Anti-NGF antibodies and methods thereof

Abstract

The present disclosure includes novel anti-NGF antibodies, antigen binding proteins and polynucleotides encoding them. The present disclosure further provides the use of the novel antibodies, antigen binding proteins and / or nucleotides of the present invention for treating and / or preventing NGF-related disorders, particularly for managing pain.

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 that has been modified to become non-immunogenic in relevant species. 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 Art

[0002] Nerve growth factor (NGF) is the first neurotrophic factor to be identified, and its role in the development and survival of peripheral and central neurons has been well characterized. NGF has been shown to be a key survival and maintenance factor in the development of peripheral sympathetic neurons and embryonic sensory neurons as well as basal forebrain cholinergic neurons (Smeyne et al., Nature 368:246-249 (1994) and Crowley et al., Cell 76:1001-1011 (1994)). NGF upregulates the expression of neuropeptides 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 general neurotrophin receptor (sometimes referred to as the "high-affinity" and "low-affinity" NGF receptors, respectively), which are 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 on the nervous system, NGF is increasingly involved in processes outside the nervous system. For example, NGF has been shown to enhance vascular permeability (Otten et al., Eur J Pharmacol. 106: 199-201 (1984)), enhance T cell and B cell immune responses (Otten et al., Proc. Natl. Acad. Sci. USA 86: 10059-10063 (1989)), induce lymphocyte differentiation and mast cell proliferation, and lead to 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 ... al., J. Bioi. 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)), bronchial epithelial cells (Kassel, et al., Clin, Exp. Allergy 31:1432-40 (2001)), renal mesangial cells (Steiner, et al., Am. J. Physiol. 261:F792-798 (1991)) and skeletal muscle myotubes (Schwartz et al., J Photochem. Photobiol. B66:195-200 (2002)). NGF receptors have been found on a variety of cell types outside the nervous system. For example, TrkA has been found on human monocytes, T and B lymphocytes, and mast cells.

[0005] A link 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. I'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)). Sustained elevated levels of NGF in peripheral tissues are associated with hyperalgesia and inflammation and have been observed in several forms of arthritis. The synovium of patients affected by rheumatoid arthritis expresses high levels of NGF, while in non-inflammatory synovium, NGF is reported to be undetectable (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)). Increased NGF levels and an increase in the number of mast cells have been reported in transgenic arthritis 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 one year of age. The development of OA is mainly secondary to trauma, joint instability and diseases such as hip dysplasia. Osteoarthritis is a disease state of the entire joint, and inflammatory and degenerative changes in all joint structures lead to disability and clinical symptoms of lameness and pain. Pain is the most important clinical manifestation of canine OA, and it is the result of a complex interaction between structural joint changes, biochemical and molecular changes, and peripheral and central pain processing mechanisms. In this network, the activation and sensitization of peripheral nociceptors by inflammatory and hyperalgesic mediators (such as cytokines, prostaglandins and neurotransmitters) is one of the main peripheral mechanisms causing joint pain. Treatment of canine pain by non-drug agents that will provide longer-lasting relief compared to traditional canine pain treatments is clearly an unmet need.

[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 class of medications prescribed by veterinarians, but are limited by their efficacy and tolerability. Market research indicates that approximately 9 million dogs in the United States are treated with NSAIDs. Corticosteroids are rarely used and are usually used for short periods of time and as a last resort. There is clearly an unmet need for a convenient, safe product that effectively treats dogs with OA.

[0008] In cats, OA is a pathological change of the diarticular synovial joints characterized by degradation of the articular cartilage, osteophyte formation, bone remodeling, soft tissue changes and low-grade non-suppurative inflammation. Although the radiographic features of feline OA have been well described, the clinical signs of the disease are rarely documented and may not be diagnosed. The difficulty in assessing lameness in cats lies in their small size and natural flexibility, which allows them to compensate. However, it is believed that the clinical signs of feline OA include weight loss, anorexia, depression, abnormal bowel habits, infrequent grooming, aggressive behavior, and a gradual decrease in the ability to jump to obvious lameness. Based on misdiagnosis, feline OA often remains untreated and is an unmet veterinary need. Summary of the invention

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

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

[0011] In one embodiment, the present invention provides an antigen binding protein comprising a light chain variable region (VL), the light chain variable region comprising a complementary determining region 1 (CDR1), the complementary determining region comprising an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO.1; a complementary determining region 2 (CDR2), which comprises an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO.2; a complementary determining region 3 (CDR3), which comprises an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO.3; and a heavy chain variable region (VH), the heavy chain variable region comprising: a complementary determining region 1 (CDR1), which comprises an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO.4; a complementary determining region 2 (CDR2), which comprises an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO.5; a complementary determining region 3 (CDR3), which comprises an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO. The amino acid sequence of NO.6 has an amino acid sequence with at least about 90% sequence identity, and any variants thereof, wherein the variants have one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 within any variable light chain region or variable heavy chain region of the antigen binding protein.

[0012] In one embodiment, the present invention provides an antigen-binding protein of the present invention comprising a light chain variable region (VL), the light chain variable region comprising a complementary determining region 1 (CDR1), the complementary determining region comprising an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO.21; a complementary determining region 2 (CDR2), which comprises an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO.22; a complementary determining region 3 (CDR3), which comprises an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO.23; and a heavy chain variable region (VH), the heavy chain variable region comprising: a complementary determining region 1 (CDR1), which comprises an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO.24; a complementary determining region 2 (CDR2), which comprises an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO.25; a complementary determining region 3 (CDR3), which comprises an amino acid sequence having at least about 90% sequence identity with an amino acid sequence comprising SEQ ID NO. The amino acid sequence of NO.26 has an amino acid sequence with at least about 90% sequence identity, and any variants thereof, wherein the variants have one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 within any variable light chain region or variable heavy chain region of the antigen binding protein.

[0013] In one aspect, the present invention provides a recombinant antigen binding protein that specifically binds to nerve growth factor (NGF), comprising a variable light chain, the variable light chain comprising an amino acid sequence having at least 90% sequence identity with 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, the variable heavy chain comprising an amino acid sequence having at least 90% sequence identity with 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 variants thereof, wherein the variants have one or more conservative amino acid substitutions in any variable light chain region or variable heavy chain region of the antigen binding protein.

[0014] In one embodiment, the present invention provides a recombinant antigen-binding protein of the present invention including a variable light chain, the variable light chain including an amino acid sequence having at least 90% sequence identity with SEQ ID NO.7; and a variable heavy chain, the variable heavy chain including an amino acid sequence having at least 90% sequence identity with SEQ ID NO.8, and any variants thereof, wherein the variant has one or more conservative amino acid substitutions in any variable light chain region or variable heavy chain region of the antigen-binding protein. In one embodiment, the present invention provides an antigen-binding protein, wherein the variable light chain includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO.27, and the variable heavy chain includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO.28, and any variants thereof, wherein the variant has one or more conservative amino acid substitutions in any variable light chain region or variable heavy chain region of the antigen-binding protein.

[0015] In one embodiment, the present invention provides a recombinant antigen-binding protein of the present invention including a variable light chain, the variable light chain including an amino acid sequence having at least 90% sequence identity with SEQ ID NO.9; and a variable heavy chain, the variable heavy chain including an amino acid sequence having at least 90% sequence identity with SEQ ID NO.10, and any variants thereof, wherein the variant has one or more conservative amino acid substitutions in any variable light chain region or variable heavy chain region of the antigen-binding protein. In one embodiment, the present invention provides a recombinant antigen-binding protein of the present invention including a variable light chain, the variable light chain including an amino acid sequence having at least 90% sequence identity with SEQ ID NO.29; and a variable heavy chain, the variable heavy chain including an amino acid sequence having at least 90% sequence identity with SEQ ID NO.30, and any variants thereof, wherein the variant has one or more conservative amino acid substitutions in any variable light chain region or variable heavy chain region of the antigen-binding protein.

[0016] In one embodiment, the present invention provides a recombinant antigen-binding protein of the present invention comprising a variable light chain, the variable light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO.55; and a variable heavy chain, the variable heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO.56, and any variants thereof, the variants having one or more conservative amino acid substitutions in any variable light chain region or variable heavy chain region of the antigen-binding protein.

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

[0018] In one embodiment, the present invention provides a recombinant antigen binding protein that specifically binds to NGF, the protein further comprising a constant region, the constant region comprising an amino acid sequence comprising SEQ ID.62. In one embodiment, the present invention provides a nucleotide sequence encoding a 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, the change in the constant region of the antigen binding protein of the present invention prevents the degradation of the antigen binding protein.

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

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

[0021] In one embodiment, the present invention provides a nucleotide sequence encoding an antigen-binding protein of the present invention, and includes nucleotides encoding a light chain variable region (VL), the light chain variable region including a complementary determining region 1 (CDR1), the complementary determining region 1 including a nucleotide sequence having at least about 90% sequence identity with a nucleotide sequence including SEQ ID NO.31; a complementary determining region 2 (CDR2), which includes a nucleotide sequence having at least about 90% sequence identity with a nucleotide sequence including SEQ ID NO.32; a complementary determining region 3 (CDR3), which includes a nucleotide sequence having at least about 90% sequence identity with a nucleotide sequence including SEQ ID NO.33; and a nucleotide sequence encoding a heavy chain variable region (VH), the heavy chain variable region including: a complementary determining region 1 (CDR1), which includes a nucleotide sequence having at least about 90% sequence identity with a nucleotide sequence including SEQ ID NO.34; a complementary determining region 2 (CDR2), which includes a nucleotide sequence having at least about 90% sequence identity with a nucleotide sequence including SEQ ID NO. NO.35 has a nucleotide sequence with at least about 90% sequence identity; a complementary determining region 3 (CDR3), which includes a nucleotide sequence with at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO.36, and any variants thereof, wherein the variants have one or more nucleic acid substitutions based on the degeneracy of the genetic code in at least one of CDR1, CDR2 or CDR3 within any variable light chain region or variable heavy chain region of the 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 to nerve growth factor (NGF), and includes nucleotides encoding a variable light chain, wherein the variable light chain includes a nucleotide sequence protein having at least 90% sequence identity with 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, wherein the variable heavy chain includes a nucleotide sequence having at least 90% sequence identity with 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.76; and SEQ ID NO.80, as well as any variants thereof, wherein the variants have one or more nucleic acid substitutions based on the degeneracy of the genetic code within any variable light chain region or variable heavy chain region of the antigen-binding protein.

[0023] In one embodiment, the present invention provides a nucleotide sequence encoding a recombinant antigen-binding protein of the present invention, including a nucleotide sequence encoding a variable light chain and a nucleotide sequence encoding a variable heavy chain, wherein the variable light chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.17, and the variable heavy chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.18, and any variants thereof, wherein the variant has one or more nucleic acid substitutions based on the degeneracy of the genetic code in any variable light chain region or variable heavy chain region of the 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, wherein the variable light chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.37; and a nucleotide sequence encoding a variable heavy chain, which includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.38, and any variants thereof, wherein the variant has one or more nucleic acid substitutions based on the degeneracy of the genetic code in any variable light chain region or variable heavy chain region of the antigen-binding protein.

[0024] In one embodiment, the present invention provides a nucleotide sequence encoding a recombinant antigen-binding protein of the present invention, including a nucleotide sequence encoding a variable light chain and a nucleotide sequence encoding a variable heavy chain, wherein the variable light chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.19, and the variable heavy chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.20, and any variants thereof, wherein the variant has one or more nucleic acid substitutions based on the degeneracy of the genetic code in any variable light chain region or variable heavy chain region of the 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, wherein the variable light chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.39; and a nucleotide sequence encoding a variable heavy chain, wherein the variable heavy chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.40, and any variants thereof, wherein the variant has one or more nucleic acid substitutions based on the degeneracy of the genetic code in any variable light chain region or variable heavy chain region of the antigen-binding protein.

[0025] In one embodiment, the present invention provides a nucleotide sequence encoding a recombinant antigen-binding protein of the present invention, including a nucleotide sequence encoding a variable light chain and a nucleotide sequence encoding a variable heavy chain, wherein the variable light chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.57, and the variable heavy chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.58, as well as any variants thereof, wherein the variant has one or more nucleic acid substitutions based on the degeneracy of the genetic code within any variable light chain region or variable heavy chain region of the antigen-binding protein.

[0026] In one embodiment, the present invention provides a nucleotide sequence encoding a recombinant antigen-binding protein of the present invention, including a nucleotide sequence encoding a variable light chain and a nucleotide sequence encoding a variable heavy chain, wherein the variable light chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.92, and the variable heavy chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.80, and any variants thereof, wherein the variant has one or more nucleic acid substitutions based on the degeneracy of the genetic code in any variable light chain region or variable heavy chain region of the 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, which includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.88; and a nucleotide sequence encoding a variable heavy chain, wherein the variable heavy chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.80, and any variants thereof, wherein the variant has one or more nucleic acid substitutions based on the degeneracy of the genetic code in any variable light chain region or variable heavy chain region of the antigen-binding protein. In one embodiment, the present invention provides a nucleotide sequence encoding a recombinant antigen-binding protein of the present invention, including a nucleotide sequence encoding a variable light chain and a nucleotide sequence encoding a variable heavy chain, wherein the variable light chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.92, and the variable heavy chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.76, and any variants thereof, wherein the variant has one or more nucleic acid substitutions based on the degeneracy of the genetic code in any variable light chain region or variable heavy chain region of the 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, which includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.88; and a nucleotide sequence encoding a variable heavy chain, wherein the variable heavy chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.76, and any variants thereof, wherein the variant has one or more nucleic acid substitutions based on the degeneracy of the genetic code in any variable light chain region or variable heavy chain region of the antigen-binding protein. In one embodiment, the present invention provides a nucleotide sequence encoding a recombinant antigen-binding protein of the present invention, including a nucleotide sequence encoding a variable light chain and a nucleotide sequence encoding a variable heavy chain, wherein the variable light chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.90, and the variable heavy chain includes a nucleotide sequence having at least 90% sequence identity with SEQ ID NO.76, as well as any variants thereof, wherein the variant has one or more nucleic acid substitutions based on the degeneracy of the genetic code within any variable light chain region or variable heavy chain region of the antigen-binding protein.

[0027] In one or more embodiments, the antigen binding proteins of the present invention inhibit the binding of NGF to the TrkA receptor. In one or more embodiments, the antigen binding proteins of the present invention inhibit the biological functions associated with the binding of NGF to the TrkA receptor. In one or more embodiments, the antigen binding proteins of the present invention inhibit the binding of NGF to the TrkA receptor. In one or more embodiments, the antigen binding proteins of the present invention inhibit the binding of NGF to the TrkA receptor. In one or more embodiments, the antigen binding proteins inhibit the biological functions associated with the binding of NGF to TrkA with or without the p75 receptor, which includes blocking the signal transduction and pathways associated with the binding of NGF to the TrkA receptor.

[0028] In one or more embodiments, the antigen binding proteins of the present invention reduce or eliminate NGF-related disorders by destroying signals associated with NGF binding to TrkA and p75 receptors. In one or more embodiments, NGF-related disorders are 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 postoperative pain, incision pain, systemic inflammatory pain, cancer pain, pain caused by trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain and gastrointestinal pain. In one embodiment, NGF-related disorders include osteoarthritis pain. In one embodiment, NGF-related disorders include surgical and postoperative pain. In one embodiment, NGF-related disorders include cancer pain.

[0029] In one or more aspects, the antigen-binding proteins of the present invention are selected from the group consisting of: monoclonal antibodies; chimeric antibodies, single-chain antibodies, tetrameric antibodies, tetravalent antibodies, multispecific antibodies, domain-specific antibodies, domain-deleted antibodies, fusion proteins, ScFc fusion proteins, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, ScFv fragments, Fd fragments, single domain antibodies, dAb fragments, small modular immune drugs (SMIP), nanobodies and IgNAR molecules. In one embodiment, the antigen-binding proteins are monoclonal antibodies. In one embodiment, the antigen-binding proteins are chimeric antibodies.

[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 equinized 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 equinized 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 composition or veterinary composition comprising a therapeutically effective amount of an antigen binding protein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition of the present invention is used to treat NGF-related disorders. In one embodiment, NGF-related disorders are selected from the group consisting of: cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain and inflammation. In one embodiment, NGF-related disorders include pain. In one embodiment, the pharmaceutical composition is used to treat pain. In one embodiment, the pharmaceutical composition is used to treat pain, and the type of pain is selected from osteoarthritis pain, rheumatoid arthritis pain, surgical and postoperative pain, incisional pain, systemic inflammatory pain, cancer pain, pain caused by trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain and gastrointestinal pain. In one embodiment, pain includes osteoarthritis pain. In one embodiment, pain includes surgical and postoperative pain. In one embodiment, pain includes cancer pain. In one or more embodiments, the pharmaceutical compositions of the present invention are used for dogs. In one or more embodiments, the pharmaceutical compositions of the present invention are used for cats. In one or more embodiments, the pharmaceutical compositions of the present invention are used for horses. In one or more embodiments, the pharmaceutical compositions of the present invention are used for humans.

[0032] In one or more embodiments, the pharmaceutical composition of the present invention has no significant side effects on the immune system of a canine. In one embodiment, the composition of the present invention has no significant side effects on the immune system of a cat. In one or more embodiments, the composition of the present invention has no significant side effects on the immune system of a horse. In one embodiment, the composition of the present invention has no significant side effects on the immune system of a human. In one embodiment, the pharmaceutical composition is a veterinary composition.

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

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

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

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

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

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

[0039] In one or more aspects, the present invention provides a method for treating a subject's NGF-related disorder, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or veterinary composition of the present invention. In one embodiment, the present invention provides NGF-related disorders selected from the group consisting of: cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation. In one embodiment, NGF-related disorders include pain. In one embodiment, NGF-related disorders are pain disorders and are selected from the group consisting of: osteoarthritis pain, rheumatoid arthritis pain, surgical and postoperative pain, incisional pain, systemic inflammatory pain, cancer pain, pain caused by trauma, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain. In one embodiment, NGF-related disorders include osteoarthritis pain. In one embodiment, NGF-related disorders include surgical and postoperative pain. In one embodiment, NGF disorders are cancer pain. In one embodiment, the subject is selected from the group consisting of: canines, cats, humans, and horses. In one embodiment, the subject includes canines. In one embodiment, the subject includes cats. In one embodiment, the subject includes horses. In one embodiment, the subject includes humans.

[0040] In one or more embodiments, the present invention provides a method for detecting or quantifying the level of NGF in a biological sample, the method comprising:

[0041] (a) incubating a clinical or biological sample containing NGF in the presence of any one of the antigen binding proteins of the present invention; and

[0042] (b) Detecting antigen binding proteins that bind to NGF in the sample.

[0043] In some embodiments, antigen-binding proteins of the present invention are detectably labeled. In some embodiments, unlabeled antigen-binding proteins 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 an antigen-binding protein of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic representation of the general structure of a mouse immunoglobulin G (IgG) molecule highlighting the antigen binding site.

[0045] Figure 2 is a schematic diagram of the general structure of mouse / canine chimeric IgG.

[0046] Figure 3 Is a diagram showing the speciation or "canization" of a mouse IgG, with the mouse CDRs grafted onto a canine framework. This figure also represents felinization, equinization, humanization, and other speciation as defined herein.

[0047] Figure 4 is a diagram of a "heterochimeric" monoclonal antibody that pairs a chimeric light chain with a fully caninized heavy chain.

[0048] Figure 5 is a diagram of an antibody variable chain showing primers for the constant region and degenerate primers for the mouse variable region.

[0049] Figure 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.

[0050] Figure 7 is a representation of aD11 mAb, negative control, and caninized version of 13L11 mAb on canine NGF-induced pERK-1 / 2 signaling in caTrkA-CHO cells.

[0051] Figure 8 Figure 5 is a representation of the anti-NGF mAb against canine NGF-induced TF-1 cell proliferation by ZTS-841 and ZTS-842 mAbs.

[0052] Fig. 9 is a representation of anti-NGF mAb use of 48L2 chimera, fe148L2VH1.1, and fe148L2VH1.2 mAbs on canine NGF-induced TF-1 proliferation.

[0053] Fig.10 is a representation of a pharmacokinetic study of the anti-NGF mAb ZTS 841 administered at a SC / SC / IV dose of 2.0 mg / kg.

[0054] Fig.11 is a representation of the pharmacokinetic study of the anti-NGF mAb ZTS 842 administered at 2.0 mg / kg SC / SC / IV doses.

[0055] Fig.12 is a schematic diagram of the rat MIA assay.

[0056] Fig.13 Is a graphical representation of mAb 841 in the rat MIA assay over the dose range of 0.1-2 mg / kg.

[0057] Fig.14 Is a graphical representation of mAb 841 in the rat MIA assay over the dose range of 0.01-2.0 mg / kg.

[0058] Fig.15 Is a graphical representation of mAb 842 dosed at 0.5 mg / kg and 2 mg / kg in the rat MIA assay.

[0059] Fig.16 is a graphical representation of mAb 841 lameness VAS for treatment groups three and five hours after induction of synovitis in the LPS synovitis model.

[0060] A brief description of the sequence

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] DETAILED DESCRIPTION

[0072] The invention disclosed herein provides anti-NGF antigen binding proteins that bind to NGF with high affinity. The present invention further provides antigen binding proteins and polypeptides that also bind to NGF as variants of the antigen binding proteins, as well as methods for preparing and using these antigen binding proteins. In some embodiments, the present invention also provides polynucleotides encoding the antigen binding proteins and / or polypeptides. The invention disclosed herein also provides a method for preventing and / or treating pain by administering a therapeutically effective amount of the anti-NGF antigen binding protein of the present invention.

[0073] General Technology

[0074] It should be understood that the present invention is not limited to the particular methodology, protocols and reagents etc. described herein and may vary accordingly. 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 limited only by the claims.

[0075] Unless otherwise defined, scientific and technical terms used in conjunction with antigen-binding proteins as described herein should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms should include plural terms, and plural terms should include singular terms. Typically, the terms used in conjunction with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization as described herein and the techniques of these aspects are those 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 can replace the described techniques.

[0076] All identified patents and other publications are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies 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.

[0077] 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 the manufacturer's instructions or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods known in the art and as described, but not limited to, in various general and more specific references cited and discussed throughout this specification, see, for example, Sambrook et al. MOLECULAR CLONING: LAB. MANUAL (3 rded.,Cold Spring Harbor Lab.Press,ColdSpring Harbor,N.Y.,2001)and Ausubel et al.Current Protocols in MolecularBiology(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)AcademicPress;Animal Cell Culture(R.1.Freshney,ed.1987);Introduction to Cell andTissue Culture(1.P.Mather and P.E.Roberts,1998)Plenum Press;Cell and TissueCulture: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 ofExperimental Immunology(D.M.Weir and C.C.Blackwell,eds.);Gene TransferVectors for Mammalian Cells(J.M.Miller and M.P.Calos,eds.,1987);CurrentProtocols in Molecular Biology(F.M.Ausubel et al.,eds.,1987);PCR:ThePolymerase Chain Reaction,(Mullis et al.,eds.,1994);Current Protocols inImmunology(E.Coligan et al.,eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal antibodies: a practical approach 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 JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (YT DeVita et al. al.,eds.,JBLippincott Company,1993).

[0078] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about".

[0079] definition

[0080] Before describing the present invention in detail, several terms used in the context of the present invention will be defined. In addition to these terms, other terms are defined as needed in other positions in the specification. Unless otherwise clearly defined herein, the technical terms used in this specification will have their art-recognized meanings.

[0081] As used in the 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.

[0082] As used herein, the term "comprising" is intended to mean that compositions and methods include the stated elements, but not excluding other elements.

[0083] 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.

[0084] "NGF receptor" refers to a polypeptide that is bound or activated by NGF. NGF receptors include the canine TrkA receptor and, to a lesser extent, the canine p75 receptor.

[0085] The "biological activity" of NGF generally refers to the ability to bind to NGF receptors and / or activate NGF receptor signaling pathways. Biological activity includes, but is not limited to, any one or more of the following: the ability to bind to NGF receptors (e.g., TrkA and / or p75); the ability to promote TrkA receptor dimerization and / or autophosphorylation; the ability to activate NGF receptor signaling pathways; the ability to promote cell differentiation, proliferation, survival, growth, and other cell physiological changes, including (in the case of neurons (including peripheral and central neurons)) changes in neuronal morphology, synapse formation, synaptic function, neurotransmitter and / or neuropeptide release, and regeneration after injury; the ability to promote the survival of mouse E13.5 trigeminal neurons; and the ability to regulate pain (including postoperative pain).

[0086] As used herein, "anti-NGF antigen binding protein" (interchangeably referred to as "anti-NGF antibody" and "anti-NGF antagonist antibody", "antigen binding fragment", "antigen binding portion", etc.) refers to an antigen binding protein that is capable of binding to NGF and inhibiting NGF biological activity and / or downstream pathways mediated by NGF signaling. Anti-NGF antigen binding proteins include binding proteins and antibodies that block, antagonize, inhibit or reduce (including significantly reduce) NGF biological activity, including downstream pathways mediated by NGF signaling and / or inhibit NGF binding to its receptor trkA, such as receptor binding and / or initiating cellular responses to NGF. For the purposes of the present invention, it will be clearly understood that the term "anti-NGF antigen binding protein" or "anti-NGF antagonist antibody" includes all previously identified terms, names, and functional states and characteristics, wherein NGF itself, NGF biological activity (including but not limited to its ability to mediate any aspect of osteoarthritis pain, inflammatory pain, postoperative pain, cancer pain, etc.) or the consequences of biological activity are substantially offset, reduced or neutralized to any meaningful extent. In some embodiments, anti-NGF antagonist antibodies bind to NGF and prevent NGF dimerization and / or binding to NGF receptors (e.g., TrkA and / or p75). In other embodiments, anti-NGF antigen binding proteins bind to NGF and prevent TrkA receptor dimerization and / or TrkA autophosphorylation. Examples of anti-NGF antagonist antibodies are provided herein.

[0087] As used herein, the terms "antigen binding protein", "antibody", "antigen binding protein" and the like are used interchangeably and refer to polypeptides or fragments thereof that include an antigen binding site. In one embodiment of the present invention, the antigen binding protein of the present invention further provides an immunoglobulin that can specifically bind to a target, such as a carbohydrate, a polynucleotide, a lipid, a polypeptide, etc., 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. Therefore, the isolated complete antibody can be isolated from a pool of polyclonal antibodies, monoclonal antibodies, synthetic antibodies, recombinant antibodies, chimeric antibodies, heterochimeric antibodies, or antibodies that are considered to be special, as defined herein. In some embodiments, the terms "antigen binding protein", "antibody", "antagonist antibody" and the like preferably refer to monoclonal antibodies and fragments thereof, as well as immunological binding equivalents that can bind to NGF proteins and fragments thereof. As used herein, the term includes not only full-length (two heavy chains and two light chains according to the standard definition) polyclonal antibodies or monoclonal antibodies, but also fragments thereof. For the purposes of the present invention, "antibodies" and "antigen binding proteins" also include antibody fragments unless otherwise specified. Exemplary antibody fragments include Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, diabodies, their antigen recognition fragments, small modular immunopharmaceuticals (SMIPs), nanobodies, IgNAR molecules, and equivalents recognized by those skilled in the art as antigen binding proteins or antibody fragments, as well as any of the foregoing 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 antigen recognition sites. Antibodies and antigen-binding proteins can be prepared, for example, by conventional 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 various other antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988, and other techniques well known to those skilled in the art.

[0088] "Monoclonal antibody" as defined herein is an antibody produced by a single cell clone (specifically, a single hybridoma clone), and is therefore a single pure homologous antibody. All monoclonal antibodies produced from the same clone are identical and have the same antigenic specificity. Monoclonal antibody is a homologous antibody group, wherein the monoclonal antibody is composed of the amino acid (naturally occurring and non-naturally occurring) that participates in the selective binding of the antigen. The colony of monoclonal antibodies is highly specific, for a single antigenic site. The term "monoclonal antibody" not only comprises complete monoclonal antibodies and full-length monoclonal antibodies, but also comprises its fragment (Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, double antibodies, their antigen recognition fragments, small module immune drugs (SMIP), nanobody, IgNAR molecules, etc.), its mutant, the fusion protein including the antibody part, and any other modified configuration of immunoglobulin molecules, which includes the antigen recognition site of required specificity and the ability to be attached to the antigen. It is not intended to be limited to the source of the antibody or its preparation method (for example, by hybridoma, phage selection, recombinant expression, transgenic animals, etc.).

[0089] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins), wherein a portion of a heavy chain and / or a light chain is identical or homologous to a corresponding sequence in an antibody from a particular species, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody from another species, and fragments of such antibodies, as long as they exhibit desired biological activity. Typically, chimeric antibodies are antibodies whose light chain and heavy chain genes are usually constructed from antibody variable regions and constant region genes belonging to different species by genetic engineering. For example, variable gene fragments from mouse monoclonal antibodies can be connected to dog constant fragments. Figure 2 is a schematic diagram of the general structure of one embodiment of a mouse:canine IgG. In this embodiment, the antigen binding site is from mouse, while the Fc portion is canine.

[0090] The term "heterochimera" as defined herein refers to an antibody in which one of the antibody chains (heavy or light) is caninized and the other is chimeric. Figure 4 An embodiment of a heterologous chimeric molecule is depicted. In this embodiment, a caninized variable heavy chain (wherein all CDRs are mouse and all FRs are dog) is paired with a chimeric variable light chain (wherein 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 a canine constant region.

[0091] For simplicity, "caninized" antibodies are described below, but can also be applied to feline, equinized, humanized or any other "specialized" antigen-binding proteins. As an example, "caninization" is defined as a method for transferring non-canine antigen binding information from donor antibodies to a less immunogenic canine antibody receptor to produce a useful treatment for canine treatment. Caninized antibodies are canine antibody sequences (wherein the hypervariable region residues of the receptor are replaced by hypervariable region residues from non-canine species (donor antibodies), such as mice, rats, rabbits, cats, dogs, goats, chickens, cattle, horses, llamas, camels, dromedaries, sharks, non-human primates, people), humanized, recombinant sequences or engineered sequences with desired properties, specificity, affinity and ability. In addition, caninized antibodies may be included in receptor antibodies or residues not found in donor antibodies. These modifications are carried out to further improve the performance of the antibody. As described herein, modifications to the hypervariable and / or framework regions are determined for each individually engineered specialized (caninized) antibody based on experiments known to those skilled in the art but not predictable prior to the experiments. The caninized antibody optionally may include an intact portion or at least a portion of an immunoglobulin constant region (Fc), typically a canine immunoglobulin constant region. Figure 3 is a diagram showing one example of speciation or caninization of mouse IgG. All descriptions of caninization of antigen binding proteins and caninized antigen binding proteins are conceptually applicable to any specialized antibody, whether caninized, felinized, equinized, humanized, etc.

[0092] The phrases "recombinant canine antibody", "recombinant feline antibody", "recombinant equine antibody", "recombinant human antibody" and the like all encompass specific antibodies prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial canine (or feline, human, etc.) antibody library, antibodies isolated from animals (e.g., mice) transgenic for canine immunoglobulin genes (see, e.g., Taylor, LD et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created or isolated by any other means involving splicing of canine (or feline, human, etc.) immunoglobulin gene sequences to other DNA sequences.

[0093] As used herein, the terms "canine antibody", "cat antibody", "horse antibody", "human antibody", etc. refer to antibodies (antigen binding proteins) generated against a target and antibodies isolated from lymphocytes from within a target species. As described herein, these antibodies have been recombinantly modified in vitro to contain specific constant regions of the target species. In addition, antibodies as described herein are identified, isolated, modified to alter antibody constant regions, and subsequently expressed and isolated from in vitro cell culture systems known and routinely used by those skilled in the art.

[0094] "Native antibodies" and "natural immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. Figure 1 is an example of the general structure of native mouse immunoglobulin G (IgG) highlighting the antigen binding site.

[0095] The "parent" antibody herein is an antibody encoded by the amino acid sequence used to prepare a variant. Preferably, the parent antibody has a canine framework region and, if present, a canine antibody constant region. For example, the parent antibody can be a caninized antibody or a canine antibody.

[0096] According to the amino acid sequence of the constant domain of the heavy chain of the antibody, immunoglobulins can be divided into different categories. There are currently five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2 (as defined by mouse and human names). The heavy chain constant domains corresponding to different classes of immunoglobulins are respectively referred to as α, δ, ε, γ and μ. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known in a variety of species. The prevalence of individual isotypes and functional activities associated with these constant domains is species-specific and must be defined experimentally.

[0097] 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.

[0098] 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, alone or in combination. The variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs on each chain are tightly bound 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: (1) methods based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Chothia et al. (1989) Nature 342:877; Al-lazikani et al (1997) J. Molec. Bioi. 273:927-948). As used herein, a CDR may refer to a CDR defined by either approach or by a combination of both approaches.

[0099] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region includes amino acid residues from "complementarity determining regions" or "CDRs" (Kabat, et al. (1991) supra) and / or amino acid residues from "hypervariable loops" (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0100] As used herein, the term "antigen binding region" refers to the portion of an antibody molecule that contains amino acid residues that interact with an antigen and confer specificity and affinity to the antibody for the antigen. The antibody binding region includes "framework" amino acid residues that are necessary to maintain the correct conformation of the antigen binding residues.

[0101] 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; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be bound to a binding domain (e.g., an antibody variable domain), and can be assessed using various assays known in the art for assessing such antibody effector functions.

[0102] A "native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" or "mutated" or "mutant" Fc region includes an amino acid sequence that differs from a native sequence Fc region due to 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 the native sequence Fc region or the Fc region of a parent polypeptide, for example, about one to about ten amino acid substitutions in the native sequence Fc region or in the Fc region of a parent polypeptide, preferably about one to about five amino acid substitutions. The variant Fc region herein will preferably have at least about 80% sequence identity with the native sequence Fc region and / or with the Fc region of a parent polypeptide, and most preferably at least about 90% sequence identity therewith, more preferably at least about 95% sequence identity therewith. The variant or mutated Fc region may also substantially eliminate the function of the Fc region of the antibody. For example, the Fc region mutation may eliminate the effector function of the antibody. In one embodiment of the invention, the antibody of the invention comprises a mutated Fc region.

[0103] As used herein, "Fc receptor" and "FcR" describe receptors that bind to the Fc region of an antibody. Preferred FcRs are native sequence FcRs. In addition, preferred FcRs are FcRs that bind to IgG antibodies (gamma receptors) and include receptors of FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. FcRs are reviewed 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 responsible for the transfer of parental IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249).

[0104] As used herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcRs) recognize bound antibodies on target cells and subsequently cause the lysis of target cells. The ADCC activity of related molecules can be assessed using in vitro ADCC assays, such as described in U.S. Patent Nos. 5,500,362 or 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 related molecules can be assessed in vivo, for example, in an animal model, such as in Clynes et al., 1998, PNAS (USA), 95: In an animal model disclosed in 652-656.

[0105] "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 can be performed, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods, 202: 163 (1996).

[0106] 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, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0107] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that some residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the name of Fab' herein, in which the cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were originally produced in the form of paired Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0108] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to form a single heavy chain variable domain. H -VL The antigen binding site is defined on the surface of the dimer. These six hypervariable regions together give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv that includes only three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of the entire binding site.

[0109] As used herein, "antigen" refers to an antigenic determinant recognized by the CDRs 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, the term "epitope" as used herein refers to the region of NGF to which an anti-NGF antigen binding protein / antibody / agent binds.

[0110] The terms "antigen binding domain", "active fragment of an antibody", etc. refer to a portion of an antibody or antigen binding protein that includes a region that specifically binds to or is complementary to a portion or all of an antigen. In the case where the antigen is large, the antibody may only bind to a specific portion of the antigen. "Epitope", "active fragment of an epitope" or "antigenic determinant" etc. is a portion of an antigen molecule that is responsible for specific interaction with the antigen binding domain of an antibody. The antigen binding domain can be provided by one or more antibody variable domains (e.g., a so-called Fd antibody fragment consisting of a VH domain). The antigen binding domain can include an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH) (U.S. Pat. No. 5,565,332).

[0111] The term "binding portion" of an antibody (or "antibody portion") or antigen-binding polypeptide, etc., comprises one or more complete domains, such as a pair of complete domains, and a fragment of an antibody that retains the ability to specifically bind to an antigen (e.g., NGF). It has been shown that the binding function of an antibody can be performed by a fragment of a full-length antibody. The binding fragment is produced by recombinant DNA technology, or by enzymatic or chemical cleavage of a complete immunoglobulin. The binding fragment comprises 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), as well as isolated complementary determining regions (CDRs). The Fab fragment is a monovalent fragment consisting of VL, VH, CL and CH1 domains. The F(ab')2 fragment is a divalent fragment that includes two Fab fragments connected by a disulfide bond in the hinge region. The Fd fragment consists of the VH and CH1 domains, and the Fv fragment consists of the VL and VH domains of a single antibody arm. The dAb fragment consists of the VH domain (Ward et al., (1989) Nature 341: 544-546). Although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be connected by a synthetic linker using recombinant methods, which enables them to be formed into a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (known as single-chain Fv (scFv) (Bird et al., 1988, Science 242: 423-426). Such single-chain antibodies are also intended to be included in the term "binding portion" of antibodies. Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and generate two antigen-binding sites (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA The antibody or binding portion thereof can also be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides.Examples of such immunoadhesion molecules include the use of a streptavidin core region to make 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 make bivalent and biotinylated scFv molecules (Kipriyanov, SM et al. (1994) Mol. Immunol. 31:1047-1058). Binding fragments (e.g., Fab and F (ab') 2 fragments) can be prepared from whole antibodies using conventional techniques (e.g., papain or pepsin digestion of whole antibodies, respectively). In addition, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein and known in the art. Except for "bispecific" or "bifunctional" antibodies, antibodies are understood to have identical each binding site. "Bispecific" or "bifunctional antibodies" are artificial hybrid antibodies with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can also contain two antigen binding regions with an intermediate constant region. Bispecific antibodies can be produced by a variety of methods (including fusion of hybridomas or connection of Fab' fragments). See, for example, Songsivilai et al., Clin. Exp. Immunol. 79: 315-321, 1990.; Kostelny et al., 1992, J. Immunol. 148, 1547-1553.

[0112] The term "reverse mutation" 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 homologous germline antibody sequences. The heavy and light chain sequences of the canine antibodies of the present invention are aligned with the germline sequences, respectively, to identify the sequences with the highest homology. The differences of the canine antibodies of the present invention are returned to the germline sequences by mutating the defined nucleotide positions encoding such different amino acids. The role of each amino acid thus identified as a candidate for reverse mutation should be studied to understand the direct or indirect role in antigen binding, and any amino acid found to affect any desired properties of the canine antibody after mutation should not be included in the final canine antibody; as an example, the activity-enhancing amino acids identified by the selective mutagenesis method will not undergo reverse mutation. In order to minimize the number of amino acids undergoing reverse mutation, those amino acid positions that are found to be different from the nearest germline sequence but the same as the corresponding amino acid in the 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. It may be necessary to reversely mutate the selected target framework residues to the corresponding donor residues to restore and / or improve affinity.

[0113] As used herein, "immunospecific" binding of an antibody refers to the antigen-specific binding interaction that occurs between the antigen binding site of an antibody and the specific antigen recognized by the antibody (i.e., the antibody reacts with proteins in ELISA or other immunoassays, but 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 known 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 binds to a specific cell or substance more frequently, more rapidly, with a longer duration, and / or with greater affinity than an alternative cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds to the target with greater affinity, avidity, more easily, and / or longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to an NGF epitope is an antibody that binds to the epitope with greater affinity, avidity, greater ease, and / or greater duration than it binds to other NGF epitopes or non-NGF epitopes. It can also be understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally speaking, but not necessarily, reference to binding means preferential binding.

[0114] In the context of antibody binding, the term "specific" refers to the high affinity and / or high avidity binding of an antibody to a specific antigen (i.e., a polypeptide or epitope). An antibody that specifically binds to an antigen binds more strongly than the same antibody to other antigens. An antibody that specifically binds to a polypeptide may be able to bind to other polypeptides at a weak but detectable level (e.g., shown as 10% or less binding to the relevant polypeptide). Such weak or background binding is easily distinguished from specific antibody binding to the subject polypeptide, for example, by using appropriate controls. Typically, specific antibodies are detected at 10% or less binding to the subject polypeptide. -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or less or 10 -13 M or smaller K d The binding affinity of the antigen is bound to the antigen.

[0115] As used herein, the term "affinity" refers to the intensity of a single antigen binding site combined with an antigenic determinant. Avidity depends on the closeness of stereochemical matching between an antibody or antigen-binding protein binding site and an 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 by surface plasmon resonance "SPR" method (such as BIACORETM). The SPR method relies on the phenomenon of surface plasmon resonance (SPR), which occurs when surface plasma waves are excited at a metal / liquid interface. Light is directed to one side of a surface that is not in contact with a sample and is reflected from the side, and at a specific angle and wavelength combination, SPR causes the intensity of reflected light to decrease. Bimolecular binding events cause changes in the refractive index at the surface layer, and this change is detected as a change in the SPR signal.

[0116] As used herein, the term "K D ” is intended to refer to the dissociation constant of the antibody-antigen interaction. The dissociation constant K D The association constant, Ka, 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 the rate constant, k a and k d Quantify the rate of each reaction. At equilibrium, ka[Ab][Ag] = kd[Ag-Ab]. Dissociation constant K d It is given by: Kd = kd / ka = [Ag][Ab] / [Ag-Ab]. D The units with concentration are typically M, mM, μM, nM, pM, etc. D When expressing antibody affinity, a greater affinity for NGF is represented by a lower value. a It is given by: Ka = ka / kd = [Ag-Ab] / [Ag] [Ab]. Ka has units of inverse concentration, the most typical being M -1 、mM -1 , μ.M -1 、nM -1 , pM -1 As used herein, the term "avidity" refers to the strength of the antigen-antibody bond after the formation of a reversible complex. Anti-NGF antibodies can be classified according to their K affinity for binding to NGF protein. D to characterize it as "at about (below K D value) to about (upper K D The dissociation constant (K D )” combination.

[0117] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may include modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as binding to a labeling component). In addition, polypeptides containing, for example, one or more analogs of amino acids (including, for example, non-natural amino acids, etc.) and other modifications known in the art are included in the definition. It should be understood that because the polypeptides of the present invention are based on antibodies, the polypeptides may exist in the form of single chains or related chains.

[0118] The term "conservative amino acid substitution" means any amino acid substitution for a given amino acid residue, wherein the substituted residue is so similar chemically to the given residue that it does not result in a significant reduction in polypeptide function (e.g., enzymatic activity). Conservative amino acid substitutions are well known in the art, and examples thereof are described in, for example, U.S. Patents 6,790,639, 6,774,107, 6,194,167, or 5,350,576. In a preferred embodiment, a conservative amino acid substitution will be any one of the following six groups:

[0119] Small aliphatic, essentially nonpolar residues: Ala, Gly, Pro, Ser and Thr;

[0120] Large aliphatic nonpolar residues: Ile, Leu and Val; Met;

[0121] Polar negatively charged residues and their amides: Asp and Glu;

[0122] Amides of polar negatively charged residues: Asn and Gin; His;

[0123] Polar, positively charged residues: Arg and Lys; His; and

[0124] Large aromatic residues: Trp and Tyr; Phe.

[0125] In a preferred embodiment, the conservative amino acid substitution will be any of the following, which are listed as pairs of natural residues (conservative substitutions): Ala (Ser); Arg (Lys); Asn (Gin; His); Asp (Glu); Gin (Asn); Glu (Asp); Gly (Pro); His (Asn; Gin); 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).

[0126] The terms "nucleic acid", "polynucleotide", "nucleic acid molecule", etc. are used interchangeably herein and refer to a series of nucleotide bases (also referred to as "nucleotides") in DNA and RNA. Nucleic acids can contain deoxyribonucleotides, ribonucleotides and / or their analogs. The term "nucleic acid" includes, for example, single-stranded and double-stranded molecules. Nucleic acids can be, for example, genes or gene fragments, exons, introns, DNA molecules (e.g., cDNA), RNA molecules (e.g., mRNA), recombinant nucleic acids, plasmids and other vectors, primers and probes. Contains 5' to 3' (sense) and 3' to 5' (antisense) polynucleotides. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or can be incorporated into any substrate in a polymer by DNA or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. If present, the nucleotide structure can be modified before or after the assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. After polymerization, the polynucleotides can be further modified, for example, by combining with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing side chain moieties (such as, for example, 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, oxidized metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides. In addition, any hydroxyl group typically present in the sugar may be substituted, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to a solid support. The 5' and 3' terminal OH may be phosphorylated or substituted with amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls can also be derived into standard protecting groups. Polynucleotides can also contain ribose or deoxyribose sugars of similar forms well known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-ribose or 2'-azido-ribose, carbocyclic sugar analogs, anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose), acyclic analogs and abasic nucleoside analogs (such as methyl riboside). One or more phosphodiester bonds can be replaced by alternative linking groups.These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("disulfide"), "(O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("methylal"), wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-2C), optionally containing an ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0127] As used herein, "vector" refers to a construct capable of delivering and preferably expressing one or more related genes or sequences in 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 production cells. As described herein, a vector has an expression control sequence, which means a nucleic acid sequence that instructs nucleic acid transcription. An expression control sequence can be a promoter (such as a constitutive or inducible promoter), or an enhancer. An expression control sequence is 'operably connected' to a nucleic acid sequence to be transcribed. When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably connected". For example, if it is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA of the presequence or secretion leader is operably connected to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a sequence, the promoter or enhancer is operably connected to a coding sequence; or if a ribosome binding site is positioned to promote translation, the ribosome binding site is operably connected to a coding sequence. Typically, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a secretory leader, continuous and in reading phase. However, enhancers do not have to be continuous. Linking is accomplished by ligating at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or joints are used according to conventional practice.

[0128] Just as a polypeptide can contain conservative amino acid substitutions, its polynucleotide can contain conservative codon substitutions. As described above, a codon substitution is considered conservative if it produces a conservative amino acid substitution when expressed. Degenerate codon substitutions (which do not result in amino acid substitutions) can also be used in polynucleotides according to the present invention. Thus, for example, a polynucleotide encoding a selected polypeptide useful in an embodiment of the present invention can be mutated by degenerate codon substitutions in order to approach the codon usage frequency exhibited by the expression host cell to be transformed therewith, or to otherwise improve its expression.

[0129] "Variant" anti-NGF antigen binding protein refers herein to a molecule that differs from the "parent" anti-NGF antibody amino acid sequence in amino acid sequence due to the addition, deletion and / or substitution of one or more amino acid residues in the parent antibody sequence, and retains at least one desired activity of the parent anti-NGF antibody. As described herein, variant anti-NGF can include conservative amino acid substitutions in the hypervariable region of the antibody. The desired activity can include the ability to specifically bind to an antigen, the ability to reduce, inhibit or neutralize NGF activity in an animal. In one embodiment, the variant includes one or more amino acid substitutions in one or more hypervariable regions and / or framework regions of the parent antibody. For example, the variant may include at least one substitution, such as about one to about ten, and preferably about two to about five substitutions, in one or more hypervariable regions and / or framework regions of the parent antibody. Typically, the variant will have an amino acid sequence having at least 50% amino acid sequence identity with the parent antibody heavy chain or light chain variable domain sequence, more preferably at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity. Identity or homology with respect to the 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 obtain the maximum percentage of sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions in the antibody sequence should not be construed as affecting sequence identity or homology. The variant retains the ability to bind to NGF and preferably has the same or superior desired activity to the parent antibody. For example, the variant may have a stronger binding affinity, an enhanced ability to reduce, inhibit or neutralize NGF activity in animals and / or an enhanced ability to inhibit the binding of NGF to TrkA and p75.

[0130] TrkA, which is considered a high affinity NGF receptor, is a member of the neurotrophic tyrosine kinase receptor (NTKR) family. This kinase is a membrane-bound receptor that, upon neurotrophin binding, autophosphorylates (autophosphorylates) and phosphorylates members of the MAPK pathway. The presence of this kinase leads to cell differentiation and may play a role in determining sensory neuron subtypes. The p75 receptor is considered a low affinity NGF receptor.

[0131] "Variant" nucleic acid refers herein to a molecule whose sequence is different from that of a "parent" nucleic acid. Polynucleotide sequence differences may be caused by changes in mutations, such as deletions, substitutions or additions of one or more nucleotides. Each of these changes may occur one or more times in a given order, either alone or in combination.

[0132] The term "isolated" refers to separating and / or recovering materials (e.g., antigen binding proteins or nucleic acids as described herein) from components of their natural environment. The pollutant components of their natural environment are materials that interfere with the diagnostic or therapeutic use of the material, and may include enzymes, hormones, and other proteins or non-protein solutes. With regard to nucleic acids, isolated nucleic acids may include nucleic acids isolated from 5' to 3' sequences, which are usually associated with them in chromosomes. In a preferred embodiment, the material will be purified to greater than 95% by weight of the material, and most preferably greater than 99% by weight. The isolated material includes the in situ material in the recombinant cell, because at least one component in the natural environment of the material will not exist. However, typically, the isolated material will be prepared by at least one purification step.

[0133] As used herein, the terms "cell", "cell line" and "cell culture" can be used interchangeably. These terms also include their offspring, which are all the next generation. It should be understood that all offspring may not be identical due to intentional or unintentional mutations. In the context of expressing heterologous nucleic acid sequences, "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 can be located in a transgenic animal. A host cell can be used as a receptor for a vector and can include any transformable organism capable of replicating a vector and / or expressing a heterologous nucleic acid encoded by a vector.

[0134] The term "label" as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody or nucleic acid. The label itself can be detectable by itself (e.g., a radioisotope label or a fluorescent label), or in the case of an enzyme label, can catalyze chemical alteration of a detectable substrate compound or composition.

[0135] "Subject" or "patient" refers to an animal in need of treatment, which may be affected by the molecules of the present invention. Animals that can be treated according to the present invention include vertebrates, among which mammals such as dogs are particularly preferred examples.

[0136] A "composition" is intended to mean a combination of an active agent, whether a chemical composition, a biological composition or a biotherapeutic agent (particularly an antigen binding protein as described herein), and another compound or composition, such as an adjuvant, which may be inert (e.g., a label) or active.

[0137] 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, alcohol / water solution, emulsion or suspension. 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 regulators 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, such as citric acid salts, citrates, ascorbates, gluconates, histidine-Hel salts, carbonates, tartrates, succinates, acetates or phthalates, Tris salts, trimethylamine hydrochloride salts or phosphate buffers. Parenteral carriers may include sodium chloride solution, Ringer's dextrose, glucose, trehalose, sucrose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous carriers may include liquid and nutrient supplements, electrolyte supplements, such as those based on Ringer's dextrose, etc. Preservatives and other additives, such as, for example, antimicrobial agents, antioxidants, chelating agents (e.g., EDTA), inert gases, etc., may also be provided in the pharmaceutical carrier. The present invention is not limited by the choice of carrier. It is within the skill of the art to prepare these pharmaceutically acceptable compositions from the above components, which have suitable pH isotonicity, stability, and other conventional characteristics. See, for example, articles such as Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, publ., 2000; and The Handbook of Pharmaceutical Excipients, 4.sup.th edit., eds. RC Rowe et al, APhA Publications, 2003.

[0138] 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 result when administered to a subject or patient. An effective amount can be administered in one or more administrations, applications, or doses. A therapeutically effective amount of a composition according to the present invention can be readily determined by a person of ordinary skill in the art. In the context of the present invention, a "therapeutically effective amount" refers to an amount that produces an objectively measured change in one or more parameters associated with a condition associated with NGF, an amount sufficient to produce a beneficial or desired result, including a clinical result, such as a reduction or decrease in pain sensation. An effective amount can be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to treat, improve, reduce the intensity of pain, and / or prevent pain, including postoperative pain, rheumatoid arthritis pain, and / or osteoarthritis pain. In some embodiments, an "effective amount" can reduce pain at rest (resting pain) or mechanically induced pain (including pain after exercise), or both, and it can be administered before, during, or after a painful stimulus. As understood in clinical settings, an effective amount of a drug, compound or pharmaceutical composition may or may not be combined with another drug, compound or pharmaceutical composition to achieve. Therefore, "effective amount" can be considered in the case of administering one or more therapeutic agents, and if combined with one or more other medicaments, a desired result can be obtained or obtained, then it can be considered to administer a single medicament in an effective amount. Of course, the therapeutically effective amount will vary according to the specific subject and the disease treated, the subject's weight and age, the severity of the disease, the specific compound selected, the dosage regimen to be followed, the administration time, the mode of administration, etc., all of which can be easily determined by a person of ordinary skill in the art.

[0139] As used herein, the term "therapeutic" encompasses the full range of treatments for a disease, disorder, or condition. The "therapeutic" agents of the invention may act in a prophylactic or preventative manner, including those combined with procedures for target animals that can be identified as being at risk (pharmacogenetics); or in an essentially ameliorative or curative manner; or may be used to slow the rate or extent of progression of at least one symptom of the disease or condition being treated.

[0140] In a further aspect, the invention features a veterinary composition in which an antibody of the invention is provided for therapeutic or prophylactic use. The invention features a method for treating a canine subject having a specific antigen (e.g., an antigen associated with a disease or condition). The method comprises administering a therapeutically effective amount of a recombinant antibody specific for a specific antigen together with a recombinant antibody described herein.

[0141] The amount of the antibody that can be used to produce the therapeutic effect can be determined by standard techniques well known to those of ordinary skill in the art. The antibody will usually be provided in a pharmaceutically acceptable buffer by standard techniques, and can be administered by any desired approach. The route of administration of the antibody or antigen-binding portion of the present invention can be oral, parenteral, by inhalation or topical. In a preferred embodiment, the route of administration is parenteral. The term parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal or intraperitoneal administration.

[0142] As used herein, "pain" refers to pain of any etiology, including acute and chronic pain, and any pain with an inflammatory component. Examples of pain include inflammatory pain, postoperative incisional pain, neuropathic pain, fracture pain, osteoporotic fracture pain, postherpetic neuralgia, cancer pain, pain caused by burns, pain associated with burns or wounds, pain associated with trauma (including traumatic head injury), neuropathic pain, pain associated with musculoskeletal disorders (e.g., rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatoid) arthropathy, non-articular rheumatism, and periarticular disorders), and cancer-related pain (including "breakthrough pain" and pain associated with advanced cancer), peripheral neuropathy, and postherpetic neuralgia.

[0143] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical outcome. For the purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: improvement or alleviation of any aspect of pain, including acute pain, chronic pain, inflammatory pain, neuropathic pain, postoperative pain, rheumatoid arthritis pain, or osteoarthritis pain. For the purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: including reduction in 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).

[0144] As described 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, osteoporotic fracture pain, postherpetic neuralgia, cancer pain, pain caused by burns, pain associated with burns or wounds, pain associated with trauma (including traumatic head injuries), neuropathic pain, pain associated with musculoskeletal disorders (e.g., rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatoid) arthritis, non-articular rheumatism and periarticular disorders) and cancer-related pain (including "breakthrough pain" and pain associated with advanced cancer), peripheral neuropathy and postherpetic neuralgia.

[0145] "Reducing the incidence of pain" refers to any of a reduction in severity (which may include reducing the need for and / or amount (e.g., exposure to other drugs and / or therapies) typically used for the condition, including, for example, opiates), duration, and / or frequency (including, for example, delaying or increasing the duration of postoperative pain in an individual). As will be appreciated by those skilled in the art, individuals may vary in their response to treatment, and thus, for example, "a method for reducing the incidence of rheumatoid arthritis pain or osteoarthritis pain in an individual" reflects the administration of an anti-NGF antagonist antibody based on a reasonable expectation that such administration may result in such a reduction in incidence in that particular individual.

[0146] "Improving" pain or one or more symptoms of pain (eg, rheumatoid arthritis pain or osteoarthritis pain) means a reduction or improvement in one or more symptoms of pain compared to not administering the anti-NGF antagonist antibody. "Improving" also encompasses shortening or reducing the duration of symptoms.

[0147] "Reducing" pain or one or more symptoms of pain (eg, rheumatoid arthritis pain or osteoarthritis pain) means reducing the extent of one or more undesirable clinical manifestations of postoperative pain in an individual or population of individuals treated with an anti-NGF antagonist antibody according to the invention.

[0148] As used herein, "delaying" the development of pain means delaying, hindering, slowing down, delaying, stabilizing and / or postponing the progress of pain (e.g., postoperative pain, rheumatoid arthritis pain, or osteoarthritis pain). This delay can be different lengths of time, depending on the history of the disease and / or the individual being treated. As is apparent to those skilled in the art, sufficient or significant delays can actually comprise prevention, because the individual will not develop pain. The method of "delaying" the development of symptoms is a method of reducing the probability of developing symptoms and / or reducing the degree of symptoms within a given time frame when compared to not using the method. Such comparisons are usually based on clinical studies, using statistically significant numbers of subjects.

[0149] "Postoperative pain" (interchangeably referred to as "post-incisional pain" or "post-traumatic pain") refers to pain caused or resulting from external trauma, such as cuts, punctures, incisions, tears or wounds in individual tissues (including trauma caused by all surgical procedures, whether invasive or non-invasive). As used herein, postoperative pain does not include pain that occurs (produced or originated) in the absence of external physical trauma. In some embodiments, postoperative pain is internal or external (including peripheral) pain, and the wound, cut, trauma, tear or incision may occur accidentally (such as traumatic wounds) or intentionally (such as surgical incisions). As used herein, "pain" includes nociception and pain sensation, and pain can be objectively and subjectively assessed using pain scores and other methods known in the art. Postoperative pain as used herein includes allodynia (i.e., an increased response to a stimulus that is usually harmless) and hyperalgesia (i.e., an increased response to a stimulus that is usually harmful or unpleasant), which in turn can be thermal or mechanical (tactile) in nature. In some embodiments, pain is characterized by thermal sensitivity, mechanical sensitivity and / or rest pain. In some embodiments, postoperative pain includes mechanically induced pain or rest pain. In other embodiments, postoperative pain includes rest pain. As is known in the art, pain can be primary or secondary pain.

[0150] Before describing the method of the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be restrictive, because the scope of the present invention will be limited only by the appended claims.

[0151] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the 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, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

[0152] The invention disclosed herein relates to antigen binding proteins (as described herein, used interchangeably with the terms "antibody", "antagonist antibody", "antibody fragment", etc.) that specifically bind to nerve growth factor (NGF), and in particular antibodies (whether they are canine, feline, equine, murine, bovine, human or any other species), caninized, feline, bovine, equine, humanized or any other specialized antibody produced by recombinant methods, hybridoma technology or phage display technology or fully "caninized" (specialized) monoclonal antibodies that specifically bind to NGF and thereby prevent NGF from binding to canine TrkA, and to a lesser extent to canine p75 receptors, thereby acting as antagonists because the signaling pathway is prevented from being activated by NGF.

[0153] NGF is the first neurotrophic factor to be identified, and its role in the development and survival of peripheral and central neurons has been well characterized. NGF has been shown to be a key survival and maintenance factor for the development of peripheral sympathetic neurons and embryonic sensory neurons as well as basal forebrain cholinergic neurons (Smeyne et al. (1994) Nature 368: 246-249; Crowley et al. (1994) Cell 76: 1001-1011). NGF upregulates the expression of neuropeptides in sensory neurons (Lindsay et al. (1989) Nature 337: 362-364) and its activity is mediated by two different membrane-bound receptors (TrkA receptor and p75 general neurotrophic factor receptor, which is considered to be of low affinity).

[0154] In NGF-related disorders, NGF has been shown to be elevated, wherein elevated amounts of NGF are present in injured or diseased tissues. NGF-related disorders can be defined as increased pain caused by elevated levels of NGF in injured, diseased or damaged tissues. Pain as used herein, as defined herein, refers to a disorder comprising chronic pain, inflammatory pain, postoperative incisional pain, neuropathic pain, fracture pain, osteoporotic fracture pain, postherpetic neuralgia, cancer pain, pain caused by burns, pain associated with burns or wounds, pain associated with trauma (including traumatic head injuries), neuropathic pain, pain associated with musculoskeletal disorders (e.g., chronic pain, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatoid) arthropathy, non-articular rheumatism, and periarticular disorders), and cancer-related pain (including "breakthrough pain" and pain associated with advanced cancer), peripheral neuropathy, and postherpetic neuralgia.

[0155] In an embodiment of the present invention, NGF disorder is defined as osteoarthritis of a subject (canine, cat, horse, human, etc.). Osteoarthritis (OA) is a slowly progressive degenerative joint disease characterized by the loss of articular cartilage and subsequent exposure of subchondral bone in dogs. This ultimately leads to a self-perpetuating recessive disorder characterized by joint pain. In an attempt to limit movement and pain, the formation of new bone occurs in response to chronic inflammation and local tissue damage. Macroscopically, there is a loss of articular cartilage, narrowing of the joint space, sclerosis of the subchondral bone, and the generation of joint osteophytes (Veterinary Focus: Vol 17 No 3; 2007).

[0156] In different species such as dogs, cats, horses, etc., the onset of primary OA depends on the breed. For example, for dogs, the average age of onset is 3.5 years in Rottweilers and 9.5 years in poodles, for example, with a wide range of average ages of onset for other breeds and mixed breeds. Developmental orthopedic diseases and related osteoarthritis are the most common joint diseases in dogs, accounting for about 70% of medical visits for joint diseases and related problems within the accessory skeleton. Twenty-two percent of cases are dogs one year old or younger. The incidence of OA is increased by trauma as well as obesity, aging and genetic abnormalities. In particular, age can be a factor in the incidence of OA, with >50% of arthritis cases observed in dogs aged 8-13 years. Musculoskeletal diseases are very common in elderly patients, and nearly 20% of elderly dogs show orthopedic conditions. In Labrador Retrievers aged >8 years, OA in several joints (elbows, shoulders, hips, knees) is typical. In addition, the size of the dog also plays a role in the onset of OA. 45% of dogs with arthritis are large breeds. Of these, >50% were large breeds, while only 28% were medium breeds, and 27% were small breeds. The need for pharmacological interventions to relieve pain in canine OA is very high.

[0157] As described herein, elevated levels of NGF indicate NGF-related disorders, particularly OA. Elevated levels of NGF have been reported in transgenic arthritic mice, as well as an increase in the number of mast cells (Aloe et al., Int. J. Tissue Reactions-Exp. Clin. Aspects 15: 139-143 (1993)). PCT Publication No. WO 02 / 096458 discloses the use of anti-NGF antibodies of certain properties in the treatment of various NGF-related disorders such as inflammatory disorders (e.g., rheumatoid arthritis). It is reported that purified anti-NGF antibodies injected into arthritic transgenic mice carrying the human tumor necrosis factor gene resulted in a decrease in the number of mast cells, as well as a decrease in the levels of histamine and substance P in the synovium of arthritic mice (Aloe et al., Rheumatol. Int. 14: 249-252 (1995)). It has been shown that exogenous administration of NGF antibodies reduces the enhanced 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-murine mammals, anti-mouse antibody responses are generated in a large number of treated individuals. Therefore, there is a great need for anti-NGF antagonist antigen binding proteins, including anti-NGF antagonist antibodies of the present invention, for canine use, particularly for the treatment of OA.

[0158] Although the properties of antibodies make them very attractive therapeutic agents, there are many limitations. As mentioned above, the vast majority of monoclonal antibodies (mAbs) are rodent-derived. When such antibodies are administered in different species, patients can produce their own antibody responses to such heterologous antibodies. Such responses may lead to the final neutralization and elimination of antibodies. As mentioned above, mice are widely used to produce monoclonal antibodies. A problem with using antibodies produced by a specific species (usually initially in mice) is that non-mouse subjects treated with the antibodies react to mouse antibodies as if they were foreign substances, thereby producing a new set of antibodies against mouse antibodies. Mouse antibodies are regarded as foreign by non-mouse (e.g., canine) immune systems, and then the subject produces an immune response to the molecule. Those skilled in the art will recognize the need to be able to treat subjects with antigen-specific antibodies, but with the specificity of the antibody species. Partial reactions produced by cross-species antibody administration (e.g., mouse monoclonal antibodies administered to dogs) can range from mild forms (e.g., rashes) to more extreme and life-threatening reactions (e.g., renal failure). This immune response can also reduce the effectiveness of the treatment, or can produce a future reaction if the subject is given a subsequent treatment containing the mouse antibody. We therefore set out to overcome this shortcoming by "caninization" of antibodies. In particular, the process focuses on the framework regions of the immunoglobulin variable domains, but can also include the complementarity determining regions (CDRs) of the variable domains. The enabling steps and practical simplifications of this process are described in this disclosure.

[0159] The process of modifying monoclonal antibodies from animals (such as antigen binding proteins, antagonist antibodies, etc. and interchangeable terms described herein) to make them less immunogenic for species therapeutic administration has been actively explored and has been described in many publications (e.g., Antibody Engineering: A practical Guide. Carl A. K. Borrebaeck ed. WH Freeman and Company, 1992). However, until recently, the process has not been applied to the development of non-human, especially canine treatments or diagnoses. In fact, there are few reports on canine variable domains. Wasserman and Capra, Biochem. 6, 3160 (1977) determined the amino acid sequences of the variable regions of canine IgM and canine IgA heavy chains. Wasserman and Capra, Immunochem. 15, 303 (1978) determined the amino acid sequence of the kappa light chain from canine IgA. McCumber and Capra, Mol. Immunol. 16, 565 (1979) disclosed the complete amino acid sequence of the canine μ chain. Tang et al., Vet. Immunology Immunopathology 80, 259 (2001) discloses a single canine IgG-A gamma chain cDNA and four canine IgG-A gamma chain protein sequences. It describes PCR amplification of a cDNA library from canine spleen using degenerate oligonucleotide primers designed from conserved regions of human, mouse, pig and bovine IgG. The lack of available information on canine antibodies has hampered their development as a therapy for treating canine diseases.

[0160] These notable limitations have promoted the development of an engineering technique known as "speciation" and are well known to those skilled in the art in terms of the "humanization" of therapeutic antibodies. As an example of a specialized molecule, caninized antibodies can be produced in the form of chimeric antibodies or fragments thereof, which contain minimal sequences derived from non-canine immunoglobulins. In most cases, caninized antibodies are canine antibodies (i.e., "receptor antibodies" or "target species antibodies"), in which residues from the complementary determining regions (CDRs) of the receptor are replaced by residues from the CDRs of non-canine species (i.e., "donor antibodies" or "source species antibodies"), such as mice, with desired properties, such as specificity, affinity, and efficacy. In some cases, the framework region (FR) residues of canine immunoglobulins are replaced by corresponding non-canine residues. This caninization strategy is referred to as "CDR grafting". It may be necessary to reversely mutate the selected target framework residues to the corresponding donor residues to restore and / or improve affinity. As described in US 7,261,890, structure-based methods can also be used for caninization and affinity maturation.

[0161] The above method has basically utilized the whole framework region of one or more antibody variable heavy chains or variable light chains from the target species, and these antibody variable heavy chains or variable light chains are engineered to receive CDR from donor species.When the antibody is felinized in the same manner as caninization so that its antigenicity is reduced when it is applied to cats, this method is also used.In some cases, the reverse mutation of selected residues in the variable region is used to enhance the expression of CDR.Design antibody (this antibody minimizes the immunogenicity reaction of the subject to the non-native sequence in the antibody) fully retains the antigen binding region of the antibody to keep effectiveness simultaneously, has been proved to be challenging.

[0162] Another challenge in developing therapeutic antibodies that target proteins is that epitopes on homologous proteins in different species are often different and have different potentials to cross-react with other proteins. Therefore, antibodies must be made, tested, and developed against specific targets in the specific species to be treated.

[0163] Antibodies target antigens by binding to specific epitopes on antigens through interactions with the variable regions of antibody molecules. In addition, antibodies have the ability to mediate, inhibit (as in the case of antagonistic anti-NGF antigen-binding proteins of the present invention) and / or initiate a variety of biological activities. Therapeutic antibodies have a wide range of functions, for example, antibodies can be used as agonists or antagonists to regulate receptor-ligand interactions. Antibody binding can initiate intracellular signaling to stimulate cell growth, cytokine production or apoptosis. Antibodies can deliver agents bound to the Fc region to specific sites. Antibodies also trigger antibody-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC) and phagocytosis. There are also antibodies that have been altered, in which ADCC, CDC, C1q binding and phagocytosis functions have been eliminated. In one embodiment of the invention, the antibodies of the present invention include changes in the Fc region of antibodies that change the effector functions of the antibodies.

[0164] Caninization and felinization

[0165] As used herein, a "caninized antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a dog and / or an antibody that has been prepared using any technique known in the art or disclosed herein. The same process is used for the felinization process and should be applied to the description here. For simplicity, caninization will be used primarily as examples, however these examples are not limited to dogs. The same concepts and designs apply to the speciation of other antigen-binding proteins, such as cats, horses, humans, etc. This definition of caninized antibodies includes antibodies that include at least one canine heavy chain polypeptide or at least one canine light chain polypeptide. The "speciation" of antibodies itself, and in particular the humanization of antibodies, are areas of research well known to those skilled in the art. Until recently, it was not known whether the speciation of antibodies other than humanization would produce therapeutic antibodies that would be effective for any other species. The present invention illustrates the caninization and felinization of anti-NGF antigen-binding proteins for therapeutic use in dogs and cats, respectively.

[0166] Chimeric antibodies include sequences from at least two different species. As an example, recombinant cloning techniques can be used to comprise variable regions containing the antigen binding site from a non-recipient antibody (ie, an antibody prepared in a donor species immunized with the antigen) and constant regions from a recipient immunoglobulin.

[0167] A specialized (caninized, feline, etc.) antibody is a chimeric antibody in which the variable region residues responsible for antigen binding (i.e., residues of the complementary determining region, abbreviated complementary determining region residues, or any other residues involved in antigen binding) are from non-canine (or non-feline) species, while the remaining variable region residues (i.e., residues of the framework region) and the constant region are at least partially from canine (or feline) antibody sequences. A subset of the framework region residues and constant region residues of the specialized antibody may be from a non-canine (or feline) source. The variable regions of specialized antibodies are also described as specialized (i.e., specialized light or heavy chain variable regions). Non-specialized species are typically species used for immunization with antigens, such as mice, rats, rabbits, non-human primates, or other non-canine or non-feline mammalian species.

[0168] Complementarity determining region (CDR) is the residue of antibody variable region that participates in antigen binding. Several numbering systems for identifying CDR are commonly used. The Kabat definition is based on the variability of sequence, while the Clothia definition is based on the position of structural loop region. The definition of AbM is a compromise between Kabat and Clothia methods. Specialized antibodies of the present invention can be constructed to include one or more CDRs. In addition, CDRs can be used alone or in combination for synthetic molecules such as SMIP and small antibody mimics.

[0169] Framework residues are those residues of the variable region of the antibody, rather than hypervariable or CDR residues. Framework residues can be derived from naturally occurring canine (e.g., but conceptually applicable to other species, such as cats, horses, humans, etc. For simplicity, dogs will be used as representative species, but examples are not limited to dogs themselves) antibodies, such as canine frameworks substantially similar to the framework regions of the antibodies of the present invention. Artificial framework sequences representing the consistency between a single sequence can also be used. When selecting a framework region for caninization, sequences widely expressed in dogs may be superior to sequences from a smaller population. Additional mutations can be made to canine framework receptor sequences to restore mouse residues believed to be involved in antigen contact and / or residues involved in the structural integrity of the antigen binding site, or to improve antibody expression.

[0170] The grafting of CDRs is performed by replacing one or more CDRs of an acceptor antibody (e.g., a caninized antibody or other antibody comprising desired framework residues) with a CDR of a donor antibody (e.g., a non-canine antibody). The acceptor antibody can be selected based on the similarity of framework residues between the candidate acceptor antibody and the donor antibody. For example, a canine framework region is identified as having substantial sequence homology with each framework region of a related non-canine antibody, and the CDRs of the non-canine antibody are grafted onto a composition of different canine framework regions.

[0171] Analysis of the three-dimensional structure of the antibody-antigen complex, combined with analysis of available amino acid sequence data, can be used to model sequence variability based on the structural differences of the amino acid residues that appear at each position within the CDR. The CDR of the present invention can also be used for small antibody mimics, which include two CDR regions and a framework region (Qui et al. Nature Biotechnology Vol 25; 921-929; August 2007).

[0172] The acceptor framework for grafting of CDRs or abbreviated 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 to the donor and / or acceptor sequences to optimize antibody binding and functionality. See, for example, International Publication No. WO 91 / 09967.

[0173] The present invention further provides cells and cell lines expressing the antibodies of the present invention. Representative host cells include bacteria, yeast, mammals and human cells, such as CHO cells, HEK-293 cells, HeLa cells, CV-1 cells and COS cells. Methods for producing stable cell lines after transforming heterologous constructs into host cells are known in the art. Representative non-mammalian host cells include insect cells (Potter et al. (1993) Int. Rev. Immunol. 10 (2-3): 103-112). Antibodies can 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).

[0174] As discussed above, monoclonal antibodies, chimeric antibodies, species-specific antibodies, and specialized antibodies are also within the scope of the invention, which have been modified, for example, by deletion, addition, or replacement of other portions of the antibody, such as the constant region. For example, an antibody can be modified as follows: (i) by deleting the constant region; (ii) by replacing the constant region with another constant region, such as a constant region that is meant 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 change, for example, the number of glycosylation sites, effector cell function, Fc receptor (FcR) binding, complement fixation, etc. In one embodiment of the invention, the antibodies of the invention include an altered Fc region that alters the effector function of the antibody. In some embodiments of the invention, the Fc region of the antigen binding protein of the invention has been replaced, modified, or removed.

[0175] Methods for altering antibody constant regions are known in the art. Antibodies with altered functions, such as altered affinity for effector ligands (e.g., FcRs on cells), or the C1 component of complement can be generated by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP 388,151A1, U.S. Pat. No. 5,624,821, and U.S. Pat. No. 5,648,260, the entire contents of which are incorporated herein by reference).

[0176] For example, the affinity of the Fc region of an antibody for FcR (e.g., FcγR1) or for C1q binding can be altered by replacing a specified residue with a residue having an appropriate functional group on its side chain, or introducing a charged functional group (e.g., glutamate or aspartate), or possibly an aromatic non-polar residue (e.g., phenylalanine, tyrosine, tryptophan, or alanine) (see, e.g., U.S. Pat. No. 5,624,821). The antibody or its binding fragment can be conjugated to a cytotoxin, a therapeutic agent, or a radioactive metal ion. In one embodiment, the conjugated protein is an antibody or fragment thereof. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Non-limiting examples include calicheamicin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothiophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (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.

[0177] Compositions, derived compositions and methods of making compositions

[0178] The present invention includes compositions (including pharmaceutical compositions) comprising antigen binding proteins (such as "antibodies", "antibody fragments", "antagonist antibodies", etc., which are used interchangeably herein), polypeptides and polynucleotides, which include sequences encoding antigen binding proteins or polypeptides of the present invention.

[0179] As used herein, compositions include 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 include suitable excipients, such as pharmaceutically / veterinarily acceptable excipients, including buffers, which are well known in the art. The present invention also includes isolated antibody, polypeptide and polynucleotide embodiments. The present invention also includes substantially pure antibody, polypeptide and polynucleotide embodiments.

[0180] 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 protein is defined as an antibody or antibody fragment. In one or more embodiments, the antigen binding protein is fully canine, fully feline, fully bovine, fully equine, fully human, caninized, feline, equine or humanized. In one or more embodiments, the antigen binding protein of the present invention binds 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 and activation to its receptor TrkA, and to a lesser extent to p75, thereby preventing the signal cascade as described herein. The antigen binding protein of the present invention is identified herein as ZTS-841.

[0181] In one or more embodiments, the present invention provides an isolated and recombinant antigen binding protein "ZTS-841", wherein the variable heavy 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 the amino acid sequence comprising SEQ ID NO.4 ("ZTS-841" 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 the amino acid sequence comprising SEQ ID NO.5 ("ZTS-841" 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 the amino acid sequence comprising SEQ ID NO.6 ("ZTS-841" VH CDR3) has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and wherein the variable light chain comprises an amino acid sequence comprising an amino acid sequence of SEQ ID NO.1 ("ZTS-841" VL CDR1) having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and an amino acid sequence comprising an amino acid sequence of SEQ ID NO.2 ("ZTS-841" VL CDR3) having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity CDR2) having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, 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 with the amino acid sequence comprising SEQ ID NO.3 ("ZTS-841" VL CDR3); and any variants thereof, wherein the variants have one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 within any variable light chain or variable heavy chain of the antigen binding protein.

[0182] In one or more embodiments, the present invention provides an isolated and recombinant antigen binding protein "ZTS-842", wherein the variable heavy 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 the 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 the amino acid sequence comprising SEQ ID NO.25 ("ZTS-842" VH 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 the amino acid sequence comprising SEQ ID NO. NO.26 ("ZTS-842" VH CDR3) has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and wherein 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 the amino acid sequence comprising SEQ ID NO.21 ("ZTS-842" VL CDR1), 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 the amino acid sequence comprising SEQ ID NO.22 ("ZTS-842" VL CDR2) having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, 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 with the amino acid sequence comprising SEQ ID NO.23 ("ZTS-842" VL CDR3); and any variants thereof, wherein the variants have one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 within any variable light chain or variable heavy chain of the antigen binding protein.

[0183] In some embodiments described herein, the invention provides recombinant antigen-binding proteins, monoclonal antibodies and antibody fragments, and their use in clinical administration and scientific procedures (including diagnostic procedures). By using the method of molecular biology and recombinant technology, antibodies and antibody-like molecules can be produced by recombinant means, and thereby the gene sequence encoding the specific amino acid sequence found in the polypeptide structure of the antibody is generated. Such antibodies can be produced by cloning the gene sequence encoding the polypeptide chain of the antibody or by directly synthesizing the polypeptide chain, and the active tetramer (H2L2) structure with affinity for specific epitopes and antigenic determinants is formed by the assembly of synthetic chains. This has allowed antibodies with sequence characteristics of neutralizing antibodies from different species and sources to be easily produced.

[0184] Regardless of the source of the antibodies, how they are recombinantly constructed, or how they are synthesized in vitro or in vivo, using transgenic animals, laboratory or commercial-scale large cell cultures, using transgenic plants, or by direct chemical synthesis without using living organisms at any stage of the process, all antibodies have a similar overall three-dimensional structure. This structure is commonly referred to as H2L2 and refers to the fact that antibodies typically include two light (L) amino acid chains and two heavy (H) amino acid chains. Both chains have regions that can interact with structurally complementary antigenic targets. The regions that interact with the target are called "variable" or "V" regions, and are characterized by different amino acid sequences from antibodies with different antigenic specificities. The variable region of the H chain or L chain contains an amino acid sequence that can specifically bind to the antigenic target.

[0185] As used herein, the term "antigen binding region" refers to the portion of an antibody molecule that contains amino acid residues that interact with the antigen and confer specificity and affinity to the antibody for the antigen. The antibody binding region comprises the "framework" amino acid residues necessary for maintaining the correct conformation of the antigen binding residues. In the variable region of the H chain or L chain providing the antigen binding region, there are smaller sequences that are referred to as "hypervariable" because they have extreme variability between antibodies of different specificities. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions. These CDR regions explain the basic specificity of antibodies to specific antigen determinant structures.

[0186] CDR represents the non-continuous extension of amino acid in variable region, but, no matter how species, it has been found that the position of these key amino acid sequences in variable heavy chain region and light chain region has similar position in the amino acid sequence of variable chain. The variable heavy chain and light chain of all antibodies have three CDR regions respectively, and each CDR region is not adjacent to other CDR regions. In all mammalian species, antibody peptides contain constant (i.e. highly conserved) region and variable region, and in the latter, there is CDR and the so-called "framework region" consisting of amino acid sequence in variable region of heavy chain or light chain but outside CDR.

[0187] The present invention further provides a vector comprising at least one of the above-mentioned nucleic acids. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which will be able to encode an amino acid. By considering the frequency of specific codons (for encoding specific amino acids) actually used in eukaryotic or prokaryotic cells expressing anti-NGF antibodies or parts, the probability that a specific oligonucleotide will actually constitute an actual coding sequence can be estimated. 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 group of nucleotide sequences can be identified, which contains the theoretically "most likely" nucleotide sequence that can encode an anti-NGF sequence. It is also intended that the antibody coding region for the present invention can also be provided by changing existing antibody genes using standard molecular biology techniques, which produce variants (agonists) of the antibodies and peptides described herein. Such variants include, but are not limited to, deletions, additions and substitutions in the amino acid sequence of anti-NGF antibodies or peptides.

[0188] For example, one type of substitution is a conservative amino acid substitution. Such a substitution is a substitution in which a given amino acid in an anti-NGF antibody peptide is replaced by another amino acid with similar characteristics. Commonly considered conservative substitutions are mutual substitutions between aliphatic amino acids Ala, Val, Leu and Ie; exchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gin, exchange of basic residues Lys and Arg, substitution between aromatic residues Phe, Tyr, etc. For guidance on which amino acid changes may be phenotypically silent, see Bowie et al., 247 Science 1306-10 (1990).

[0189] Variant anti-NGF antigen binding proteins or antibody fragments may be fully functional, or may lack functionality in one or more activities. Fully functional variants typically contain only conservative variants or variants in non-critical residues or non-critical regions. Functional variants may also contain similar amino acid substitutions, which result in unchanged or no significant changes in function. Alternatively, such substitutions may affect function to a certain extent, either positively or negatively. 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 key residues or key regions.

[0190] 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 critical 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).

[0191] In addition, polypeptides typically contain amino acids other than the twenty "natural" amino acids. In addition, many amino acids (including terminal amino acids) can be modified by natural processes (e.g., processing and other post-translational modifications), or by chemical modification techniques 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 phosphatidylinositol, 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. Several particularly common modifications (e.g., glycosylation, lipid attachment, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation) are described in most basic texts, such as Proteins-Structure and Molecular Properties (2nd ed., TECreighton, WH Freeman & Co., NY, 1993). Many detailed reviews on this topic are available, for example, Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983); Seifter et al., 182 Meth. Enzymol. 626-46 (1990); and Rattan et al., 663 Ann. NY Acad. Sci. 48-62 (1992).

[0192] Therefore, the antibodies and peptides of the present invention also include derivatives or analogs in which the substituted amino acid residues are not amino acid residues encoded by the genetic code. Similarly, the additions and substitutions in the amino acid sequence just described as well as variants and modifications can also be applied to the amino acid sequence of the NGF antigen and / or its epitope or peptide, and are therefore covered by the present invention. As described above, the gene encoding the monoclonal antibody according to the present invention is particularly effective in the recognition of NGF.

[0193] Antibody derivatives

[0194] Included within the scope of the present invention are antibody derivatives. "Derivatives" of antibodies contain additional chemical moieties that are not usually part of the protein. Covalent modifications of proteins are included within the scope of the present invention. Such modifications can be introduced into the molecule by reacting the targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents well known in the art can be used to crosslink antibodies or fragments to a water-insoluble carrier matrix or other macromolecular carrier.

[0195] Derivatives also include radiolabeled monoclonal antibodies. For example, radioactive iodine (251, 1311), carbon (4C), sulfur (35S), indium, tritium (H 3 ) etc.; conjugates of monoclonal antibodies with biotin or avidin, conjugates with enzymes such as horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid dehydratase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase; and conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemiluminescent agents (such as acridinium esters) or fluorescent agents (such as phycobiliproteins).

[0196] Another kind of derivative bifunctional antibody of the present invention is a bispecific antibody, which is generated by combining parts of two separate antibodies that recognize two different antigen groups. This can be achieved by crosslinking or recombinant technology. In addition, a part can be added to an antibody or a part thereof to increase the half-life in vivo (for example, by extending the time of removal from the bloodstream). Such technology includes, for example, adding a PEG part (also referred to as pegylation), and is well known in the art. See U.S. Patent Application Publication No. 20030031671.

[0197] Recombinant expression of antibodies

[0198] In certain embodiments, nucleic acid encoding subject's monoclonal antibody is directly introduced into host cell, and the cell is incubated under the condition of being enough to induce the expression of the antibody of coding.After subject's nucleic acid has been introduced into cell, the cell is incubated for about 1-24 hours at 37 ℃ (sometimes under selection) to allow the expression of antibody.In one embodiment, antibody is secreted into the supernatant of the culture medium where cell is growing.Traditionally, monoclonal antibody has been produced as the natural molecule in mouse hybridoma cell line.In addition to this technology, the present invention also provides the recombinant DNA expression of monoclonal antibody.This allows to produce caninized antibody and a series of antibody derivatives and fusion protein in the host species selected.

[0199] The nucleic acid sequence encoding at least one anti-NGF antibody, part or polypeptide of the present invention can be recombined with a vector DNA according to conventional techniques, including blunt or staggered ends for ligation, restriction enzyme digestion to provide suitable ends, filling of sticky ends when appropriate, alkaline phosphatase treatment to avoid undesired ligation, and ligation with a suitable ligase. Techniques for such operations 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), which can be used to construct nucleic acid sequences encoding monoclonal antibody molecules or their antigen binding regions.

[0200] A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains a nucleotide sequence that includes transcriptional and translational regulatory information, and such sequence is "operably linked" to a nucleotide sequence encoding the polypeptide. An operable linkage is one in which the regulatory DNA sequence and the DNA sequence sought to be expressed are linked in such a way as to allow for gene expression in recoverable quantities as part of the anti-NGF peptide or antibody. The precise nature of the regulatory regions required for gene expression may vary from organism to organism, as is well known in similar fields. See, e.g., Sambrook et al., 2001; Ausubel et al., 1993, supra.

[0201] Therefore, the present invention includes the expression of anti-NGF antibodies or peptides in prokaryotic cells or eukaryotic cells. Suitable hosts include bacteria or eukaryotic hosts, including bacteria, yeast, insects, fungi, birds and mammalian cells in vivo or in situ, or host cells of mammalian, insect, bird or yeast origin. Mammalian cells or tissues can be human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cells can be used.

[0202] In one embodiment, the nucleotide sequence of the present invention will be incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host. Any of a variety of vectors can be used for this purpose. See, for example, Ausubel et al., 1993 above. Important factors in selecting a specific plasmid or viral vector include: the ease with which a recipient cell containing the vector can be identified and selected from a recipient cell that does not contain the vector; the number of copies of the vector required in a specific host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0203] Exemplary prokaryotic vectors known in the art include plasmids, such as those capable of replication in E. coli (e.g., but not limited to, pBR322, ColE1, pSC101, pACYC 184, etc.). Such plasmids are disclosed, for example, by Maniatis et al., 1989; Ausubel et al, 1993, supra. Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan in THE MOLEC. BIO. OF THE BACILLI 307-329 (Academic Press, NY, 1982). Suitable Streptomyces plasmids include pIJ101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987), and Streptomyces phage such as phLC31 (Chater et al., in SIXTH INT'L SYMPOSIUM ONACTINOMYCETALES 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., 1993 (supra).

[0204] Alternatively, gene expression elements that can be used to express cDNA encoding anti-NGF antibodies or peptides include but are not limited to (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl. Acad. Sci., USA 6777 (1982)) and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splicing regions and polyadenylation sites, such as the splicing region and polyadenylation site from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites, such as the polyadenylation site in SV40 (Okayama et al., 1983).

[0205] Immunoglobulin cDNA genes can be expressed as described in Weidle et al., 51 Gene 21 (1987) using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit S-globin insertion sequence, the immunoglobulin and rabbit S-globin polyadenylation sites, and the SV40 polyadenylation element as expression elements. For immunoglobulin genes composed of part cDNA and part 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.

[0206] In one embodiment, to express cDNA genes in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancer is one 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 natural chromosomal sequences corresponding to the immunoglobulin chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the above-mentioned expression elements to achieve protein expression in mammalian cells.

[0207] 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 anti-NGF peptide or chimeric H or chimeric L chain encoding genes alone, or co-transfected with chimeric H and chimeric L chain genes. The transfected recipient cells are cultured under conditions that allow expression of the integrated genes, and the expressed immunoglobulin chains or complete antibodies or fragments are recovered from the culture.

[0208] In one embodiment, the fusion gene encoding the anti-NGF peptide or chimeric H chain and L chain or its part is assembled in a separate expression vector, which is then used to co-transfect the receptor cell. Alternatively, the fusion gene encoding the chimeric H chain and L chain can be assembled on the same expression vector. In order to transfect the expression vector and produce chimeric antibodies, the receptor cell line can be a myeloma cell. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes, and have a glycosylated mechanism of immunoglobulins. Myeloma cells can be grown in culture or in the peritoneal cavity of mice, where the secreted immunoglobulins can be obtained from ascites. Other suitable receptor cells include lymphocytes, such as B lymphocytes of human or non-human origin, hybridoma cells of human or non-human origin, or interspecies heterologous hybridoma cells.

[0209] 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 microjet bombardment. Johnston et al, 240 Science 1538 (1988).

[0210] Yeast can provide significant advantages over bacteria for the production of immunoglobulin H and L chains. Yeast performs post-translational peptide modifications (including glycosylation). There are several recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids that can be used to produce the desired protein in yeast. Yeast recognizes the leader sequence of cloned mammalian gene products and secretes peptides (i.e., propeptides) with the leader sequence. Hitzman et al., 11th Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0211] Yeast gene expression systems can routinely assess the levels of production, secretion and stability of anti-NGF peptides, antibodies and assembled mouse and chimeric, heterochimeric, caninized antibodies, fragments and regions thereof. When yeast is grown in a medium rich in glucose, any of a range of yeast gene expression systems can be used that incorporates promoter and termination elements from genes encoding active expression of glycolytic enzymes produced in large quantities. Known glycolytic genes can also provide very effective transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Several methods can be used to assess the optimal expression plasmid for expressing cloned immunoglobulin cDNAs in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) IRL Press, Oxford, UK (1985).

[0212] Bacterial strains can also be used as hosts to produce antibody molecules or peptides described in the present invention. Plasmid vectors containing replicons and control sequences are used in combination with these bacterial hosts, and the replicons and control sequences are from species compatible with host cells. The vector carries a replication site and a specific gene that can provide phenotypic selection in transformed cells. A variety of methods can be used to evaluate expression plasmids for producing antibodies, fragments and regions or antibody chains encoded by cloned immunoglobulin cDNAs in bacteria (see: (Glover, 1985 above; Ausubel, 1993; Sambrook, 2001;) Colligan et al., eds. Current Protocols in Immunology, John Wiley & Sons, NY, NY (1994-2001); Colligan et al., eds. Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001)).

[0213] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including the removal of leader peptides, the folding and assembly of H chains and L chains, the glycosylation of antibody molecules, and the secretion of functional antibody proteins. In addition to the above-mentioned lymphoid cells, mammalian cells that can be used as hosts for producing antibody proteins also include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Many vector systems can be used to express cloned anti-NGF peptide H chains and L chain genes in mammalian cells (see Glover, 1985 above). Different methods can be followed to obtain complete H2L2 antibodies. H chains and L chains can be co-expressed in the same cell to achieve intracellular binding and connection of H chains and L chains in complete tetrameric H2L2 antibodies and / or anti-NGF peptides. Co-expression can occur by using the same or different plasmids in the same host. The genes for the H chain and the L chain and / or the anti-NGF peptide can be placed into the same plasmid and then transfected into the cells, thereby directly selecting cells expressing both chains. Alternatively, the cells can be first transfected with a plasmid encoding one chain (e.g., the L chain), and the resulting cell line can then be transfected with an H chain plasmid containing a second selective marker. Cell lines producing anti-NGF peptides and / or H2L2 molecules by either approach can be transfected with plasmids encoding additional copies of the peptide, H, L, or H+L chains, along with additional selective markers, to produce cell lines with enhanced properties, such as higher yields of assembled H2L2 antibody molecules or enhanced stability of the transfected cell lines.

[0214] For the long-term high yield production of recombinant antibodies, stable expression can be used.For example, the cell line of the antibody molecule can be stably expressed by engineering.Host cells can be transformed with immunoglobulin expression cassettes and selective markers, rather than using expression vectors containing viral replication origins.After introducing foreign DNA, engineered cells can be allowed to grow in enriched medium for 1-2 days, and then switch to selective medium.The selective marker in the recombinant plasmid has given resistance to selection, and allows cells to stably integrate plasmids into chromosomes, and grow to form lesions, which can then be cloned and expanded in the cell line.Such engineered cell lines may be particularly useful in screening and evaluating compounds / components that interact directly or indirectly with antibody molecules.

[0215] Once the antibodies of the invention have been produced, the antibodies may be purified by any method known in the art for purifying immunoglobulin molecules (e.g., by chromatography (e.g., ion exchange, affinity, particularly for a specific antigen following protein A, and size column chromatography), centrifugation, differential solubility), or by any other standard technique for purifying proteins. In many embodiments, the antibodies are secreted from the cells into the culture medium and harvested from the culture medium.

[0216] Pharmaceutical and veterinary applications

[0217] The anti-NGF antigen binding proteins or antibody fragments of the present invention as described herein can be used, for example, to treat NGF-related disorders in dogs and cats. More specifically, the present invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and an antibody or antibody fragment according to the present invention as an active ingredient. The antibodies can be chimeric, heterochimeric, caninized, felineized, equineized, humanized or specialized to suit different species. Complete immunoglobulins or binding fragments thereof such as Fab are also contemplated. The antibodies of the present invention and their pharmaceutical compositions can be used for parenteral administration, such as subcutaneous injection, intramuscular injection or intravenous injection.

[0218] The anti-NGF antibodies and / or peptides of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents. They can be administered alone, but are usually administered together with a pharmaceutical carrier selected according to the selected route of administration and standard pharmaceutical practice. The administration of the antibodies disclosed herein can be carried out by any suitable means, including parenteral injection (e.g., intraperitoneal, subcutaneous or intramuscular injection), oral administration, or by topical administration of the antibody (usually carried in a pharmaceutical preparation) to the airway surface. Topical administration to the airway surface can be performed by intranasal administration (e.g., by using a dropper, swab or inhaler). Topical administration of antibodies to the airway surface can also be performed by inhalation administration, for example, by producing inhalable particles of a pharmaceutical preparation (comprising solid and liquid particles) containing the antibody as an aerosol suspension, and then causing the subject to inhale the inhalable particles. Methods and equipment for administering inhalable particles of pharmaceutical preparations are well known, and any conventional technique can be used.

[0219] In some desirable embodiments, the antibody is administered by parenteral injection. For parenteral administration, the anti-NGF antibody or peptide can be formulated into a solution, suspension, emulsion or lyophilized powder together with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle can be a solution of the antibody or a mixture thereof dissolved in an acceptable carrier, such as an aqueous carrier, such a vehicle being water, saline, Ringer's solution, glucose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine, etc. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. These solutions are sterile and generally do not contain particulate matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances required to approach physiological conditions, such as pH adjustment and buffering agents, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antibody in these preparations can vary greatly, for example, from less than about 0.5% by weight, typically or at least about 1% to as much as 15% or 20%, and will be selected based primarily on the volume of fluid, viscosity, etc. according to the selected specific mode of administration. The vehicle or lyophilized powder may contain additives to maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffer and preservatives). The preparation is sterilized by conventional techniques. The actual method for preparing parenteral compositions is known or apparent to those skilled in the art, and is described in more detail in, for example, REMINGTON'S PHARMA.SCI. (15th ed.Mack Pub.Co., Easton, Pa., 1980).

[0220] Antibodies of the present invention can be lyophilized for preservation and reconstructed in a suitable carrier before use. This technology has been proven to be effective for conventional immunoglobulins. Any suitable lyophilization and reconstructing techniques can be used. Those skilled in the art will appreciate that lyophilization and reconstructing can result in varying degrees of antibody activity loss, and may have to adjust the use level to compensate. Compositions containing antibodies of the present invention or their mixtures can be used to prevent recurrence and / or therapeutic treatment of existing diseases. Suitable drug carriers are described in the latest version of the standard reference REMINGTON'S PHARMACEUTICAL SCIENCES in this area. In therapeutic applications, compositions are applied to subjects who have already suffered from the disease in an amount sufficient to cure or at least partially suppress or alleviate the disease and its complications. An amount sufficient to achieve this is defined as a "therapeutically effective dose" or "therapeutically effective amount". The effective amount used for this purpose will depend on the severity of the disease and the general state of the subject's own immune system, but generally ranges from 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. Given the minimization of foreign matter and the lower probability of "foreign matter" rejection achieved by the canines and antibodies of the invention, it is possible to administer a large excess of these antibodies.

[0221] The dosage administered will, of course, vary according to 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 symptoms, kind of concurrent treatment, frequency of treatment and the effect desired.

[0222] As a non-limiting example, treatment of NGF-related diseases in dogs and cats can be provided as biweekly or monthly doses of the anti-NGF antibodies of the invention within the required dosage range. According to the present invention, exemplary antibodies for canine therapeutic use are high affinity (these can also be high avidity) antibodies, and fragments, regions and derivatives thereof having effective in vivo anti-NGF activity. Single or multiple administrations of the composition can be carried out at a dosage level and pattern selected by the treating veterinarian. In any event, the pharmaceutical formulation should provide an amount of the antibody of the invention sufficient to effectively treat the subject.

[0223] Diagnostic Applications

[0224] The present invention also provides the above-mentioned anti-NGF antibodies and peptides for use in diagnostic methods for detecting NGF in species (particularly canines and cats) known to suffer from or suspected of suffering from NGF-related disorders. 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 can be used for immunoassays for detecting or quantifying NGF or anti-NGF antibodies in samples. Immunoassays for NGF generally include incubating clinical or biological samples in the presence of detectably labeled high-affinity (or high-affinity) anti-NGF antibodies or polypeptides of the present invention that can selectively bind to NGF, and detecting the labeled peptides or antibodies bound 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 assays as described below. Therefore, anti-NGF antibodies or polypeptides can be fixed to nitrocellulose or another solid support capable of fixing cells, cell particles or soluble proteins. The support can then be washed with a suitable buffer and subsequently treated with a detectably labeled NGF-specific peptide, antibody or antigen-binding protein. The solid support can then be washed again with a buffer to remove unbound peptides or antibodies. The amount of the labeled binding on the solid support can then be detected by known method steps.

[0225] "Solid support" or "carrier" refers to any support capable of binding a peptide, antigen or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride (PVDF), dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. For the purposes of the present invention, the nature of the carrier can be soluble or insoluble to some extent. The support material can have virtually any possible structural configuration, as long as the coupled molecule is capable of binding to NGF or anti-NGF antibodies. Thus, the support configuration can be spherical, such as beads, or cylindrical, such as the inner surface of a test tube, or the outer surface of a rod. Alternatively, the surface can be flat, such as a sheet, a culture dish, a test strip, etc. For example, the support can comprise polystyrene beads. Those skilled in the art will be aware of many other suitable carriers for binding antibodies, peptides or antigens, or can determine the carrier by routine experimentation. Well-known method steps can determine the binding activity of a given batch of anti-NGF peptides and / or antibodies or antigen-binding proteins. Those skilled in the art can determine operating and optimal assay conditions by routine experimentation.

[0226] Detectably labeled NGF-specific peptides and / or antibodies can be achieved by being linked to an enzyme for enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA). The linked enzyme reacts with the exposed substrate to generate a chemical moiety that can be detected, for example, by spectrophotometry, fluorescence analysis, or by 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, δ5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. By radioactively labeling the NGF-specific antibody, NGF can be detected by using a radioimmunoassay (RIA). See Work et al., LAB.TECHNIQUES & BIOCHEM. INMOLEC. BIO (No. Holland Pub. Co., NY, 1978). Radioactive isotopes can be detected, for example, by means of a gamma counter or a scintillation counter or by autoradiography. Particularly useful isotopes for the purposes of the present invention include: 3 H. 125 I. 131 I. 35 S and 14 C.

[0227] The NGF-specific antibody can also be labeled with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected due to fluorescence. The most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine. The NGF-specific antibody or antigen binding protein can also be labeled with a metal that emits fluorescence, such as 125 Eu or other lanthanides can be detectably labeled. These metals can be attached to NGF-specific antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0228] NGF-specific antibodies can also be detectably labeled by coupling to a chemiluminescent compound. The presence of the chemiluminescent labeled antibody is then determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of useful chemiluminescent labeled compounds are luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalates.

[0229] Likewise, 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.

[0230] Detection of NGF-specific antibodies, portions, fragments, polypeptides or derivatives can be accomplished by a scintillation counter (e.g., if the detectable label is a radioactive gamma emitter), or by a fluorimeter (e.g., if the label is a fluorescent material). In the case of an enzyme label, detection can be accomplished by a colorimetric method using a substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of the enzymatic reaction of the substrate with a similarly prepared standard.

[0231] For the purposes of the present invention, the NGF detected by the above-mentioned assay can be present in a biological sample. Any sample containing NGF can be used. For example, the sample is a biological fluid, such as, for example, 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 only these samples, however, based on this specification, a person of ordinary skill in the art can determine appropriate conditions that allow the use of other samples.

[0232] In situ detection can be achieved by removing a histological sample from an animal subject and providing such a sample with a combination of labeled antibodies of the present invention. The antibody (or portion thereof) can be provided by applying or covering the labeled antibody (or portion) onto a biological sample. By using such a procedure, not only the presence of NGF can be determined, but also the distribution of NGF in the examined tissue can be determined. Using the present invention, a person of ordinary skill will readily appreciate that any of a variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[0233] The antibodies, fragments or derivatives of the invention may be suitable for use in immunoassays, also known as "two-site" or "sandwich" assays. In a typical immunoassay, an amount of unlabeled antibody (or fragment of an antibody) is bound to a solid support that is insoluble in the fluid being tested, and an amount of a detectably labeled soluble antibody is added to allow detection and / or quantification of the ternary complex formed between the solid phase antibody, antigen and labeled antibody.

[0234] Antibodies can 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 (using fluorescently labeled antibodies (see below)) in combination with fluorescence microscopy, flow cytometry or fluorescence detection. For diagnostic purposes, antibodies can be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (secondary antibodies) that react with the antibody, such as antibodies specific to canine immunoglobulin constant regions. Alternatively, antibodies can be labeled directly. A variety of labels can be used, such as radionuclides, fluoresces, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), etc. Many types of immunoassays (such as those discussed above) are available and are well known to those skilled in the art. Importantly, the antibodies of the present invention may be helpful in diagnosing NGF-related disorders in canines. More specifically, the antibodies of the present invention can recognize overexpression of NGF in companion animals. Therefore, the antibodies of the present invention can provide an important immunohistochemical tool. The antibodies of the present invention can be used in antibody arrays, which are very suitable for measuring gene expression profiles.

[0235] Reagent test kit

[0236] The kit for implementing the subject method is also included in the scope of the present invention. The kit comprises at least one or more antibodies of the present invention, nucleic acids encoding the antibodies or cells containing the antibodies. The antibodies of the present invention can be provided in a container, usually in a lyophilized form. Antibodies (which can be conjugated with a label or toxin, or can be unconjugated with a label or toxin) are usually contained in a kit with a buffer (e.g., Tris, phosphate, carbonate, etc.), a stabilizer, a biocide, an inert protein (e.g., serum albumin), etc. Usually, these materials will be present in a total amount of at least about 0.001wt% based on the amount of the active antibody, and usually again based on the antibody concentration. Usually, it is desirable to include an inert extender or excipient to dilute the active ingredient, wherein the excipient can be present in about 1wt% to 99wt% of the total composition. In the case of using a second antibody capable of binding to the first antibody in the assay, this will usually be present in a separate vial. The second antibody is usually conjugated with a label and prepared in a manner similar to the above-mentioned antibody preparation. The kit will also usually include a set of instructions for use.

[0237] The invention will now be further described by way of the following non-limiting examples.

[0238] Example

[0239] The present invention is further illustrated and supported by the following examples. However, these examples should never be considered to further limit the scope of the present invention. On the contrary, it will be readily appreciated by those skilled in the art that other embodiments, modifications and equivalents of the present invention exist without departing from the spirit of the present invention and / or the scope of the appended claims.

[0240] Example 1

[0241] Synthesis and Purification of Canine NGF (cNGF)

[0242] PCR primers are designed to have appropriate restriction sites to amplify canine pre-β-NGF (SEQ ID NO: 59). The β-NGF gene is cloned into plasmid pCTV927 (Chromos targeting plasmid) through the EcoRV / Kpnl site. The pCTV927 / β-NGF plasmid is co-transfected into CHOK1SV cells together with a plasmid encoding the Chromos system integrase pSIO343 using Lipofectamine 2000 transfection reagent. The expression of a single stable clone is analyzed, and high-expression clones are selected for amplification and expression for subsequent purification. Canine β-NGF (cNGF) produced by these transfections is purified using ion exchange chromatography. The initial cleanup is performed in a flow-through batch mode on Q Sepharose FF (GE Healthcare #17-0510-01). The clarified supernatant is diluted 1:1 with water, and the pH is adjusted to 8.5 with 1M Tris. The diluted sample was mixed with QSepharose FF at a ratio of 150:1 for >1.5 hours. The resin was allowed to settle and the unbound fraction was collected. cNGF was further purified by cation exchange chromatography; diluted again with water at 1:1 and loaded onto SP-Sepharose FF (GE Healthcare #17-0729-01) pre-equilibrated with 20mM Tris, pH 8.5. After loading, the column was washed and then eluted by a linear gradient from 0 to 210mM ​​NaCl (each 20mM Tris, pH 8.5) over 20 column volumes. The fractions were analyzed by SDS-PAGE, combined, and dialyzed again with PBS at 4°C (3.5K mwco). The dialysate was collected, sterile filtered, and the concentration was measured by absorbance at 280nm (1mg / ml=1.48A 280 ).

[0243] Example 2:

[0244] Canine Immunization

[0245] Canine immunization can be performed 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 administered directly to a dog together with an adjuvant to stimulate an immune response. In order to obtain the best anti-antigen response, booster injections are administered to the dogs, and serum samples are collected regularly. The antibody immune response from immunized dogs is monitored and determined using a standard antigen direct binding enzyme-linked immunosorbent assay (ELISA) method as known to those skilled in the art and described below.

[0246] Example 3:

[0247] Primary antigen binding and B cell activation

[0248] To evaluate the titer of canine anti-NGF antibodies, 100 μL of recombinant canine NGF (10 μg / mL) was coated overnight in Immunolon2Hb plates at 4°C. The wells were washed three times with PBS-T (PBS + 0.1% Tween) and incubated with 200 μL of PBS + 5% skim milk at room temperature for 1 hour to block nonspecific binding. After washing the plates three times with 300 μL of PBD-T, serial dilutions of canine serum were incubated for one hour. The binding of canine anti-NGF IgG was detected using a 100 μL mixture of 0.2 μg / mL Bethyl anti Dog IgG1 (A40-120P) and anti-Dog IgG2 (A40-121 P). After adding a chromogenic substrate (SureBlue Reserve TMB 1 component microporous peroxidase substrate, KPL53-00-01) and incubating at room temperature for ten minutes, the reaction was terminated by adding 100 μL of 0.1N HCl. The absorbance of each well was determined at optical density (OD) of 450 nm.

[0249] Activation Protocol for Canine Memory B Cells

[0250] Using Ficoll TM Gradient separation is separated by centrifugation to separate peripheral blood mononuclear cells (PBMC). After PBMC is separated from the sample, the specific selection of antibody secreting cells is performed according to the expression of specific antibody cell surface markers known to those skilled in the art and as described in US 2014 / 0287402 and Callard and Kotowicz "Cytokine Cell Biology: A practical approach" Oxford University Press, 2000, pg 17-31 (which is incorporated herein by reference). Before B cells are deposited on the sorting chip, cells are activated in vitro. After separation and freezing, cells (PBMC, about 107 Cells / vial) were removed from liquid nitrogen and quickly 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 the cells were centrifuged at 1000 rpm for 10 min, the pellet was resuspended in 10 ml of complete medium and centrifuged again at 1000 rpm for 10 min. Finally, the cells were resuspended in 4 ml of culture medium.

[0251] Example 4

[0252] DNA sequences encoding 9L12 (ZTS-841), 48L2 (ZTS-842) and 13L11 antibodies

[0253] Relevant single cells are recovered from the microarray by micromanipulation and deposited into a microtube containing lysis buffer and magnetic beads for mRNA capture. cDNA is prepared from total RNA, wherein a mixture of gene-specific primers hybridizes in the early constant domains of γHC, κLC and λLC. Terminal deoxynucleotidyl transferase (TdT) is used for 3' end tailing of the first strand cDNA product. For the subsequent first PCR, a mixture of gene-specific reverse primers and universal primer forward primers is used. Subsequently, nested PCR is performed for each VH and VL chain to amplify the antibody variable region. The reverse primer used for this purpose is located in the HC or LC constant domain together with the universal forward primer. The fragments amplified from PCR are separated by gel electrophoresis on an agarose gel. The full-length VH and VL amplicons isolated from single cells are cloned into expression vectors containing the constant portion of the corresponding HC or LC. The canine variable domain sequences are 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).

[0254] The constant region of the separated antibody separated as described above is not used for the subsequent construction of the antibody of the present invention. The Fc region of the recombinant antibody of the present invention includes a modified version of canine IgGB (Bergeron et al., Vet Immunol Immunopathol 2014 Jan 15: 157 (1-2): 31-41), and is selected according to its half-life, biophysical properties and lack of effector functions. As reported by Bergeron et al., canine IgGB has good affinity for canine FcRn and has biophysical properties suitable for downstream processing. The differential scanning calorimetry (DSC) performed only on the canine Fc region shows that the thermal stability of the constant region is about 70 ° C and 83 ° C. These melting temperatures are similar to or higher than the melting temperatures reported for the humanized mAb sold.

[0255] The CH2 domain of canine IgGB was subjected to three point mutations to eliminate ADCC and CDC activity. The mutated Fc is referred to herein as IgGB (e-) (SEQ ID NO.43). Although NGF is a soluble target, effector functions are eliminated from anti-NGF antibodies to protect them from any potential nonspecific targets or adverse effects associated with effector functions. These mutations do not appear to affect the immunogenicity of this mAb. In addition, mutations in the Fc region to eliminate effector functions do not affect FcRn or protein A binding. Reduced binding to canine FcγRI and FcγRIII and reduced ADCC activity were observed. C1q protein is the first protein in the complement cascade and is necessary for cells to undergo complement-dependent cytotoxicity (CDC). It has been shown that IgGB (e-) lacks binding to C1q protein. As described above, the amino acid sequence of canine constant HC-65 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.

[0256] Example 5:

[0257] Antigen binding affinity determination

[0258] Antibody binding affinities for antibodies against canine NGF were determined by surface plasmon resonance (SPR) on a Biacore system (Biocore Life Sciences (GE Healthcare), Uppsala, Sweden). Immobilization of canine and rat NGF was obtained by amine coupling of 5 μg / mL of 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 for binding to immobilized NGF was evaluated. Different concentrations of canine and feline anti-NGF antibodies were injected on the NGF surface, and the binding 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.

[0259] Table 1: Summary of canine and rat NGF binding kinetics

[0260] antigen name ZTS ka(M-1s-1) kd(s-1) KD(M) Canine NGF 48L2 ZTS-842 7.03E+05±2.72E+05 2.27E-05±5.30E-05 2.17E-11±5.13E-11 Canine NGF 9L12 ZTS-841 5.27E+05±1.44E+05 2.36E-05±2.84E-05 5.06E-11±6.73E-11 Canine NGF fel48L2 1.1 ZTS-205 4.26E+05±2.21E+05 8.93E-05±2.14E-04 1.11E-10±2.56E-10 Canine NGF fel48L2 1.2 ZTS-206 4.22E+05±2.10E+05 8.64E-05±2.04E-04 1.13E-10±2.55E-10 Canine NGF fel48L2 chimera 4.71E+05±1.26E+05 2.64E-06±1.71E-06 6.25E-12±5.40E-12 Rat NGF 48L2 ZTS-842 5.73E+05±2.65E+05 1.42E-06±1.45E-06 3.25E-12±3.43E-12 Rat NGF 9L12 ZTS-841 5.87E+05±2.32E+05 4.61E-05±4.69E-05 1.08E-10±1.21E-10 Rat NGF fel48L2 1.1 ZTS-205 6.72E+05±3.41E+05 2.9E-04±5.01E-04 2.77E-10±4.77E-10 Rat NGF fel48L2 1.2 ZTS-206 6.42E+05±3.13E+05 2.91E-04±4.83E-04 3.09E-10±4.80E-10 Rat NGF fel48L2 chimera 3.87E+05 1.10E-07 2.85E-13

[0261] Example 6:

[0262] Construction of 9L12(841) and 48L2(842) chimeric antibodies

[0263] The antibody variable domain is responsible for antigen binding. Antibodies consist of homodimer pairings of two heterodimeric proteins. Each protein chain (one heavy chain and one light chain) of the heterodimer consists of a variable domain and a constant domain. Each variable domain contains three complementary determining regions (CDRs) that contribute to antigen binding. The CDRs are separated in the variable domains by framework regions, which provide a scaffold for the proper spatial display of binding sites on the antibody. Together, the CDRs and framework regions contribute to the ability of the antibody to bind to its cognate antigen. Grafting the full variable domains to the corresponding constant regions is expected to have little or no effect on the ability of the antibody to bind to NGF. In order to simultaneously confirm that the correct sequence of the heavy and light chain variable regions is recognized and to produce homologous materials, expression vectors are designed to produce recombinant chimeric antibodies or canine antibodies in a mammalian expression system. The chimeric antibodies described in the text consist of variable sequences (CDRs and frameworks) from host species antibodies, which are grafted to the corresponding heavy and light constant regions of canine IgG molecules. The chimeric antibodies described herein are composed of the variable fragments (CDRs and frameworks) of the canine molecule, which are grafted onto the corresponding heavy constant region and light constant region of the cat IgG molecule. Since the variable domains are responsible for antigen binding, grafting the full canine variable domains onto the cat constant domains is expected to have little or no effect on the ability of the antibody to bind to NGF. The chimeric variable domain sequences are as follows: canfel_chimera9L12 (841) variable light chain (SEQ ID NO: 9), corresponding nucleotide sequence (SEQ ID NO: 19); canfel_chimera9L12 (841) variable heavy chain (SEQ ID NO: 10), corresponding nucleotide sequence (SEQ ID NO: 21); canfel_chimera48L2 (842) variable light chain (SEQ ID NO: 29), corresponding nucleotide sequence (SEQ ID NO: 39); canfel_chimera48L2 (842) heavy chain (SEQ ID NO: 30), corresponding nucleotide sequence (SEQ ID NO: 40). Each variable fragment was cloned into a mammalian expression plasmid containing a feline IgG heavy or light chain. The amino acid sequence of the feline heavy constant region is represented by SEQ ID NO: 62, and its corresponding nucleotide sequence is represented by SEQ ID NO: 63. The amino acid sequence of the feline light constant region is represented by SEQ ID NO: 64, and its corresponding nucleotide sequence is represented by SEQ ID NO: 65.

[0264] Example 7:

[0265] Felinization of 48L2 and 9L12 antibodies

[0266] The generation of anti-drug antibodies (ADA) can be associated with the loss of efficacy of any biotherapeutic protein, including monoclonal antibodies. A comprehensive review of the literature has shown that species formation of monoclonal antibodies can reduce the tendency of mAbs to be immunogenic. In order to help mitigate the risks associated with ADA formation of the canine anti-NGF monoclonal antibodies provided herein, a felinization strategy was used for the final use of the antibody in cats. This felinization strategy is based on identifying the most suitable feline antibody sequences to be used for CDR grafting. After extensive analysis of all available feline germline sequences (heavy and light chains), the germline candidates were selected based on their homology with the canine mAb, and CDRs from canine progenitor cell fragments were used to replace natural cat CDRs. Felinized mAbs were expressed and characterized for their ability to bind to NGF. The purpose is to use a feline antibody framework to maintain high affinity and cell-based activity to minimize the potential for in vivo immunogenicity. Synthetic constructs representing felinized variable heavy and light chains of mAb 48L2 (ZTS-842) using SEQ ID NOs 21-26 and felinized variable heavy and light chains of 9L12 (ZTS-841) using SEQ ID NOs 1-6 were prepared. After subcloning each variable chain into a plasmid containing a feline constant heavy chain (SEQ ID NO: 62) and a feline constant light chain (SEQ ID NO: 64) region, the plasmids were co-transfected for antibody expression in HEK293 cells. Chimeric, heterochimeric, and felinized versions of mAb 48L2 and 9L12 were expressed and characterized for their ability to bind NGF by SPR.

[0267] Example 8:

[0268] Antibody production from Glutamine Synthetase (GS) plasmid

[0269] The genes encoding canine and feline 9L12 and 48L2 (ZTS-841 and ZTS-842, respectively) and 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 according to the manufacturer's protocol and ligated together to form a single mammalian expression plasmid. To demonstrate the transient production of each antibody, each plasmid was used to transfect HEK 293 cells and expressed in cultures of varying sizes. Protein A affinity chromatography was used to separate proteins from conditioned HEK culture media according to standard protein purification methods. The culture media was loaded onto a chromatographic resin and eluted by pH changes. The eluted protein was pH adjusted, dialyzed, and sterile filtered before use. The affinity and potency of the antibodies were tested.

[0270] In order to generate a stable cell line producing candidate antibodies, before transfection with restriction enzyme Pvul, GS plasmid was linearized, which was cut at a single site in the plasmid backbone. GS-CHOK1SV (clone 144E12) cells were transfected with linearized plasmid DNA by electroporation. After transfection, cells were placed in 48-well plates (48WP) to produce stable pools. When the pool was at least 50% confluent in 48WP, the IgG expression of the supernatant of 100 μl was analyzed using ForteBio Octet and protein A biosensor (PallForteBio, Fremont, CA). The clones of the best expression were amplified to 6 well plates (6WP) and then amplified to 125mL shake flasks (SF). Once cells were adapted to suspension culture in 125mL flasks, 2 vials in each cell line pool were stored for LN storage. Since the manufacturing cell line must be cloned, the top 3 highest expression pools were subcloned by limiting dilution in 96-well culture plates. To demonstrate clonality and avoid a second round of limiting dilution, 96-well plates were imaged using a Molecular Devices Clonal Selection Imager (CSI) (Molecular Devices LLC, San Jose, CA), which captures images of individual cells and their subsequent growth. Clones were selected based on successful CSI images, growth in 96WP, and yield.

[0271] To evaluate cell culture growth and productivity, the top expression pool was further evaluated in a 14-day feed batch in 125 ml of SF. Cells were inoculated in platform medium and feed consisting of CD CHO from Life Technologies plus 4 amino acids, proprietary feed CDF v6.2, and 10% glucose. After the 14-day feed batch, the pool was centrifuged and the mAbs produced by CD CHO were separated by filtering the supernatant through a 0.20 μm polyethersulfone (PES) membrane before purification.

[0272] Typical purification includes loading two liters of conditioned medium (from CHO cell culture, 0.2 μm filtered) onto a 235 mL column of MabSelect (GE healthcare, cat#17-5199-02). The column has been pre-equilibrated with PBS. The sample is loaded with a residence time of>2.5 minutes. After loading, the column is washed with PBS again, and then washed with 25mM sodium acetate (pH is about neutral). The column is eluted with 25mM acetic acid (pH 3.6), and then stripped with 250mM acetic acid, 250mM sodium chloride (pH is about 2.2). Fractions (50 mL) are collected during the elution and stripping steps. The UV absorbance at A280 is monitored throughout the process. Peak fractions are merged, the pH is adjusted to about 5.5 by adding 20mM sodium acetate, and then dialyzed for three buffer exchanges. The dialysate is collected, sterile filtered, and stored at 4 ° C.

[0273] Example 9:

[0274] Neutralization of canine NGF biological activity in vitro

[0275] As described in Example 5, the affinity of each canine and felinized anti-NGF antibody for canine NGF was measured using SPR (surface plasmon resonance) on a Biacore system (Biocore Life Sciences (GE Healthcare), Uppsala, Sweden). In addition, a functional in vitro assay was developed to measure the inhibition constant of the ability of the mAb to inhibit the binding of NGF to TrkA. In order to determine whether the anti-NGF mAbs of the present invention block downstream cellular signaling due to inhibition of NGF binding to TrkA, the purified antibodies were evaluated in an assay measuring canine NGF-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1 / 2). The cells used in the assay were CHO-K1 cells expressing canine TrkA (Life Technologies) that were cultured in DMEM / F12+GlutaMAX TM -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 plated at 5.0×10 4Cells were seeded in 96-well tissue culture plates (Costar) and incubated overnight at 37°C to allow adhesion. The cells were then serum starved for 2 hours in HBSS (Life Technologies) containing calcium chloride and magnesium chloride. Anti-NGF antibodies were serially diluted in HBSS and pre-incubated with recombinant canine NGF diluted in HBSS / 0.1% BSA for 1 hour at room temperature before addition to the cells. The final concentrations of canine NGF and BSA in the assay were 15 ng / ml (EC90) and 0.025%, respectively. After removing the assay mixture and adding 100 μl of cell lysis buffer (the buffer was set with pERK 1 / 2 Ultra assay kit (PerkinElmer)) at 37°C for 10 min. Cell lysates were then processed according to the manufacturer's instructions and Plates were read on a plate reader (PerkinElmer). The maximum response in this assay was defined as the ERK 1 / 2 phosphorylation measured in the presence of canine NGF alone (no mAb). The minimum response was defined as the basal level of ERK 1 / 2 phosphorylation (no stimulation). The calculated inhibition values ​​for the anti-NGF antibody were expressed as a percentage of the minimum and maximum responses. The resulting percentage inhibition data were plotted using GraphPad Prism 5 for IC50 determination (4 parameter curve fit). Please refer to Figure 6-7 .

[0276] TF-1 cell proliferation assay

[0277] 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.). The 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 per 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 CellTiter-GLO luminescent assay kit (Promega) was used to evaluate the effect of anti-NGF antibodies on canine NGF-induced cell proliferation. The maximum response in this assay is defined as the proliferation in the presence of canine NGF alone (no antibody). The minimum response is defined as the proliferation measured in the absence of canine NGF. The calculated inhibition (NGF neutralization) values ​​of anti-NGF antibodies were expressed as a percentage of the minimum and maximum responses. The obtained percentage inhibition data were plotted using GraphPad Prism5 for IC50 determination (4-parameter curve fitting). Please refer to Figure 8-9 .

[0278] Example 10:

[0279] Pharmacokinetics

[0280] Pharmacokinetics of canine anti-NGF mAbs 48L2 (ZTS-842) and 9L12 (ZTS-841). Dogs were studied following two subcutaneous (SC) and one intravenous (IV) doses of 2.0 mg / kg, 28 days apart. IV data showed a half-life of 13.3 ± 3.4 days (mean ± standard deviation) and a lower clearance of 3.9 ± 0.2 mL / d / kg. Following SC administration, peak serum concentrations were observed 1-7 days after dosing. SC absolute bioavailability averaged 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, 10 pg / mL. Following 48L2(ZTS-842) dosing, total NGF concentrations increased in all animals, averaging 1300±500pg / mL on day 84 of the study. 48L2(ZTS-842) concentrations were in substantial excess throughout the study, averaging 7.8±1.3μg / mL on days 84 and 28 after the last dose, suggesting that even smaller doses are sufficient to capture endogenous NGF for at least one month following dosing. 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 in the three-dose, 84-day study. Please refer to Fig.11 . In addition, ZTS-841 was studied using the same parameters as above; 20 mg / kg SC / SC / IV doses, 28 days apart, showed a half-life of 11.8 + 4.1 days. SC bioavailability was 94% + 12%. Please refer to Figure 10-11 .

[0281] The PK of the felinized anti-NGF mAb fel48L21.1 (ZTS-205) was studied in three male and three female cats following two subcutaneous (SC) and one intravenous (IV) doses of 1.5 mg / kg administered 28 days apart. IV data showed a half-life of 10.8 ± 2.5 days (mean ± SD) and a lower clearance of 3.0 ± 1.0 mL / d / kg. Following SC dosing, peak serum concentrations were observed 2-7 days after dosing. The absolute bioavailability of the SC averaged 88% ± 17%. Concentrations of fel48L21.1 (ZTS-205) were high throughout the study, averaging 7.2 ± 4.0 μg / mL on days 84 and 28 after the last dose, suggesting that even smaller doses are sufficient to capture endogenous NGF for at least one month after dosing. Although immunogenicity was not directly assessed, there was no indication from ZTS-842 concentration-time data that any anti-drug antibodies were induced in the six cats in the three-dose, 84-day study.

[0282] Bioanalytical Assay Methodology

[0283] Biotinylated canine NGF based on streptavidin Gyrolab TM Free mAb was captured on the disk and then added with AlexaFluor TM A free 48L2 (ZTS-842) ligand binding assay was developed based on biotinylated canine NGF in the presence of streptavidin and Gyrolab TM Free mAb was captured on the disk and then added with AlexaFluor TM A ligand binding assay for free feli48L21.1 (ZTS-205) was developed using labeled goat anti-cat IgG polyclonal antibody followed by fluorescence detection.

[0284] Example 11:

[0285] Evaluation of canine and feline anti-NGF antibodies in the rat MIA model

[0286] Osteoarthritis (OA) is a degenerative joint disease characterized by joint pain and progressive loss of articular cartilage. Intra-articular injection of MIA results in loss of articular cartilage accompanied by progression of subchondral bone damage that resembles OA damage. This model provides a rapid and minimally invasive method to replicate OA-like damage in rodent species.

[0287] During the study period on study days 7 and 14, the analgesic effect of specialized (e.g., canine, feline, etc.) anti-NGF antibodies at one dose of MIA in the rat MIA model of osteoarthritis was confirmed by administering monoclonal antibodies ZTS-841 and ZTS-842, respectively. Pain was assessed using a weight-bearing test for sustained pain and a joint compression (Randall Selitto) test for mechanical hyperalgesia test using an algometer (Ugo Basile). The test was performed by applying pressure to the hind paw. By pressing the pedal that starts the motor, the force increases in linear proportion at a constant rate. When pain is shown, pay attention to the withdrawal or vocalization of the paw, release the pedal immediately, and read the nociceptive threshold on the scale. A critical value of 400g is used to avoid potential injury. The Randall-Selitto test was performed on study days 1 (baseline), 20, and 28. For a schematic diagram of the rat MIA procedure, see Fig.12 .

[0288] Cartilage loss was induced by administering the metabolic inhibitor monoiodoacetate (MIA). Rats were anesthetized with isoflurane (3-5% in 100% O2). Once the animals were fully anesthetized, 50 μL injections of 40 mg of MIA / ml saline were injected into the joint cavity of the left hind ankle using a 1cc syringe equipped with a 27G needle. The animals were removed from isoflurane and allowed to fully recover and then returned to their home cages.

[0289] In order to evaluate the effect of the anti-NGF mAb of the present invention in these animals, the Incapacitance Tester is used to evaluate the weight bearing of the animal. The animal is placed in an acrylic test box, and when it is in the correct position, the force is evaluated. Three evaluations are performed at each time point. The following formula is used to calculate the weight bearing percentage score (WBS) of each evaluation:

[0290]

[0291] The mean value of 3 WBS is obtained as the WBS at this time point. On the 21st day, WBS was calculated before inducing MIA, and MIA was instilled into the left knee joint with a dosage of 2mg / 0.05mL. WBS was measured on the 1st day of randomization. On the 0th day, anti-NGF mAb or placebo was used, and then weight bearing was assessed on the 7th, 14th, 21st and 28th days. Body weight was recorded on the day of weight bearing assessment every week.

[0292] Serum samples were collected by cardiac terminal puncture on day 28 post-dose. After euthanasia by CO2 asphyxiation, whole blood was collected from cardiac puncture and placed in serum separator tubes, coagulated at room temperature, then centrifuged (3500 rpm, 15 min) and transferred to 96-well plates in two aliquots of 300 μL each as listed in the table below. Samples were frozen at ≤-10°C until analysis. For a graphical representation of ZTS-841 and ZTS-842 as tested in the rat MIA assay, please refer to Figure 13-15 .

[0293] Example 12

[0294] Effects on lameness: evaluation of caninized antibodies in a dog synovitis model

[0295] The inflammatory process of soft tissues is recognized as an important component of osteoarthritis. In the synovitis pain model, transient inflammation of the synovium in a single asphyxial fluid is induced by intra-articular injection of bacterial lipopolysaccharide (LPS). Quantifiable lameness occurs within 2 hours of synovitis induction, peaks at 3-4 hours, gradually disappears until 6 hours, and completely disappears after 24 hours. This model has been commonly used to study the goal of pain control.

[0296] In the canine LPS synovitis model, ZTS-841 reduced lameness by a single intravenous administration of 5 mg / kg dose to intact male beagle dogs compared to saline placebo. As can be seen from Table 2 below, ZTS-841 demonstrated efficacy at 3 hours and 12 hours after induction of LPS synovitis.

[0297] Table 2 and Fig.16 The least square means (with standard errors) of the VAS for lameness in the treatment groups at three hours and three hours after synovitis induction are presented. The difference between 5 mg / kg ZTS-841 and placebo was statistically significant.

[0298] Table 2

[0299] Visual analogue score (cm)

[0300] Hours after administration

[0301]

[0302]

[0303] Example 13

[0304] Humanization of antibodies 48L2 (ZTS-842) and 9L12 (ZTS-841)

[0305] Similar to the felinization strategy described and well known to those skilled in the art, suitable germline antibody sequences were identified from all human sequences available for CDR grafting from mAb 48L2 and 9L12. Variable light and variable heavy chains were selected based on the highest homology to their respective canine frameworks. The CDRs of the native human fragments were removed and replaced with the parent canine CDRs. Recombinant humanized 48L2 and 9L12 were produced using selected variable regions linked to their respective canine constant heavy IgG chain sequences. The antibodies were produced by HEK cells, purified as described above, and then evaluated for their ability to bind to human NGF, as shown in Table 3 below. Synthetic constructs representing humanized variable heavy and light chains of mAb 48L2 (ZTS-842) and 9L12 (ZTS-841) were constructed. Different combinations of variable heavy and light chains in the two groups were synthesized and binding was determined (see below). During construction, the CDR sequences were not changed, only the framework sequences were changed.

[0306] The antibody binding affinity of antibodies against 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 described humanized anti-NGF antibodies were injected onto the surface of human NGF, and the binding 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.

[0307] Table 3

[0308]

[0309]

[0310] Example 14:

[0311] Paratope scanning mutagenesis of antibodies 48L2 9L12 (ZTS-841) and 48L2 (ZTS-842)

[0312] The region of the antibody responsible for antigen recognition represents the paratope. The paratope is produced by the combination of amino acids in the complementary determining regions (CDRs) of the heavy and light chain variable regions. The binding between the antibody and the antigen is usually mediated by the side chains of the CDR residues and the side chains or carbohydrate moieties of the antigen. In order to help define the key side chains involved in antibody recognition, alanine scanning mutagenesis is performed on each CDR residue in the heavy and light chains, and the technique is described as by Cunningham and Wells (1989) Science, Vol.244, Issue 4908, pp.1081-1085. Then, Biacore was used to test the ability of these mutants to NGF, respectively. The binding affinity to human NGF (hN hereinafter), canine NGF (cN hereinafter) and rat NGF (rN hereinafter) was measured, and KD values ​​were generated by the same scheme as described in Examples 5 and 9 above. These values ​​were then compared with wild-type antibodies and expressed in a table as a percentage of wild-type binding. The data presented in Tables 4 and 5 below are shown as "percent similarity scores" compared to wild type.

[0313] To determine the relative affinity of the alanine scanning mutant mAb to the binding profile of the parent mAb to the NGF-coated chip, Biacore T200 was used to measure at 100nM. The average response units of four replicates of the parent mAb + / - 3 standard deviations were used to generate parameters to define the thresholds of response units for antibody binding including association rate and dissociation rate. The percentage of data points for each mutant that fell within the threshold was then used to define the "similarity score %". In Tables 4 and 5, the similarity scores obtained by replacing the heavy and light chains with alanine at each heavy and light chain CDR position of ZTS-841 and ZTS-842, respectively, are shown as "percent inhibition relative to the parent". The results of the substitution of alanine at each CDR position.

[0314] The sequences in Table 4 relate to alanine mutagenesis of the variable heavy and variable light CDR amino acid sequences of ZTS-841 (9L12). Table 5 relates to alanine substitutions of the variable heavy and light CDR amino acid sequences of ZTS-842 (48L2). The mutated amino acids are described in the table according to the wild-type numbering of the variable heavy and variable light sequences, as previously described and included below. Amino acid positions 1, 25, 50, 75, and 100 are labeled as follows. In the "Sample Name" column, the heavy or light variable sequence is listed with the numbered amino acid position alanine substitution.

[0315] ZTS-841 VH: SEQ ID NO.8:

[0316] E 1 VQLVESGGDLVKPGGSLRLSCVAS25 GFTFSSHGMHWVRQSPGKGLQWVAV 50 INSGGSSTYYTDAVKGRFTISRDNA 75 KNTVYLQMNSLRAEDTAMYYCAKES 100 VGGWEQLVGPHFDYWGQGTLVIVSS 124

[0317] ZTS-841 VL: SEQ ID NO. 7:

[0318] Q 1 SVLTQPTSVSGSLGQRVTISCSGS 25 TNNIGILGASWYQLFPGKAPKLLVY 50 GNGNRPSGVPDRFSGADSGDSVTLT 75 ITGLQAEDEADYYCQSFDTTLGAHV 100 FGGGTHLTVL 110

[0319] Table 4

[0320]

[0321]

[0322] For Table 5:

[0323] ZTS-842 VH: SEQ ID NO.28

[0324] E 1 VQLVESGGDLVKPGGSLRLSCVAS 25 GFTFSTYGINWVRQAPGKGLQWVAY 50 ISSGGSSTYYADPVKGRFTI 75 SRDDAKNMLYLQMNSLRAEDTAIYYCAGSRY 100 TYAYGGGYEFHFWGQGTLVTVSS 124

[0325] ZTS-842 VL: SEQ ID NO. 27

[0326] Q 1 AVLNQPASVSGALGQKVTISCSGS 25 TMDIDIFGVSWYQQLPGKAPKLLVD 50SDGDRPSGIPDRFSGSRSGNSGTLT 75 ITGLQAEDEADYHCQSGDSTLGALAI 100 FGGGTHVTVL 110

[0327] Table 5

[0328]

[0329]

[0330]

[0331] The values ​​of percent similarity less than 50% generated in Tables 4 and 5 indicate the amino acid positions where the antibody paratope is essential for binding to NGF. Mutation of the wild-type amino acid to alanine at the indicated position resulted in reduced or complete loss of binding to NGF, indicating which amino acids are required for binding and which amino acids can be replaced with at least conservative amino acid substitutions. Sequence Listing <110> Zoetis Services LLC Steiniger, Sebastian CJ Dunkle, William Rugg, Catherine Dunham, Steven A. <120> Anti-NGF antibodies and methods thereof <130> ZP000167 <140> 62 / 641,538 <141> 2018-03-12 <160> 92 <170> PatentIn Version 3.5 <210> 1 <211> 8 <212> PRT <213> Domestic dog <400> 1 Thr Asn Asn Ile Gly Ile Leu Gly 1 5 <210> 2 <211> 3 <212> PRT <213> Domestic dog <400> 2 Gly Asn Gly 1 <210> 3 <211> 11 <212> PRT <213> Domestic dog <400> 3 Gln Ser Phe Asp Thr Thr Leu Gly Ala His Val 1 5 10 <210> 4 <211> 8 <212> PRT <213> Domestic dog <400> 4 Gly Phe Thr Phe Ser Ser His Gly 1 5 <210> 5 <211> 8 <212> PRT <213> Domestic dog <400> 5 Ile Asn Ser Gly Gly Ser Ser Thr 1 5 <210> 6 <211> 18 <212> PRT <213> Domestic dog <400> 6 Ala Lys Glu Ser Val Gly Gly Trp Glu Gln Leu Val Gly Pro His Phe 1 5 10 15 Asp Tyr <210> 7 <211> 110 <212> PRT <213> Domestic dog <400> 7 Gln Ser Val Leu Thr Gln Pro Thr Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Asn Asn Ile Gly Ile Leu 20 25 30 Gly Ala Ser Trp Tyr Gln Leu Phe Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Val Tyr Gly Asn Gly Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ala Asp Ser Gly Asp Ser Val Thr Leu Thr Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Thr Thr Leu 85 90 95 Gly Ala His Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> 8 <211> 125 <212> PRT <213> Domestic dog <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Gly Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ala Val Ile Asn Ser Gly Gly Ser Ser Thr Tyr Tyr Thr Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Val Gly Gly Trp Glu Gln Leu Val Gly Pro His Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Ile Val Ser Ser 115 120 125 <210> 9 <211> 110 <212> PRT <213> Domestic dog <400> 9 Gln Ser Val Leu Thr Gln Pro Thr Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Asn Asn Ile Gly Ile Leu 20 25 30 Gly Ala Ser Trp Tyr Gln Leu Phe Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Val Tyr Gly Asn Gly Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ala Asp Ser Gly Asp Ser Val Thr Leu Thr Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Thr Thr Leu 85 90 95 Gly Ala His Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> 10 <211> 125 <212> PRT <213> Domestic dog <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Gly Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ala Val Ile Asn Ser Gly Gly Ser Ser Thr Tyr Tyr Thr Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Val Gly Gly Trp Glu Gln Leu Val Gly Pro His Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Ile Val Ser Ser 115 120 125 <210> 11 <211> 24 <212> DNA <213> Canis familiaris <400> 11 acgaacaaca tcggtattct tggt 24 <210> 12 <211> 9 <212> DNA <213> Canis familiaris <400> 12 ggtaatggg 9 <210> 13 <211> 36 <212> DNA <213> Canis familiaris <400> 13 cagtcctttg ataccacgct tggtgctcat gtgttc 36 <210> 14 <211> 24 <212> DNA <213> Canis familiaris <400> 14 ggattcacct tcagtagcca cggc 24 <210> 15 <211> 24 <212> DNA <213> Canis familiaris <400> 15 attaacagcg gtggaagtag caca 24 <210> 16 <211> 54 <212> DNA <213> Canis familiaris <400> 16 gcaaaggagt ccgtcggggg gtgggagcaa ctggtcggac ctcattttga ctac 54 <210> 17 <211> 330 <212> DNA <213> Canis lupus familiaris <400> 17 cagtctgtgc tgactcagcc gacctcagtg tcagggtccc ttggccagag ggtcaccatc 60 tcctgctctg gaagcacgaa caacatcggt attcttggtg cgagctggta ccaactgttc 120 ccaggaaagg cccctaaact cctcgtgtac ggtaatggga atcgaccgtc aggggtccct 180 gaccggtttt ccggcgccga ctctggcgac tcagtcaccc tgaccatcac tgggcttcag 240 gctgaggacg aggctgatta ttactgccag tcctttgata ccacgcttgg tgctcatgtg 300 ttcggcggag gcacccacct gaccgtcctt 330 <210> 18 <211> 375 <212> DNA <213> Canis lupus familiaris <400> 18 gaggtgcagc tggtggagtc tgggggagat ttggtgaagc ctggggggtc cttgagactg 60 tcctgtgtgg cctctggatt caccttcagt agccacggca tgcactgggt ccgtcagtct 120 ccagggaagg gactgcagtg ggtcgcagtt attaacagcg gtggaagtag cacatactac 180 acagacgctg tgaagggccg attcaccatc tccagagaca acgccaagaa cacagtgtat 240 ctacagatga acagcctgag agccgaggac acggccatgt attactgtgc aaaggagtcc 300 gtcggggggt gggagcaact ggtcggacct cattttgact actggggcca gggaaccctg 360 gtcatcgtct cgagc 375 <210> 19 <211> 333 <212> DNA <213> Canis familiaris <400> 19 caggcggtgc tgaaccagcc ggcgagcgtg agcggcgcgc tgggccagaa agtgaccatt 60 agctgcagcg gcagcaccat ggatattgat atttttggcg tgagctggta tcagcagctg 120 ccgggcaaag cgccgaaact gctggtggat agcgatggcg atcgcccgag cggcattccg 180 gatcgcttta gcggcagccg cagcggcaac agcggcaccc tgaccattac cggcctgcag 240 gcggaagatg aagcggatta tcattgccag agcggcgata gcaccctggg cgcgctggcg 300 atttttggcg gcggcaccca tgtgaccgtg ctg 333 <210> 20 <211> 375 <212> DNA <213> Domestic dog <400> 20 gaagtgcagc tggtggaaag cggcggcgat ctggtgaaac cgggcggcag cctgcgcctg 60 agctgcgtgg cgagcggctt tacctttagc agccatggca tgcattgggt gcgccagagc 120 ccgggcaaag gcctgcagtg ggtggcggtg attaacagcg gcggcagcag cacctattat 180 accgatgcgg tgaaaggccg ctttaccatt agccgcgata acgcgaaaaa caccgtgtat 240 ctgcagatga acagcctgcg cgcggaagat accgcgatgt attattgcgc gaaagaaagc 300 gtgggcggct gggaacagct ggtgggcccg cattttgatt attggggcca gggcaccctg 360 gtgattgtct cgagc 375 <210> 21 <211> 8 <212> PRT <213> Domestic dog <400> 21 Thr Met Asp Ile Asp Ile Phe Gly 1 5 <210> 22 <211> 3 <212> PRT <213> Domestic dog <400> 22 Ser Asp Gly 1 <210> 23 <211> 12 <212> PRT <213> Domestic dog <400> 23 Gln Ser Gly Asp Ser Thr Leu Gly Ala Leu Ala Ile 1 5 10 <210> 24 <211> 8 <212> PRT <213> Canis familiaris <400> 24 Gly Phe Thr Phe Ser Thr Tyr Gly 1 5 <210> 25 <211> 8 <212> PRT <213> Canis familiaris <400> 25 Ile Ser Ser Gly Gly Ser Ser Thr 1 5 <210> 26 <211> 17 <212> PRT <213> Canis familiaris <400> 26 Ala Gly Ser Arg Tyr Thr Tyr Ala Tyr Gly Gly Gly Tyr Glu Phe His 1 5 10 15 Phe <210> 27 <211> 111 <212> PRT <213> Canis familiaris <400> 27 Gln Ala Val Leu Asn Gln Pro Ala Ser Val Ser Gly Ala Leu Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Thr Met Asp Ile Asp Ile Phe 20 25 30 Gly Val Ser Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Val Asp Ser Asp Gly Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Asn Ser Gly Thr Leu Thr Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr His Cys Gln Ser Gly Asp Ser Thr Leu 85 90 95 Gly Ala Leu Ala Ile Phe Gly Gly Gly Thr His Val Thr Val Leu 100 105 110 <210> 28 <211> 124 <212> PRT <213> Domestic dog <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Tyr Thr Tyr Ala Tyr Gly Gly Gly Tyr Glu Phe His 100 105 110 Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 29 <211> 110 <212> PRT <213> Canis familiaris <400> 29 Gln Ser Val Leu Thr Gln Pro Thr Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Asn Asn Ile Gly Ile Leu 20 25 30 Gly Ala Ser Trp Tyr Gln Leu Phe Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Val Tyr Gly Asn Gly Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ala Asp Ser Gly Asp Ser Val Thr Leu Thr Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Thr Thr Leu 85 90 95 Gly Ala His Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> 30 <211> 124 <212> PRT <213> Domestic dog <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Tyr Thr Tyr Ala Tyr Gly Gly Gly Tyr Glu Phe His 100 105 110 Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 31 <211> 24 <212> DNA <213> Domestic dog <400> 31 acaatggaca ttgatatatt tggt 24 <210> 32 <211> 9 <212> DNA <213> Domestic dog <400> 32 agtgatggg 9 <210> 33 <211> 36 <212> DNA <213> Domestic dog <400> 33 cagtctggtg attccacgct tggtgccctt gctatt 36 <210> 34 <211> twenty four <212> DNA <213> Domestic dog <400> 34 ggattcacct tcagtaccta tggc 24 <210> 35 <211> twenty four <212> DNA <213> Domestic dog <400> 35 attagtagtg gtggaagtag caca 24 <210> 36 <211> 51 <212> DNA <213> Domestic dog <400> 36 gcgggtagta gatatacata tgcatacgga ggaggatatg agtttcactt c 51 <210> 37 <211> 333 <212> DNA <213> Domestic dog <400> 37 caggctgtgc tgaatcagcc ggcctcagtg tctggggccc tgggccagaa ggtcaccatc 60 caggctgtgc tgaatcagcc ggcctcagtg tctggggccc tgggccagaa ggtcaccatc 60 tcctgctctg gaagcacaat ggacattgat atatttggtg tgagctggta ccaacagctc 120 tcctgctctg gaagcacaat ggacattgat atatttggtg tgagctggta ccaacagctc 120 ccaggaaagg cccctaaact cctcgtggac agtgatgggg atcgaccctc agggatccct 180 ccaggaaagg cccctaaact cctcgtggac agtgatgggg atcgaccctc agggatccct 180 gacagatttt ctggctccag gtctggcaac tcaggcaccc tgaccatcac tgggctccag 240 gacagatttt ctggctccag gtctggcaac tcaggcaccc tgaccatcac tgggctccag 240 gctgaggacg aggctgatta tcactgtcag tctggtgatt ccacgcttgg tgcccttgct 300 gctgaggacg aggctgatta tcactgtcag tctggtgatt ccacgcttgg tgcccttgct 300 attttcggcg gaggcaccca tgtgaccgtc ctt 333 attttcggcg gaggcaccca tgtgaccgtc ctt 333 <210> 38<210> 38 <211> 372<211> 372 <212> DNA<212> DNA <213> 家犬<213> Domestic dog <400> 38 <400> 38 gaggtacaac tggtggaatc tgggggagac ctggtgaagc ctgggggatc cctgagactc 60 gaggtacaac tggtggaatc tgggggagac ctggtgaagc ctgggggatc cctgagactc 60 tcctgtgtgg cctctggatt caccttcagt acctatggca tcaactgggt ccgccaggct 120 tcctgtgtgg cctctggatt caccttcagt acctatggca tcaactgggt ccgccaggct 120 ccagggaagg ggctgcagtg ggtcgcatac attagtagtg gtggaagtag cacatactat 180 ccagggaagg ggctgcagtg ggtcgcatac attagtagtg gtggaagtag cacatactat 180 gcagatcctg tgaagggccg gttcaccatc tccagagacg acgccaagaa catgctgtat 240 gcagatcctg tgaagggccg gttcaccatc tccagagacg acgccaagaa catgctgtat 240 cttcagatga acagcctgag agccgaggac acggccatat attactgtgc gggtagtaga 300 cttcagatga acagcctgag agccgaggac acggccatat attactgtgc gggtagtaga 300 tatacatatg catacggagg aggatatgag tttcacttct ggggccaggg aaccctggtc 360 accgtctcga gc 372 <210> 39 <211> 333 <212> DNA <213> Canis familiaris <400> 39 caggcggtgc tgaaccagcc ggcgagcgtg agcggcgcgc tgggccagaa agtgaccatt 60 agctgcagcg gcagcaccat ggatattgat atttttggcg tgagctggta tcagcagctg 120 ccgggcaaag cgccgaaact gctggtggat agcgatggcg atcgcccgag cggcattccg 180 gatcgcttta gcggcagccg cagcggcaac agcggcaccc tgaccattac cggcctgcag 240 gcggaagatg aagcggatta tcattgccag agcggcgata gcaccctggg cgcgctggcg 300 atttttggcg gcggcaccca tgtgaccgtg ctg 333 <210> 40 <211> 372 <212> DNA <213> Canis familiaris <400> 40 gaagtgcagc tggtggaaag cggcggcgat ctggtgaaac cgggcggcag cctgcgcctg 60 agctgcgtgg cgagcggctt tacctttagc acctatggca ttaactgggt gcgccaggcg 120 ccgggcaaag gcctgcagtg ggtggcgtat attagcagcg gcggcagcag cacctattat 180 gcggatccgg tgaaaggccg ctttaccatt agccgcgatg atgcgaaaaa catgctgtat 240 ctgcagatga acagcctgcg cgcggaagat accgcgattt attattgcgc gggcagccgc 300 tatacctatg cgtatggcgg cggctatgaa tttcattttt ggggccaggg caccctggtg 360 accgtctcga gc 372 <210> 41 <211> 335 <212> PRT <213> Canis lupus familiaris <400> 41 Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly 1 5 10 15 Ser Thr Ser Gly Ser Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr 65 70 75 80 Phe Thr Cys Asn Val Ala His Pro Ala Ser Lys Thr Lys Val Asp Lys 85 90 95 Pro Val Pro Lys Arg Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys 100 105 110 Pro Lys Cys Pro Ala Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile 115 120 125 Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu 130 135 140 Val Thr Cys Val Val Val Asp Leu Asp Pro Glu Asp Pro Glu Val Gln 145 150 155 160 Ile Ser Trp Phe Val Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln 165 170 175 Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu 180 185 190 Pro Ile Gly His Gln Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys 195 200 205 Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys 210 215 220 Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser 225 230 235 240 Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys 245 250 255 Asp Phe Phe Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln 260 265 270 Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu 275 280 285 Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg 290 295 300 Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu 305 310 315 320 His Asn His Tyr Thr Gln Glu Ser Leu Ser His Ser Pro Gly Lys 325 330 335 <210> 42 <211> 1005 <212> DNA <213> Canis lupus familiaris <400> 42 gcctcaacaa ctgctcctag cgtgtttccc ctggccccta gctgcggaag tacctcaggc 60 agcacagtgg ccctggcttg tctggtgtct ggatatttcc ctgagccagt gaccgtgagt 120 tggaacagcg gctctctgac ctccggggtg cacacatttc catctgtgct gcagtctagt 180 ggcctgtact ccctgtcaag catggtgact gtgccttcct ctaggtggcc atcagaaact 240 ttcacctgca acgtggccca tcccgccagc aagaccaaag tggacaagcc cgtgcctaaa 300 agggagaatg gaagggtgcc aagaccacct gattgcccta agtgtccagc tccagaaatg 360 ctgggaggac caagcgtgtt catctttcca cccaagccca aagacacact gctgattgct 420 agaactcccg aggtgacctg cgtggtggtg gacctggatc cagaggaccc cgaagtgcag 480 atctcctggt tcgtggatgg gaagcagatg cagacagcca aaactcagcc tcgggaggaa 540 cagtttaacg gaacctatag agtggtgtct gtgctgccaa ttggacacca ggactggctg 600 aagggcaaac agtttacatg caaggtgaac aacaaggccc tgcctagtcc aatcgagagg 660 actatttcaa aagctagggg acaggctcat cagccttccg tgtatgtgct gcctccatcc 720 cgggaggaac tgtctaagaa cacagtgagt ctgacttgtc tgatcaaaga tttctttccc 780 cctgacattg atgtggagtg gcagagcaat gggcagcagg agccagaatc caagtacaga 840 accacaccac cccagctgga cgaagatggc tcctatttcc tgtacagtaa gctgtcagtg 900 gacaaatcta ggtggcagcg cggggatacc tttatctgcg ccgtgatgca cgaggctctg 960 cacaatcatt acacacaaga aagtctgtca catagccccg gcaag 1005 <210> 43 <211> 335 <212> PRT <213> Domestic dog <400> 43 Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly 1 5 10 15 Ser Thr Ser Gly Ser Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr 65 70 75 80 Phe Thr Cys Asn Val Ala His Pro Ala Ser Lys Thr Lys Val Asp Lys 85 90 95 Pro Val Pro Lys Arg Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys 100 105 110 Pro Lys Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Ile 115 120 125 Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu 130 135 140 Val Thr Cys Val Val Val Asp Leu Asp Pro Glu Asp Pro Glu Val Gln 145 150 155 160 Ile Ser Trp Phe Val Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln 165 170 175 Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu 180 185 190 Pro Ile Gly His Gln Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys 195 200 205 Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys 210 215 220 Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser 225 230 235 240 Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys 245 250 255 Asp Phe Phe Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln 260 265 270 Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu 275 280 285 Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg 290 295 300 Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu 305 310 315 320 His Asn His Tyr Thr Gln Glu Ser Leu Ser His Ser Pro Gly Lys 325 330 335 <210> 44 <211> 1005 <212> DNA <213> Domestic dog <400> 44 gcctcaacaa ctgctcctag cgtgtttccc ctggccccta gctgcggaag tacctcaggc 60 agcacagtgg ccctggcttg tctggtgtct ggatatttcc ctgagccagt gaccgtgagt 120 tggaacagcg gctctctgac ctccggggtg cacacatttc catctgtgct gcagtctagt 180 ggcctgtact ccctgtcaag catggtgact gtgccttcct ctaggtggcc atcagaaact 240 ttcacctgca acgtggccca tcccgccagc aagaccaaag tggacaagcc cgtgcctaaa 300 agggagaatg gaagggtgcc aagaccacct gattgcccta agtgtccagc tccagaagcg 360 gcgggagcac caagcgtgtt catctttcca cccaagccca aagacacact gctgattgct 420 agaactcccg aggtgacctg cgtggtggtg gacctggatc cagaggaccc cgaagtgcag 480 atctcctggt tcgtggatgg gaagcagatg cagacagcca aaactcagcc tcgggaggaa 540 cagtttaacg gaacctatag agtggtgtct gtgctgccaa ttggacacca ggactggctg 600 aagggcaaac agtttacatg caaggtgaac aacaaggccc tgcctagtcc aatcgagagg 660 actatttcaa aagctagggg acaggctcat cagccttccg tgtatgtgct gcctccatcc 720 cgggaggaac tgtctaagaa cacagtgagt ctgacttgtc tgatcaaaga tttctttccc 780 cctgacattg atgtggagtg gcagagcaat gggcagcagg agccagaatc caagtacaga 840 accacaccac cccagctgga cgaagatggc tcctatttcc tgtacagtaa gctgtcagtg 900 gacaaatcta ggtggcagcg cggggatacc tttatctgcg ccgtgatgca cgaggctctg 960 cacaatcatt acacacaaga aagtctgtca catagccccg gcaag 1005 <210> 45 <211> 6 <212> PRT <213> Canis lupus familiaris <400> 45 Asn Ile Gly Ser Lys Asp 1 5 <210> 46 <211> 3 <212> PRT <213> Domestic dog <400> 46 Ser Asp Ser 1 <210> 47 <211> 11 <212> PRT <213> Domestic dog <400> 47 Gln Val Trp Asp Ile Ser Ala Asp Ala Ile Val 1 5 10 <210> 48 <211> 8 <212> PRT <213> Domestic dog <400> 48 Gly Tyr Thr Phe Thr Asp Tyr Tyr 1 5 <210> 49 <211> 8 <212> PRT <213> Domestic dog <400> 49 Ile Asp Pro Gly Asn Gly Ala Thr 1 5 <210> 50 <211> 13 <212> PRT <213> Domestic dog <400> 50 Ala Pro Leu Gly Tyr Val Pro Ala Ser Thr Ser Glu Tyr 1 5 10 <210> 51 <211> 108 <212> PRT <213> Domestic dog <400> 51 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Thr Val Thr Leu Arg Gln 1 5 10 15 Thr Ala His Ile Thr Cys Gly Gly Asp Asn Ile Gly Ser Lys Asp Val 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Ile Ile Tyr 35 40 45 Ser Asp Ser Lys Arg Pro Thr Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Met Ala Thr Leu Thr Ile Ser Gly Ala Leu Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ile Ser Ala Asp Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 <210> 52 <211> 120 <212> PRT <213> Canis familiaris <400> 52 Glu Val Gln Leu Val Gln Ser Ala Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Gln Gln Ala Pro Gly Ala Gly Leu Asn Trp Met 35 40 45 Gly Arg Ile Asp Pro Gly Asn Gly Ala Thr Arg Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Leu Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Pro Leu Gly Tyr Val Pro Ala Ser Thr Ser Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Ser Val Ser Ser 115 120 <210> 53 <211> 324 <212> DNA <213> Canis lupus familiaris <400> 53 tcctatgtgc tgacccagcc accatcagtg actgtgaccc tgaggcagac ggcccacatc 60 acctgtgggg gagacaacat tggaagtaaa gatgtttatt ggtaccagca gaagccgggc 120 caggcccccg tgttgattat ctatagtgat agcaagaggc cgacagggat ccctgagcga 180 ttctccggct ccaactcggg gaacatggcc accctgacca tcagtggggc cttggcggag 240 gatgaggctg actattactg ccaggtatgg gacatcagtg ctgatgctat tgtgttcggc 300 ggaggcaccc atctgaccgt cctt 324 <210> 54 <211> 360 <212> DNA <213> Canis lupus familiaris <400> 54 gaggtccagc tggtgcagtc tgcagctgag gttaagaagc caggggcatc tgtaaaggtc 60 tcctgcaaga cctctggata caccttcact gactactata tgcactgggt acaacaggct 120 ccaggagcag ggctcaattg gatgggacgg attgatcctg gaaatggtgc cacaaggtat 180 gcacagaagt tccagggcag actcaccctg acggcagaca catccacaag cacagcctac 240 atggagctga gcggtctgag agctgaggac acagctgtgt actactgtgc gcccctaggg 300 tacgtgcctg catcaacatc tgagtactgg ggccagggca ccctggtcag cgtctcgagc 360 <210> 55 <211> 111 <212> PRT <213> Felis catus <400> 55 Gln Ala Val Leu Asn Gln Pro Ser Ser Val Ser Gly Ala Leu Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Met Asp Ile Asp Ile Phe 20 25 30 Gly Val Ser Trp Tyr Gln Gln Ile Pro Gly Met Ala Pro Lys Thr Ile 35 40 45 Ile Asp Ser Asp Gly Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Ser Thr Gly Thr Leu Thr Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Gly Asp Ser Thr Leu 85 90 95 Gly Ala Leu Ala Ile Phe Gly Gly Gly Thr His Val Thr Val Leu 100 105 110 <210> 56 <211> 124 <212> PRT <213> Domestic cat <400> 56 Asp Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Val Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Tyr Thr Tyr Ala Tyr Gly Gly Gly Tyr Glu Phe His 100 105 110 Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 57 <211> 333 <212> DNA <213> Domestic cat <400> 57 caggcggtgc tgaaccagcc gagcagcgtg agcggcgcgc tgggccagcg cgtgaccatt 60 agctgcagcg gcagcaccat ggatattgat atttttggcg tgagctggta tcagcagatt 120 ccgggcatgg cgccgaaaac cattattgat agcgatggcg atcgcccgag cggcgtgccg 180 gatcgcttta gcggcagcaa aagcggcagc accggcaccc tgaccattac cggcctgcag 240 gcggaagatg aagcggatta ttattgccag agcggcgata gcaccctggg cgcgctggcg 300 atttttggcg gcggcaccca tgtgaccgtg ctg 333 <210> 58 <211> 372 <212> DNA <213> Domestic cat <400> 58 gaggtacaac tggtggaatc tgggggagac ctggtgaagc ctgggggatc cctgagactc 60 tcctgtgtgg cctctggatt caccttcagt acctatggca tcaactgggt ccgccaggct 120 ccagggaagg ggctgcagtg ggtcgcatac attagtagtg gtggaagtag cacatactat 180 gcagatcctg tgaagggccg gttcaccatc tccagagacg acgccaagaa catgctgtat 240 cttcagatga acagcctgag agccgaggac acggccatat attactgtgc gggtagtaga 300 tatacatatg catacggagg aggatatgag tttcacttct ggggccaggg aaccctggtc 360 accgtctcga gc 372 <210> 59 <211> 201 <212> PRT <213> Domestic dog <400> 59 Gln His Ser Leu Asp Thr Ala Leu Arg Arg Ala Arg Ser Ala Pro Ala 1 5 10 15 Gly Ala Ile Ala Ala Arg Val Thr Gly Gln Thr Arg Asn Ile Thr Val 20 25 30 Asp Pro Lys Leu Phe Lys Lys Arg Arg Leu Arg Ser Pro Arg Val Leu 35 40 45 Phe Ser Thr His Pro Pro Pro Val Ala Ala Asp Ala Gln Asp Leu Asp 50 55 60 Leu Glu Ala Gly Ser Thr Ala Ser Val Asn Arg Thr His Arg Ser Lys 65 70 75 80 Arg Ser Ser Pro His Pro Val Phe His Arg Gly Glu Phe Ser Val Cys 85 90 95 Asp Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile 100 105 110 Lys Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser 115 120 125 Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Thr Pro 130 135 140 Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr 145 150 155 160 Cys Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys 165 170 175 Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val 180 185 190 Leu Ser Arg Lys Ala Gly Arg Arg Ala 195 200 <210> 60 <211> 106 <212> PRT <213> Domestic dog <400> 60 Gly Gln Pro Lys Ala Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gly Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Ser Gly Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Thr Gln Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Asp Lys Trp Lys 65 70 75 80 Ser His Ser Ser Phe Ser Cys Leu Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Lys Val Ala Pro Ala Glu Cys Ser 100 105 <210> 61 <211> 318 <212> DNA <213> Domestic dog <400> 61 ggacaaccga aggcctcccc ctcggtcaca ctcttcccgc cctcctctga ggagctcggc 60 gccaacaagg ccaccctggt gtgcctcatc agcgacttct accccagcgg cgtgacggtg 120 gcctggaagg cagacggcag ccccgtcacc cagggcgtgg agaccaccaa gccctccaag 180 cagagcaaca acaagtacgc ggccagcagc tacctgagcc tgacgcctga caagtggaaa 240 tctcacagca gcttcagctg cctggtcacg cacgagggga gcaccgtgga gaagaaggtg 300 gcccccgcag agtgctct 318 <210> 62 <211> 335 <212> PRT <213> Domestic cat <400> 62 Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly 1 5 10 15 Thr Thr Ser Gly Ala Thr Val Ala Leu Ala Cys Leu Val Leu Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ala Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Leu Ser Asp Thr 65 70 75 80 Phe Thr Cys Asn Val Ala His Pro Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Arg Lys Thr Asp His Pro Pro Gly Pro Lys Pro Cys Asp Cys 100 105 110 Pro Lys Cys Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile Phe Ile 115 120 125 Phe Pro Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser Arg Thr Pro Glu 130 135 140 Val Thr Cys Leu Val Val Asp Leu Gly Pro Asp Asp Ser Asp Val Gln 145 150 155 160 Ile Thr Trp Phe Val Asp Asn Thr Gln Val Tyr Thr Ala Lys Thr Ser 165 170 175 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 180 185 190 Pro Ile Leu His Gln Asp Trp Leu Lys Gly Lys Glu Phe Lys Cys Lys 195 200 205 Val Asn Ser Lys Ser Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys 210 215 220 Ala Lys Gly Gln Pro His Glu Pro Gln Val Tyr Val Leu Pro Pro Ala 225 230 235 240 Gln Glu Glu Leu Ser Arg Asn Lys Val Ser Val Thr Cys Leu Ile Lys 245 250 255 Ser Phe His Pro Pro Asp Ile Ala Val Glu Trp Glu Ile Thr Gly Gln 260 265 270 Pro Glu Pro Glu Asn Asn Tyr Arg Thr Thr Pro Pro Gln Leu Asp Ser 275 280 285 Asp Gly Thr Tyr Phe Val Tyr Ser Lys Leu Ser Val Asp Arg Ser His 290 295 300 Trp Gln Arg Gly Asn Thr Tyr Thr Cys Ser Val Ser His Glu Ala Leu 305 310 315 320 His Ser His His Thr Gln Lys Ser Leu Thr Gln Ser Pro Gly Lys 325 330 335 <210> 63 <211> 1005 <212> DNA <213> Domestic cat <400> 63 gcctccacca cggccccatc ggtgttccca ctggccccca gctgcgggac cacatctggc 60 gccaccgtgg ccctggcctg cctggtgtta ggctacttcc ctgagccggt gaccgtgtcc 120 tggaactccg gcgccctgac cagcggtgtg cacaccttcc cggccgtcct gcaggcctcg 180 gggctgtact ctctcagcag catggtgaca gtgccctcca gcaggtggct cagtgacacc 240 ttcacctgca acgtggccca cccgcccagc aacaccaagg tggacaagac cgtgcgcaaa 300 acagaccacc caccgggacc caaaccctgc gactgtccca aatgcccacc ccctgagatg 360 cttggaggac cgtccatctt catcttcccc ccaaaaccca aggacaccct ctcgatttcc 420 cggacgcccg aggtcacatg cttggtggtg gacttgggcc cagatgactc cgatgtccag 480 atcacatggt ttgtggataa cacccaggtg tacacagcca agacgagtcc gcgtgaggag 540 cagttcaaca gcacctaccg tgtggtcagt gtcctcccca tcctacacca ggactggctc 600 aaggggaagg agttcaagtg caaggtcaac agcaaatccc tcccctcccc catcgagagg 660 accatctcca aggccaaagg acagccccac gagccccagg tgtacgtcct gcctccagcc 720 caggaggac tcagcaggaa caaagtcagt gtgacctgcc tgatcaaatc cttccacccg 780 cctgacattg ccgtcgagtg ggagatcacc ggacagccgg agccagagaa caactaccgg 840 acgaccccgc cccagctgga cagcgacggg acctacttcg tgtacagcaa gctctcggtg 900 gacaggtccc actggcagag gggaaacacc tacacctgct cggtgtcaca cgaagctctg 960 cacagccacc acacacagaa atccctcacc cagtctccgg gtaaa 1005 <210> 64 <211> 106 <212> PRT <213> Domestic cat <400> 64 Gly Gln Pro Lys Ser Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Asn 1 5 10 15 Glu Glu Leu Ser Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Ser Gly Leu Thr Val Ala Trp Lys Ala Asp Gly Thr Pro 35 40 45 Ile Thr Gln Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Ser Pro Asn Glu Trp Lys 65 70 75 80 Ser Arg Ser Arg Phe Thr Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Asn Val Val Pro Ala Glu Cys Ser 100 105 <210> 65 <211> 318 <212> DNA <213> Domestic cat <400> 65 ggccagccca agagcgctcc ctccgtgacc ctgttccccc caagcaacga ggaactgagc 60 ggccagccca agagcgctcc ctccgtgacc ctgttccccc caagcaacga ggaactgagc 60 gccaacaagg ccaccctggt gtgcctgatc agcgacttct accccagcgg cctgaccgtg 120 gccaacaagg ccaccctggt gtgcctgatc agcgacttct accccagcgg cctgaccgtg 120 gcctggaagg ccgatggcac ccctatcacc cagggcgtgg aaaccaccaa gcccagcaag 180 gcctggaagg ccgatggcac ccctatcacc cagggcgtgg aaaccaccaa gcccagcaag 180 cagagcaaca acaaatacgc cgccagcagc tacctgagcc tgagccccaa cgagtggaag 240 cagagcaaca acaaatacgc cgccagcagc tacctgagcc tgagccccaa cgagtggaag 240 tcccggtccc ggttcacatg ccaggtgaca cacgagggca gcaccgtgga aaagaacgtg 300 tcccggtccc ggttcacatg ccaggtgaca cacgagggca gcaccgtgga aaagaacgtg 300 gtgcccgccg agtgcagc 318 gtgcccgccg agtgcagc 318 <210> 66<210> 66 <211> 240<211> 240 <212> PRT<212> PRT <213> 智人<213> Homo sapiens <400> 66<400> 66 Met Ser Met Leu Phe Tyr Thr Leu Ile Thr Ala Phe Leu Ile Gly Ile Met Ser Met Leu Phe Tyr Thr Leu Ile Thr Ala Phe Leu Ile Gly Ile 1 5 10 15 1 5 10 15 Gln Ala Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Gln Ala Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile 20 25 30 20 25 30 Pro Gln Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Pro Gln Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu 35 40 45 35 40 45 Arg Arg Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Arg Arg Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala 50 55 60 50 55 60 Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg 65 70 75 80 Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu 85 90 95 Ala Ala Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro 100 105 110 Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser Ser His Pro Ile Phe 115 120 125 His Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly 130 135 140 Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu 145 150 155 160 Gly Glu Val Ser Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu 165 170 175 Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile 180 185 190 Asp Ser Lys His Trp Asn Ser Cys Thr Thr Thr His Thr Phe Val Lys 195 200 205 Ala Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile 210 215 220 Asp Thr Ala Cys Met Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala 225 230 235 240 <210> 67 <211> 125 <212> PRT <213> Homo sapiens <400> 67 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Asn Ser Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Val Gly Gly Trp Glu Gln Leu Val Gly Pro His Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Ile Val Ser Ser 115 120 125 <210> 68 <211> 375 <212> DNA <213> Homo sapiens <400> 68 gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggctt tacctttagc agccatggca tgagctgggt gcgccaggcg 120 ccgggcaaag gcctggaatg ggtgagcgtg attaacagcg gcggcagcag cacctattat 180 gcggatagcg tgaaaggccg ctttaccatt agccgcgata acagcaaaaa caccctgtat 240 ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gaaagaaagc 300 gtgggcggct gggaacagct ggtgggcccg cattttgatt attggggcca gggcaccctg 360 gtgattgtct cgagc 375 <210> 69 <211> 125 <212> PRT <213> Homo sapiens <400> 69 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Asn Ser Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Val Gly Gly Trp Glu Gln Leu Val Gly Pro His Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Ile Val Ser Ser 115 120 125 <210> 70 <211> 375 <212> DNA <213> Homo sapiens <400> 70 caggtgcagc tggtggaaag cggcggcggc gtggtgcagc cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggctt tacctttagc agccatggca tgcattgggt gcgccaggcg 120 ccgggcaaag gcctggaatg ggtgagcgtg attaacagcg gcggcagcag cacctattat 180 gcggatagcg tgaaaggccg ctttaccatt agccgcgata acagcaaaaa caccctgtat 240 ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gaaagaaagc 300 gtgggcggct gggaacagct ggtgggcccg cattttgatt attggggcca gggcaccctg 360 gtgattgtct cgagc 375 <210> 71 <211> 110 <212> PRT <213> Homo sapiens <400> 71 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Thr Asn Asn Ile Gly Ile Leu 20 25 30 Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Gly Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Thr Thr Leu 85 90 95 Gly Ala His Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> 72 <211> 330 <212> DNA <213> Homo sapiens <400> 72 cagagcgtgc tgacccagcc gccgagcgtg agcggcgcgc cgggccagcg cgtgaccatt 60 agctgcaccg gcagcaccaa caacattggc attctgggcg tgcattggta tcagcagctg 120 ccgggcaccg cgccgaaact gctgatttat ggcaacggca accgcccgag cggcgtgccg 180 gatcgcttta gcggcagcaa aagcggcacc agcgcgagcc tggcgattac cggcctgcag 240 gcggaagatg aagcggatta ttattgccag agctttgata ccaccctggg cgcgcatgtg 300 tttggcggcg gcacccatct gaccgtgctg 330 <210> 73 <211> 110 <212> PRT <213> Homo sapiens <400> 73 Gln Ser Val Leu Thr Gln Pro Thr Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Thr Asn Asn Ile Gly Ile Leu 20 25 30 Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Gly Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ala Asp Ser Gly Asp Ser Val Ser Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Thr Thr Leu 85 90 95 Gly Ala His Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> 74 <211> 330 <212> DNA <213> Homo sapiens <400> 74 cagagcgtgc tgacccagcc gaccagcgtg agcggcgcgc cgggccagcg cgtgaccatt 60 agctgcaccg gcagcaccaa caacattggc attctgggcg tgcattggta tcagcagctg 120 ccgggcaccg cgccgaaact gctgatttat ggcaacggca accgcccgag cggcgtgccg 180 gatcgcttta gcggcgcgga tagcggcgat agcgtgagcc tggcgattac cggcctgcag 240 gcggaagatg aagcggatta ttattgccag agctttgata ccaccctggg cgcgcatgtg 300 tttggcggcg gcacccatct gaccgtgctg 330 <210> 75 <211> 124 <212> PRT <213> Homo sapiens <400> 75 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Gly Gly Ser Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Tyr Thr Tyr Ala Tyr Gly Gly Gly Tyr Glu Phe His 100 105 110 Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 76 <211> 372 <212> DNA <213> Homo sapiens <400> 76 gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggctt tacctttagc acctatggca tgaactgggt gcgccaggcg 120 ccgggcaaag gcctggaatg ggtgagctat attagcagcg gcggcagcag catttattat 180 gcggatagcg tgaaaggccg ctttaccatt agccgcgata acgcgaaaaa cagcctgtat 240 ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gggcagccgc 300 tatacctatg cgtatggcgg cggctatgaa tttcattttt ggggccaggg caccctggtg 360 attgtctcga gc 372 <210> 77 <211> 124 <212> PRT <213> Homo sapiens <400> 77 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ser Tyr Ile Ser Ser Gly Gly Ser Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Tyr Thr Tyr Ala Tyr Gly Gly Gly Tyr Glu Phe His 100 105 110 Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 78 <211> 372 <212> DNA <213> Homo sapiens <400> 78 gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggctt tacctttagc acctatggca tgaactgggt gcgccaggcg 120 ccgggcaaag gcctgcagtg ggtgagctat attagcagcg gcggcagcag catttattat 180 gcggatagcg tgaaaggccg ctttaccatt agccgcgata acgcgaaaaa cagcctgtat 240 ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gggcagccgc 300 tatacctatg cgtatggcgg cggctatgaa tttcattttt ggggccaggg caccctggtg 360 attgtctcga gc 372 <210> 79 <211> 124 <212> PRT <213> Homo sapiens <400> 79 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Tyr Thr Tyr Ala Tyr Gly Gly Gly Tyr Glu Phe His 100 105 110 Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 80 <211> 372 <212> DNA <213> Homo sapiens <400> 80 gaagtgcagc tggtggaaag cggcggcggc ctggtgaaac cgggcggcag cctgcgcctg 60 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly agctgcgcgg cgagcggctt tacctttagc acctatggca ttaactgggt gcgccaggcg 120 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val ccgggcaaag gcctggaatg ggtgagctat attagcagcg gcggcagcag cacctattat 180 Pro Gly Lys Gly Leu Gln Trp Val Ser Tyr Ile Ser Ser Gly Gly Ser Ser Thr Tyr Tyr gcggatagcg tgaaaggccg ctttaccatt agccgcgata acgcgaaaaa cagcctgtat 240 Ala Asp Ser Val Glu Lys Ala Ala Phe Thr Ile Ser Ala Arg Asn Ala Glu Lys Gln Pro Tyr ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gggcagccgc 300 Leu Gln Met Asn Gln Pro Cys Arg Arg Glu Asp Thr Arg Gly Val Ile Ile Cys Arg Gly Ser Arg tatacctatg cgtatggcgg cggctatgaa tttcattttt ggggccaggg caccctggtg 360 Tyr Thr Tyr Ala Tyr Gly Gly Gly Tyr Glu Phe His Phe Phe Gly Pro Gly Thr Leu Val attgtctcga gc 372 Ile Val Ser Glu <210> 81 <211> 124 <212> PRT <213> Homo sapiens <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ser Tyr Ile Ser Ser Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Ser Tyr Ile Ser Ser Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Tyr Thr Tyr Ala Tyr Gly Gly Gly Tyr Glu Phe His 100 105 110 Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 82 <211> 372 <212> DNA <213> Homo sapiens <400> 82 gaagtgcagc tggtggaaag cggcggcggc ctggtgaaac cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggctt tacctttagc acctatggca ttaactgggt gcgccaggcg 120 ccgggcaaag gcctgcagtg ggtgagctat attagcagcg gcggcagcag cacctattat 180 gcggatagcg tgaaaggccg ctttaccatt agccgcgata acgcgaaaaa cagcctgtat 240 ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gggcagccgc 300 tatacctatg cgtatggcgg cggctatgaa tttcattttt ggggccaggg caccctggtg 360 attgtctcga gc 372 <210> 83 <211> 110 <212> PRT <213> Homo sapiens <400> 83 Gln Ser Val Leu Thr Gln Pro Ala Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Met Asp Ile Asp Ile Phe 20 25 30 Gly Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asp Gly Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr His Cys Gln Ser Gly Asp Ser Thr Leu 85 90 95 Gly Ala Leu Ala Ile Phe Gly Gly Gly Thr His Val Thr Val 100 105 110 <210> 84 <211> 333 <212> DNA <213> Homo sapiens <400> 84 cagagcgtgc tgacccagcc ggcgagcgcg agcggcaccc cgggccagcg cgtgaccatt 60 agctgcagcg gcagcaccat ggatattgat atttttggcg tgaactggta tcagcagctg 120 AGCTGCAGCG GCAACACCAT GGATATTGAT ATTTTTGGC G TGAACTGGTA TCAGCAGCTG 120 ccgggcaccg cgccgaaact gctgatttat agcgatggcg atcgcccgag cggcgtgccg 180 CCGGGACCGA CGGCCGAAAC TGCTGATATA TAGCGATGGC G ATCGCCCGAG CGGCGTGCCG 180 gatcgcttta gcggcagcaa aagcggcacc agcgcgagcc tggcgattag cggcctgcag 240 GATCGCTTTA GCGGCAGCAA AAGCGGCACC AGCAGCGAGC CTGGCGAATA GCGGCCTGCA G 240 agcgaagatg aagcggatta tcattgccag agcggcgata gcaccctggg cgcgctggcg 300 AGCGAAGATG AAGCGGATTA TCATTGCCAG AGCAGCGATA GCACCCGGGC GCGCTGGC G 300 atttttggcg gcggcaccca tgtgaccgtg ctg 333 ATTTTTGGCG GCGGCACCCA TGTGACCGTG CTG 333 <210> 85 <211> 111 <212> PRT <213> Homo sapiens <400> 85 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Leu Gly Gln Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Leu Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Met Asp Ile Asp Ile Phe Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Met Asp Ile Asp Ile Phe 20 25 30 Gly Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Gly Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asp Gly Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Ile Tyr Ser Asp Gly Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr His Cys Gln Ser Gly Asp Ser Thr Leu 85 90 95 Gly Ala Leu Ala Ile Phe Gly Gly Gly Thr His Val Thr Val Leu 100 105 110 <210> 86 <211> 333 <212> DNA <213> Homo sapiens <400> 86 cagagcgtgc tgacccagcc gccgagcgcg agcggcaccc tgggccagcg cgtgaccatt 60 agctgcagcg gcagcaccat ggatattgat atttttggcg tgaactggta tcagcagctg 120 ccgggcaccg cgccgaaact gctgatttat agcgatggcg atcgcccgag cggcgtgccg 180 gatcgcttta gcggcagcaa aagcggcacc agcgcgagcc tggcgattag cggcctgcag 240 agcgaagatg aagcggatta tcattgccag agcggcgata gcaccctggg cgcgctggcg 300 atttttggcg gcggcaccca tgtgaccgtg ctg 333 <210> 87 <211> 111 <212> PRT <213> Homo sapiens <400> 87 Gln Ser Val Val Thr Gln Pro Ala Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Thr Met Asp Ile Asp Ile Phe 20 25 30 Gly Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asp Gly Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Ala Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr His Cys Gln Ser Gly Asp Ser Thr Leu 85 90 95 Gly Ala Leu Ala Ile Phe Gly Gly Gly Thr His Val Thr Val Leu 100 105 110 <210> 88 <211> 333 <212> DNA <213> Homo sapiens <400> 88 cagagcgtgg tgacccagcc ggcgagcgtg agcggcgcgc cgggccagcg cgtgaccatt 60 agctgcaccg gcagcaccat ggatattgat atttttggcg tgagctggta tcagcagctg 120 ccgggcaccg cgccgaaact gctgatttat ggcgatggcg atcgcccgag cggcgtgccg 180 gatcgcttta gcggcagcaa aagcggcgcg agcgcgagcc tggcgattac cggcctgcag 240 gcggaagatg aagcggatta tcattgccag agcggcgata gcaccctggg cgcgctggcg 300 atttttggcg gcggcaccca tgtgaccgtg ctg 333 <210> 89 <211> 111 <212> PRT <213> Homo sapiens <400> 89 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Gly Ala Leu Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Thr Met Asp Ile Asp Ile Phe 20 25 30 Gly Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asp Gly Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Ala Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr His Cys Gln Ser Gly Asp Ser Thr Leu 85 90 95 Gly Ala Leu Ala Ile Phe Gly Gly Gly Thr His Val Thr Val Leu 100 105 110 <210> 90 <211> 333 <212> DNA <213> Homo sapiens <400> 90 cagagcgtgg tgacccagcc gccgagcgtg agcggcgcgc tgggccagcg cgtgaccatt 60 agctgcaccg gcagcaccat ggatattgat atttttggcg tgagctggta tcagcagctg 120 ccgggcaccg cgccgaaact gctgatttat ggcgatggcg atcgcccgag cggcgtgccg 180 gatcgcttta gcggcagcaa aagcggcgcg agcgcgagcc tggcgattac cggcctgcag 240 gcggaagatg aagcggatta tcattgccag agcggcgata gcaccctggg cgcgctggcg 300 atttttggcg gcggcaccca tgtgaccgtg ctg 333 <210> 91 <211> 111 <212> PRT <213> Canis lupus familiaris <400> 91 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Thr Met Asp Ile Asp Ile Phe 20 25 30 Gly Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asp Gly Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Ala Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr His Cys Gln Ser Gly Asp Ser Thr Leu 85 90 95 Gly Ala Leu Ala Ile Phe Gly Gly Gly Thr His Val Thr Val Leu 100 105 110 <210> 92 <211> 333 <212> DNA <213> Canis familiaris <400> 92 cagagcgtgg tgacccagcc gccgagcgtg agcggcgcgc cgggccagcg cgtgaccatt 60 agctgcaccg gcagcaccat ggatattgat atttttggcg tgagctggta tcagcagctg 120 ccgggcaccg cgccgaaact gctgatttat ggcgatggcg atcgcccgag cggcgtgccg 180 gatcgcttta gcggcagcaa aagcggcgcg agcgcgagcc tggcgattac cggcctgcag 240 gcggaagatg aagcggatta tcattgccag agcggcgata gcaccctggg cgcgctggcg 300 atttttggcg gcggcaccca tgtgaccgtg ctg 333

Claims

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

6.

2. The recombinant antigen-binding protein of claim 1, wherein the variable light chain and the variable heavy chain comprise an amino acid sequence selected from the group consisting of: a. SEQ ID NO.7 and SEQ ID NO.8 respectively; b. SEQ ID NO.9 and SEQ ID NO.10 respectively; c. SEQ ID NO.7 and SEQ ID NO.67 respectively; d. SEQ ID NO.7 and SEQ ID NO.69, respectively; and e. SEQ ID NO. 8 and SEQ ID NO. 73 respectively. 3 . The recombinant antigen-binding protein according to claim 1 , wherein the variable light chain comprises the amino acid sequence of SEQ ID NO. 7, and the variable heavy chain comprises the amino acid sequence of SEQ ID NO.

8.

4. A recombinant antigen binding protein that specifically binds to nerve growth factor (NGF), the recombinant antigen binding protein comprising a variable light chain and a variable heavy chain consisting of an amino acid sequence selected from the group consisting of: a. SEQ ID NO.7 and SEQ ID NO.8 respectively; b. SEQ ID NO.9 and SEQ ID NO.10 respectively; c. SEQ ID NO.7 and SEQ ID NO.67 respectively; d. SEQ ID NO.7 and SEQ ID NO.69, respectively; and e. SEQ ID NO. 8 and SEQ ID NO. 73 respectively. 5 . The recombinant antigen-binding protein according to claim 4 , wherein the variable light chain consists of the amino acid sequence of SEQ ID NO. 7, and the variable heavy chain consists of the amino acid sequence of SEQ ID NO.

8.

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

7. The antigen binding protein according to any one of claims 1-5, wherein the binding protein reduces or eliminates an NGF-related disorder, wherein the NGF-related disorder is pain.

8. The antigen binding protein of claim 7, wherein the NGF-related disorder comprises osteoarthritis pain.

9. The antigen binding protein according to any one of claims 1 to 5, wherein the binding protein is selected from a monoclonal antibody or a multispecific antibody.

10. The antigen binding protein according to any one of claims 1 to 5, wherein the binding protein is selected from a chimeric antibody.

11. The antigen binding protein according to any one of claims 1 to 5, wherein the binding protein is selected from a tetrameric antibody.

12. The antigen binding protein according to any one of claims 1 to 5, wherein the binding protein is selected from a tetravalent antibody.

13. The antigen binding protein according to any one of claims 1-5, wherein the binding protein is selected from a domain-specific antibody or a domain-deleted antibody.

14. The antigen binding protein according to any one of claims 1 to 5, wherein the binding protein is selected from a fusion protein.

15. The antigen binding protein according to claim 14, wherein the binding protein is selected from ScFc fusion protein.

16. The antigen binding protein according to any one of claims 1-5, wherein the binding protein is selected from the group consisting of: a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment and a ScFv fragment.

17. The antigen binding protein according to any one of claims 1 to 5, wherein the binding protein is selected from small modular immunopharmaceuticals (SMIPs).

18. The antigen binding protein of claim 9, wherein the antigen binding protein is a monoclonal antibody.

19. The antigen binding protein of claim 18, wherein the monoclonal antibody is a canine monoclonal antibody, a caninized monoclonal antibody, a feline monoclonal antibody, a feline monoclonal antibody, a human monoclonal antibody, a humanized monoclonal antibody, an equine monoclonal antibody, or an equinized monoclonal antibody.

20. A pharmaceutical composition or veterinary composition comprising a therapeutically effective amount of the antigen binding protein according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. The pharmaceutical or veterinary composition according to claim 20, for use in treating pain in a subject.

22. The pharmaceutical composition of claim 21, wherein the pain comprises osteoarthritis pain.

23. A pharmaceutical composition according to any one of claims 20 to 22 for use in canines, felines, horses or humans.

24. The pharmaceutical composition according to claim 23, for use in dogs.

25. The pharmaceutical composition according to claim 23, for use in cats.

26. A host cell comprising the antigen binding protein according to any one of claims 1-19.

27. An isolated nucleic acid comprising a nucleic acid sequence encoding the antigen binding protein of any one of claims 1-19.

28. A vector comprising the nucleic acid sequence according to claim 27.

29. A host cell comprising the vector according to claim 28.

30. A host cell comprising the nucleic acid of claim 27.

31. A method for producing an antigen binding protein according to any one of claims 1 to 19, comprising culturing a host cell according to any one of claims 26, 29 or 30 under conditions that result in the production of the antigen binding protein, and isolating the antigen binding protein from the host cell or the culture medium of the host cell.

32. Use of a therapeutically effective amount of a pharmaceutical composition according to any one of claims 20-25 in the preparation of a medicament for treating an NGF-related disorder in a subject, wherein the NGF-related disorder is pain.

33. The use according to claim 32, wherein the NGF-related disorder comprises osteoarthritis pain.

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