WO2014093387A1 - Protéines hybrides du récepteur de vegf pour utilisation vétérinaire - Google Patents

Protéines hybrides du récepteur de vegf pour utilisation vétérinaire Download PDF

Info

Publication number
WO2014093387A1
WO2014093387A1 PCT/US2013/074196 US2013074196W WO2014093387A1 WO 2014093387 A1 WO2014093387 A1 WO 2014093387A1 US 2013074196 W US2013074196 W US 2013074196W WO 2014093387 A1 WO2014093387 A1 WO 2014093387A1
Authority
WO
WIPO (PCT)
Prior art keywords
companion animal
fusion protein
igg
seq
vegf
Prior art date
Application number
PCT/US2013/074196
Other languages
English (en)
Inventor
Richard Chin
Original Assignee
Kindred Biosciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kindred Biosciences, Inc. filed Critical Kindred Biosciences, Inc.
Publication of WO2014093387A1 publication Critical patent/WO2014093387A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention provides methods and compositions for treating diseases such as cancer in companion animals with vascular endothelial growth factor (VEGF) receptor fusion proteins and so relates to the fields of biology, molecular biology, and veterinary medicine.
  • diseases such as cancer in companion animals with vascular endothelial growth factor (VEGF) receptor fusion proteins and so relates to the fields of biology, molecular biology, and veterinary medicine.
  • VEGF vascular endothelial growth factor
  • VEGF also known as vascular permeability factor
  • cancer inflammatory disease
  • pre-eclampsia autoimmune disease (such as rheumatoid arthritis and psoriasis)
  • atherosclerosis diabetic retinopathy
  • retrolental fibroplasia neovascular glaucom
  • VEGF associated conditions such as cancer and macular degeneration in humans.
  • aflibercept marketed under the trade name Zaltrap (Regeneron/Sanofi-Aventis): see US Patents No. 7,070,959 and 6,100,071.
  • Aflibercept has been approved for the treatment of wet macular degeneration and colorectal cancer.
  • Another agent is bevacizumab, marketed under the trade name Avastin (Genentech), approved for similar indications in humans.
  • Companion animals such as cats, dogs, and horses, suffer from many diseases similar to human diseases, including cancer, macular degeneration, and autoimmune diseases.
  • aflibercept or bevacizumab could be used to treat such diseases in companion animals.
  • proteins with significant human-derived amino acid sequence content can be immunogenic in non-human animals, and aflibercept and bevacizumab might not bind sufficiently well to companion animal VEGF if the companion animal VEGF differs in sequence from human VEGF.
  • the present invention provides companion animal VEGF receptor fusion proteins capable of binding to companion animal VEGF and thereby exerting an inhibitory effect thereon.
  • These fusion proteins comprise tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2, and 3 and an amino acid sequence or other molecule that extends half-life of the fusion protein in vivo.
  • the tyrosine kinase receptor-derived immunoglobulin-like domains are from the extracellular ligand-binding region of the companion animal flt- 1, flt-4, and KDR receptors.
  • the amino acid sequence or other molecule that extends half-life in vivo is the Fc region of companion animal immunoglobulin gamma, wherein said companion animal VEGF receptor protein and said companion animal immunoglobulin gamma Fc region are derived from the same companion animal.
  • the present invention provides pharmaceutical compositions comprising the fusion proteins of the present invention admixed with one or more pharmaceutically acceptable excipients.
  • the pharmaceutical compositions are suitable for parenteral administration.
  • the present invention provides nucleic acids encoding companion animal VEGF receptor fusion proteins, including expression vectors for expressing such proteins.
  • the present invention also provides recombinant host cells transfected with those expression vectors.
  • the present invention provides methods for treating disease in companion animals that comprise administering therapeutically effective amounts of the fusion proteins or pharmaceutical compositions of the invention to the companion animal.
  • the present invention provides methods for preparing the fusion proteins and pharmaceutical compositions of the invention. [0010] These and other aspects and various embodiments of them are described more fully below.
  • the present invention provides companion animal VEGF receptor fusion proteins comprising amino acid sequences derived from the companion animal vascular endothelial growth factor (VEGF) receptors fit- 1 and KDR that bind to companion animal VEGF.
  • the present invention also provides nucleic acids and expression vectors encoding the companion animal VEGF receptor fusion proteins, host cells harboring such expression vectors, pharmaceutically compositions comprising such proteins, methods of preparing such proteins, and methods utilizing such proteins and pharmaceutically compositions for the treatment of conditions associated with undesired effects of companion animal VEGF.
  • the VEGF receptor fusion proteins of the present invention further include peptide constructs comprising VEGF receptors that are linked to the Fc regions of various canine immunoglobulin ⁇ chains, namely, IgG-A, IgG-B, IgG-C, and IgG-D.
  • amino acid sequence means a sequence of amino acids in a protein, and includes sequences of amino acids in which one or more amino acids of the sequence have had their side-groups chemically modified, as well as those in which, relative to a known sequence, one or more amino acids have been replaced or deleted, without thereby eliminating a desired property, such as VEGF binding ability.
  • “Companion animal” means cat, dog, or horse.
  • Companion animal fit- 1 receptor means the companion animal homologs of the human flt-1 receptor, also known as VEGF-R1.
  • Companion animal VEGF means companion animal vascular endothelial growth factor, also known as vascular permeability factor.
  • Companion animal immunoglobulin-like domain and “companion animal Ig-like domain” means any of the seven independent and distinct domains in the extracellular ligand-binding region of the companion animal flt-1, KDR, and flt-4 receptors.
  • Companion animal KDR receptor means the companion animal homologs of the human KDR receptor, designated FLK- 1.
  • Companion animal VEGF receptor fusion protein means a molecule having amino acid sequences derived from companion animal tyrosine kinase receptor- derived immunoglobulin-like domain and includes fusion proteins further comprising either an amino acid sequence derived from a heterologous protein or a non-protein molecule, such as a polyethylene glycol, that confers extended half-life in vivo on the fusion protein to which it is attached.
  • IgG Fc means the Fc region of an immunoglobulin ⁇ chain.
  • IgG-N-Fc means the Fc region of a specific immunoglobulin ⁇ chain that has been identified by a specific letter, wherein the specific letter is substituted for "N”.
  • the "Fc region” is that portion of the immunoglobulin ⁇ chain amino acid sequence comprising the hinge, CH2, and CH3 domains.
  • IgG Fc and IgG-N-Fc regions may be derived from any companion animal, such as dogs, cats, and horses.
  • IgG Fc regions are isolated from canine immunoglobulin ⁇ heavy chains IgG-A, IgG-B, IgG- C, and IgG-D, represented by SEQ ID NOs: 7, 11, 3, and 15, respectively.
  • IgG-N-Fc region of IgG-A is represented as "IgG-A-Fc.”
  • Constants in companion animals associated with companion animal VEGF means diseases in companion animals associated with, or caused by, or characterized by, elevated levels or altered gradients of companion animal VEGF concentration. Such diseases include, but are not limited to, cancer and macular degeneration. Other such diseases are listed in the background section above.
  • Companion animal tyrosine kinase receptor means companion animal fit- 1 receptor or companion animal KDR receptor or companion animal flt-4 receptor.
  • Ig-like domain means the complete wild- type domain and insertion, deletion, and substitution variants thereof that substantially retain the functional characteristics of the intact domain.
  • the present invention provides companion animal VEGF receptor fusion proteins capable of binding to companion animal VEGF and exerting an inhibitory effect thereon, wherein said companion animal VEGF receptor fusion proteins comprise Ig-like domains 1, 2, and 3 of the companion animal fit- 1 and/or KDR receptor and/or flt-4 receptor (in any combination) or functional equivalents thereof.
  • the fusion proteins further comprise a heterologous protein or other compound that confers a longer half-life to the fusion protein to which it is attached.
  • the Ig-like domains 1, 2, and 3 have an amino acid sequence consisting essentially of the corresponding Ig-like domain of the companion animal fit- 1 or KDR or flt-4 receptor.
  • the three domains are linked by short (one to ten amino acid residue) sequences that can either be derived from the corresponding companion animal protein but can be any non-immunogenic sequence that does not substantially negatively impact the VEGF binding properties of the fusion protein.
  • the companion animal VEGF receptor fusion proteins of the present invention comprise canine fit- 1 amino acid receptor sequences corresponding to Ig-like domains 1, 2, and 3 of the extracellular ligand-binding region thereof, and a canine IgG Fc amino acid sequence, which confers extended in vivo half-life on the fusion protein.
  • various linker sequences may be present between domains 1 and 2, domains 2 and 3, and domain 3 and the canine IgG Fc amino acid sequence.
  • the IgG Fc amino acid sequence is not present, but a poly-glycine moiety (of from 50 to 2,000 glycine residues) instead serves the purpose of providing a longer half-life to the fusion protein.
  • SEQ ID NO: l provides the amino acid sequence for canine fit- 1 , a sequence of 1337 amino acids.
  • Residues 32 to 128 constitute Ig-like domain 1.
  • Residues 134 to 226 constitute Ig-like domain 2.
  • Residues 232 to 331 constitute Ig-like domain 3.
  • Suitable linkers to link domains 1 and 2 and 2 and 3 thus include residues 129 to 133 and 227 to 231, respectively, for canine fusion proteins of the invention.
  • SEQ ID NO:2 provides the amino acid sequence for canine KDR, a sequence of 1355 amino acids.
  • Residues 32 to 118 constitute Ig-like domain 1.
  • Residues 124 to 220 constitute Ig-like domain 2.
  • Residues 226 to 327 constitute Ig-like domain 3.
  • Suitable linkers to link domains 1 and 2 and 2 and 3 thus include residues 119 to 123 and 221 to 225, respectively, for canine fusion proteins of the invention.
  • Amino acid sequences from canine IgG-C (SEQ ID NO:3), IgG-A (SEQ ID NO:7), IgG-B (SEQ ID NO: 11), and IgG-D (SEQ ID NO: 15) are suitable for use in the fusion proteins of the invention to provide the fusion proteins with longer in vivo half-life.
  • Fusion proteins of the invention comprising an Fc region of IgG-A, IgG-B, and IgG-D may provide for higher expression levels in recombination production systems, such as plant cell-based recombinant expression systems, and may be purified using a Protein-A resin column more readily than fusion proteins comprising the Fc region of IgG-C.
  • Illustrative VEGF receptor fusion proteins of the invention suitable for use in canines comprising such sequences include the fusion proteins defined by SEQ ID NOs:4-6, 8-10, 12-14, and 16-18.
  • the VEGF fusion proteins of the present invention comprise various independent amino acid sequences linked by amide bonds.
  • Some embodiments of the present invention include a fusion protein comprising a VEGF receptor, more typically a portion of a VEGF receptor, linked to an isolated Fc region of a companion animal immunoglobulin gamma (IgG) chain via an amino acid or peptide.
  • a linker can provide flexibility between the fused moieties, thereby allowing the molecule to bend and contour for improved efficacy.
  • the linker is a single amino acid residue, such as a glycine, alanine, or serine.
  • various fusion proteins of the invention utilize a linker that is a single glycine residue.
  • there is no linker, or the linker is an amino acid or sequence of 10 or fewer amino acids, typically amino acids that do not participate in disulfide bond formation or impart unusual structural features, i.e., cysteine and proline amino acids may be avoided in linkers of the invention.
  • SEQ ID NOs: 4-6, 8-10, 12-14, and 16-18 provide non-limiting examples of various IgG Fc fusion proteins of the invention.
  • Each of these fusion proteins includes sequences derived from the canine VEGF receptor fit- 1.
  • the methods and compositions described herein can be adapted to provide IgG Fc fusion proteins of the invention that utilize alternative VEGF receptor sequences, such as those derived from the feline or equine VEGF receptors.
  • the fit- 1 sequence is, in some embodiments, substituted with homologous sequences derived from flt-4 and KDR to provide other fusion proteins of the invention.
  • SEQ ID NO:4 is a canine flt-l-IgG-C Fc fusion protein of the invention. Residues 1-303 of SEQ ID NO:4 correspond to the canine fit- 1 peptide (and correspond to residues 26-328 of SEQ ID NO: l). Residues 1-303 of SEQ ID NO:4 are linked to linker IEGRMD (SEQ ID NO: 19). The linker of SEQ ID NO:4 is linked to the Fc region of canine IgG-C (residues 310-553 of SEQ ID NO:4, corresponding to SEQ ID NO: 20, as well as residues 231-474 of SEQ ID NO:3).
  • SEQ ID NO:5 is another canine flt-l-IgG-C Fc fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO:5 correspond to the flt-1 peptide (residues 26-328 of SEQ ID NO: l).
  • Residues 1-303 of SEQ ID NO:5 are linked to a glycine residue linker.
  • the glycine is further linked to the Fc region of canine IgG-C (residues 305- 548 of SEQ ID NO:5, corresponding to SEQ ID NO:20 and residues 231-474 of SEQ ID NO:3).
  • SEQ ID NO:6 is another canine flt-l-IgG-C Fc fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO:6 correspond to the flt-1 peptide (residues 26-328 of SEQ ID NO: l) and are linked to the linker GGGGSGGGGSGGGGS (SEQ ID NO: 21), which is in turn linked to the Fc region of canine IgG-C (residues 319- 562 of SEQ ID NO:6, corresponding to SEQ ID NO:20 and to residues 231-474 of SEQ ID NO:3).
  • SEQ ID NO: 8 is a canine flt-l-IgG-A Fc fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO:8 correspond to the flt-1 peptide (residues 26-328 of SEQ ID NO: l) and are linked to linker IEGRMD (SEQ ID NO: 19), which in turn is linked to Fc region of canine IgG-A (residues 310-542 of SEQ ID NO:8, corresponding to SEQ ID NO:22 and to residues 236-468 of SEQ ID NO: 7).
  • SEQ ID NO:9 is another canine flt-l-IgG-A Fc fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO:9 correspond to the flt-1 peptide (residues 26-328 of SEQ ID NO: l) and are linked to a glycine residue linker that is linked to the Fc region of canine IgG-A (residues 305-546 of SEQ ID NO:9, corresponding to SEQ ID NO:22 and to residues 227-468 of SEQ ID NO:7).
  • SEQ ID NO: 10 is another canine flt-l-IgG-A Fc fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO: 10 correspond to the flt-1 peptide (residues 26-328 of SEQ ID NO: l) and are fused to the linker GGGGSGGGGSGGGGS (SEQ ID NO: 21) that is in turn linked to the Fc region of canine IgG-A (residues 319-551 of SEQ ID NO: 10, correlating to SEQ ID NO:22 and to residues 236-468 of SEQ ID NO:7).
  • SEQ ID NO: 12 is a canine flt-l-IgG-B Fc fusion protein fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO: 12 correspond to the flt-1 peptide (residues 26-328 of SEQ ID NO: l) and are linked to the linker IEGRMD (SEQ ID NO: 19) that is in turn linked to the Fc region of canine IgG-B (residues 310-546 of SEQ ID NO: 12, correlating to SEQ ID NO: 23 and residues 237-473 of SEQ ID NO: 11).
  • SEQ ID NO: 13 is another canine flt-l-IgG-B Fc fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO: 13 correspond to the fit- 1 peptide (residues 26-328 of SEQ ID NO: l) and are linked by a glycine residue that is in turn linked to the Fc region of canine IgG-B (residues 305-550 of SEQ ID NO: 13, correlating to SEQ ID NO: 23 and to residues 228-473 of SEQ ID NO: 11).
  • SEQ ID NO: 14 is another canine flt-l-IgG-B Fc fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO: 14 correspond to the fit- 1 peptide (residues 26-328 of SEQ ID NO: l) and are linked to the linker GGGGSGGGGSGGGGS (SEQ ID NO: 21) that is in turn linked to the Fc region of canine IgG-B (residues 319-555 of SEQ ID NO: 14, correlating to SEQ ID NO: 23 and to residues 237-473 of SEQ ID NO: l l).
  • SEQ ID NO: 16 is another canine fit- 1 -IgG-D Fc fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO: 16 correspond to the fit- 1 peptide (residues 26-328 of SEQ ID NO: l) and are linked to the linker IEGRMD (SEQ ID NO: 19) that is in turn linked to the Fc region of canine IgG-D (residues 310-542 of SEQ ID NO: 16, correlating to SEQ ID NO:24 and to residues 238-470 of SEQ ID NO: 15).
  • SEQ ID NO: 17 is another canine fit- 1 -IgG-D Fc fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO: 17 correspond to the fit- 1 peptide (residues 26-328 of SEQ ID NO: l) that is linked to a glycine residue that is in turn linked to the Fc region of Canine IgG-D (residues 305-546 of SEQ ID NO: 17, correlating to SEQ ID NO:24 and to residues 229-470 of SEQ ID NO: 15).
  • SEQ ID NO: 18 is another canine flt-l-IgG-D fc fusion protein of the invention.
  • Residues 1-303 of SEQ ID NO: 18 correspond to the fit- 1 peptide (residues 26-328 of SEQ ID NO: l) and are linked to the linker GGGGSGGGGSGGGGS (SEQ ID NO: 21) that is in turn linked to the Fc region of canine IgG-D (residues 319-551 of SEQ ID NO: 18, correlating to SEQ ID NO:24 and to residues 238-470 of SEQ ID NO: 15).
  • Additional VEGF receptor fusion proteins include the feline and equine counterparts of the canine fusion proteins described above and the canine, feline, and equine counterparts of the canine fusion proteins described above in which the VEGF receptor sequences are replaced with analogous sequences from the KDR and flt-4 genes.
  • the companion animal VEGF receptor fusion proteins of the present invention comprise feline fit- 1 amino acid sequences corresponding to Ig-like domains 1, 2, and 3 of the extracellular ligand-binding region thereof and feline IgG Fc amino acid sequence, which confers extended in vivo half- life on the protein.
  • various linker sequences may be present between domains 1 and 2 and 2 and 3 and 3 and the feline IgG Fc amino acid sequence.
  • the IgG Fc amino acid sequence is not present but a poly-glycine (of from 50 to 2,000 glycine residues) instead serves the purpose of providing a longer half-life to the fusion protein.
  • the companion animal VEGF receptor fusion proteins of the present invention comprise equine fit- 1 amino acid sequences corresponding to Ig-like domains 1, 2, and 3 of the extracellular ligand-binding region thereof and equine IgG Fc amino acid sequence, which confers extended in vivo half- life on the protein.
  • various linker sequences may be present between domains 1 and 2, domains 2 and 3, and domain 3 and the equine IgG Fc amino acid sequence.
  • the IgG Fc amino acid sequence is not present but a poly-glycine (of from 50 to 2,000 glycine residues) instead serves the purpose of providing a longer half-life to the fusion protein.
  • the companion animal VEGF receptor fusion proteins of the present invention comprise Ig-like domains 1, 2, and 3 of the extracellular ligand-binding region of the companion animal fit- 1 or KDR receptor in addition to one or more of the remaining four immunoglobulin-like domains thereof.
  • the Ig-like domains employed are derived from the same receptor; however, a combination of Ig-like domains derived from one or more of the companion animal fit- 1 and KDR receptors also provide benefit.
  • Various embodiments of the present invention comprise one or more companion animal VEGF receptor fusion proteins, DNA sequences that encode them, recombinant expression vectors that comprise the DNA sequences and transcription and translation control elements suitable for use in recombinant expression systems, which may include, without limitation, plant cell-based recombinant expression systems, and methods for purification of the fusion proteins from such systems. In one embodiment, purification is accomplished using a Protein A resin column.
  • a VEGF receptor fusion protein comprising a companion animal tyrosine kinase receptor selected from fit- 1 , flt-4, and KDR, wherein the companion animal receptor is linked to an IgG Fc region of one or more of the VEGF receptors represented by SEQ ID NOs: 3, 7, 11, and 15.
  • VEGF receptor fusion proteins which include Fc regions selected from, and represented by SEQ ID NOs: 7, 11 and 15 are expressed, purified, and demonstrate increased expression, higher yields, and better purification, as compared to VEGF receptor fusion proteins which include the IgG-C-Fc region of the fit- 1 receptor represented by SEQ ID NO:3.
  • the VEGF receptor fusion proteins of the invention reduce blood vessel formation.
  • a fit- 1 VEGF receptor fusion protein composed of the amino acid sequence glycine 26 to isoleucine 328 of canine fit- 1 followed by a linker sequence which couples the receptor peptide to an IgG Fc region selected from SEQ ID NOs: 7, 11, or 15.
  • these flt-l-Fc VEGF receptor fusion proteins are expressed in plant cells and subsequently purified via a Protein A resin column.
  • the present invention also provides pharmaceutical compositions comprising an animal VEGF receptor fusion protein compounded with a pharmaceutically acceptable carrier, as discussed further below.
  • a pharmaceutically acceptable carrier such as a pharmaceutically acceptable carrier.
  • two or more companion animal VEGF receptor fusion proteins of the invention are combined together to provide a pharmaceutical composition of the invention.
  • the fusion proteins are formulated as multiverse aggregates.
  • the pharmaceutical formulation can comprise homo-dimers or homo- tetramers of a fusion protein of the invention.
  • the present invention also provides nucleic acid sequences encoding the companion animal VEGF receptor fusion proteins of the invention as well as expression vectors comprising such sequences, host cells transfected with those expression vectors, and methods for culturing such host cells to produce the fusion proteins of the invention.
  • the latter methods for producing the companion animal VEGF receptor fusion proteins include the steps of introducing an expression vector encoding the desired protein into an appropriate expression systems and effecting the expression of said protein.
  • Nucleic acids provided by the invention encode the desired companion animal VEGF receptor fusion protein and optionally include one or more additional sequences such as (i) sequences providing restriction enzyme sites to facilitate cloning and constructing expression vectors; (ii) sequences to direct homologous recombination; (iii) promoter, enhancer, terminator, or other expression control sequences; (iv) sequences that encode amino acid sequences, such as His tags, to assist in purification of the fusion protein (in this instance, the fusion protein is expressed as a protein that includes the His tag or other purification facilitating sequence, which is typically located on the amino or carboxy terminus).
  • additional sequences such as (i) sequences providing restriction enzyme sites to facilitate cloning and constructing expression vectors; (ii) sequences to direct homologous recombination; (iii) promoter, enhancer, terminator, or other expression control sequences; (iv) sequences that encode amino acid sequences, such as His tags, to assist in purification of the
  • Nucleic acids of the invention may be constructed by any of several methods known to those of skill in the art. For example, PCR may be performed upon a DNA sequence known to contain a desired sequence(s) to amplify that sequence(s) or the DNA may be synthesized de novo. Using such techniques, one can obtain all or part of the desired sequence as one or more fragments. Sets of fragments can be assembled into a larger fragment using primers for PCR designed such that the appropriate DNA sequence is created via the amplification process. In many embodiments, the desired sequence may be synthesized chemically, in entirety or in several fragments that are ligated together.
  • the present invention also provides expression vectors that encode the fusion proteins of the invention.
  • the expression vectors of the invention include plasmid and virus-derived expression vectors that, when introduced into an appropriate expression system, drive expression of the fusion protein (or fragment thereof, in those embodiments in which the fusion protein is assembled from multiple protein subcomponents).
  • Suitable expression systems include eukaryotic cell-based systems, including but not limited to the CHO cell, yeast, insect cell, transgenic animal, plant cell, and plant (including tobacco) expression systems.
  • Suitable expression systems include permanent and transient systems.
  • the expression system is a cell-free translation system.
  • the expression system is a plant cell-based system.
  • the present invention also provides methods for purifying the resulting fusion proteins.
  • the fusion protein is purified using chromatography such as size exclusion or ion exchange chromatography, protein A column chromatography, and His tag affinity column chromatography.
  • the fusion protein once purified, can be formulated into a pharmaceutical composition of the invention by admixing it with one or more pharmaceutically acceptable carriers or excipients in accordance with the methods of the invention.
  • suitable carriers include alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffers such as phosphate buffers, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol.
  • buffers such as phosphate buffers, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose
  • the companion animal VEGF receptor fusion protein pharmaceutical composition will typically be stored in lyophilized form; thus, in some embodiments, the preparation process includes a lyophilization step.
  • the lyophilized composition is then reformulated, typically as an aqueous composition suitable for parenteral administration, prior to administration to the companion animal.
  • the pharmaceutical composition can be stored as a liquid, i.e., aqueous, composition, which may be administered directly, or with appropriate dilution, to the companion animal.
  • the invention provides pharmaceutical compositions in both solid and liquid form.
  • the pH of the companion animal VEGF receptor fusion protein pharmaceutical compositions typically will be in the range of from about pH 6 to pH 8 when administered to the companion animal.
  • the companion animal VEGF receptor fusion protein formulations of the invention are sterile if they are to be used for therapeutic purposes. Sterility can be achieved by any of several means know in the art, including by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).
  • the pharmaceutical formulations of the invention are useful in the methods of the invention for treating inflammatory, oncologic, immunologic, or other conditions in companion animals associated with altered levels or gradients companion animal VEGF. These methods generally comprise administering a therapeutically effective dose of a fusion protein of the invention to said companion animal. In many embodiments, particularly where the animal companion is suffering from cancer, the therapeutically effective dose is administered parenterally, including by intravenous infusion.
  • a companion animal VEGF receptor fusion protein of the present invention or a pharmaceutical composition comprising it is administered in a therapeutically effective dose to a companion animal.
  • the therapeutically effective dose can be administered as a bolus or by continuous infusion over a period of time, and can be administered by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intrasynovial, intrathecal, and inhalation routes.
  • the companion animal VEGF receptor fusion proteins of the present invention can also be administered by intratumoral, peritumoral, intralesional, and perilesional routes, to exert local as well as systemic effects.
  • the appropriate dose of companion animal VEGF receptor fusion protein can depend upon the type of disease to be prevented or treated, the severity and course of the disease, whether the administration is for preventive or therapeutic purposes, any previous therapy, the clinical history, the response to prior treatment, and the discretion of the attending veterinarian.
  • the therapeutically effective dose of the companion animal VEGF receptor fusion protein can be administered to the animal at one time or over a series of treatments.
  • the "therapeutically effective amount" of a companion animal VEGF receptor fusion protein is an amount that is effective to prevent, lessen the worsening of, alleviate, or cure the disease condition.
  • the therapeutically effective dose is a dose sufficient to reduce or inhibit the proliferation of vascular endothelium in vivo.
  • the therapeutically effective dose is a dose of from 0.1 mg/kg to 100 mg/kg, and this dose is typically administered more than once.
  • the therapeutically effective dose is administered once per week for at least two or three consecutive weeks, and in some embodiments, this cycle of treatment is repeated two or more times, optionally interspersed with one or more weeks of no treatment. In other embodiments, the therapeutically effective dose is administered once per day for two to five consecutive days, and in some embodiments, this cycle of treatment is repeated two or more times, optionally interspersed with one or more days or weeks of no treatment.
  • the fusion protein and pharmaceutical compositions comprising it can be utilized in accordance with the methods of the invention to treat conditions correlated with elevated levels of or altered gradient of companion animal VEGF, such as cancer in dogs, cancer in cats, cancer in horses, inflammatory conditions in dogs, inflammatory conditions in cats, and inflammatory conditions in horses.
  • VEGF companion animal VEGF
  • Neoplasms and related conditions that are amenable to treatment include carcinomas of the skin, breast, lung, esophagus, gastric anatomy, colon, rectum, liver, ovary, cervix, endometrium, and osteosarcoma.
  • DNA sequence encoding canine fit- 1 amino acid sequences corresponding to Ig-like domains 1, 2, and 3 of the extracellular ligand-binding region thereof, a linker amino acid sequence consisting of glycine, and canine flt-l-IgG-C-Fc amino acid sequence (SEQ ID NO:5) is synthesized chemically, along with flanking DNA sequence corresponding to an appropriate restriction enzyme site.
  • the DNA sequence is ligated into an expression vector designed to result in integration of the DNA sequence encoding the transcription and translation of the encoded protein in a CHO cell.
  • the expression vector is placed inside CHO cells via transfection.
  • the CHO cells are selected for high yield and stability of expression of the fusion protein.
  • the CHO cells are cultured until sufficient quantities of the companion animal fusion VEGF receptor fusion protein are produced.
  • the fusion protein is purified by various steps including Protein A column.
  • the purified protein is admixed with excipients, and sterilized by filtration to prepare a pharmaceutical composition of the invention.
  • the pharmaceutical composition is administered to a dog with cancer in a dose sufficient to bind to inhibit canine VEGF. Similar procedures can be carried out to construct the VEGF fusion proteins represented by SEQ ID NOs: 4, 6, 8-10, 12-14, and 16-18.
  • Example 2 Synthesis and Purification of flt-l-fc Fusion Protein from Plant Cells
  • DNA sequence, optimized for expression in plants, encoding canine fit- 1 amino acid sequences corresponding to Ig-like domains 1, 2, and 3 of the extracellular ligand-binding region thereof, a linker amino acid sequence consisting of IEGRMD, and canine IgG-C-Fc amino acid sequence (SEQ ID NO:4) is synthesized chemically, along with flanking DNA sequence corresponding to an appropriate restriction enzyme site.
  • the sequence is sub-cloned into the Tobacco mosaic virus (TMV) vector-based 'launch vector' .
  • TMV Tobacco mosaic virus
  • the resulting expression vector is transformed into Agrobacterium tumefaciens as a delivery vehicle for introducing them into plants. Recombinant A.
  • tumefaciens strains carrying 'launch vectors' encoding each target are introduced into aerial tissues of healthy non-genetically modified Nicotiana benthamiana by the process of vacuum infiltraton.
  • Target expression is monitored over a time course by Western blot analysis. Once the time of peak expression has been identified, larger scale infiltrations are conducted. Once larger scale infiltration is performed, the plants are harvested at a point of high expression (of the companion animal VEGF receptor protein), homogenized or otherwise processed to release the protein, and the resulting material purified by various steps including Protein A column chromatography.
  • the purified protein is admixed with excipients, and sterilized by filtration to prepare a pharmaceutical composition of the invention.
  • the pharmaceutical composition is administered to a dog with cancer in a dose sufficient to bind to inhibit canine VEGF. Similar procedures can be carried out for the VEGF fusion proteins represented by SEQ ID NOs: 5, 6, 8-10, 12-14, and 16-18.
  • VEGF receptor fusion proteins are purified by affinity chromatography.
  • a Protein A column is used to bind, with high specificity, the Fc region of the fusion proteins.
  • the affinity-purified fusion proteins are then concentrated and passed over a SEC column.
  • the fusion proteins are then eluted into a formulation buffer.
  • a preparation containing VEGF receptor fusion proteins derived from the expression system is applied to a Protein A Fast Flow column that has been equilibrated with PBS.
  • the column is washed with PBS containing 350 mM NaCl and 0.02% CHAPS and the bound protein eluted with 20 mM citric acid containing 10 mM Na 2 HP0 4 .
  • the single peak elution is collected and its pH raised to neutrality with 1M NaOH.
  • the eluate fractions are concentrated using 10K regenerated cellulose membranes by both tangential flow filtration and by stirred cell concentration.
  • the contracted protein is applied to a column packed with Superdex 200 preparation grade resin and run in PBS containing 5% glycerol.
  • the main peak fractions are pooled, sterile filtered, aliquoted and stored at -80°C.
  • Fusion proteins of the invention such as the Fltl-Fc proteins can be demonstrated to have the desired biological properties using an assay that evaluates their ability to bind to VEGF; fusion proteins of the invention specifically bind VEGF.
  • an assay that evaluates their ability to bind to VEGF; fusion proteins of the invention specifically bind VEGF.
  • One such assay is commercially available from Biocore (Sweden).
  • fusion proteins of the invention such as the Fltl-Fc proteins are immobilized on the surface of a Biacore chip (see Biacore Instruction Manual, GE Healthcare, for standard procedures) and a sample containing 0.2 ⁇ g/mL VEGF and Fltl-Fc (at 0, 0.5, 1.0, 5.0, 10.0, or 25.0 ⁇ g/mL) is passed over the Fltl-Fc-coated chip.
  • a sample containing 0.2 ⁇ g/mL VEGF and Fltl-Fc (at 0, 0.5, 1.0, 5.0, 10.0, or 25.0 ⁇ g/mL) is passed over the Fltl-Fc-coated chip.
  • the bound samples are washed with a 0.5 M NaCl wash.
  • Fltl-Fc is mixed with heparin. Heparin is a negatively charged molecule and the Fltl-Fc protein is a positively charged molecule.
  • a second method for evaluating unmodified Fltl-Fc is achieved by letting the VEGF/Fltl-Fc mixture bind to the chip and then wash the chip with 0.5 M NaCl, thereby minimizing non-specific binding to the chip.
  • Biacore format can also be used whereby Protein-A (Pierce, Rockford, IL) is linked to the chip surface using (l-Ethyl-3-[3- dimethylaminopropyl]carbodi-imide hydrochloride)/N-hydroxysuccinimide (EDC/NHS) coupling chemistry.
  • Binding of a fusion protein of the invention such as an flt-l-Fc fusion protein to VEGF can also be demonstrated in an ELISA format.
  • a suitable ELISA assay is performed by coating an ELISA plate with VEGF, binding varying concentrations of the unmodified flt-l-Fc to the plate, washing, and detecting bound flt-l-Fc with a reporter detection reagent, such as anti-canine Fc antibody linked to HRP.
  • Example 5 Corneal Neovascularization Model
  • Fusion proteins of the invention can be demonstrated to have the desired biological activity using a corneal neovascularization model.
  • a fusion protein with the desired biological activity will inhibit the neovascularization induced by VEGF.
  • Both sides of the mesh are covered with a thin layer of Hydron to stabilize the pellet hydrophobically and are allowed to dry on a sterile petri dish for 30 minutes. Subsequently, the fibers of the mesh are pulled apart under a microscope, and, among the approximately 200 pellets produced, only 30 to 40 uniformly sized pellets of 0.4 X 0.4 X 0.2 mm are selected (with the aid of a dissecting microscope) for implantation. All procedures are performed under sterile conditions.
  • Graefe knife with small edge and 45 -angle blade.
  • the implant is introduced through the keratotomy line, parallel to the corneal epithelium and under it, in the external third of the stroma, up to 0.5 mm from the limbus.
  • One single pellet is introduced in the corneal pocket, close to the corneoscleral limbus, at the base of the pocket, with the knife blade.
  • an antibiotic solution is instilled, to prevent suprainfection and reduce irritation.
  • VEGF receptor fusion protein represented by SEQ ID NOs: 4-6, 8- 10, 12-14, and 16-18 are injected intravenously.
  • the eyes are routinely examined by slit lamp biomicroscopy (Nikon [Tokyo, Japan] FS-2) on postoperative days 3 through 8 after pellet implantation.
  • Rabbits are anesthetized with methoxyflurane, the eyes are proptosed, and the maximum vessel length (VL) of the neovascularization zone, extending from the base of the limbal vascular plexus toward the pellet, is measured with a linear reticule through the slit lamp.
  • the contiguous circumferential zone of neovascularization is measured as clock hours with a 360° reticule (where 30° of arc equals 1 clock hour).
  • VEGF receptor fusion protein injected intravenously induces a dose-dependent inhibition of the vessel length.
  • GTYRVVSVLP IEHQDWLTGK EFKCRVNHID LPSPIERTIS

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des protéines hybrides, d'un récepteur du facteur de croissance endothélial vasculaire (VEGF) d'animaux de compagnie, qui comportent des séquences d'acides aminés issues des récepteurs fit-1 et KDR du VEGF d'animaux de compagnie et une séquence d'acides aminés hétérologue qui étend la demi-vie de la protéine hybride in vivo, qui se lient au VEGF d'animaux de compagnie et qui peuvent être utilisées pour traiter des maladies, comprenant, mais sans s'y limiter, le cancer et la dégénérescence maculaire, associées à des effets indésirables du VEGF d'animaux de compagnie.
PCT/US2013/074196 2012-12-10 2013-12-10 Protéines hybrides du récepteur de vegf pour utilisation vétérinaire WO2014093387A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261735411P 2012-12-10 2012-12-10
US61/735,411 2012-12-10

Publications (1)

Publication Number Publication Date
WO2014093387A1 true WO2014093387A1 (fr) 2014-06-19

Family

ID=50934905

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/074196 WO2014093387A1 (fr) 2012-12-10 2013-12-10 Protéines hybrides du récepteur de vegf pour utilisation vétérinaire

Country Status (1)

Country Link
WO (1) WO2014093387A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180009869A1 (en) * 2016-07-08 2018-01-11 AskGene Pharma, Inc. Fusion Protein Comprising Leptin and Methods for Producing and Using the Same
CN113840831A (zh) * 2019-03-20 2021-12-24 金德雷德生物科学股份有限公司 医用ngf拮抗剂
US20220017590A1 (en) * 2018-06-29 2022-01-20 Akston Biosciences Corporation Ultra-long acting insulin-fc fusion proteins and methods of use
EP3902564A4 (fr) * 2018-12-27 2022-09-28 Kindred Biosciences, Inc. Variants d'igg fc à usage vétérinaire
WO2024165857A1 (fr) * 2023-02-08 2024-08-15 Levicept Limited Protéine de fusion

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6100071A (en) * 1996-05-07 2000-08-08 Genentech, Inc. Receptors as novel inhibitors of vascular endothelial growth factor activity and processes for their production
US7070959B1 (en) * 1999-06-08 2006-07-04 Regeneron Pharmaceuticals, Inc. Modified chimeric polypeptides with improved pharmacokinetic properties
US20070178112A1 (en) * 2006-01-27 2007-08-02 Novagen Holding Corporation Recombinant human EPO-Fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
US20100331250A1 (en) * 2009-03-18 2010-12-30 Joe Zhou Anti-angiogenesis fusion proteins

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6100071A (en) * 1996-05-07 2000-08-08 Genentech, Inc. Receptors as novel inhibitors of vascular endothelial growth factor activity and processes for their production
US7070959B1 (en) * 1999-06-08 2006-07-04 Regeneron Pharmaceuticals, Inc. Modified chimeric polypeptides with improved pharmacokinetic properties
US20070178112A1 (en) * 2006-01-27 2007-08-02 Novagen Holding Corporation Recombinant human EPO-Fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
US20100331250A1 (en) * 2009-03-18 2010-12-30 Joe Zhou Anti-angiogenesis fusion proteins

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI 30 August 2005 (2005-08-30), accession no. P_534520.2 *
TANG, L. ET AL.: "Cloning and characterization of cDNAs encoding four different canine immunoglobulin gamma chains", VET. IMMUNOL. IMMUNOPATHOL., vol. 80, no. 3-4, 10 August 2001 (2001-08-10), pages 259 - 270 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180009869A1 (en) * 2016-07-08 2018-01-11 AskGene Pharma, Inc. Fusion Protein Comprising Leptin and Methods for Producing and Using the Same
US20220017590A1 (en) * 2018-06-29 2022-01-20 Akston Biosciences Corporation Ultra-long acting insulin-fc fusion proteins and methods of use
US11773151B2 (en) * 2018-06-29 2023-10-03 Akston Biosciences Corporation Ultra-long acting insulin-Fc fusion proteins and methods of use
EP3902564A4 (fr) * 2018-12-27 2022-09-28 Kindred Biosciences, Inc. Variants d'igg fc à usage vétérinaire
CN113840831A (zh) * 2019-03-20 2021-12-24 金德雷德生物科学股份有限公司 医用ngf拮抗剂
EP3941933A4 (fr) * 2019-03-20 2023-07-26 Kindred Biosciences, Inc. Antagonistes du ngf à usage médical
WO2024165857A1 (fr) * 2023-02-08 2024-08-15 Levicept Limited Protéine de fusion

Similar Documents

Publication Publication Date Title
JP7421338B2 (ja) 免疫調節融合タンパク質
JP6110091B2 (ja) Ｆｇｆｒ融合タンパク質によって疾患を治療するための組成物および方法
US8926972B2 (en) Anti-angiogenesis fusion proteins
JP5787757B2 (ja) Ｆｇｆｒ細胞外ドメイン酸性領域突然変異タンパク質
JP3171268B2 (ja) 治療抗体基融合タンパク質
JP7427046B2 (ja) Ｂｄｎｆを含む融合蛋白質
CZ302303B6 (cs) Homodimerní fúzní protein vykazující inhibicní aktivitu na angiogenezi, zpusob jeho produkce, molekula DNA a replikovatelný expresní vektor
AU2018202982A1 (en) Fusion immunomodulatory proteins and methods for making same
KR102696275B1 (ko) 염증을 치료 및 예방하기 위한 조성물 및 방법
JP2008222711A6 (ja) Ｆｇｆｒ融合タンパク質によって疾患を治療するための組成物および方法
JP2005530484A (ja) アルブミン融合抗血管形成ペプチド
WO2014093387A1 (fr) Protéines hybrides du récepteur de vegf pour utilisation vétérinaire
WO2011139629A2 (fr) Molécules ciblant light et ses utilisations
CN112262158B (zh) 新型血管生成素2，vegf双特异性拮抗剂
KR20030048041A (ko) 표적-특이적 프로드럭으로서 사용하기 위한항체-사이토카인-사이토카인 억제제로 이루어진 융합단백질(셀렉토카인)
KR100559918B1 (ko) 강화 백신
WO2011084711A2 (fr) Procédés favorisant la pousse des cheveux à l'aide des domaines extracellulaires de fgfr3
CA2939639C (fr) Proteines de fusion d'uti
KR20010015868A (ko) 피치아 효모 발현계를 사용하여 항혈관형성 단백질인엔도스타틴, 안지오스타틴 또는 레스틴을 생성시키는 방법
CN104974262A (zh) 新型重组双功能融合蛋白及其制法和用途
AU2016226097A1 (en) sFv-Fc dimers that bind Transforming Growth Factor-Beta1 with high affinity, avidity and specificity
JP2013253079A (ja) 生物学的に活性なヒト尿中トリプシンインヒビターのＦｃ融合タンパク質並びにその調製および使用
CN112574315B (zh) 改变效应功能的Fc变体及其融合蛋白
WO2022256820A1 (fr) Antagonistes multispécifiques
KR20220006013A (ko) 보체 경로 억제제를 포함하는 융합단백질 및 이의 용도

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13863216

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13863216

Country of ref document: EP

Kind code of ref document: A1