Patent Application
20250188139
The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Mar. 8, 2023, is named 251609_000089_SL.xml and is 347,984 bytes in size.
The present invention relates generally to vascular endothelial growth factor C (VEGF-C) muteins having selective binding for vascular endothelial growth factor receptor-3 (VEGFR-3) over vascular endothelial growth factor receptor-2 (VEGFR-2). The present invention relates also 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 relates also to a method for treating a 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.
Immune surveillance against pathogens and tumors in the central nervous system is thought to be limited owing to the lack of lymphatic drainage. It has been shown that the meningeal lymphatic vasculature can be manipulated to mount better immune responses against brain tumors. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is very limited when the tumor is confined to the central nervous system, resulting in uncontrolled 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 the tumor, rapid clearance of the glioblastoma and a long-lasting antitumor memory response. Furthermore, transfection of an mRNA construct that expresses VEGF-C works synergistically with checkpoint blockade therapy to eradicate existing glioblastoma.
Vascular endothelial growth factor C (VEGF-C) binds to vascular endothelial growth factor receptor-2 (VEGFR-2) promoting the growth of blood vessels (angiogenesis) and regulating vascular permeability, and to vascular endothelial growth factor receptor-3 (VEGFR-3) promoting the growth of lymphatic vessels (lymphangiogenesis). VEGF-C acts on lymphatic endothelial cells (LECs) primarily via VEGFR-3 promoting survival, growth and migration. Angiogenesis is a necessity for growth of both primary tumors and metastases. There exists a need to reduce unwanted angiogenesis while promoting lymphangiogenesis.
Various non-limiting aspects and embodiments of the invention are described below.
In one aspect, provided herein is 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 a reduced or no ability to stimulate blood endothelial cell proliferation, as compared to a wild-type VEGF-C protein from the same species but preserves the ability to stimulate lymphatic endothelial cell proliferation.
In one aspect, provided herein is 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 (i) has a reduced binding affinity to vascular endothelial growth factor receptor-2 (VEGFR-2) as compared to a wild-type VEGF-C protein from the same species, (ii) has the ability to bind and generate signaling through vascular endothelial growth factor receptor-3 (VEGFR-3), and (iii) comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the wild-type VEGF-C protein from the same species.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof generates reduced or no signaling through VEGFR-2 as 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 generate 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 level in blood endothelial cells, by a wound healing assay, by a proliferation assay, or by 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 level in lymphatic endothelial cells, by a wound healing assay (scratch assay), by a proliferation assay, or by an angiogenesis assay.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof does not induce angiogenesis.
In some embodiments of any of the above methods, the VEGF-C mutein protein is a mutein of a wild-type VEGF-C protein comprising amino acids 111-211 of SEQ ID NO: 4, or a polypeptide defined by the corresponding positions at the wild-type VEGF-C protein of another species. In some embodiments of any of the above methods, the wild-type VEGF-C protein comprises amino acids 111-211 of SEQ ID NO: 4.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises one or more mutations selected from mutations at residues T112, L115, D119, Q126, T144, N145, K149, N163, S164, E165, I184, V186, L188, and P192, wherein the positions of said residues are defined in relation to SEQ ID NO: 4, or mutations at the corresponding residues within the wild-type VEGF-C protein of another species, or one or more mutations at residues T116, L119, D123, Q130, T148, N149, K153, N167, S168, E169, I188, V190, L192, and P196 wherein the positions of said residues are defined in relation to SEQ ID NO: 1.
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, wherein the position of said residue is defined in relation to SEQ ID NO: 4, or a mutation at the corresponding residue within the wild-type VEGF-C protein of another species. In some embodiments, the mutation at residue C133 is C133A mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein is a mutein of a wild-type VEGF-C protein comprising amino acids 115-215 of SEQ ID NO: 1, or a polypeptide defined by the corresponding positions within the wild-type VEGF-C protein of another species. In some embodiments, the wild-type VEGF-C protein comprises amino acids 115-215 of SEQ ID NO: 1.
In some embodiments of any of the above 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 wherein the positions of said residues are defined in relation to SEQ ID NO: 1.
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, wherein the position of said residue is defined in relation to SEQ ID NO: 1, or mutations at the corresponding residues within the wild-type VEGF-C protein of other species. In some embodiments, the mutation at residue C137 is C137A mutation.
In some embodiments of any of the above methods, the mutation at residue L119 is L119E mutation, or L119M mutation; the mutation at residue D123 is D123N mutation; the mutation at residue Q130 is Q130K mutation; the mutation at residue N167 is N167R mutation, N167I mutation, N167Q mutation, or N167H mutation; the mutation at residue S168 is S168G mutation, or S168R mutation; the mutation at residue V190 is V190T mutation; and/or the mutation at residue L192 is L192I mutation.
In some embodiments of any of the above 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.
In some embodiments of any of the above methods, the mutation at residue N167 is N167I mutation, N167Q mutation, or N167H mutation; the mutation at residue S168 is S168G mutation, or S168R mutation; and/or the mutation at residue L192 is L192I mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises N167Q mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 160 or SEQ ID NO: 56.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 160 or SEQ ID NO: 56.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises N167Q mutation and S168G mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO: 57.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO: 57.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises N167Q mutation and L192I mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 58.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 58.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises N167Q mutation, S168G mutation, and L192I mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 163 or SEQ ID NO: 59.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 163 or SEQ ID NO: 59.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises N167I mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 166 or SEQ ID NO: 62.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 166 or SEQ ID NO: 62.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises N167I mutation and S168G mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 167 or SEQ ID NO: 63.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 167 or SEQ ID NO: 63.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises N167I mutation and L192I mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 168 or SEQ ID NO: 64.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 168 or SEQ ID NO: 64.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises N167I mutation, S168G mutation, and L192I mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 169 or SEQ ID NO: 65.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 169 or SEQ ID NO: 65.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises S168G mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 68.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 68.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises S168G mutation and L192I mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 70.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 70.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises N167H mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO: 80.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO: 80.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof comprises N167I mutation and S168R mutation.
In some embodiments of any of the above methods, the VEGF-C mutein protein comprises the amino acid sequence of SEQ ID NO: 192 or SEQ ID NO: 88.
In some embodiments of any of the above methods, the VEGF-C mutein protein consists of the amino acid sequence of SEQ ID NO: 192 or SEQ ID NO: 88.
In one aspect, provided herein is a fusion protein or conjugate comprising the VEGF-C mutein protein or functional fragment thereof disclosed herein, wherein the mutein protein or a functional fragment thereof, is fused and/or conjugated to one of more heterologous moieties.
In some embodiments, the one of 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.
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 a 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 one aspect, provided herein is an isolated polynucleotide molecule encoding the VEGF-C mutein protein or functional fragment thereof of the present disclosure or the fusion proteins of the present disclosure.
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 functional fragment thereof which is operably linked to a promoter.
In some embodiments, the polynucleotide molecule is an mRNA.
In some embodiments, the polynucleotide molecule comprises one or more nucleotide modifications. In some embodiments, the one or more nucleotide modifications are a 5′cap, a 5-methylcytosine, or a pseudo-uridine.
In one aspect, provided herein is a vector comprising the polynucleotide molecules of the present disclosure.
In some embodiments, the vector is a viral vector. In 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.
In one aspect, provided herein is a particle comprising the polynucleotide molecules of the present disclosure. In some embodiments, the particle is a nanoparticle, lipid particle, microparticle, lipid nanoparticle, polymer particle, or virus-like particle (VLP).
In one aspect, provided herein is a host cell comprising the polynucleotides of the present disclosure or the vectors of the present disclosure.
In one aspect, provided herein is a method of producing a VEGF-C mutein protein or functional fragment thereof, or fusion protein thereof, comprising culturing the host cell of the present disclosure under conditions at which the VEGF-C mutein protein or functional fragment thereof, or fusion protein thereof is expressed.
In one aspect, provided herein is a VEGF-C mutein protein or functional fragment thereof, or fusion protein thereof, produced by the methods of the present disclosure.
In one aspect, provided herein is a kit comprising the VEGF-C mutein proteins or functional fragments thereof of the present disclosure or the fusion proteins or conjugates of the present disclosure, and optionally instructions for use.
In one aspect, provided herein is a kit comprising the polynucleotides of the present disclosure or the vectors of the present disclosure, or the particles of the present disclosure, and optionally instructions for use.
In one aspect, provided herein is a pharmaceutical composition comprising a VEGF-C mutein protein or functional fragment thereof of the present disclosure or the fusion protein or conjugate of the present disclosure, or the polynucleotide molecule of the present disclosure, or the vector of the present disclosure, or the particle of the present disclosure, and a pharmaceutically acceptable carrier or diluent.
In some embodiments, the composition comprises mRNA encoding the VEGF-C mutein protein or functional fragment thereof, or fusion protein thereof as mRNA-nanoparticle formulation.
In some embodiments, the pharmaceutical composition further comprising an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, BTLA, GITR, 4-1BB, or Ox-40. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-BLTA antibody, an anti-GITR antibody, an anti-4-IBB antibody, or an anti-Ox-40 antibody.
In some embodiments, the pharmaceutical composition is formulated for intrathecal administration. In some embodiments, the pharmaceutical composition is formulated for intratumoral administration. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for intracisternal administration. In some embodiments, the pharmaceutical composition is formulated for eye-drop administration. In some embodiments, the pharmaceutical composition is formulated for intraocular administration.
In 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 the VEGF-C mutein proteins or functional fragments thereof of the present disclosure or the fusion proteins or conjugate of the present disclosure, or the polynucleotide molecules of the present disclosure, or the vectors of the present disclosure, or the particles of the present disclosure, or the pharmaceutical compositions of the present disclosure.
In some embodiments of any of the above methods, the administration said 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 corresponding fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition. In some embodiments, the one or more side effects are angiogenesis and/or increased intraocular pressure (IOP).
In some embodiments of any of the above 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, via eye drops, subcutaneously, intradermally, via inhalation, via long-dwelling catheter, orally, topically, or systemically.
In some embodiments of any of the above methods, the VEGF-C mutein protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered to the cisterna magna or directly into the lymphatic system.
In some embodiments of any of the above methods, the subject has a disease or condition selected from cancer, coronary vessel function, osmoregulation, heart ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, diseases or conditions associated with lymph node transplant, 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.
In some embodiments of any of the above methods, the diseases or conditions associated with lymph node transplant are breast cancer associated lymphedema, idiopathic lymphedema, and/or heart failure associated lymphedema.
In one aspect, provided herein is a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a VEGF-C mutein proteins or functional fragments thereof of the present disclosure or the fusion proteins or conjugate of the present disclosure, or the polynucleotide molecules of the present disclosure, or the vectors of the present disclosure, or the particles of the present disclosure, or the pharmaceutical compositions of the present disclosure.
In some embodiments, wherein the method provided herein is a method of treating a disease or condition in a subject in need thereof, the disease or condition is cancer, coronary vessel function, osmoregulation, heart ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, diseases or conditions associated with lymph node transplant, 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. In some embodiments, the cancer is melanoma, lung cancer, breast cancer, stomach cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, head and neck squamous cell carcinoma, thyroid cancers, liquid cancer, kidney cancers, urothelial bladder cancers, prostate cancers, pheochromocytoma, cholangiocarcinoma, liver hepatocellular carcinoma, pancreatic ductal adenocarcinoma, thymoma, sarcoma, mesothelioma, testicular cancer, or colorectal cancer. In some embodiments, the cancer is in the brain or the 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 melanoma and rhabdoid tumor.
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-BLTA 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, via eye drops, subcutaneously, intradermally, via inhalation, via long-dwelling 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 to the cisterna magna or directly into the lymphatic system.
In some embodiments, wherein the method provided herein is a method of treating a disease or condition in a subject in need thereof, and wherein the disease is cancer, the method further comprising administering an additional anti-cancer treatment to the subject. In some embodiments, the additional anti-cancer treatment is selected from surgery, radiation therapy, administration of a chemotherapeutic agent, an immunotherapy, and any combinations thereof.
In one aspect, provided herein is a method for modulating intraocular pressure in a subject in need thereof comprising administering to the subject an effective amount of the VEGF-C mutein protein or functional fragment thereof, the fusion protein or conjugate, the polynucleotide molecule, the vector, the particle, or the 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 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, or the fusion protein or conjugate thereof, or the polynucleotide molecule encoding said wild-type VEGF-C protein or functional fragment thereof, or the vector or particle comprising said polynucleotide molecule, or the pharmaceutical composition comprising any of the above, is administered to the posterior eye. In some embodiments, the administration is intraocular. In some embodiments, the intraocular administration is intravitreal, via eye drops, or subretinal.
In one aspect, provided herein is a method for removing unwanted fluid in an eye of a subject in need thereof comprising administering to the subject an effective amount of the VEGF-C mutein protein or functional fragment thereof, the fusion protein or conjugate, the polynucleotide molecule, the vector, the particle, or the 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, retinal, subretinal, choroidal, or suprachoroidal fluid. In some embodiments, the subject has 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, via eye drops, or subretinal.
In one aspect, provided herein is a method for providing neuroprotection in a subject in need thereof comprising administering to the subject an effective amount of the VEGF-C mutein protein or functional fragment thereof, the fusion protein or conjugate, the polynucleotide molecule, or the vector, the particle, or the 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 one aspect, provided herein is a vaccine comprising the VEGF-C mutein protein or functional fragment thereof, the fusion protein or conjugate, the polynucleotide molecule, the vector, the particle, the pharmaceutical composition of the present disclosure.
In one aspect, provided herein is a method inducing an immune response in a subject in need thereof in a subject in need thereof, the method comprising administering to the subject an effective amount of the vaccine of the present disclosure.
In some embodiments of any of the above methods, the subject is a human.
In a further aspect, provided herein is a method of generating a library of VEGF-C muteins having selective binding to VEGFR-3, wherein amino acid residues comprising the shared binding interface on VEGF-C which binds both VEGFR-3 and VEGFR-2 are diversified to other amino acids via mutagenesis of the corresponding nucleic acid sequence.
In a further aspect, provided herein is a yeast cell library for selection of VEGF-C mutein proteins, comprising a plurality of yeast cells comprising a cell wall peptide anchor sequence, a linker peptide, and the VEGF-C mutein sequence.
These and other aspects described herein will be apparent to those of ordinary skill in the art in the following description, claims and drawings.
The present invention provides VEGF-C muteins and methods of inducing lymphangiogenesis in a subject in need thereof, the method comprising administering to the subject an effective amount of a VEGF-C mutein.
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 items.
The term “about” when used before a numerical value indicates that the value may vary within a reasonable range, such as within ±10%, ±5% or ±1% of the stated value, and include the stated value.
The terms “patient,” “individual,” “subject,” “mammal,” and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, rabbits, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.
The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
As used herein, the terms “mutein protein,” “mutein polypeptide” and “mutein” are used interchangeably to refer to a protein with an altered amino acid sequence as compared to its wild-type counterpart. Amino acid sequence alterations may comprise amino acid substitutions, deletions or additions.
As used herein, the term “lymphangiogenesis” refers to the process of the formation of lymphatic vessels and/or stimulation of lymphatic vasculature function.
In one aspect, provided herein is an isolated vascular endothelial growth factor C (VEGF-C) mutein protein or a functional fragment thereof, having selective binding for VEGFR-3 over VEGFR-2.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof has a reduced or no ability to stimulate blood endothelial cell proliferation, as compared to a wild-type VEGF-C protein from the same species but preserves the ability to stimulate lymphatic endothelial cell proliferation.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof (i) has a reduced binding affinity to VEGFR-2 as compared to a wild-type VEGF-C protein from the same species, (ii) has the ability to bind 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 from the same species.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof has a binding affinity to VEGFR-2 that is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more as compared to a wild-type VEGF-C protein from the same species.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof has an ability to bind and generate signaling through VEGFR-3 which is about the same, increased, or mildly reduced as compared to a wild-type VEGF-C protein from the same species.
In a cell, 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 generating pro-VEGF-C. Pro-VEGF-C is able to bind VEGFR-3 but does not activate it. The second proteolytic cleavage by A disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) removes both terminal domains resulting in 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 case of the wild-type human VEGF-C, 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 case of the wild-type murine VEGF-C, mature VEGF-C protein corresponds to amino acids 111-211 (SEQ ID NO: 292) of the murine VEGF-C propeptide (SEQ ID NO: 4).
In some embodiments, the VEGF-C mutein protein may be a mutein protein of a human VEGF-C protein. 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 amino acids 115-215 of the following polypeptide sequence:
The 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 343-645 of the following VEGF-C-encoding sequence:
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:
The 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:
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, wherein the positions of mutations are defined in relation 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.
In some embodiments, the VEGF-C mutein protein may be a mutein protein of a murine VEGF-C protein. In certain embodiments, 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%, or 99% identical to amino acids 111-211 of the following polypeptide sequence:
The 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 331-631 of the following VEGF-C-encoding sequence:
In some embodiments, the VEGF-C mutein protein may be a mutein protein of a murine VEGF-C protein. In certain embodiments, 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 the following polypeptide sequence:
The polynucleotide molecule encoding a murine 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
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, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the mutation at N167 is N167I mutation, N167Q mutation, or N167H mutation; the mutation at S168 is S168G mutation, or S168R mutation; and/or the mutation at L192 is L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises N167Q mutation, wherein the position of the mutation is defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises N167Q mutation and S168G mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises N167Q mutation and L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises N167Q mutation, S168G mutation, and L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises N167I mutation, wherein the position of the mutation is defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises N167I mutation and S168G mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises N167I mutation and L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises N167I mutation, S168G mutation, and L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises S168G mutation, wherein the position of the mutation is defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises S168G mutation and L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises N167H mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises N167I mutation and S168R mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In certain embodiments, the VEGF-C mutein may comprise a functional fragment of the human VEGF-C.
In certain embodiments, the 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 polypeptide sequence of any of the SEQ ID NOs: 154-159, SEQ ID NOs: 164-165, SEQ ID NOs: 170-171, SEQ ID NO: 173, SEQ ID NOs: 175-183, SEQ ID NOs: 185-191, or SEQ ID NOs: 193-203.
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with at least or at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the any of SEQ ID NOs: 204-209, SEQ ID NOs: 214-215, SEQ ID NOs: 220-221, SEQ ID NO: 223, SEQ ID NOs: 225-233, SEQ ID NOS: 235-241, or SEQ ID NOs: 243-253.
In certain embodiments, the VEGF-C mutein may comprise a functional fragment of the murine VEGF-C. In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
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, wherein the positions of mutations are defined in relation 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 some embodiments, the VEGF-C mutein protein comprises mutations N163I.
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the 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:
In some embodiments, the VEGF-C mutein protein comprises mutations N163R, V186T and L188I, wherein the positions of mutations are defined in relation to SEQ ID NO: 4.
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the VEGF-C mutein comprises a mutation at residue C133, wherein the position of the mutation is defined in relation to SEQ ID NO: 4. In certain embodiments, the mutation at residue C133 is a C133A mutation.
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the VEGF-C mutein protein comprises mutations C133A and N163I, wherein the positions of mutations are defined in relation to SEQ ID NO: 4.
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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
In some embodiments, the VEGF-C mutein protein comprises mutations C133A, N163R, V186T and L188I, wherein the positions of mutations are defined in relation to SEQ ID NO: 4.
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the VEGF-C mutein comprises a mutation at residue C137, wherein the position of the mutation is defined in relation to SEQ ID NO: 1. In certain embodiments, the mutation at residue C137 is a C1337A mutation.
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
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, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the mutation at C137 is C137A, the mutation at N167 is N167I mutation, N167Q mutation, or N167H mutation; the mutation at S168 is S168G mutation, or S168R mutation; and/or the mutation at L192 is L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation and N167Q mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation, N167Q mutation and S168G mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation, N167Q mutation and L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation, N167Q mutation, S168G mutation, and L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation and N167I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation, N167I mutation and S168G mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation, N167I mutation and L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation, N167I mutation, S168G mutation, and L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation and S168G mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation, S168G mutation and L192I mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation and N167H mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In some embodiments, the VEGF-C mutein protein or functional fragment thereof comprises C137A mutation, N167I mutation and S168R mutation, wherein the positions of mutations are defined in relation to SEQ ID NO: 1.
In certain embodiments, the 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 polypeptide sequence of any of the 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.
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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.
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.
These variants may include: (i) variants in which one or more amino acid residues are substituted with conservative or nonconservative 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 group, and (iv) variants in which the described VEGF-C mutein or fragment thereof is fused or conjugated with 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 mutein or fragment thereof, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. VEGF-C muteins or fragments thereof described herein may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In other embodiments, amino acid residues at nonconserved positions are substituted with conservative or nonconservative 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.
Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. A skilled person would understand which amino acids share similar chemical properties. For example, the following groups of amino acids share 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).
In some embodiments, the VEGF-C mutein protein is modified to extend its circulating half-life. Strategies to extend the half-life of recombinant proteins include, but are not limited to fusion to immunoglobulin of a fragment thereof of immunoglobulin such as the Fc domain of IgG; fusion to albumin or an albumin fragment thereof; fusion to an albumin-binding antibody or antibody fragment thereof such as an scFv, Fab, or single-domain antibody (VHH); or chemical modification with polyethylene glycol (PEG).
In some embodiments, the VEGF-C mutein, or a fragment thereof or a variant thereof, is fused to and/or conjugated to one or more heterologous moieties, such as, but not limited to, peptides, polypeptides, small molecules, polymers, nucleic acids, lipids, sugars, etc.
In certain embodiments, the VEGF-C muteins are fused to and/or conjugated to a moiety that provides longer half-life to the VEGF-C mutein. In some embodiments, the VEGF-C muteins are fused to and/or conjugated to a moiety that specifically binds albumin including but not limited to, a small molecule, a peptide, a polypeptide, or a lipid that bind albumin. In some embodiments, the VEGF-C muteins are fused to and/or conjugated to an immunoglobulin constant region (an Fc domain), an scFv, an Fab, a single-domain antibody (VHH), an immunoglobulin, or a heavy or light chain thereof.
In certain embodiments, the VEGF-C muteins are fused to and/or conjugated to a polymer including, but not limited to, lipid polymers, polyethylene glycol (PEG), biodegradable polymers such as PLA (poly (lactic acid)) and PLGA (poly (lactic-glycolic acid)), polysaccharides, polysaccharides, poly (propylene glycol), polyoxy ethylated polyols, polyvinyl ethers, copolymers of ethylene glycol and propylene glycolpolyvinyl alcohols, dextran, hyaluronic acid, chitin, and the like.
In certain embodiments, the VEGF-C muteins are fused to and/or conjugated to an Fc domain. In certain embodiments, the Fc sequence to be fused or conjugated to the VEGF-C muteins 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:
In certain embodiments, the polynucleotide molecule encoding the Fc sequence may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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
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
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In some embodiments, the present invention relates to modifying the IgG Fc domain to reduce a Fc effector function. Such modification can be achieved by various techniques, including, but not limited to, amino acid substitutions, deletions, or additions to the Fc domain. For example, the modification may involve replacing one or more amino acid residues in the Fc domain with non-natural amino acids, altering glycosylation patterns, or introducing steric hindrance to the Fc domain. In some embodiments, the modified Fc domain exhibits reduced binding affinity to Fc receptors or complement proteins, resulting in reduced Fc effector function. Examples of modified Fc domains that exhibit reduced Fc effector function include, but are not limited to, Fc variants with point mutations such as N297A, L234A/L235A, S239D/1332E, S298A/IgG1/IgG3, L309D/1332E, 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. The modified Fc domains may 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 a 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 a mutation one or more mutations at residues F234, or L235. In some embodiments, the IgG Fc domain is an IgG4 variant comprising a mutation one or more mutations F234A, or L235A. In some embodiments, the IgG Fc domain is an IgG1 variant comprising a mutation one or more mutations at residues L234, or L235. In some embodiments, the IgG Fc domain is an IgG1 variant comprising one or more mutations L234A, or L235A. In some embodiments, the IgG Fc domain can be a cross-subclass domain. For example, but no limited to, the IgG Fc domain can be IgG2 variant with point mutations from IgG4 (e.g., H268Q/V309L/A330S/P331S).
In some embodiments, the Fc domain comprises one or more mutations. In some embodiments, the Fc domain is mutated, glycoengineered, or otherwise modified to reduce Fc effector functions.
In some embodiments, the Fc domain comprises one or more mutations that ablate a critical glycosylation site required for effector function.
In some embodiments, the Fc domain comprises a mutation at residue N82, wherein the positions of said residues are defined in relation to SEQ ID NO: 21. In some embodiments, the mutation at N82, defined in relation to SEQ ID NO: 21, is N82Q mutation, N82A mutation, or N82G mutation.
In certain embodiments, the VEGF-C muteins are fused to and/or conjugated to albumin. In certain embodiments, the albumin is human albumin.
In some embodiments, VEGF-C muteins are fused to and/or conjugated to albumin or to an Fc domain though a linker. Examples of linkers include, but not limited to, proline-rich linkers, acidic linkers, basic linkers, cleavable linkers or rigid linkers. Examples of proline-rich linkers include, but not limited to, (Pro-Pro-Gly) 3 (SEQ ID NO: 295), (Pro-Gly) 5 (SEQ ID NO: 296), or (Pro-Pro-Pro-Pro-Gly) 3 (SEQ ID NO: 297). Examples of acidic linkers include, but 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 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 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 not limited to, (Azido-Lys-Arg-Pro) m which contains the non-natural amino acid azido-lysine, which can form a triazole linkage with an alkyne-containing molecule, or (Cyclohexyl-Ala-Pro-Pro), which contains the non-natural amino acid cyclohexyl-alanine, which can form a rigid cyclohexane structure. Example of such linker, but not limited to, includes a linker with the amino acid sequence: GGGGGSGGGGSGGGGS (SEQ ID NO: 294).
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In certain embodiments, the 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:
In certain embodiments, the polynucleotide molecule encoding the VEGF-C mutein may comprise a sequence with 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:
In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein or the functional fragment thereof is a mRNA.
mRNA can provide several advantages to AAV and other gene delivery systems, which include, but are not limited to one or more of the following: mRNA can be highly customizable, mRNA can prevent recognition from pattern recognition receptors and nucleases to allow for sustained expression, mRNA can provide for well-controlled expression kinetics with the option of repeated dosing, and mRNA can provide for low risk of integration into the genome due to its localization in the cytosol. mRNA can also be cost-effective.
In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein protein or the functional fragment thereof comprises a modified nucleotide such as 5-methyl-cytosine and pseudo-uridine substitutions that can increase stability, decrease deamination, decrease nuclease activity, decrease innate recognition, or increase translation efficiency of the polynucleotide molecule. In some embodiments, the modified nucleotide is a 5-methyl-cytosine or a pseudo-uridine. In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein comprises a 5′ cap.
The mRNA may comprise a modified nucleotide. In some embodiments, the modified nucleotide is a 5-methyl-cytosine or a pseudo-uridine. In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein or the functional fragment thereof comprises a 5′ cap. In some embodiments, the 5′ cap is added using the CleanCap Reagent AG. CleanCap is made up of C32H43N15024P4 and allows for high capping efficiencies resulting in more active mRNA. Cap 1 does not activate Pattern Recognition Receptors and is important for proficient in vivo expression. Without wishing to be bound by theory, any one or more of 5-methyl-cytosine, pseudo-uridine, and the 5′ cap may improve stability of the mRNA, which in turn can prolong expression of the VEGF-C mutein or the functional fragment thereof.
In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein or the functional fragment thereof is comprised within a viral vector. Exemplary viral vectors include, but are not limited to, herpes virus, cytomegalovirus, poliovirus, alphavirus, vaccinia virus, rabies virus, adeno-associated virus (AAV), a retrovirus, a 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.
In some embodiments, the VEGF-C mutein or the functional fragment thereof can be administered in a dosage regimen involving a combination of mRNA and AAV. One or more administrations of mRNA can be undertaken to quickly obtain high expression, such as within 2 hours post delivery of the mRNA. The expression of VEGF-C mutein or the functional fragment thereof provided by AAV may take 7-14 days, or even up to four weeks, depending on the serotype. Administration of protein or the functional fragment thereof can be undertaken to get instantaneous expression. Administration of both mRNA and AAV, conjointly or in short succession, can provide a sustained expression of the VEGF-C mutein or the functional fragment thereof. Without wishing to be bound by theory, administering protein can provide for instantaneous expression and controlled expression kinetics, with multiple doses possible. Without wishing to be bound by theory, administering mRNA can provide for instantaneous expression, controlled expression kinetics, and high expression, with multiple doses possible. Without wishing to be bound by theory, administering AAV can provide for delayed expression and high levels of expression. The expression kinetics can be effectively and sensitively measured using ELISA and Western blotting.
In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein or the functional fragment thereof is comprised within a liposome. The VEGF-C mutein or the functional fragment thereof may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the polynucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. The liposome comprising the VEGF-C mutein or the functional fragment thereof may be present in a bilayer structure, as micelles, or with a “collapsed” structure. The liposomes may also simply be interspersed in a solution, possibly forming aggregates which are not uniform in either size or shape. For example, a nucleotide (e.g., siRNA) may be encapsulated in a neutral liposome using a method involving ethanol and calcium. The shape may be that of a spherical vesicle. In various embodiments, the liposomes may comprise one or more concentric layers of lipid bilayer molecules. In some embodiments, the lipid components include a combination of Cl 2-200, XTC, MC3, NC98-5, DLinDMA, HGT5001cis, HGT5001trans, HGT5000, HGT4003, DLinKC2DMA, ALNY100, ICE, DLinKC2DMA, CHOL, DOPE, DMG-PEG-2000, Cl 2-200, DOPE, CHOL, and DMGPEG2K.
In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein or the functional fragment thereof is attached to a nanoparticle or a polymer. In certain embodiments, present nanoparticles further comprise at least one agent that specifically binds a particular type or category of cells and/or other particular type compounds, (e.g., a moiety that targets a specific ceil or type of cell). In some embodiments, the nanoparticle is a nanosphere. In some embodiments, the polymer is dextran, poly (amine-co-ester), poly (beta-amino-ester), polyethylenimine, poly-L-Lysine, polyethylene glycol, or dendrimers.
In some embodiments, the polynucleotide molecule encoding the VEGF-C mutein or the functional fragment thereof is comprised within a recombinant viral particle or within a virus like particle (VLP).
In some embodiments, the VEGF-C mutein is produced and administered as a “masked” prodrug that is activated after administration to a patient. In some cases, this may confer desirable tissue or tumor-specific activity of the mutein, for example by virtue of restricted expression of endogenous proteases that cleave the prodrug and release an active “mature” fragment. In some embodiments, the VEGF-C mutein is a variant of the full-length VEGF-C propeptide and activated by proteases such as, e.g., ADAMTS2, plasmin, furin, Cathepsin D, thrombin, and/or KLK3/PSA. In other embodiments, the VEGF-C mutein is fused to an inhibitory peptide that is released by proteolysis by an endogenous protease. The inhibitory peptide can take many forms, including by not limited to, an antibody or antibody fragment that binds to VEGF-C mutein to obscure its interaction with VEGFR-3, the VEGFR-3 ectodomain or fragment thereof that competitively inhibits interaction of VEGF-C with the VEGFR-3-on cells, and any other binder (such as, e.g., a VHH, fibronectin domain, knottin, lipocalin, leucine-rich repeat, etc). A non-limiting set of examples of protease cleavage sites is described below. The inhibitory peptide may be separated from the biologically active VEGF-C mutein by an enterokinase cleavage site (EKCS) peptide, which is recognized and cleaved by the endogenous protease enterokinase present in the small intestine. In this way, the prodrug can be designed to be inactive until it reaches the small intestine, where it can be activated by enterokinase cleavage. Alternatively, the inhibitory peptide can be 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. By designing the prodrug to be cleaved specifically by furin, the activation of the prodrug can be localized to the tumor microenvironment, resulting in targeted therapy. Furthermore, the prodrugs can be designed to be cleaved by other endogenous proteases that are overexpressed in specific tumors. For example, the prodrug can be designed to be cleaved by matrix metaloproteases MMPs or urokinase-type plasminogen activator uPA, which are overexpressed in many tumors. In this way, the activation of the prodrug can be localized to the tumor microenvironment, resulting in a targeted VEGF-C mutein therapy.
In one aspect is provided 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 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 fusion protein thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.
In one aspect, provided herein is a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a VEGF-C mutein proteins or functional fragments thereof of the present disclosure or the fusion proteins of the present disclosure, or the polynucleotide molecules of the present disclosure, or the vectors of the present disclosure, or the particles of the present disclosure, or the pharmaceutical compositions of the present disclosure.
In some embodiments, the disease or condition is cancer, coronary vessel function, osmoregulation, heart ischemia, restenosis, fibrosis, colitis, chronic liver disease, polycystic kidney disease, diseases or conditions associated with lymph node transplant, 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.
In some embodiments, the cancer is in the brain or the 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 melanoma and rhabdoid tumor.
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, via eye drops, subcutaneously, intradermally, via inhalation, via long-dwelling catheter, orally, topically, or systemically. In some embodiments, the pharmaceutical composition is formulated for intrathecal administration. In some embodiments, the pharmaceutical composition is formulated for intratumoral administration. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for intracisternal administration. In some embodiments, the pharmaceutical composition is formulated for eye-drop administration. In some embodiments, the pharmaceutical composition is formulated for intraocular administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intradermal administration. In some embodiments, the pharmaceutical composition is formulated for administration by inhalation. In some embodiments, the pharmaceutical composition is formulated for administration via long-dwelling catheters. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for topical administration (e.g., as a cream or gel).
In one aspect is provided a method of treating a cancer 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 functional fragment thereof or fusion protein thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.
In one aspect is provided a pharmaceutical composition comprising a VEGF-C mutein or a functional fragment thereof and optionally an immunotherapeutic agent.
In one aspect is provided a method of generating a library of VEGF-C muteins or functional fragments thereof, wherein the VEGF-C muteins or functional fragments thereof have (i) specific binding to VEGF; and (ii) reduced binding to VEGFR-2 compared to the wild-type VEGF-C.
In certain embodiments, the cancer is a melanoma. In certain embodiments the cancer is in the brain or the central nervous system of the subject. Examples of such cancer include, but are not limited to, glioma (e.g., astrocytoma, glioblastoma, oligodendroglioma, brain stem glioma, juvenile pilocytic astrocytoma, and optic nerve glioma), ependymoma, subependymoma, primitive neuroectodermal tumor, ganglioglioma, Schwannoma, germinoma, craniopharyngioma, meningioma, CNS lymphoma, pineal tumor, retinoblastoma, uveal melanoma, and rhabdoid tumor.
The glioma can be any tumor that arises from the glia tissue of the brain. In some embodiments the glioma can be a mixed glioma. The glioma can be a low grade glioma or high grade glioma. The glioma can be supratentorial, infratentorial, or pontine. Examples of glioma include, but are not limited to, glioblastoma.
In certain embodiments, the cancer is glioblastoma. In certain embodiments, the cancer is glioblastoma multiforme (GBM). An initial diagnosis of GBM is generally made using CT or MRI, in which the glioblastomas generally appear as ring-enhancing lesions. Confirmation of the diagnosis can be made based on a biopsy, e.g., a stereotactic biopsy or a craniotomy with tumor resection.
In certain embodiment, the cancer is a metastatic cancer. In certain embodiment, the cancer is a metastatic cancer that has spread into the brain or the central nervous system of the subject. In certain embodiment, the cancer is a metastatic brain cancer.
In certain embodiment, the cancer is melanoma, lung cancer, breast cancer, stomach cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, head and neck squamous cell carcinoma, thyroid cancer, liquid cancer (such as, e.g., acute myeloid leukemia), kidney cancer, urothelial bladder cancer, prostate cancer, pheochromocytoma, cholangiocarcinoma, liver hepatocellular carcinoma, pancreatic ductal adenocarcinoma, thymoma, sarcoma, mesothelioma, testicular cancer and colorectal cancer.
In certain embodiments, the VEGF-C muteins or the 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 or regulation of cardiac functions or diseases. Examples of such cardiac functions or diseases include, but are not limited to, coronary vessel function, osmoregulation, heart ischemia, and restenosis.
In certain embodiments, the VEGF-C muteins or the 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 pulmonology.
In certain embodiments, the VEGF-C muteins or the 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.
In certain embodiments, the VEGF-C muteins or the 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.
In certain, non-limiting, embodiments, the VEGF-C muteins or the 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 lymph node transplants.
In certain embodiments, the VEGF-C muteins or the 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 gut health. In certain, non-limiting, embodiments, the VEGF-C muteins or the functional fragments thereof can be used for the treatment of colitis and chronic liver disease.
In certain embodiments, the VEGF-C muteins or the 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.
In certain, non-limiting, embodiments, the VEGF-C muteins or the 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.
In certain, non-limiting, embodiments, the VEGF-C muteins or the 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 with lung injury. In certain embodiments, the VEGF-C muteins or the functional fragments thereof have neuro-regenerative properties.
In certain, non-limiting, embodiments, the VEGF-C muteins or the 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.
In certain, non-limiting, embodiments, the VEGF-C muteins or the 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 lymphedema. Non-limiting examples of lymphedema include, but are not limited to, primary lymphedema, secondary lymphedema, and hereditary lymphedema.
In certain embodiments, the VEGF-C muteins or the 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 genetic conditions. Non-limiting examples of such genetic conditions include, but are not limited to, Milroy's disease, Hennekam syndrome and Turner syndrome.
In certain embodiments, the VEGF-C muteins or the 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 ocular diseases. Non-limiting examples of such ocular diseases include, but are not limited to, age related macular degeneration, glaucoma, diabetic retinopathy, central serous chorioretinopathy, macular edema, and retinal edema.
In certain embodiments, the VEGF-C muteins or the 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.
In certain embodiments, the VEGF-C muteins or the 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 cancer vaccines.
In some embodiments, the method does not comprise administering a tumor-specific antigen to the subject.
In certain embodiments, the VEGF-C muteins or the 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 an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. The immune checkpoint inhibitor may target PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, BTLA, GITR, 4-1BB, or Ox-40. The immune checkpoint inhibitor may be an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-BTLA antibody, an anti-GITR antibody, an anti-4-IBB antibody, or an anti-Ox-40 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody.
The VEGF-C mutein or the functional fragment 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) and the immunotherapeutic agent may be administered conjointly. For example, the VEGF-C mutein and the immunotherapeutic agent are administered in the same composition.
Alternatively, the VEGF-C mutein or the functional fragment 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) and the immunotherapeutic agent may be administered sequentially.
In various embodiments, the VEGF-C mutein or the functional fragment 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) is administered prior to administering the immunotherapeutic agent. The VEGF-C mutein or the functional fragment 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 administered locally to the brain or central nervous system (e.g., to the cisterna magna) and then the immunotherapeutic agent can be administered systemically (e.g., intravenously). The VEGF-C mutein or the functional fragment thereof can be administered intratumorally (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) and then the immunotherapeutic agent can be administered systemically (e.g., intravenously). The VEGF-C mutein or the functional fragment 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 administered intrathecally and then the immunotherapeutic agent can be administered systemically (e.g., intravenously). The VEGF-C mutein or the functional fragment 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 administered directly into the lymphatic system and then the immunotherapeutic agent can be administered systemically (e.g., intravenously).
In various embodiments, the methods for treating cancer further comprise administering an additional anti-cancer treatment to the subject. Examples of the additional anti-cancer treatments include, but are not limited to, surgery, radiation therapy, administration of a chemotherapeutic agent, and any combinations thereof. These additional anti-cancer treatments may be administered before, conjointly with, or after the administration of the VEGF-C mutein or the functional fragment 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).
In various embodiments, the subject is a human patient. The human patient can be a child or an adult.
In various embodiments, the method is effective to treat the cancer in the subject. In some embodiments, the method is effective to induce lymphangiogenesis in the tumor in the brain or the central nervous system of the subject. In various embodiments, lymphangiogenesis can be confirmed through MRI imaging, e.g., in which the diameter of lymphatic vasculature can be calculated using a contrast agent. In various embodiments, lymphangiogenesis can be confirmed through serial CSF collection to measure VEGFA, VEGFB, VEGFC or VEGFD concentrations. In various embodiments, lymphangiogenesis can be confirmed through serial CSF collection to measure VEGFC concentrations. The method may be effective to reduce tumor volume. In some embodiments, the method is effective to reduce the volume of a tumor in the brain or the central nervous system of the subject. In various embodiments, the method is effective to provide an immune memory against the tumor. Without wishing to be bound by theory, low clinical efficacy of immunotherapy for GBM patients may be due to a low antigen sampling from the CNS at steady state and during initial stages of tumor development. The administration of a VEGF-C mutein or a functional fragment thereof may increase the amount of antigen sampling that occurs in the brain, which in turn could improve the efficacy and outcome of any other immunotherapy (e.g., anti-CTLA-4 antibody) administered. Although VEGFC's role in cancer has been thought to promote metastasis through (lymph) angiogenesis, the inventors have surprisingly shown that in the brain, VEGF-C can reduce tumor size through an increase in immunosurveillance. VEGF-C may stimulate lymphatic endothelial cell proliferation through VEGFR-3 and increase lymphatic vessel functions.
In one aspect, provided herein is a method for modulating intraocular pressure in a subject in need thereof comprising administering to the subject an effective amount of the VEGF-C mutein protein 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 fusion protein 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 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 one aspect, provided herein is a method for removing unwanted fluid in an eye of a subject in need thereof comprising administering to the subject an effective amount of the 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 fusion protein 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 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, retinal, subretinal, choroidal, or suprachoroidal fluid.
In some embodiments, wherein the method of the present disclosure is a method for modulating intraocular pressure in a subject in need thereof or a method for removing unwanted fluid in an eye of a subject in need thereof, the subject has glaucoma, macular edema, central serous chorioretinopathy, retinal edema, papilledema, macular degeneration, or diabetic retinopathy.
In some embodiments, wherein the method of the present disclosure is a method for modulating intraocular pressure in a subject in need thereof or a method for removing unwanted fluid in an eye of a subject in need thereof, the VEGF-C mutein protein or functional fragment thereof, fusion protein, conjugate, polynucleotide molecule, vector, particle, or pharmaceutical composition is administered to the posterior eye. In some embodiments, the administration is intraocular. In some embodiments, the intraocular administration is intravitreal, via eye drops, or subretinal.
In one aspect, provided herein is a method for providing neuroprotection in a subject in need thereof comprising administering to the subject an effective amount of the 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 fusion protein 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 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 neuroprotection is provided, but not limited to, for macular degeneration, glaucoma, stroke, Alzheimer's disease, or Parkinson's disease. In some embodiments, the administration is intraocular. In some embodiments, the intraocular administration is intravitreal, via eye drops, or subretinal.
In one aspect, provided herein is a vaccine comprising the 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 fusion protein thereof, or a vector or particle comprising said polynucleotide molecule, or a pharmaceutical composition comprising any of the above.
In one aspect, provided herein is a method inducing an immune response in a subject in need thereof in a subject in need thereof, the method comprising administering to the subject an effective amount of the vaccine of the present disclosure. In some embodiments, the immune response is, but not limited to, an anti-cancer mediated immune response, a vaccine mediated immune response, an anti-viral, immune response, an anti-bacterial immune response or any other anti-pathogen immune response.
The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.
Therapeutic stimulation of lymph vessel development (lymphangiogenesis) holds promise as an emerging treatment paradigm for a wide range of indications spanning cardiovascular disease to cancer immunotherapy. However, there are currently no specific pharmacologic agents that promote lymphangiogenesis that do not also stimulate potentially harmful angiogenesis. Notably, the principle physiological driver of lymphangiogenesis, Vascular endothelial growth factor C (VEGF-C), agonizes pro-lymphangiogenic signaling via VEGFR-3, but also signals through the pro-angiogenic VEGFR-2. Here, directed evolution with yeast-surface display was used to generate a lymphatic-specific VEGF-C variant that only engages VEGFR-3 but does not signal through VEGFR-2 (LS-VEGF-C). Compared to wild-type VEGF-C, LS-VEGF-C demonstrated superior preclinical efficacy in models of lymphangiogenesis and anti-PD-1 cancer immunotherapy. The biased impact of LS-VEGF-C on lymphangiogenesis versus angiogenesis was further determined herein by studying its effects on augmenting posterior lymphatic drainage in the eye. LS-VEGF-C markedly lowered intraocular pressure in normotensive mice and in two distinct ocular hypertensive mouse models, but without increasing angiogenesis and vascular permeability as seen with WT-VEGF-C treatment. LS-VEGF-C also demonstrated neuroprotective properties independent of intraocular pressure in a model of retinal ganglion cell death from excitotoxic injury. Collectively, these results highlight the feasibility and preclinical efficacy of therapeutic lymphangiogenesis via VEGFR-3-specific agonism and reveal an unexpected impact of lymphangiogenesis in the control of intraocular pressure.
Extracellular domain of murine VGFR2 (first three Ig domains, amino acids 20-326), VGFR3 (amino acids 25-329), human VGFR2 (amino acids 19-325) and VGFR3 (amino acids 25-329) were cloned into the pEZT vector with an N-terminal H7 signal peptide, a C-terminal AviTag and hexahistidine tag (SEQ ID NO: 25), and expressed by transient transfection of Expi293 cells (Thermo Fisher) per manufacturer's instructions. Proteins were enriched from cell supernatant via Ni-NTA chelating resin and further purified by size exclusion chromatography (Column SEC650, Bio-rad) into a final buffer of HEPES buffered saline (HBS; 10 mM HEPES, pH 7.5, 150 mM NaCl). Protein biotinylation was carried out at room temperature for 2 h with 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.
Murine VEGF-C (amino acids 112-215) was cloned into a C-terminal displayed vector pCT-GCN42 with a yeast GCN4 sequence RMKQLEDKIEELLSKIYHLENEIARLKKLIGER (SEQ ID NO: 26) to promote in-situ dimerization, and displayed on the surface of yeast strain Saccharomyces cerevisiae EBY100. Yeasts were maintained and expanded in liquid synthetic dextrose medium with casamino acids (SDCAA) at 30° C. and then induced for expression in liquid synthetic glucose medium with casamino acids (SGCAA) at 20° C. for 24-48 h. The displayed protein level was verified by staining with a C-terminal Myc tag antibody (Cell Signaling Technology, #3739S). Biotinylated VEGFR-2 and VEGFR-3 binding was detected using a fluorescent streptavidin phycoerythrin secondary and quantified by flow cytometry using a Sony SA3800 flow cytometer.
14 residues were identified from analysis of the three dimensional structures of the VEGF-C: VEGFR3 and VEGF-C: VEGFR2 complexes. A library randomizing these residues (
Experiments were conducted using a Biacore T100 and carried out at 25° C. Protein concentrations were quantified by 280 nm absorbance with a Nanodrop2000 spectrometer (Thermo Scientific). Biotinylated proteins, VEGFR-2 or VEGFR-3, were immobilized onto a Biacore biotin CAPture sensor chip (Cytiva Life Sciences). An unrelated biotinylated protein was immobilized to act as a reference surface for nonspecific binding. Measurements were made with serial dilutions of WT-VEGF-C, LS-VEGF-C, and C125S in Hepes buffer saline-P+ buffer (GE Healthcare) using single cycle kinetics. The VEGFR-2 and VEGFR-3 surfaces were regenerated using the reagents provided in the CAPture kit according to the manufacturer's instructions. All data was analyzed with the Biacore T100 evaluation software 2.0 with a 1:1 Langmuir binding model.
Crystal structures of 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 m VEGF-C which were in contact with both mVEGFR-2 and m VEGFR-3 were identified to create mutation library (Table 1). Synthesized degeneration primers covering these mutated residues were used to assemble the library (Table 2). The PCR products were further amplified with primers containing homology to the vector and co-electroporated into EBY100 competent yeast together with linearized pCT-GCN42 vector. The resulting library was later measured to contain 108 transformants.
Transformed yeasts were recovered and expanded in SDCAA medium at 30° C. and induced in SGCAA medium at 20° C. for 24-48 h. Naïve libraries were selected with 2 rounds of 1 μM mVEGFR-3 to enrich mVEGFR-3 positive binders using LS column (Miltenyi, #130-042-401) and magnetic selection. Starting from round 3, yeast populations were counter-selected with 1 μM m VEGFR-2 monomer and selected with 100 nM m VEGFR-3 using flow cytometer sorting with Sony SH800 cell sorter. For rounds 4-6, counter-selection reagent m VEGFR-2 concentration was gradually increased to 0.25 μM (round 4) and 0.5 M (round 5 & 6) tetramer, while m VEGFR-3 concentration was decreased to 10 nM (round 4), 2 nM (round 5), or 1 nM (round 6). Yeasts post each round of selection were kept and boosted simultaneously to check their binding affinity towards VEGFR-2 and VEGFR-3 when all selections were completed. After the final round of selection, VEGF-C plasmids were extracted from expanded SDCAA cultured yeasts and transformed into E. coli for colony sequencing (Table 3). Converged unique clones were re-transformed into yeast to titrate their binding preference to VEGFR-2 and VEGFR-3. The final selected VEGF-C mutants were expressed by insect cell pFastbac expression system and purified in FPLC with SEC columns. Mono disperse proteins were finally cleared with endotoxin removing for in vivo animal treatment. As shown in
Increased lymphatic drainage demonstrates therapeutic benefit for pathologies such as lymphedema, in which the build-up of fluid due to destroyed lymphatic infrastructure is directly addressed2. With a more immunological perspective, leveraging meningeal lymphatics, increased brain-antigen drainage by lymphangiogenesis and facilitated enhanced immunological response against glioblastoma3. These applications not only demonstrate the therapeutic potential of harnessing lymphangiogenesis, but also its versatility of application even in immune-privileged spaces.
Previous studies show that VEGFR-2 primarily facilitates angiogenesis4 while VEGFR-3 facilitates lymphangiogenesis5-8. Vascular endothelial growth factor C (VEGF-C) is the main growth factor able to promote lymphatic growth and pump, however its pleiotropism, binding to both VEGFR-2 and VEGFR-3 (
Towards overcoming these limitations, it was sought to engineer a more selective, yet potent VEGF-C mutein. To develop a mutant VEGF-C that would only bind to VEGFR-3 and prompt lymphangiogenesis, 14 residues at the receptor binding interface were identified by structural analysis for randomization (
To evaluate whether functionality was conserved, HUVEC and HDLEC cells were used, which are vascular endothelial and lymphatic endothelial cells respectively. Upon administering VEGF-A, LS-VEGF-C, and WT-VEGF-C, which demonstrate binding to VEGFR-2, VEGFR-3, or both (
mRNA/Nanoparticle
VEGF-C wild type and mutein coding mRNA was synthesized by TriLink Bio Technologies with full substitution of pseudouridine and 5-methylcytosine bases, capped using CleanCap reagent AG and poly-adenylated (120A). mRNA was mixed at a ratio of 1 ug per 0.1 μL of in vivo JETPEI (Polyplus Transfection) and vortexed for 30 seconds and incubated in room temperature for 15 minutes before use.
Human umbilical vascular endothelial cells (HUVECs) and human dermal lymphatic endothelial cells (HDLECs) were obtained from (Promocell). They were cultured in MV media (Promocell) with supplements (0.05 mL fetal calf serum/mL, 0.004 mL endothelial cell growth supplement/mL, 10 ng recombinant human EGF/mL, 90 ug heparin/mL, 1 ug hydrocortisone/mL).
HEK293T cells were transfected with the VEGFC mRNA constructs combined with lipofectamine. Supernatant was taken from these cells and HUVEC, HDLEC-j, or HDLEC-a cells were incubated with it. In other experiments, VEGF-C or VEGF-A proteins were directly added to media and put on top of cells. Samples were lysed in RIPA buffer and boiled for 5 minutes with sample buffer. In other experiments, HUVEC and HDLEC cells were treated with 100 ng/ml of VEGF-A, WT-VEGF-C, or LS-VEGF-C in RPMI medium with 1% FBS supplement. Samples were trypsinized (0.05%), quenched with RPMI medium (1% FBS supplement). Supernatant was aspirated, and cell pellets were resuspended and subsequently lysed in RIPA buffer, after which they were boiled for 5 minutes with sample buffer.
Western blotting was performed in a manner similar to that previously reported3. In short, 10% gels were used and run at 10 mA per gel for 30 min and 40 mA per gel until appropriate separation of ladder. Wet transfer was performed at 120 mA per gel for 90 min on ice. After blocking with milk-TBST and 3 washes with TBST, anti-pERK was used at a concentration of 1:1000 and incubated overnight in the cold room. After washing, HRP-conjugated anti-rabbit secondary antibodies were used at a concentration of 1:500 at room temperature for 2 h and imaged using the ChemiDoc MP imaging system (Bio-Rad).
Six-to-ten-week-old mixed sex C57BL/6J (WT), DBA2J and DBA/2J-Gpnmb+/SjJ mice were purchased from Jackson Laboratory, and subsequently bred and housed at Yale University. All procedures used in this study (sex-matched, age-matched) complied with federal guidelines and the institutional policies of the Yale School of Medicine Animal Care and Use Committee.
Using a 96 well plate, 10,000 cells per well were seeded. Cells were first starved for 12 hours in RPMI medium without supplements, before undergoing a wash with PBS and then being treated to experimental conditions. Negative controls were treated to RPMI medium with 1% FBS supplement. Experimental conditions consisted of the addition of 100 ng/ml of VEGF-A, WT-VEGF-C, or LS-VEGF-C to the negative control media (RPMI medium with 1% FBS supplement). A standard curve was made with cell titration. 10 uL of a 12 mM MTT stock solution was then added to each sample. After homogenizing each well by pipetting up and down, absorbance was made at 570 nm and cell count was determined by matching to the standard curve.
The cornea, retina, choroid and optic nerve was isolated for single cell dissociation. These tissues were digested with 1 mg ml−1 collagenase D (Roche) and 30 ug ml−1 DNase I (Sigma-Aldrich) in RPMI at 37° C. for 45 min. Samples were then pipetted up and down to mechanically dissociate the tissue and filtered through a 70-um filter. Samples were then spun down at 5 minutes at 500× g. Cell pellet was then resuspended in FACS buffer (PBS with 2% FBS and 1 mM EDTA) for staining.
Nonspecific binding was blocked using a Fc receptor-blocking solution (TruStain FcX™, 101320, BioLegend) for 10 minutes at 4° C. prior to immunostaining. Subsequently, the 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 run on an Attune NxT flow cytometer and then analyzed using FlowJo software (10.8.1, Tree Star).
For AKT phosphorylation staining, surface markers were first stained on ice for 30 min. Cells were then fixed, and stained following the directions of the BD Phosflow kit. Samples were run on an Attune NxT flow cytometer and then analyzed using FlowJo software (10.8.1, Tree Star).
Intraocular pressures were measured following the consensus recommendations (https://iovs.arvojournals.org/article.aspx?articleid=2778419). Mice were exposed to brief exposure of isoflurane for sedation, after which intraocular pressure was measured by an iCare tonometer. IOP measurement is an average of 6 measurements in one sedation session. Mice were positioned consistently, such that the probe was perpendicular to the surface of the eye when the measurements were taken.
Administrations into the Eye
After intraperitoneal injection of a mixture of ketamine (50 mg kg−1) and xylazine (5 mg kg−1), mice then received topical, intravitreal, or intracameral administration. Topical administration involved placing 5 uL of 1 ug/uL solutions onto the eye. Intravitreal and intracameral administration involved a small puncture along the edge of the cornea to allow access for a Hamilton syringe. For intracameral administration, the needle travels into the anterior chamber. For intravitreal administration, the needle enters the vitreous humor space. Both intravitreal and intracameral administration involved 1 ug/uL solutions. After administration, the eyes were then covered with an artificial tear ointment. Any eyes not being evaluated was also covered with an artificial tear ointment to avoid drying out.
After IVT administration of LS-VEGF-C, mice were then anesthetized with an intraperitoneal injection of ketamine (25 mg kg−1) and xylazine (2.5 mg kg−1) mixture two days later. Subsequently, eyes received topical administration of an FDA-approved drug to assess for combinatorial effect.
Adapted from Sappington et al.18, the mice were anesthetized through intraperitoneal injection of ketamine (50 mg kg−1) and xylazine (5 mg kg−1) and then received intracameral administration of polysterene beads. Artificial tear ointment was then placed topically to avoid drying out the cornea.
WT mice were intravitreally injected with AAVs (dose) with PBS, VEGF-A (concentration), WT-VEGF-C (concentration), or LS-VEGF-C (concentration). After 1 day, the mice were anesthetized intraperitoneally injecting a mixture of ketamine (50 mg kg−1) and xylazine (5 mg kg−1). 100 uL of dye at a concentration of 20 mg/mL was also injected intraperitoneally. Eyes were dilated with 1% tropicamide. After 5 minutes incubation, the mice were placed upon a mount for fundus, fluorescein angiography, OCT imaging on Phoenix Micron IV.
For Evans blue readouts, instead of the dye, Evans blue was injected intraperitoneally. After 4 hours, the mouse was then euthanized and perfused with PBS. The eyes were then isolated and homogenized with beads, before running on a plate reader (Abs_max at 620 nm).
For retinal wholemounts, mice were first enucleated, and the eyes were then fixed in 1% formaldehyde. Upon careful removal of the optic nerve, cornea, and the sclera, the isolated retina was then dissected into quarters with cuts halfway to the optic nerve After staining with Brn3a and DAPI, confocal imaging was done on a LeicaSP8 microscope.
For cross-section of the optic nerve, the optic nerve was fixed in 4% before being processed and embedded in resin/OTC/paraffin mixtures. Blocks were then sectioned on an microtome/cryostat. Cross-sections were then visualized on a transmission electron microscope.
Adapted from Schlüter et al.31, 10 nmol of NMDA was delivered intravitreally and RGCs were evaluated at day 1 post injection by confocal microscopy of retinal wholemounts, as described above.
All statistical analysis was performed using GraphPad Prism software. Data were analyzed with a two-tailed unpaired Student's t-test or paired Student's t-test with Prism software. Statistical significance is defined as *P<0.05, **P<0.01, and ***P<0.001.
Mice were anaesthetized using ketamine and xylazine. One drop of 0.5% Tropicamide was applied to the eyes. Mouse was positioned to expose the superior nasal region of the eye and using a 33 g needle, the superior nasal sclera at the level of the pars plana was punctured. Mouse head was secured, and the needle was positioned at a 45 degree angle. Once the tip was inserted, 2 μL of mRNA-nanoparticle formulation was injected and needle was left in for 5 seconds to prevent backflow. Antibacterial ophthalmic ointment was applied afterwards to prevent infection and mice were placed in a heated cage until full recovery.
Mice were anaesthetized using a mixture of ketamine (50 mg kg−1) and xylazine (5 mg kg−1), injected intraperitoneally. Mice heads were shaved and then 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 bregma. A 10-μl Hamilton syringe loaded with tumor cells was inserted into the burr hole at a depth of 2.5 mm from the surface of the brain and left to equilibrate for 1 minute before infusion. A micro-infusion pump (World Precision Instruments) was used to infuse 3 μl of tumor cells at 1 μl min−1. Once the infusion was finished, the syringe was left in place for another minute before removal of the syringe. Bone wax was used to fill the burr hole and the skin was stapled and cleaned. Following intramuscular administration of analgesic (meloxicam and buprenorphine, 1 mg kg−1), mice were placed in a heated cage until full recovery.
For intracisterna-magna injections, mice were anaesthetized using ketamine and xylazine, and the dorsal neck was shaved and cleaned with alcohol. A 2-cm incision was made at the base of the skull, and the dorsal neck muscles were separated using forceps. After visualization of the cisterna magna, a Hamilton syringe with a 15-degree, 33-gauge needle was used to puncture the dura. Three microliters of mRNA vector (4-5 ug) was administered per mouse at a rate of 1 μl min−1. After completion of the injection, the needle was left in to prevent backflow for an additional 3 minutes. The skin was stapled and cleaned and the same postoperative procedures were performed as for the tumor inoculations.
To confirm that LS-VEGF-C acts through VEGFR-3 signaling in vivo, LS-VEGF-C and WT-VEGF-C were first administered into wild-type mice and then endothelial cells (CD45−CD31+) were evaluated for activation of downstream signaling using phos-flow. WT-VEGF-C showed significant increase of AKT-phosphorylation in endothelial cells regardless of VEGFR-3 expression (
Since its discovery, VEGF-C's potential of lymphatic modulation was demonstrated in vivo in many pathological processes. LS-VEGF-C's ability to provide therapeutic lymphangiogenesis was tested herein in several of these models. First, a mouse model of hind limb lymphedema10 was induced through local lymphatic ablation by surgical ablation. Administration of LS-VEGF-C was able to resolve swelling more effectively than WT-VEGF-C and to an extent comparable to normal (
Previous studies show that the eye has a lymphatic system draining the anterior compartment; lymph vessels in the conjunctiva11 and ciliary body12 have previously been described, as well as a posterior glymphatic clearance system.13 It was recently identified by our team that ocular lymphatics are compartmentalized and established anterior and posterior part of the eye have distinct lymphatic drainage systems. By expanding these lymphatic structures through lymphangiogenesis, it was hypothesized herein that ocular administration of LS-VEGF-C would lead to increased drainage and drop intraocular pressure in the eye.
WT-VEGF-C lead to lower intraocular pressure (IOP) measurements by intracameral injection, similar to previous reports14, and after intravitreal injection (
To evaluate the off-target effects and vascular changes from VEGF growth factors, retinal vasculature was imaged after intravitreal injection of VEGF-A, WT-VEGF-C, and LS-VEGF-C. Looking at fundus photos, compared to the PBS control, VEGF-A and WT-VEGF-C demonstrated spots of hyper-reflection along with increased vascular tortuosity (
Having demonstrated IOP decreasing efficacy in wild-type mice, it was examined whether LS-VEGF-C could offer therapeutic benefits in glaucoma models. Glaucoma is characterized as a neurodegenerative disease, due to retinal ganglion cell (RGC) death, and is often associated with elevated intraocular pressure17. A microbead model was first used, which blocks outflow pathways in the eye and induces elevated IOP in a short time period18, reminiscent of acute closed angle glaucoma (
To further evaluate the effects of LS-VEGF-C in glaucoma, a spontaneous glaucoma mouse was utilized, the DBA2J pigment dispersion model.19 As a negative control to establish baseline, a D2-Gpnmb+ model was used, which has a normally functioning Gpnmb gene and therefore no iris pigment dispersion that leads to spontaneous glaucoma. Although the mechanical injury to the anterior chamber is similar to those of the microbead injection, this model presents a more chronic disease state with a lead time of 6-8 months to demonstrate significantly elevated IOP (
Although the exact mechanism of neuronal death in ocular-neurodegenerative diseases are still unknown, a unifying theory includes the build-up of metabolites or excitatory neuronal signals that are neurotoxic20,21. Given that VEGF-C is implicated in lymphatic drainage, which allows for clearance of metabolites and macromolecules in organs22,23, it was theorized herein that LS-VEGF-C may have neuroprotective properties in glaucoma beyond its ability to decrease IOP. Intravitreal injection of NMDA resulted in widespread RGC death, which was prevented with co-administration of LS-VEGF-C intravitreally (
Yeast display of LS-hVEGF-C variants: Candidate human LS-VEGF-C sequences (SEQ IDs: 1-50 and 100) and WT hVEGF-C were synthesized by Twist Biosciences and inserted into a yeast display system as described above. All clones contained a C137A mutation for enhanced biochemical stability (independent of 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 one hour at 4° C. Yeast were then washed with FACS buffer (PBS+0.5% BSA+0.5 mM EDTA) and stained with a fluorescent streptavidin secondary. Receptor binding was then quantified by flow cytometry. For the binding titration studies in
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 and protein yield was quantified in milligrams per liter (Table 6). Quality was then assessed by size exclusion chromatography using a Superose 6 column (GE Healthcare) (
VEGFR3fl/fl mice were bred with Cdh5aCRE, NESTINCRE and PROXICRE (all ERT2, tamoxifen inducible) mice to generate tamoxifen inducible, conditional knockout mice. In the Control settings, mice received just corn oil, in the other settings, mice received tamoxifen daily for 7 days. All mice were then given ocular hypertension with bead injection into the anterior chambers. After increases in intraocular pressure was confirmed (baseline), all mice were treated with RTI-Fc intravitreally and eye pressures were measured 7 days later.
Data, shown in
Wildtype mice were either treated with intravitreal LS-VEGF-C in the form of a monomer, with albumin conjugation or Fc conjugation and evaluated for intraocular pressure drops.
The monomer had almost no effect while the albumin conjugation and Fc conjugation had similar peak decreases in IOP with the Fc conjugate showing superiority overtime (
The creation of a lymphatic-specific VEGF-C that provides a new molecular tool to study lymphatic biology is provided herein. Although lymphatic vasculature dysregulation is implicated in many disease processes, translation of these findings into clinic is limited by lack of methods to specifically stimulate lymphatics. Previous efforts identified muteins that lost VEGFR-2 binding, but also significantly lost binding towards VEGFR-324-26. To address these clinical barriers, a library of VEGF-C muteins was created herein to generate LS-VEGF-C proteins that demonstrate picomolar binding to VEGFR-3 without binding VEGFR-2. By utilizing this new property, it was first highlighted herein the potential of this new molecule to treat various diseases known to have lymphatic dysfunction. By completely abrogating the angiogenic activity, LS-VEGF-C's activity could be further expanded into new disease spaces. The superior efficacy of LS-VEGF-C as a therapy for glaucoma was shown herein, a disease with clearly identifiable biophysical dysfunction that can benefit from lymphatic therapy-WT-VEGF-C is precluded as a possible candidate due to the dangers of angiogenesis in the eye. LS-VEGF-C not only demonstrated strong in vivo activity consistent with its binding properties, but also resulted in no angiogenesis or vascular permeability.
This is not the first time that lymphatic-based therapy was proposed for glaucoma. Trabecular meshwork and Schlemm's canal show signatures of lymphatic vasculature and have been identified as regions that can be stimulated by lymphatic signaling14,27,28. This concept has also been applied to aqueous mapping in NHPs and humans29. In addition, uveoscleral lymphatic pathways are also thought to be involved in aqueous humor drainage12. As stated above, the focus on decreasing ocular pressure has been focused specifically in the anterior compartment, with all drugs and surgical procedures targeting components of the anterior eye. However, the pathology of glaucoma occurs in the posterior eye, with damage to the optic nerve being the primary sign of progression. It was decided herein that IOP decreasing focused in this compartment will provide a unique approach to complement current therapeutic strategies. As a therapeutic molecule, LS-VEGF-C demonstrated herein potent effects in decreasing IOP in two models of ocular hypertension which resulted in significant neuronal health preservation. Beyond this, it was demonstrated herein that stimulation of the optic nerve lymphatics allows for drainage of neurotoxic molecules from the eye that allows for neuroprotection.
Most importantly, LS-VEGF-C is not only a new pharmacological agent but a tool to answer questions regarding lymphatic biology. Previous reports have tried to specifically stimulate VEGFR-3 and concluded that without VEGFR-2 signaling, effects were not as potent30. In contrast, LS-VEGF-C demonstrated stronger phenotypic differences compared to WT-VEGF-C in vivo. This is likely due to a combination of factors including 1) without VEGFR-2 binding, there is no VEGF-C being sequestered away from its lymphatic binding partner VEGFR-3 and 2) LS-VEGF-C retains picomolar concentration binding affinity to VEGFR-3. This makes our mutein not only an excellent candidate for clinical implications requiring lymphatic stimulation, but also a unique tool to decouple VEGFR-2 (angiogenic) and VEGFR-3 (lymphangiogenic) signaling. Its utility can be imagined for diseases with obvious indications such as lymphedema2 to recent discoveries in neurodegeneration22,23, having potential to make significant impact in human health and disease. In summary, the present results from developing a VEGFR-3-specific ligand establish the therapeutic potential and versatility of lymphangiogenesis, even in immune-privileged spaces.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. It is further to be understood that all values are approximate, and are provided for description.
Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.
This application claims benefit of Provisional U.S. Application No. 63/317,614, filed Mar. 8, 2022, the contents of which is incorporated by reference in its entirety for all purposes.
This invention was made with government support under CA239444 awarded by National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/063952 | 3/8/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63317614 | Mar 2022 | US |
