Anti-NGF antibodies and uses thereof

Abstract

The invention provides novel anti-NGF proteins, antibodies, and NGF -binding fragments thereof which inhibit association of NGF with TrkA and / or p75 and are suitable for administration to a canine or feline subject. The invention also provides novel compositions and methods of treating pain or eliciting an analgesic effect in a canine or feline subject, comprising administering an effective amount of an anti-NGF protein, antibody or fragment thereof. The methods and compositions are used to treat or prevent NGF-related disorders.

Description

ANTI-NGF ANTIBODIES AND USES THEREOFRELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims priority to US provisional application Serial No. 63 / 649,637, filed May 20, 2024, incorporated by reference herein in its entirety.

[0002] The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.FIELD OF THE INVENTION

[0003] The invention provides novel anti-NGF proteins, antibodies, and NGF-binding fragments thereof which inhibit association of NGF with TrkA and / or p75 and are suitable for administration to humans, companion animals or livestock. The invention also provides novel compositions and methods of treating pain or eliciting an analgesic effect comprising administering an effective amount of an anti-NGF protein, antibody or fragment thereof. The methods and compositions are used to treat or prevent NGF-related disorders.SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said .xml copy, created on May 19, 2025, is named Y9432-99010, and is 73,723 bytes in size.BACKGROUND OF THE INVENTION

[0004] Nerve growth factor (NGF) is critical in the development and maintenance of peripheral sympathetic and embryonic sensory neurons and of basal forebrain cholinergic neurons. NGF upregulates expression of neuropeptides in sensory neurons and its activity is mediated through two different membrane-bound receptors. Several neurotropins (NTs) including NGF bindto a low-affinity receptor identified as p75. NGF selectively binds to and displays a high affinity for the high affinity neurotrophin receptor TrkA.

[0005] Upon neurotrophin binding, TrkA undergoes autophosphorylation as well as phosphorylates members of the MAPK pathway. The presence of this kinase leads to cell differentiation and may play a role in specifying sensory neuron subtypes.

[0006] NGF plays a role in several diseases and disorders, including but not limited to pain associated with a broad range of diseases and disorders, such as pain associated with cancers, neuropathic pain, and neurogenic pain. Due to the involvement of NGF in a wide range of pain- related diseases and disorders, there is a need in the art for compositions and methods useful for preventing or treating diseases and disorders associated with NGF, particularly those associated with pain, including in canines, felines and other animals. Particularly preferred anti-NGF compositions are those having minimal or minimized adverse reactions, such as inflammation when administered to a subject.

[0007] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.SUMMARY OF THE INVENTION

[0008] The invention provides novel anti-NGF binding protein for treatment or amelioration of NGF-related disorders, particularly adapted for use in dogs and cats but not limited thereby.

[0009] The invention provides binding proteins that specifically binds to NGF. In certain embodiments, the binding proteins are optimized for administration to a canine. In certain embodiments, the binding proteins are optimized for administration to a feline.

[0010] In an aspect, the invention provides a binding protein that specifically binds to nerve growth factor (NGF), which comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising TDNMGVG (SEQ ID NO:25); (b) a heavy chain complementarity determining region 2 (HCDR2) comprising IWWDDDK (SEQ ID NO:27); (c) a heavy chain complementarity determining region 3 (HCDR3) comprising VYGXiGHYFDH (SEQ ID NO:56), wherein Xi comprises I, L, or M; (d) a light chain complementarity determining region 1 (LCDR1) comprising QNVGNY (SEQ ID NO:32); (e) a light chain complementarity determining region 2 (LCDR2) comprising YAS (SEQ ID NO:35); and (f) a light chain complementarity determining region 3 (LCDR3) comprising QRIYISPWT (SEQ ID NO:38).

[0011] In certain embodiments, the binding protein comprises canine or caninized frameworks. In certain embodiments, the binding protein comprises human or humanized frameworks. In certain embodiments, the binding protein comprises feline or felinized frameworks.

[0012] In certain embodiments, the antigen binding protein comprises a heavy chain framework (FR1H+FR2H+FR3H+FR4H) at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 18. In certain embodiments, the binding protein comprises a light chain framework (FR1L+FR2L+FR3L+FR4L) at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 14. In certain embodiments, the antigen binding protein comprises a VH domain at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 18, and a VL domain at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 14.

[0013] In an aspect, the invention provides a binding protein that specifically binds to nerve growth factor (NGF), which comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising NYDMA (SEQ ID NO:40); (b) a heavy chain complementarity determining region 2 (HCDR2) comprising XiSPGGGSI (SEQ ID NO:59), wherein Xi comprises I, L, or M; (c) a heavy chain complementarity determining region 3 (HCDR3) comprising EGELGPFX2Y (SEQ ID NO:62), wherein X2 comprises D, E, Q, or S; (d) a light chain complementarity determining region 1 (LCDR1) comprising QSVGIN (SEQ ID NO:47); (e) a light chain complementarity determining region 2 (LCDR2) comprising GAS (SEQ ID NO:50); and (f) a light chain complementarity determining region 3 (LCDR3) comprising LQYGSIPWT (SEQ ID NO:52).

[0014] In certain embodiments, the binding protein comprises canine or caninized frameworks. In certain embodiments, the binding protein comprises human or humanized frameworks. In certain embodiments, the binding protein comprises feline or felinized frameworks.

[0015] In certain embodiments, the binding protein comprises a heavy chain framework (FR1H+FR2H+FR3H+FR4H) at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 15. In certain embodiments, the binding proteincomprises a light chain framework (FR1L+FR2L+FR3L+FR4L) at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 16.

[0016] In certain embodiments, the binding protein comprises a VH domain at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 15 and a VL domain at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 16.

[0017] In an aspect, the invention provides an isolated nucleic acid sequence encoding an anti- NGF antibody or antibody fragment set forth above. In an aspect, the invention provides a vector that comprises the nucleic acid encoding an anti-NGF antibody or antibody fragment set forth above.

[0018] In an aspect, the invention provides a recombinant cell which comprises the nucleic acid encoding an anti-NGF antibody or antibody fragment set forth above.

[0019] In an aspect, the invention provides a method of producing an anti-NGF antibody or antibody fragment which comprises culturing the cell under conditions that result in production of the anti-NGF antibody or antibody fragment set forth above.

[0020] In an aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-NGF protein set forth above.

[0021] In an aspect, the invention provides a method of treating or reducing pain in a subject which comprises administering to the subject a therapeutically effective amount of an anti-NGF protein set forth above. In certain embodiments, the pain comprises inflammatory pain, postoperative incision pain, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, pain resulting from burn, pain from trauma, musculoskeletal pain, rheumatic pain, or osteoporosis pain. In certain embodiments, the subject is a canine. In certain embodiments the subject comprises a feline. In certain embodiments the subject comprises a human.

[0022] In an aspect, the invention provides a method of detecting NGF in a sample comprising incubating a sample comprising NGF in the presence of an anti-NGF protein set forth above and detecting the anti-NGF protein bound to NGF in the sample.

[0023] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0024] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” “comprising,” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes,” “included,” “including,” and the like; and that terms such as “consisting essentially of’ and “consists essentially of’ have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0025] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0027] FIG. 1 depicts amino acid sequences of antibody 2178 VH (SEQ ID NO:3) and VK (SEQ ID NO:4) variable domains, showing extent of CDRs (see Table 3) according to IMGT and Kabat. CDRs for Grafting are the amino acids grafted to canine frameworks to generate 2178 clone 006.

[0028] FIG. 2 depicts amino acid sequences of antibody 2141 VH (SEQ ID NO: 1) and VK (SEQ ID NO:2) variable domains, showing extent of CDRs (see Table 2) according to IMGT and Kabat. CDRs for Grafting are the amino acids grafted to canine frameworks to generate 2141 clone Oi l .

[0029] FIG. 3 depicts inhibition of proliferation of TF-1 cells treated with 10 ng / mL canine NGF and varying concentrations of caninized antibodies 2141 01 l_M103L (ICso = 108 pM) and 2178_Clone 006 (ICso = 335 pM).

[0030] FIG. 4 depicts a chemically induced Monosodium lodoacetate (MIA) model of osteoarthritis and dynamic weight bearing (DWB) in treated mice. Mice with MIA induced osteoarthritis were treated with one of three anti-NGF antibodies: Antibody 2141 01 l_M103L, 2178 clone 006, or bedinvetmab, or dexamethasone.DETAILED DESCRIPTION OF THE INVENTION

[0031] The invention provides binding proteins that specifically binds to NGF. In certain embodiments, the binding proteins are optimized for administration to a canine. In certain embodiments, the binding proteins are optimized for administration to a feline.

[0032] In an aspect, the invention provides a binding protein that specifically binds to nerve growth factor (NGF), which comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising TDNMGVG (SEQ ID NO:25); (b) a heavy chain complementarity determining region 2 (HCDR2) comprising IWWDDDK (SEQ ID NO:27); (c) a heavy chain complementarity determining region 3 (HCDR3) comprising VYGXiGHYFDH (SEQ ID NO:56), wherein Xi comprises I, L, or M; (d) a light chain complementarity determining region 1 (LCDR1) comprising QNVGNY (SEQ ID NO:32); (e) a light chain complementarity determining region 2 (LCDR2) comprising YAS (SEQ ID NO:35); and (f) a light chain complementarity determining region 3 (LCDR3) comprising QRIYISPWT (SEQ ID NO:38).

[0033] In certain embodiments, the binding protein comprises canine or caninized frameworks. In certain embodiments, the binding protein comprises human or humanized frameworks. In certain embodiments, the binding protein comprises feline or felinized frameworks.

[0034] In certain embodiments, the antien binding protein comprises a heavy chain framework (FR1H+FR2H+FR3H+FR4H) at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 18. In certain embodiments, the binding protein comprises a light chain framework (FR1L+FR2L+FR3L+FR4L) at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 14. In certain embodiments, the antigen binding protein comprises a Vn domain at least 80%, or at least 85%, orat least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 18, and a VL domain at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 14.

[0035] In an aspect, the invention provides a binding protein that specifically binds to nerve growth factor (NGF), which comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising NYDMA (SEQ ID NO:40); (b) a heavy chain complementarity determining region 2 (HCDR2) comprising XiSPGGGSI (SEQ ID NO:59), wherein Xi comprises I, L, or M; (c) a heavy chain complementarity determining region 3 (HCDR3) comprising EGELGPFX2Y (SEQ ID NO:62), wherein X2 comprises D, E, Q, or S; (d) a light chain complementarity determining region 1 (LCDR1) comprising QSVGIN (SEQ ID NO:47); (e) a light chain complementarity determining region 2 (LCDR2) comprising GAS (SEQ ID NO:50); and (f) a light chain complementarity determining region 3 (LCDR3) comprising LQYGSIPWT (SEQ ID NO:52).

[0036] According to certain exemplary embodiments of the present invention, the NGF binding protein is an anti-NGF antibody or antigen-binding fragment thereof. The term "antibody," as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, Ciil, CII2 and Cn3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-NGF antibody (or antigen-binding portion thereof) may be identical to the canine germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0037] Antibody residues that have a substantial impact on affinity and specificity of binding to target antigen are primarily located in CDRs. Kabat et al. compiled and aligned immunoglobulin heavy and light chain sequences and were the first to propose a standardized numbering schemefor the variable regions of immunoglobulins identifying conserved and hypervariable regions and residues. (Kabat EA et al., 1979, Sequences of Immunoglobulin Chains: Tabulation and Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain and BP-Microglobulins, Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health). While the Kabat system is a widely adopted standard for numbering antibody residues, the hypervariable regions defined by Kabat do not exactly match with the structural aspects of antigen-binding loops. Chothia and Lesk developed a structure-based numbering scheme by aligning crystal structures of antibody variable regions and classified CDR loops in a small number of “canonical” classes (Chothia C, et al., 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196:901-17. doi: 10.1016 / 0022-2836(87)90412-8). An advantage of the Chothia numbering scheme is that topologically aligned residues from different antibodies are localized at the same position number and the Chothia CDR definition corresponds in most antibody sequences to the structural antigen-binding loop. Lefranc introduced a new system based on germ-line sequences intended to standardize numbering for all proteins of the immunoglobulin superfamily, including T cell receptor chains. (Giudicelli V et al., 1997, IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 25:206-11), which was then extended to entire variable domains (Lefranc M-P et al., 2003, IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. 27:55-77. doi: 10.1016 / S0145-305X(02)00039-3). Additional numbering systems have been proposed to align unconventional frameworks (Abhinandan KR et al., 2008, Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol. 45:3832-9. doi: 10.1016 / j.molimm.2008.05.022) and to subdivide variable chain sequences into multiple fragments including structurally invariant “cores” (Gelfand et al., 1998, Algorithmic determination of core positions in the VL and VH domains of immunoglobulin molecules. J Comput Biol. (1998) 5:467-77). In certain embodiments of the invention, CDR residues are identified according to such a standard system as set forth above. In certain embodiments, antibodies of the invention can be identified by all or a subset of Kabat CDR residues of the antibody sequences set forth herein. In certain embodiments, antibodies of the invention can be identified by all or a subset of Chothia CDR residues of the antibody sequences set forth herein. In certain embodiments, antibodies of the invention can be identified by all or a subset of IMGT CDR residues of the antibody sequences set forth herein. In certain embodiments,antibodies of the invention can be identified by CDR residues defined by two or more systems, comprising e g., but not limited to, all or a subset of residues of VH-CDR1 according to Kabat, all or a subset of residues of VH-CDR2 according to Chothia, all or a subset of residues of VH-CDR3 according to Kabat, all or a subset of residues of VL-CDR1 according to Kabat, all or a subset of residues of VL-CDR2 according to IMGT, and all or a subset of residues of VL-CDR3 according to Chothia.

[0038] Frameworks comprise the amino acid residues of variable domains located outside the CDRs. For example, if all three CDRs of a heavy chain variable domain are referred to as IMGT CDRs, the heavy chain variable domain frameworks would comprise the amino acid residues not included in the IMGT CDRs, i.e. IMGT frameworks. Similarly, in instances where some CDRs are referred to according to one system and other CDRs according to another system, or CDRs are referred to by a range of amino acid locations, the frameworks comprise the amino acids that are not CDR amino acids. All four VH framework regions may be referred to herein as “FR1H+FR2H+FR3H+FR4H.” All four VL framework regions may be referred to herein “FR1L+FR2L+FR3L+FR4L.”

[0039] In certain embodiments, an amino acid residue is mutated into one that allows the properties of the amino acid side-chain to be conserved. Examples of the properties of amino acid side chains comprise: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids comprising the following side chains: aliphatic side-chains (G, A, V, L, I, P); hydroxyl group-containing side-chains (S, T, Y); sulfur atom-containing side-chains (C, M); carboxylic acid- and amide-containing side-chains (D, N, E, Q); base-containing side-chains (R, K, H); and aromatic-containing side-chains (H, F, Y, W). The letters within parenthesis indicate the one-letter amino acid codes. Amino acid substitutions within each group are called conservative substitutions. It is well known that a polypeptide comprising a modified amino acid sequence in which one or more amino acid residues is deleted, added, and / or substituted can retain the original biological activity (Mark D. F. et al., Proc. Natl. Acad. Sci. U.S.A. 81 :5662-5666 (1984); Zoller M. J. and Smith M., Nucleic Acids Res. 10: 6487-6500 (1982); Wang A. et al., Science 224: 1431-1433; Dalbadie-McFarland G. et al., Proc. Natl. Acad. Sci. U.S.A. 79: 6409-6413 (1982)). The number of mutated amino acids is not limited, but in general, the number falls within 40% of amino acids of each CDR, and preferably within 35%, andstill more preferably within 30% (e.g., within 25%). The identity of amino acid sequences can be determined as described herein.

[0040] The invention provides recombinant antibodies designed or modified to minimize antigenicity in canines and felines. In certain embodiments, the antibodies are further modified to remove T cell epitopes.

[0041] As used herein, the term “canine” includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise indicated.

[0042] As used herein, the term “feline” refers to any member of the Felidae family. Domestic cats, pure-bred and / or mongrel companion cats, and wild or feral cats are all felines.

[0043] As used herein the term “canine framework” or “feline framework” refers to the amino acid sequence of the heavy chain and light chain of a canine antibody other than the hypervariable region residues defined herein as CDR residues. With regard to a caninized antibody, in certain embodiments, canine CDRs are identified in canine antibody heavy and light chains variable domain sequences that closely match CDRs of NGF -binding antibodies originating in other species. In certain embodiments, native canine CDRs are replaced with the corresponding foreign CDRs (e.g ., those from a rat or a mouse antibody) in both chains. With regard to a felinized antibody, in certain embodiments, feline CDRs are identified in feline antibody heavy and light chains variable domain sequences that closely match CDRs of NGF-binding antibodies originating in other species. In certain embodiments, native feline CDRs are replaced with the corresponding foreign CDRs (e.g ., those from a rat or a mouse antibody) in both chains. Optionally the heavy and / or light chains of the caninized or felinized antibody may contain some mutated or foreign non-CDR residues, e.g., framework amino acid residues that vary among germline antibody sequence or mutations that preserve the conformation of the foreign CDRs within the antibody.

[0044] Five major isotypes (IgA, IgG, IgM, IgD, IgE) and two forms of light chain (K and 1) are present in dogs. In the dog, there are four subtypes for IgG, which are IgGA, IgGB, IgGC, and IgGD (Bergeron etal al, 2014, Comparative functional characterization of canine IgG subclasses. Veterinary Immunology and Immunopathology. 157:31-41). For the cat, there are three subtypes of IgG which are lgGla, IgGlb, and IgG2 (Streitzel et al. 2014, In vitro functional characterization of feline IgGs. Vet Immunol Immunopathol 158, 214-223, doi.org / 10.1016 / j.vetimm.2014.01.012).

[0045] The invention provides caninized and felinized antibodies engineered to modulate one or more effector functions or circulation half-life. Hinge and constant domains of an antibody engage host receptors or complement protein to mediate effector functions and regulate antibody circulation. In certain embodiments, one or more effector functions is enhanced. In certain embodiments, one or more effector functions are reduced or eliminated. In certain embodiments, antibodies of the invention comprise modifications to modulate antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). A non-limiting example involves engineering of canine IgGB constant region residues Met242 and / or Leu243 (EU numbering) to reduce effector function (see, e.g., Lund et al., Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG. J Immunol., 1991, 147:2657-62). In certain embodiments, a IgGB constant region of the invention comprises M242A and L243A substitution. In certain embodiments, the second constant domain (CH2) and / or the third constant domain (CH3) comprises mutations and combinations of mutations from wild-type designed to modulate binding to FcRn (neonatal Fc) receptor. In canine constant regions, such mutations include, without limitation substitutions of Ala426, for example A426Y or A426H, substitutions of Thr286, for example T286L or T286Y, substitutions of Tyr436, for example Y436H, and combinations of such mutations including but not limited to A426Y + T286L, A426Y + Y436H, A426H + T286L, and A426H + T286Y. In certain embodiments a chimeric or caninized antibody of the invention comprises a substitution at amino acid Asn434, such as but not limited to N434H. In feline constant regions, such mutations include, without limitation substitutions of Ser428, including but not limited to S428Y or S428L, substitutions of Gln311, including but not limited to Q311V, substitutions of Leu309, including but not limited to L309V, substitutions of Thr286, including but not limited to T286E, substitutions of Glu380, including but not limited to E380T, and combinations of such mutations including but not limited to S428Y + Q311V, S428Y + L309V, S428Y + Q311V + T286E, S428Y + Q311V + E380T, and S428Y +L309V + E380T. In certain embodiments a chimeric or felinized antibody of the invention comprises a substitution at amino acid Ser428 and / or Ser434 including but not limited to S428L and / or S434H.

[0046] The term "antibody," as used herein, includes antigen-binding fragments of full antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein thatspecifically binds an antigen to form a complex. As used herein, the term "specifically binds" or "binds specifically" means that an NGF binding protein of the invention reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with NGF than it does with alternative antigens. For example, NGF binding protein binds to NGF with materially greater affinity (e.g., at least 2-fold or 5-fold or 10-fold or 20-fold or 50-fold or 100-fold or 500-fold or 1000-fold or 10,000-fold or greater) than it does to other proteins or peptides. In certain embodiments, the NGF-binding proteins binds to NGF with an equilibrium dissociation constant KD for the epitope or target to which it binds of, e.g., 10'4M or smaller, e.g., IO'5M, 10'6M, 10'7M, 10'8M, 10'9M, IO'10M, 10'11M, or 10'12M. The term “high affinity” for an IgG antibody refers to an antibody having a Kn of l.OxlO'6M or less, or 3.0x 10’7M or less, or l.OxlO’7M or less, or 3.0* 10'8M or less, or l.Ox lO'8M or less, or 3.0x l0'9M or less, or l.Ox lO'9M or less, or from l.Ox lO'6M to 3.0x l0'7M, or from 3.0x l0'7M to l.Ox lO'7M, or from l.Ox lO'7M to 3.0x l0'8M, or from 3.0x l0'8M to l.Ox lO'8M, or from l.Ox lO'8M to 3.0x l0'9M, or from 3.0x 1 O'9M to l.Ox 10'9M for a target antigen. The term “very high affinity” for an IgG antibody refers to an antibody having a Ko of 3.0x 1 O'10M or less, or l.Ox lO'10M or less, or 3.0x 1 O'11M or less, or l.Ox lO'11M or less, or 3.0x l0'12M or less or l.Ox lO'12M or less, or from l.Ox lO'10M to 3.Ox lO'10M, or from 3.Ox lO'10M or to l.OxlO'10M or from l.Ox lO'10M to 3.0x l0'nM, or from 3.0X 10'11M or to l.Ox lO'11M, or from l.Ox lO'11M to 3.0x l0'12M, or from 3.0x l0'12M to l.Ox lO'12M. It will be recognized by one of skill that an antibody that specifically binds to a target (e.g., NGF) from one species may also specifically bind to orthologs of NGF.

[0047] Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0048] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi)dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0049] In certain embodiments, an antigen-binding fragment of an antibody comprises at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0050] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigenbinding fragment of an antibody of the present invention include: (i) Vii-Cnl; (ii) VII-CII2; (iii) VH-CH3; (iv) VH-CH 1 -CH2; (V) VH-CH1 -CH2-CH3 ; (vi) VH-CH2-CH3 ; (vii) VH-CL; (viii) VL-CH1 ; (ix) VL-CH2, (x) VL-CH3 ; (xi) VL-CH 1 -CH2; (xii) VL-CH1 -CH2-CH3 ; (xiii) VL-CH2-CH3 ; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

[0051] The term "diabody (Db)" refers to a bivalent antibody fragment constructed by gene fusion (for example, P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP 404,097, WO 93 / 11161). In general, a diabody is a dimer of two polypeptide chains. In the each of the polypeptide chains, a light chain variable region (VL) and a heavy chain variable region (VH) in an identical chain are connected via a short linker, for example, a linker of about five residues, so that they cannot bind together. Because the linker between the two is too short, the VL and VH in the same polypeptide chain cannot form a single chain V region fragment, but instead form a dimer. Thus, a diabody has two antigen-binding domains. When the VL and VH regions against the two types of antigens (a and b) are combined to form VLa-Vnb and VLb-Vna via a linker of about five residues, and then co-expressed, they are secreted as bispecific Dbs. The antibodies of the present invention may be such Dbs.

[0052] A single-chain antibody (also referred to as "scFv") can be prepared by linking a heavy chain V region and a light chain V region of an antibody (for a review of scFv see Pluckthun "The Pharmacology of Monoclonal Antibodies" Vol. 113, eds. Rosenburg and Moore, Springer Verlag, N.Y., pp. 269-315 (1994)). Methods for preparing single-chain antibodies are known in the art (see, for example, U.S. Pat. Nos. 4,946,778; 5,260,203; 5,091,513; and 5,455,030). In such scFvs, the heavy chain V region and the light chain V region are linked together via a linker, preferably, a polypeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A, 1988, 85, 5879-5883). The heavy chain V region and the light chain V region in a scFv may be derived from the same antibody, or from different antibodies. The peptide linker used to ligate the V regions may be any single-chain peptide consisting of 12 to 19 residues. A DNA encoding a scFv can be amplified by PCR using, as a template, either the entire DNA, or a partial DNA encoding a desired amino acid sequence, selected from a DNA encoding the heavy chain or the V region of the heavy chain of the above antibody, and a DNA encoding the light chain or the V region of the light chain of the above antibody; and using a primer pair that defines the two ends. Further amplification can be subsequently conducted using a combination of the DNA encoding the peptide linker portion, and the primer pair that defines both ends of the DNA to be ligated to the heavy and light chain respectively. After constructing DNAs encoding scFvs, conventional methods can be used to obtain expression vectors comprising these DNAs, and hosts transformed by these expression vectors. Furthermore, scFvs can be obtained according to conventional methods using the resulting hosts. These antibody fragments can be produced in hosts by obtaining genes that encode theantibody fragments and expressing these as outlined above. Antibodies bound to various types of molecules, such as polyethylene glycols (PEGs), may be used as modified antibodies. Methods for modifying antibodies are already established in the art. The term "antibody" in the present invention also encompasses the above-described antibodies.

[0053] The term "Kd" as used herein, refers to the dissociation constant of an antibody-antigen interaction. The dissociation constant, Kd, and the association constant, Ka, are quantitative measures of affinity. At equilibrium, free antigen (Ag) and free antibody (Ab) are in equilibrium with antigen-antibody complex (Ag-Ab), and the rate constants, ka and kd, quantitate the rates of the individual reactions. At equilibrium, ka [Ab][Ag]=kd [Ag-Ab], The dissociation constant, Kd, is given by: Kd=kd / ka=[Ag][Ab] / [Ag-Ab], Kd has units of concentration, most typically M, mM, nM, pM, etc. When comparing antibody affinities expressed as Kd, having greater affinity for NGF is indicated by a lower value. The association constant, Ka, is given by: Ka=ka / kd=[Ag- Ab] / [Ag][Ab], Ka has units of inverse concentration, most typically M'1, mM'1, nM'1, pM'1, etc. As used herein, the term "avidity" refers to the strength of the antigen-antibody binding taking valency into account.

[0054] The antibodies obtained can be purified to homogeneity. The antibodies can be isolated and purified by a method routinely used to isolate and purify proteins. The antibodies can be isolated and purified by the combined use of one or more methods appropriately selected from column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectro-focusing, for example (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Such methods are not limited to those listed above. Chromatographic methods include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography. These chromatographic methods can be practiced using liquid phase chromatography, such as HPLC and FPLC. Columns to be used in affinity chromatography include protein A columns and protein G columns. For example, protein A columns include Hyper D, POROS, and Sepharose F. F. (Pharmacia). Antibodies can also be purified by utilizing antigen binding, using carriers on which antigens have been immobilized.

[0055] Exemplary and non-limiting amino acid sequences of certain binding proteins, antigens and ligands described herein are provided in Table 1.

[0056] Unless otherwise indicated, CDR locations are defined for antibody 2141 and its derivatives in Table 2 and for antibody 2178 and its derivatives in Table 3.

[0057] As used herein, the term “therapeutic agent” refers to any agent or material that has a beneficial effect on the mammalian recipient. Thus, “therapeutic agent” embraces both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0058] “Treating” as used herein refers to ameliorating at least one symptom of, curing and / or preventing the development of a given disease or condition.

[0059] The anti-NGF proteins described herein, including antibodies or fragments thereof, are useful for ameliorating, or reducing the symptoms of, or treating, or preventing, diseases and disorders associated with NGF. The anti-NGF proteins or fragments, as well as combinations with other agent, are to be administered in a therapeutically effective amount to subjects in need of treatment of diseases and disorders associated with NGF in the form of a pharmaceutical composition as described herein.

[0060] In certain embodiments the method comprises ameliorating, or reducing the symptoms of, or treating, or preventing pain in a subject. In certain embodiments, the anti-NGF proteins, antibodies, or fragments thereof inhibit the association of NGF with TrkA and / or p75, for example administered alone or in conjunction with a second agent and are used to treat, ameliorate, reduce the symptoms of, or prevent inflammatory pain, post-operative incision pain, complex, cancer pain (including but not limited to primary or metastatic bone cancer pain), fracture pain, osteoporotic fracture pain, pain from osteoporosis, pain resulting from burn, and other nociceptic pain.

[0061] In certain embodiments the antibody compositions and methods are used for ameliorating, or reducing the symptoms of, or treating, or preventing pain of osteoarthritis (OA). OA is a slowly progressive degenerative joint disease characterized by whole-joint structural changes including articular cartilage, synovium, subchondral bone and periarticular components,leading to pain and loss of joint function. Chronic pain and OA are common in dogs and cats. 20- 30% of dogs are affected clinically and have signs of OA. Up to 40% of all cats being affected clinically, with 90% of all cats over 12 years of age have signs of OA.

[0062] In dogs the most common site of OA is the hip, followed by stifle (knee), shoulder and carpus. In cats hip, stifle, carpus or spine are most commonly affected.

[0063] The anti-NGF proteins, antibodies or antibody fragments, are optionally administered in combination with one or more active agents including other analgesic agents. Such active agents include analgesic, anti -histamine, antipyretic, anti-inflammatory, antibiotic, antiviral, and anticytokine agents. Active agents include agonists, antagonists, and modulators of TNF-a, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-a, IFN-y, BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (HGF), Hepcidin, including antibodies reactive against any of the foregoing, and antibodies reactive against any of their receptors. Active agents also include, without limitation, 2-arylpropionic acids, aceclofenac, acemetacin, acetylsalicylic acid (Aspirin), alclofenac, alminoprofen, amoxiprin, ampyrone, arylalkanoic acids, azapropazone, benorylate / benorilate, benoxaprofen, bromfenac, carprofen, celecoxib, choline magnesium salicylate, clofezone, COX-2 inhibitors, dexibuprofen, dexketoprofen, diclofenac, diflunisal, droxicam, ethenzamide, etodolac, etoricoxib, faislamine, fenamic acids, fenbufen, fenoprofen, flufenamic acid, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indometacin, indoprofen, kebuzone, ketoprofen, ketorolac, lomoxicam, loxoprofen, lumiracoxib, magnesium salicylate, meclofenamic acid, mefenamic acid, meloxicam, metamizole, methyl salicylate, mofebutazone, nabumetone, naproxen, n-arylanthranilic acids, nerve growth factor (NGF), oxametacin, oxaprozin, oxicams, oxyphenbutazone, parecoxib, phenazone, phenylbutazone, phenylbutazone, piroxicam, pirprofen, profens, proglumetacin, pyrazolidine derivatives, rofecoxib, salicyl salicylate, salicylamide, salicylates, sulfinpyrazone, sulindac, suprofen, tenoxicam, tiaprofenic acid, tolfenamic acid, tolmetin, and valdecoxib.

[0064] An anti-histamine can be any compound that opposes the action of histamine or its release from cells (e.g., mast cells). Anti -histamines include but are not limited to acrivastine, astemizole, azatadine, azelastine, betatastine, brompheniramine, buclizine, cetirizine, cetirizine analogues, chlorpheniramine, clemastine, CS 560, cyproheptadine, desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609, hydroxyzine, levocabastine,loratidine, methscopolamine, mizolastine, norastemizole, phenindamine, promethazine, pyrilamine, terfenadine, and tranilast.

[0065] Antibiotics include but are not limited to amikacin, aminoglycosides, amoxicillin, ampicillin, ansamycins, arsphenamine, azithromycin, azlocillin, aztreonam, bacitracin, carbacephem, carbapenems, carbenicillin, cefaclor, cefadroxil, cefalexin, cefalothin, cefalotin, cefamandole, cefazolin, cefdinir, cefditoren, cefepime, cefixime, cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cephalosporins, chloramphenicol, cilastatin, ciprofloxacin, clarithromycin, clindamycin, cioxacillin, colistin, co-trimoxazole, dalfopristin, demeclocycline, dicloxacillin, dirithromycin, doripenem, doxycycline, enoxacin, ertapenem, erythromycin, ethambutol, flucloxacillin, fosfomycin, furazolidone, fusidic acid, gatifloxacin, geldanamycin, gentamicin, glycopeptides, herbimycin, imipenem, isoniazid, kanamycin, levofloxacin, lincomycin, linezolid, lomefloxacin, loracarbef, macrolides, mafenide, meropenem, meticillin, metronidazole, mezlocillin, minocycline, monobactams, moxifloxacin, mupirocin, nafcillin, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oxacillin, oxytetracycline, paromomycin, penicillin, penicillins, piperacillin, platensimycin, polymyxin B, polypeptides, prontosil, pyrazinamide, quinolones, quinupristin, rifampicin, rifampin, roxithromycin, spectinomycin, streptomycin, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, sulfonamides, teicoplanin, telithromycin, tetracycline, tetracyclines, ticarcillin, tinidazole, tobramycin, trimethoprim, trimethoprim-sulfamethoxazole, troleandomycin, trovafloxacin, and vancomycin.

[0066] Active agents also include aldosterone, beclometasone, betamethasone, corticosteroids, cortisol, cortisone acetate, deoxycorticosterone acetate, dexamethasone, fludrocortisone acetate, glucocorticoids, hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids, and triamcinolone. Any suitable combination of these active agents is also contemplated.

[0067] The most common form of current treatment for OA and pain related to OA is NSAIDs (which are also anti-pain medications). NSAIDs are not always sufficiently effective, typically need to be administered daily and none are approved for long-term use in cats in the US. Additionally, there are safety and tolerability concerns with the use of NSAIDS in both dogs and cats, especially with long-term treatment. NSAIDs are not recommended to be co-administered with anti-NGF mAbs for long periods.

[0068] In certain embodiments, treatment comprises coadministration of dietary supplements containing Omega-3 fatty acids, microlactin, and / or glucosamine / chondroitin as an aid to joint health. Adequan (polysulfated glycosaminoglycan) is an FDA-approved disease modifying drug that inhibits cartilage loss and may also be co-administered.

[0069] Formulations and Methods of Administration

[0070] For in vivo use, a therapeutic agent as described herein is generally incorporated into a pharmaceutical composition prior to administration. Within such compositions, one or more therapeutic compounds as described herein are present as active ingredient(s) (i.e., are present at levels sufficient to provide a statistically significant effect on the symptoms of cystic fibrosis, as measured using a representative assay). A pharmaceutical composition comprises one or more such compounds in combination with any pharmaceutically acceptable carrier(s) known to those skilled in the art to be suitable for the particular mode of administration. In addition, other pharmaceutically active ingredients (including other therapeutic agents) may, but need not, be present within the composition.

[0071] The antibodies of the present invention can be formulated according to standard methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A), and may comprise pharmaceutically acceptable carriers and / or additives. The present invention relates to compositions (including reagents and pharmaceuticals) comprising the antibodies of the invention, and pharmaceutically acceptable carriers and / or additives. Exemplary carriers include surfactants (for example, PEG and Tween), excipients, antioxidants (for example, ascorbic acid), coloring agents, flavoring agents, preservatives, stabilizers, buffering agents (for example, phosphoric acid, citric acid, and other organic acids), chelating agents (for example, EDTA), suspending agents, isotonizing agents, binders, disintegrators, lubricants, fluidity promoters, and corrigents. However, the carriers that may be employed in the present invention are not limited to this list. In fact, other commonly used carriers can be appropriately employed: light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmelose calcium, carmelose sodium, hydroxypropylcellulose, hydroxypropylmethyl cellulose, polyvinylacetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salt, and so on. The composition may also comprise other low-molecular-weight polypeptides, proteins such as serum albumin, gelatin, andimmunoglobulin, and amino acids such as glycine, glutamine, asparagine, arginine, and lysine. When the composition is prepared as an aqueous solution for injection, it can comprise an isotonic solution comprising, for example, physiological saline, dextrose, and other adjuvants, including, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride, which can also contain an appropriate solubilizing agent, for example, alcohol (for example, ethanol), polyalcohol (for example, propylene glycol and PEG), and non-ionic detergent (polysorbate 80 and HCO-50).

[0072] If necessary, antibodies of the present invention may be encapsulated in microcapsules (microcapsules made of hydroxycellulose, gelatin, polymethylmethacrylate, and the like), and made into components of colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nano-particles, and nano-capsules) (for example, see "Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980)). Moreover, methods for making sustained- release drugs are known, and these can be applied for the antibodies of the present invention (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981); Langer, Chem. Tech. 12: 98-105 (1982); U.S. Pat. No. 3,773,919; EP Patent Application No. 58,481; Sidman et al., Biopolymers 22: 547- 556 (1983); EP: 133,988).

[0073] A preferred route of administration in both canines and felines is by subcutaneous injection usually into the skin at the base of the neck. In certain embodiments, the anti-NGF protein is packaged in an integrated delivery system such as a pen or prefilled syringe for subcutaneous administration. Ghil et al. describes administration of the adalimumab biosimilar, SB5, via prefilled syringe (PFS) and autoinjector (Al) pen based on injection site pain, patient preference, and safety in rheumatoid arthritis (RA) (See Ghil et al., Usability and safety of SB5 (an adalimumab biosimilar) prefilled syringe and autoinjector in patients with rheumatoid arthritis. Curr Med Res Opin 2019 Mar;35(3):497-502.) Compositions of the invention are similarly administered to canines, felines, and other mammals.

[0074] The term “therapeutically effective amount,” in reference to treating a disease state / condition, refers to an amount of a compound either alone or as contained in a pharmaceutical composition that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state / condition when administered as a single dose or in multiple doses. Such effect need not be absolute to be beneficial.

[0075] The terms “treat,” “treating” and “treatment” as used herein include administering a compound prior to the onset of clinical symptoms of a disease state / condition so as to prevent anysymptom, as well as administering a compound after the onset of clinical symptoms of a disease state / condition so as to reduce or eliminate any symptom, aspect or characteristic of the disease state / condition. Such treating need not be absolute to be useful.

[0076] In certain embodiments, the present therapeutic agent may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard- or soft-shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0077] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0078] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0079] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0080] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0081] Useful dosages of the compounds of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. In certain embodiments, a useful dose is from about 0.1 mg / kg to about 5 mg / kg or from about 0.5 mg / kg to about 2 mg / kg. Methods for the extrapolation of effective dosages in humans and animals of different sizes are known to the art; for example, see U.S. Pat. No. 4,938,949.

[0082] The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route ofadministration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0083] In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg / kg, e.g., from about 10 to about 75 mg / kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg / kg / day, most preferably in the range of 15 to 60 mg / kg / day.

[0084] The compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.

[0085] Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 pM, preferably, about 1 to 50 pM, most preferably, about 2 to about 30 pM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg / kg / hr or by intermittent infusions containing about 0.4-15 mg / kg of the active ingredient(s).

[0086] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0087] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0088] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.ExamplesExample 1

[0089] Generation and characterization of rat antibodies 2141 and 2178 that binds to canineNGF

[0090] Lewis rats were immunized with human NGF (R&D Systems, 256-GF-100 / CF) on a weekly basis for eight weeks. The titers were measured in a flow cytometry assay using human NGF-coated beads. Beads were conjugated with human NGF (R&D Systems, 256-GF-100 / CF) and incubated with different dilutions of serum (1 : 100, 1 :500, 1 :2500) for 30 minutes. Beads were washed and binding was detected by using a fluorescently labeled anti-rat IgG secondary antibody. Fluorescence was measured using the Intellicyte iQue Screener Plus. Titers were measured at a 1 :2500 dilution for all three rats and they were ~100-fold greater than the values of normal Lewis rat serum.

[0091] Lymph nodes (brachial, axillary, inguinal, popliteal and sciatic) and bone marrow from femur, tibia and pelvis were collected from rats with significant NGF titers. Cells from both tissues were isolated and enriched for plasma cells using flow cytometry. Enriched plasma cell suspension was injected into AbCellera’s microfluidic screening devices with either 91,000 or 153,000 individual nanoliter-volume reaction chambers. Single cells secreting NGF-specific antibodies were identified and isolated using a bead-based assay. Beads coated with anti-rat IgG antibody were flowed onto microfluidic screening devices and incubated with single antibody-secreting cells. The IgG secreted by plasma cells were captured on beads using the constant region. Binding to secreted IgG immobilized onto beads was subsequently assessed using fluorescently labeled human NGF antigen. Positive hits were identified using machine vision and recovered using automated robotics-based protocols. Approximately 269,000 individual B cells were screened in the NGF binding assay and 592 cells expressed antibodies recognized NGF. From these positive cells, 190 unique antibody sequences were identified. Eighty-eight antibodies were selected from the 190 antibodies based on the diversity of the clonotypes.

[0092] Single cell polymerase chain reaction and custom molecular biology protocols generated NGS sequencing libraries (MiSeq, Illumina) using automated workstations (Bravo, Agilent). Sequencing data were analyzed using a custom bioinformatics pipeline to yield paired heavy and light chain sequences for each recovered antibody-secreting cell (Jones et al., 2020, bioRxiv 2020.09.30.318972. doi: 10.1101 / 2020.09.30.318972). The amino acid sequences of the heavy and light variable domains of rat antibody 2141 (SEQ ID NO: 1, SEQ ID NO:2) and 2178 (SEQ ID NO:3, SEQ ID NO:4) are shown in FIG. 1 and FIG. 2 respectively and the CDRs are indicated. The variable (V(D)J) region of each antibody chain was synthesized and inserted into mammalian expression plasmids using a custom, automated high-throughput cloning pipeline. Theexpression vectors were transfected into Expi293-F cells (Gibco, ThermoFisher Scientific) in 24 deep well plates using the manufacturer’s recommended protocol. Four days post-transfection, the conditioned medium was purified with protein A beads and the antibody was eluted by the addition of 100 mM glycine, pH 2.0 and neutralized to pH 7.0 by the addition of 1 M Tris-HCL, pH 8.0. The neutralized antibodies were buffer exchanged into PBS, pH 7.2.

[0093] The analytics for the purified antibodies included CE-SDS (denaturing capillary sodium dodecyl sulfate gel electrophoresis). The CE-SDS was used to determine the purity of the purified antibodies and was completed by using the LabChip GXII Touch instrument (Perkin Elmer). Two microliters of antibody solution at a concentration of 350 pg / mL in PBS was mixed with a non-reducing denaturing buffer solution (Perkin Elmer) and incubated at 70 °C for 10 minutes. Separation and detection were performed using the HT Antibody Analysis 200 assay setting on the LabChip instrument (Perkin Elmer). The fluorescence data was analyzed using the LabChip GX Reviewer Software (Perkin Elmer), with percent purity. The percent purity of the rat monoclonal antibody 2141 and 2178 was 99%.

[0094] A binding assay was completed to confirm binding of the antibodies to NGF (R&D Systems, 256-GF-100 / CF). In addition, the specificity of the antibodies was determined by testing the binding of the antibodies to NT-3 and BDNF which are closely related proteins. Unique antibody sequences were confirmed to bind the screening target using a multiplexed bead assay on a high throughput flow cytometer. Different optically encoded beads were conjugated to either human NGF (R&D Systems, 256-GF-100 / CF), NT-3 (R&D Systems, 267-N3-025 / CF) or BDNF (R&D Systems, 248-BDB-050 / CF). Purified antibodies were incubated with the multiplexed beads at different antibody concentrations for 30 minutes at room temperature. Beads were washed and binding was detected by using a fluorescently labelled secondary antibody. Fluorescence was measured using high throughput plate-based flow cytometry on an Intellicyt® iQue Screener Plus.

[0095] Median fluorescence intensity of each antibody was normalized over the median fluorescence intensity of the appropriate isotype control for individual bead types. Antibody values greater than 10-fold over isotype were considered as binders. Antibody 2141 bound to NGF greater than 186-fold higher than background levels and the binding of this antibody to NT-3 and BDNF was at background levels. Antibody 2178 bound to NGF greater than 135-fold higher than background levels and the binding of this antibody to NT-3 and BDNF was at background levels.

[0096] A functional assay with TF-1 cells was used to determine if the binding of the 2141 and 2178 antibodies to canine NGF blocks the ability of canine NGF to induce signaling with human TrkA which is the high affinity receptor for NGF (Chevalier et al., 1994. Blood, 83 : 1479). For these studies, canine NGF (Genbank NP 001181879.1) was used for the NGF source. Canine NGF with a strep-tag (WSHPQFEK; SEQ ID NO:53) at the C-terminus (SEQ ID NO:5) was stably expressed in Dmel-2 cells and purified using StrepTactinXT chromatography followed by a polishing step with Superdex 200 16 / 600 chromatography. The proliferation of TF-1 cells can be stimulated by different growth factors such as GM-CSF and NGF. TF-1 cells (ATCC-CRL2003) were cultured in RPMI-1640 media containing 10% fetal bovine serum, 100 U / mL Penicillin, 100 pg / ml Streptomycin and 2 ng / rnL recombinant human GM-CSF. Cells were maintained between 3 X 104and 5 X 105viable cells / mL and passaged every 48 hours. Each condition was run in triplicate wells. Cells were resuspended in media without GM-CSF at 1.75xl05cells / ml and incubated in a flask in a humidified 37 °C, 5% CO2 incubator for 4 hours. During the incubation, NGF / antibody mixtures were prepared in media as 2x media solutions in full media without GM- CSF and with 10 ng / mL canine NGF. Antibodies were added to the appropriate 2x media solutions and the NGF / antibody solutions were incubated for at least 1 hour at room temperature before being added to the cells. Cells were then collected and resuspended in appropriate media volume to achieve a 0.5 x 106cells / ml suspension in media without GM-CSF. 50 pl of the cell suspension was added per well in a 96-well plate, to which 50 pl of the 2x NGF / antibody media was added per well to the cell plate. Cells were incubated in a humidified 37 °C, 5% CO2 incubator for 48 hours, then 20 pl of Aqueous One solution Reagent (Promega) was added per well. Cells were incubated for further 4 hours in a humidified 37 °C, 5% CO2 incubator and then absorbance was read at 490 nm on a BioTek Synergy Neo2. Data was analyzed by subtracting the blank well from all measured values. Percent inhibition was calculated using the following formula: % inhibition = 100 x [1 - (X - MIN) / (MAX - MIN)], where X = signal at a given concentration, MAX = 0% inhibition = Canine NGF only and MIN = 100% inhibition = No NGF control. The average of the triplicates for each condition was calculated which included rat antibody 2141, 2178 and an isotype rat antibody control. The proliferation data demonstrated that the rat antibodies 2141 and 2178 effectively blocks NGF from binding to TrkA.

[0097] The VH domain of antibodies 2141 (SEQ ID NO:6) and 2178 (SEQ ID NO:8) were fused with the canine IgGB constant domains (Tang et al. 2001. Vet. Immunol. Immunopathol.80:259) and the VL domain of both antibodies (SEQ ID NO:7, SEQ ID NO:9) were fused to the canine kappa constant domain to generate a canine chimeric antibody. Two residue changes (AA) were made in the Fc (underlined and in bold font) to eliminate effector activity and these changes are analogous to the “LALA” mutation described for human IgGl Fc (Tamm & Schmidt, 1997. Int. Rev. Immunol. 16:57). These two constructs were subcloned into pcDNA3.4 (ThermoFisher Scientific) and an equal ratio of heavy chain and light chain plasmids for each were co-transfected using the ExpiCHO system. The IgG in the conditioned medium was purified using MabSelect SuRe chromatography. The antibodies were buffer exchanged into PBS, pH 7.4.

[0098] The affinity for canine NGF of rodent 2141 and 2178 were determined using a Biacore T200 instrument. The antibodies were captured using a CM5 Series S chip amine coupled with an anti-dog Fc antibody (lackson Immunoresearch). For these studies, canine NGF (Genbank NP 001181879.1) was generated by fusing the C-terminus with the Flag tag (DYKDDDDK; SEQ ID NO:54), expressing the canine NGF construct (SEQ ID NO: 10) with baculovirus technology and then purifying the NGF with Anti -DYKDDDDK (SEQ ID NO: 54) G1 affinity chromatography followed by HiLoad26 / 600 Superdex 200pg. Canine NGF binding was then assessed at multiple concentrations starting at 50 nM using PBSP+ running buffer (Cytiva) with a flow rate of 30 pL / min. The length of the association time was 120s and the dissociation time was run for 600s. The chip surface was regenerated with 10 mM glycine. Double reference-subtracted sensorgrams were fitted to a 1 : 1 binding model using Biacore T200 Evaluation software. The kinetics of 2141 and 2178 for canine NGF are shown in Table 4.

[0099] NGF receptor blocking was assessed using recombinant canine TrkA and canine p75 NGF receptors in an SPR experiment on the Biacore T200. The extracellular domain of canine TrkA (Genbank number XP 038527745.1) was cloned with an AviTag (GLNDIFEAQKIEWHE; SEQ ID NO:55) and 8X His tag (SEQ ID NO:75) at the C-terminus (SEQ ID NO: 11) and expressed in HEK293 cells. The recombinant canine TrkA protein was purified from the conditioned medium using nickel chromatography. The extracellular domain of canine p75 (Genbank numberXP_038475022.1) was cloned with an AviTag (GLNDIFEAQKIEWHE SEQ ID NO:55) and 8X His tag (SEQ ID NO:75) at the C-terminus and expressed in HEK293 cells (SEQ ID NO: 12). The recombinant canine p75 protein was purified from the conditioned medium using nickel chromatography. Both receptors were biotinylated at the AviTag site using the BirA biotin protein ligase reaction kit (Avidity). Biotinylated receptors were captured on a Series S CAP chip and Biotin CAPture reagent (Cytiva). Antibodies were titrated in running buffer (1 X PBSP+, Cytiva) and pre-incubated 50 nM NGF at the indicated ratios. Binding was assessed by injecting these samples over the captured receptor for 180s. The relative response units (RU) at the binding report point were used to calculate the inhibition percent by dividing the relative binding response of the cycles with antibody samples by an average of the NGF-only relative binding response (with no antibody present) that were collected throughout the assay. The ability of each antibody to block binding of NGF to canine TrkA and p75 are described below in Table 5.

[0100] The results demonstrate that 2141 and 2178 show high percent blocking of NGF to TrkA and p75 at a 1 : 1 ratio of mAb to NGF.Example 2

[0101] Caninization of Rodent Antibodies

[0102] The rodent antibodies 2141 and 2178 were caninized by replacing the human frameworks with canine frameworks. Using a proprietary informatics approach, twelve heavy chain canine frameworks were selected along with four light chain frameworks for caninizing the rodent 2141 antibody. Six heavy chain canine frameworks were selected along with eight light chain frameworks for caninizing the rodent 2178 antibody. The heavy and light CDRs, as defined in FIG. 1 and FIG. 2 and Table 2 and Table 3, were used for grafting into the selected canine heavy and light chain frameworks. The DNA for these caninized variable domains weresynthesized with the canine IgGB constant and canine kappa constant domains. The Fc contained the MALA variant as described above, and the LY variant which is T286L and A426Y (EU numbering). The LY variant increases the IgG half-life in vivo (US11434276B2). The constructs were cloned into a proprietary mammalian expression vector. Each of the different heavy and light chains were paired and co-transfected into HEK 293 cells and the IgGs in the conditioned medium were purified with Mab Select SuRe protein A resin. The antibodies were buffered exchanged into PBS, pH 7.4. An SPR assay was initially completed on the caninized antibodies using the Carterra instrument with canine NGF (SEQ ID NO: 10). The binding kinetics for the caninized clone with the highest affinity to NGF for rodent mAb 2141 was Clone Oi l (SEQ ID NO: 13, SEQ ID NO: 14) and the highest affinity to NGF for rodent mAb 2178 was Clone 006 (SEQ ID NO: 15, SEQ ID NO:16). Clone 011 has the IGHV3-47*01 and IGKV3-18*01 germline frameworks for the heavy and light chains, respectively. Clone 006 has the IGHV3-4 0 I and IGKV3-18*01 germline frameworks for the heavy and light chains, respectively.Example 3

[0103] Sequence Liability Evaluation

[0104] The caninized clones with the best affinity were further evaluated for potential sequence liabilities in the CDRs. The caninized 2141_Clone 011 contained a methionine in the HCDR3 which is a potential oxidation site. Two variants were created to remove this site, M103I (SEQ ID NO:17) and M103L (SEQ ID NO: 18). The caninized 2178_Clone 006 contained a methionine in the HCDR2 which is a potential oxidation site and a potential deamidation site, DY in HCDR3. Two variants were created to remove the methionine, M51I (SEQ ID NO:19) and M51L (SEQ ID NO:20) and three variants were made to remove the deamidation site, D106E (SEQ ID NO:21), D106S (SEQ ID NO:22), and D106Q (SEQ ID NO:23). These variants along with the caninized 2141_Clone 011 and 2178_Clone 006 sequences were subcloned with a canine IgGB heavy chain constant containing the MALA and LY variants and canine kappa light chain constant into pcDNA 3.4 (ThermoFisher). An equal ratio of heavy chain and light chain plasmids for each were co-transfected into ExpiCHO cells and the IgG in the conditioned medium was purified using MabSelect SuRe chromatography. The antibodies were buffer exchanged into PBS, pH 7.4.

[0105] The affinity for the caninized clones and variants were determined using a Biacore T200 instrument. Bedinvetmab was also run as a control. The antibodies were captured using a CM5 Series S chip amine coupled with an anti-dog Fc antibody (Jackson Immunoresearch). Canine NGF binding was then assessed using the Flag-tagged protein (SEQ ID NO: 10) at multiple concentrations starting at 100 nM using PBSP+ running buffer (Cytiva) with a flow rate of 30 pL / min. The length of the association time was 120s and the dissociation time was run for 600s. The chip surface was regenerated with 10 mM glycine. Double reference-subtracted sensorgrams were fitted to a 1 : 1 binding model using Biacore T200 Evaluation software. The kinetics of the caninized clones and sequence liability variants for canine NGF are shown in Table 6.

[0106] The results demonstrate that variant 2141 01 l_M103L retains binding affinity for canine NGF and this methionine can successfully be removed. It also demonstrates that for caninized 2178_Clone 006, both M51 and DI 06 are important residues for maintenance of affinity.

[0107] Variant 2141 011 M103L and 2178_Clone 006 were further characterized. The functional assay with TF-1 cells, previously described, was used to confirm that binding of the caninized 2141 and 2178 antibodies to canine NGF still blocks the ability of canine NGF to induce signaling with the high affinity receptor for NGF. For these studies, 10 ng / mL of canine NGF with a C-terminus Flag tag was used (SEQ ID NO: 10) and absorbance was read on Varioskan PlateReader (ThermoFisher Scientific). The proliferation data is shown in FIG. 3 and demonstrates that the caninized antibodies effectively blocks NGF from binding to TrkA with an IC50 of 108 pM for 2141 01 l_M103L and an IC50 of 335 pM 2178_Clone 006.

[0108] NGF receptor blocking was assessed using recombinant canine TrkA and canine p75 NGF receptors in an SPR experiment on the Biacore T200, as previously described. The ability of each antibody to block binding of NGF to canine TrkA and p75 are described below in Table 7.

[0109] The results demonstrate that 2141 011 M103L and 2178_Clone 006 are potent blockers of NGF binding to TrkA and p75 at a 1 : 1 ratio of mAb to NGF after caninization.Example 4

[0110] In vivo efficacy of caninized antibodies

[0111] To provide further confirmation on the efficacy of the selected clones, an experimental model of OA was conducted in rats (Charles River Laboratories). The chemically-induced Monosodium lodoacetate (MIA) model of Osteoarthritis is known to produce a robust pain response. In this model, MIA is injected intra-articularly into the right knee joint. As a glycolytic inhibitor MIA causes gradual joint cartilage damage and other biochemical and behavioral changes in the rodents mimicking OA observed in humans as well as dogs and cats. MIA-induced chronic joint pain in rats as assessed by measuring dynamic weight bearing (DWB) deficit in the MIA- injected limb using the BioSeb® DWB system.

[0112] Male Sprague Dawley rats 6-8 weeks old were administered 3 mg of monosodiumiodoacetate (MIA) into the right hind limb knee joint on Day 0. Animals were then treated on Day 3 and Day 10 subcutaneously with 1 mg / kg of antibody (2141 01 l_M103L, 2178_Clone 006, or bedinvetmab) or a similar volume of vehicle. One additional control group was treated daily with 0.5 mg / kg of dexamethasone by oral gavage from Day 3 to Day 22. On days 3, 6, 10, 14, and 22,DWB was evaluated using the BioSeb® automated DWB system according to the manufacturer’s manual. DWB was performed 3 hours after dosing. The system consists of an arena box made from pressure-sensitive sensor mat on the bottom and an attached high-resolution camera on the top. The rat can freely move inside the arena box. A five-minute recording was done for each rat. Analysis of dynamic weight bearing data was done off-line using the BioSeb® software. The system automatically calculates the weight borne by each limb. Body weight was measured for each rat immediately before the DWB for each time of testing.

[0113] The results of the MIA study are shown in FIG. 4. Antibody 2141 01 l_M103L was significantly better than vehicle on Days 6, 10, 14, and 22. Antibody 2178_Clone 006 was significantly better than vehicle on Days 14 and 22.

[0114] Antibodies were also screened for cross-reactivity to rat NGF using a Biacore T200 instrument. The antibodies were captured using a CM5 Series S chip amine coupled with an antidog Fc antibody (lackson Immunoresearch). Rat [3-NGF binding was then assessed (R&D Systems, 556-NG-100 / CF) at multiple concentrations starting at 25 nM using PBSP+ running buffer (Cytiva) with a flow rate of 30 pL / min. The length of the association time was 120s and the dissociation time was run for 600s. The chip surface was regenerated with 10 mM glycine. Double reference-subtracted sensorgrams were fitted to a 1 : 1 binding model using Biacore T200 Evaluation software. The kinetics of the caninized clones and sequence liability variants for rat NGF are shown in Table 8.

[0115] The SPR results demonstrate that 2141 011 M103L retains affinity to rat NGF while 2178_Clone 006 has a lower affinity for rat NGF compared to canine NGF (Table 4) which may explain the lower efficacy in the rodent model.* * *

[0116] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. An antigen binding protein that specifically binds to nerve growth factor (NGF), which comprises:(a) a heavy chain complementarity determining region 1 (HCDR1) comprising TDNMGVG (SEQ ID NO:25);(b) a heavy chain complementarity determining region 2 (HCDR2) comprising IWWDDDK (SEQ ID NO:27);(c) a heavy chain complementarity determining region 3 (HCDR3) comprisingVYGXiGHYFDH (SEQ ID NO:56), wherein Xi comprises I, L, or M;(d) a light chain complementarity determining region 1 (LCDR1) comprising QNVGNY (SEQ ID NO:32);(e) a light chain complementarity determining region 2 (LCDR2) comprising YAS (SEQ ID NO:35); and(f) a light chain complementarity determining region 3 (LCDR3) comprising QRIYISPWT (SEQ ID NO:38).

2. The antigen binding protein of claim 1, wherein the HCDR3 comprises VYGMGHYFDH (SEQ ID NO: 31).

3. The antigen binding protein of claim 1, wherein the HCDR3 comprises VYGIGHYFDH. (SEQ ID NO:57)4. The antigen binding protein of claim 1 , wherein the HCDR3 comprises VYGLGHYFDH (SEQ ID NO:58).

5. The antigen binding protein of any of claims 1 to 4, wherein the antigen binding protein comprises canine or caninized frameworks.

6. The antigen binding protein of any of claims 1 to 4, which comprises a heavy chain framework (FR1H+FR2H+FR3H+FR4H) at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 13.

7. The antigen binding protein of claims 1 to 4, which comprises a light chain framework (FR1L+FR2L+FR3L+FR4L) at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 14.

8. The antigen binding protein of any one of claims 1 to 7, which comprises a VH domain at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical, or identical to SEQ ID NO: 13, and a VL domain at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical, or identical to SEQ ID NO: 14.

9. The antigen binding protein of any one of claims 1 to 7, wherein the VH domain comprises SEQ ID NO: 13 and the VL domain comprises SEQ ID NO: 14; or the VH domain comprises SEQ ID NO: 17 and the VL domain comprises SEQ ID NO: 14; or the VH domain comprises SEQ ID NO:18 and the VL domain comprises SEQ ID NO:14..

10. The antigen binding protein of any of claims 1 to 4, wherein the antigen binding protein comprises human or humanized frameworks.

11. The antigen binding protein of any of claims 1 to 4, wherein the antigen binding protein comprises feline or felinized frameworks.

12. An antigen binding protein that specifically binds to nerve growth factor (NGF), which comprises:(a) a heavy chain complementarity determining region 1 (HCDR1) comprising NYDMA (SEQ ID NO:40);(b) a heavy chain complementarity determining region 2 (HCDR2) comprising XiSPGGGSI (SEQ ID NO:59), wherein Xi comprises I, L, or M;(c) a heavy chain complementarity determining region 3 (HCDR3) comprising EGELGPFX2Y (SEQ ID NO 62), wherein X2comprises D, E, Q, or S;(d) a light chain complementarity determining region 1 (LCDR1) comprising QSVGIN (SEQ ID NO:47);(e) a light chain complementarity determining region 2 (LCDR2) comprising GAS (SEQ ID NO:50); and(f) a light chain complementarity determining region 3 (LCDR3) comprising LQYGSIPWT (SEQ ID NO:52).

13. The antigen binding protein of claim 12, wherein the HCDR2 comprises MSPGGGSI (SEQ ID NO:42), or ISPGGGSI (SEQ ID NO:60), or LSPGGGSI (SEQ ID NO:61).

14. The antigen binding protein of claim 12, wherein the HCDR3 comprises EGELGPFDY (SEQ ID NO:46), or EGELGPFEY (SEQ ID NO:63), or EGELGPFQY (SEQ ID NO:64), or EGELGPFSY (SEQ ID NO:65).

15. The antigen binding protein of any of claims 12 to 14, wherein the antigen binding protein comprises canine or caninized frameworks.

16. The antigen binding protein of any of claims 12 to 14 which comprises a heavy chain framework (FR1H+FR2H+FR3H+FR4H) at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:15.

17. The antigen binding protein of any of claims 12 to 14 which comprises a light chain framework (FR1L+FR2L+FR3L+FR4L) at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 16.

18. The antigen binding protein of any one of claims 12 to 17, which comprises a VH domain at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 15 and a VL domain at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO: 16.

19. The antigen binding protein of any one of claims 12 to 17, wherein the Vn domain comprises SEQ ID NO: 15 and the VL domain comprises SEQ ID NO: 16; or the VH domain comprises SEQ ID NO: 19 and the VL domain comprises SEQ ID NO: 16; or the VH domain comprises SEQ ID NO:20 and the VL domain comprises SEQ ID NO: 16; or the VH domain comprises SEQ ID NO:21 and the VL domain comprises SEQ ID NO: 16; or the VH domain comprises SEQ ID NO:22 and the VL domain comprises SEQ ID NO: 16; or the VH domain comprises SEQ ID NO:23 and the VL domain comprises SEQ ID NO: 16.

20. The antigen binding protein of any of claims 12 to 14, wherein the antigen binding protein comprises human or humanized frameworks.

21. The antigen binding protein of any of claims 12 to 14, wherein the antigen binding protein comprises feline or felinized frameworks.

22. An isolated nucleic acid sequence encoding an anti-NGF antibody or antibody fragment of any one of claims 1 to 21.

23. A vector that comprises the nucleic acid of claim 22.

24. A recombinant cell which comprises the nucleic acid of any one of claims 22 or 23.

25. A cell that expresses the antigen binding protein of any one of claims 1 to 21, or the nucleic acid of any one of claims 22 or 23.

26. A method of producing the antigen binding protein of any one of claims 1 to 21, which comprises culturing the cell of claim 25 under conditions that result in production of the antigen binding protein.

27. A pharmaceutical composition comprising a therapeutically effective amount of the anti-NGF protein of any one of claims 1 to 21.

28. A method of treating or reducing pain in a subject which comprises administering to the subject a therapeutically effective amount of the anti-NGF protein of any one of claims 1 to 21.

29. The method of claim 28, wherein the pain comprises inflammatory pain, postoperative incision pain, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, pain resulting from burn, pain from trauma, musculoskeletal pain, rheumatic pain, or osteoporosis pain.

30. The method of claim 28, wherein the subject comprises a canine.

31. The method of claim 28, wherein the subject comprises a feline.

32. The method of claim 28, wherein the subject comprises a human.

33. A method of detecting NGF in a sample comprising incubating a sample comprising NGF in the presence of an anti -NGF protein of any one of claims 1 to 21 and detecting the anti-NGF protein bound to NGF in the sample.

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