Anti-NGF antibodies and methods thereof

Abstract

To provide anti-nerve growth factor (NGF) antibodies and antigen binding proteins used for the treatment or prevention of NGF related disorders.SOLUTION: The present invention provides a recombinant antigen binding protein comprising at least one complementarity determining region (CDR) selected from the group consisting of: a variable light chain (VL) comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3) which comprise specific amino acid sequences; a variable heavy chain (VH) comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3) which comprise specific amino acid sequences; and any variants thereof having one or more conservative amino acid substitutions in at least one of CDR1, CDR2 and CDR3 in any of the variable light or heavy chain regions of the antigen binding protein.SELECTED DRAWING: Figure 15

Description

[Technical Field]

[0001] The present invention relates to the field of immunology. More specifically, the present invention relates to anti-NGF antigen-binding proteins that specifically bind to NGF and have been modified to be non-immunogenic in a species of interest. The present invention further relates to the use of such antigen-binding proteins in the treatment and / or prevention of NGF-related disorders, particularly pain. [Background technology]

[0002] Nerve growth factor (NGF) was the first neurotrophin identified, and its role in the development and survival of both peripheral and central neurons has been well characterized. NGF has been shown to be an essential survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons and basal forebrain cholinergic neurons (Smeyne, et al., Nature 368:246-249 (1994) and Crowley, et al., Cell 76:1001-101 I (1994)). NGF upregulates neuropeptide expression in sensory neurons (Lindsay, et al., Nature 337:362-364 (1989)), and its activity is mediated through two distinct membrane-bound receptors, the TrkA tyrosine kinase receptor and the p75 common neurotrophin receptor (sometimes referred to as the "high-affinity" and "low-affinity" NGF receptor, respectively), the latter of which is structurally related to other members of the tumor necrosis factor receptor family (Chao, et al., Science 232:518-521 (1986)).

[0003] In addition to its effects in the nervous system, NGF has been increasingly implicated in processes outside the nervous system. For example, NGF promotes vascular permeability (Otten, et al., Eur J Pharmacol. 106:199-201 (1984)), enhances T and B cell immune responses (Otten, et al., Proc. Natl. Acad. Sci. USA 86:10059-10063 (1989)), induces lymphocyte differentiation and mast cell proliferation, and causes the release of soluble biological signals from mast cells (Matsuda, et al., Proc. Natl. Acad. Sci. USA 85:6508-6512 (1988); Pearce, et al., J. Physiol. 372:379-393 (1986); Bischoff, et al., Blood 79:2662-2669 (1992); Horigome, et al., J. Biol. Chem. 268:14881-14887 (1993)).

[0004] NGF is produced by several cell types, including mast cells (Leon, et al., Proc. Natl. Acad. Sci. USA 91:3739-3743 (1994)), B lymphocytes (Torcia, et al., Cell 85:345-356 (1996)), keratinocytes (Di Marco, et al., J. Biol. Chem. 268:22838-22846)), smooth muscle cells (Ueyama, et al., J. Hypertens. 11:1061-1065 (1993)), fibroblasts (Lindholm, et al., Eur. J. Neurosci. 2:795-801 (1990)), and bronchial epithelial cells (Kassel, et al., Clin. Exp. Allergy 1990). 31:1432-40(2001)), renal mesangial cells (Steiner, et al., Am. J. Physiol. 261:F792-798(1991)), and skeletal myotube cells (Schwartz, et al., J. Photochem. Photobiol. B66:195-200(2002)). NGF receptors have been found in various cell types outside the nervous system. For example, TrkA has been found on human monocytes, T and B lymphocytes, and mast cells.

[0005] The association between increased NGF levels and various inflammatory conditions has been observed in human patients as well as in several animal models. These include systemic lupus erythematosus (Bracci-Laudiero, et al., Neuroreport 4:563-565 (1993)), multiple sclerosis (Bracci-Laudiero, et al., Neurosci. Lett. 147:9-12 (1992)), psoriasis (Raychaudhuri, et al., Acta Derm. l'enereol. 78:84-86 (1998)), arthritis (Falcim, et al., Ann. Rheum. Dis. 55:745-748 (1996)), interstitial cystitis (Okragly, et al., J. Urology 161:438-441 (1999)), and asthma (Braun, et al., Eur. J. Immunol. 28:3240-3251 (1998)). Consistently elevated levels of NGF in peripheral tissues are associated with hyperalgesia and inflammation and have been observed in some forms of arthritis. It has been reported that synovial membranes from patients with rheumatoid arthritis express high levels of NGF, whereas non-inflamed synovial membranes contain undetectable NGF (Aloe, et al., Arch. Rheum. 35:351-355 (1992)). Similar results were observed in rats with experimentally induced rheumatoid arthritis (Aloe, et al., Clin. Exp. Rheumatol. 10:203-204 (1992)). Elevated levels of NGF, along with increased numbers of mast cells, have been reported in transgenic arthritic mice (Aloe, et al., Int. J. Tissue Reactions-Exp. Clin. Aspects 15:139-143(1993)).

[0006] Osteoarthritis (OA) is one of the most common chronic musculoskeletal diseases in dogs, affecting 20% ​​of the canine population over the age of 1 year. The development of OA is primarily secondary to trauma, joint instability, and conditions such as hip dysplasia. Osteoarthritis is a global disease state in which both inflammatory and degenerative changes in all joint structures result in impaired and clinical signs of lameness and pain. Pain is the most important clinical symptom of canine OA and is the result of a complex interplay between structural joint changes, biochemical and molecular alterations, as well as peripheral and central pain-processing mechanisms. Within this network, activation and sensitization of peripheral nociceptors by inflammatory and hyperalgesic mediators (e.g., cytokines, prostaglandins, and neuromediators) is one of the main peripheral mechanisms involved in joint pain. There is a clear unmet need for non-pharmaceutical treatments for canine pain that would provide relief for longer periods of time than classical pain treatments.

[0007] Approximately 14.5 million dogs suffer from OA in the United States alone (2010 market research). Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most common category of medication prescribed by veterinarians, but are limited by their effectiveness and tolerability. Market research indicates that approximately 9 million dogs are treated with NSAIDs in the United States. Corticosteroids are used infrequently, typically as a short-term last resort. There remains a clear unmet need for convenient and safe products to effectively treat dogs with OA.

[0008] In cats, OA is a pathological change of synovial diarthrodial joints characterized by articular cartilage deterioration, osteophyte formation, bone remodeling, soft tissue changes, and low-grade nonsuppurative inflammation. Although the radiographic features of feline OA have been well described, clinical signs of the disease are poorly documented and may go undiagnosed. The difficulty in assessing lameness in cats is a result of their small size and innate agility, which allows them to compensate. However, clinical signs of feline OA may include weight loss, anorexia, depression, abnormal elimination habits, poor grooming, aggressive behavior, and a gradual decrease in jumping ability leading to overt lameness. Due to misdiagnosis, feline OA remains largely untreated and unmet. There is a need for veterinary medicines that do not Summary of the Invention

[0009] The present invention provides novel anti-NGF antigen-binding proteins (antibodies, antibody fragments, antigen-binding fragments, antigen-binding portions, antagonist antibodies, etc., as defined and used interchangeably herein) and polynucleotides encoding same. The present invention further provides methods for producing and using said antigen-binding proteins and / or nucleotides in the treatment and / or prevention of NGF-related disorders, particularly pain, in a subject. The present invention further provides pharmaceutical compositions and uses for the treatment of NGF-related disorders, particularly pain, in a subject.

[0010] In one aspect, the present invention provides a recombinant antigen binding protein that specifically binds nerve growth factor (NGF), comprising a Complementary Determining Region 1 (CDR) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 21. a variable light chain comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:22; a complementarity determining region 1 (CDR2) comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:3 or SEQ ID NO:23; and a complementarity determining region 1 (CDR1) comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:4 or SEQ ID NO:24; a complementarity determining region 1 (CDR2) comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:5 or SEQ ID NO:25; and a variable heavy chain (VH) comprising a complementarity determining region 1 (CDR3) comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:6 or SEQ ID NO:26; and any variant thereof having one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0011] In one embodiment, the present invention provides an antigen binding protein comprising a Complementary Determining Region 1 (CDR) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:1. a light chain variable region comprising: a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:3; and a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:4; a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:5; a heavy chain variable region comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:6; and any variant thereof having one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 in either the variable light chain or variable heavy chain region of the antigen binding protein.

[0012] In one embodiment, the invention provides an antigen binding protein of the invention comprising a light chain variable region (VL) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:21; a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:22; a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:23; and a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:24; a heavy chain variable region (VH) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:26; and any variant thereof having one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0013] In one aspect, the present invention provides a recombinant antigen binding protein which specifically binds to nerve growth factor (NGF), said recombinant antigen binding protein comprising a variable light chain comprising an amino acid sequence with at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:55, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:91; and a variable heavy chain comprising an amino acid sequence with at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:56, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, and SEQ ID NO:81; and any variant thereof with one or more conservative amino acid substitutions in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0014] In one embodiment, the present invention provides that a recombinant antigen binding protein of the present invention comprises a variable light chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 7 and a variable heavy chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 8, and any variant thereof having one or more conservative amino acid substitutions in either the variable light or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides an antigen binding protein wherein the variable light chain comprises an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 27, and the variable heavy chain comprises an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 28, and any variant thereof. wherein the antigen binding protein has one or more conservative amino acid substitutions in either the variable light or variable heavy chain region of said antigen binding protein.

[0015] In one embodiment, the present invention provides that the recombinant antigen binding protein of the present invention comprises a variable light chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 9 and a variable heavy chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 10, and any variant thereof with one or more conservative amino acid substitutions in either the variable light chain or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides that the recombinant antigen binding protein of the present invention comprises a variable light chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 29 and a variable heavy chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 30, and any variant thereof with one or more conservative amino acid substitutions in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0016] In one embodiment, the present invention provides that a recombinant antigen binding protein of the present invention comprises a variable light chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 56, and any variant thereof having one or more conservative amino acid substitutions in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0017] In one embodiment, the present invention relates to a recombinant antigen binding protein of the present invention, wherein the recombinant antigen binding protein comprises a variable light chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 91 and a variable heavy chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 79; and any variant thereof with one or more conservative amino acid substitutions in either the variable light or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides that a recombinant antigen binding protein of the present invention comprises a variable light chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 87 and a variable heavy chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 79, and any variant thereof with one or more conservative amino acid substitutions in either the variable light or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides that a recombinant antigen binding protein of the present invention comprises a variable light chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 91 and a variable heavy chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 75, and any variant thereof with one or more conservative amino acid substitutions in either the variable light or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides that the recombinant antigen binding protein of the present invention comprises a variable light chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 87 and a variable heavy chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 89, and any variant thereof which has one or more conservative amino acid substitutions in either the variable light chain or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides that the recombinant antigen binding protein of the present invention comprises a variable light chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 91 and a variable heavy chain comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 75, and any variant thereof which has one or more conservative amino acid substitutions in either the variable light chain or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides a recombinant antigen binding protein which specifically binds to nerve growth factor (NGF), further comprising a constant region comprising an amino acid selected from either SEQ ID NO: 41 or 43.In one embodiment, the present invention provides a nucleotide sequence encoding a constant region selected from the group consisting of SEQ ID NO: 42 or SEQ ID NO: 44. In one embodiment, the constant region of the antigen binding protein of the present invention lacks effector function. In one embodiment, modifications to the constant region of the antigen binding protein of the present invention prevent degradation of the antigen binding protein.

[0018] In one embodiment, the present invention provides a recombinant antigen binding protein that specifically binds to NGF, further comprising a constant region comprising an amino acid sequence comprising SEQ ID NO: 62. In one embodiment, the present invention provides a nucleotide sequence encoding the constant region comprising SEQ ID NO: 63. In one embodiment, the constant region of the antigen binding protein of the present invention lacks effector function. In one embodiment, modifications to the constant region of the antigen binding protein of the present invention prevent degradation of the antigen binding protein.

[0019] In one aspect, the present invention provides a variable light (VL) chain comprising a nucleotide sequence encoding a recombinant antigen binding protein of the invention that specifically binds nerve growth factor (NGF), the variable light (VL) chain comprising a complementarity determining region 1 (CDR1) nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:11 or SEQ ID NO:31; a complementarity determining region 1 (CDR2) comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:12 or SEQ ID NO:32; a complementarity determining region 1 (CDR3) comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:13 or SEQ ID NO:33; and a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:14 or SEQ ID NO:34. a variable heavy chain (VH) comprising a complementarity determining region 1 (CDR1) comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 35; a complementarity determining region 1 (CDR2) comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 35; and a complementarity determining region 1 (CDR3) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 36; and one or more nucleic acid substitutions based on the degeneracy of the genetic code in at least one of CDR1, CDR2 or CDR3 in either the variable light chain or variable heavy chain region of said antigen binding protein. The nucleotide sequence is provided, including any variants thereof.

[0020] In one embodiment, the present invention provides a light chain variable region (VL) comprising a nucleotide sequence encoding an antigen binding protein of the present invention, the light chain variable region (VL) comprising a complementarity determining region 1 (CDR1) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 11; a complementarity determining region 2 (CDR2) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 12; a complementarity determining region 3 (CDR3) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 13; and a complementarity determining region 1 (CDR1) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 14; a complementarity determining region 2 (CDR2) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 15; a nucleic acid sequence comprising SEQ ID NO: 16. and any variant thereof having one or more nucleic acid substitutions based on the degeneracy of the genetic code in at least one of CDR1, CDR2 or CDR3 in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0021] In one embodiment, the present invention provides a nucleotide sequence encoding an antigen binding protein of the present invention, the nucleotide sequence encoding a light chain variable region (VL) comprising: complementarity determining region 1 (CDR1) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 31; complementarity determining region 2 (CDR2) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 32; complementarity determining region 3 (CDR3) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 33; and a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 34. a nucleotide sequence encoding a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 35; a complementarity determining region 2 (CDR2) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 35; a complementarity determining region 3 (CDR3) comprising a nucleotide sequence having at least about 90% sequence identity to a nucleotide sequence comprising SEQ ID NO: 36; and any variant thereof having one or more nucleic acid substitutions based on the degeneracy of the genetic code in at least one of CDR1, CDR2 or CDR3 in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0022] In one aspect, the present invention provides a nucleotide sequence encoding a recombinant antigen binding protein of the present invention which specifically binds nerve growth factor (NGF), wherein the nucleotide sequence comprises nucleotides encoding a variable light chain comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:57, SEQ ID NO:88, SEQ ID NO:90, and SEQ ID NO:92; and nucleotides encoding a variable heavy chain comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:76, and SEQ ID NO:80; and any variant thereof having one or more nucleic acid substitutions based on the degeneracy of the genetic code in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0023] In one embodiment, the present invention provides a method for producing a recombinant antigen binding protein of the present invention, comprising the steps of: and a nucleotide sequence encoding a variable heavy chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18, as well as any variant thereof having one or more nucleic acid substitutions based on the degeneracy of the genetic code in either the variable light chain or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides a nucleotide sequence encoding an antigen binding protein of the present invention, wherein the nucleotide sequence encoding a variable light chain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37, and the nucleotide sequence encoding a variable heavy chain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 38; as well as any variant thereof having one or more nucleic acid substitutions based on the degeneracy of the genetic code in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0024] In one embodiment, the present invention provides that nucleotide sequences encoding the recombinant antigen binding protein of the present invention include a nucleotide sequence encoding a variable light chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19 and a nucleotide sequence encoding a variable heavy chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 20, and any variant thereof having one or more nucleic acid substitutions based on degeneracy of the genetic code in either the variable light chain or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides a nucleotide sequence encoding the antigen binding protein of the present invention, wherein the nucleotide sequence encodes a variable light chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 39, and the nucleotide sequence encodes a variable heavy chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 40; and any variant thereof having one or more nucleic acid substitutions based on degeneracy of the genetic code in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0025] In one embodiment, the present invention provides that the nucleotide sequence encoding the recombinant antigen binding protein of the present invention comprises a nucleotide sequence encoding a variable light chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 57 and a nucleotide sequence encoding a variable heavy chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 58, and any variant thereof having one or more nucleic acid substitutions based on the degeneracy of the genetic code in either the variable light or variable heavy chain region of said antigen binding protein.

[0026] In one embodiment, the present invention provides that nucleotide sequences encoding a recombinant antigen binding protein of the present invention comprise a nucleotide sequence encoding a variable light chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 92 and a nucleotide sequence encoding a variable heavy chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 80, and any variant thereof having one or more nucleic acid substitutions based on degeneracy of the genetic code in either the variable light chain or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides a nucleotide sequence encoding an antigen binding protein of the present invention, wherein the nucleotide sequence encodes a variable light chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 88, and the nucleotide sequence encodes a variable heavy chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 80; and any variant thereof having one or more nucleic acid substitutions based on degeneracy of the genetic code in either the variable light chain or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention relates to a recombinant antigen binding protein of the present invention, wherein the nucleotide sequence encoding the variable light chain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 92 and a nucleotide sequence encoding a variable light chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 76. In one embodiment, the present invention provides a nucleotide sequence encoding an antigen binding protein of the present invention, wherein the nucleotide sequence encodes a variable light chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 88 and a variable heavy chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 76; and any variant thereof having one or more nucleic acid substitutions based on the degeneracy of the genetic code in either the variable light chain or variable heavy chain region of said antigen binding protein. In one embodiment, the present invention provides a nucleotide sequence encoding a recombinant antigen binding protein of the present invention, wherein the nucleotide sequence encodes a variable light chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 90 and a nucleotide sequence encoding a variable heavy chain comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 76; and any variant thereof having one or more nucleic acid substitutions based on the degeneracy of the genetic code in either the variable light chain or variable heavy chain region of said antigen binding protein.

[0027] In one or more embodiments, the antigen binding proteins of the invention inhibit the binding of NGF to the TrkA receptor. In one or more embodiments, the antigen binding proteins of the invention inhibit the biological functions associated with NGF binding to the TrkA receptor. In one or more embodiments, the antigen binding proteins of the invention inhibit the binding of NGF to both TrkA receptors. In one or more embodiments, the antigen binding proteins inhibit the biological functions associated with NGF binding to TrkA with or without the p75 receptor, including blocking signaling and pathways associated with NGF binding to the TrkA receptor.

[0028] In one or more embodiments, the antigen binding proteins of the invention reduce or eliminate NGF-related disorders by disrupting signals associated with NGF binding to the TrkA and p75 receptors. In one or more embodiments, the NGF-related disorder is selected from the group consisting of cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation. In one embodiment, the NGF-related disorder is pain. In one embodiment, the NGF-related disorder is a pain disorder and is selected from the group consisting of osteoarthritis pain, rheumatoid arthritis pain, surgical and post-operative pain, incisional pain, systemic inflammatory pain, cancer pain, pain from trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic disease, pain associated with musculoskeletal disease, visceral pain, and gastrointestinal pain. In one embodiment, the NGF-related disorder comprises osteoarthritis pain. In one embodiment, the NGF-related disorder comprises surgical and post-operative pain. In one embodiment, the NGF-related disorder comprises cancer pain.

[0029] In one or more aspects, the antigen-binding protein of the present invention is selected from the group consisting of a monoclonal antibody; a chimeric antibody, a single-chain antibody, a tetrameric antibody, a tetravalent antibody, a multispecific antibody, a domain-specific antibody, a domain-deleted antibody, a fusion protein, an ScFc-fusion protein, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, an ScFv fragment, an Fd fragment, a single-domain antibody, a dAb fragment, a small modular immunopharmaceutical (SMIP), a nanobody, and an IgNAR molecule. In one embodiment, the antigen-binding protein is a monoclonal antibody. In one embodiment, the antigen-binding protein is a chimeric antibody.

[0030] In one embodiment, the antigen binding protein of the present invention is selected from a canine or caninized monoclonal antibody, a feline monoclonal antibody, an equine monoclonal antibody, or a humanized monoclonal antibody. In one embodiment, the antigen binding protein is a canine or caninized antibody. In one embodiment, the antigen binding protein of the present invention is a feline antibody. In one embodiment, the antigen binding protein of the present invention is an equine antibody. In one embodiment, the antigen binding protein of the present invention is a humanized antibody.

[0031] In one or more aspects, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an antigen binding protein and a pharmaceutically acceptable carrier. In one embodiment, the present invention provides a veterinary composition comprising a therapeutically effective amount of an antigen binding protein and a pharmaceutically acceptable carrier. In one embodiment, the present invention provides a pharmaceutical or veterinary composition comprising a therapeutically effective amount of an antigen binding protein and a pharmaceutically acceptable carrier. In one embodiment, a pharmaceutical composition of the present invention is used in the treatment of an NGF-related disorder. In one embodiment, the NGF-related disorder is selected from the group consisting of cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation. In one embodiment, the NGF-related disorder includes pain. In one embodiment, the pharmaceutical composition is used in the treatment of pain. In one embodiment, the pharmaceutical composition is used for the treatment of pain, and the type of pain is selected from osteoarthritis pain, rheumatoid arthritis pain, surgical and post-surgical pain, incisional pain, systemic inflammatory pain, cancer pain, pain from trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic disease, pain associated with musculoskeletal disease, visceral pain, and gastrointestinal pain. In one embodiment, the pain comprises osteoarthritis pain. In one embodiment, the pain comprises surgical and post-surgical pain. In one embodiment, the pain comprises cancer pain. In one or more embodiments, the pharmaceutical composition of the present invention is for use in dogs. In one or more embodiments, the pharmaceutical composition of the present invention is for use in cats. In one or more embodiments, the pharmaceutical composition of the present invention is for use in horses. In one or more embodiments, the pharmaceutical composition of the present invention is for use in humans.

[0032] In one or more embodiments, the pharmaceutical compositions of the present invention have no significant adverse effect on the canine immune system. In one embodiment, the compositions of the present invention have no significant adverse effect on the feline immune system. In one or more embodiments, the compositions of the present invention have no significant adverse effect on the equine immune system. In one embodiment, the compositions of the present invention have no significant adverse effect on the human immune system. In one embodiment, the pharmaceutical composition is a veterinary composition.

[0033] In one or more embodiments, the invention provides host cells that produce any one or more of the antigen binding proteins of the invention.

[0034] In one or more embodiments, the invention provides vectors comprising any one or more of the nucleic acids of the invention.

[0035] In one or more embodiments, the invention provides a host cell comprising any one or more of the nucleic acids of the invention.

[0036] In one or more embodiments, the invention provides a host cell comprising a vector comprising any one or more of the nucleic acids of the invention.

[0037] In one or more embodiments, the invention provides a host cell comprising any one or more of the nucleic acids of the invention.

[0038] In one or more embodiments, the invention provides methods of producing the antigen binding protein of the invention by culturing a host cell of the invention under conditions that result in the production of the antigen binding protein, and then isolating the antigen binding protein from the host cell or the host cell culture medium.

[0039] In one or more aspects, the present invention provides a method of treating a subject for an NGF-related disorder, comprising administering to the subject a therapeutically effective amount of a pharmaceutical or veterinary composition of the present invention. In one embodiment, the present invention provides that the NGF-related disorder is selected from the group consisting of cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation. In one embodiment, the NGF-related disorder includes pain. In one embodiment, the NGF-related disorder is a pain disorder and is selected from the group consisting of osteoarthritis pain, rheumatoid arthritis pain, surgical and post-operative pain, incisional pain, systemic inflammatory pain, cancer pain, pain from trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic disease, pain associated with musculoskeletal disease, visceral pain, and gastrointestinal pain. In one embodiment, the NGF-related disorder includes osteoarthritis pain. In one embodiment, the NGF-related disorder includes surgical and post-operative pain. In one embodiment, the NGF-related disorder is cancer pain. In one embodiment, the subject is selected from the group consisting of a dog, a cat, a human, and a horse. In one embodiment, the subject comprises a dog. In one embodiment, the subject comprises a cat. In one embodiment, the subject comprises a horse. In one embodiment, the subject comprises a human.

[0040] In one or more embodiments, the present invention provides a method for detecting or quantifying NGF levels in a biological sample, comprising: (a) incubating a clinical or biological sample containing NGF in the presence of any one of the antigen binding proteins of the invention; and (b) detecting an antigen-binding protein bound to NGF in the sample; The present invention provides a method comprising:

[0041] In some embodiments, the antigen binding proteins of the present invention are detectably labeled. In some embodiments, the antigen binding proteins are unlabeled and are used in combination with a second antigen binding protein or fragment that is detectably labeled. In one embodiment, the present invention includes a kit comprising the antigen binding proteins of the present invention. [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 is a schematic diagram of the overall structure of the mouse immunoglobulin G (IgG) molecule, highlighting the antigen-binding site. [Figure 2] FIG. 2 is a schematic diagram of the overall structure of mouse / dog chimeric IgG. [Figure 3] Figure 3 is an illustration showing the speciation or "caninization" of mouse IgG, where the mouse CDRs are grafted onto a dog framework. This diagram also represents feline, equine, humanized, and other speciations as defined herein. [Figure 4] FIG. 4 is an illustration of a "heterochimeric" monoclonal antibody in which a chimeric light chain is paired with a fully caninized heavy chain. [Figure 5] FIG. 5 is an illustration of an antibody variable chain showing primers to the constant region and degenerate primers targeting the mouse variable region. [Figure 6] FIG. 6 is a representation of the effects of anti-NGF mAbs ZTS-841 and ZTS-842 on canine NGF-induced pERK-1 / 2 signaling in caTrkA-CHO cells. [Figure 7] FIG. 7 is a representation of caninized aD11 mAb, negative control, and 13L11 mAb on canine NGF-induced pERK-1 / 2 signaling in caTrkA-CHO cells. [Figure 8] FIG. 8 shows the effect of ZTS-841 and ZTS-842 mAbs on canine NGF-induced TF-1 cell proliferation against anti-NGF mAbs. [Figure 9] FIG. 9 is a representation of anti-NGF mAb responses to canine NGF-induced TF-1 proliferation using 48L2 chimera, fel48L2VH1.1 and fel48L2VH1.2 mAbs. [Figure 10] FIG. 10 is a representation of anti-NGF mAb ZTS 841 administered SC / SC / IV at 2.0 mg / kg for a pharmacokinetic study. [Figure 11]FIG. 11 is a representation of anti-NGF mAb ZTS 842 administered SC / SC / IV at 2.0 mg / kg for a pharmacokinetic study. [Figure 12] FIG. 12 is a schematic diagram of the rat MIA assay. [Figure 13] FIG. 13 is a graphical representation of mAb 841 at doses ranging from 0.1 to 2 mg / kg in a rat MIA assay. [Figure 14] FIG. 14 is a graphical representation of mAb 841 at doses ranging from 0.01 to 2.0 mg / kg in a rat MIA assay. [Figure 15] FIG. 15 is a graphical representation of mAb 842 at doses of 0.5 and 2 mg / kg in a rat MIA assay. [Figure 16] FIG. 16 is a graphical representation of mAb 841 lameness VAS for treatment groups at 3 and 5 hours after synovitis induction in the LPS synovitis model.

[0043] A brief description of arrays [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] DETAILED DESCRIPTION OF THE INVENTION

[0044] The invention disclosed herein provides anti-NGF antigen-binding proteins that bind to NGF with high affinity. The invention further provides antigen-binding proteins and polypeptides that are variants of the antigen-binding proteins and that also bind to NGF, as well as methods for making and using these antigen-binding proteins. In some embodiments, the invention also provides polynucleotides encoding the antigen-binding proteins and / or polypeptides. The invention disclosed herein also provides methods for preventing and / or treating pain by administering a therapeutically effective amount of an anti-NGF antigen-binding protein of the invention.

[0045] General technology It is to be understood that this invention is not limited to, and as such may vary, the particular methodology, protocols, and reagents, etc., described herein. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0046] Unless otherwise defined, scientific and technical terms used in connection with the antigen-binding proteins described herein should have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms include pluralities, and plural terms include singularities. Generally, the technical terms and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art and are not limited to a single description. It is well known in the art that different techniques may be substituted for those described.

[0047] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application.

[0048] Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transfection (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described, including but not limited to the various general and more specific references referenced and discussed throughout this specification. See, for example, Sambrook et al., MOLECULAR CLONING: LAB. MANUAL (3 rd ed., Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY, 2001) and Ausubel et al. Current Protocols in Molecular Biology (New York: Greene Publishing Association J Wiley Interscience),Oligonucleotide Synthesis(M.J.Gait,ed.,1984);Methods in Molecular Biology,Humana Press;Cell Biology:A Laboratory Notebook(J.E.Cellis,ed.,1998)Academic Press;Animal Cell Culture(R.1.Freshney,ed.1987);Introduction to Cell and Tissue Culture(1.P.Mather and P.E.Roberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,J.B.Griffiths,and D.G.Newell,eds.,1993-1998)J.Wiley and Sons;Methods in Enzymology(Academic Press,Inc.);Handbook of Experimental Immunology(D.M.Weir and C.C.Blackwell,eds.);Gene Transfer Vectors for Mammalian Cells(J.M.Miller and M.P.Calos,eds.,1987);Current Protocols in Molecular Biology(F.M.Ausubel et al.,eds.,1987);PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.,1994);Current Protocols in Immunology(E.Coligan et al.,eds.,1991);Short Protocols in Molecular Biology(Wiley and Sons,1999);Immunobiology(C.A.Janeway and P.Travers,1997);Antibodies(P.Finch,1997);Antibodies:a practical approach(D.Catty.,ed.,IRL Press,1988-1989);Monoclonal antibodies:a practical approach(P.Shepherd and C.Dean,eds.,Oxford University Press,2000);Using antibodies:a laboratory manual(E.Harlow and D.Lane(Cold Spring Harbor Laboratory Press,1999);The Antibodies(M.Zanetti and JDCapra,eds.,Harwood Academic Publishers, 1995); and Cancer: Principles and. See Practice of Oncology (YT DeVita et al., eds., J.B. Lippincott Company, 1993).

[0049] Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about."

[0050] definition Before describing the present invention in detail, some terms used in the context of the present invention will be defined. In addition to these terms, other terms will be defined elsewhere in this specification as necessary. Unless otherwise expressly defined herein, technical terms used herein have their art-recognized meanings.

[0051] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, reference to "an antibody" includes a plurality of such antibodies.

[0052] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others.

[0053] As used herein, the terms "nerve growth factor" and "NGF" refer to nerve growth factor and variants thereof that retain at least some of the biological activity of NGF.

[0054] "NGF receptor" refers to a polypeptide that is bound to or activated by NGF. NGF receptors include the TrkA receptor and, to a lesser extent, the canine p75 receptor.

[0055] The "biological activity" of NGF generally refers to its ability to bind to NGF receptor and / or activate NGF receptor signaling pathway.Non-limiting biological activity includes any one or more of the following: its ability to bind to NGF receptor (for example, TrkA and / or p75); its ability to promote the dimerization and / or autophosphorylation of TrkA receptor; its ability to activate NGF receptor signaling pathway; its ability to promote cell differentiation, proliferation, survival, growth and other changes in cell physiology, including (in the case of neurons, including peripheral and central neurons) changes in neuronal morphology, synapse formation, synaptic function, release of neurotransmitters and / or neuropeptides and regeneration after injury; its ability to promote the survival of mouse E13.5 trigeminal neurons; and its ability to mediate pain, including postoperative pain.

[0056] As used herein, "anti-NGF antigen binding protein" (interchangeably referred to as "anti-NGF antibody" and "anti-NGF antagonist antibody," "antigen-binding fragment," and "antigen-binding portion," etc.) refers to an antigen binding protein that can bind to NGF and inhibit the biological activity of NGF and / or downstream pathways mediated by NGF signaling. Anti-NGF antigen binding proteins include binding proteins and antibodies that block, antagonize, suppress, or reduce (including significantly reduce) the biological activity of NGF, including downstream pathways mediated by NGF signaling, and / or inhibit NGF binding to its receptor trkA, such as receptor binding and / or elicitation of a cellular response to NGF. For purposes of the present invention, the term "anti-NGF antigen binding protein" or "anti-NGF antagonist antibody" is expressly understood to encompass all previously identified terms, titles, and functional states and characteristics whereby NGF itself, the biological activity of NGF (including, but not limited to, the ability to mediate any aspect of osteoarthritis pain, inflammatory pain, post-surgical pain, and cancer pain), or the consequences of biological activity, are substantially abolished, reduced, or neutralized to any meaningful extent. In some embodiments, the anti-NGF antagonist antibody binds to NGF and prevents NGF dimerization and / or binding to NGF receptors (e.g., TrkA and / or p75). In other embodiments, the anti-NGF antigen binding protein binds to NGF and prevents TrkA receptor dimerization and / or TrkA auto-reactivation. Examples of anti-NGF antagonist antibodies are provided herein.

[0057] As used herein, terms such as "antigen-binding protein," "antibody," and "antigen-binding protein," which may be used interchangeably, refer to a polypeptide or a fragment thereof comprising an antigen-binding site. In one embodiment of the present invention, the antigen-binding protein of the present invention further provides an immunoglobulin capable of specifically binding to a target, such as a carbohydrate, a polynucleotide, a lipid, or a polypeptide, through at least one antigen recognition site located in one or more variable regions of the immunoglobulin molecule. In some embodiments, the antibody has two light chains and two heavy chains. Thus, an isolated intact antibody may be isolated from a pool of polyclonal antibodies, monoclonal antibodies, synthetic antibodies, recombinant antibodies, chimeric antibodies, heterochimeric antibodies, or antibodies considered to be speciated as defined herein. In some embodiments, terms such as "antigen-binding protein," "antibody," and "antagonist antibody" preferably refer to monoclonal antibodies and fragments thereof, as well as immunologically binding equivalents thereof capable of binding to NGF protein and fragments thereof. As used herein, the terms encompass not only full-length (meaning two heavy chains and two light chains by standard definition) polyclonal or monoclonal antibodies, but also fragments thereof. For purposes of the present invention, "antibody" and "antigen-binding protein" also include antibody fragments, unless otherwise specified. Exemplary antibody fragments include Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, diabodies, antigen-recognition fragments thereof, small modular immunopharmaceuticals (SMIPs), nanobodies, IgNAR molecules, and equivalents recognized by those skilled in the art as antigen-binding proteins or antibody fragments, and any of the above fragments and their chemically or genetically manipulated counterparts, as well as other antibody fragments and mutants thereof, fusion proteins comprising antibody portions, and any other modified configurations of immunoglobulin molecules comprising an antigen-recognition site.Antibodies and antigen-binding proteins can be produced, for example, via traditional hybridoma technology (Kohler et al., Nature 256:495-499 (1975)), recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage display technology using antibody libraries (Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991)). For a variety of other antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988, as well as other techniques known to those skilled in the art.

[0058] As defined herein, a "monoclonal antibody" is an antibody produced by a single clone of cells (specifically, a single clone of hybridoma cells), and is therefore a single, pure, homogeneous type of antibody. All monoclonal antibodies produced from the same clone are identical and have the same antigen specificity. A monoclonal antibody is a homogeneous antibody population, and a monoclonal antibody consists of amino acids (naturally occurring and non-naturally occurring) involved in selective binding of an antigen. A population of monoclonal antibodies is highly specific and targets a single antigen site. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, diabody, antigen-recognition fragment thereof, small modular immunopharmaceutical (SMIP), nanobody, and IgNAR molecule), mutants thereof, fusion proteins containing antibody portions, and any other modified configuration of an immunoglobulin molecule that contains an antigen-recognition site of the required specificity and the ability to bind to an antigen. It is not intended to be limited by the source of the antibody or the manner in which it is made (eg, by hybridoma, phage selection, recombinant expression, transgenic animals, etc.).

[0059] The term "monoclonal antibodies" as used herein specifically includes "chimeric" antibodies (immunoglobulins) in which portions of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Typically, chimeric antibodies are antibodies whose light and heavy chain genes are constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of genes from a mouse monoclonal antibody may be linked to canine constant segments. Figure 2 is a schematic diagram of the overall structure of one embodiment of a mouse:canine IgG. In this embodiment, the antigen-binding site is derived from the mouse, while the Fc portion is derived from the dog.

[0060] The term "heterochimeric" as defined herein refers to an antibody in which one antibody chain (heavy or light) is caninized and the other is chimeric. Figure 4 depicts one embodiment of a heterochimeric molecule. In this embodiment, a caninized variable heavy chain (all CDRs are mouse and all FRs are canine) is paired with a chimeric variable light chain (all CDRs are mouse and all FRs are mouse). In this embodiment, both the variable heavy chain and the variable light chain are fused to canine constant regions.

[0061] For simplicity, the following will refer to "caninized" antibodies; however, the same can be applied to felineized, equineized, humanized, or any other "speciesized" antigen-binding proteins. By way of example, "caninization" is defined as a method of transferring non-canine antigen-binding information from a donor antibody to a less immunogenic canine antibody acceptor to generate therapeutics useful as therapeutics in dogs. Caninized antibodies are canine antibody sequences in which hypervariable region residues of the recipient have been replaced with hypervariable region residues from a non-canine species (donor antibody), such as mouse, rat, rabbit, cat, dog, goat, chicken, cow, horse, llama, camel, dromedary, shark, non-human primate, human, humanized, recombinant, or engineered sequence, possessing the desired properties, specificity, affinity, and capacity. Furthermore, caninized antibodies may contain residues that are not found in either the recipient or donor antibody. These modifications are made to further refine antibody performance. Modifications to hypervariable and / or framework regions as described herein are determined for each separately engineered speciation (caninization) antibody based on experiments known to those skilled in the art, but still cannot be predicted prior to said experiments. A caninized antibody may optionally comprise all or at least a portion of an immunoglobulin constant region (Fc), typically the constant region (Fc) of a canine immunoglobulin. Figure 3 is an illustration of one embodiment showing the speciation or caninization of a mouse IgG. All descriptions of caninization of an antigen-binding protein and caninized antigen-binding proteins can be conceptually applicable to any speciation antibody, whether caninized, feline, equine, humanized, etc.

[0062] The phrases "recombinant canine antibody," "recombinant feline antibody," "recombinant equine antibody," and "recombinant human antibody" all include speciation antibodies prepared, expressed, produced, or isolated by recombinant means, e.g., antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial canine (or feline, human, etc.) antibody libraries, antibodies isolated from animals (e.g., mice) that are transgenic for canine immunoglobulin genes (see, e.g., Taylor, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means involving splicing canine (or feline, human, etc.) immunoglobulin gene sequences into other DNA sequences.

[0063] Terms such as "canine antibody," "feline antibody," "equine antibody," and "human antibody" are used herein. As used herein, the term refers to antibodies (antigen-binding proteins) generated against a target and isolated from lymphocytes within a target species. These antibodies, as described herein, have been recombinantly modified in vitro to contain a target species-specific constant region. Additionally, antibodies, as described herein, have been identified, isolated, and modified to alter the antibody constant region, and then expressed and isolated from in vitro cell culture systems known and routinely used by those skilled in the art.

[0064] "Native antibodies" and "native immunoglobulins" are typically heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light (I) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. Figure 1 is an example of the overall structure of native mouse immunoglobulin G (IgG), highlighting the antigen-binding site.

[0065] A "parent" antibody herein is an antibody encoded by an amino acid sequence used to prepare a variant. Preferably, the parent antibody has canine framework regions and, if present, canine antibody constant regions. For example, the parent antibody may be a caninized or canine antibody.

[0066] Depending on the amino acid sequence of the constant domain of the antibody's heavy chain, immunoglobulins can be assigned to different classes. Currently, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2 (defined by mouse and human designations). The heavy-chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in multiple species. The frequency of individual isotypes and the functional activities associated with these constant domains are species-specific and must be experimentally defined.

[0067] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino acid sequences of their constant domains.

[0068] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (I) an approach based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda, Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., 1991). (1989)Nature 342:877;AI-Iazikani et al. al (1997) J. Molec. Biol. 273:927-948). As used herein, CDRs can refer to CDRs defined by either approach or by a combination of both approaches.

[0069] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions include amino acid residues from the "complementarity-determining regions" or "CDRs" (Kabat, et al. (1991) supra) and / or residues from the "hypervariable loops" (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or "FR" residues are variable domain residues other than the hypervariable region residues as herein defined.

[0070] As used herein, the term "antigen-binding region" refers to the portion of an antibody molecule containing amino acid residues that interact with an antigen to confer specificity and affinity to the antibody for the antigen. The antibody-binding region includes "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues.

[0071] A "functional Fc region" possesses at least one effector function of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding; neonatal receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors; BCRs), and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain), and can be assessed using a variety of assays known in the art for assessing such antibody effector functions.

[0072] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. A "variant Fc region," or "mutant" or "mutant" Fc region, comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, and may or may not retain at least one effector function of the native-sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of a parent polypeptide. A variant Fc region herein preferably has at least about 80% sequence identity with a native-sequence Fc region and / or the Fc region of a parent polypeptide, most preferably at least about 90% sequence identity, and more preferably at least about 95% sequence identity. A variant or mutant Fc region may also essentially eliminate a function of an antibody Fc region. For example, mutations in the Fc region may eliminate effector functions of the antibody. In one embodiment of the invention, the antibodies of the invention comprise a mutated Fc region.

[0073] As used herein, "Fc receptor" and "FcR" refer to a receptor that binds to the Fc region of an antibody. Preferred FcRs are native sequence FcRs. Further, preferred FcRs are those that bind IgG antibodies (gamma receptors), including receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcRs are described in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4 :25-34, and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587, and Kim, et al., 1994, J. Immunol., 24:249).

[0074] As used herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, resulting in lysis of the target cells. The ADCC activity of a molecule of interest can be evaluated using an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo in an animal model, such as that disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.

[0075] "Complement-dependent cytotoxicity" and "CDC" refer to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may be performed.

[0076] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0077] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0078] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. The three hypervariable regions of each variable domain interact to form the V H -V L It is in this configuration that defines an antigen-binding site on the surface of the dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0079] "Antigen," as used herein, refers to an antigenic determinant recognized by the CDR of an antigen-binding protein or antibody as described herein. In other words, an epitope refers to a portion of any molecule that can be recognized and bound by an antibody. Unless otherwise indicated, as used herein, the term "epitope" refers to an antigenic determinant recognized by the CDR of an antigen-binding protein or antibody as described herein. This refers to the region of NGF to which the GF antigen-binding protein / antibody / agent binds.

[0080] Terms such as "antigen-binding domain" or "active fragment of an antibody" refer to a portion of an antibody or antigen-binding protein that comprises an area that specifically binds to or is complementary to a portion or all of an antigen. If the antigen is large, an antibody may bind only to a specific portion of the antigen. An "epitope," "active fragment of an epitope," or "antigenic determinant" is a portion of an antigen molecule that is involved in specific interactions with the antigen-binding domain of an antibody. An antigen-binding domain may be provided by one or more antibody variable domains (e.g., a so-called Fd antibody fragment consisting of a VH domain). An antigen-binding domain may comprise an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH) (U.S. Pat. No. 5,565,332).

[0081] The term "binding portion," such as an antibody (or "antibody portion") or antigen-binding polypeptide, includes not only one or more intact domains, e.g., a pair of intact domains, that retain the ability to specifically bind to an antigen, e.g., NGF, but also antibody fragments. It has been shown that the binding function of an antibody can be performed by fragments of a full-length antibody. Binding fragments are produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab')2, F(abc), Fd, dAb, Fv, single chain, single-chain antibodies, e.g., scFv, and single-domain antibodies (Muyldermans et al., 2001, 26:230-5), and isolated complementarity-determining regions (CDRs). Fab fragments are monovalent fragments consisting of the VL, VH, CL, and CH1 domains. F(ab')2 fragments are bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region. An Fd fragment consists of the VH and CH1 domains, and an Fv fragment consists of the VL and VH domains of a single arm of an antibody. A dAb fragment consists of the VH domain (Ward et al., (1989) Nature 341:544-546). Although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker using recombinant techniques, allowing them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv)) (Bird et al., 1988, Science 242:423-426). Such single-chain antibodies are also intended to be encompassed by the term "binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites by using a linker that is too short to allow pairing between the two domains on the same chain (see, e.g., Holliger, et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448).An antibody or binding portion thereof may also be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058). Binding fragments such as Fab and F(ab')2 fragments can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies, respectively. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein and known in the art. Other than "bispecific" or "bifunctional" antibodies, an antibody is understood to have each of its binding sites identical. A "bispecific antibody" is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. A bispecific antibody can also contain two antigen-binding regions with an intervening constant region. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. For example, Songsivilai et al., Clin. Exp. Immunol. 79:315-321, 1990; Kostelny et al., 1992, J. Immunol. 148, 1547-1553.

[0082] The term "backmutation" refers to a process in which some or all of the somatically mutated amino acids of a canine antibody are replaced with corresponding germline residues from a homologous germline antibody sequence. The heavy and light chain sequences of the canine antibody of the present invention are separately aligned with the germline sequence to identify the sequence with the highest homology. Differences in the canine antibody of the present invention are restored to the germline sequence by mutating the defined nucleotide positions encoding such different amino acids. The role of each amino acid so identified as a candidate for backmutation should be examined for its direct or indirect role in antigen binding, and any amino acid found after mutation that affects any desirable characteristics of the canine antibody should not be included in the final canine antibody; for example, activity-enhancing amino acids identified by a selective mutagenesis approach are not subjected to backmutation. To minimize the number of amino acids subjected to backmutation, amino acid positions that differ from the closest germline sequence but are found to be identical to the corresponding amino acid in a second germline sequence can be retained, as long as the second germline sequence is identical and colinear with the sequence of the canine antibody of the present invention. Backmutation of selected target framework residues to the corresponding donor residues may be required to restore and / or improve affinity.

[0083] As used herein, "immunospecific" binding of an antibody refers to an antigen-specific binding interaction that occurs between the antigen-binding site of an antibody and the specific antigen recognized by that antibody (i.e., the antibody reacts with the protein in an ELISA or other immunoassay and does not detectably react with unrelated proteins). An epitope that "specifically binds" or "preferentially binds" (used interchangeably herein) to an antibody or polypeptide is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with a higher affinity than with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to an NGF epitope is one that binds to this epitope with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other NGF epitopes or non-NGF epitopes. For example, it is understood by reading this definition that an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although can include) exclusive binding. Generally, but not necessarily, reference to binding refers to preferential binding.

[0084] The term "specifically" in the context of antibody binding refers to high avidity and / or high affinity binding of an antibody to a particular antigen, i.e., polypeptide, or epitope. An antibody that specifically binds to an antigen is stronger than the binding of the same antibody to other antigens. An antibody that specifically binds to a polypeptide may bind at a weak but detectable level (e.g., to the polypeptide of interest). A specific antibody may have the ability to bind to other polypeptides at a binding affinity of at least 10% (10% or less) of the binding exhibited by the specific antibody. Such weak binding, or background binding, is readily discernible from the binding of the specific antibody to the polypeptide of interest, e.g., by use of appropriate controls. Generally, a specific antibody will bind to other polypeptides at a binding affinity of at least 10% (10% or less) of the binding exhibited by the specific antibody. -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, or 10 -13 M or less, etc. d The antigen binds to the antigen with a binding affinity having a

[0085] As used herein, the term "affinity" refers to the strength of binding of a single antigen-binding site to an antigenic determinant. Affinity depends on the tightness of the stereochemical fit between the binding site of an antibody or antigen-binding protein and the antigenic determinant, the size of the contact area between them, the distribution of charged and hydrophobic groups, etc. Antibody affinity can be measured by equilibrium analysis or surface plasmon resonance (SPR) methods (e.g., BIACORE™). SPR methods rely on the phenomenon of surface plasmon resonance (SPR), which occurs when surface plasmon waves are excited at a metal / liquid interface. Light is directed to the side of the surface that is not in contact with the sample and reflected from it, and SPR causes a reduction in the reflected light intensity at a specific combination of angle and wavelength. A bimolecular binding event causes a change in the refractive index at the surface layer, which is detected as a change in the SPR signal.

[0086] "K D The term " as used herein is intended to refer to the dissociation constant of an antibody-antigen interaction. The dissociation constant K D , and the association constant K a is a quantitative measure of affinity. At equilibrium, free antigen (Ag) and free antibody (Ab) are in equilibrium with the antigen-antibody complex (Ag-Ab), and there is a rate constant, k a and kd quantifies the rate of each reaction. At equilibrium, ka[Ab][Ag] = kd[Ag-Ab]. The dissociation constant, K d is given by Kd = kd / ka = [Ag][Ab] / [Ag-Ab]. D has units of concentration, most typically M, mM, μM, nM, pM, etc. D When comparing antibody affinities, expressed as the association constant K, a higher affinity for NGF is indicated by a lower value. a is given by Ka = ka / kd = [Ag-Ab] / [Ag][Ab]. a is the unit of the reciprocal of concentration, most typically M -1 , mM -1 , μM -1 , nM -1 , pM -1 As used herein, the term "avidity" refers to the strength of antigen-antibody binding after the formation of a reversible complex. Anti-NGF antibodies have an avidity of about (lower limit K D value) ~ approx. (upper limit K D Dissociation constant (K value) within the range D ) for binding to NGF protein. D It may be characterized in terms of:

[0087] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Because the polypeptides of the invention are based on antibodies, it is understood that the polypeptides can occur as single chains or associated chains.

[0088] The term "conservative amino acid substitution" refers to any amino acid substitution for a given amino acid residue where the substituted residue is chemically similar to the given residue to the extent that it does not result in a substantial decrease in polypeptide function (e.g., enzymatic activity). Conservative amino acid substitutions are generally known in the art, and examples are described, for example, in U.S. Pat. Nos. 6,790,639, 6,774,107, 6,194,167, or 5,350,576. In a preferred embodiment, the conservative amino acid substitutions are those that occur within one of the following six groups: · Small aliphatic, substantially non-polar residues: Ala, Gly, Pro, Ser, and Thr; · Large aliphatic, non-polar residues: Lie, Leu, and Val; Met; · Polar, negatively charged residues and their amides: Asp and Glu; · Amides of polar, negatively charged residues: Asn and Gin; His; Polar, positively charged residues: Arg and Lys; His; and · Large aromatic residues: Trp and Tyr; Phe.

[0089] In preferred embodiments, the conservative amino acid substitution is any one of the following, listed as natural residue (conservative substitution) pairs: Ala (Ser); Arg (Lys); Asn (Gin; His); Asp (Glu); Gin (Asn); Glu (Asp); Gly (Pro); His (Asn; Gln); Ile (Leu; Val); Leu (Ile; Val); Lys (Arg; Gin; Glu); Met (Leu; Ile); Phe (Met; Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe); and Val (Ile; Leu).

[0090] Terms such as "nucleic acid," "polynucleotide," and "nucleic acid molecule" are sometimes used interchangeably herein and refer to a series of nucleotide bases (also called "nucleotides") in DNA and RNA. A nucleic acid may contain deoxyribonucleotides, ribonucleotides, and / or their analogs. The term "nucleic acid" includes, for example, single-stranded and double-stranded molecules. A nucleic acid can be, for example, a gene or gene fragment, an exon, an intron, a DNA molecule (e.g., cDNA), an RNA molecule (e.g., mRNA), a recombinant nucleic acid, a plasmid and other vector, a primer, and a probe. Both 5' to 3' (sense) and 3' to 5' (antisense) polynucleotides are included. A nucleotide can be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and / or its analog, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.Other types of modifications include, for example, "capping," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties (e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides. Additionally, any of the hydroxyl groups normally present in the sugar may be replaced by, for example, phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or attached to a solid support. The 5' and 3' terminal OH groups can be phosphorylated, or replaced by amines or organic capping groups of 1 to 20 carbon atoms. The hydroxyl groups may be replaced with a moiety. Other hydroxyls may also be derivatized with standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, anomeric sugars, epimeric sugars (such as arabinose, xylose, or lyxose), pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs (such as methyl riboside). One or more phosphodiester linkages may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C), optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0091] As used herein, "vector" refers to a construct capable of delivering and preferably expressing one or more genes or sequences of interest into a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. Vectors as described herein have expression control sequences, i.e., nucleic acid sequences that direct the transcription of a nucleic acid. The expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. An expression control sequence is "operably linked" to a nucleic acid sequence to be transcribed. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0092] Just as a polypeptide may contain conservative amino acid substitutions, the polynucleotide may contain conservative codon substitutions. A codon substitution is considered conservative if it results in a conservative amino acid substitution as described above when expressed. Degenerate codon substitutions that do not result in amino acid substitutions are also useful in polynucleotides according to the present invention. Thus, for example, a polynucleotide encoding a selected polypeptide useful in embodiments of the present invention may be mutated by degenerate codon substitutions to approximate the codon usage exhibited by an expression host cell transformed with it, or to otherwise improve its expression.

[0093] A "variant" anti-NGF antigen binding protein differs from the "parent" anti-NGF antibody amino acid sequence by the addition, deletion, and / or substitution of one or more amino acid residues in the parent antibody sequence. A variant anti-NGF antibody is herein referred to as a molecule that differs in amino acid sequence and retains at least one desired activity of a parent anti-NGF antibody. Variant anti-NGFs may contain conservative amino acid substitutions in the hypervariable regions of the antibody, as described herein. Desired activities may include the ability to specifically bind to an antigen and the ability to reduce, inhibit, or neutralize NGF activity in an animal. In one embodiment, the variant contains one or more amino acid substitutions in one or more hypervariable and / or framework regions of the parent antibody. For example, the variant may contain at least one, e.g., about one to about ten, preferably about two to about five, substitutions in one or more hypervariable and / or framework regions of the parent antibody. Typically, the variant has an amino acid sequence that shares at least 50% amino acid sequence identity, more preferably at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity, with the heavy or light chain variable domain sequence of the parent antibody. The identity or homology for this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the parent antibody residues after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence identity percentage.Neither N-terminal, C-terminal, nor internal extensions, deletions, nor insertions into the antibody sequence are considered to affect sequence identity or homology.Variants retain the ability to bind to NGF and preferably have a desired activity that is equal to or superior to that of the parent antibody.For example, variants may have stronger binding affinity, an improved ability to reduce, inhibit, or neutralize NGF activity in animals, and / or an improved ability to inhibit NGF binding to TrkA and p75.

[0094] TrkA, which is thought to be the high-affinity NGF receptor, is a member of the neurotrophic tyrosine kinase receptor (NTKR) family. This kinase is a membrane-bound receptor that phosphorylates itself (autophosphorylation) and members of the MAPK pathway upon neurotrophin binding. The presence of this kinase leads to cell differentiation and may play a role in the specification of sensory neuron subtypes. The p75 receptor is thought to be the low-affinity NGF receptor.

[0095] A "variant" nucleic acid is herein referred to as a molecule that differs in sequence from a "parent" nucleic acid. Differences in polynucleotide sequence can result from mutational changes, such as deletion, substitution, or addition of one or more nucleotides. Each of these changes may occur alone or in combination, one or more times in a given sequence.

[0096] The term "isolated" means that a material (e.g., an antigen-binding protein or nucleic acid as described herein) has been separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses of the material, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. With respect to nucleic acids, isolated nucleic acids can include those separated from the 5' to 3' sequences with which they are normally associated in chromosomes. In preferred embodiments, the material is purified to greater than 95% by weight, and most preferably greater than 99% by weight. Isolated material includes material in situ within recombinant cells, since at least one component of the material's natural environment will not be present. Typically, however, isolated material will be prepared by at least one purification step.

[0097] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. These terms also include all subsequent generations, that is, their progeny. It is understood that all progeny may not be identical due to intentional or unintentional mutations. In the context of expression of heterologous nucleic acid sequences, a "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial cell, yeast cell, mammalian cell, and insect cell), whether located in vitro or in vivo. For example, a host cell may be located in a transgenic animal. A host cell may contain a recombinant vector for a vector. The vector may be used as a recipient and may include any transformable organism that is capable of replicating a vector and / or expressing a heterologous nucleic acid encoded by the vector.

[0098] As used herein, the term "label" refers to a detectable compound or composition that is directly or indirectly attached to an antibody or nucleic acid. The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0099] "Subject" or "patient" refers to an animal in need of treatment that may be affected by a molecule of the invention. Animals that can be treated in accordance with the present invention include vertebrates, with mammals such as dogs being particularly preferred examples.

[0100] A "composition" is intended to mean a combination of an active agent, whether a chemical composition, a biological composition, or a biologic (particularly an antigen binding protein as described herein), with another compound or composition, which may be inert (e.g., a label) or active (e.g., an adjuvant).

[0101] As defined herein, "pharmaceutically acceptable carriers" suitable for use in the present invention are well known to those skilled in the art. Such carriers include, but are not limited to, water, saline, buffered saline, phosphate buffer, alcoholic / aqueous solutions, emulsions, or suspensions. Other conventionally used diluents, adjuvants, and excipients may be added according to conventional techniques. Such carriers may include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusters may also be used. Buffers include, but are not limited to, salts prepared from organic acids or bases. Representative buffers include, but are not limited to, organic acid salts (e.g., citric acid, citrates, ascorbic acid, gluconic acid, histidine-Hel, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid salts), Tris, trimethanmine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, and sodium chloride, lactated Ringer's, or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents (e.g., EDTA), and inert gases, may also be provided in the pharmaceutical carrier. The present invention is not limited by the choice of carrier. The preparation of these pharmaceutically acceptable compositions from the above ingredients with appropriate pH, isotonicity, stability, and other conventional characteristics is within the skill of the art.See, for example, texts such as Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott Williams & Wilkins, publ., 2000; and The Handbook of Pharmaceutical Excipients, 4th sup.th edit., eds. R.C. Rowe et al., APhA Publications, 2003.

[0102] A "therapeutically effective amount" (or "effective amount") refers to an amount of an active ingredient, e.g., an agent according to the present invention, sufficient to produce a beneficial or desired effect when administered to a subject or patient. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition according to the present invention can be readily determined by one of skill in the art. In the context of the present invention, a "therapeutically effective amount" refers to a therapeutically effective amount that produces a beneficial or desired effect, including clinical results such as relief or reduction of pain sensation. The effective amount is one that produces an objectively measured change in one or more parameters associated with an NGF-related condition sufficient to produce the desired result. An effective amount may be administered in one or more administrations. For purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to treat, relieve, reduce the intensity of, and / or prevent pain, including postoperative pain, rheumatoid arthritis pain, and / or osteoarthritis pain. In some embodiments, an "effective amount" may reduce pain at rest (rest pain) or mechanically induced pain (including post-exercise pain), or both, and may be administered before, during, or after a painful stimulus. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can or is achieved in combination with one or more other agents. Of course, the therapeutically effective amount will vary depending on the particular subject and condition being treated, the subject's weight and age, the severity of the condition, the particular compound selected, the administration regimen to be followed, the timing of administration, and the mode of administration, etc., all of which can be readily determined by one of ordinary skill in the art.

[0103] As used herein, the term "therapeutic" encompasses the full spectrum of treatments for diseases, conditions, or disorders. The "therapeutic" agents of the present invention may act in a prophylactic or preventive manner, including incorporating procedures designed for target animals that can be identified as being at risk (pharmacogenetics); or may act in a manner that is palliative or curative in nature; or may act to slow the rate or extent of progression of at least one symptom of the disease or disorder being treated.

[0104] In a further aspect, the invention features veterinary compositions in which the antibodies of the invention are provided for therapeutic or prophylactic use. The invention features methods of treating a canine subject having a particular antigen, e.g., a particular antigen associated with a disease or condition. The method includes administering a therapeutically effective amount of a recombinant antibody specific for the particular antigen, the recombinant antibody being described herein.

[0105] The amount of antibody useful for producing a therapeutic effect can be determined by standard techniques well known to those skilled in the art. The antibody is generally provided in a pharmaceutically acceptable buffer by standard techniques and may be administered by any desired route. The route of administration of the antibody or antigen-binding portion of the present invention may be oral, parenteral, inhalation, or topical. In a preferred embodiment, the route of administration is parenteral. As used herein, the term parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration.

[0106] " Pain " used herein refers to the pain of any etiology, including acute and chronic pain and any pain that has inflammatory components.Examples of pain include inflammatory pain, postoperative incision pain, neuropathic pain, fracture pain, osteoporosis fracture pain, postherpetic neuralgia, cancer pain, the pain resulting from burns, the pain associated with burns or wounds, the pain associated with trauma (including traumatic head injury), neuropathic pain, the pain associated with musculoskeletal disorders (such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatic) arthropathy, non-articular rheumatoid arthritis and periarticular disorders), and cancer (including " breakthrough pain " and the pain associated with terminal cancer), the pain associated with peripheral neuropathy and postherpetic neuralgia.

[0107] As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include: Amelioration or alleviation of any aspect of pain, including, but not limited to, acute, chronic, inflammatory, neuropathic, post-operative, rheumatoid arthritis, or osteoarthritic pain. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reduction in the severity, alleviation of one or more symptoms associated with pain, including any aspect of pain (e.g., shortening the duration of pain, reducing pain sensitivity or sensation).

[0108] As used herein, NGF-related disorders refer to disorders including cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain and inflammation.In some embodiments of the present invention, NGF-related disorders refer to pain, particularly chronic pain, inflammatory pain, postoperative incision pain, neuropathic pain, fracture pain, osteoporosis fracture pain, postherpetic neuralgia, cancer pain, pain resulting from burns, pain associated with burns or wounds, pain associated with trauma (including traumatic head injury), neuropathic pain, pain associated with musculoskeletal disorders (for example, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatic) arthropathy, non-articular rheumatoid arthritis and periarticular disorders), and cancer (including "breakthrough pain" and pain associated with terminal cancer), pain associated with peripheral neuropathy and postherpetic neuralgia.

[0109] "Reducing the incidence" of pain means either reducing the severity (which can include reducing the need for and / or the amount (e.g., exposure to) other drugs and / or therapies commonly used for this condition, including, for example, sedatives), reducing the duration, and / or reducing the frequency (including, for example, delaying or increasing the time to post-operative pain in an individual). As will be understood by those skilled in the art, individuals may differ with respect to their response to treatment, so, for example, a "method of reducing the incidence of rheumatoid arthritis pain or osteoarthritis pain in an individual" reflects administering an anti-NGF antagonist antibody with the reasonable expectation that such administration will cause such a reduction in incidence in that particular individual.

[0110] "Alleviation" of pain or one or more symptoms of pain (e.g., rheumatoid arthritis pain or osteoarthritis pain) refers to a decrease or improvement in one or more symptoms of pain compared to when the anti-NGF antagonist antibody is not administered. "Alleviation" also includes a shortening or reduction in the duration of symptoms.

[0111] "Relief" of pain or one or more symptoms of pain (e.g., rheumatoid arthritis pain or osteoarthritis pain) means a decrease in the magnitude of one or more undesirable clinical symptoms of post-operative pain in an individual or population of individuals treated with an anti-NGF antagonist antibody according to the invention.

[0112] As used herein, "delaying" the onset of pain means to postpone, prevent, slow, delay, stabilize, and / or postpone the progression of pain, such as postoperative pain, rheumatoid arthritis pain, or osteoarthritis pain. This delay can be for a variable length of time, depending on the history of the disease being treated and / or the individual. As will be apparent to those skilled in the art, a sufficient or significant delay can essentially encompass prevention, in that the individual does not develop pain. A method of "delaying" the onset of symptoms is a method that reduces the probability of developing symptoms within a given time frame and / or reduces the severity of symptoms within a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of subjects.

[0113] "Postoperative pain" (interchangeably referred to as "post-incision pain" or "post-traumatic pain") is pain caused by trauma (invasive or non-invasive) such as a cut, puncture, incision, laceration, or wound to the tissues of an individual. "Postoperative pain" refers to pain resulting from or resulting from a surgical procedure, whether surgical or non-surgical, including that resulting from all surgical procedures. As used herein, postoperative pain does not include pain that occurs (arises from or originates) without external physical trauma. In some embodiments, postoperative pain is internal or external (including peripheral) pain, and wounds, cuts, trauma, lacerations, or incisions may be accidental (as in a traumatic wound) or intentional (as in a surgical incision). As used herein, "pain" includes nociception and sensation of pain, and pain can be assessed objectively and subjectively using pain scores and other methods well known in the art. Postoperative pain, as used herein, includes allodynia (i.e., increased response to normally non-noxious stimuli) and hyperalgesia (i.e., increased response to normally noxious or unpleasant stimuli), which may be thermal or mechanical (tactile) in nature. In some embodiments, the pain is characterized by thermal sensitivity, mechanical sensitivity, and / or resting pain. In some embodiments, the postoperative pain comprises mechanically induced pain or resting pain. In other embodiments, the postoperative pain comprises resting pain. Pain can be primary or secondary pain, as is well known in the art.

[0114] Before describing the methods of the present invention, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, and thus, the scope of the present invention will be limited only by the appended claims.

[0115] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety.

[0116] The invention disclosed herein relates to antigen binding proteins (used interchangeably with terms such as "antibody," "antagonist antibody," and "antibody fragment," as described herein) that specifically bind to nerve growth factor (NGF), in particular antibodies produced by recombinant methods, hybridoma technology, or phage display technology, caninized, feline, bovine, equine, humanized, or any other speciation antibody, or fully "caninized" (speciation) monoclonal antibody, that specifically binds to NGF, whether canine, feline, equine, murine, bovine, human, or any other species, and thereby acts as an antagonist in that it prevents NGF from binding to the canine TrkA and, to a lesser extent, the canine p75 receptor, thereby preventing signal transduction pathways from being activated by NGF.

[0117] NGF was the first neurotrophin to be identified, and its role in the development and survival of both peripheral and central neurons has been well characterized. NGF has been shown to be an essential survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons and basal forebrain cholinergic neurons (Smeyne et al. (1994) Nature 368:246-249; Crowley et al. (1995) Nature 368:246-249). NGF upregulates neuropeptide expression in sensory neurons (Lindsay et al. (1989) Nature 337:362-364), and its activity is mediated through two distinct membrane-bound receptors, the TrkA receptor and what is thought to be the low-affinity p75 common neurotrophin receptor.

[0118] NGF has been shown to be elevated in NGF-associated disorders, in which: Elevated amount of NGF exists in injured or diseased tissue.NGF-related disorder can be defined as the increase in pain caused by the increase of NGF in injured, diseased or damaged tissue.Pain as used herein is defined as described herein, and refers to disorders including chronic pain, inflammatory pain, postoperative incision pain, neuropathic pain, fracture pain, osteoporosis fracture pain, postherpetic neuralgia, cancer pain, the pain resulting from burns, the pain associated with burns or wounds, the pain associated with trauma (including traumatic head injury), neuropathic pain, the pain associated with musculoskeletal disorders (for example, chronic pain, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatic) arthropathy, non-articular rheumatoid arthritis and periarticular disorders), and cancer (including " breakthrough pain " and the pain associated with terminal cancer), the pain associated with peripheral neuropathy and postherpetic neuralgia.

[0119] In one embodiment of the present invention, NGF disorder is defined as osteoarthritis in subjects (dogs, cats, horses, humans, etc.).Osteoarthritis (OA) is a slowly progressing joint degenerative disease in dogs, characterized by the loss of articular cartilage and the subsequent exposure of subchondral bone.This ultimately leads to a self-perpetuating, insidious disorder characterized by joint pain.New bone formation occurs in response to chronic inflammation and local tissue damage to limit both movement and pain.Macroscopically, there is the loss of articular cartilage, narrowing of joint space, sclerosis of subchondral bone, and the production of osteophytes in joints (Veterinary Focus: Vol 17 No 3; 2007).

[0120] In different species, such as dogs, cats, and horses, the onset of primary OA depends on breed. For example, in dogs, the average age of onset is 3.5 years in Rottweilers and 9.5 years in Poodles, with a wide range of incidence for mixed breeds as well as other breeds. Developmental orthopedic disease and associated osteoarthritis are the most common joint diseases in dogs, accounting for approximately 70% of cases due to joint disease and related problems within the bones of the appendages. Twenty-two percent of cases occurred in dogs aged 1 year or less. The incidence of OA increases not only due to trauma but also due to obesity, aging, and genetic abnormalities. Age may be a factor in the incidence of OA, with >50% of arthritis cases observed in dogs aged 8 to 13 years. Musculoskeletal disorders are very common in geriatric patients, with nearly 20% of older dogs exhibiting orthopedic disorders. OA in several joints (elbows, shoulders, hips, and knees) is typical in Labrador retrievers >8 years of age. Additionally, dog size also plays a role in OA development. 45% of dogs with arthritis are large breeds. Of these, >50% are giant breeds, while only 28% are medium breeds and 27% are small breeds. There is a great need for pharmaceutical interventions to relieve OA pain in dogs.

[0121] As described herein, elevated NGF levels are indicative of NGF-related disorders, particularly OA. Elevated NGF levels, along with increased mast cell numbers, have been reported in transgenic arthritic mice (Aloe et al., Int. J. Tissue Reactions-Exp. Clin. Aspects 15:139-143 (1993)). PCT International Publication No. 02 / 096458 discloses the use of anti-NGF antibodies with specific properties in the treatment of various NGF-related disorders, such as inflammatory conditions (e.g., rheumatoid arthritis). It has been reported that purified anti-NGF antibodies injected into arthritic transgenic mice carrying human tumor necrosis factor genes not only reduced the number of mast cells, but also reduced the histamine and substance P levels in the synovial membranes of arthritic mice (Aloe et al., Rheumatol. Int. 14:249-252 (1995)). Exogenous administration of NGF antibodies has been shown to reduce the elevated levels of TNFα that occur in arthritic mice (Marmi et al., Rheumatol. Int. 18:97-102 (1998)). Rodent anti-NGF antagonist antibodies have been reported. See, for example, Hongo et al., Hybridoma ( 2000) 19(3):215-227; Ruberti et al. (1993) Cell. Molec. Neurobiol. 13(5):559-568. However, when rodent antibodies are used therapeutically in non-mouse mammals, anti-mouse antibody responses develop in a significant number of treated individuals. Thus, there is a serious need for anti-NGF antagonist antigen binding proteins, including the anti-NGF antagonist antibodies of the present invention, for canine use, particularly for use in the treatment of OA.

[0122] Although the properties of antibodies make them very attractive therapeutic agents, they have numerous limitations. As mentioned above, the majority of monoclonal antibodies (mAbs) are of rodent origin. When such antibodies are administered in a different species, the patient may mount their own antibody response against the xenoantibody. Such a response can result in the eventual neutralization and elimination of the antibody. As mentioned above, mice are extensively used in the production of monoclonal antibodies. One problem with using antibodies initially produced in a specific species, typically mice, is that non-mouse subjects being treated with the antibody react to the mouse antibodies as if they were foreign, thereby generating a new set of antibodies against the mouse antibodies. The mouse antibodies are "seen" as foreign by non-mouse immune systems, such as the canine immune system, and the subject then mounts an immune response against the molecule. Those skilled in the art recognize that while antigen-specific antibodies can be used to treat subjects, the antibodies need to be species-specific. The portion of the reaction generated from interspecies antibody administration, e.g., administration of a murine monoclonal antibody to a dog, can range from mild forms such as a rash to more extreme and life-threatening reactions such as renal failure. This immune response can also reduce the effectiveness of the treatment or lead to future reactions if the subject is given subsequent treatments containing murine antibodies. Therefore, we describe overcoming this drawback by "caninizing" antibodies. In particular, this process focuses on the framework regions of immunoglobulin variable domains, but not on the complementarity determinant regions of the variable domains. The enabling steps and reductions for implementing this process are described in this disclosure.

[0123] The process of modifying monoclonal antibodies (antigen-binding proteins, antagonist antibodies, etc., as described herein, and terms used interchangeably) from animals to make them less immunogenic for therapeutic administration to a species has been actively pursued and described in numerous publications (e.g., Antibody Engineering: A Practical Guide. Carl A.K. Borrebaeck ed. W.H. Freeman and Company, 1992). However, until recently, this process has not been applied to the development of therapeutics or diagnostics for non-humans, particularly canines. In fact, there has been little published work on canine variable domains. Wasserman and Capra, Biochem. 6, 3160 (1977), determined the amino acid sequences of the variable regions of both canine IgM and canine IgA heavy chains. Wasserman and Capra, Immunochem. 15, 303 (1978) determined the amino acid sequence of the K light chain from canine IgA. McCumber and Capra, Mol. Immunol. 16, 565 (1979) disclose the complete amino acid sequence of the canine mu chain. Tang et al., Vet. Immunology Immunopathology 80, 259 (2001) discloses a single canine IgG-A y chain cDNA and four canine IgG-A y chain protein sequences. It describes PCR amplification of a canine spleen cDNA library using degenerate oligonucleotide primers designed from conserved regions of human, mouse, porcine, and bovine IgG. The limited information available about canine antibodies has hindered their development as therapeutics for the treatment of canine diseases.

[0124] These stated limitations have prompted the development of an engineering technique known as "speciation," well known to those skilled in the art for the "humanization" of therapeutic antibodies. An example of a speciated molecule is a caninized antibody. Chimeric antibodies or fragments thereof can be generated that contain minimal sequence derived from a non-canine immunoglobulin. For the most part, caninized antibodies are canine antibodies (i.e., "recipient antibodies" or "target species antibodies") in which residues from the recipient's complementarity-determining regions (CDRs) have been replaced with residues from the CDRs of a non-canine species, such as mouse (i.e., "donor antibody" or "originating species antibody"), that have the desired properties (e.g., specificity, affinity, and potency). In some cases, framework region (FR) residues of the canine immunoglobulin are replaced with corresponding non-canine residues. This caninization strategy is referred to as "CDR grafting." Backmutation of selected target framework residues to the corresponding donor residues may be required to restore and / or improve affinity. Structure-based methods may also be used for caninization and affinity maturation, as described in U.S. Pat. No. 7,261,890.

[0125] The above approach involves the use of a target species that is engineered to accept CDRs from a donor species. Essentially the entire framework region from one or more antibody variable heavy or light chains is utilized. This approach is also utilized to felineize antibodies in the same manner as caninization, making them less antigenic when administered to cats. In some cases, backmutation of selected residues in the variable regions is used to enhance presentation of the CDRs. Designing antibodies that minimize immunogenic responses in subjects to non-native sequences in the body while simultaneously preserving enough of the antigen-binding region of the antibody to maintain efficacy has proven difficult.

[0126] Another challenge in developing therapeutic antibodies that target proteins is that the epitopes on homologous proteins in different species often differ, as do the potential for cross-reactivity with other proteins. As a result, antibodies need to be generated, tested, and developed for specific targets in the particular species being treated.

[0127] Antibodies target antigens through binding to specific epitopes on the antigen through interactions with the variable region of the antibody molecule. Furthermore, antibodies have the ability to mediate, inhibit, and / or initiate (as with the antagonist anti-NGF antigen-binding proteins of the present invention) various biological activities. The functions of therapeutic antibodies are broad; for example, antibodies can modulate receptor-ligand interactions as agonists or antagonists. Antibody binding can initiate intracellular signaling to stimulate cell proliferation, cytokine production, or apoptosis. Antibodies can deliver agents bound to the Fe domain to specific sites. Antibodies also induce antibody-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and phagocytosis. Some antibodies have been modified to eliminate ADCC, CDC, C1q binding, and phagocytic functions. In one embodiment of the present invention, the antibodies of the present invention contain alterations in the Fc region of the antibody that alter the effector function of the antibody.

[0128] Dogification and catification As used herein, "caninized antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a dog and / or produced using any of the techniques known in the art or disclosed herein. The same process should be adopted for the felineization process and applied to the description herein. For simplicity, caninization will be used primarily as an example, but these examples are not limited to dogs. The same concept and design apply to the speciation of other antigen-binding proteins, such as cats, horses, and humans. This definition of a caninized antibody includes antibodies comprising at least one canine heavy chain polypeptide or at least one canine light chain polypeptide. Antibody "speciation" itself, particularly antibody humanization, is a field of research well known to those skilled in the art. Until recently, it was unknown whether antibody speciation other than humanization would result in therapeutic antibodies that could be effective in any other species. The present invention relates to the development of anti-N antibodies for therapeutic use in dogs and cats, respectively. 1 illustrates caninization and felinization of a GF antigen binding protein.

[0129] Chimeric antibodies contain sequences from at least two different species. As an example, recombinant cloning techniques may be used to include a variable region containing the antigen-binding site from a non-recipient antibody (i.e., an antibody prepared in a donor species immunized with the antigen) and a constant region derived from a recipient immunoglobulin.

[0130] Speciation (caninized, feline, etc.) antibodies are a type of chimeric antibody in which the variable region residues involved in antigen binding (i.e., complementarity-determining regions, truncated complementarity-determining region residues, or any other residues participating in antigen binding) are derived from a non-canine (or non-feline) species, while the remaining variable region residues (i.e., framework region residues) and constant region residues are at least partially derived from canine (or feline) antibody sequences. A subset of the framework and constant region residues of a speciation antibody may be derived from a non-canine (or feline) source. The variable region of a speciation antibody is also described as speciation (i.e., speciation light or heavy chain variable region). The non-speciation species is typically the species used for immunization with the antigen, such as mouse, rat, rabbit, non-human primate, or other non-canine or non-feline mammalian species.

[0131] Complementarity-determining regions (CDRs) are residues of antibody variable regions that participate in antigen binding. Several numbering systems for identifying CDRs are commonly used. The Kabat definition is based on sequence variability, while the Clothia definition is based on the location of structural loop regions. The AbM definition is a compromise between the Kabat and Clothia approaches. Speciation antibodies of the present invention may be constructed to contain one or more CDRs. Furthermore, CDRs may be used separately or in combination in synthetic molecules such as SMIPs and small antibody mimetics.

[0132] Framework residues are residues of antibody variable regions other than hypervariable or CDR residues. Framework residues may be derived from naturally occurring canine (this is an example and is conceptually applicable to other species, such as cats, horses, and humans. For simplicity, canine is used as a representative species, but the example is not limited to canine) antibodies, for example, canine frameworks substantially similar to the framework regions of the antibodies of the present invention. Artificial framework sequences that represent a consensus among individual sequences may also be used. When selecting framework regions for caninization, sequences that are more commonly represented in canine may be preferred over sequences that are less commonly represented. Additional mutations of the canine framework acceptor sequence may be made to restore murine residues thought to be involved in antigen contact and / or residues involved in the structural integrity of the antigen-binding site, or to improve antibody expression.

[0133] CDR grafting is performed by replacing one or more CDRs of an acceptor antibody (e.g., a caninized or other antibody comprising the desired framework residues) with the CDRs of a donor antibody (e.g., a non-canine antibody). The acceptor antibody may be selected based on the similarity of framework residues between the candidate acceptor antibody and the donor antibody. For example, canine framework regions are identified as having substantial sequence homology to each framework region of a related non-canine antibody, and the CDRs of the non-canine antibody are grafted onto a composite of the different canine framework regions.

[0134] Analysis of the three-dimensional structure of the antibody-antigen complex, combined with analysis of available amino acid sequence data, may be used to model sequence variability based on the structural differences in amino acid residues that occur at each position within the CDRs. The CDRs of the present invention may also be utilized in small antibody mimetics comprising two CDR regions and framework regions (Qui et al., 2004). al.Nature Biotechnology Vol 25;921-929; August 2007).

[0135] The acceptor framework for grafting CDRs or truncated CDRs can be further modified to introduce desired residues. For example, the acceptor framework can include a heavy chain variable region of a canine consensus sequence, optionally with non-canine donor residues at one or more positions. After grafting, additional changes can be made in the donor and / or acceptor sequences to optimize antibody binding and functionality. See, for example, WO 91 / 09967.

[0136] The present invention further provides cells and cell lines that express the antibodies of the present invention. Representative host cells include bacteria, yeast, mammalian and human cells, such as CHO cells, HEK-293 cells, HeLa cells, CV-1 cells, and COS cells. Methods for generating stable cell lines after transformation of heterologous constructs into host cells are known in the art. Representative non-mammalian host cells include insect cells (Potter et al., 2004). al. (1993) Int. Rev. Immunol. 10(2-3):103-112). Antibodies may also be produced in transgenic animals (Houdebine (2002) Curr. Opin. Biotechnol. 13(6):625-629) and transgenic plants (Schillberg et al. (2003) Cell Mol. Life Sci. 60(3):433-45).

[0137] As discussed above, monoclonal, chimeric, species-specific, and speciation-specific antibodies that have been modified, for example, by deleting, adding, or substituting other portions of the antibody, such as the constant region, are also within the scope of the present invention. For example, antibodies can be modified as follows: (i) by deleting the constant region; (ii) by replacing the constant region with another constant region, for example, a constant region intended to increase the half-life, stability, or affinity of the antibody, or a constant region from another species or antibody class; or (iii) by modifying one or more amino acids in the constant region to, for example, change the number of glycosylation sites, effector cell function, Fc receptor (FcR) binding, complement fixation, among others. In one embodiment of the present invention, the antibody of the present invention comprises an altered Fc region that alters the effector function of the antibody. In some embodiments of the present invention, the Fc region of the antigen-binding protein of the present invention is substituted, modified, or removed.

[0138] Methods for altering antibody constant regions are known in the art. Antibodies with altered function, for example, altered affinity for effector ligands such as FcR on cells or the C1 component of complement, can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP 388,151 A1, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260; the contents of all of which are incorporated herein by reference).

[0139] For example, it is possible to alter the affinity of the Fc region of an antibody for FcR (e.g., Fc.gamma.R1) or C1q binding by substituting a specified residue with a residue having appropriate functionality on its side chain, or by introducing a charged functional group (e.g., glutamate or aspartate), or perhaps an aromatic non-polar residue (e.g., phenylalanine, tyrosine, tryptophan, or alanine) (see, e.g., U.S. Pat. No. 5,624,821). An antibody or binding fragment thereof may be conjugated with a cytotoxin, a therapeutic agent, or a radioactive metal ion. In one embodiment, the conjugated protein is an antibody or a fragment thereof. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Non-limiting examples include calicheamicin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, Therapeutic agents include etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof. Therapeutic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil), and the like. Examples of suitable anti-inflammatory drugs include, but are not limited to, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP), cisplatin, anthracyclines (e.g., daunorubicin and doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mithramycin, and anthramycin), and antimitotic agents (e.g., vincristine and vinblastine). Techniques for conjugating such moieties to proteins are well known in the art.

[0140] Compositions, derived compositions, and methods for making compositions The present invention encompasses compositions, including pharmaceutical compositions, comprising antigen binding proteins (such as "antibodies," "antibody fragments," and "antagonist antibodies," as used interchangeably herein), polypeptides, and polynucleotides comprising sequences encoding the antigen binding proteins or polypeptides of the invention.

[0141] As used herein, a composition comprises one or more antibodies, antigen-binding proteins, or polypeptides (which may or may not be antibodies) that bind to NGF, and / or one or more polynucleotides comprising sequences encoding one or more antibodies or polypeptides that bind to NGF. These compositions may further comprise a suitable excipient, such as a pharmaceutically / veterinarily acceptable excipient including a buffer, which excipients are well known in the art. The present invention also encompasses isolated antibody, polypeptide, and polynucleotide embodiments. The present invention also encompasses substantially pure antibody, polypeptide, and polynucleotide embodiments.

[0142] In one or more embodiments, the present invention provides novel antigen-binding proteins that specifically bind to NGF. In one or more embodiments, the antigen-binding proteins are defined as antibodies or antibody fragments. In one or more embodiments, the antigen-binding proteins are fully canine, fully feline, feline-bovine, fully equine, fully human, caninized, feline, equine, or humanized. In one or more embodiments, the antigen-binding proteins of the present invention bind to canine, feline, equine, or human NGF. In one embodiment, the antigen-binding protein is a monoclonal antibody. In one embodiment, the monoclonal antibody of the present invention binds to NGF and prevents its binding to and activation of the receptor Trk A and, to a lesser extent, p75, thereby preventing the signaling cascade described herein. The antigen-binding protein of the present invention is identified herein as ZTS-841.

[0143] In one or more embodiments, the present invention provides an isolated recombinant antigen binding protein, "ZTS-841," wherein the variable heavy chain has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence comprising SEQ ID NO: 5 ("ZTS-841" VH CDR1). and the variable light chain comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence comprising SEQ ID NO: 6 ("ZTS-841" VH CDR3); and the variable light chain comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence comprising SEQ ID NO: 1 ("ZTS-841" VH CDR3). an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence comprising SEQ ID NO:2 ("ZTS-841" VL CDR2), and an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence comprising SEQ ID NO:3 ("ZTS-841" VL CDR3). and any variant thereof having one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 in either the variable light chain or variable heavy chain of said antigen binding protein.

[0144] In one or more embodiments, the present invention provides an isolated recombinant antigen binding protein "ZTS-842," wherein the variable heavy chain has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence comprising SEQ ID NO: 24 ("ZTS-842" VH CDR1), an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence comprising SEQ ID NO: 25 ("ZTS-842" VH CDR2), an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence comprising SEQ ID NO: 26 ("ZTS-842" VH CDR2), and the variable light chain comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence comprising SEQ ID NO:211 ("ZTS-842" VL CDR1), SEQ ID NO:22 ("ZTS-842" VL CDR3). and an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence comprising SEQ ID NO: 23 ("ZTS-842" VL CDR3); and any variant thereof having one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 in either the variable light chain or variable heavy chain of the antigen binding protein.

[0145] The present invention provides recombinant antigen-binding proteins, in some embodiments described herein, monoclonal antibodies, and antibody fragments, and their use in scientific procedures, including clinical administration and diagnostic procedures. Using molecular biology methods and recombinant technology, it is possible to produce antibodies and antibody-like molecules by recombinant means, thereby generating gene sequences that encode specific amino acid sequences found in the polypeptide structure of an antibody. Such antibodies can be produced by cloning the gene sequences encoding the antibody polypeptide chains, or by directly synthesizing the polypeptide chains and synthesizing the synthesized chains. These antibodies can be produced by assembling them to form active tetrameric (H2L2) structures with affinity for specific epitopes and antigenic determinants, allowing the rapid production of antibodies with sequences characteristic of neutralizing antibodies from different species and sources.

[0146] Regardless of the source of the antibody, how it is recombinantly constructed or synthesized, whether in vitro or in vivo, using transgenic animals, large laboratory cell cultures or commercial sizes, using transgenic plants, or by direct chemical synthesis without the use of living organisms at any stage of the process, all antibodies have a similar overall three-dimensional structure. This structure is often given as H2L2, which refers to the fact that antibodies generally contain two light (L) amino acid chains and two heavy (H) amino acid chains. Both chains have regions that can interact with structurally complementary antigen targets. The target-interacting regions are referred to as "variable" or "V" regions and are characterized by differences in amino acid sequence between antibodies of different antigen specificities. The variable regions of the H or L chains contain amino acid sequences that can specifically bind to antigen targets.

[0147] As used herein, the term "antigen-binding region" refers to the portion of an antibody molecule containing amino acid residues that interact with an antigen to confer its specificity and affinity for the antigen to the antibody. The antibody-binding region includes "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Within the variable regions of the heavy or light chains that provide the antigen-binding region, there are smaller sequences called "hypervariable" due to the extreme variability between antibodies of different specificities. Such hypervariable regions are also referred to as "complementarity-determining regions" or "CDR" regions. These CDR regions account for the basic specificity of antibodies for specific antigenic determinant structures.

[0148] Although CDRs represent non-contiguous stretches of amino acids within the variable region, the positional locations of these essential amino acid sequences within the variable heavy and light chain regions are found to have similar positions within the amino acid sequences of the variable chains, regardless of species. The variable heavy and light chains of all antibodies each have three CDR regions, each of which is non-contiguous with the others. In all mammalian species, antibody peptides contain constant (i.e., highly conserved) regions and variable regions, within the latter of which there are CDRs and so-called "framework regions" made up of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.

[0149] The present invention further provides a vector containing at least one of the above-described nucleic acids. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which can encode an amino acid. The actual probability that a particular oligonucleotide constitutes an actual coding sequence can be estimated by considering unusual base-pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic or prokaryotic cells expressing an anti-NGF antibody or portion. Such "codon usage rules" are disclosed by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). Using Lathe's "codon usage rules," a single nucleotide sequence, or a set of nucleotide sequences, containing the theoretically "most probable" nucleotide sequence capable of encoding an anti-NGF sequence can be identified. It is also contemplated that antibody coding regions for use in the present invention can also be provided by altering existing antibody genes using standard molecular biology techniques, resulting in the antibody and peptide variants (agonists) described herein. Such variants include, but are not limited to, deletions, additions and substitutions in the amino acid sequence of the anti-NGF antibody or peptide.

[0150] For example, one type of substitution is conservative amino acid substitution.This type of substitution is to replace a given amino acid in anti-NGF antibody peptide with another amino acid of similar characteristics.Conservative substitutions typically include the substitution between aliphatic amino acids Ala, Val, Leu and Lie; the exchange of hydroxyl residues Ser and Thr, the exchange of acidic residues Asp and Glu, the exchange between amide residues Asn and Gin, the exchange of basic residues Lys and Arg, and the exchange between aromatic residues Phe, Tyr, etc. Guidance on which amino acid changes are likely to be phenotypically silent can be found in Bowie et al., 247 Science 1306-10 (1990).

[0151] A variant anti-NGF antigen-binding protein or antibody fragment may be fully functional or may lack function in one or more activities. Fully functional variants typically contain only conservative variations or variations in non-essential residues or non-essential regions. Functional variants may also contain substitutions of similar amino acids that do not result in a change in function or result in an insignificant change in function. Alternatively, such substitutions may have a positive or negative effect on function to some extent. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or substitutions, insertions, inversions, or deletions in essential residues or essential regions.

[0152] Amino acids essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as epitope binding or in vitro ADCC activity. Sites essential for ligand-receptor binding can also be determined by structural analysis, such as crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224 J. Mol. Biol. 899-904 (1992); de Vos et al., 255 Science 306-12 (1992).

[0153] Moreover, polypeptides often contain amino acids other than the 20 "naturally occurring" amino acids. Moreover, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques which are well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those skilled in the art and have been described in great detail in the scientific literature. Some particularly common modifications, such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, are described in Proteins - Structure and Molecular Properties (2nd ed., TE Creighton, W.H. Freeman & Co., NY, 1993). ld, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Many detailed reviews are available on this subject, such as (1990) Seifter et al. Meth. Enzymol. 626-46, (1990) and Rattan et al. Ann. NY Acad. Sci. 48-62, (1992).

[0154] Therefore, the antibodies and peptides of the present invention also encompass derivatives or analogs in which the substituted amino acid residues are not encoded by the genetic code. Similarly, the additions and substitutions in the amino acid sequence described immediately above, as well as variations and modifications, may be equally applicable to the amino acid sequence of the NGF antigen and / or its epitopes or peptides, and are therefore encompassed by the present invention. As mentioned above, the genes encoding the monoclonal antibodies of the present invention are particularly effective in recognizing NGF.

[0155] antibody derivative Antibody derivatives are included within the scope of the present invention. A "derivative" of an antibody contains additional chemical moieties not normally part of the protein. Covalent modifications of proteins are included within the scope of the present invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents, well known in the art, is useful for crosslinking antibodies or fragments to a water-insoluble support matrix or other polymeric carrier.

[0156] Derivatives also include, for example, radioactive iodine (251, 1311), carbon (4C), sulfur (35S), indium, or tritium (H 3); conjugates of monoclonal antibodies with enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose 6-phosphate dehydrogenase), with biotin or avidin; and conjugates of monoclonal antibodies with bioluminescent agents (e.g., luciferase), chemoluminescent agents (e.g., acridine esters), or fluorescent agents (e.g., phycobiliproteins).

[0157] Another derivative bifunctional antibody of the present invention is a bispecific antibody generated by combining portions of two separate antibodies that recognize two different antigenic groups. This may be achieved by crosslinking or recombinant techniques. Additionally, moieties may be added to the antibody or portion thereof to increase its half-life in vivo (e.g., by increasing the time until clearance from the bloodstream). Such techniques include, for example, the addition of PEG moieties (also called pegylation), and are well known in the art. See U.S. Patent Application Publication No. 20030031671.

[0158] Recombinant expression of antibodies In some embodiments, nucleic acids encoding a subject monoclonal antibody are directly introduced into host cells, and the cells are incubated under conditions sufficient to induce expression of the encoded antibody. After the subject nucleic acids are introduced into the cells, the cells are typically incubated, usually at 37°C, optionally under selection, for a period of about 1 to 24 hours to allow expression of the antibody. In one embodiment, the antibody is expressed on the medium in which the cells are grown. The antibodies are secreted into the supernatant. Traditionally, monoclonal antibodies have been produced as natural molecules in murine hybridoma lines. In addition to that technology, the present invention provides recombinant DNA expression of monoclonal antibodies. This allows for the production of not only caninized antibodies, but also a wide range of antibody derivatives and fusion proteins in a selected host species.

[0159] Nucleic acid sequences encoding at least one anti-NGF antibody, portion, or polypeptide of the present invention can be recombined with vector DNA according to conventional techniques, including blunting or cohesive ends for ligation, restriction enzyme digestion to provide suitable ends, filling in any sticky ends, alkaline phosphatase treatment to avoid undesired ligation, and ligation with an appropriate ligase. Techniques for such manipulation are disclosed, for example, by Maniatis et al., MOLECULAR CLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al., 1993, supra, can be used to construct nucleic acid sequences encoding monoclonal antibody molecules or antigen-binding regions thereof.

[0160] A nucleic acid molecule such as DNA contains a nucleotide sequence containing transcriptional and translational regulatory information, and is said to be "capable of expressing" a polypeptide when such a sequence is "operably linked" to a nucleotide sequence encoding the polypeptide.Operable linkage is a linkage in which the regulatory DNA sequence and the DNA sequence to be expressed are connected in such a way that it allows gene expression as an anti-NGF peptide or antibody moiety in recoverable amounts.The exact nature of the regulatory region required for gene expression may vary from organism to organism, as is well known in the art.For example, see Sambrook et al., supra. al.; see Ausubel et al., 1993, supra.

[0161] The present invention therefore encompasses the expression of anti-NGF antibodies or peptides in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including in vivo or in situ bacterial, yeast, insect, fungal, avian, and mammalian cells, or host cells of mammalian, insect, avian, or yeast origin. Mammalian cells or tissues may be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, although any other mammalian cells may be used.

[0162] In one embodiment, the nucleotide sequence of the present invention is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a variety of vectors may be used for this purpose. See, for example, Ausubel et al., 1993, supra. Important factors in selecting a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells that do not contain the vector; the copy number of the vector desired in the particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0163] Exemplary prokaryotic vectors known in the art include plasmids such as those capable of replicating in E. coli (e.g., but not limited to, pBR322, ColE1, pSC101, and pACYC184). Such plasmids are disclosed, for example, by Maniatis et al., 1989, supra, and Ausubel et al., 1993, supra. Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are also disclosed by Gryczan in THE MOLEC. BIO. OF THE BACILLI 307-329 (Academic Press, NY, 1988). 2). Suitable Streptomyces plasmids include plJ101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987)) and phLC31 (Chater et al., 1987). Streptomyces bacteriophages such as John et al., SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido, Budapest, Hungary 1986) Pseudomonas plasmids are reviewed in John et al., 8 Rev. Infect. Dis. 693-704 (1986), Izaki, 33 Jpn. J. Bacteriol. 729-42 (1978), and Ausubel et al., supra, 1993.

[0164] Alternatively, gene expression elements useful for expressing cDNA encoding an anti-NGF antibody or peptide include (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), the Rous sarcoma virus long terminal repeat (Gorman et al., 79 Proc. Natl. Acad. Sci., USA 6777 (1982)), and the Moloney murine leukemia virus long terminal repeat (Grosschedl et al., 41 Cell 885 (1985); (b) splice regions and polyadenylation sites, e.g., those derived from the SV40 late region (Okayarea et al. (c) polyadenylation sites, such as those in SV40 (Okayama et al., 1983), and (d) polyadenylation sites, such as those in SV40 (Okayama et al., 1983).

[0165] Immunoglobulin cDNA genes may be expressed using the SV40 early promoter and enhancer, mouse immunoglobulin heavy chain promoter-enhancer, SV40 late region mRNA splicing, rabbit S-globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation element as expression elements, as described by Weidle et al., 51 Gene 21 (1987). For immunoglobulin genes containing partial cDNA and partial genomic DNA (Whittle et al., 1 Protein Engin. 499 (1987)), the transcription promoter can be human cytomegalovirus, the promoter-enhancer can be cytomegalovirus and mouse / human immunoglobulin, and the mRNA splicing and polyadenylation regions can be native chromosomal immunoglobulin sequences.

[0166] In one embodiment, for expression of cDNA genes in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancer is either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, the splice region contains an intron greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the immunoglobulin chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the above expression elements to achieve protein expression in mammalian cells.

[0167] Each fusion gene can be assembled into or inserted into an expression vector. Recipient cells capable of expressing the chimeric immunoglobulin chain gene products are then transfected with the anti-NGF peptide or chimeric H or L chain encoding genes alone, or co-transfected with the chimeric H and L chain genes. The transfected recipient cells are cultured under conditions that allow expression of the integrated genes, and the expressed immunoglobulin chains or intact antibodies or fragments are recovered from the culture.

[0168] In one embodiment, fusion genes encoding anti-NGF peptides or chimeric H and L chains, or portions thereof, are assembled into separate expression vectors, which are then used to co-transfect recipient cells. Alternatively, fusion genes encoding chimeric H and L chains can be assembled into the same expression vector. For transfection of the expression vector and production of chimeric antibodies, the recipient cell line may be myeloma cells. Myeloma cells are capable of synthesizing, assembling, and secreting immunoglobulins encoded by the transfected immunoglobulin genes and possess the machinery for immunoglobulin glycosylation. Myeloma cells can be grown in culture or in the peritoneal cavity of mice; in the latter case, secreted immunoglobulins can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells, such as B lymphocytes of human or non-human origin, hybridoma cells of human or non-human origin, or interspecies heterohybridoma cells.

[0169] Expression vectors carrying the chimeric, caninized antibody constructs or anti-NGF polypeptides of the invention can be introduced into suitable host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and particle bombardment. Johnston et al., 240 Science 1538 (1988).

[0170] Yeast may offer substantial advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeast carries out post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies currently exist that utilize strong promoter sequences and high-copy-number plasmids that can be used for the production of desired proteins in yeast. Yeast recognizes leader sequences in cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., prepeptides). Hitzman et al., 11th International Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0171] Yeast gene expression systems can be routinely evaluated for the levels of production, secretion, and stability of anti-NGF peptides, antibodies, and assembled mouse, chimeric, heterochimeric, and caninized antibodies, fragments, and regions thereof. Any of a range of yeast gene expression systems incorporating promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium can be utilized. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Several approaches can be taken to evaluate the optimal expression plasmid for expression of cloned immunoglobulin cDNA in yeast. See Vol. II DNA Cloning, pp. 45-66, (Glover, ed.) IRL Press, Oxford, UK 1985).

[0172] Bacterial strains can also be utilized as hosts for producing the antibody molecules or peptides described by this invention. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these bacterial hosts. The vector carries not only a replication site but also specific genes that are capable of providing phenotypic selection in transformed cells. A number of approaches can be taken to evaluate expression plasmids for the production in bacteria of murine, chimeric, heterochimeric, caninized antibodies, fragments and regions, or antibody chains encoded by cloned immunoglobulin cDNAs (Glover, 1985, supra; Ausubel, 1993, supra; Sambr, 1994, supra). ook,2001、Colligan et al.,eds.Current Protocols in Immunology,John Wiley & Sons,NY,NY(1994-2001); Sons,NY,NY(1997-2001)

[0173] Host mammalian cells may be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including leader peptide removal, folding and assembly of heavy and light chains, glycosylation of antibody molecules, and secretion of functional antibody proteins. In addition to the lymphoid cells described above, mammalian cells that may be useful as hosts for antibody protein production include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Many vector systems are available for the expression of cloned anti-NGF peptide heavy and light chain genes in mammalian cells (see Glover, 1985, supra). Complete H2L2 antibodies can be obtained according to different approaches. Heavy and light chains can be coexpressed in the same cell to achieve intracellular assembly and linkage of heavy and light chains into complete tetrameric H2L2 antibodies and / or anti-NGF peptides. Coexpression can be achieved by using the same or different plasmids in the same host. Genes for both H and L chains and / or anti-NGF peptides can be placed on the same plasmid, which is then transfected into cells, thereby directly selecting cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, such as the L chain, and then the resulting cell line can be transfected with an H chain plasmid containing a second selectable marker. Cell lines producing anti-NGF peptides and / or H2L2 molecules via either route can be transfected with plasmids encoding additional copies of the peptide, H, L, or H+L chain in combination with additional selectable markers to generate cell lines with improved properties, such as increased production of assembled H2L2 antibody molecules or increased stability of the transfected cell line.

[0174] Stable expression may be used for long-term, high-yield production of recombinant antibodies. For example, cell lines that stably express antibody molecules may be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with an immunoglobulin expression cassette and a selectable marker. After introduction of the foreign DNA, engineered cells may be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and expanded into cell lines. Such engineered cell lines may be particularly useful in screening and evaluating compounds / components that interact directly or indirectly with antibody molecules.

[0175] Once an antibody of the invention is produced, it may be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen named after Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. In many embodiments, the antibody is secreted from the cells into the culture medium and recovered from the culture medium.

[0176] Pharmaceutical and veterinary applications The anti-NGF antigen binding proteins or antibody fragments as described herein of the present invention can be used for the treatment of NGF-related disorders in, for example, dogs and cats. More specifically, the present invention further provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or diluent and, as an active ingredient, an antibody or antibody fragment according to the present invention. The antibody can be chimeric, heterochimeric, caninized, felineized, equineized, humanized, or speciated to suit different species. Intact immunoglobulins or binding fragments thereof, such as Fabs, are also contemplated. The antibodies and pharmaceutical compositions thereof of the present invention are useful for parenteral administration, e.g., subcutaneous, intramuscular, or intravenous administration.

[0177] The anti-NGF antibodies and / or peptides of the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected based on the chosen route of administration and standard pharmaceutical practice. Administration of the antibodies disclosed herein can be carried out by any suitable means, including parenteral injection (e.g., intraperitoneal, subcutaneous, or intramuscular injection), orally, or by topical administration of the antibody (typically contained in a pharmaceutical formulation) to the respiratory tract surface. Topical administration to the respiratory tract surface can be carried out by intranasal administration (e.g., using a dropper, swab, or inhaler). Topical administration of the antibody to the respiratory tract surface can also be carried out by inhalation, for example, by producing respirable particles (including both solid and liquid particles) of a pharmaceutical formulation containing the antibody as an aerosol suspension and then having the subject inhale the respirable particles. Methods and devices for administering respirable particles of a pharmaceutical formulation are well known, and any conventional techniques can be used.

[0178] In some desirable embodiments, the antibody is administered by parenteral injection. For parenteral administration, the anti-NGF antibody or peptide can be formulated with a pharmaceutically acceptable parenteral vehicle as a solution, suspension, emulsion, or lyophilized powder. For example, the vehicle can be a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, such as an aqueous carrier, such as water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, and 0.3% glycine. Non-aqueous vehicles, such as liposomes and fixed oils, can also be used. These solutions are sterile and generally free of particulate matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances, such as pH adjusters, buffers, and toxicity adjusters, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, as needed to approximate physiological conditions. The concentration of antibody in these formulations can vary widely, e.g., from less than about 0.5%, usually about 1% or at least about 1%, up to as much as 15% or 20% by weight, and is selected primarily based on liquid volume, viscosity, etc., according to the particular mode of administration selected. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques. Actual methods for preparing parenterally administrable compositions are known or apparent to those skilled in the art and are described in more detail, for example, in REMINGTON'S PHARMA. SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980).

[0179] The antibodies of the present invention can be lyophilized for storage and reconstitution in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins. Any suitable lyophilization and reconstitution technique can be used. Those skilled in the art will appreciate that lyophilization and reconstitution may lead to varying degrees of antibody activity loss and that adjustment of usage levels may be required to compensate. Compositions containing the antibodies of the present invention or a cocktail thereof can be administered for the prevention of recurrence and / or therapeutic treatment of existing disease. Suitable pharmaceutical carriers can be prepared according to standard reference methods in the art. The most recent edition of REMINGTON'S PHARMACEUTICAL SCIENCES is a reference reference. In therapeutic applications, compositions are administered to a subject already suffering from a disease in an amount sufficient to cure or at least partially arrest or alleviate the disease and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective dose" or "therapeutically effective amount." Amounts effective for this use will depend on the severity of the disease and the general state of the subject's own immune system, but generally will be in the range of about 0.1 mg antibody / kg body weight to about 10 mg antibody / kg body weight, preferably about 0.3 mg antibody / kg body weight to about 5 mg antibody / kg body weight. In view of the minimized foreign material and lower probability of "foreign material" rejection achieved with the canine-like antibodies of the present invention, it may be possible to administer substantial excesses of these antibodies.

[0180] The dosage administered will, of course, vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health, and weight of the recipient; the nature and extent of the condition, type of concurrent treatment, frequency of treatment, and the desired effect.

[0181] As a non-limiting example, treatment of NGF-related pathologies in dogs and cats can be provided by biweekly or monthly administration of an anti-NGF antibody of the present invention within a dosage range as needed. Exemplary antibodies for therapeutic use in dogs are high-affinity (which may also be high-avidity) antibodies with potent in vivo anti-NGF activity according to the present invention, as well as fragments, regions, and derivatives thereof. Single or multiple administrations of the composition can be carried out, with dose levels and patterns selected by the treating veterinarian. In either case, the pharmaceutical formulation should provide a sufficient quantity of the antibody of the present invention to effectively treat the subject.

[0182] Diagnostic applications The present invention also provides the above-described anti-NGF antibodies and peptides for use in diagnostic methods for detecting NGF in species known to or suspected of having an NGF-related disorder, particularly dogs and cats. In an embodiment of the present invention, the NGF-related disorder is pain. In another embodiment, the NGF-related disorder is osteoarthritis. The anti-NGF antibodies and / or peptides of the present invention are useful for immunoassays to detect or quantify NGF or anti-NGF antibodies in a sample. An immunoassay for NGF typically involves incubating a clinical or biological sample in the presence of a detectably labeled, high-affinity (or high-avidity) anti-NGF antibody or polypeptide of the present invention capable of selectively binding to NGF, and detecting the bound, labeled peptide or antibody in the sample. Various clinical assay procedures are well known in the art. See, for example, Immunoassays for the 80's (Voller et al., eds., Univ. Park, 1981). Such samples include tissue biopsies, blood, serum, and fecal samples, or liquids collected from animal subjects and subjected to ELISA analysis as described below.Therefore, anti-NGF antibody or polypeptide can be fixed to nitrocellulose or another solid support that can fix cells, cell particles, or soluble proteins.The support can then be washed with a suitable buffer, and then treated with detectably labeled NGF-specific peptide, antibody, or antigen-binding protein.The solid support can then be washed a second time with buffer to remove unbound peptide or antibody.The amount of label bound to the solid support can then be detected by known method steps.

[0183] A "solid phase support" or "carrier" refers to any support capable of binding a peptide, antigen, or antibody. Well-known supports or carriers include glass, polystyrene, poly Examples of suitable carriers include propylene, polyethylene, polyvinylidene fluoride (PVDF), dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be soluble or insoluble to some extent for purposes of the present invention. The support material can have virtually any possible structural configuration, so long as the attached molecule can bind to NGF or anti-NGF antibodies. Thus, the support configuration can be spherical, as in beads, or cylindrical, as in the interior surface of a test tube, or a rod-like external surface. Alternatively, the surface can be flat, such as a sheet, culture dish, test strip, etc. For example, the support may comprise polystyrene beads. Many other suitable carriers for binding antibodies, peptides, or antigens are known to those skilled in the art, or can be ascertained by routine experimentation. The binding activity of a given lot of anti-NGF peptide and / or antibody or antigen-binding protein can be determined by well-known method steps. Those skilled in the art can determine functional and optimal assay conditions by routine experimentation.

[0184] Detectably labeling NGF-specific peptides and / or antibodies can be achieved by linking them to enzymes for use in enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA). The linked enzymes react with exposed substrates to produce chemical moieties that can be detected, for example, by spectrophotometry, fluorimetry, or visual means. Enzymes that can be used to detectably label the NGF-specific antibodies of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Radioactively labeling NGF-specific antibodies allows NGF to be detected through the use of radioimmunoassays (RIA). See Work et al., LAB. TECHNIQUES & BIOCHEM. IN MOLEC. BIO (No. Holland Pub. Co., NY, 1978). Radioactive isotopes can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. Isotopes that are particularly useful for the purposes of the present invention include: 3 H, 125 I, 131 I, 35 S and 14 C is one example.

[0185] It is also possible to label NGF-specific antibodies with fluorescent compounds. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine. NGF-specific antibodies or antigen-binding proteins can also be labeled with fluorescent compounds. 125 Fluorescence-emitting metals such as Eu or others of the lanthanide series can be used to delectably label NGF-specific antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0186] An NGF-specific antibody can also be detectably labeled by linking it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0187] Similarly, bioluminescent compounds can be used to label the NGF-specific antibodies, portions, fragments, polypeptides, or derivatives of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase, and aequorin.

[0188] Detection of NGF-specific antibodies, parts, fragments, polypeptides, or derivatives can be achieved, for example, by a scintillation counter if the detectable label is a radioactive gamma emitter, or by a fluorometer if the label is a fluorescent material. In the case of an enzyme label, detection can be achieved by colorimetric methods using a substrate for the enzyme. Detection can also be achieved by visual comparison of the extent of enzymatic reaction of the substrate compared to a similarly prepared standard.

[0189] For the purposes of the present invention, the NGF detected by the above assay can be present in a biological sample. Any sample containing NGF can be used. For example, the sample can be a biological fluid, such as blood, serum, lymph, urine, feces, inflammatory exudate, cerebrospinal fluid, amniotic fluid, tissue extract or homogenate, etc. The present invention is not limited to assays using these samples, and those skilled in the art can determine suitable conditions that allow the use of other samples in light of this specification.

[0190] In situ detection can be achieved by removing a histological specimen from an animal subject and combining such specimen with a labeled antibody of the present invention. The antibody (or a portion thereof) may be provided by applying or overlaying the labeled antibody (or portion) on a biological sample. Through the use of such procedures, not only the presence of NGF but also the distribution of NGF in the examined tissue can be determined. Using the present invention, those skilled in the art will readily recognize that any of a variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[0191] The antibodies, fragments, or derivatives of the present invention can be adapted for use in immunometric assays, also known as "two-site" or "sandwich" assays. In a typical immunometric assay, a quantity of unlabeled antibody (or antibody fragment) is bound to a solid support that is insoluble in the liquid being tested, and a quantity of detectably labeled soluble antibody is added to allow for detection and / or quantitation of the ternary complex formed between the solid-phase antibody, antigen, and labeled antibody.

[0192] Antibodies may be used to quantitatively or qualitatively detect NGF in a sample or to detect the presence of cells expressing NGF. This can be achieved by immunofluorescence techniques (see below) using fluorescently labeled antibodies coupled with fluorescence microscopy, flow cytometry, or fluorometric detection. For diagnostic purposes, antibodies may be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) reactive with the antibody, such as antibodies specific for canine immunoglobulin constant regions. Alternatively, antibodies can be directly labeled. A variety of labels may be used, including radionuclides, fluoresceins, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (especially haptens). Numerous types of immunoassays, such as those previously discussed, are available and well known to those skilled in the art. Importantly, the antibodies of the present invention may be useful in diagnosing NGF-related disorders in dogs. More specifically, the antibodies of the present invention may identify overexpression of NGF in companion animals. Thus, the antibodies of the present invention may provide an important immunohistochemical tool. The antibody of the present invention , may be used in antibody arrays and are highly suitable for measuring gene expression profiles.

[0193] kit Kits for carrying out the subject methods are also within the scope of the present invention. The kits include at least one or more of the antibody of the present invention, a nucleic acid encoding it, or cells containing it. The antibody of the present invention may be provided in a container, usually in lyophilized form. The antibody, which may or may not be conjugated to a label or toxin, is typically included in the kit along with a buffer such as Tris, phosphate, or carbonate, a stabilizer, a killing agent, or an inert protein such as serum albumin. Generally, these materials will be present in an amount of less than 5% by weight based on the amount of active antibody, and usually will be present in a total amount of at least about 0.001% by weight, again based on the antibody concentration. Frequently, it will be desirable to include an inert bulking agent or excipient to dilute the active ingredient; the excipient may be present in an amount of about 1% to 99% by weight of the total composition. If a second antibody capable of binding to the primary antibody is used in the assay, it is usually present in a separate vial. The second antibody is typically conjugated to a label and formulated in a manner similar to the antibody formulation described above. The kit also typically includes a set of instructions. The present invention includes, but is not limited to, the following aspects. [Aspect 1] 1. A recombinant antigen-binding protein that specifically binds to nerve growth factor (NGF), comprising: ai. Complementarity-determining region 1 (CDR1) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 21; ii. Complementarity determining region 1 (CDR2) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 22; iii. a variable light chain (VL) comprising a complementarity determining region 1 (CDR3) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 23; and bi complementarity-determining region 1 (CDR1) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4 or SEQ ID NO:24; ii. Complementarity determining region 1 (CDR2) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 25; and iii. a variable heavy chain (VH) comprising a complementarity determining region 1 (CDR3) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 26; and and any variant thereof having one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 in either said variable light or variable heavy chain region of said antigen binding protein. 1. A recombinant antigen-binding protein comprising: [Aspect 2] a. the light chain variable region (VL) is i. Complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:1 ii. Complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:2 iii. A complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO: 3; and b. the heavy chain variable region (VH) i. Complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:4 ii. Complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:5 iii. comprises a complementarity-determining region 3 (CDR3) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO:6; and any variant thereof having one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 in either said variable light or variable heavy chain region of said antigen binding protein; 2. An antigen-binding protein according to embodiment 1. [Aspect 3] c. the light chain variable region (VL) is i. Complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO: 21 ii. Complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO: 22 iii. A complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO: 23; and d. The heavy chain variable region (VH) is i. Complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO: 24 ii. Complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO: 25 iii. comprises a complementarity-determining region 3 (CDR3) comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence comprising SEQ ID NO: 26; and any variant thereof having one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 in either said variable light or variable heavy chain region of said antigen binding protein; 2. An antigen-binding protein according to embodiment 1. [Aspect 4] 1. A recombinant antigen-binding protein that specifically binds to nerve growth factor (NGF), comprising: a. a variable light chain comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:55, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:91; and b. a variable heavy chain comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:56, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, and SEQ ID NO:81; and any variant thereof having one or more conservative amino acid substitutions in either the variable light or variable heavy chain region of said antigen binding protein, Recombinant antigen-binding proteins. [Aspect 5] 5. A recombinant antigen binding protein according to aspect 4, wherein said variable light chain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 7 and said variable heavy chain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 8; and any variant thereof comprises one or more conservative amino acid substitutions in either the variable light or variable heavy chain regions of said antigen binding protein. [Aspect 6] 5. A recombinant antigen binding protein according to aspect 4, wherein said variable light chain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 27 and said variable heavy chain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 28; and any variant thereof comprises one or more conservative amino acid substitutions in either the variable light chain or variable heavy chain regions of the antigen binding protein. [Aspect 7] 7. The antigen-binding protein of any one of aspects 1 to 6, wherein said binding protein inhibits binding of NGF to the TrkA receptor. [Aspect 8] 8. The antigen-binding protein of any one of aspects 1 to 7, wherein said binding protein reduces or eliminates an NGF-associated disorder. [Aspect 9] 9. The antigen binding protein of embodiment 8, wherein said NGF-related disorder is selected from the group consisting of cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain and inflammation. [Aspect 10] 10. The antigen binding protein of aspect 9, wherein said NGF-related disorder is pain. [Aspect 11] The NGF-related disorder is a pain disorder, and is selected from the group consisting of osteoarthritis pain, rheumatoid arthritis pain, surgical and post-operative pain, incision pain, systemic inflammatory pain, cancer pain, pain from trauma, neuropathic pain, 11. The antigen-binding protein of embodiment 10, wherein the pain is selected from the group consisting of pain, neurological pain, diabetic neuropathy pain, pain associated with a rheumatic disease, pain associated with a musculoskeletal disease, visceral pain, and gastrointestinal pain. [Aspect 12] 12. The antigen binding protein of aspect 11, wherein said NGF-related disorder comprises osteoarthritis pain. [Aspect 13] 13. The antigen-binding protein of any one of aspects 1 to 12, wherein the binding protein is selected from the group consisting of a monoclonal antibody; a chimeric antibody, a single-chain antibody, a tetrameric antibody, a tetravalent antibody, a multispecific antibody, a domain-specific antibody, a domain-deleted antibody, a fusion protein, an ScFc-fusion protein, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, an ScFv fragment, an Fd fragment, a single domain antibody, a dAb fragment, a small modular immunopharmaceutical (SMIP), a nanobody, and an IgNAR molecule. [Aspect 14] 14. The antigen-binding protein of embodiment 13, which is a monoclonal antibody. [Aspect 15] 15. The antigen-binding protein of embodiment 14, wherein said monoclonal antibody is a canine monoclonal antibody, a caninized monoclonal antibody, a feline monoclonal antibody, a felineized monoclonal antibody, a human monoclonal antibody, a humanized monoclonal antibody, an equine monoclonal antibody or an equine-enhanced monoclonal antibody. [Aspect 16] 16. A pharmaceutical or veterinary composition comprising a therapeutically effective amount of the antigen-binding protein of any one of aspects 1 to 15 and a pharmaceutically acceptable carrier. [Aspect 17] 20. The pharmaceutical or veterinary composition according to embodiment 18 for use in treating pain in a subject. [Aspect 18] 20. The pharmaceutical composition of embodiment 19, wherein the pain is selected from the group consisting of osteoarthritis pain, rheumatoid arthritis pain, surgical and post-operative pain, incisional pain, systemic inflammatory pain, cancer pain, pain from trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain. [Aspect 19] 19. The pharmaceutical composition of embodiment 18, wherein the pain comprises osteoarthritis pain. [Aspect 20] 20. The pharmaceutical composition according to embodiment 18, wherein the pain comprises surgical and post-surgical pain. [Aspect 21] 20. The pharmaceutical composition of embodiment 18, wherein the pain comprises cancer pain. [Aspect 22] 22. A pharmaceutical composition according to any one of aspects 16 to 21 for use in dogs, cats, horses or humans. [Aspect 23] 23. The pharmaceutical composition according to aspect 22, for use in a dog. [Aspect 24] 23. The pharmaceutical composition according to aspect 22, for use in a cat. [Aspect 25] A host cell that produces the antigen-binding protein according to any one of aspects 1 to 15. [Aspect 26] 16. An isolated nucleic acid comprising a nucleic acid sequence encoding the antigen-binding protein of any one of aspects 1 to 15. [Aspect 27] A vector comprising the nucleic acid sequence of embodiment 26. [Aspect 28] A host cell comprising the vector of embodiment 27. [Aspect 29] 27. A host cell comprising nucleic acid according to embodiment 26. [Aspect 30] A method of producing an antigen binding protein according to any one of aspects 1 to 15, said method comprising culturing a host cell according to any one of aspects 26, 29 or 30 under conditions that result in the production of said antigen binding protein, and isolating said antigen binding protein from said host cell or from a culture medium of said host cell. [Aspect 31] 17. A method of treating a subject for an NGF-related disorder, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 16. [Aspect 32] 32. The method of embodiment 31, wherein the NGF-related disorder is selected from the group consisting of cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation. [Aspect 33] 33. The method of embodiment 32, wherein the NGF-associated disorder comprises pain. [Aspect 34] 34. The method of embodiment 33, wherein the NGF-related disorder is a pain disorder and is selected from the group consisting of osteoarthritis pain, rheumatoid arthritis pain, surgical and post-operative pain, incisional pain, systemic inflammatory pain, cancer pain, pain from trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic disease, pain associated with musculoskeletal disease, visceral pain, and gastrointestinal pain. [Aspect 35] 35. The method of embodiment 34, wherein the NGF-associated disorder comprises osteoarthritis pain. [Aspect 36] 35. The method of embodiment 34, wherein said NGF-related disorder comprises surgery and post-operative pain. [Aspect 37] 35. The method of embodiment 34, wherein the NGF-associated disorder comprises cancer pain. [Aspect 38] 20. A method of inhibiting NGF activity in a subject by administering to said subject the pharmaceutical composition of aspect 17. [Aspect 39] Aspect 39. The method of any one of aspects 31 to 38, wherein said subject is selected from the group consisting of a dog, a cat, a human, and a horse. [Aspect 40] 40. The method of embodiment 39, wherein the subject comprises a dog. [Aspect 41] 40. The method of embodiment 39, wherein the subject comprises a cat. [Aspect 42] 40. The method of embodiment 39, wherein the subject comprises a human.

[0194] The invention will now be further described by the following non-limiting examples. [Example]

[0195] The present invention is further illustrated and supported by the following examples. However, these examples should in no way be construed as further limiting the scope of the present invention. On the contrary, those skilled in the art will readily recognize that there are other embodiments, modifications, and equivalents of the present invention without departing from the spirit of the present invention and / or the scope of the appended claims.

[0196] Example 1 Synthesis and purification of canine NGF (cNGF) PCR primers were designed with appropriate restriction sites to amplify canine pre-pro-β-NGF (SEQ ID NO: 59). The β-NGF gene was cloned into the plasmid pCTV927 (Chromos targeting plasmid) via the EcoRV / KpnI sites. The pCTV927 / β-NGF plasmid was cotransfected with the Chromos system integrase-encoding plasmid pSIO343 into CHOK1SV cells using Lipofectamine 2000 transfection reagent. Individual stable clones were analyzed for expression, and high-expressing clones were selected for expansion and subsequent expression for purification. Canine β-NGF (cNGF) produced from these transfections was purified using ion exchange chromatography. Initial cleanup was performed on Q Sepharose FF (GE Healthcare #17-0510-01) in flow-through batch mode. The clarified supernatant was diluted 1:1 with water and the pH was adjusted to 8.5 with 1 M Tris. The diluted sample was mixed with Q Sepharose FF at a 150:1 ratio for >1.5 hours. The resin was allowed to settle, and the unbound portion was collected. cNGF was further purified by cation exchange chromatography; it was again diluted 1:1 with water and loaded onto SP-Sepharose FF (GE Healthcare #17-0729-01) pre-equilibrated with 20 mM Tris, pH 8.5. After loading, the column was washed and then eluted via a linear gradient of 0 to 210 mM NaCl (each in 20 mM Tris, pH 8.5) over 20 column volumes. Fractions were analyzed by SDS-PAGE, pooled, and dialyzed (3.5K mwco) against PBS at 4°C. The dialysate was collected, sterile filtered, and the concentration was measured via absorbance at 280 nm (1 mg / mL = 1.48 A). 280 ).

[0197] Example 2 : Immunization of dogs Dog immunization can be carried out by methods known in the art, and is not limited to any one method.In one example, canine NGF (as described in Example 1) is directly administered to dogs together with adjuvants to stimulate immune response.To obtain optimal anti-antigen response, dogs are given booster injections, and serum samples are collected periodically.The antibody immune response from immunized dogs is monitored and determined using standard antigen direct-binding enzyme-linked immunosorbent assay (ELISA) method, as well known to those skilled in the art and described below.

[0198] Example 3 : Primary antigen binding and B cell activation To assess the titer of canine anti-NGF antibodies, 100 μL of recombinant canine NGF (10 μg / mL) was coated onto Immunolon 2Hb plates overnight at 4°C. Wells were washed three times with PBS-T (PBS + 0.1% Tween) and nonspecific binding was blocked using 200 μL of PBS + 5% nonfat skim milk, which was incubated for 1 hour at room temperature. After washing the plate three times with 300 μL of PBS-T, serial dilutions of canine serum were incubated for 1 hour. Canine anti-NGF IgG binding was detected using 100 μL of a cocktail of 0.2 μg / mL Bethyl anti-canine IgG1 (A40-120P) and anti-canine IgG2 (A40-121P). After addition of chromogenic substrate (SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate, KPL 53-00-01) and incubation for 10 min at RT, the reaction was stopped by adding 100 μL of 0.1 N HCl. The absorbance of each well was determined at an optical density (OD) of 450 nm.

[0199] Protocol for Activating Canine Memory B Cells Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll™ gradient separation by centrifugation. After isolation of PBMCs from the sample, specific selection of antibody-secreting cells was performed based on the expression of specific antibody cell surface markers as known to those skilled in the art and described in U.S. Patent Publication No. 2014 / 0287402 and Callard and Kotowicz, "Cytokine Cell Biology: A practical approach," Oxford University Press, 2000, pp. 17-31 (incorporated herein by reference). Before placing B cells on the sorting chip, the cells were activated in vitro. After isolation and freezing, the cells (PBMCs, approximately 10 7 The cells (per vial) were removed from liquid nitrogen and rapidly thawed in a water bath. The cells were transferred to a 15 ml centrifuge tube and 12 ml of complete medium was added dropwise. After centrifuging the cells at 1000 rpm for 10 minutes, the pellet was resuspended in 10 ml of complete medium and centrifuged again at 1000 rpm for 10 minutes. Finally, the cells were resuspended in 4 ml of medium.

[0200] Example 4 DNA sequences encoding the 9L12 (ZTS-841), 48L2 (ZTS-842), and 13L11 antibodies Single cells of interest were removed from the microarray by micromanipulation and placed in a microtube containing lysis buffer and magnetic beads for mRNA capture. cDNA was prepared from total RNA using a mix of gene-specific primers hybridizing to the early constant domains of gamma HC, kappa LC, and lambda LC. Terminal deoxynucleotidyl transferase (TdT) enzyme was used to tail the 3' end of the first-strand cDNA product. A mix of gene-specific reverse primers and universal forward primers was then used for the first PCR. Nested PCR was then performed separately for each VH and VL chain to amplify the antibody variable regions. The reverse primer used for PCR amplification, along with the universal forward primer, is located in the HC or LC constant domain. The amplified fragments from PCR were separated by gel electrophoresis on an agarose gel. Full-length VH and VL amplicons isolated from single cells were cloned into expression vectors containing the corresponding HC or LC constant domains. The canine variable domain sequences were as follows: 9L12(841) variable light chain (SEQ ID NO: 7), corresponding nucleotide sequence (SEQ ID NO: 17); 9L12(841) variable heavy chain (SEQ ID NO: 8), corresponding nucleotide sequence (SEQ ID NO: 18); 48L2(842) variable light chain (SEQ ID NO: 27), corresponding nucleotide sequence (SEQ ID NO: 36); 48L2(842) variable heavy chain (SEQ ID NO: 28), corresponding nucleotide sequence (SEQ ID NO: 38); 13L11 variable light chain (SEQ ID NO: 51), corresponding nucleotide sequence (SEQ ID NO: 53); 13L11 variable heavy chain (SEQ ID NO: 52), corresponding nucleotide sequence (SEQ ID NO: 54).

[0201] The constant regions of the isolated antibodies isolated as described above were not used in the subsequent construction of the antibodies of the present invention. The Fc regions of the recombinant antibodies of the present invention comprise a modified canine IgGB (Bergeron et al., Vet Immunol Immunopathol 2014 Jan 15:157(1-2):31-41) and was selected for its half-life, biophysical properties, and lack of effector function. Bergeron et al. As reported in

[1999] et al., canine IgGB has good affinity for canine FcRn and favorable biophysical properties for downstream processing. Differential scanning calorimetry (DSC) performed on the canine Fc region alone indicated that the thermal stability of the constant region was approximately 70°C and 83°C. These melting temperatures are similar to or higher than those reported for commercially available humanized mAbs.

[0202] Three point mutations were made in the CH2 domain of canine IgGB to eliminate ADCC and CDC activity. The mutant Fc is referred to herein as IgGB(e-) (SEQ ID NO: 43). Although NGF is a soluble target, effector function was removed from the anti-NGF antibody to protect against any potential nonspecific target or effector function-related adverse effects. These mutations did not appear to affect the immunogenicity of this mAb. Additionally, mutations in the Fc region to remove effector function did not affect FcRn or protein A binding. Not only was reduced binding to canine FcγRI and FcγRIII observed, but ADCC activity was also reduced. C1q protein is the first protein in the complement cascade and is required for cells to undergo complement-dependent cytotoxicity (CDC). IgGB(e-) has been shown to lack binding to C1q protein. The amino acid sequence of canine constant HC-65 as described is represented as SEQ ID NO: 41, and its corresponding nucleotide sequence is represented as SEQ ID NO: 42. The amino acid sequence of canine constant lambda is represented as SEQ ID NO: 60, and its corresponding nucleotide sequence is represented as SEQ ID NO: 61.

[0203] Example 5 : Determination of antigen-binding affinity The antibody binding affinity of the antibody against canine NGF was determined using the Biacore system (Biacore The binding affinity of canine and rat NGF was determined by surface plasmon resonance (SPR) at GE Life Sciences (GE Healthcare, Uppsala, Sweden). Immobilization of canine and rat NGF was achieved by amine coupling of 5 μg / mL NGF using N-hydroxysuccinimide (NHS) / 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry. The chip was quenched with ethanolamine, and the affinity of all candidate mAbs to the immobilized NGF was evaluated. Varying concentrations of canine and feline anti-NGF antibodies were injected over the NGF surface, and the association of the antibody to the antigen and the dissociation of the formed complex were monitored in real time. Kinetic analysis was performed to obtain the equilibrium dissociation constant (KD). The results are shown in Table 1 below.

[0204] [Table 2]

[0205] Example 6 : Construction of 9L12(841) and 48L2(842) chimeric antibodies Antibody variable domains are involved in antigen binding. Antibodies consist of a homodimeric pairing of two heterodimeric proteins. Each protein chain (one heavy and one light chain) of the heterodimer consists of a variable domain and a constant domain. Each variable domain contains three complementarity-determining regions (CDRs) that contribute to antigen binding. The CDRs are separated within the variable domain by framework regions, which provide a scaffold for the proper spatial presentation of the binding site on the antibody. Together, the CDRs and framework regions contribute to the antibody's ability to bind to its cognate antigen. Grafting the entire variable domain onto each constant region is expected to have little or no effect on the antibody's ability to bind to NGF. To simultaneously confirm that the correct sequences of the heavy and light chain variable regions were identified and to produce homogeneous material, expression vectors for producing recombinant chimeric or canine antibodies in a mammalian expression system were designed. The chimeric antibodies described herein consist of variable sequences (both CDRs and frameworks) from a host species antibody grafted onto the respective heavy and light chain constant regions of a canine IgG molecule. The chimeric antibodies described herein consist of variable segments (both CDRs and frameworks) from a canine molecule grafted onto the respective heavy and light chain constant regions of a feline IgG molecule. Because the variable domains are involved in antigen binding, grafting a complete canine variable domain onto a feline constant domain is expected to have little or no effect on the antibody's ability to bind NGF. The chimeric variable domain sequences were as follows: canfel_chimeric 9L12(841) variable light chain (SEQ ID NO: 9), corresponding nucleotide sequence (SEQ ID NO: 19); canfel_chimeric 9L12(841) variable heavy chain (SEQ ID NO: 10), corresponding nucleotide sequence (SEQ ID NO: 21); canfel_chimeric 48L2(842) variable light chain (SEQ ID NO: 29), corresponding nucleotide sequence (SEQ ID NO: 39); canfel_chimeric 48L2(842) heavy chain (SEQ ID NO: 30), corresponding nucleotide sequence (SEQ ID NO: 40). Each variable segment was cloned into a mammalian expression plasmid containing either the feline IgG heavy or light chain.The amino acid sequence of the feline heavy chain constant region is represented as SEQ ID NO: 62, and its corresponding nucleotide sequence is represented as SEQ ID NO: 63. The amino acid sequence of the feline light chain constant is represented as SEQ ID NO: 64. and the corresponding nucleotide sequence is represented as SEQ ID NO:65.

[0206] Example 7: Felinization of 48L2 and 9L12 antibodies The generation of anti-drug antibodies (ADAs) can be associated with a loss of efficacy for any biologic protein, including monoclonal antibodies. A comprehensive review of the literature has shown that speciation of monoclonal antibodies can reduce the tendency of mAbs to be immunogenic. To help mitigate the risk associated with ADA formation for the canine anti-NGF monoclonal antibody provided herein, a felinization strategy was used for the antibody's eventual use in cats. This felinization strategy is based on identifying the most appropriate feline germline antibody sequence to be used for CDR grafting. After extensive analysis of all available feline germline sequences for both the heavy and light chains, germline candidates were selected based on homology to canine mAbs, and CDRs from canine precursor segments were used to replace the native feline CDRs. The felinized mAbs were expressed and characterized for their ability to bind NGF. The goal was to retain high affinity and cell-based activity using a feline antibody framework to minimize the potential for in vivo immunogenicity. Synthetic constructs were generated displaying felinized variable heavy and light chains of mAb 48L2 (ZTS-842) using SEQ ID NOS: 21-26 and 9L12 (ZTS-841) using SEQ ID NOS: 1-6. After subcloning each variable chain into a plasmid containing the feline constant heavy chain (SEQ ID NOS: 62) and feline constant light chain (SEQ ID NOS: 64) regions, the plasmids were cotransfected into HEK293 cells for antibody expression. Chimeric, heterochimeric, and felinized forms of mAb 48L2 and 9L12 were expressed and characterized for their ability to bind NGF via SPR.

[0207] Example 8 : Production of antibodies from glutamine synthetase (GS) plasmids Genes encoding canine and feline 9L12 and 48L2 (ZTS-841 and ZTS-842, respectively), as well as the feline 9L12 heavy and light chains, as described herein, were cloned into GS plasmids pEE 6.4 and pEE 12.4 (Lonza, Basel, Switzerland) according to standard molecular biology techniques well known to those skilled in the art. The resulting individual plasmids were digested and ligated together to form a single mammalian expression plasmid according to the manufacturer's protocol. To demonstrate transient production of each antibody, each plasmid was used to transfect HEK 293 cells, and expression was carried out in cultures of various sizes. Proteins were isolated from conditioned HEK medium using Protein A affinity chromatography according to standard protein purification methods. The medium was loaded onto the chromatography resin and eluted by pH shift. The pH of the eluted protein was adjusted, dialyzed, and sterile filtered before use. Antibodies were tested for affinity and potency.

[0208] To generate stable cell lines producing candidate antibodies, the GS plasmid was linearized and then transfected with the restriction enzyme PvuI, which cuts at a single site in the plasmid backbone. GS-CHOK1SV (clone 144E12) cells were transfected with linearized plasmid DNA via electroporation. After transfection, cells were plated into 48-well plates (48WP) to generate stable pools. When pools were at least 50% confluent in 48WP, 100 μl of supernatant was analyzed for IgG expression using a ForteBio Octet and Protein A biosensor (Pall ForteBio, Fremont, CA). The best-expressing clones were scaled up to 6-well plates (6WP) and then 125 mL shake flasks (SF). Once cells were adapted to suspension culture in 125 mL flasks, two vials of each cell line pool were banked for LN storage. Because cell line production must be clonal, the top three expressing pools were cultured in 96-well culture plates. To prove clonality and avoid a second round of limiting dilution, a Molecular Imaging System (MMS) was used to capture images of single cells and their subsequent proliferation. 96-well plates were imaged using a Devices Clone-Select Imager (CSI) (Molecular Devices LLC, San Jose, CA). Clones were selected based on successful CSI imaging, growth, and production in 96WP.

[0209] To assess cell culture growth and productivity, the top expressing pools were further evaluated in 125 mL of SF for 14 days in fed-batch. Cells were seeded in platform medium, and the feed consisted of Life Technologies' CD CHO + four amino acids, proprietary feed CDF v6.2, and 10% glucose. After 14 days of fed-batch, the pools were centrifuged, and CD CHO-produced mAbs were isolated by filtering the supernatant through a 0.20 μm polyethersulfone (PES) membrane, followed by purification.

[0210] A typical purification consisted of loading 2 liters of conditioned medium (from 0.2 μm-filtered CHO cell culture) onto a 235 mL MabSelect (GE Healthcare, catalog number 17-5199-02) column. The column was pre-equilibrated with PBS. The sample was loaded with a residence time of >2.5 minutes. After loading, the column was again washed with PBS and then with 25 mM sodium acetate at approximately neutral pH. The column was eluted with 25 mM acetic acid at pH 3.6, followed by a strip with 250 mM acetic acid, 250 mM sodium chloride at pH 2.2. Fractions (50 mL) were collected during the elution and strip steps. UV absorbance at A280 was monitored throughout. Peak fractions were pooled and dialyzed against three changes of buffer after adjusting the pH to approximately 5.5 with the addition of 20 mM sodium acetate. The dialysate was collected, sterile filtered, and stored at 4°C.

[0211] Example 9: Neutralization of the biological activity of canine NGF in vitro. The affinity of each canine and feline anti-NGF antibody for canine NGF was measured using SPR (surface plasmon resonance) on a Biacore system (Biocore Life Sciences (GE Healthcare), Uppsala, Sweden) as described in Example 5. In addition, a functional in vitro assay was developed to measure the inhibition constant for the ability of the mAbs to inhibit NGF binding to TrkA. To determine whether the anti-NGF mAbs of the present invention blocked downstream cell signaling as a result of inhibiting NGF binding to TrkA, purified antibodies were evaluated in an assay measuring canine NGF-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK 1 / 2). The cells used in the assay were CHO-K1 (Life Technologies) cells expressing canine TrkA, grown in DMEM / F12 + GlutaMAX™-1 medium (Life Technologies) supplemented with 10% dialyzed FBS, 20 mM HEPES, 500 μg / ml Geneticin, and 1× antibiotic-antimycotic μg / ml (Life Technologies) at 37°C in a humidified 5% CO2, 95% air incubator. For the pERK 1 / 2 assay, cells were seeded at 5.0 × 104 cells / well in 96-well tissue culture plates (Costar) and incubated overnight at 37°C to allow attachment. Cells were then serum-starved for 2 hours in HBSS containing calcium chloride and magnesium chloride (Life Technologies). Anti-NGF antibodies were serially diluted in HBSS and preincubated with recombinant canine NGF diluted in HBSS / 0.1% BSA for 1 hour at room temperature before being added to the cells. The final concentrations of canine NGF and BSA in the assay were 15 ng / ml (EC90) and 0.025%, respectively. Cells were stimulated for 10 min at 37°C. After stimulation, the assay mixture was removed and 100 μl of cell lysis buffer provided with the pERK 1 / 2 AlphaLISA® SureFire® Ultra assay kit (PerkinElmer) was added. The cell lysates were then processed according to the manufacturer's instructions, and the plates were read on an EnSpire® plate reader (PerkinElmer). The maximal response in the assay was defined as the measured ERK 1 / 2 phosphorylation in the presence of canine NGF alone (no mAb). The minimal response was defined as the basal level of ERK 1 / 2 phosphorylation (unstimulated). Calculated inhibition values ​​for the anti-NGF antibodies are expressed as a percentage of the minimal and maximal responses. The resulting percent inhibition data were plotted using GraphPad Prism 5 for IC50 determination (four-parameter curve fitting). See Figures 6-7.

[0212] TF-1 cell proliferation assay TF-1 cells (ATCC) were routinely grown in ATCC-modified RPMI 1640 medium (Life Technologies) supplemented with 10% FBS and 2 ng / ml recombinant human GM-CSF (R&D Systems Inc.). TF-1 proliferation assay medium was RPMI 1640 supplemented with 10% BIT 9500 (Stemcell Technologies) and 10 μg / ml gentamicin. TF-1 proliferation was performed in 96-well microplates (Costar) by incubating 15,000 cells / well with the indicated concentrations of canine and feline anti-NGF antibodies and 2 ng / ml recombinant canine NGF. After a 65-hour culture period, the effect of anti-NGF antibodies on canine NGF-induced cell proliferation was assessed using the CellTiter-GLO luminescent assay kit (Promega). The maximal response in the assay was defined as proliferation in the presence of canine NGF alone (no antibody). The minimal response was defined as proliferation measured without canine NGF. Calculated inhibition (NGF neutralization) values ​​for anti-NGF antibodies are expressed as a percentage of the minimum and maximum response. The resulting percent inhibition data were plotted using GraphPad Prism 5 for IC50 determination (four-parameter curve fit). See Figures 8-9.

[0213] Example 10: Pharmacokinetics Pharmacokinetics (PK) of both canine anti-NGF mAbs 48L2 (ZTS-842) and 9L12 (ZTS-841) was studied in dogs after two subcutaneous (SC) and one intravenous (IV) dose of 2.0 mg / kg administered 28 days apart. IV data demonstrated a half-life of 13.3 ± 3.4 days (mean ± standard deviation) and a slow clearance of 3.9 ± 0.2 mL / day / kg. After SC administration, peak serum concentrations were observed 1 to 7 days after administration. The mean SC absolute bioavailability was 88% ± 41%. In vivo binding to NGF was confirmed using a highly sensitive total NGF (free + NGF-mAb complex) assay. Prior to administration of 48L2 (ZTS-842), NGF concentrations were below the lower limit of quantitation of 10 pg / mL. After administration of 48L2 (ZTS-842), total NGF concentrations increased in all animals, averaging 1300 ± 500 pg / mL on day 84 of the study. 48L2 (ZTS-842) concentrations were in the high excess throughout the study, averaging 7.8 ± 1.3 μg / mL on day 84, 28 days after the last dose, suggesting that even lower doses were sufficient to capture endogenous NGF for at least one month after administration. Although immunogenicity was not directly assessed, there was no indication from the 48L2 (ZTS-842) concentration-time data that any anti-drug antibodies were induced in the four dogs during the three-dose, 84-day study. See Figure 11. Additionally, ZTS-841 was also studied using the same parameters as above, administered SC / SC / IV at 20 mg / kg at 28-day intervals, and showed a half-life of 11.8 ± 4.1 days. 94% ± 12 % SC bioavailability. See Figures 10-11.

[0214] The PK of the felinized anti-NGF mAb fel48L21.1 (ZTS-205) was studied in three male and three female cats after two subcutaneous (SC) and one intravenous (IV) doses of 1.5 mg / kg administered 28 days apart. IV data demonstrated a half-life of 10.8 ± 2.5 days (mean ± standard deviation) and a slow clearance of 3.0 ± 1.0 mL / day / kg. After SC administration, peak serum concentrations were observed 2–7 days after dosing. The mean SC absolute bioavailability was 88% ± 17%. fel48L21.1 (ZTS-205) concentrations were high throughout the study, averaging 7.2 ± 4.0 μg / mL on Day 84, 28 days after the last dose, suggesting that even lower doses are sufficient to capture endogenous NGF for at least 1 month after administration. Although immunogenicity was not directly assessed, there was no indication from the ZTS-842 concentration-time data that any anti-drug antibodies were induced in the six cats during the three-dose, 84-day study.

[0215] Bioanalytical Assay Methodology A free 48L2 (ZTS-842) ligand-binding assay was developed based on capture of free mAb by biotinylated canine NGF on streptavidin Gyrolab™ disks and fluorescent detection after addition of an AlexaFluor™-labeled mouse anti-dog IgG monoclonal antibody. A free fel48L21.1 (ZTS-205) ligand-binding assay was developed based on capture of free mAb by biotinylated canine NGF on streptavidin Gyrolab™ disks and fluorescent detection after addition of an AlexaFluor™-labeled goat anti-cat IgG polyclonal antibody.

[0216] Example 11: Evaluation of canine and feline anti-NGF antibodies in a rat MIA model Osteoarthritis (OA) is a degenerative joint disease characterized by joint pain and progressive loss of articular cartilage. Intra-articular injection of MIA induces articular cartilage loss accompanied by the development of subchondral bone lesions that mimic those of OA. This model provides a rapid and minimally invasive method to reproduce OA-like lesions in rodent species.

[0217] The analgesic effects of speciated (e.g., caninized and feline) anti-NGF antibodies at one dose of MIA in a rat MIA model of osteoarthritis were demonstrated by separately administering monoclonal antibodies ZTS-841 and ZTS-842 on study days 7 and 14 of the study. Pain was assessed using a weight-bearing test for persistent pain and a joint compression (Randall-Selitto) test for mechanical hyperalgesia using an analgesiometer (Ugo Basile). Testing was performed by applying pressure to the hindpaw. Force was increased at a constant rate on a linear scale by pressing a pedal that activated the motor. If pain was indicated and paw withdrawal or vocalization was observed, the pedal was immediately released and the nociceptive threshold was read on a scale. A 400 g cutoff was used to avoid potential injury. The Randall-Selitto test was performed on study days -1 (baseline), 20, and 28. See Figure 12 for a schematic diagram of the rat MIA procedure.

[0218] Cartilage loss was induced via administration of the metabolic inhibitor monoiodoacetic acid (MIA). Rats were anesthetized with isoflurane (3-5% in 100% O). Once the animals were adequately anesthetized, 50 μl of 40 mg MIA / milliliter saline injection was injected into the intra-articular space of the left stifle joint using a 1 cc syringe fitted with a 27G needle. The animals were then removed from isoflurane and allowed to fully recover before being returned to their home cage.

[0219] To evaluate the efficacy of the anti-NGF mAb of the present invention in these animals, Animals were assessed for weight bearing using an acitance tester. Animals were placed in an acrylic test chamber and a force rating was obtained when in position. Three ratings were obtained at each time point. The formula was:

number

[0220] Serum samples were collected 28 days after dose administration via terminal cardiac puncture. After euthanasia via CO2 asphyxiation, whole blood was collected via cardiac puncture, placed into serum separator tubes, and allowed to clot at room temperature before centrifugation (3500 rpm, 15 minutes) and transfer to a 96-well plate in two aliquots of 300 μl each, as listed in the table below. Samples were frozen at ≦−10°C until analysis. See Figures 13-15 for graphical representations of ZTS-841 and ZTS-842 as tested in the rat MIA assay.

[0221] Example 12 Efficacy on lameness: Evaluation of caninized antibodies in a canine synovitis model Inflammatory processes in soft tissues are well recognized as a prominent component of osteoarthritis. In the synovitis pain model, transient inflammation of the synovium in a single stifle joint is induced via intra-articular injection of bacterial lipopolysaccharide (LPS). Quantifiable lameness occurs within 2 h of synovitis induction, peaks at 3–4 h, gradually decreases by 6 h, and completely resolves after 24 h. This model has been routinely used to investigate targets for pain control.

[0222] A 5 mg / kg dose of ZTS-841 administered as a single intravenous injection to healthy male beagles reduced lameness compared to a saline placebo in a canine LPS synovitis model. As can be seen in Table 2 below, ZTS-841 demonstrated efficacy 3 hours after LPS synovitis induction.

[0223] Table 2 and Figure 16 present the least squares means (and standard errors) of the claudication VAS for the treatment groups at 3 and 5 hours after synovitis induction. The difference between 5 mg / kg ZTS-841 and placebo was statistically significant.

[0224] [Table 3]

[0225] Example 13 Humanization of antibodies 48L2 (ZTS-842) and 9L12 (ZTS-841) Similar to the felinization strategy described and well known to those skilled in the art, suitable germline antibody sequences were identified from all available human sequences for CDR grafting from mAbs 48L2 and 9L12. Variable light and heavy chains were selected based on the highest homology to the respective canine frameworks. The CDRs of the native human segments were removed and replaced with the parent canine CDRs. Recombinant humanized 48L2 and 9L12 were produced using the selected variable regions linked to the respective canine constant IgG heavy chain sequences. The antibodies were produced in HEK cells, purified as previously described, and then evaluated for their ability to bind to human NGF, as shown in Table 3 below. Synthetic constructs displaying humanized variable heavy and light chains of mAbs 48L2 (ZTS-842) and 9L12 (ZTS-841) were constructed. Different combinations of variable heavy and light chains within both sets were synthesized and assayed for binding (see below). The CDR sequences were not altered during construction; only the framework sequences were altered.

[0226] The antibody binding affinity of the antibody to human NGF (SEQ ID NO: 66) was determined by surface plasmon resonance (SPR). Human NGF was immobilized on the surface of a BIACORE chip by direct amine coupling. Various concentrations of the indicated humanized anti-NGF antibodies were injected over the human NGF surface, and the association of the antibody to the antigen and the dissociation of the formed complex were monitored in real time. Kinetic analysis was performed to obtain the equilibrium dissociation constant (KD). The results are shown in Table 3 below.

[0227] [Table 4]

[0228] Example 14 : Paratope scanning mutagenesis of antibodies 48L2 9L12 (ZTS-841) and 48L2 (ZTS-842) The region of an antibody involved in antigen recognition represents the paratope. The paratope is created by the combination of amino acids in the complementarity-determining regions (CDRs) of both the heavy and light chain variable regions. Binding of an antibody to an antigen is often mediated by the side chains of CDR residues with the antigen's side chain or carbohydrate moiety. To help define the essential side chains involved in antibody recognition, alanine scanning mutagenesis was performed at each CDR residue in both the heavy and light chains using the technique described in Cunningham and Wells (1989) Science, Vol. 244, Issue 4908, pp. 1081-1085. These mutants were then individually tested for their ability to bind NGF using Biacore. Binding affinities to human NGF (hN), canine NGF (cN), and rat NGF (rN) were measured, and KD values ​​were generated using the same protocol as described in Examples 5 and 9 above. The values ​​are then compared to the wild-type antibody and tabulated as a percent of wild-type binding. The data presented in Tables 4 and 5 below are presented as "percent similarity scores" compared to wild-type.

[0229] To determine the relative affinity of the alanine scanning mutant mAbs to the parent mAb, binding profiles to NGF-coated chips were determined at 100 nM using a Biacore T200. The average response units of four replicates of the parent mAb + / - 3 standard deviations were used to generate parameters for defining a threshold response unit, which includes both the on-rate and off-rate of antibody binding. The percentage of data points for each mutant falling within this threshold was then used to define the "% similarity score." The similarity scores resulting from alanine substitutions at each heavy and light chain CDR position of ZTS-841 and ZTS-842 for the heavy and light chains are shown in Tables 4 and 5, respectively, as "percent inhibition relative to parent." Results from alanine substitutions at each CDR position.

[0230] The sequences in Table 4 are directed to alanine mutagenesis of the variable heavy and variable light chain CDR amino acid sequences of ZTS-841 (9L12). Table 5 is directed to alanine substitutions of the variable heavy and light chain CDR amino acid sequences of ZTS-842 (48L2). The mutated amino acids are listed in the table according to the wild-type numbering of both the variable heavy and variable light chain sequences as previously described and included below. Amino acid positions 1, 25, 50, 75, and 100 are marked below. In the "Sample Name" column, the variable sequence of either the heavy or light chain is listed along with the alanine substitution at the numbered amino acid position. ZTS-841 VH:Sequence number 8: E 1 VQLVESGGDLVKPGGSLRLSCVAS 25 GFTFSSHGMHWVRQSPGKGLQWVAV 50 INSGGSSTYYTDAVKGRFTISRDNA 75 KNTVYLQMNSLRAEDTAMYYCAKES 100 VGGWEQLVGPHFDYWGQGTLVIVSS 124 ZTS-841 VL: SEQ ID NO: 7: Q 1SVLTQPTSVSGSLGQRVTISCSGS 25 TNNIGILGASWYQLFPGKAPKLLVY 50 GNGNRPSGVPDRFSGADSGDSVTLT 75 ITGLQAEDEADYYCQSFDTTLGAHV 100 FGGGTHLTVL 110

[0231]

Table 5-1

Table 5-2

[0232] Regarding Table 5: ZTS-842 VH:Sequence number 28 E 1 VQLVESGGDLVKPGGSLRLSCVAS 25 GFTFSTYGINWVRQAPGKGLQWVAY 50 ISSGGSSTYYADPVKGRFTI 75 SRDDAKNMLYLQMNSLRAEDTAIYYCAGSRY 100 TYAYGGGYEFHFWGQGTLVTVSS 124 ZTS-842 VL: SEQ ID NO: 27 Q 1 AVLNQPASVSGALGQKVTISCSGS 25 TMDIDIFGVSWYQQLPGKAPKLLVD 50 SDGDRPSGIPDRFSGSRSGNSGTLT 75 ITGLQAEDEADYHCQSGDSTLGALAI 100 FGGGTHVTVL 110

[0233]

Table 6-1

Table 6-2

[0234] Values ​​generated in Tables 4 and 5 with percent similarity less than 50% suggest amino acid positions essential for binding of the antibody paratope to NGF. Mutation of the wild-type amino acid at the indicated position with alanine, which leads to reduced or total absence of binding to NGF, suggests which amino acids are required for binding and which may be substituted, at a minimum, with conservative amino acid substitutions.

Claims

1. 1. A recombinant antigen binding protein that specifically binds to nerve growth factor (NGF), comprising: a. The light chain variable region (VL) is i. Complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1 ii. Complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2 iii. Complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3; and b. The heavy chain variable region (VH) is i. Complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:4 ii. Complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 5 iii. An antigen-binding protein comprising a complementarity-determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO:

6.

2. 1. A recombinant antigen binding protein that specifically binds to nerve growth factor (NGF), comprising: a. The light chain variable region (VL) is i. Complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 21 ii. Complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 22 iii. A complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 23; and b. The heavy chain variable region (VH) is i. Complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 24 ii. Complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 25 iii. Complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 26; Antigen-binding proteins.

3. 1. A recombinant antigen binding protein that specifically binds to nerve growth factor (NGF), comprising: a. SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 55, SEQ ID NO: a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 85; and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:56, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:79, and SEQ ID NO:81; or, b. a light chain variable region comprising an amino acid sequence having 1 to 10 conservative amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:55, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:83, and SEQ ID NO:85; and a heavy chain variable region comprising an amino acid sequence having 1 to 10 conservative amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:56, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:79, and SEQ ID NO:81; Including, 3. The recombinant antigen-binding protein of claim 1 or 2.

4. the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8; or, the light chain variable region comprises an amino acid sequence having 1 to 10 conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 7, and the heavy chain variable region comprises an amino acid sequence having 1 to 10 conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 8; The recombinant antigen-binding protein of claim 3.

5. the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:27, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:28; or, 4. The recombinant antigen binding protein of claim 3, wherein said light chain variable region comprises an amino acid sequence having one to ten conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO:27, and said heavy chain variable region comprises an amino acid sequence having one to ten conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO:

28.

6. 6. The antigen binding protein of any one of claims 1 to 5, wherein said binding protein inhibits binding of NGF to the TrkA receptor.

7. 7. The antigen binding protein of any one of claims 1 to 6, wherein the binding protein reduces or eliminates an NGF-associated disorder.

8. 8. The antigen-binding protein of claim 7, wherein the NGF-related disorder is selected from the group consisting of cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation.

9. 9. The antigen binding protein of claim 8, wherein the NGF-associated disorder is pain.

10. The NGF-related disorder is a pain disorder, and is characterized by osteoarthritis pain, rheumatoid arthritis pain, hand pain, or the like.

10. The antigen binding protein of claim 9, which is selected from the group consisting of surgical and post-surgical pain, incisional pain, systemic inflammatory pain, cancer pain, pain from trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain.

11. 11. The antigen binding protein of claim 10, wherein the NGF-related disorder comprises osteoarthritis pain.

12. The binding protein is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a single chain antibody, a tetrameric antibody, a tetravalent antibody, a multispecific antibody, a domain-specific antibody, a domain-deleted antibody, a fusion protein, an ScFc fusion protein, a Fab fragment, a Fab' fragment, a F(ab') ２ 12. The antigen-binding protein of any one of claims 1 to 11, which is selected from the group consisting of a fragment, an Fv fragment, an ScFv fragment, and a small modular immunopharmaceutical (SMIP).

13. 13. The antigen-binding protein of claim 12, which is a monoclonal antibody.

14. 14. The antigen-binding protein of claim 13, wherein the monoclonal antibody is a canine monoclonal antibody, a caninized monoclonal antibody, a feline monoclonal antibody, a humanized monoclonal antibody, or an equine monoclonal antibody.

15. 15. A pharmaceutical or veterinary composition comprising a therapeutically effective amount of the antigen binding protein of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. 16. A pharmaceutical or veterinary composition according to claim 15 for use in treating pain in a subject.

17. 17. The pharmaceutical or veterinary composition of claim 16, wherein the pain is selected from the group consisting of osteoarthritis pain, rheumatoid arthritis pain, surgical and post-operative pain, incisional pain, systemic inflammatory pain, cancer pain, pain from trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic disease, pain associated with musculoskeletal disease, visceral pain, and gastrointestinal pain.

18. 18. The pharmaceutical or veterinary composition of claim 17, wherein the pain comprises osteoarthritis pain.

19. 18. The pharmaceutical or veterinary composition of claim 17, wherein the pain includes surgical and post-surgical pain.

20. 18. The pharmaceutical or veterinary composition of claim 17, wherein the pain comprises cancer pain.

21. A pharmaceutical or veterinary composition according to any one of claims 15 to 20 for use in dogs, cats, horses or humans.

22. 22. A pharmaceutical or veterinary composition according to claim 21 for use in dogs.

23. 22. A pharmaceutical or veterinary composition according to claim 21 for use in a cat.

24. A host cell producing the antigen-binding protein of any one of claims 1 to 14.

25. An isolated nucleic acid comprising a nucleic acid sequence encoding the antigen-binding protein of any one of claims 1 to 14.

26. A vector comprising the nucleic acid of claim 25.

27. A host cell comprising the vector of claim 26.

28. 26. A host cell comprising the nucleic acid of claim 25.

29. 29. A method of producing an antigen binding protein according to any one of claims 1 to 14, said method comprising culturing a host cell according to any one of claims 24, 27 or 28 under conditions which result in the production of said antigen binding protein, and isolating said antigen binding protein from said host cell or from culture medium of said host cell.

30. 16. A method of treating a non-human subject for an NGF-related disorder, comprising administering to said subject a therapeutically effective amount of the pharmaceutical or veterinary composition of claim 15.

31. 31. The method of claim 30, wherein the NGF-related disorder is selected from the group consisting of cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation.

32. 32. The method of claim 31, wherein the NGF-associated disorder comprises pain.

33. 33. The method of claim 32, wherein the NGF-related disorder is a pain disorder and is selected from the group consisting of osteoarthritis pain, rheumatoid arthritis pain, surgical and post-operative pain, incisional pain, systemic inflammatory pain, cancer pain, pain from trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain.

34. 34. The method of claim 33, wherein the NGF-associated disorder comprises osteoarthritis pain.

35. 34. The method of claim 33, wherein the NGF-related disorder comprises surgery and post-operative pain.

36. 34. The method of claim 33, wherein the NGF-associated disorder comprises cancer pain.

37. 16. A method of inhibiting NGF activity in a non-human subject by administering to said subject the pharmaceutical or veterinary composition of claim 15.

38. 38. The method of any one of claims 30 to 37, wherein the subject is selected from the group consisting of a dog, a cat, and a horse.

39. 39. The method of claim 38, wherein the subject comprises a dog.

40. 39. The method of claim 38, wherein the subject comprises a cat.

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