucleic acid sequence.

[0093] In a further aspect, provided herein is a yeast cell library for selection of a VEGF-C mutein protein, the yeast cell library including a plurality of yeast cells comprising a cell wall peptide anchor sequence, a linker peptide, and a sequence of the VEGF-C mutein.

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

[0095] [Figure 1A] 1A-1B provide an overview of the pleiotropism of VEGF-C: Figure 1A is a schematic diagram showing that VEGF-C binds to both VEGFR-2 and VEGFR-3, which promote angiogenesis and lymphangiogenesis, respectively. [Figure 1B] FIG. 1B is a schematic diagram of engineered VEGF-C muteins that lose binding to VEGFR-2 and exhibit specific binding to VEGFR-3. [Figure 2A]Figures 2A-2B show VEGF-C display constructs for dimerization and screening. Figure 2A shows multiple methods for displaying VEGF-C on the yeast surface that were utilized to optimize dimerization of VEGF-C on the surface. The final combination displayed wild-type VEGF-C with high binding affinity to VEGFR-3 and low affinity to VEGFR-2. [Figure 2B] FIG. 2B is a schematic showing yeast display library selection directed to the discovery of VEGFR-3-specific VEGF-C muteins. [Diagram 3] FIG. 3 shows the design of the VEGF-C mutein library. A structure-based primer library was designed to generate approximately 103 VEGF-C mutein combinations. The structure of VEGF-C bound to VEGFR-3 and VEGFR-2 was studied to identify key residues on the surface of the binding site. Amino acids that could potentially cause significant polarity changes at these residues were selected for primer design. The figure shows SEQ ID NOs: 27-34 and 34-39, respectively, in order of appearance. [Figure 4] FIG. 4 shows that VEGF-C muteins were positively selected for VEGFR-3 binding and negatively selected for VEGFR-2 binding. Wild-type VEGF-C showed binding to yeast cells expressing both VEGFR-2 and VEGFR-3 (top row, WT). The first two rounds selected for binding to VEGFR-3. Subsequent rounds exploited the reduced amount of VEGFR-3 to select for muteins with high binding affinity to VEGFR-3. Concurrently, VEGFR-2 was included and negatively selected to enrich for muteins with high binding affinity to VEGFR-3 but lost binding affinity to VEGFR-2. [Diagram 5]Figure 5 shows confirmation of receptor affinity preferences after the final selection round. Muteins positively selected for VEGFR-3 and negatively selected for VEGFR-2 show high binding to VEGFR-3 and no binding to VEGFR-2, even at 100-fold higher receptor concentrations compared to wild-type binding. [Figure 6-1] Figure 6 shows confirmation of receptor affinity preferences after the final selection round. Clonal muteins after selection rounds 5 and 6 were sequenced to identify unique clones enriched within the mutein population. Unique mutein residues were identified and specific mutations were made in wild-type VEGF-C for validation. [Figure 6-2] Continued from Figure 6-1. [Figure 7] Figure 7 shows an overview of the sequence, mutation rate, and affinity of the selected mouse clones and the final LS-VEGF-C. The positions and IDs of important residues are listed in the first two columns. The shaded columns indicate mutations at contact positions, all of which facilitate the final mutations in WT-VEGF-C that create LS-VEGF-C. Affinities for VEGFR-2 and VEGFR-3 were determined by SPR for WT-VEGF-C and LS-VEGF-C. [Figure 8] FIG. 8 shows VEGFR-2 and VEGFR-3 affinity of WT VEGF-C and after round 6 of selection. [Figure 9]Figure 9 shows that isolated VEGF-C muteins showed specific signaling through VEGFR-3 in vitro. Signaling of VEGF-A, wild-type VEGF-C, and VEGF-C muteins (163I and RTI) was evaluated in vitro using human umbilical vein endothelial cells (HUVEC) and human dermal lymphatic endothelial cells (young HDLEC-j and adult HDLEC-a). HUVEC specifically express VEGFR-2, while HDLEC express both VEGFR-2 and VEGFR-3, allowing for evaluation of signaling of each receptor. Cells were stimulated with each protein, and protein lysates were collected and subjected to Western blot to detect ERK phosphorylation (downstream of VEGFR-2 and VEGFR-3). [Figure 10] Figure 10 shows the use of VEGF-C muteins in vivo. A single intravitreal administration of VEGF-C muteins sustainedly reduced intraocular pressure (IOP) in wild-type mice compared to their counterparts. [Figure 11] Figure 11 shows the use of VEGF-C muteins in vivo. Wild-type VEGF-C and VEGF-C muteins were evaluated in vivo for the treatment of brain tumors. When combined with anti-PD-1 antibodies, VEGF-C muteins showed significant therapeutic benefit in treating brain tumors. [Figure 12] FIG. 12 shows the VEGFR-2 and VEGFR-3 affinity of WT VEGF-C and the previously established VEGFR-3 specific ligand, VEGF-C152S / C-156S. [Figure 13]FIG. 13 shows that isolated VEGF-C muteins showed specific signaling through VEGFR-3 in vivo. Signaling of wild-type VEGF-C and VEGF-C mutein (RTI) was evaluated in vivo. These vectors were injected into the eye of mice. After 24 hours, the eyes were enucleated and single cell suspensions were made to check AKT phosphorylation. Wild-type VEGF-C retained signaling through VEGFR-2 in vascular endothelial cells, whereas mutant VEGF-C (RTI) no longer had signaling. Both wild-type protein and RTI mutein signaled through VEGFR-3 in lymphatic endothelial cells. [Figure 14] Figure 14 shows that the route of administration resulted in different IOP reductions in vivo. VEGF-C (wt) or VEGF-C mutein (rti) were administered by eye drop, by injection into the anterior chamber (AC), or intravitreally. While both wild type (wt) and mutein (rti) had similar activity in the AC, the mutein showed a selective ability to reduce intraocular pressure when administered by eye drop or intravitreally. [Figure 15A] Figures 15A-15D show directed evolution of lymphoid-specific VEGFR-3 binding. Figure 15A shows mVEGFR2 affinity of WT VEGF-C and muteins after round 6 of iterative selection. [Figure 15B] FIG. 15B shows the mVEGFR3 affinity of WT VEGF-C and the muteins after round 6 of iterative selection. [Figure 15C] FIG. 15C shows downstream ERK phosphorylation in HUVEC (R2+) following treatment with control (n=3), VEGF-A (n=3), WT VEGF-C (n=3), and LS-VEGF-C (n=3). [Figure 15D]Figure 15D shows proliferation of HUVEC (R2+) and HDLEC (R2+R3+) following treatment with control (n=3), VEGF-A (n=3), WT VEGF-C (n=3), and LS-VEGF-C (n=3). In Figures 15C and 15D, data are presented as mean ± sem, *P<0.05, **P<0.01, ***P<0.005, ****P<0.001, and ns means "nonsignificant." [Figure 16A] 16A-16B show that administration of LS-VEGF-C reduced lymphedema in a mouse model of lymphedema. LS-VEGF-C was administered subcutaneously near the lymphatic ligation site in mice, resulting in a significant reduction in ankle size compared to controls. To demonstrate remedyability of LS-VEGF-C, mice were treated twice. [Figure 16B] Continued from Figure 16A. [Figure 16C] In Figures 16C-16D, tumor-bearing mice were treated with tumor vaccines and PD-1 together with LS-VEGF-C or WT-VEGF-C. Figure 16C shows the tumor growth curves. [Figure 16D] FIG. 16D shows the survival rate of the mice. [Figure 17A] Figures 17A-17E show that LS-VEGF-C altered lymphangiogenesis without increasing vascular permeability in the eye. Figure 17A shows intraocular pressure measurements after topical, intracameral, and intravitreal administration of WT-VEGF-C in WT mice. [Figure 17B] FIG. 17B shows intraocular pressure measurements following topical (eye drop), intracameral, and intravitreal administration of LS-VEGF-C in WT mice. [Figure 17C] FIG. 17C shows representative fundus images (left column), fluorescence angiography images (middle column), and OCT images (right column) following intravitreal administration of VEGF-A, WT-VEGF-C, and LS-VEGF-C. [Figure 17D]FIG. 17D shows normalized measurements of fluorescence intensity across two multiple fluorescein angiography images, indicating vascular permeability to extravascular regions; a sharp peak above baseline (normalized value approx. 20) indicates the presence of leaky vessels, while persistently elevated values ​​indicate increased permeability. [Figure 17E] Figure 17E shows a comparison of repeated measures 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 blood vessels to the extravascular space. For Figure 17E, ***P<0.005, ****P<0.001, *P<0.05, and ns means "nonsignificant." [Figure 18A] Figures 18A-18H show that LS-VEGF-C provides neuroprotection in a glaucoma model. Figure 18A is a schematic representation of the microbead mouse model of glaucoma and assessment by IOP measurements. [Figure 18B] FIG. 18B shows summary (mean±sem, left) and individual IOP measurements (right) over a 28-day period for WT mice (control, n=12), a microbead mouse model of glaucoma (induced glaucoma, n=12), a microbead mouse model of glaucoma with IVT administration of LS-VEGF-C (induced glaucoma+LS-VEGF-C(IVT), n=12), a microbead mouse model of glaucoma with intravitreal administration of LS-VEGF-C (induced glaucoma+LS-VEGF-C(IVT), n=12), a microbead mouse model of glaucoma with topical administration of LS-VEGF-C (induced glaucoma+LS-VEGF-C(eye drops), n=12), and WT mice with intravitreal administration of LS-VEGF-C (LS-VEGF-C(IVT), n=12). [Figure 18C]Figure 18C shows Brn3a and DAPI labeled confocal images of the retina after intravitreally injected NMDA excitotoxicity studies comparing WT mice (control), microbead glaucoma model (induced glaucoma), and microbead glaucoma models with intravitreous (+LS-VEGF-C IVT) and topical (+LS-VEGF-C eye drops) administration of LS-VEGF-C. [Figure 18D] FIG. 18D shows the number of Brn3a cells observed within the image field (control, n=7; microbead glaucoma model, n=6; microbead glaucoma model+IVT LS-VEGF-C, n=7; microbead glaucoma model+LS-VEGF-C eye drops, n=8). [Figure 18E] FIG. 18E is a schematic diagram of the DBA2J pigment dispersion mouse model of glaucoma. [Figure 18F] FIG. 18F shows summary (mean±sem, left) and individual IOP measurements (right) over a 28-day period for WT mice (control, n=12), a microbead mouse model of glaucoma (induced glaucoma, n=12), a microbead mouse model of glaucoma with IVT administration of LS-VEGF-C (induced glaucoma+LS-VEGF-C(IVT), n=12), a microbead mouse model of glaucoma with intravitreal administration of LS-VEGF-C (induced glaucoma+LS-VEGF-C(IVT), n=12), a microbead mouse model of glaucoma with topical administration of LS-VEGF-C (induced glaucoma+LS-VEGF-C(eye drops), n=12), and WT mice with intravitreal administration of LS-VEGF-C (LS-VEGF-C(IVT), n=12). [Figure 18G] FIG. 18G shows images of RGC axons in the optic nerve of control D2-Gpnmb+ mice and DBA2J mice that received the following different treatments: no treatment (spontaneous glaucoma), +LS-VEGF-C IVT, +LS-VEGF-C eye drops. [Figure 18H] FIG. 18H shows the number of RGC axon cells observed within the image field (control, n=3; DBA2J glaucoma, n=4; LS-VEGF-C IVT, n=4; LS-VEGF-C eye drops, n=4). [Figure 19] FIG. 19 shows data from VEGFR2 and VEGFR3 binding assays of WT VEGF-C to VEGFR2 and VEGFR3. [Figure 20A] 20A-20B show that administration of LS-VEGF-C reduced lymphedema in a mouse model of lymphedema. LS-VEGF-C was administered subcutaneously near the lymphatic ligation site in mice, resulting in a significant reduction in ankle size compared to controls. To demonstrate remedyability of LS-VEGF-C, mice were treated twice. [Figure 20B] Continued from Figure 20A. [Figure 20C] We evaluated LS-VEGF-C as an adjuvant to anti-PD-1 cancer immunotherapy in a mouse model of melanoma. LS-VEGF-C showed superior preclinical efficacy compared to WT-VEGF-C in both tumor volume reduction and survival. [Figure 20D] Continued from Figure 20C. [Figure 20E] LS-VEGF-C was utilized as an adjuvant therapy to checkpoint inhibitor therapy for the treatment of glioblastoma. LS-VEGF-C demonstrated comparable activity to WT-VEGF-C and resulted in survival of the majority of treated animals that would otherwise have had a fatal outcome. [Figure 20F] Continued from Figure 20E. [Figure 21A] Figures 21A-21D show that LS-VEGF-C provides a unique mechanism of action with observed sustained effects: Figure 21A shows IOP measurements (mean ± sem) over a 10-day period following topical (left), intracameral (center), and intravitreal (right) administration of WT-VEGF-C and LS-VEGF-C. [Figure 21B] FIG. 21B is a schematic diagram including arrows showing the flow of fluids into and out of the eye. [Figure 21C] Figure 21C shows IOP measurements after topical administration of current FDA-approved medications for glaucoma. Data points from the first hour have been excluded due to the misleading IOP-lowering effect of light anesthesia. [Figure 21D]FIG. 21D shows a comparison of IOP measurements (mean±sem) for control mice, mice treated with FDA-approved topical glaucoma drugs at the time point when the maximum IOP reduction was observed (dorzolamide, Dz, at 2 hours; rhopressor, Rho, at 2 hours; brimonidine, Bri, at 2 hours; latanoprost, Lat, at 3 hours; timolol, at 2 hours), mice 2 days after IV injection of LS-VEGF-C, and mice treated with topical administration of FDA-approved drugs at the time point when the maximum IOP reduction was observed in mice that had received intravitreal LS-VEGF-C 2 days prior. Data are presented as mean±sem, *P<0.05, **P<0.01, ***P<0.005, ****P<0.001, and ns means "nonsignificant." [Figure 22A] Figures 22A-22D show images of the anterior side of the eye, which show similar signs as the posterior side. Figure 22A shows images of a control WT mouse focused on the cornea and iris (n=3). [Figure 22B] Figure 22B shows images of WT mice after IVT administration of VEGF-A, focusing on the cornea and iris (n=3). Neovasculature and corneal haze were observed. [Figure 22C] Figure 22C shows images of WT mice after IVT administration of WT-VEGF-C, focusing on the cornea and iris (n=3). Corneal haze was observed. [Figure 22D] FIG. 22D shows images of WT mice following IVT administration of LS-VEGF-C, focusing on the cornea and iris (n=3). [Figure 23A] Figures 23A-23B show that LS-VEGF-C provides neuroprotection in a glaucoma model. Figure 23A shows Brn3a and DAPI labeled confocal images of the retina after intravitreally injected NMDA excitotoxicity studies. [Figure 23B] FIG. 23B shows the number of RGC cells observed within the image field from the NMDA-facilitated excitability study. [Figure 24]Figure 24 shows a VEGFR2 binding summary for hLS-VEGF-C variants, summarizing the binding of hVEGF-C variants to hVEGFR2 compared to WT VEGF-C, expressed as a % of the yeast population that stained positive, with lower values ​​indicating less VEGFR2 engagement being desirable. [Diagram 25] Figure 25 shows the relative bias between VEGFR3 and VEGFR2 for LS-hVEGF-C clones. Figure 25 includes plots of hVEGFR3 binding (represented as mean fluorescence intensity MFI) versus hVEGFR2 binding (represented as % of yeast population that stained positive). In the top graph, 100 nM VEGFR3 was used. In the bottom graph, 10 nM hVEGFR3 was used. Selected points are labeled with the number of LS-hVEGF-C muteins. Clones in the lower right quadrant of these plots are preferred as they showed biased binding towards VEGFR3 over VEGFR2. [Figure 26] FIG. 26 shows VEGFR2 binding and VEGFR3 binding for LS-hVEGF-C; specific values ​​for hVEGFR2 and hVEGFR3 for each clone are shown in FIGS. [Figure 27] Figure 27 shows the binding isotherms of selected LS-hVEGF-C muteins to hVEGFR3. Specific EC50 and R2 values ​​for curve fitting are provided at the bottom of Figure 27. [Figure 28A] Figures 28A-28M show the quality of human VEGF-C muteins fused to human IgG1 Fc. Figure 28A shows the quality of the human C137A clone fusion. [Figure 28B] FIG. 28B shows the quality of the human clone 7 fusion. [Figure 28C] FIG. 28C shows the quality of the human clone 8 fusion. [Figure 28D] FIG. 28D shows the quality of the human clone 9 fusion. [Figure 28E] FIG. 28E shows the quality of the human clone 10 fusion. [Figure 28F]FIG. 28F shows the quality of the human clone 13 fusion. [Figure 28G] FIG. 28G shows the quality of human clone 14 fusions. [Fig. 28H] FIG. 28H shows the quality of the human clone 15 fusion. [Figure 28I] FIG. 28I shows the quality of the human clone 16 fusion. [Figure 28J] FIG. 28J shows the quality of the human clone 19 fusion. [Figure 28K] FIG. 28K shows the quality of the human clone 21 fusion. [Figure 28L] FIG. 28L shows the quality of the human clone 31 fusion. [Figure 28M] FIG. 28M shows the quality of the human clone 39 fusion. [Figure 29] FIG. 29 shows the effect on mice intravitreally treated with RTI-Fc muteins and their intraocular pressure. [Diagram 30] FIG. 30 shows the reduction in intraocular pressure in mice treated with intravitreal LS-VEGF-C in the monomeric form, albumin conjugate, or Fc conjugate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0096] The present invention provides a VEGF-C mutein and 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 mutein protein.

[0097] 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.

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

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

[0100] The term "treat" a state, disorder, or condition, or "treatment" thereof, includes: (1) preventing, delaying, or reducing the occurrence and / or likelihood of at least one clinical or subclinical symptom of said condition, disorder, or condition occurring in a subject who may be suffering from or predisposed to said condition, disorder, or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of said condition, disorder, or condition; or (2) inhibiting said condition, disorder, or condition, i.e., preventing, reducing, or delaying the progression of the disease or (in the case of maintenance therapy) its recurrence, or at least one clinical or subclinical symptom thereof; or (3) palliating the disease, i.e., causing regression of said condition, disorder, or condition, or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated is statistically significant or at least perceptible to the patient or physician.

[0101] As used herein, the terms "mutein protein," "mutein polypeptide," and "mutein" are used interchangeably and refer to a protein that has an altered amino acid sequence compared to its wild-type counterpart. The alteration in amino acid sequence can include amino acid substitutions, deletions, or additions.

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

[0103] In one aspect, provided herein is an isolated vascular endothelial growth factor C (VEGF-C) mutein protein, or functional fragment thereof, that selectively binds to VEGFR-3 over VEGFR-2.

[0104] In some embodiments, the VEGF-C mutein protein or functional fragment thereof has reduced or no ability to stimulate vascular endothelial cell proliferation compared to a wild-type VEGF-C protein of the same species but which retains the ability to stimulate lymphatic endothelial cell proliferation.

[0105] In some embodiments, the VEGF-C mutein protein or functional fragment thereof (i) has reduced binding affinity to VEGFR-2 compared to a wild-type VEGF-C protein of the same species, (ii) has the ability to bind to VEGFR-3 and generate signaling 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 of the same species.

[0106] In some embodiments, the VEGF-C mutein protein or 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 percentage compared to a wild-type VEGF-C protein from the same species.

[0107] In some embodiments, the VEGF-C mutein protein or functional fragment thereof has the ability to bind to VEGFR-3 and produce signal transduction through VEGFR-3 that is approximately the same, increased, or slightly decreased compared to wild-type VEGF-C protein from the same species.

[0108] Intracellularly, VEGF-C is produced as an inactive propeptide. Convertases such as Furin, PC5, or PC7 cleave between the VEGF homology domain and the C-terminal silk homology domain to generate proVEGF-C. ProVEGF-C can bind to, but not activate, VEGFR-3. A second proteolytic cleavage by A disintegrin and metalloproteinase with thrombospondin motif 3 (ADAMTS3) removes both terminal domains to yield the mature, active VEGF-C protein. See, e.g., 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 #P49767). In the case of wild-type mouse VEGF-C, the mature VEGF-C protein corresponds to amino acids 111-211 (SEQ ID NO:292) of the mouse VEGF-C propeptide (SEQ ID NO:4).

[0109] In some embodiments, the VEGF-C mutein protein can be a mutein protein of a human VEGF-C protein. The human VEGF-C mutein 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: MHLLGFFSVACSLLAAAALLPGPREAPAAAAAFESGLDLSDAEPDAGEATAYASKDLEEQLRSVSSVDELMTVLYPEYWKMYKCQLRKGGWQHNREQANLNSRTEETIKFAAAHYNTEILKSIDNE WRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLDVYRQVHSIIRRSLPATLPQCQAANKTCPTNYMWNN HICRCLAQEDFMFSSDAGDDSTDGFHDICGPNKELDEETCQCVCRAGLRPASCGPHKELDRNSCQCVCKNKLFPSQCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFSQPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFS (SEQ ID NO: 1; wild-type VEGF-C propeptide; UniProt database accession number P49767).

[0110] A polynucleotide molecule encoding a human VEGF-C mutein 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 to 645 of the following VEGF-C encoding sequence:

[0111] A human VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (wild-type human mature VEGF-C protein; SEQ ID NO: 101).

[0112] A polynucleotide molecule encoding a human VEGF-C mutein 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-encoding sequence: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (SEQ ID NO: 153; nucleotide sequence encoding human mature VEGF-C protein).

[0113] In certain embodiments, the VEGF-C mutein comprises one or more mutations at residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, I188, V190, L192, P196, and combinations thereof, where the positions of the mutations are defined with reference to SEQ ID NO: 1. In certain embodiments, the mutation at L119 is a L119E mutation. In certain embodiments, the mutation at L119 is a 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 a N167R mutation. In certain embodiments, the mutation at N167 is a N167I mutation. In certain embodiments, the mutation at N167 is a N167Q mutation. In certain embodiments, the mutation at N167 is a 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.

[0114] In some embodiments, the VEGF-C mutein protein can be a mutein protein of a mouse VEGF-C protein. In certain embodiments, the mouse VEGF-C mutein 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: MHLLCFLSLACSLLAAALIPSPREAPATVAAFESGLGFSEAEPDGGEVKAFEGKDLEEQLRSVSSVDELMSVLYPDYWKMYKCQLRKGGWQQPTLNTRTGDSVKFAAAHY NTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLDVYRQVHSI IRRSLPATLPQCQAANKTCPTNYVWNNYMCRCLAQQDFIFYSNVEDDSTNGFHDVCGPNKELDEDTCQCVCKGGLRPSSCGPHKELDRDSCQCVCKNKLFPNSCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTENTQKCFLKGKKFHHQTCSCYRRPCANRLKHCDPGLSFSEEVCRCVPSYWKRPHLN (mouse wild-type VEGFC propeptide; SEQ ID NO: 4).

[0115] A polynucleotide molecule encoding a murine VEGF-C mutein 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 to 631 of the following VEGF-C encoding sequence:

[0116] In some embodiments, the VEGF-C mutein protein can be a mutein protein of a mouse VEGF-C protein. In certain embodiments, the mouse VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (wild-type mouse mature VEGF-C protein; SEQ ID NO: 292).

[0117] A polynucleotide molecule encoding a mouse VEGF-C mutein 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 encoding sequence: AACACAGAGATCCTGAAAAGTATTGATAATGAGTGGAGAAAGACTCAATGCATGCCACGTGAGGTGTGTATAGATGTGGGGAAGGAGTTTGGAGCAGCCACAAACACCTTCTTTAAACCTCCATGTGTGTCCGTCTACAGATGTGGGGGTTGCTGCAACAGCGAGGGGCTGCAGTGCATGAACACCAGCACAGGTTACCTCAGCAAGACGTTGTTTGAAATTACAGTGCCTCTCTCACAAGGCCCCAAACCAGTCACAATCAGTTTTGCCAATCACACTTCCTGCCGGTGCATGTCTAAACTG (nucleotide sequence encoding wild-type mouse mature VEGF-C; SEQ ID NO: 293).

[0118] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167Q VEGF-C mutein protein; SEQ ID NO: 160).

[0119] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGTCTGAGGGGCTCCAATGTATGAACACGAGTACGtctTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167Q VEGF-C mutein protein; SEQ ID NO:210).

[0120] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167Q and S168G VEGF-C mutein protein; SEQ ID NO: 161).

[0121] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGGAGGTAGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167Q and S168G VEGF-C mutein protein; SEQ ID NO:211).

[0122] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human N167Q and L192I VEGF-C mutein protein; SEQ ID NO: 162).

[0123] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167Q and L192I VEGF-C mutein protein; SEQ ID NO:212).

[0124] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human N167Q, S168G, and L192I VEGF-C mutein protein; SEQ ID NO: 163).

[0125] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGGAGGTAGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167Q, S168G, and L192I VEGF-C mutein protein; SEQ ID NO:213).

[0126] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167I VEGF-C mutein protein; SEQ ID NO: 166).

[0127] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGGAGGTAGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCTCTGAGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167I VEGF-C mutein protein; SEQ ID NO:216).

[0128] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167I and S168G VEGF-C mutein protein; SEQ ID NO: 167).

[0129] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCGGCGAGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167I and S168G VEGF-C mutein protein; SEQ ID NO: 217).

[0130] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human N167I and L192I VEGF-C mutein protein; SEQ ID NO: 168).

[0131] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCTCTGAGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167I and L192I VEGF-C mutein protein; SEQ ID NO:218).

[0132] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human N167I, S168G and L192I VEGF-C mutein protein; SEQ ID NO: 169).

[0133] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCGGCGAGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167I, S168G and L192I VEGF-C mutein protein; SEQ ID NO:219).

[0134] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human S168G VEGF-C mutein protein; SEQ ID NO: 172).

[0135] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGGAGGTAGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human S168G VEGF-C mutein protein; SEQ ID NO: 222).

[0136] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human S168G and L192I VEGF-C mutein protein; SEQ ID NO: 174).

[0137] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGGAGGTAGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human S168G and L192I VEGF-C mutein protein; SEQ ID NO: 224).

[0138] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCHSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167H VEGF-C mutein protein; SEQ ID NO: 184).

[0139] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGGAGGTAGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCACTCTGAGGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167H VEGF-C mutein protein; SEQ ID NO: 234).

[0140] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCIREGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human N167I and S168R VEGF-C mutein protein; SEQ ID NO: 192).

[0141] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGGAGGTAGTATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCAGAGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human N167I and S168R VEGF-C mutein protein; SEQ ID NO:242).

[0142] In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises one or more mutations selected from mutations at residues N167, S168, and / or L192, where the positions of the mutations are defined relative to SEQ ID NO:1.

[0143] In some embodiments, the mutation at N167 is an N167I mutation, an N167Q mutation, or an N167H mutation; the mutation at S168 is an S168G mutation, or an S168R mutation; and / or the mutation at L192 is an L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0144] In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises a N167Q mutation, where the position of the mutation is defined relative to SEQ ID NO:1.

[0145] In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises an N167Q mutation and an S168G mutation, where the positions of the mutations are defined relative to SEQ ID NO:1.

[0146] In some embodiments, the VEGF-C mutein protein or 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.

[0147] In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises a N167Q mutation, a S168G mutation, and a L192I mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0148] In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises an N167I mutation, wherein the position of the mutation is defined relative to SEQ ID NO:1.

[0149] In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises an N167I mutation and an S168G mutation, where the positions of the mutations are defined relative to SEQ ID NO:1.

[0150] In some embodiments, the VEGF-C mutein protein or 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.

[0151] In some embodiments, the VEGF-C mutein protein or 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.

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

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

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

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

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

[0157] In certain embodiments, a VEGF-C mutein 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 any of the polypeptide sequences of SEQ ID NOs: 154-159, 164-165, 170-171, 173, 175-183, 185-191, or 193-203.

[0158] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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 any of the sequences set forth in SEQ ID NOs: 204-209, 214-215, 220-221, 223, 225-233, 235-241, or 243-253.

[0159] In certain embodiments, a VEGF-C mutein may comprise a functional fragment of mouse VEGF-C. In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (mouse VEGF-C mutein protein, SEQ ID NO: 23).

[0160] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTATGTATCGACGTTGGTAAAGAATTTGGTGCGGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATTCTGAGGGGCTCCAATGTATGAACACGAGTACGGGTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding mouse VEGF-C mutein protein, SEQ ID NO:24).

[0161] In certain embodiments, the VEGF-C mutein comprises one or more mutations at residues T112, L115, D119, Q126, T144, N145, K149, N163, S164, E165, I184, V186, L188, P192, and combinations thereof, where the positions of the mutations are defined with reference to SEQ ID NO: 4. In certain embodiments, the mutation at L115 is a L115E mutation. In certain embodiments, the mutation at L115 is a 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 a N163R mutation. In certain embodiments, the mutation at N163 is a N163I mutation. In certain embodiments, the mutation at N163 is a N163Q mutation. In certain embodiments, the mutation at S164 is a 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 certain embodiments, the VEGF-C mutein protein comprises a N163I mutation.

[0162] In certain embodiments, a VEGF-C mutein 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: MHLLCFLSLACSLLAAALIPSPREAPATVAAFESGLGFSEAEPDGGEVKAFEGKDLEEQLRSVSSVDELMSVLYPDYWKMYKCQLRKGGWQQPTLNTRTGDSVKFAAAHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLD VYRQVHSIIRRSLPATLPQCQAANKTCPTNYVWNNYMCRCLAQQDFIFYSNVEDDSTNGFHDVCGPNKELDEDTCQCVCKGGLRPSSCGPHKELDRDSCQCVCKNKLFPNSCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTENTQKCFLKGKKFHHQTCSCYRRPCANRLKHCDPGLSFSEEVCRCVPSYWKRPHLN (mouse N163I VEGF-C mutein protein; SEQ ID NO: 6).

[0163] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0164] In some embodiments, the VEGF-C mutein protein comprises the mutations N163R, V186T and L188I, where the positions of the mutations are defined relative to SEQ ID NO:4.

[0165] In certain embodiments, a VEGF-C mutein 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: MHLLCFLSLACSLLAAALIPSPREAPATVAAFESGLGFSEAEPDGGEVKAFEGKDLEEQLRSVSSVDELMSVLYPDYWKMYKCQLRKGGWQQPTLNTRTGDSVKF AAAHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKL DVYRQVHSIIRRSLPATLPQCQAANKTCPTNYVWNNYMCRCLAQQDFIFYSNVEDDSTNGFHDVCGPNKELDEDTCQCVCKGGLRPSSCGPHKELDRDSCQCVCK NKLFPNSCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTENTQKCFLKGKKFHHQTCSCYRRPCANRLKHCDPGLSFSEEVCRCVPSYWKRPHLN(Mouse RTI VEGFC mutein protein, sequence number 8).

[0166] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0167] In certain embodiments, the VEGF-C mutein comprises a mutation at residue C133, where the position of the mutation is defined with reference to SEQ ID NO: 4. In certain embodiments, the mutation at residue C133 is a C133A mutation.

[0168] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (Mouse VEGF-C mutein protein with C133A mutation, SEQ ID NO: 9)

[0169] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGCGGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATTCTGAGGGGCTCCAATGTATGAACACGAGTACGGGTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding a VEGF-C mutein protein having a C133A mutation, SEQ ID NO: 10).

[0170] In a particular embodiment, the VEGF-C mutein protein comprises the mutations C133A and N163I, where the positions of the mutations are defined relative to SEQ ID NO:4.

[0171] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (mouse VEGF-C mutein protein with C133A and N163I mutations, SEQ ID NO: 17).

[0172] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGCAAAACTCAATGTATGCCACGGGAGGTTGCAATAGACGTGGGAAAGGAATTTGGCGCCGCCACGAATACCTTTTTCAAGCCTCCCTGTGTGAGTGTTTACAGATGTGGTGGTTGTTGCATATCAGAGGGATTGCAGTGCATGAACACAAGTACAGGTTACTTGAGTAAAACATTGTTTGAAATCACAGTACCATTGTCCCAAGGCCCTAAGCCGGTTACGATCTCTTTCGCCAATCATACGTCATGCCGCTGTATGAGTAAGTTG (nucleotide sequence encoding mouse VEGF-C mutein protein having C133A and N163I mutations, SEQ ID NO: 18).

[0173] In some embodiments, the VEGF-C mutein protein comprises the mutations C133A, N163R, V186T and L188I, where the positions of the mutations are defined relative to SEQ ID NO:4.

[0174] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKL (mouse VEGF-C mutein protein having C133A, N163R, V186T, and L188I mutations, SEQ ID NO: 13).

[0175] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGAAAAACCCAATGTATGCCACGCGAGGTAGCGATAGATGTGGGCAAAGAATTCGGCGCCGCGACGAACACCTTTTTCAAGCCCCCTTGCGTCTCCGTATATAGATGCGGTGGATGTTGCCGATCCGAGGGCCTTCAGTGTATGAACACATCTACTGGCTATTTGAGCAAGACGCTCTTTGAGATTACAACACCAATTAGTCAAGGTCCCAAGCCTGTTACCATCTCTTTCGCTAACCACACTTCATGCCGCTGTATGAGTAAGTTG (nucleotide sequence encoding mouse VEGF-C mutein protein having C133A, N163R, V186T, and L188I mutations, SEQ ID NO: 14).

[0176] In certain embodiments, the VEGF-C mutein comprises a mutation at residue C137, where the position of the mutation is defined with reference to SEQ ID NO: 1. In certain embodiments, the mutation at residue C137 is a C137A mutation.

[0177] In certain embodiments, a human VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human VEGF-C mutein protein with a C133A mutation; SEQ ID NO: 100).

[0178] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding a human VEGF-C mutein protein having a C133A mutation, SEQ ID NO: 152).

[0179] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A and N167Q VEGF-C mutein protein; SEQ ID NO:56).

[0180] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A and N167Q VEGF-C mutein protein; SEQ ID NO: 108).

[0181] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A, N167Q and S168G VEGF-C mutein protein; SEQ ID NO:57).

[0182] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167Q and S168G VEGF-C mutein protein; SEQ ID NO: 109).

[0183] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human C137A, N167Q and L192I VEGF-C mutein protein; SEQ ID NO:58).

[0184] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGTCTGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167Q, and L192I VEGF-C mutein protein; SEQ ID NO:110).

[0185] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human C137A, N167Q, S168G and L192I VEGF-C mutein protein; SEQ ID NO:59).

[0186] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCAGGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167Q, S168G, and L192I VEGF-C mutein protein; SEQ ID NO:111).

[0187] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A and N167I VEGF-C mutein protein; SEQ ID NO: 62).

[0188] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCTCTGAGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A and N167I VEGF-C mutein proteins; SEQ ID NO:114).

[0189] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A, N167I and S168G VEGF-C mutein protein; SEQ ID NO: 63).

[0190] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCGGCGAGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167I and S168G VEGF-C mutein protein; SEQ ID NO: 115).

[0191] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human C137A, N167I and L192I VEGF-C mutein protein; SEQ ID NO: 64).

[0192] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCTCTGAGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167I and L192I VEGF-C mutein protein; SEQ ID NO:116).

[0193] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human C137A, N167I, S168G and L192I VEGF-C mutein protein; SEQ ID NO: 65).

[0194] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGGAGGGTGTTGCATCGGCGAGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167I, S168G and L192I VEGF-C mutein protein; SEQ ID NO: 117).

[0195] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A and S168G VEGF-C mutein protein; SEQ ID NO: 68).

[0196] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A and S168G VEGF-C mutein protein; SEQ ID NO: 120).

[0197] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKL (human C137A, S168G and L192I VEGF-C mutein protein; SEQ ID NO: 70).

[0198] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCAATGGCGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCAATCTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, S168G and L192I VEGF-C mutein protein; SEQ ID NO: 122).

[0199] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCHSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A and N167H VEGF-C mutein protein; SEQ ID NO: 80).

[0200] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCCACTCTGAGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A and N167H VEGF-C mutein proteins; SEQ ID NO: 132).

[0201] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIREGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKL (human C137A, N167I and S168R VEGF-C mutein protein; SEQ ID NO: 88).

[0202] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACGGAAATCCTCAAGTCTATAGATAATGAGTGGCGAAAGACACAATGTATGCCGCGCGAGGTAGCAATCGACGTTGGTAAAGAATTTGGTGTAGCAACGAACACATTCTTCAAACCCCCTTGTGTGAGTGTATATAGATGTGGAGGGTGTTGCATCAGAGAGGGGCTCCAATGTATGAACACGAGTACGTCTTACTTGAGTAAGACCTTGTTCGAAATTACAGTACCACTTTCCCAAGGACCGAAGCCTGTGACTATCAGCTTTGCTAACCATACAAGCTGTCGCTGTATGAGCAAACTG (nucleotide sequence encoding human C137A, N167I and S168R VEGF-C mutein protein; SEQ ID NO: 140).

[0203] In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises one or more mutations selected from mutations at residues C137, N167, S168, and / or L192, where the positions of the mutations are defined relative to SEQ ID NO:1.

[0204] In some embodiments, the mutation at C137 is C137A, 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.

[0205] In some embodiments, the VEGF-C mutein protein or 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.

[0206] In some embodiments, the VEGF-C mutein protein or 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.

[0207] In some embodiments, the VEGF-C mutein protein or 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.

[0208] In some embodiments, the VEGF-C mutein protein or 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.

[0209] In some embodiments, the VEGF-C mutein protein or 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.

[0210] In some embodiments, the VEGF-C mutein protein or 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.

[0211] In some embodiments, the VEGF-C mutein protein or 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.

[0212] In some embodiments, the VEGF-C mutein protein or 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.

[0213] In some embodiments, the VEGF-C mutein protein or 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.

[0214] In some embodiments, the VEGF-C mutein protein or 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.

[0215] In some embodiments, the VEGF-C mutein protein or 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.

[0216] In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises a C137A mutation, an N167I mutation and an S168R mutation, wherein the positions of the mutations are defined relative to SEQ ID NO:1.

[0217] In certain embodiments, a VEGF-C mutein 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 a polypeptide sequence of any of SEQ ID NOs:50-55, SEQ ID NOs:60-61, SEQ ID NOs:66-67, SEQ ID NO:69, SEQ ID NOs:71-79, SEQ ID NOs:81-87, or SEQ ID NOs:89-99.

[0218] In certain embodiments, a polynucleotide encoding a VEGF-C mutein 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 any of SEQ ID NOs: 102-107, SEQ ID NOs: 112-113, SEQ ID NOs: 118-119, SEQ ID NO: 121, SEQ ID NOs: 123-131, SEQ ID NOs: 133-139, or SEQ ID NOs: 141-151.

[0219] The VEGF-C muteins or fragments thereof described herein include variants having single or multiple amino acid substitutions, deletions or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described VEGF-C muteins or fragments thereof.

[0220] These variants include: (i) variants in which one or more amino acid residues are replaced with conservative or non-conservative amino acids, (ii) variants in which one or more amino acids are added to or deleted from the polypeptide, (iii) variants in which one or more amino acids include a substituent, and (iv) variants in which the described VEGF-C muteins or fragments thereof are fused or conjugated to another peptide or polypeptide (e.g., a fusion partner, a protein tag) or other chemical moiety that may confer useful properties to the VEGF-C muteins or fragments thereof, such as, for example, an antibody epitope, a polyhistidine sequence, a biotin site, etc. The VEGF-C muteins or fragments thereof described herein may include variants in which amino acid residues of one species are replaced with the corresponding residue of another species, either at the conserved or non-conserved positions. In other embodiments, amino acid residues at non-conserved positions are replaced with conservative or non-conserved residues. Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

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

[0222] In some embodiments, the VEGF-C mutein protein is modified to increase its circulating half-life. Strategies to increase the half-life of recombinant proteins include, but are not limited to, fusion to an immunoglobulin or a fragment of an immunoglobulin, such as the Fc domain of IgG; fusion to albumin or an albumin fragment; fusion to an albumin-binding antibody or an antibody fragment thereof, such as scFv, Fab, or single domain antibody (VHH); or chemical modification with polyethylene glycol (PEG).

[0223] In some embodiments, the VEGF-C mutein or fragment or variant thereof is fused and / or conjugated to one or more heterologous moieties, including but not limited to peptides, polypeptides, small molecules, polymers, nucleic acids, lipids, sugars, etc.

[0224] In certain embodiments, the VEGF-C mutein is fused and / or conjugated to a moiety that provides the VEGF-C mutein with a longer half-life. In some embodiments, the VEGF-C mutein is fused and / or conjugated to a moiety that specifically binds to albumin, including but not limited to a small molecule, peptide, polypeptide, or lipid that binds albumin. In some embodiments, the VEGF-C mutein is fused and / or conjugated to an immunoglobulin constant region (Fc domain), scFv, Fab, single domain antibody (VHH), immunoglobulin, or a heavy or light chain thereof.

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

[0226] In certain embodiments, the VEGF-C muteins are fused and / or conjugated to an Fc domain. In certain embodiments, the Fc sequence fused or conjugated to a VEGF-C mutein 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: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 21).

[0227] In certain embodiments, a polynucleotide molecule encoding an 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: (Sequence number 22).

[0228] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (mouse VEGF-C mutein protein with C133A mutation and Fc conjugation, SEQ ID NO: 11).

[0229] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0230] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKLA AAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (mouse VEGF-C mutein protein with C133A, N163R, V186T, and L188I mutations and Fc conjugation, SEQ ID NO: 15).

[0231] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0232] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (mouse VEGF-C mutein protein with C133A and N163I mutations and Fc conjugation, SEQ ID NO: 19).

[0233] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0234] In some embodiments, the IgG Fc domain comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the VEGF-C mutein protein 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.

[0235] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (Human C137A VEGF-C protein with Fc conjugation; SEQ ID NO: 266).

[0236] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0237] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (Human C137A and N167Q VEGF-C mutein protein with Fc conjugation; SEQ ID NO: 267).

[0238] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0239] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (human C137A, N167Q and S168G with Fc conjugation VEGF-C mutein protein; SEQ ID NO:268).

[0240] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0241] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQSEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (human C137A, N167Q and L192I with Fc conjugation VEGF-C mutein protein; SEQ ID NO:269).

[0242] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0243] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCQGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (human C137A, N167Q, S168G and L192I with Fc conjugation VEGF-C mutein protein; SEQ ID NO:).

[0244] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0245] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (human C137A and N167I VEGF-C mutein protein with Fc conjugation; SEQ ID NO: 271).

[0246] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0247] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (human C137A, N167I and S168G with Fc conjugation VEGF-C mutein protein; SEQ ID NO:272).

[0248] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0249] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (human C137A, N167I and L192I with Fc conjugation VEGF-C mutein protein; SEQ ID NO:273).

[0250] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0251] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIGEGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (human C137A, N167I, S168G and L192I with Fc conjugation VEGF-C mutein protein; SEQ ID NO:274).

[0252] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0253] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (Human C137A and S168G VEGF-C mutein protein with Fc conjugation; SEQ ID NO: 275).

[0254] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0255] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCNGEGGLQCMNTSTSYLSKTLFEITVPISQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (human C137A, S168G and L192I with Fc conjugation VEGF-C mutein protein; SEQ ID NO:276).

[0256] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0257] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCHSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (human C137A and N167H VEGF-mutein protein with Fc conjugation; SEQ ID NO: 277).

[0258] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0259] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGVATNTFFKPPCVSVYRCGGCCIREGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHH (human C137A, N167I and S168R with Fc conjugation VEGF-C mutein protein; SEQ ID NO:278).

[0260] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

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

[0262] In some embodiments, the Fc domain comprises one or more mutations, hi some embodiments, the Fc domain is mutated, glycoengineered, or otherwise modified to reduce Fc effector function.

[0263] In some embodiments, the Fc domain contains one or more mutations that remove critical glycosylation sites required for effector function.

[0264] In some embodiments, the Fc domain comprises a mutation at residue N82, wherein said residue position is defined with reference to SEQ ID NO: 21. In some embodiments, the mutation at N82 defined with reference to SEQ ID NO: 21 is an N82Q mutation, an N82A mutation, or an N82G mutation.

[0265] In certain embodiments, the VEGF-C mutein is fused and / or conjugated to albumin, hi certain embodiments, the albumin is human albumin.

[0266] In some embodiments, the VEGF-C mutein 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-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 is cleavable by proteases such as trypsin, chymotrypsin, or thrombin, or (Leu-Val-Pro-Arg) (SEQ ID NO: 305), which is cleavable by the protease Factor Xa. Examples of rigid linkers include, but are not limited to, the unnatural amino acid azido-lysine, which can form a triazole bond with an alkyne-containing molecule (azido-Lys-Arg-Pro)m, or the unnatural amino acid cyclohexyl-alanine, which can form a rigid cyclohexane structure (cyclohexyl-Ala-Pro-Pro). Examples of such linkers include, but are not limited to, a linker having the amino acid sequence: GGGGGSGGGGSGGGGS (SEQ ID NO: 294).

[0267] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLHHHHHHH (mouse VEGF-C mutein protein having 8xHis (SEQ ID NO: 306) aa sequence and C133A and N163I mutations; SEQ ID NO: 254).

[0268] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGCAAAACTCAATGTATGCCACGGGAGGTTGCAATAGACGTGGGAAAGGAATTTGGCGCCGCCACGAATACCTTTTTCAAGCCTCCCTGTGTGAGTGTTTACAGATGTGGTGGTTGTTGCATATCAGAGGGATTGCAGTGCATGAACACAAGTACAGGTTACTTGAGTAAAACATTGTTTGAAATCACAGTACCATTGTCCCAAGGCCCTAAGCCGGTTACGATCTCTTTCGCCAATCATACGTCATGCCGCTGTATGAGTAAGTTGCATCACCATCACCATCATCACCAT (8xHis (SEQ ID NO: 306) aa sequence and nucleotide sequence encoding a C mutein protein having the C133A and N163I mutations; SEQ ID NO: 260).

[0269] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCRSEGLQCMNTSTGYLSKTLFEITTPISQGPKPVTISFANHTSCRCMSKLHHHHHHH (mouse VEGF-C mutein protein having 8xHis (SEQ ID NO: 306) aa sequence and C133A, N163R, V186T and L188I mutations; SEQ ID NO: 255).

[0270] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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: AACACCGAAATCTTGAAATCAATAGATAACGAATGGCGAAAAACCCAATGTATGCCACGCGAGGTAGCGATAGATGTGGGCAAAGAATTCGGCGCCGCGACGAACACCTTTTTCAAGCCCCCTTGCGTCTCCGTATATAGATGCGGTGGATGTTGCCGATCCGAGGGCCTTCAGTGTATGAACACATCTACTGGCTATTTGAGCAAGACGCTCTTTGAGATTACAACACCAATTAGTCAAGGTCCCAAGCCTGTTACCATCTCTTTCGCTAACCACACTTCATGCCGCTGTATGAGTAAGTTGCATCACCATCACCATCATCACCAT (nucleotide sequence encoding a mouse VEGF-C mutein protein having 8xHis (SEQ ID NO: 306) aa sequence and C133A, N163R, V186T and L188I mutations; SEQ ID NO: 261).

[0271] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (C133A and N163I mutations AAA cloning scar Mouse VEGF-C mutein protein with Fc(N297Q) fusion 6xHis (SEQ ID NO: 25) aa sequence; SEQ ID NO: 256).

[0272] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0273] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (C133A, N163R, V186T and L188I mutations AAA cloning scar Fc (N297Q) fusion 6xHis Mouse VEGF-C mutein protein having the aa sequence; SEQ ID NO: 257).

[0274] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0275] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (C133A and N163I mutations AAA cloning scar and linker mouse serum albumin fusion GS Mouse VEGF-C mutein protein having 8xHis (SEQ ID NO: 306) aa sequence; SEQ ID NO: 258).

[0276] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0277] In certain embodiments, a VEGF-C mutein 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: NTEILKSIDNEWRKTQCMPREVAIDVGKEFGAATNTFFKPPCVSVYRCGGCCISEGLQCMNTSTGYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (C133A, N163R, V186T and L188I mutations AAA cloning scar and linker mouse serum albumin fusion GS Mouse VEGF-C mutein protein having 8xHis (SEQ ID NO: 306) aa sequence; SEQ ID NO: 259).

[0278] In certain embodiments, a polynucleotide molecule encoding a VEGF-C mutein 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:

[0279] In some embodiments, the polynucleotide molecule encoding a VEGF-C mutein or functional fragment thereof is an mRNA.

[0280] mRNA can offer multiple benefits over AAV and other gene delivery systems, including but not limited to one or more of the following: mRNA is highly customizable; mRNA can prevent recognition from pattern recognition receptors and nucleases, allowing for sustained expression; mRNA can provide well-controlled expression kinetics with the option for repeated dosing; and mRNA can be localized in the cytoplasm, reducing the risk of integration into the genome. mRNA can also be cost-effective.

[0281] In some embodiments, the polynucleotide molecule encoding a VEGF-C mutein protein or functional fragment thereof comprises modified nucleotides such as 5-methylcytosine and pseudouridine substitutions, which can improve the stability of the polynucleotide molecule, reduce deamination, reduce nuclease activity, reduce innate recognition, or improve translation efficiency. In some embodiments, the modified nucleotide is 5-methylcytosine or pseudouridine. In some embodiments, the polynucleotide molecule encoding a VEGF-C mutein comprises a 5'cap.

[0282] The mRNA may include modified nucleotides. In some embodiments, the modified nucleotides are 5-methylcytosine or pseudouridine. In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein or functional fragment thereof includes a 5'cap. In some embodiments, the 5'cap is added using CleanCap Reagent AG. CleanCap is composed of C32H43N15O24P4, which allows for high capping efficiency, resulting in more active mRNA. Cap1 does not activate pattern recognition receptors and is important for good in vivo expression. Without wishing to be bound by theory, any one or more of 5-methylcytosine, pseudouridine, and 5'cap may improve the stability of the mRNA, thereby prolonging the expression of the VEGF-C mutein or functional fragment thereof.

[0283] In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein or functional fragment thereof is contained within a viral vector. Exemplary viral vectors include, but are not limited to, herpesvirus, cytomegalovirus, poliovirus, alphavirus, vaccinia virus, rabies virus, adeno-associated virus (AAV), retrovirus, lentivirus, and adenovirus. The retrovirus may be a lentivirus. The recombinant viral particle may be derived from an adeno-associated virus (AAV). In some embodiments, the AAV is AAV2. In some embodiments, the AAV is AAV5. In some embodiments, the AAV is AAV9.

[0284] In some embodiments, the VEGF-C mutein or functional fragment thereof can be administered in a dosing regimen that requires a combination of mRNA and AAV. One or more administrations of mRNA can provide high expression quickly, for example within 2 hours after delivery of the mRNA. Expression of the VEGF-C mutein or functional fragment thereof provided by AAV can take 7-14 days, or up to 4 weeks, depending on the serotype. Administration of a protein or functional fragment thereof can provide immediate expression. Both mRNA and AAV can be administered together or in succession over a short period of time to provide sustained expression of the VEGF-C mutein or functional fragment thereof. Without wishing to be bound by theory, administration of a protein can provide immediate expression and controlled expression kinetics, and multiple doses are possible. Without wishing to be bound by theory, administration of mRNA can provide immediate expression, controlled expression kinetics, and high expression, and multiple doses are possible. Without wishing to be bound by theory, administration of AAV can provide delayed expression and high levels of expression. Expression kinetics can be effectively and sensitively measured using ELISA and Western blotting.

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

[0286] In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein or functional fragment thereof is attached to a nanoparticle or polymer. In certain embodiments, the nanoparticles of the invention further comprise at least one agent that specifically binds to a particular type or category of cells and / or other particular types of compounds (e.g., a moiety that targets a particular cell or cell type). In some embodiments, the nanoparticle is a nanosphere. In some embodiments, the polymer is dextran, poly(amine coester), poly(beta amino ester), polyethyleneimine, poly-L-lysine, polyethylene glycol, or a dendrimer.

[0287] In some embodiments, a polynucleotide molecule encoding a VEGF-C mutein or functional fragment thereof is contained within a recombinant viral particle or virus-like particle (VLP).

[0288] In some embodiments, VEGF-C muteins are produced and administered as "masked" prodrugs that are activated following administration to a patient. In some cases, this can provide desirable tissue or tumor specific activity of the mutein, for example, by limited expression of endogenous proteases that cleave the prodrug to release an active "mature" fragment. In some embodiments, the VEGF-C muteins are variants of the full-length VEGF-C propeptide and are activated by proteases such as, for example, ADAMTS2, plasmin, furin, cathepsin D, thrombin, and / or KLK3 / PSA. In other embodiments, the VEGF-C muteins are fused to inhibitory peptides that are released by proteolysis by endogenous proteases. Inhibitory peptides can take many forms, including but not limited to: antibodies or antibody fragments that bind to VEGF-C muteins and attenuate their interaction with VEGFR-3; VEGFR-3 extracellular domains or fragments thereof that competitively inhibit the interaction of VEGF-C with VEGFR-3 on cells; and any other binding factors (e.g., VHHs, fibronectin domains, knottins, lipocalins, leucine-rich repeats, etc.). A non-limiting set of examples of protease cleavage sites is described below. Inhibitory peptides can be separated from biologically active VEGF-C muteins by an enterokinase cleavage site (EKCS) peptide that is recognized and cleaved by enterokinase, an endogenous protease present in the small intestine. This allows the prodrug to be designed so that it is inactive until it reaches the small intestine, where it can be activated by enterokinase cleavage. Alternatively, the inhibitory peptide can be separated from the biologically active VEGF-C mutein by a furin cleavage site (FCS) peptide that is recognized and cleaved by furin, an endogenous protease that is overexpressed in many tumor cells.The prodrug can be designed to be specifically cleaved by furin, thereby allowing activation of the prodrug to be localized to the tumor microenvironment, resulting in targeted therapy. Furthermore, the prodrug can be designed to be cleaved by other endogenous proteases that are overexpressed in certain tumors. For example, the prodrug can be designed to be cleaved by matrix metalloproteases MMPs or urokinase-type plasminogen activators uPA, which are overexpressed in many tumors. This allows activation of the prodrug to be localized to the tumor microenvironment, resulting in targeted VEGF-C mutein therapy.

[0289] In one aspect, there is provided 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 mutein protein or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or a functional fragment thereof, or a fusion protein thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

[0290] In one aspect, provided herein is a method of treating a disease or condition in a subject in need of such treatment, comprising administering to the subject an effective amount of a VEGF-C mutein protein or functional fragment thereof of the present disclosure, 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.

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

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

[0293] In some embodiments, the VEGF-C mutein protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisternally, intravitreally, ophthalmic, subcutaneously, intradermally, by inhalation, via a chronic 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 intratumor 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 ophthalmic 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 via a chronic indwelling catheter. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical compositions are formulated for topical administration (eg, as a cream or gel).

[0294] In one aspect, a method of treating cancer in a subject in need of cancer treatment is provided, comprising administering to the subject an effective amount of a VEGF-C mutein protein or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or a functional fragment thereof, or a fusion protein thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

[0295] In one aspect, a pharmaceutical composition is provided comprising a VEGF-C mutein or a functional fragment thereof, and optionally an immunotherapeutic agent.

[0296] In one aspect, a method is provided for generating a library of VEGF-C muteins or functional fragments thereof, the VEGF-C muteins or functional fragments thereof having: (i) specific binding to VEGF; and (ii) reduced binding to VEGFR-2 compared to wild-type VEGF-C.

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

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

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

[0300] 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.

[0301] In certain embodiments, the cancer is melanoma, lung cancer, breast cancer, gastric cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, squamous cell carcinoma of the head and neck, thyroid cancer, liquid cancer (e.g., acute myeloid leukemia), renal cancer, urothelial cancer, prostate cancer, pheochromocytoma, cholangiocarcinoma, hepatocellular carcinoma, invasive pancreatic ductal carcinoma, thymoma, sarcoma, mesothelioma, testicular cancer, and colorectal cancer.

[0302] In certain embodiments, the disclosed VEGF-C muteins or functional fragments thereof (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used for the treatment or regulation of cardiac function or disease. Examples of such cardiac function or disease include, but are not limited to, coronary vascular function, osmoregulation, cardiac ischemia, and restenosis.

[0303] In certain embodiments, the VEGF-C muteins or functional fragments thereof of the present disclosure (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used in respiratory medicine.

[0304] In certain embodiments, the VEGF-C muteins or functional fragments thereof of the present disclosure (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used for the treatment of fibrosis.

[0305] In certain embodiments, the VEGF-C muteins or functional fragments thereof of the present disclosure (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used in immunology.

[0306] In certain non-limiting embodiments, the VEGF-C muteins or functional fragments thereof disclosed herein (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used for lymph node transplantation.

[0307] In certain embodiments, the disclosed VEGF-C muteins or functional fragments thereof (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used for gut health. In certain non-limiting embodiments, the VEGF-C muteins or functional fragments thereof can be used for the treatment of colitis and chronic liver disease.

[0308] In certain embodiments, the VEGF-C muteins or functional fragments thereof of the present disclosure (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used in nephrology.

[0309] In certain non-limiting embodiments, the VEGF-C muteins or functional fragments thereof of the present disclosure (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used for the treatment of polycystic kidney disease.

[0310] In certain non-limiting embodiments, the VEGF-C muteins or functional fragments thereof of the present disclosure (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used for the treatment of Alzheimer's disease, Parkinson's disease, stroke, and cerebral ischemia associated with lung injury. In certain embodiments, the VEGF-C muteins or functional fragments thereof have neuroregenerative properties.

[0311] In certain non-limiting embodiments, the VEGF-C muteins or functional fragments thereof of the present disclosure (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used for wound healing.

[0312] In certain non-limiting embodiments, the disclosed VEGF-C muteins or functional fragments thereof (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used for the treatment of lymphedema. Non-limiting examples of lymphedema include primary lymphedema, secondary lymphedema, and hereditary lymphedema.

[0313] In certain embodiments, the disclosed VEGF-C muteins or functional fragments thereof (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used for the treatment of genetic diseases. Non-limiting examples of such genetic diseases include Milroy's disease, Hennekam's syndrome, and Turner's syndrome.

[0314] In certain embodiments, the disclosed VEGF-C muteins or functional fragments thereof (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used for the treatment of ocular diseases. Non-limiting examples of such ocular diseases include age-related macular degeneration, glaucoma, diabetic retinopathy, central serous chorioretinopathy, macular edema, and retinal edema.

[0315] In certain embodiments, the VEGF-C muteins or functional fragments thereof of the present disclosure (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used in combination with chimeric antigen receptor (CAR) T cells.

[0316] In certain embodiments, the VEGF-C muteins or functional fragments thereof disclosed herein (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used in combination with a cancer vaccine.

[0317] In some embodiments, the method does not include administering a tumor-specific antigen to the subject.

[0318] In certain embodiments, the disclosed VEGF-C muteins or functional fragments thereof (or fusion proteins or conjugates thereof, or polynucleotide molecules encoding said VEGF-C mutein proteins or functional fragments thereof or fusion proteins thereof, or vectors or particles comprising said polynucleotide molecules, or pharmaceutical compositions comprising any of the above) can be used in combination with immunotherapeutic agents. In some embodiments, 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.

[0319] The VEGF-C mutein or functional fragment thereof (or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or a functional fragment thereof or a fusion protein thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above) and the immunotherapeutic agent may be administered together, for example, the VEGF-C mutein and the immunotherapeutic agent are administered in the same composition.

[0320] Alternatively, the VEGF-C mutein or functional fragment thereof (or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or functional fragment thereof or a fusion protein thereof, or a vector or particle containing said polynucleotide molecule, or a pharmaceutical composition comprising any of the above) and the immunotherapeutic agent may be administered sequentially.

[0321] In various embodiments, the VEGF-C mutein or functional fragment thereof (or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or a functional fragment thereof or a fusion protein thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above) is administered prior to administration of an immunotherapeutic agent. The VEGF-C mutein or functional fragment thereof (or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or a functional fragment thereof or a fusion protein thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be administered locally to the brain or central nervous system (e.g., into the cisterna magna), followed by systemic (e.g., intravenous) administration of an immunotherapeutic agent. A VEGF-C mutein or a functional fragment thereof (or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or a functional fragment thereof or a fusion protein thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be administered intratumorally, followed by systemic (e.g., intravenous) administration of an immunotherapeutic agent. A VEGF-C mutein or a functional fragment thereof (or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or a functional fragment thereof or a fusion protein thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be administered intrathecally, followed by systemic (e.g., intravenous) administration of an immunotherapeutic agent. A VEGF-C mutein or a functional fragment thereof (or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or a functional fragment thereof or a fusion protein thereof, or a vector or particle containing said polynucleotide molecule, or a pharmaceutical composition comprising any of the above) can be administered directly into the lymphatic system, followed by systemic (e.g., intravenous) administration of an immunotherapeutic agent.

[0322] In various embodiments, the method for treating cancer further comprises administering to the subject an additional anti-cancer treatment. Examples of the additional anti-cancer treatment include, but are not limited to, surgery, radiation therapy, administration of a chemotherapeutic agent, and any combination thereof. The additional anti-cancer treatment may be administered before, simultaneously with, or after administration of a VEGF-C mutein or a functional fragment thereof (or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding the VEGF-C mutein 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).

[0323] In various embodiments, the subject is a human patient. The human patient may be a child or an adult.

[0324] In various embodiments, the method is effective for treating the cancer in the subject. In some embodiments, the method is effective for inducing lymphangiogenesis in a tumor in the brain or central nervous system of the subject. In various embodiments, lymphangiogenesis can be confirmed by MRI imaging, where, for example, a contrast agent can be used to calculate the diameter of the lymphatic vasculature. In various embodiments, lymphangiogenesis can be confirmed by serial CSF collection to measure VEGFA, VEGFB, VEGFC, or VEGFD concentrations. In various embodiments, lymphangiogenesis can be confirmed by serial CSF collection to measure VEGFC concentrations. The method can be effective for reducing tumor volume. In some embodiments, the method is effective for reducing tumor volume in the brain or central nervous system of the subject. In various embodiments, the method is effective for providing immune memory against the tumor. Without wishing to be bound by theory, the poor clinical efficacy of immunotherapy in GBM patients may be due to poor antigen sampling from the CNS at steady state and in early stages of tumor development. Administration of VEGF-C muteins or functional fragments thereof can increase the amount of antigen sampling that occurs in the brain, thereby improving the efficacy and outcome of any other immunotherapy administered (e.g. anti-CTLA-4 antibodies). The role of VEGF-C in cancer has been thought to be to promote metastasis via (lymphatic) angiogenesis, but the inventors have surprisingly shown that in the brain, VEGF-C can reduce tumor size by increasing immune surveillance. VEGF-C can stimulate the proliferation of lymphatic endothelial cell proliferation via VEGFR-3 and enhance lymphatic function.

[0325] In one aspect, provided herein is a method of regulating intraocular pressure in a subject in need thereof, comprising the step of administering to the subject an effective amount of a VEGF-C mutein protein or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or a functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above, or a corresponding wild-type VEGF-C protein or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said wild-type VEGF-C protein or a functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

[0326] In one aspect, provided herein is a method of removing unwanted intraocular fluid in a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutein protein of the present disclosure or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above, or a corresponding wild-type VEGF-C protein or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said wild-type VEGF-C protein or functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

[0327] In some embodiments, the unwanted fluid is optic nerve fluid, retinal fluid, subretinal fluid, choroidal fluid, or suprachoroidal fluid.

[0328] In some embodiments where the disclosed method is a method of regulating intraocular pressure in a subject in need of regulation of intraocular pressure or a method of removing unwanted intraocular fluid in a subject in need of removal of unwanted intraocular fluid, the subject is afflicted with glaucoma, macular edema, central serous chorioretinopathy, retinal edema, papilledema, macular degeneration, or diabetic retinopathy.

[0329] In some embodiments, where the method of the disclosure is a method of regulating intraocular pressure in a subject in need of regulating intraocular pressure or a method of removing unwanted intraocular fluid in a subject in need of removing unwanted intraocular fluid, the VEGF-C mutein protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered to the posterior segment of the eye. In some embodiments, the administration is intraocular administration. In some embodiments, the intraocular administration is intravitreal administration, ocular administration, or subretinal administration.

[0330] In one aspect, provided herein is a method of providing neuroprotection to a subject in need thereof, comprising administering to the subject an effective amount of a VEGF-C mutein protein of the present disclosure or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or a functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above, or a corresponding wild-type VEGF-C protein or a functional fragment thereof, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said wild-type VEGF-C protein or a functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above. In some embodiments, neuroprotection is provided for, but not limited to, macular degeneration, glaucoma, stroke, Alzheimer's disease, or Parkinson's disease. In some embodiments, the administration is intraocular administration. In some embodiments, the intraocular administration is intravitreal administration, eye drop administration, or subretinal administration.

[0331] In one aspect, provided herein is a vaccine comprising a VEGF-C mutein protein or functional fragment thereof of the present disclosure, or a fusion protein or conjugate thereof, or a polynucleotide molecule encoding said VEGF-C mutein protein or functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

[0332] In one aspect, provided herein is a method of inducing an immune response in a subject in need thereof, 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 other anti-pathogen immune response. EXAMPLES

[0333] To further illustrate some of the embodiments disclosed herein, the following examples are provided, which are intended to illustrate, but not to limit, embodiments of the present disclosure.

[0334] Example 1. Generation of VEGF-C muteins Therapeutic stimulation of lymphatic vessel development (lymphangiogenesis) is a promising new therapeutic paradigm for a wide range of indications, from cardiovascular disease to cancer immunotherapy. However, specific pharmacological agents that promote lymphangiogenesis without also stimulating potentially deleterious angiogenesis are currently lacking. In particular, vascular endothelial growth factor C (VEGF-C), the major physiological driver of lymphangiogenesis, stimulates pro-lymphangiogenic signaling through pro-VEGFR-3 as well as pro-angiogenic VEGFR-2-mediated signaling. Here, we used directed evolution by yeast surface display to generate a lymphatic-specific VEGF-C variant (LS-VEGF-C) that binds exclusively to VEGFR-3 and does not signal through VEGFR-2. Compared to wild-type VEGF-C, LS-VEGF-C showed superior preclinical efficacy in models of lymphangiogenesis and anti-PD-1 cancer immunotherapy. The biased influence of LS-VEGF-C towards lymphangiogenesis over angiogenesis was further determined herein by studying its effect on enhancing lymphatic drainage in the posterior segment of the eye. LS-VEGF-C significantly reduced intraocular pressure in normotensive mice and in two separate mouse models of ocular hypertension without the increased angiogenesis and vascular permeability seen with WT-VEGF-C treatment. LS-VEGF-C also exhibited neuroprotective properties independent of intraocular pressure in a model of retinal ganglion cell death due to excitotoxic injury. Taken together, these results highlight the feasibility and preclinical efficacy of therapeutic lymphangiogenesis via VEGFR-3-specific agonism and reveal an unexpected impact of lymphangiogenesis in controlling intraocular pressure.

[0335] Protein expression, purification, and biotinylation The extracellular domains of mouse VGFR2 (first three Ig domains, amino acids 20-326), VGFR3 (amino acids 25-329), human VGFR2 (amino acids 19-325), and VGFR3 (amino acids 25-329) were cloned into pEZT vector with an N-terminal H7 signal peptide, a C-terminal AviTag, and a hexahistidine tag (SEQ ID NO:25) and expressed by transient transfection of Expi293 cells (Thermo Fisher) 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) in the final buffer HEPES-buffered saline (HBS; 10 mM HEPES, pH 7.5, 150 mM NaCl). Biotinylation of proteins was carried out for 2 hours at room temperature using in-house purified BirA ligase enzyme in 0.1 mM bicine (pH 8.3), 10 mM ATP, 10 mM magnesium acetate, and 0.5 mM biotin (Avidity, #BIO500). Biotinylated proteins were then purified by gel filtration as described above. Biotinylation efficiency was assessed using an SDS / PAGE streptavidin shift assay.

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

[0337] Figure 2A shows multiple strategies for displaying VEGF-C on the yeast surface that were utilized to optimize surface VEGF-C dimerization. The final combination displayed wild-type VEGF-C with high binding affinity to VEGFR-3 and low affinity to VEGFR-2. Figure 2B is a schematic showing yeast display library selection aimed at discovering VEGFR-3-specific VEGF-C muteins.

[0338] Analysis of the three-dimensional structures of the VEGF-C:VEGFR3 and VEGF-C:VEGFR2 complexes identified 14 residues. A library randomizing these residues (Figure 3) was constructed using assembly PCR. To enable homologous recombination, the PCR products were further amplified with primers with homology to the yeast display vector backbone. Transformed yeast were harvested and grown in SDCAA medium.

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

[0340] Construction and selection of a mouse VEGF-C library The crystal structures of the VEGF-C / VEGFR-2 complex (PDB ID 2XIW) and VEGF-C / VEGFR-3 (PDB ID 4BSK) were aligned to analyze VEGF-C and its receptor interface. Fourteen positions in mVEGF-C that contact both mVEGFR-2 and mVEGFR-3 were identified to generate a mutation library (Table 1). The library was assembled using synthetic degenerate primers that cover these mutated residues (Table 2). The PCR products were further amplified using primers with homology to the vector and co-electroporated with the linearized pCT-GCN42 vector into EBY100 competent yeast. The resulting library was subsequently measured to have a 10 8 Figure 3 shows the design of the VEGF-C mutein library. 3To generate a combination of VEGF-C muteins, we designed a structure-based primer library. We studied the structures of VEGF-C bound to VEGFR-3 and VEGFR-2 and identified key residues on the surface of the binding site. Amino acids that could potentially cause significant polarity changes at these residues were selected for primer design.

[0341] [Table 1]

[0342] [Table 2]

[0343] Transformed yeast were harvested and grown in SDCAA medium at 30 °C and induced for 24-48 h in SGCAA medium at 20 °C. The naive library was selected using two rounds of 1 μM mVEGFR-3 and mVEGFR-3 positive binders were enriched using LS columns (Miltenyi, #130-042-401) and magnetic selection. From round 3, yeast populations were counterselected with 1 μM mVEGFR-2 monomer and selected with 100 nM mVEGFR-3 using flow cytometer sorting on a Sony SH800 cell sorter. For rounds 4-6, the concentration of the counterselection reagent mVEGFR-2 was gradually increased to 0.25 μM (round 4) and 0.5 μM (rounds 5, 6) tetramer, while the mVEGFR-3 concentration was decreased to 10 nM (round 4), 2 nM (round 5), or 1 nM (round 6). Yeast after each selection round was retained and boosted simultaneously to check the binding affinity to VEGFR-2 and VEGFR-3 when all selections were completed. After the final selection round, the VEGF-C plasmid was extracted from the grown SDCAA cultured yeast and transformed into E. coli for colony sequencing (Table 3). The collected unique clones were retransformed into yeast and titrated for binding preference to VEGFR-2 and VEGFR-3. The final selected VEGF-C mutants were expressed by insect cell pFastbac expression system and purified by FPLC using a SEC column. Monodisperse proteins were finally removed by endotoxin removal for in vivo animal treatment. As shown in FIG. 4, wild-type VEGF-C showed binding to yeast expressing both VEGFR-2 and VEGFR-3 (top row, WT). The first two rounds selected for binding to VEGFR-3. Subsequent rounds exploited the reduced amount of VEGFR-3 to select for high binding affinity muteins to VEGFR-3. Concurrently, VEGFR-2 was included and negatively selected to enrich for muteins that had high binding to VEGFR-3 but lost binding affinity to VEGFR-2. FIG. 5 shows confirmation of receptor affinity preference after the final selection round.Muteins positively selected for VEGFR-3 and negatively selected for VEGFR-2 show high binding to VEGFR-3 and no binding to VEGFR-2, even at 100-fold higher receptor concentration than wild-type binding. Figure 6 shows confirmation of receptor affinity preference after the final selection round. Clonal muteins after selection rounds 5 and 6 were sequenced to identify unique clones enriched within the mutein population. Unique mutein residues were identified and specific mutations were made in wild-type VEGF-C for validation.

[0344] Figure 7 shows confirmation of receptor affinity preference after the final selection round. Clonal muteins after selection rounds 5 and 6 were sequenced to identify unique clones enriched within the mutein population. Unique mutein residues were identified and mutations specific to wild-type VEGF-C were purified for validation.

[0345] Figure 8 shows confirmation of receptor affinity preference after the final round of selection for human VEGF-receptors. Muteins positively selected for VEGFR-3 and negatively selected for VEGFR-2 show high binding to VEGFR-3 and no binding to VEGFR-2, even at 100-fold higher receptor concentrations compared to wild-type binding.

[0346] [Table 3]

[0347] Creation of lymphatic-specific VEGF-C (LS-VEGF-C) Increasing lymphatic drainage has therapeutic benefit in conditions such as lymphedema, where fluid accumulation due to disruption of lymphatic infrastructure is directly addressed. 2 From a more immunological perspective, utilizing meningeal lymphatics may promote enhanced immune responses against glioblastoma by increasing drainage of brain antigens through lymphangiogenesis. 3These applications demonstrate not only the therapeutic potential of harnessing lymphangiogenesis but also its versatility, even in immune-privileged areas.

[0348] Previous studies have shown that VEGFR-2 primarily promotes angiogenesis. 4 , VEGFR-3 promotes lymphangiogenesis 5~8 It has been shown that vascular endothelial growth factor C (VEGF-C) is a major growth factor that can promote lymphatic vessel growth and pumping, but its multitropic nature of binding to both VEGFR-2 and VEGFR-3 (Figure 19) limits its use as a therapeutic and molecular tool for studying in vivo signaling. Furthermore, the ability of VEGF-C to promote sufficient lymphangiogenesis is variable, with high doses often required for significant effects. 9 collectively, these shortcomings prevent the full realization of the therapeutic potential of VEGF-C-driven lymphangiogenesis.

[0349] To overcome these limitations, the generation of more selective, yet still potent, VEGF-C muteins was sought. To develop mutant VEGF-C that binds only VEGFR-3 and promotes lymphangiogenesis, 14 residues in the receptor binding interface were identified by structural analysis for randomization (Figure 3). Using a yeast display directed evolution platform (Figure 2B), a library was created that underwent six rounds of selection for selectivity against VEGFR-3 and counterselectivity against VEGFR-2 (Figure 4). After this final selection, the population of variants showed conserved exclusive binding to VEGFR-3, but no binding to VEGFR-2, in both the human (Figure 8) and mouse versions of the receptor (Figures 15A and 15B). Notably, VEGFR-3 binding of these variants exceeds that of the previously established VEGFR-3-specific ligand VEGF-C152S / C-156S (Figure 12). Examination of clones identified seven key residues, 115, 119, 126, 163, 164, 186, and 188, resulting in the final variant of the present invention (LS-VEGF-C); biophysical characterization by surface plasmon resonance demonstrated that LS-VEGF-C retained picomolar-level affinity for VEGFR-3 while completely suppressing VEGFR-2 affinity compared to WT-VEGF-C (Figures 6 and 7).

[0350] To assess whether functionality was preserved, HUVEC and HDLEC cells, vascular and lymphatic endothelial cells, respectively, were used. Upon administration of VEGF-A, LS-VEGF-C, and WT-VEGF-C, which exhibit binding to VEGFR-2, VEGFR-3, or both (Figure 1B), downstream signaling was detected by measuring intracellular ERK activation. HUVEC expressing VEGFR-2 showed similar enhanced ERK activation by VEGF-A and WT-VEGF-C, but not LS-VEGF-C (Figure 15C). In contrast, HDLEC showed similar ERK phosphorylation between WT-VEGF-C and LS-VEGF-C (Figure 15C), reflecting the preserved VEGFR-3 binding and signaling in the mutant proteins. Examination of cell proliferation upon VEGF treatment confirms this trend; vascular endothelial cells were increased in proliferation by VEGF-A and WT-VEGF-C, whereas lymphatic endothelial cells were increased in proliferation by both VEGFs, but most statistically significantly by LS-VEGF-C (Figure 15D). Because lymphatic endothelial cells express both VEGFR-2 and VEGFR-3, it is not entirely surprising that VEGF-A and WT-VEGF-C also increased lymphatic endothelial cell proliferation. 5 Together, these data indicate that LS-VEGF-C is a novel mutant form of VEGF-C with high specificity for VEGFR-3 binding and lymphangiogenesis.

[0351] mRNA / Nanoparticles mRNA encoding VEGF-C wild type and muteins was synthesized by TriLink Bio Technologies with complete substitution of pseudouridine and 5-methylcytosine bases, capped with CleanCap reagent AG, and polyadenylated (120A). The mRNA was mixed with 0.1 μL of in vivo-jetPEI (Polyplus Transfection) per ug, vortexed for 30 seconds, and incubated at room temperature for 15 minutes before use.

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

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

[0354] Western blotting has been reported previously. 3The assay was performed in a similar manner to that described in Example 1. Briefly, 10% gels were used and processed at 10 mA per gel for 30 min and 40 mA per gel until adequate separation of the ladder. Wet transfer was performed on ice for 90 min at 120 mA per gel. After blocking with nonfat dry milk-TBST and washing three times with TBST, anti-pERK was used at a concentration of 1:1000 and incubated overnight in a cold room. After washing, HRP-conjugated anti-rabbit secondary antibody was used at a concentration of 1:500 for 2 h at room temperature and imaged using a ChemiDoc MP imaging system (Bio-Rad).

[0355] mouse Six- to 10-week-old mixed-sex C57BL / 6J (WT), DBA2J, and DBA / 2J-Gpnmb+ / SjJ mice were purchased from Jackson Laboratory and then bred and housed at Yale University. All procedures (sex-matched, age-matched) used in this study were in accordance with federal guidelines and the institutional policies of the Yale School of Medicine Animal Care and Use Committee.

[0356] MTT assay 10,000 cells were seeded per well in a 96-well plate. Cells were first starved for 12 hours in RPMI medium without supplements, washed with PBS, and then processed to the experimental conditions. Negative controls were processed to RPMI medium with 1% FBS supplement. Experimental conditions consisted of the addition of 100ng / mL VEGF-A, WT-VEGF-C, or LS-VEGF-C to the negative control medium (RPMI medium with 1% FBS supplement). A standard curve was generated by cell titration. Each sample was then supplemented with 10uL of 12mM MTT stock solution. After homogenization of each well by pipetting, the absorbance was measured at 570nm and the cell number was determined by comparison with the standard curve.

[0357] Isolation of endothelial cells For single cell dissociation, the cornea, retina, choroid, and optic nerve were isolated. These tissues were cultured at 1 mg ml in RPMI at 37 °C. -1 of collagenase D (Roche) and 30 μg ml -1 The tissue was digested with 100 mL of DNase I (Sigma-Aldrich) for 45 min. The samples were then pipetted to mechanically dissociate the tissue and filtered through a 70 um filter. The samples were then centrifuged at 500xg for 5 min. The cell pipettes were then resuspended in FACS buffer (PBS containing 2% FBS and 1 mM EDTA) for staining.

[0358] Flow cytometry Prior to immunostaining, non-specific binding was blocked using Fc receptor blocking solution (TruStain FcX™, 101320, BioLegend) for 10 min at 4°C. Then, cells were stained with corresponding antibodies for 30 min at 4°C. Cells were then washed to remove excess antibodies and resuspended in FACS buffer. Samples were processed on an Attune NxT flow cytometer and analyzed using FlowJo software (10.8.1, Tree Star).

[0359] For AKT phosphorylation staining, surface markers were first stained for 30 min on ice. Cells were then fixed and stained according to the BD Phosflow kit instructions. Samples were processed on an Attune NxT flow cytometer and analyzed using FlowJo software (10.8.1, Tree Star).

[0360] Measurement of intraocular pressure Intraocular pressure was measured according to consensus-based recommendations (https: / / iovs.arvojournals.org / article.aspx?articleid=2778419). Mice were briefly exposed to isoflurane for sedation, after which intraocular pressure was measured with an iCare tonometer. IOP measurements are the average of six measurements from one sedation session. Mice were positioned so that the probe was perpendicular to the ocular surface when measurements were taken.

[0361] Intraocular administration Ketamine (50 mg kg -1 ) and xylazine (5 mg kg -1 ) was injected intraperitoneally, followed by topical, intravitreal, or intracameral dosing in mice. Topical dosing consisted of placing 5uL of a 1ug / uL solution on the eye. Intravitreal and intracameral dosing consisted of creating a small hole along the edge of the cornea to allow access for a Hamilton syringe. For intracameral dosing, the needle entered the anterior chamber. For intravitreal dosing, the needle entered the vitreous humor cavity. For both intravitreal and intracameral dosing, a 1ug / uL solution was used. After dosing, the eye was covered with artificial tear ointment. The non-evaluated eye was also covered with artificial tear ointment to prevent drying.

[0362] Combination therapy Two days after IVT administration of LS-VEGF-C, ketamine (25 mg kg -1 ) and xylazine (2.5 mg kg -1 Mice were anesthetized by intraperitoneal injection of a mixture of 5-aminopropyl 1,2-diphenylmethane (5-HT) and 5-methyl-1,2-diphenylmethane (5-HT) and then administered FDA-approved drugs topically to the eye to evaluate the effects of the combination.

[0363] Microbead Model Sappington et al. 18 Based on the above, ketamine (50 mg kg -1 ) and xylazine (5 mg kg -1 Mice were anesthetized by intraperitoneal injection of a mixture of 1,2-dichlorophenyl phthalate (1,2,3-trimethylphenyl phthalate) and then administered polystyrene beads into the anterior chamber. Artificial tear ointment was then placed topically to prevent drying.

[0364] Intravitreal AAV injection and imaging WT mice were intravitreally injected with AAV (dose) with 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 -1Mice were anesthetized by intraperitoneal injection of a mixture of 100μL of 20mg / mL dye. 100uL of 20mg / mL dye was also injected intraperitoneally. Pupils were dilated with 1% tropicamide. After 5 minutes of incubation, mice were mounted for fundus imaging, fluorescein angiography, and OCT imaging on a Phoenix Micron IV.

[0365] Evans Blue absorbance measurement For Evans Blue readings, Evans Blue was injected intraperitoneally instead of the dye. After 4 hours, mice were euthanized and perfused with PBS. Eyeballs were isolated and homogenized with beads before processing on a plate reader (Abs_max at 620 nm).

[0366] Tissue processing and microscopy For retinal whole mount preparations, mouse eyes were first enucleated and then fixed in 1% formaldehyde. After carefully removing the optic nerve, cornea, and sclera, the isolated retina was cut into quarters by cutting the optic nerve in half. After staining with Brn3a and DAPI, confocal imaging was performed with a Leica SP8 microscope.

[0367] For cross-sectioning of the optic nerve, the nerve was fixed in 4%, then processed and embedded in a resin / OTC / paraffin mixture. The blocks were then sectioned on a microtome / cryostat. The cross-sections were visualized by transmission electron microscopy.

[0368] Research on NMDA-induced excitation Schluter et al. 31 Based on this, 10 nmol of NMDA was delivered intravitreally, and RGCs were evaluated 1 day after injection by confocal microscopy of retinal whole mount preparations as described above.

[0369] statistical analysis All statistical analyses were performed using GraphPad Prism software. Data were analyzed with unpaired two-tailed 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.

[0370] Intravitreal injection Mice were anesthetized with ketamine and xylazine. One drop of 0.5% tropicamide was applied to the eye. Mice were positioned so that the superonasal region of the eye was exposed, and a 33 g needle was used to puncture the superonasal sclera at the level of the pars plana. The mouse's head was fixed and the needle was positioned at a 45° angle. After inserting the tip, 2 μL of the mRNA-nanoparticle formulation was injected, and the needle was left in place for 5 seconds to prevent backflow. Ophthalmic antibacterial ointment was then applied to prevent infection, and the mice were placed in a warmed cage until fully recovered. Figure 10 shows the use of VEGF-C muteins in vivo. A single intravitreal administration of VEGF-C muteins caused a sustained reduction in intraocular pressure (IOP) in wild-type mice compared to their counterparts.

[0371] Tumor inoculation Ketamine (50 mg kg) injected intraperitoneally -1 ) and xylazine (5 mg kg -1 Mice were anesthetized with a mixture of 100 mM NaCl and 100 mM NaCl. The heads of mice were shaved and placed in a stereotaxic frame. After sterilization of the scalp with alcohol and betadine, a midline scalp incision was made to expose the coronal and sagittal sutures, and a burr hole was drilled 2 mm lateral to the sagittal suture and 0.5 mm posterior to the buglema. A 10 μl Hamilton syringe loaded with tumor cells was inserted into the burr hole at a depth of 2.5 mm from the brain surface and allowed to equilibrate for 1 min before injection. A microinfusion pump (World Precision Instruments) was used to infuse 3 μl of tumor cells at 1 μl min. -1After completion of the injection, the syringe was left in place for an additional minute before being removed. The burr hole was filled with bone wax, and the skin was stapled and cleaned. Analgesics (meloxicam and buprenorphine, 1 mg kg -1 After intramuscular administration of VEGF-C, mice were placed in a heated cage until fully recovered. FIG. 11 shows the use of VEGF-C muteins in vivo. Wild-type VEGF-C and VEGF-C muteins were evaluated in vivo for the treatment of brain tumors. When combined with anti-PD-1 antibodies, VEGF-C muteins showed significant therapeutic benefits for treating brain tumors. Furthermore, as shown in FIG. 12, VEGF-C mutants reported in the literature have lost binding affinity to both VEGFR-2 and VEGFR-3. Mutants such as C152S previously reported in the literature have lost activity to both VEGFR-2 and VEGFR-3, and have lost their potent lymphangiogenic activity. As shown in FIG. 13, the isolated VEGF-C muteins of the present disclosure showed specific signaling through VEGFR-3 in vivo. Signaling of wild-type VEGF-C and VEGF-C muteins (RTI) was evaluated in vivo. These vectors were injected into the eyes of mice. After 24 hours, eyes were enucleated and single cell suspensions were made to check for AKT phosphorylation. Wild-type VEGF-C retained signaling through VEGFR-2 in vascular endothelial cells, whereas mutant VEGF-C (RTI) no longer signaled. Both the wild-type protein and the RTI mutein signaled through VEGFR-3 in lymphatic endothelial cells.

[0372] Intracisternal injection For intracisternal injection, mice were anesthetized with ketamine and xylazine, and the dorsal neck was shaved and washed with alcohol. A 2 cm incision was made at the base of the skull, and the dorsal neck muscles were separated using forceps. After the cisterna magna was visualized, the dura was punctured using a Hamilton syringe with a 15° 33-gauge needle. Three microliters of mRNA vector (4–5 μg) per mouse was infused at 1 μl min. -1After the injection was completed, the needle was left in place for an additional 3 minutes to prevent backflow. The skin was stapled and cleaned, and the same post-operative care as for tumor inoculation was performed.

[0373] Example 2. LS-VEGF-C exhibits potent therapeutic effects in multiple conditions. To confirm that LS-VEGF-C acts through VEGFR-3 signaling in vivo, we first administered LS-VEGF-C and WT-VEGF-C to wild-type mice and then used Phosflow to monitor endothelial cells (CD45 - CD31 + ) was evaluated. WT-VEGF-C showed a significant increase in AKT phosphorylation in endothelial cells, regardless of VEGFR-3 expression (Figure 13). In contrast, LS-VEGF-C showed a significant increase in AKT phosphorylation in endothelial cells, regardless of VEGFR-3 expression (CD45 - CD31 + VEGFR3 + ) but not VEGFR-3 expressing cells (CD45 - CD31 + VEGFR3 - ), but not LS-VEGF-C, suggesting that the in vivo activity of LS-VEGF-C mirrors that observed in vitro.

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

[0375] Example 3. Use of ocular lymphatics for biophysical regulation. Previous studies have shown that the eye has a lymphatic system that drains the anterior compartment; the conjunctiva. 11 and ciliary body 12 lymphatics in the posterior part of the lymphatic system, as well as the posterior glymphatic clearance system 13 has been previously described. Recently, our team has determined that the ocular lymphatics are compartmentalized, establishing that the anterior and posterior portions of the eye have separate lymphatic drainage systems. It is hypothesized that by expanding these lymphatic structures through lymphangiogenesis, intraocular administration of LS-VEGF-C will lead to increased drainage and reduce intraocular pressure in the eye.

[0376] WT-VEGF-C was 14 Similarly, LS-VEGF-C produced low intraocular pressure (IOP) measurements by intracameral injection and after intravitreal injection (FIG. 17A). In contrast, LS-VEGF-C produced sustained and enhanced reductions in IOP after any administration method, whether topical, intracameral, or intravitreal (FIG. 17B). The impact and versatility of LS-VEGF-C may stem from its VEGFR-3 specificity, whereas WT-VEGF-C has two potential receptors to bind, namely VEGFR-2, which acts as a VEGF-C sink. 15 This may also contribute to the lack of efficacy of WT-VEGF-C when administered topically, as well as the rapid clearance and multiple intraocular barriers that must be overcome, which preclude multiple topical applications. 16 Interestingly, in mRNA form, WT-VEGF-C was ineffective in lowering IOP when administered topically or intravitreally, but performed similarly to the more effective LS-VEGF-C when administered intracamerally (FIG. 21A), highlighting the importance of both the delivery method and the vehicle. Importantly, LS-VEGF-C showed a long-lasting effect in lowering intraocular pressure, with IOP reduction observable within hours and sustained for up to several days (FIGS. 21B and 21C), compared to commonly used glaucoma eye drops that only show activity for a few hours. The activity of LS-VEGF-C as a single agent was significantly higher than other glaucoma drugs, and combination therapy was not required to achieve a plateau in IOP reduction (FIG. 21D). The efficacy of LS-VEGF-C in a normotensive IOP model demonstrated the translation of our in vitro findings to a physiological phenomenon.

[0377] Example 4. LS-VEGF-C eliminates adverse vascular effects in vivo. To assess off-target effects and vascular changes due to VEGF growth factors, retinal vasculature was imaged after intravitreal administration of VEGF-A, WT-VEGF-C, and LS-VEGF-C. Fundus photographs showed hyperreflective spots along with increased vascular tortuosity in VEGF-A and WT-VEGF-C compared to PBS controls (Figure 17C, left column). Fluorescein angiography showed similar vascular anatomy with improved visibility of permeability into the retinal stroma. VEGF-A and WT-VEGF-C increased the presence of dye in extravascular regions (Figure 17C, center column), which could be quantified by measuring the fluorescence intensity throughout the image (Figure 17D). Intensities above 40 were not observed in control and LS-VEGF-C treated retinas except in areas overlying major blood vessels, whereas multiple large localized patches showed intensities above 40 in VEGF-A and WT-VEGF-C treated retinas. Optical coherence tomography (OCT) showed findings consistent with angiography, with pockets of fluid near the choroid / retinal pigment epithelium layer after administration of VEGF-A or WT-VEGF-C to eyes, but not LS-VEGF-C (Figure 17C, right column). As a parallel method to validate 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 angiography data, these data revealed that LS-VEGF-C was able to attenuate the increase in vascular permeability, indicating an unaltered healthy retinal vasculature (Figure 17E). At the anterior side of the eye, VEGF-A led to uncontrolled neovascularization in the iris and cornea, along with corneal haze indicative of edema or leakage of fluid into the anterior chamber (Figure 22B). WT-VEGF-C produced similar corneal haze but without evident neovascularization, whereas LS-VEGF-C showed similar physical findings as controls (Figures 22C and 22D). LS-VEGF-C not only demonstrates its utility by showing mitigation of undesirable vascularity, but also its properties as a novel molecular tool elucidate the contrasting effects of VEGFR-2 and VEGFR-3 signaling in vivo.

[0378] Example 5. LS-VEGF-C provides neuroprotection in ocular hypertension. Having demonstrated IOP-reducing effects 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) and is often associated with elevated intraocular pressure. 17 First, we used a microbead model, which induces a short-term increase in IOP by blocking the intraocular outflow pathway. 18 , reminiscent of acute angle-closure glaucoma (FIG. 18A). Mice showed an average 20 mmHg increase in IOP after administration of beads into the anterior chamber. LS-VEGF-C was administered by two different routes to evaluate its efficacy. Intravitreal (IVT) administration of LS-VEGF-C was able to return the elevated IOP to baseline on average. Looking more closely, the effect was observed to be bimodal, with mice experiencing a 10 mmHg reduction in IOP, comparable to wild-type mice treated with LS-VEGF-C or mice with a less pronounced response and persistently elevated IOP. Notably, topically administered LS-VEGF-C provided limited benefit. Mice experienced a reduction in intraocular pressure, but this was not sustained and never below baseline measurements (FIG. 18B). Imaging of RGCs, as shown by Brn3a staining, demonstrated that LS-VEGF-C provided neuroprotection to RGCs. In induced glaucomatous conditions, a clear reduction in RGCs was evident, which was ameliorated by intravitreal administration of LS-VEGF-C (Figure 18C). As a group, eye drop treatment did not appear to have a statistically significant effect, but a bimodal effect was observed in retinas that had similar numbers of Brn3a cells as controls and similar numbers of other cells as glaucomatous eyes (Figure 18D).

[0379] To further evaluate the effect of LS-VEGF-C in glaucoma, we utilized the DBA2J pigment dispersion model, a mouse model of spontaneous glaucoma. 19As a negative control to establish a baseline, we used the D2-Gpnmb+ model. It has a normally functioning Gpnmb gene and therefore does not have the pigment dispersion of the iris that leads to spontaneous glaucoma. Although mechanical damage to the anterior chamber is similar to that of microbead injection, this model shows a more chronic pathology with a lead time of 6-8 months to show significant elevation of IOP (Figure 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 lowered IOP within the range of control mice (Figure 18F). RGC axons were assessed in the optic nerves of these mice, which showed that LS-VEGF-C was somehow able to preserve axonal integrity in this glaucoma model. Interestingly, whereas IVT administration showed a significant pressure drop and corresponding optic nerve axonal health, topical administration did not show significant preservation of axonal integrity, despite a similar drop in IOP (Figures 18G and 18H).

[0380] Although the exact mechanism of neuronal death in ocular neurodegenerative diseases remains unclear, a unifying theory involves the accumulation of neurotoxic metabolites or excitatory neural signals. 20,21 VEGF-C is involved in lymphatic drainage, thereby allowing clearance of metabolites and macromolecules within the organ. 22,23 It was therefore theorized that LS-VEGF-C may have neuroprotective properties in glaucoma beyond its ability to reduce IOP. Intravitreal injection of NMDA led to widespread RGC death, which was prevented by concomitant intravitreal administration of LS-VEGF-C (FIGS. 23A and 23B). Together, these results demonstrate that LS-VEGF-C can provide neuroprotection in multiple models of glaucoma through multimodal mechanisms targeting IOP and clearance of neurotoxins.

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

[0382] [Table 4] TIFF2025510570000005.tif174170

[0383] [Table 5]

[0384] Example 7. Characterization of human LS-VEGF-C-Fc. Human VEGFC muteins fused to human IgG1 Fc (N297Q) were transiently expressed in Expi293 cells using the Expifectamine 293 transfection kit (Thermo-Fisher Scientific) according to the manufacturer's instructions. Expression supernatants were then subjected to Ni-NTA chromatography to quantify protein yield in milligrams per liter (Table 6). Quality was subsequently assessed by size-exclusion chromatography using a Superose 6 column (GE Healthcare) (Figures 28A-28M).

[0385] [Table 6]

[0386] Example 8. VEGFR3 expression in lymphatic vessels is likely responsible for mediating the reduction of intraocular pressure. VEGFR3fl / fl mice were crossed with Cdh5aCRE, NESTINCRE, and PROX1CRE (all ERT2, tamoxifen inducible) mice to generate tamoxifen-inducible conditional knockout mice. In the control condition, mice received only corn oil, while in the other conditions, mice received tamoxifen daily for 7 days. All mice were then made ocular hypertensive by bead injection into the anterior chamber. After elevated intraocular pressure was confirmed (baseline), all mice were treated intravitreally with RTI-Fc, and intraocular pressure was measured 7 days later.

[0387] The data shown in Figure 29 demonstrates that expression of VEGFR3 in prox1 and cdh5a positive cells results in a reduction in intraocular pressure, but not in Nestin cells. Since prox1 is expressed in neurons and lymphatic vessels, cdh5a is expressed in lymphatic vessels and blood vessels, and Nestin is expressed in neurons, this demonstrates that expression of VEGFR in lymphatic vessels is likely responsible for mediating the reduction in intraocular pressure. It has been shown that RTI activity is particularly important in lymphatic endothelial cells.

[0388] Example 9. Evaluation of VEGF-C mutein conjugates for reducing intraocular pressure. Wild-type mice were treated with intravitreal LS-VEGF-C in monomeric form, albumin conjugated, or Fc conjugated, and intraocular pressure reduction was assessed.

[0389] The monomer had little effect, while albumin and Fc conjugations produced similar peak reductions in IOP, with the Fc conjugate showing superiority over time (Figure 30).

[0390] Consider Provided herein is the creation of a lymphatic-specific VEGF-C that provides a new molecular tool for the study of lymphatic biology. Dysregulation of the lymphatic vasculature is involved in many disease processes, but translation of these findings to the clinic has been limited by the lack of methods to specifically stimulate lymphatic vessels. Previous efforts have identified muteins that not only lost VEGFR-2 binding, but also significantly lost binding to VEGFR-3. 24~26 To address these clinical barriers, a library of VEGF-C muteins was generated herein to generate LS-VEGF-C protein that exhibits picomolar levels of binding to VEGFR-3 but not VEGFR-2. By utilizing this new property, the potential of this new molecule to treat a variety of diseases known to involve lymphatic dysfunction is highlighted herein for the first time. The activity of LS-VEGF-C could be further extended to new disease areas by completely eliminating angiogenic activity. The excellent efficacy of LS-VEGF-C as a treatment for glaucoma is demonstrated herein. Glaucoma is a disease with clearly identifiable biophysical dysfunction that can benefit from lymphatic therapy. WT-VEGF-C has been ruled out as a possible candidate due to the risk of neovascularization in the eye. LS-VEGF-C not only exhibited potent in vivo activity consistent with its binding properties, but also did not result in neovascularization or vascular permeability.

[0391] This is not the first time that a lymphatic-based therapy has been proposed for glaucoma: the trabecular meshwork and Schlemm's canal display signatures 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 aqueous humor mapping in NHPs and humans. 29 In addition, the uveoscleral outflow pathway is also thought to be involved in drainage of aqueous humor. 12 As mentioned above, the focus in lowering intraocular pressure is specifically on the anterior segment, and all drugs and surgical procedures target components of the anterior segment. However, glaucoma pathology occurs in the posterior segment, and optic nerve damage is the main sign of progression. Herein, we have determined that reducing IOP focused on this compartment provides a unique approach to complement current treatment strategies. As a therapeutic molecule, LS-VEGF-C herein showed a strong effect in reducing IOP in two models of ocular hypertension, resulting in the maintenance of neuronal health. Furthermore, it has been demonstrated herein that stimulating the optic nerve lymphatics can drain neurotoxic molecules from the eye, providing neuroprotection.

[0392] Most importantly, LS-VEGF-C is not only a new pharmacological agent but also a tool to address questions about lymphatic biology. Previous reports sought to specifically stimulate VEGFR-3 and concluded that the effects were less potent in the absence of VEGFR-2 signaling. 30In contrast, LS-VEGF-C showed stronger phenotypic differences compared to WT-VEGF-C. This is likely due to a combination of factors including 1) the absence of VEGF-C being sequestered from its lymphatic binding partner, VEGFR-3, in the absence of VEGFR-2 binding, and 2) LS-VEGF-C retains its binding affinity for VEGFR-3 at picomolar concentrations. This makes our mutein not only an excellent candidate for clinical indications requiring lymphatic stimulation, but also a unique tool to uncouple VEGFR-2 (angiogenesis) and VEGFR-3 (lymphangiogenesis) signaling. One potential application of this utility is lymphedema. 2 From diseases with clear symptoms such as 22,23 These include, but are not limited to, VEGFR-3 specific ligands, which have the potential to have a profound impact on human health and disease. In summary, the results herein from the development of VEGFR-3 specific ligands establish therapeutic potential and versatility, even in immune privileged areas.

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

[0395] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated by reference in their entireties into this application for all purposes.

Claims

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

2. An isolated vascular endothelial growth factor C (VEGF-C) mutein protein or a functional fragment thereof, the VEGF-C mutein protein or the functional fragment thereof (i) has a reduced binding affinity to vascular endothelial growth factor receptor-2 (VEGFR-2) as compared to a wild-type VEGF-C protein derived from the same species, (ii) has the ability to bind to vascular endothelial growth factor receptor-3 (VEGFR-3) and generate signal transduction via 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 derived from the same species.

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

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

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

6. The VEGF-C mutein protein or its functional fragment according to any one of claims 2 to 5, wherein the signal transduction via 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 angiogenesis assay.

7. The VEGF-C mutein protein or a functional fragment thereof according to any one of claims 1 to 6, wherein the VEGF-C mutein protein or a functional fragment thereof does not induce angiogenesis.

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

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

4.

10. 10. The VEGF-C mutein protein or functional fragment thereof according to any one of claims 1 to 9, comprising 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 said positions of said residues are defined with reference to SEQ ID NO:4; or a mutation at a corresponding residue in another wild-type VEGF-C protein.

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

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

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

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

1.

15. 15. The VEGF-C mutein protein or functional fragment thereof according to any one of claims 1 to 7 and 13 to 14, wherein the VEGF-C mutein 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 said residues are defined with reference to SEQ ID NO:

1.

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

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

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

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

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

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

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

56.

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

56.

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

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

57.

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

57.

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

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

58.

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

58.

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

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

59.

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

59.

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

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

62.

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

62.

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

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

63.

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

63.

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

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

64.

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

64.

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

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

65.

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

65.

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

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

68.

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

68.

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

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

70.

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

70.

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

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

80.

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

80.

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

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

88.

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

88.

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

58. 58. The fusion protein or conjugate of claim 57, wherein the one or more heterologous moieties are selected from an immunoglobulin or a functional fragment thereof, an albumin or a functional fragment thereof, an albumin-binding antibody or a functional fragment thereof, and a polyethylene glycol (PEG) polymer.

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

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

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

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

63. An isolated polynucleotide molecule encoding a VEGF-C mutein 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.

64. 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. 64. 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. 64. 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. 64. 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. 64. 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 of claim 63, wherein the polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO:234 ​​or SEQ ID NO:

132.

75. 64. 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 to 75, comprising a nucleotide sequence encoding the VEGF-C mutein protein or a functional fragment thereof operably linked to a promoter.

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

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

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

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

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

82. 82. The vector of claim 81, wherein the viral vector is derived from a herpesvirus, 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 a polynucleotide molecule according to any one of claims 63 to 79.

84. 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 a polynucleotide according to any one of claims 63 to 79 or a vector according to any one of claims 80 to 82.

86. A method for producing a VEGF-C mutein protein or a functional fragment thereof, or a fusion protein thereof, comprising culturing a host cell described in claim 85 under conditions in which the VEGF-C mutein protein or a functional fragment thereof, or a fusion protein thereof is expressed.

87. A VEGF-C mutein protein or a functional fragment thereof, or a fusion protein thereof, produced by the method according to claim 86.

88. A kit comprising a VEGF-C mutein 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, and optionally instructions for use.

89. A kit comprising a polynucleotide 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 84, and optionally instructions for use.

90. A pharmaceutical composition comprising a VEGF-C mutein 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 84, and a pharma- ceutically acceptable carrier or diluent.

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

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

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

94. 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-BTLA 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 to 95, wherein the pharmaceutical composition is formulated for intrathecal administration.

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

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

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

100. The pharmaceutical composition of any one of claims 90 to 95, wherein the pharmaceutical composition is formulated for ophthalmic administration.

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

102. 102. A method of inducing lymphangiogenesis in a subject in need thereof, said method comprising the step of administering to said subject an effective amount of a VEGF-C mutein 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 84, or a pharmaceutical composition according to any one of claims 90 to 101.

103. The method of claim 102, wherein the administration of the VEGF-C mutein protein or 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. 104. The method of claim 103, wherein the one or more side effects are neovascularization and / or elevated intraocular pressure (IOP).

105. The method of any one of claims 102 to 104, wherein the VEGF-C mutein protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisternally, intravitreally, ophthalmically, subcutaneously, intradermally, by inhalation, via a chronic indwelling catheter, orally, topically, or systemically.

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

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

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

109. 102. A method of treating a disease or condition in a subject in need thereof, said method comprising the step of administering to the subject an effective amount of a VEGF-C mutein 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 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, osmoregulation, cardiac ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, diseases or conditions associated with lymph node transplantation, Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, wound healing, lymphedema, Hennekam syndrome, Milroy's disease, Turner syndrome, age-related macular degeneration, glaucoma, central serous chorioretinopathy, diabetic retinopathy, macular edema, and retinal edema.

111. 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, renal cancer, urothelial cancer, prostate cancer, pheochromocytoma, cholangiocarcinoma, hepatocellular carcinoma, invasive pancreatic ductal carcinoma, thymoma, sarcoma, mesothelioma, testicular cancer, or colorectal cancer.

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

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

114. 114. The method of any one of claims 109 to 113, further comprising the step of 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-BTLA 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 to 117, wherein the VEGF-C mutein protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered intrathecally, intraocularly, intratumorally, intracisternally, intravitreally, ophthalmically, subcutaneously, intradermally, by inhalation, via a chronic indwelling catheter, orally, topically, or systemically.

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

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

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

122. 103. A method of modulating intraocular pressure in a subject in need thereof, said method comprising the step of administering to said subject an effective amount of a VEGF-C mutein 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 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 said wild-type VEGF-C protein or functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

123. The method of claim 122, wherein the VEGF-C mutein protein or the corresponding wild-type VEGF-C protein, or a functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition, 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, is administered to the posterior segment of the eye.

124. 124. The method of claim 122 or 123, wherein the administration is intraocular administration.

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

126. 103. A method of removing unwanted intraocular fluid in a subject in need thereof, said method comprising the step of administering to said subject an effective amount of a VEGF-C mutein 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 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 said wild-type VEGF-C protein or functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

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

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

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

130. 130. The method of claim 129, wherein the intraocular administration is intravitreal administration, eye drop administration, or subretinal administration.

131. 103. A method of providing neuroprotection to a subject in need thereof, the method comprising the step of administering to the subject an effective amount of a VEGF-C mutein 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 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 said wild type VEGF-C protein or functional fragment thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.

132. 102. A vaccine comprising a VEGF-C mutein 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 84, or a pharmaceutical composition according to any one of claims 90 to 101.

133. 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 to 133, wherein the subject is a human.

135. A method for generating a library of VEGF-C muteins that selectively bind to VEGFR-3, wherein amino acid residues that constitute a covalent binding interface on VEGF-C 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 selection of a VEGF-C mutein protein, the yeast cell library comprising a cell wall peptide anchor sequence, a linker peptide, and a plurality of yeast cells comprising the sequence of said VEGF-C mutein protein.

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