WO2014093387A1 - Vegf receptor fusion proteins for veterinary use - Google Patents

Abstract

Companion animal vascular endothelial growth factor (VEGF) receptor fusion proteins comprising amino acid sequences derived from the companion animal VEGF receptors fit- 1 and KDR and a heterologous amino acid sequence that extends half-life of the fusion protein in vivo bind to companion animal VEGF and can be used to treat diseases, including but not limited to cancer and macular degeneration, associated with undesired effects of companion animal VEGF.

Description

VEGF RECEPTOR FUSION PROTEINS FOR VETERINARY USE

BACKGROUND OF THE INVENTION

[0001] 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.

[0002] VEGF, also known as vascular permeability factor, plays an important role in many biological processes, including angiogenesis, and has multiple biological effects, including promotion of vascular growth, vascular permeability, and inflammation. Higher than normal levels of human VEGF, or abnormal concentration gradients of human VEGF, have been reported in certain human diseases, such as cancer, inflammatory disease, pre-eclampsia, autoimmune disease (such as rheumatoid arthritis and psoriasis), atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, hemangiomas, immune rejection of transplanted corneal tissue and other tissues, and chronic inflammation. By administering agents that bind to human VEGF, it is possible to treat human VEGF associated conditions, such as cancer and macular degeneration in humans. One such agent is 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.

[0003] Companion animals such as cats, dogs, and horses, suffer from many diseases similar to human diseases, including cancer, macular degeneration, and autoimmune diseases. However, there has been no demonstration to date that aflibercept or bevacizumab could be used to treat such diseases in companion animals. Moreover, proteins with significant human-derived amino acid sequence content, especially if those sequences are derived from antibodies, 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. [0004] There remains a need, therefore, for methods and compounds that can be used to bind companion animal VEGF in companion animals for treating VEGF associated conditions in companion animals. Ideally, the compounds would bind specifically to companion animal VEGF and have a half-life in plasma sufficiently long to be practicable for therapy, but would not be highly immunogenic in companion animals. The present invention meets this need.

SUMMARY OF THE INVENTION

[0005] In a first aspect, 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. In various embodiments 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. In various embodiments, 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.

[0006] In a second aspect, the present invention provides pharmaceutical compositions comprising the fusion proteins of the present invention admixed with one or more pharmaceutically acceptable excipients. In various embodiments, the pharmaceutical compositions are suitable for parenteral administration.

[0007] In a third aspect, 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.

[0008] In a fourth aspect, 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.

[0009] In other aspects, 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.

DETAILED DESCRIPTION OF THE INVENTION

[0011] 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 γ chains, namely, IgG-A, IgG-B, IgG-C, and IgG-D.

[0012] For the convenience of the reader, the following definitions of terms used herein are provided.

[0013] "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.

[0014] "Companion animal" means cat, dog, or horse.

[0015] "Companion animal fit- 1 receptor" means the companion animal homologs of the human flt-1 receptor, also known as VEGF-R1.

[0016] "Companion animal VEGF" means companion animal vascular endothelial growth factor, also known as vascular permeability factor.

[0017] "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.

[0018] "Companion animal KDR receptor" means the companion animal homologs of the human KDR receptor, designated FLK- 1. [0019] "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.

[0020] "IgG Fc" means the Fc region of an immunoglobulin γ chain. "IgG-N-Fc" means the Fc region of a specific immunoglobulin γ 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 γ 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. In some instances, IgG Fc regions are isolated from canine immunoglobulin γ heavy chains IgG-A, IgG-B, IgG- C, and IgG-D, represented by SEQ ID NOs: 7, 11, 3, and 15, respectively. Thus, the "IgG-N-Fc" region of IgG-A is represented as "IgG-A-Fc."

[0021] "Conditions 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.

[0022] "Companion animal tyrosine kinase receptor" means companion animal fit- 1 receptor or companion animal KDR receptor or companion animal flt-4 receptor.

[0023] "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.

[0024] 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. In various embodiments, 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. [0025] In one embodiment, 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. In various embodiments, 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.

[0026] In various embodiments, 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. In these embodiments, 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. In other embodiments, 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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. Generally, 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. In some embodiments, the linker is a single amino acid residue, such as a glycine, alanine, or serine. For example, various fusion proteins of the invention utilize a linker that is a single glycine residue. In other embodiments, 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.

[0031] 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. One having skill in the art will appreciate that 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. Further, one skilled in the art will appreciate that 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.

[0032] 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). [0033] 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).

[0034] 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).

[0035] 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).

[0036] 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).

[0037] 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).

[0038] 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). [0039] 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).

[0040] 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).

[0041] 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).

[0042] 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).

[0043] 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).

[0044] Additional VEGF receptor fusion proteins provided by the invention 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. [0045] In various embodiments, 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. In these embodiments, 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. In other embodiments, 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.

[0046] In various embodiments, 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. In these embodiments, 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. In other embodiments, 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.

[0047] In various embodiments, 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. In various embodiments, 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.

[0048] 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. [0049] In other embodiments, a VEGF receptor fusion protein is provided 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. In some embodiments, 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.

[0050] In some embodiments, a fit- 1 VEGF receptor fusion protein is provided 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. In some embodiments, these flt-l-Fc VEGF receptor fusion proteins are expressed in plant cells and subsequently purified via a Protein A resin column.

[0051] The present invention also provides pharmaceutical compositions comprising an animal VEGF receptor fusion protein compounded with a pharmaceutically acceptable carrier, as discussed further below. In some embodiments, two or more companion animal VEGF receptor fusion proteins of the invention are combined together to provide a pharmaceutical composition of the invention. In some embodiments, the fusion proteins are formulated as multiverse aggregates. For example, the pharmaceutical formulation can comprise homo-dimers or homo- tetramers of a fusion protein of the invention.

[0052] 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. In various embodiments, 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. [0053] 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).

[0054] 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.

[0055] 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. In one embodiment, the expression system is a cell-free translation system. In another embodiment, the expression system is a plant cell-based system.

[0056] The present invention also provides methods for purifying the resulting fusion proteins. In various methods, 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. Examples of 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.

[0057] 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. In other embodiments, particularly where the fusion protein is highly stable to thermal and oxidative denaturation, 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. Thus, the invention provides pharmaceutical compositions in both solid and liquid form.

[0058] 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).

[0059] 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.

[0060] Thus, in accordance with the methods of the invention, 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.

[0061] For the prevention or treatment of disease, 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. For purposes herein, 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. In various embodiments, the therapeutically effective dose is a dose sufficient to reduce or inhibit the proliferation of vascular endothelium in vivo. Generally, 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.

[0062] In some embodiments, 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. [0063] 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. 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.

[0064] The following examples illustrate suitable methods materials for practicing the invention; other methods and materials for practicing the invention will be readily apparent to those of skill in the art in view of this disclosure.

EXAMPLES

Example 1: Synthesis and Purification of flt-l-fc Fusion Protein from CHO Cells

[0065] 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

[0066] 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' . 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.

Example 3: Purification of VEGF Receptor Fusion Proteins Obtained from Recombinant Expression Systems

[0067] 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.

Materials and Methods

[0068] 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 Na₂HP0₄. 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. To remove aggregates and other contaminants, 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.

Example 4: Binding of VEGF to Fltl-Fc in a Biacore-Based Assay

[0069] 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. One such assay is commercially available from Biocore (Sweden). In the Biacore assay, 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 μg/mL VEGF and Fltl-Fc (at 0, 0.5, 1.0, 5.0, 10.0, or 25.0 μg/mL) is passed over the Fltl-Fc-coated chip. To minimize the effects of non-specific binding, the bound samples are washed with a 0.5 M NaCl wash. In one sample, Fltl-Fc is mixed with heparin. Heparin is a negatively charged molecule and the Fltl-Fc protein is a positively charged molecule. When the two molecules are mixed together, they undergo an interaction through their respective charges that essentially neutralizes the positive charge of the Fltl-Fc, reducing its tendency to bind non- specifically via charge interactions. 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.

[0070] An alternate 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.

[0071] 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.

[0072] Example 5: Corneal Neovascularization Model

[0073] Fusion proteins of the invention can be demonstrated to have the desired biological activity using a corneal neovascularization model. In this model, a fusion protein with the desired biological activity will inhibit the neovascularization induced by VEGF.

Methods

[0074] Suspensions of sterile saline containing the appropriate amount of recombinant canine VEGF with doses ranging from 5 to 40 ug, plus 10 mg of sucralfate (Bukh Meditec, Vaerlose, Denmark) are made and are speed vacuumed for 5 minutes. To this suspension, 10 microliters of 12% Hydron in ethanol are added. The suspension is then deposited onto an autoclave, sterilized, and placed on a 15 X 15-mm piece of nylon mesh (Tetko [Lancaster, NY] 3-300/50, approximate pore size 0.4 X 0.4 mm), thereby becoming embedded between the fibers, resulting in a grid of 15 X 15 squares. 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.

[0075] Implantation of the pellets. Twelve rabbits are anesthetized with 5% ketamine hydrochloride (Calypsol) 15 mg/kbw, and injected intra- muscularly in the hind leg. After 20 minutes, 20% chloralhydrate 0.25 mg/kbw is intraperitoneally injected. A local anesthetic, 2% lydocaine or 4% cocaine, is instilled intraocularly. A binocular surgical microscope (Carl Zeiss) and a surgical table with a rotative stand are used. The pellet implantation procedure starts with a linear intrastromal incision, parallel to the corneoscleral limbus (linear keratotomy), using a surgical blade (Bard-Parker). The preparation of the corneal pocket for the pellet implant is made with a Von

o

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. Finally, an antibiotic solution is instilled, to prevent suprainfection and reduce irritation.

[0076] Three of the rabbits serve as controls, and nine of the rabbits receive various effective doses of 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).

Results

[0077] VEGF receptor fusion protein injected intravenously induces a dose-dependent inhibition of the vessel length.

SEQUENCE LISTING

SEQ ID NO: l - Amino Acid Sequence for Canine flt-1

1 MVSCRDTGVL LCALLGGLLL TGSSSGSKLK GPELSLKGTQ 1 HVMQAGQTLY LKCRGEAAHS WSLPETVRKE SKRLSITKSA

81 CGRNGKQFCS TLTLNMAQAN HTGFYSCKYL SIPASKKKKT

121 ESTIYIFIND TGRPFLEMHS EIPEIIYMTE GRQIVIPCRV

161 TSPNITVTLK KFPLDTLIPD GKRITWDSRK GFIIANATYR 01 EIGLLTCETT VNGHLYKTNY LTYRQTNTIR DVQINTPSPV 41 KLLRGHTLTL NCTATTALNT RVQMNWSYPG EKNKRASIRQ 81 RIGQRSSHAN VFYSVLIIDK VQNKDKGLYT CHVKSGPSFK

321 SVNTSVHIYD KAFITVKHRK EHVLETIAGK RSYRLSVKVK

361 AFPSPEVLWL KDGFPVTEKS ARYLVHGYSL IIKDVAAEDA 01 GDYMILLAIK QSSVFKNLTT TLIVNVKPQI YEKAVSSFPD 41 PTLYPLGSRQ ILTCTIYGIP QPTVRWFWRP CNHNHS KARY 81 DFCSSKEESL IVELDSNIGN RIESITQRTA IIEGKNKTAS

521 TLVVADARIS GIYSCMASNK IGTVERNISF YVTDVPNGFH

561 INLEKMPTEG EDLKLSCTVN KFLYRDITWI LLRTVKNRTM

601 HHSISKQKMA TTKEYSITLN VTIKNVSLED SGTYACRARN

641 IYTGEEILQK KEVTIRDQEA PHLLRNLSDH TVAISSSTTL

681 DCPATGVPEP EITWFKNNHK IQQEPGIILG PGSSTLFIER 21 VTEEDEGVYH CKATNQKGSV ESSAYLTVQG TSDKSNLELI 61 TLTCTCVAAT LFWLLLTLFI RKLKRSSSEI KTDYLSIIMD

801 PDEVPLDEQC ERLP YD AS KW EFARERLKLG KSLGRGAFGK

841 VVQASAFGIK KSPTCRTVAV KMLKEGATAS EYKALMTELK

881 ILTHIGHHLN VVNLLGACTK QGGPLMVIVE YCKYGNLSNY

921 LKSKRDLFFL NKDAALHMEP KKEKMEPDPE QDKKQRLDSV

961 TSSESFASSG FQEDKSLSDV EEEEDSDDFY KQPITMEDLI

1001 SYSFQVARGM EFLSSRKCIH RDLAARNILL SENNVVKICD

1041 FGLARDIYKN PDYVRKGDTR LPLKWMAPES IFDKIYSTKS

1081 DVWSYGVLLW EIFSLGGSPY PGVQMDEDFC SRLKEGMRMR

1121 APEYATPEIY QIMLDCWHKD PKERPRFAEL VEKLGDLLQA

1161 NVQQDGKDYI PLNAILTGNS GFTYSSPAFS DDFFKEDISA

1201 PKFNSGSSDN VRYVNAFNFM SLERIKTFEE LSPNTTSIFD

1241 DYQVDSSTLL ASPLLKRFTW AESKPKASLK IDLRVTSKSK

1281 ESGLSDLTRP FCHSRCGHIN RGRRRFTYDN SELEKKISCC

1321 SPPPDYNSVV LYSTPPV

SEP ID NO:2 - Amino Acid Sequence for Canine KDR

1 MESKALLAIA LWLCVETGAA SGGLPSVSLH PPRLSIQKDI 1 LTIMANTTLQ ITCRGQRDLD WLWPNNQSGS EKRVEVTECS

81 DSFFCKMLTI PKVMGNDTGA YKCFYRDTDM ASVIYVYVQD

121 YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS

161 LCARYPEKRF VPDGNRISWD SKKGFTIPSY MISYAGMVFC 01 EAKINDESYQ SIMYIVVVVG YKIYDVILSP PHGVELSVGE 41 RLVLNCTVRT ELNVGLDFNW EYPSLKHQHK KLVNRDLKTQ 81 SGSEMKKFLS TLTIDGVTRS DQGWYTCAAS SGLMTKRNST

321 FIRVHEKPFV AFGSGMESSV EATVGDRVRI PVKYLGYPPP

361 EIKWYKNGRP IESNHTIKVG HVLTIMEVSE KDTGNYTVIL 01 TNPISKEKQS HVISLVVNVP PQIGEKSLIS PVDSYQYGTT 41 QSLTCTVYAV PPPHHIRWYW QLEECAYKPA QAVLMTNPYT 81 CKEWRNVDDF QGGNKIEVNK NQFALIEGKN KTVSTLVIQA

521 ANVSALYKCE AVNKVGRGER VISFHVIRGP EITLQPDTQP

561 TEQESVSLWC TADRTTFENL TWYKSGPQAL AVHAGEVPTP

601 VCKNLDALWR MNATTFSNGT SDILILELQN VSLQDQGDYV

641 CFAQDRKTKK RHCVVRQLTV LERVAPMITG NLENQTTSIG

681 ETIEVSCTTS GNPPPQITWF KDNETLVEDS GIILKDGNRN 21 LTIRRVRKED EGLYTCQACS VLGCAKVEAF FIVEGAQEKT 61 NLEVIILVGT AVIAMFFWLL LVIVLRTVKR ANGGELKTGY

801 LSIVMDPDEL PLDEHCERLP YD AS KWEFPR DRLKLGKPLG

841 RGAFGQVIEA DAFGIDKTAT CKTVAVKMLK EGATHSEHRA

881 LMSELKILIH IGHHLNVVNL LGACTKPGGP LMVIVEFCKF

921 GNLSTYLRSK RNEFVPYKTK GARFRQGKEY VGEITMDPKR

961 RLDSITSSQS SASSGFVEEK SLSDVEEEEV SEDLYKNFLT

1001 LEHLICYSFQ VAKGMEFLAS RKCIHRDLAA RNILLSEKNV

1041 VKICDFGLAR DIYKDPDYVR KGDARLPLKW MAPETIFDRV

1081 YTIQSDVWSF GVLLWEIFSL GASPYPGVKI DEEFCRRLKE

1121 GTRMRAPDYT TPEMYQTMLD CWHGEPNQRP TFSELVEHLG

1161 NLLQANAQQD GKDYIVLPIS ETLSMEEDSG LSLPTSPVSC

1201 MEEEEVCDPK FHYDNTAGIS QYLQNSKRKS RPVSVKTFED

1241 IPLEEPEVKV IPDDNQTDSG MVLASEELKT LEDRTKLAPS

1281 FSGLMPSKSK ESVASEGSNQ TSGYQSGYHS DDTDTTVYSS

1321 EEAELLKLME IGPQAGSAAQ ILQPDSGPTL SSPPV

SEP ID NO:3 - Canine IgG-C VEGF Receptor MESVLYWVFL VAILKGVQGD VQLVESGGDL VKPGGSLRLS CVASGFTFSS CAMSWVRQSP GKGPQWVATI RYDGSDIYYA DAVKGRFSIS RDNAKNTVYL QMNSLRAEDT AVYYCAKAPP YDSYHYGMDY WGPGTSLFVS SASTTAPSVF PLAPSCGSQS GSTVALACLV SGYIPEPVTV SWNSVSLTSG VHTFPSVLQS SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA TNTKVDKPVA KECECKCNCN NCPCPGCGLL GGPSVFIFPP KPKDILVTAR TPTVTCVVVD LDPENPEVQI SWFVDSKQVQ TANTQPREEQ SNGTYRVVSV LPIGHQDWLS GKQFKCKVNN KALPSPIEEI ISKTPGQAHQ PNVYVLPPSR DEMSKNTVTL TCLVKDFFPP EIDVEWQSNG QQEPESKYRM TPPQLDEDGS YFLYSKLSVD KSRWQRGDTF ICAVMHEALH NHYTQISLSH SPGK

SEP ID NP:4 - Canine flt-l-IgG-C Fc Fusion Protein GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRrr WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD KGLYTCHVKS GPSFKSVNTS VHIIEGRMDT NTKVDKPVAK ECECKCNCNN CPCPGCGLLG GPSVFIFPPK PKDILVTART PTVTCVVVDL DPENPEVQIS WFVDSKQVQT ANTQPREEQS NGTYRVVSVL PIGHQDWLSG KQFKCKVNNK ALPSPIEEII SKTPGQAHQP NVYVLPPSRD EMSKNTVTLT CLVKDFFPPE IDVEWQSNGQ QEPESKYRMT PPQLDEDGSY FLYSKLSVDK SRWQRGDTFI CAVMHEALHN HYTQISLSHS PGK

SEP ID NP:5 - Canine flt-l-IgG-C Fc Fusion Protein GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRIT WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD KGLYTCHVKS GPSFKSVNTS VHIGTNTKVD KPVAKECECK CNCNNCPCPG CGLLGGPSVF IFPPKPKDIL VTARTPTVTC VVVDLDPENP EVQISWFVDS KQVQTANTQP REEQSNGTYR VVSVLPIGHQ DWLSGKQFKC KVNNKALPSP IEEIISKTPG QAHQPNVYVL PPSRDEMSKN TVTLTCLVKD FFPPEIDVEW QSNGQQEPES KYRMTPPQLD EDGSYFLYSK LSVDKSRWQR GDTFICAVMH EALHNHYTQI SLSHSPGK SEP ID NO: 6 - Canine flt-l-IgG-C Fc Fusion Protein GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRrr WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD KGLYTCHVKS GPSFKSVNTS VHIGGGGSGG GGSGGGGSTN TKVDKPVAKE CECKCNCNNC PCPGCGLLGG PSVFIFPPKP KDILVTARTP TVTCVVVDLD PENPEVQISW FVDSKQVQTA NTQPREEQSN GTYRVVSVLP IGHQDWLSGK QFKCKVNNKA LPSPIEEIIS KTPGQAHQPN VYVLPPSRDE MSKNTVTLTC LVKDFFPPEI DVEWQSNGQQ EPESKYRMTP PQLDEDGSYF LYSKLSVDKS RWQRGDTFIC AVMHEALHNH YTQISLSHSP GK

SEP ID NO: 7 - Canine IgG-A VEGF Receptor MESVFCWVFL VVILKGVQGE VQLVESGGDL VKPGGSLRLS CVASGFTFSS YYMHWIRQAP GKGLQRVAHI RGDGRTTHYA DAMKGRFTIS RDNAKNTLYL QMNSLTVEDT AIYYCVKDIY YGVGDYWGQG TLVTVSSAST TAPSVFPLAP SCGSTSGSTV ALACLVSGYF PEPVTVSWNS GSLTSGVHTF PSVLQSSGLH SLSSMVTVPS SRWPSETFTC NVVHPASNTK VDKPVFNECR CTDTPPCPVP EPLGGPSVLI FPPKPKDILR ITRTPEVTCV VLDLGREDPE VQISWFVDGK EVHTAKTQSR EQQFNGTYRV VSVLPIEHQD WLTGKEFKCR VNHIDLPSPI ERTISKARGR AHKPSVYVLP PSPKELSSSD TVSITCLIKD FYPPDIDVEW QSNGQQEPER KHRMTPPQLD EDGSYFLYSK LSVDKSRWQQ GDPFTCAVMH ETLQNHYTDL SLSHSPGK

SEP ID NP:8 - Canine flt-l-IgG-A Fc Fusion Protein

GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE

TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY

SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI

IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRIT

WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ

TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN

WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD

KGLYTCHVKS GPSFKSVNTS VHIIEGRMDF NECRCTDTPP

CPVPEPLGGP SVLIFPPKPK DILRITRTPE VTCVVLDLGR

EDPEVQISWF VDGKEVHTAK TQSREQQFNG TYRVVSVLPI

EHQDWLTGKE FKCRVNHIDL PSPIERTISK ARGRAHKPSV

YVLPPSPKEL SSSDTVSITC LIKDFYPPDI DVEWQSNGQQ EPERKHRMTP PQLDEDGSYF LYSKLSVDKS RWQQGDPFTC AVMHETLQNH YTDLSLSHSP GK

SEP ID NO:9 - Canine flt-l-IgG-A Fc Fusion Protein GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRIT WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD KGLYTCHVKS GPSFKSVNTS VHIGSNTKVD KPVFNECRCT DTPPCPVPEP LGGPSVLIFP PKPKDILRIT RTPEVTCVVL DLGREDPEVQ ISWFVDGKEV HTAKTQSREQ QFNGTYRVVS VLPIEHQDWL TGKEFKCRVN HIDLPSPIER TISKARGRAH KPSVYVLPPS PKELSSSDTV SrrCLIKDFY PPDIDVEWQS NGQQEPERKH RMTPPQLDED GSYFLYSKLS VDKSRWQQGD PFTCAVMHET LQNHYTDLSL SHSPGK

SEP ID NP:10 - Canine flt-l-IgG-A Fc Fusion Protein GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRIT WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD KGLYTCHVKS GPSFKSVNTS VHIGGGGSGG GGSGGGGSFN ECRCTDTPPC PVPEPLGGPS VLIFPPKPKD ILRITRTPEV TCVVLDLGRE DPEVQISWFV DGKEVHTAKT QSREQQFNGT YRVVSVLPIE HQDWLTGKEF KCRVNHIDLP SPIERTISKA RGRAHKPSVY VLPPSPKELS SSDTVSITCL IKDFYPPDID VEWQSNGQQE PERKHRMTPP QLDEDGSYFL YSKLSVDKSR WQQGDPFTCA VMHETLQNHY TDLSLSHSPG K

SEP ID NO: 11 - Canine IgG-B VEGF Receptor MESVLFWVFL VTILKGVQGE VRLVESGGTL VKPGGSLKLS CVASGFTFRR YSMDWVRQAP GKSLQWVAGI NGDGTGTSYS QTVKGRFTIS RDNAKNTLYL QINSLRAEDS AVYYCAKSWS RNGDLDYWGQ GTLVTVSSAS TTAPSVFPLA PSCGSTSGST VALACLVSGY FPEPVTVSWN SGSLTSGVHT FPSVLQSSGL YSLSSMVTVP SSRWPSETFT CNVAHPASKT KVDKPVPKRE NGRVPRPPDC PKCPAPEMLG GPSVFIFPPK PKDTLLIART PEVTCVVVDL DPEDPEVQIS WFVDGKQMQT AKTQPREEQF NGTYRVVSVL PIGHQDWLKG KQFTCKVNNK ALPSPIERTI SKARGQAHQP SVYVLPPSRE ELSKNTVSLT CLIKDFFPPD IDVEWQSNGQ QEPESKYRTT PPQLDEDGSY FLYSKLSVDK SRWQRGDTFI CAVMHEALHN HYTQESLSHS PGK

SEP ID NO: 12 - Canine flt-l-IgG-B Fc Fusion Protein GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRIT WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD KGLYTCHVKS GPSFKSVNTS VHIIEGRMDP KRENGRVPRP PDCPKCPAPE MLGGPSVFIF PPKPKDTLLI ARTPEVTCVV VDLDPEDPEV QISWFVDGKQ MQTAKTQPRE EQFNGTYRVV SVLPIGHQDW LKGKQFTCKV NNKALPSPIE RTISKARGQA HQPSVYVLPP SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK

SEQ ID NO: 13 - Canine flt-l-IgG-B Fc Fusion Protein GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRIT WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD KGLYTCHVKS GPSFKSVNTS VHIGSKTKVD KPVPKRENGR VPRPPDCPKC PAPEMLGGPS VFIFPPKPKD TLLIARTPEV TCVVVDLDPE DPEVQISWFV DGKQMQTAKT QPREEQFNGT YRVVSVLPIG HQDWLKGKQF TCKVNNKALP SPIERTISKA RGQAHQPSVY VLPPSREELS KNTVSLTCLI KDFFPPDIDV EWQSNGQQEP ESKYRTTPPQ LDEDGSYFLY SKLSVDKSRW QRGDTFICAV MHEALHNHYT QESLSHSPGK

SEP ID NO: 14 - Canine flt-l-IgG-B Fc Fusion Protein GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRIT WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN 241 WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD

281 KGLYTCHVKS GPSFKSVNTS VHIGGGGSGG GGSGGGGSPK

321 RENGRVPRPP DCPKCPAPEM LGGPSVFIFP PKPKDTLLIA

361 RTPEVTCVVV DLDPEDPEVQ ISWFVDGKQM QTAKTQPREE

401 QFNGTYRVVS VLPIGHQDWL KGKQFTCKVN NKALPSPIER

441 TISKARGQAH QPSVYVLPPS REELS KNTVS LTCLIKDFFP

481 PDIDVEWQSN GQQEPESKYR TTPPQLDEDG SYFLYSKLSV

521 DKSRWQRGDT FICAVMHEAL HNHYTQESLS HSPGK

SEO ID NO: 15 - Canine IsG-D VEGF Receptor

1 MESVLCWVFL VSILKGVQGE VQLVESGGDL VKPGGSLRLS

41 CVASGFTFSD YGMSWVRQSP GKGLQWVAAV SNRGDTYYAD

81 AVKGRFTISR DNAKNTLYLQ MSSLKAEDTA IYHCVTGVWP

121 RHYYGMDHWG NGTSLFVSSA STTAPSVFPL APSCGSTSGS

161 TVALACLVSG YFPEPVTVSW NSGSLTSGVH TFPSVLQSSG

201 LYSLSSTVTV PSSRWPSETF TCNVVHPASN TKVDKPVPKE

241 STCKCISPCP VPESLGGPSV FIFPPKPKDI LRITRTPEIT

281 CVVLDLGRED PEVQISWFVD GKEVHTAKTQ PREQQFNSTY

321 RVVSVLPIEH QDWLTGKEFK CRVNHIGLPS PIERTISKAR

361 GQAHQPSVYV LPPSPKELSS SDTVTLTCLI KDFFPPEIDV

401 EWQSNGQPEP ESKYHTTAPQ LDEDGSYFLY SKLSVDKSRW

441 QQGDTFTCAV MHEALQNHYT DLSLSHSPGK

SEO ID NO: 16 - Canine flt-l-IgG-D Fc Fusion Protein

1 GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE

41 TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY

81 SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI

121 IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRIT

161 WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ

201 TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN

241 WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD

281 KGLYTCHVKS GPSFKSVNTS VHIIEGRMDP KESTCKCISP

321 CPVPESLGGP SVFIFPPKPK DILRITRTPE ITCVVLDLGR

361 EDPEVQISWF VDGKEVHTAK TQPREQQFNS TYRVVSVLPI

401 EHQDWLTGKE FKCRVNHIGL PSPIERTISK ARGQAHQPSV

441 YVLPPSPKEL SSSDTVTLTC LIKDFFPPEI DVEWQSNGQP

481 EPESKYHTTA PQLDEDGSYF LYSKLSVDKS RWQQGDTFTC

521 AVMHEALQNH YTDLSLSHSP GK

SEO ID NO: 17 - Canine flt-l-IsG-D Fc Fusion Protein

1 GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE

41 TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY

81 SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI

121 IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRIT 161 WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ

201 TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN

241 WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD

281 KGLYTCHVKS GPSFKSVNTS VHIGSKTKVD KPVPKESTCK

321 CISPCPVPES LGGPSVFIFP PKPKDILRIT RTPEITCVVL

361 DLGREDPEVQ ISWFVDGKEV HTAKTQPREQ QFNSTYRVVS

401 VLPIEHQDWL TGKEFKCRVN HIGLPSPIER TISKARGQAH

441 QPSVYVLPPS PKELSSSDTV TLTCLIKDFF PPEIDVEWQS

481 NGQPEPESKY HTTAPQLDED GSYFLYSKLS VDKSRWQQGD

521 TFTCAVMHEA LQNHYTDLSL SHSPGK

SEO ID NO: 18 - i Canine flt-l-IgG-D Fc Fusion Protein

1 GSKLKGPELS LKGTQHVMQA GQTLYLKCRG EAAHSWSLPE

41 TVRKESKRLS ITKSACGRNG KQFCSTLTLN MAQANHTGFY

81 SCKYLSIPAS KKKKTESTIY IFINDTGRPF LEMHSEIPEI

121 IYMTEGRQIV IPCRVTSPNI TVTLKKFPLD TLIPDGKRIT

161 WDSRKGFIIA NATYREIGLL TCETTVNGHL YKTNYLTYRQ

201 TNTIRDVQIN TPSPVKLLRG HTLTLNCTAT TALNTRVQMN

241 WSYPGEKNKR ASIRQRIGQR SSHANVFYSV LIIDKVQNKD

281 KGLYTCHVKS GPSFKSVNTS VHIGGGGSGG GGSGGGGSPK

321 ESTCKCISPC PVPESLGGPS VFIFPPKPKD ILRITRTPEI

361 TCVVLDLGRE DPEVQISWFV DGKEVHTAKT QPREQQFNST

401 YRVVSVLPIE HQDWLTGKEF KCRVNHIGLP SPIERTISKA

441 RGQAHQPSVY VLPPSPKELS SSDTVTLTCL IKDFFPPEID

481 VEWQSNGQPE PESKYHTTAP QLDEDGSYFL YSKLSVDKSR

521 WQQGDTFTCA VMHEALQNHY TDLSLSHSPG K

SEO ID NO: 19 - Peptide Linker

1 IEGRMD

SEO ID NO: 20 - IgG-C Fc Region

1 TNTKVDKPVA KECECKCNCN NCPCPGCGLL GGPSVFIFPP

41 KPKDILVTAR TPTVTCVVVD LDPENPEVQI SWFVDSKQVQ

81 TANTQPREEQ SNGTYRVVSV LPIGHQDWLS GKQFKCKVNN

121 KALPSPIEEI ISKTPGQAHQ PNVYVLPPSR DEMSKNTVTL

161 TCLVKDFFPP EIDVEWQSNG QQEPESKYRM TPPQLDEDGS

201 YFLYSKLSVD KSRWQRGDTF ICAVMHEALH NHYTQISLSH

241 SPGK

SEO ID NO: 21 - Peptide Linker

1 GGGGSGGGGS GGGGS SEO ID NO: 22 - IgG-A Fc Region

FNECRCTDTP PCPVPEPLGG PSVLIFPPKP KDILRITRTP

EVTCVVLDLG REDPEVQISW FVDGKEVHTA KTQSREQQFN

GTYRVVSVLP IEHQDWLTGK EFKCRVNHID LPSPIERTIS

KARGRAHKPS VYVLPPSPKE LSSSDTVSIT CLIKDFYPPD

IDVEWQSNGQ QEPERKHRMT PPQLDEDGSY FLYSKLSVDK

SRWQQGDPFT CAVMHETLQN HYTDLSLSHS PGK

SEO ID NO: 23 - IgG-B Fc Region

PKRENGRVPR PPDCPKCPAP EMLGGPSVFI FPPKPKDTLL

IARTPEVTCV VVDLDPEDPE VQISWFVDGK QMQTAKTQPR

EEQFNGTYRV VSVLPIGHQD WLKGKQFTCK VNNKALPSPI

ERTISKARGQ AHQPSVYVLP PSREELSKNT VSLTCLIKDF

FPPDIDVEWQ SNGQQEPESK YRTTPPQLDE DGSYFLYSKL

SVDKSRWQRG DTFICAVMHE ALHNHYTQES LSHSPGK

SEO ID NO: 24 - IgG-D Fc Region

SNTKVDKPVF NECRCTDTPP CPVPEPLGGP SVLIFPPKPK

DILRITRTPE VTCVVLDLGR EDPEVQISWF VDGK EVHTAK

TQSREQQFNG TYRVVSVLPI EHQDWLTGKE FKCRVNHIDL

PSPIERTISK ARGRAHKPSV YVLPPSPKEL SSSDTVSrrC

LIKDFYPPDI DVEWQSNGQQ EPERKHRMTP PQLDEDGSYF

LYSKLSVDKS RWQQGDPFTC AVMHETLQNH YTDLSLSHSP

GK

Claims

1. A companion animal vascular endothelial growth factor (VEGF) receptor fusion protein capable of binding to companion animal VEGF and thereby exerting an inhibitory effect thereon, said companion animal VEGF receptor fusion protein fusion comprising tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2, and 3, wherein said

immunoglobulin-like domains all come from the same genus of companion animal, and an amino acid sequence or other molecule that extends half-life of said fusion protein in vivo.

2. The fusion protein of claim 1, wherein said amino acid sequence or other molecule that extends half-life of said fusion protein in vivo is an Fc region of an immunoglobulin gamma of said companion animal.

3. The fusion protein of claim 1 or 2, wherein said domains 1, 2, and 3 are derived from tyrosine kinase receptor fit- 1, flt-4, or KDR or a combination of any of them.

4. The fusion protein of claim 3, wherein said fusion protein comprises:

(a) either amino acids 32-128 of canine animal fit- 1 tyrosine kinase receptor or amino acids 32-118 of the companion animal KDR tyrosine kinsase receptor; and

(b) either amino acids 134-226 of the companion animal fit- 1 tyrosine kinsase receptor or amino acids 124-220 of the companion animal KDR tyrosine kinase receptor; and

(c) amino acids 232-331 of the companion animal fit- 1 tyrosine kinase receptor or

amino acids 226-327 of the companion animal KDR tyrosine kinase receptor.

5. The fusion protein of claim 4 that is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

6. The fusion protein of claim 2, wherein said companion animal is a canine and said Fc region is derived from IgG-A, IgG-B, IgG-C, and IgG-D.

7. The fusion protein of claim 6, wherein said Fc region is derived from IgG-A, IgG-B, or IgG-D.

8. The fusion protein of claim 1 or 2 wherein the companion animal species is dog.

9. The fusion protein of claim 1 or 2 wherein the companion animal species is cat.

10. The fusion protein of claim 1 or 2 wherein the companion animal species is horse.

11. A pharmaceutical composition that comprises the fusion protein of claim 1 or 2 admixed with one or more pharmaceutically acceptable carriers, excipients, and/or diluents.

12. A nucleic acid that encodes the fusion protein of any one of claims 1 through 7.

13. An expression vector comprising the nucleic acid of claim 12.

14. A host cell comprising the expression vector of claim 13.

15. Use of companion animal VEGF receptor fusion protein of any one of claims 1 through 7 or composition of claim 11 for treatment of a disease or condition selected from the group consisting of inflammatory, oncologic, and immunologic diseases or conditions in a companion animal associated with altered levels or gradients companion animal VEGF.