Antigen-binding molecules and uses thereof

Abstract

The present disclosure relates to antigen-binding molecules that specifically bind to nerve growth factor (NGF) and uses thereof.

Description

[Technical field]

[0001] (Related Applications) This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 239,054, filed August 31, 2021. The contents of the aforementioned patent application are incorporated herein by reference in their entirety.

[0002] FIELD OF THEINVENTION The present disclosure relates generally to antigen binding molecules. In particular, the present invention relates to antigen binding molecules that specifically bind to nerve growth factor (NGF) and their use for the treatment of conditions associated with abnormal NGF expression and / or activity, such as pain. [Background technology]

[0003] All references, including any patents or patent applications cited in this specification are incorporated herein by reference to enable a complete understanding of the present invention. Nevertheless, such references should not be read as constituting an admission that any of these documents form part of the common general knowledge in the art in Australia or any other country.

[0004] Pain, including chronic pain, can be a debilitating condition with far-reaching social and economic consequences. Many analgesic compounds are prescribed for the treatment or prevention of pain in both humans and non-human animals, including local and general anesthetics, opioid analgesics, alpha2 agonists, non-steroidal anti-inflammatory drugs (NSAIDs), and steroids, although their efficacy may vary. Furthermore, current analgesics typically require frequent administration over long periods of time, which contributes, at least in part, to some of the adverse side effects associated with long-term use, including addiction and reduced efficacy.

[0005] As described by Enomoto et al. (2019, Veterinary Record; 184(1):23), current pharmacological treatment of pain is primarily focused on nonsteroidal anti-inflammatory drugs (NSAIDs) to reduce pain and promote functional improvement. Globally, several NSAIDs have been approved for use in dogs, but only two NSAIDs have been approved for long-term use in cats, and only in certain countries. Despite their widespread use and apparent benefits in many cases, NSAIDs are not always fully effective when used as monotherapy. In addition, Enomoto et al. describe that there are safety and tolerability concerns with their use in both dogs and cats. Outside of cyclooxygenase-inhibiting NSAIDs and the recently approved piperant NSAID, the prostaglandin receptor antagonist, grapiprant, treatment options for pain control are very limited. There is also limited evidence of efficacy for so-called adjunctive analgesics. The authors note that there are few proven non-pharmacological therapies, and none have been shown to provide rapid pain relief. This includes pain associated with inflammatory conditions such as osteoarthritis, which remains a difficult clinical entity to treat and is one of the most common reasons for euthanasia in dogs. Therefore, there is an urgent need for improved painkillers that are effective for both human and veterinary use, and that also avoid or at least partially alleviate some of the aforementioned problems associated with existing painkillers.

[0006] Nerve growth factor (NGF) is a secreted polypeptide and a member of the neurotrophin family that is involved in several different signal transduction pathways. For example, NGF has been shown to promote the survival and differentiation of sensory and sympathetic neurons through two membrane-bound receptors, p75, a low-affinity NGF receptor, and TrkA, a transmembrane tyrosine kinase and high-affinity NGF receptor. Binding of NGF to TrkA or p75 results in the upregulation of neuropeptides in sensory neurons, which typically results in pain perception or nociception.

[0007] NGF antagonists have been used to treat pain and pain sensitivity in humans, dogs, and cats. For example, Cattaneo (2010, Curr. Op. Mol. Ther. 12(1):94-106) and WO 2006 / 131951 both describe the use of a humanized form of rat alpha D11 (αD11) monoclonal antibody, which retains binding specificity for mouse NGF but also binds to human and rat forms of NGF. The primary rationale for humanizing donor antibodies, such as rat αD11 monoclonal antibodies, is to minimize the production of neutralizing antibodies that would otherwise result from a human anti-rat antibody response to rodent-derived antibodies after administration to a human subject, for example, during antibody therapy. In Cattaneo (2010) and WO 2006 / 131951, the CDR regions of a rat-derived αD11 monoclonal antibody were grafted onto framework regions from human immunoglobulin sequences, with the human framework sequences selected for closest sequence identity to the corresponding framework regions of the rat αD11 antibody. CDR grafting removes FR sequences that would otherwise be foreign to the immunoglobulin and generate an immune response against the immunoglobulin, which is often associated with a loss of binding specificity and selectivity to the target antigen. The loss of binding specificity and selectivity is typically ameliorated by backmutating one or more amino acid residues across the FR sequence from the target species, i.e., by replacing one or more amino acid residues across the modified framework region of the target species with the corresponding residue from the same position in the framework region of the donor antibody. However, while this can rescue binding specificity and selectivity, the introduction of amino acid residues from the donor antibody into the framework region is likely to introduce amino acid residues that would be foreign to the target species, i.e., to the species to which the modified antibody is to be administered.The methods described in WO 2012 / 153121 seek to minimize problems associated with back mutations by comparing amino acid residues spanning the framework regions of a donor anti-NGF antibody (such as the rat-derived αD11 monoclonal antibody) with the corresponding framework region sequences of one or more antibodies from a target species (e.g., dog) and replacing only those residues spanning the framework regions identified as foreign at the corresponding positions with respect to the framework regions from the target species, such that the modified antibody no longer contains any amino acid residues in its framework regions that would be foreign to the target species.

[0008] However, although modifying the framework region of immunoglobulin molecules for compatibility with target species advantageously minimizes the possibility of generating immunogenic responses in the target species to which the modified molecule is administered, the presence of foreign epitopes remains when CDR sequences are derived from species other than the target species.The presence of foreign epitopes in CDR sequences may contribute to anti-drug-antibody responses after administration, especially when multiple-dose therapy is required.Therefore, there is an urgent need for improved anti-NGF binding molecules that can be used in therapy, including the treatment and prevention of pain in humans and non-human animals, that overcome or at least partially alleviate one or more of the above-mentioned difficulties associated with existing therapeutic modalities. Summary of the Invention

[0009] The present disclosure is based, at least in part, on the inventors' surprising discovery that an amino acid substitution at a position corresponding to position 14 of the heavy chain CDR2 sequence of the rat αD11 anti-NGF binding molecule (SEQ ID NO: 8, as previously described in WO 2006 / 131951) unexpectedly and significantly reduces the anti-drug antibody response to the modified anti-NGF binding molecule when administered to species other than rats, while advantageously preserving NGF binding activity. The resulting anti-NGF binding molecule also exhibits clinical efficacy 60 days after administration, suggesting that the interval between administrations may advantageously not be less than 60 days, but may be longer, perhaps 90 or even 120 days between doses.

[0010] Accordingly, in an aspect disclosed herein, there is provided an antigen binding molecule that specifically binds to nerve growth factor (NGF), wherein the antigen binding molecule comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6: VH CDR1 GFSLTNNNVN (SEQ ID NO: 1) VH CDR2 GVWAGGATDYNSAVKS (SEQ ID NO:2) VH CDR3 DGGYSSSTLYAMDA (SEQ ID NO: 3) VL CDR1 RASEDIYNALA (SEQ ID NO: 4) VL CDR2 NTDTLHT (SEQ ID NO:5) VL CDR3 QHYFHYPRT (SEQ ID NO: 6)

[0011] In one embodiment, the antigen binding molecule comprises: (a) a VH framework region 1 (FR1) comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 36, 40, 44, 48, 52, and 72 to 127; (b) a VH FR2 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 37, 41, 45, 49, and 53; (c) a VH FR3 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 38, 42, 46, 50, and 54; (d) a VH FR4 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 39, 43, 47, 51, and 55; (e) a VL FR1 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 56, 60, 64, 68, and 129; (f) a VL FR2 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 57, 61, 65, and 69; (g) a VL FR3 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 58, 62, 66, and 70; and (h) a VL FR4 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 59, 63, 67, and 71.

[0012] In an embodiment, (a) VH comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NOs: 27 to 31, and (b) VL comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 128, and SEQ ID NO: 32-35.

[0013] In embodiments, the antigen binding molecule comprises: (a) a VH FR1 comprising an amino acid sequence having at least 80% sequence identity to a VH FR1 amino acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NOs: 72 to 127; (b) a VH FR2 comprising an amino acid sequence having at least 80% sequence identity to the VH FR2 amino acid sequence of SEQ ID NO: 14; (c) a VH FR3 comprising an amino acid sequence having at least 80% sequence identity to the VHFR3 amino acid sequence of SEQ ID NO: 15; (d) a VH FR4 comprising an amino acid sequence having at least 80% sequence identity to the VHFR4 amino acid sequence of SEQ ID NO: 16; (e) a VLFR1 comprising an amino acid sequence having at least 80% sequence identity to the VLFR1 amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 129; (f) a VLFR2 comprising an amino acid sequence having at least 80% sequence identity to the VLFR2 amino acid sequence of SEQ ID NO: 18; (g) a VL FR3 comprising an amino acid sequence having at least 80% sequence identity to the VLFR3 amino acid sequence of SEQ ID NO: 19; and (h) a VL FR4 comprising an amino acid sequence having at least 80% sequence identity to the VHFR4 amino acid sequence of SEQ ID NO:20.

[0014] In embodiments, the antigen binding molecule comprises: (a) the VH comprises an amino acid sequence having at least 80% sequence identity to the VH amino acid sequence of SEQ ID NO: 12; and (b) the VL comprises an amino acid sequence having at least 80% sequence identity to the VL amino acid sequence of SEQ ID NO:10 or SEQ ID NO:128.

[0015] In embodiments, the antigen binding molecule comprises: (a) VH comprises an amino acid sequence having at least 80% sequence identity to any one of the VH amino acid sequences of SEQ ID NOs: 27 to 31; and (b) VL comprises an amino acid sequence having at least 80% sequence identity to any one of the VL amino acid sequences of SEQ ID NOs: 32 to 35.

[0016] In embodiments, the antigen-binding molecule is an antibody or an NGF-binding fragment thereof. Suitable NGF-binding fragments are well known to those skilled in the art, and illustrative examples thereof include Fab fragments, scFab, Fab', single chain variable fragments (scFv), and single-arm antibodies. Thus, in embodiments disclosed herein, the NGF-binding fragment is selected from the group consisting of Fab fragments, scFab, Fab', single chain variable fragments (scFv), and single-arm antibodies.

[0017] In embodiments, the antigen binding molecule is a humanized, canine, feline, or equine antibody, or an NGF binding fragment thereof.

[0018] In another aspect disclosed herein, an isolated nucleic acid molecule is provided comprising a nucleic acid sequence encoding an antigen-binding molecule described herein.

[0019] Also disclosed herein are expression constructs comprising a nucleic acid sequence encoding an antigen-binding molecule described herein, wherein the nucleic acid sequence is operably linked to one or more regulatory sequences.

[0020] The present disclosure also extends to host cells containing the expression constructs described herein.

[0021] The present disclosure also encompasses a vector comprising the nucleic acid sequence encoding the antigen-binding molecule described herein.Suitable vectors are well known to those skilled in the art.In an embodiment, the vector is an AAV vector.

[0022] The present disclosure also extends to a pharmaceutical composition comprising an antigen-binding molecule described herein and a pharma- ceutically acceptable carrier.

[0023] In another aspect disclosed herein, a method for treating or preventing a condition associated with increased expression and / or activity of NGF is provided, comprising administering to a subject in need thereof an antigen-binding molecule, vector, or pharmaceutical composition described herein.

[0024] Conditions associated with increased expression and / or activity of NGF are well known to those of skill in the art, illustrative examples of which include pain, arthritis, and cancer.

[0025] Illustrative examples of pain associated with increased expression and / or activity of NGF include neuropathic pain, inflammatory pain, pruritic pain, perioperative pain, postoperative pain, and post-surgical pain.

[0026] Illustrative examples of arthritis associated with increased expression and / or activity of NGF include immune-mediated polyarthritis, rheumatoid arthritis, and osteoarthritis.

[0027] In another aspect disclosed herein, a method for treating or preventing tumors and associated conditions whose proliferation is induced by NGF is provided, comprising administering to a subject in need thereof an antigen-binding molecule, vector, or pharmaceutical composition as described herein. An illustrative example of a tumor and associated conditions whose proliferation is induced by NGF is osteosarcoma.

[0028] Also disclosed herein are kits comprising the antigen-binding molecules, vectors, or pharmaceutical compositions described herein.

[0029] The present disclosure also extends to the use of the antigen-binding molecules or vectors described herein in the manufacture of a medicament for treating or preventing a condition associated with increased expression and / or activity of NGF in a subject in need thereof.

[0030] The present disclosure also extends to the use of the antigen-binding molecule or vector described herein in the manufacture of a medicament for treating or preventing tumors whose growth is induced by NGF and conditions associated therewith in a subject in need thereof.

[0031] The present disclosure also extends to an antigen-binding molecule, vector, or pharmaceutical composition described herein for use in treating or preventing a condition associated with increased expression and / or activity of NGF in a subject in need thereof.

[0032] The present disclosure also extends to the antigen-binding molecule, vector, or pharmaceutical composition described herein for use in treating or preventing tumors whose growth is induced by NGF and conditions associated therewith in a subject in need thereof. [Brief description of the drawings]

[0033] Embodiments of the invention will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Figure 1-1] Figure 1 shows the pharmacokinetic profile of felinized anti-NGF monoclonal antibodies fe1_αD11, fe1_αD11_HCCDR1(F / L), and fe1_αD11_HCCDR2(L / V) in cats after subcutaneous administration. Antibodies were administered subcutaneously twice to each of four cats at 2 mg / kg body weight on days 0, 21, 42, and 63. Serum concentrations of felinized anti-NGF antibodies were determined at the indicated times using a quantitative NGF binding ELISA as described elsewhere herein. Data shown are mean + / - SD. [Figure 1-2] Figure 1 shows the pharmacokinetic profile of felinized anti-NGF monoclonal antibodies fe1_αD11, fe1_αD11_HCCDR1(F / L), and fe1_αD11_HCCDR2(L / V) in cats after subcutaneous administration. Antibodies were administered subcutaneously twice to each of four cats at 2 mg / kg body weight on days 0, 21, 42, and 63. Serum concentrations of felinized anti-NGF antibodies were determined at the indicated times using a quantitative NGF binding ELISA as described elsewhere herein. Data shown are mean + / - SD. [Diagram 2]Immunogenicity profile of felinized anti-NGF monoclonal antibodies fe1_αD11, fe1_αD11_HCCDR1(F / L), and fe1_αD11_HCCDR2(L / V) in cat serum after subcutaneous administration. The three felinized anti-NGF antibodies were administered subcutaneously at 2 mg / kg body weight to four cats on days 0, 21, 42, and 63. The presence of anti-drug antibodies (ADA) at various time points was determined using a bridging immunoassay. Data for each animal is shown. [Diagram 3] Pharmacokinetic (PK) profile of caninized anti-NGF antibody ca_αD11_HCCDR2(L / V) (CaNGF) after subcutaneous administration to dogs. ca_αD11_HCCDR2(L / V) antibody was administered subcutaneously at 1 mg / kg body weight to four dogs on days 0 and again on day 28. Concentrations of ca_αD11_HCCDR2(L / V) were determined at the indicated times by quantitative NGF binding ELISA. Data shown are mean + / - SD. [Figure 4] Expression of fe_αD11_HCCDR2(L / V) in feline serum after IM administration of an AAV construct containing a transgene encoding fe_αD11_HCCDR2(L / V) at 1×1012 gc / kg body weight on day 14 of the study. Concentrations of fe_αD11_HCCDR2(L / V) were determined on days 30 and 90 by quantitative NGF binding ELISA. Data shown are mean + / - SD (n=14 on day 30 and n=6 on day 90). [Figure 5-1]Figure 1 shows the percentage of cats that achieved clinical success after (i) subcutaneous administration of placebo on study days 0 and 30, (ii) subcutaneous administration of monoclonal antibody fe_αD11_HCCDR2(L / V) at 2 mg / kg body weight on study days 0 and 30, and (iii) intramuscular administration of an AAV construct containing a transgene encoding fe_αD11_HCCDR2(L / V) administered at 1 x 1012 gc / kg body weight on study day 14. Y-axis: reduction in client-specific outcome measure (CSOM) score of ≥ 2 (A) or CSOM score of ≥ 3 (B) on study days 30 and 90 (LS mean, 95% confidence interval). [Figure 5-2] Figure 1 shows the percentage of cats that achieved clinical success after (i) subcutaneous administration of placebo on study days 0 and 30, (ii) subcutaneous administration of monoclonal antibody fe_αD11_HCCDR2(L / V) at 2 mg / kg body weight on study days 0 and 30, and (iii) intramuscular administration of an AAV construct containing a transgene encoding fe_αD11_HCCDR2(L / V) administered at 1 x 1012 gc / kg body weight on study day 14. Y-axis: reduction in client-specific outcome measure (CSOM) score of ≥ 2 (A) or CSOM score of ≥ 3 (B) on study days 30 and 90 (LS mean, 95% confidence interval). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] As described elsewhere herein, the present disclosure is based, at least in part, on the inventors' surprising discovery that an amino acid substitution at a position corresponding to position 14 of the heavy chain CDR2 sequence of the rat αD11 anti-NGF binding molecule (SEQ ID NO:8, as previously described in WO 2006 / 131951) unexpectedly and significantly reduces the anti-drug antibody response to the modified anti-NGF binding molecule when administered to species other than rats, while advantageously preserving NGF binding activity.

[0035] Accordingly, disclosed herein is an antigen-binding molecule capable of specifically binding to nerve growth factor (NGF), the antigen-binding molecule comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6: VH CDR1 GFSLTNNNVN (SEQ ID NO: 1) VH CDR2 GVWAGGATDYNSAVKS (SEQ ID NO:2) VH CDR3 DGGYSSSTLYAMDA (SEQ ID NO: 3) VL CDR1 RASEDIYNALA (SEQ ID NO: 4) VL CDR2 NTDTLHT (SEQ ID NO:5) VL CDR3 QHYFHYPRT (SEQ ID NO: 6)

[0036] A "conservative amino acid substitution" should be understood to mean a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and can generally be subclassified as shown in the "Amino Acid Classifications" table below.

[0037] [Table 1]

[0038] Conservative amino acid substitutions also include groupings based on side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine. The group of amino acids with aliphatic hydroxyl side chains is serine and threonine. The group of amino acids with amide-containing side chains is asparagine and glutamine. The group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan. The group of amino acids with basic side chains is lysine, arginine, and histidine. And the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to expect that the replacement of leucine with isoleucine or valine, the replacement of aspartic acid with glutamic acid, the replacement of threonine with serine, or similar replacement of amino acids with structurally related amino acids will not significantly affect the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can be easily determined by assaying its activity.

[0039] Conservative substitutions are also shown in the following table (exemplary preferred amino acid substitutions). Amino acid substitutions that fall within the scope of the present invention are generally achieved by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of ​​substitution, (b) the charge or hydrophobicity of the molecule in the target site, or (c) the bulk of the side chain. After substitutions are introduced, variants can be screened for their ability to specifically bind to NGF using methods known to those skilled in the art, including those described elsewhere herein.

[0040] [Table 2]

[0041] "Antigen binding molecule" refers to a molecule that has binding affinity for a target antigen. It is understood that the term extends to immunoglobulins, immunoglobulin fragments, and non-immunoglobulin derived protein frameworks that exhibit antigen binding activity. Illustrative examples of suitable antigen binding molecules include antibodies and antigen binding fragments thereof. Preferably, the antigen binding molecule specifically binds to NGF and neutralizes or substantially neutralizes its activity. The term "neutralizing" is understood to mean that the antigen binding molecule binds to NGF and inhibits, reduces, abrogates, blocks, or otherwise prevents the ability of the NGF molecule to bind to its natural receptor (e.g., p75 or TrkA). In some embodiments, the antigen binding molecule completely neutralizes the activity of NGF (in vivo or in vitro), resulting in no or negligible NGF activity compared to the absence of the antigen binding molecule. In other embodiments, the antigen binding molecule partially neutralizes the activity of NGF (in vivo or in vitro), resulting in less NGF activity compared to the absence of the antigen binding molecule.

[0042] In embodiments, the antigen binding molecules described herein are conjugated to another molecule or moiety, including functional moieties (e.g., toxins), detectable moieties (e.g., fluorescent molecules, radioisotopes), small molecule drugs, and polypeptides.

[0043] The term "antibody", as used herein, is understood to mean any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or specifically interacts with a target antigen. The term "antibody" includes full-length immunoglobulin molecules containing two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (HCVR, VH, or V H and a heavy chain constant region. The heavy chain constant region typically comprises three domains -C H 1. C H 2, and CH Each light chain comprises a light chain variable region (LCVR, VL, VK, V K , or V L The light chain constant region typically comprises one domain (C L 1) is included. H and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, also called framework regions (FRs). H and V L Typically, the FRs comprise three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antigen-binding molecules described herein may be identical to the FRs of the germline sequence of the target species (i.e., the species to which the antigen-binding molecules or antigen-binding fragments thereof described herein are administered). In some embodiments, the FRs may be modified naturally or artificially. Although it is generally desirable that each of the FR sequences is identical to the FR sequence derived from an immunoglobulin molecule of the target species, such as to minimize an immune response against the binding molecule upon administration to a subject of the target species, in some embodiments, the antigen-binding molecule or antigen-binding fragment thereof may comprise one or more amino acid residues across one or more of its FR sequences that would be foreign at the corresponding positions in one or more FRs from the target species. Preferably, when an antigen-binding molecule or antigen-binding fragment thereof comprises one or more amino acid residues across one or more of its FR sequences that would be foreign at the corresponding positions in the target species, the "foreign" amino acid residues (i) do not adversely affect the binding specificity of the antigen-binding molecule or antigen-binding fragment thereof for NGF, including native NGF, and / or (ii) do not generate an immune response against the antigen-binding molecule or antigen-binding fragment thereof when administered to a subject of the target species.

[0044] Suitable antibodies include antibodies of any class, such as IgG, IgA, or IgM (including subclasses thereof). There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, characterized by heavy chain constant regions α, δ, ε, γ, and μ, respectively. Some antibody classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

[0045] As used herein, the term "complementarity determining region" (CDR) refers to the region of an immunoglobulin variable domain that recognizes and binds to a target antigen. Each variable domain may contain up to three CDR sequences, identified as CDR1, CDR2, and CDR3. The amino acid sequence of each CDR is often numbered according to Kabat numbering (e.g., about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and about residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or Chothia numbering (e.g., about residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and about residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and (see Lesk J. Mol. Biol. 196:901-917 (1987)). As disclosed elsewhere herein, the inventors have unexpectedly demonstrated that amino acid positions along the CDR sequences of an NGF-binding molecule can be substituted with one or more conservative or non-conservative amino acids while retaining the ability to specifically bind to its target antigen, NGF. Thus, the present disclosure extends to functional variants of the NGF-binding molecules disclosed herein. The term "functional variant", as used herein, should be understood to mean an NGF-binding molecule that comprises a CDR sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-6 and that retains the ability to specifically bind to NGF and neutralize or otherwise inhibit the activity of NGF.

[0046] The present disclosure extends to antigen-binding molecules that specifically bind to any species of NGF. In an embodiment, the NGF is selected from the group consisting of human NGF, canine NGF, feline NGF, and equine NGF. In an embodiment, the NGF is human NGF. In another embodiment, the NGF is canine NGF. In another embodiment, the NGF is feline NGF. In yet another embodiment, the NGF is equine NGF. The present disclosure extends to antigen-binding molecules that specifically bind to native NGF (i.e., naturally occurring NGF), and variants thereof. Such variants may include NGF molecules that differ from the naturally occurring (wild-type) molecule by one or more amino acid substitutions, deletions, and / or insertions. Variant NGF molecules of this type may be naturally occurring or synthetic (e.g., recombinant). However, it should be understood that in a preferred embodiment, the antigen-binding molecules described herein specifically bind to the native form of NGF, whether human or non-human species.

[0047] The terms "antigen-binding fragment", "antigen-binding portion", "antigen-binding domain", "antigen-binding site" and the like are used interchangeably herein and refer to a portion of an antigen-binding molecule that retains the ability to bind to a target antigen, i.e., NGF, including native NGF. These terms include naturally occurring, enzymatically obtainable, synthetic or genetically engineered (recombinant) polypeptides and glycoproteins that specifically bind to NGF to form a complex.

[0048] Antigen-binding fragments can be derived, for example, from naturally occurring immunoglobulin molecules using any suitable method known to those of skill in the art, illustrative examples of which include proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of nucleic acid sequences encoding antibody variable domains and optionally constant domains. Suitable nucleic acid sequences are known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. Nucleic acid sequences can be sequenced and engineered chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0049] Non-limiting examples of suitable 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 amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated CDRs, such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, single arm antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), and small modular immunopharmaceuticals (SMIPs), are also encompassed by the term "antigen-binding fragment" as used herein.

[0050] In embodiments, the antigen-binding fragment comprises at least one immunoglobulin variable domain. The variable domain may comprise an amino acid sequence of any suitable length or composition and generally comprises at least one CDR adjacent to or in frame with one or more framework sequences. H Domain and V LIf domain is included, V H and V L The domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer, with the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of the antibody may comprise a monomeric V H Or V L It may include a domain.

[0051] In some embodiments, an antigen-binding fragment may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting configurations of variable and constant domains that may be found in an antigen-binding fragment include: (i) a V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -CL, (viii)V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V LIn any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be either directly linked to each other or linked by a full or partial hinge or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which provides a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. In some embodiments, the antigen-binding fragments described herein can be linked to each other and / or to one or more monomeric V H Or V L The variable and constant domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, non-covalently linked (e.g., by disulfide bonds) with the domains. Multispecific antigen-binding molecules typically comprise at least two different variable domains, each of which can specifically bind to a separate antigen or a different epitope on the same antigen. Any multispecific antigen-binding molecule format, including bispecific antigen-binding molecule formats, can be adapted for use in the context of the antigen-binding fragments of the antibodies of the present disclosure using routine techniques available in the art.

[0052] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin heavy or light chain that is involved in binding to a target antigen. The heavy and light chain variable domains (V and V, respectively) of a native immunoglobulin molecule H A single V domain (VL and VL) generally has a similar structure, with each domain containing four conserved framework regions and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). H Or V L The domain may be sufficient to confer antigen-binding specificity.

[0053] As described elsewhere herein, the amino acid substitutions identified as advantageously reducing anti-drug antibody responses to modified anti-NGF binding molecules in target species other than rats, while preserving NGF binding activity, are in the heavy chain CDR sequences corresponding to the CDR sequences of the rat αD11 anti-NGF binding molecule. The anti-NGF binding molecules described herein may suitably include further modifications, such as in their framework and / or constant regions, for compatibility with the target species. As an illustrative example, one or more of the framework regions of the modified anti-NGF binding molecules suitably include amino acid sequences that are native (not foreign) to the target species to which the modified anti-NGF binding molecule is administered. It should be understood that the framework regions of the anti-NGF binding molecules described herein may be modified for compatibility with any target species (other than rat). Such modified binding molecules are often referred to as referring to the species to which they are targeted. For example, a binding molecule comprising a framework region modified for compatibility with cats may be referred to as a felineized antigen binding molecule, a binding molecule comprising a framework region modified for compatibility with dogs may be referred to as a caninized antigen binding molecule, a binding molecule comprising a framework region modified for compatibility with horses may be referred to as an equineized antigen binding molecule, a binding molecule comprising a framework region modified for compatibility with humans may be referred to as a humanized antigen binding molecule, etc.Suitable target species will be well known to those of skill in the art, and illustrative examples thereof include primates (e.g., humans, monkeys, and apes, including monkey species from the genus Macaca (e.g., cynomolgus monkeys, such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatta)) and baboons (Papio ursinus), as well as ape species such as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri), and tamarins (species from the genus Saguinus), and chimpanzees (Pan troglodytes)), rodents (e.g., mice, Any member of the subphylum Chordata, including mammals such as rats, guinea pigs, lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), birds (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), as well as fish.

[0054] In embodiments, the antigen-binding molecule or antigen-binding fragment thereof is humanized, canine, feline, or equine.

[0055] "Humanized" means that the antigen-binding molecule comprises an amino acid sequence that is compatible with humans, such that the amino acid sequence is unlikely to be viewed as foreign by the immune system of a human subject. In embodiments, the humanized antigen-binding molecule comprises one or more immunoglobulin framework regions derived from one or more human immunoglobulin molecules. In some embodiments, all of the framework regions of the humanized antigen-binding molecule are derived from one or more human immunoglobulin molecules. The humanized antibody may optionally comprise an immunoglobulin heavy chain constant region derived from a human immunoglobulin molecule.

[0056] "Caninized" means that the antigen-binding molecule comprises an amino acid sequence that is compatible with dogs, such that the amino acid sequence is unlikely to be seen as foreign by the immune system of a canine subject. In embodiments, the caninized antigen-binding molecule comprises one or more immunoglobulin framework regions derived from one or more canine immunoglobulin molecules. In some embodiments, all of the framework regions of the caninized antigen-binding molecule are derived from one or more canine immunoglobulin molecules. The caninized antibody may optionally comprise an immunoglobulin heavy chain constant region derived from a canine immunoglobulin molecule.

[0057] "Fenconized" means that the antigen-binding molecule comprises an amino acid sequence that is compatible with cats, such that the amino acid sequence is unlikely to be seen as foreign by the immune system of a feline subject. In embodiments, the felined antigen-binding molecule comprises one or more immunoglobulin framework regions derived from one or more feline immunoglobulin molecules. In some embodiments, all of the framework regions of the felined antigen-binding molecule are derived from one or more feline immunoglobulin molecules. The felined antibody may optionally comprise an immunoglobulin heavy chain constant region derived from a feline immunoglobulin molecule.

[0058] "Equidized" means that the antigen-binding molecule comprises an amino acid sequence that is compatible with horses, such that the amino acid sequence is unlikely to be viewed as foreign by the immune system of an equine subject. In embodiments, the equine-made antigen-binding molecule comprises one or more immunoglobulin framework regions derived from one or more equine immunoglobulin molecules. In some embodiments, all of the framework regions of the equine-made antigen-binding molecule are derived from one or more equine immunoglobulin molecules. The equine-made antibody may optionally comprise an immunoglobulin heavy chain constant region derived from an equine immunoglobulin molecule.

[0059] As described elsewhere herein, the present disclosure extends to antigen-binding molecules that are compatible with species other than humans, dogs, cats, and horses. In this regard, antigen-binding molecules can be said to be "specialized" with respect to the target species to which the molecule is administered.

[0060] Suitable methods for designing and producing recombinant antibodies or antigen-binding molecules that are compatible with the target species are well known to the skilled artisan, and illustrative examples thereof are described in Cattaneo (2010, supra), WO 2006 / 131951, WO 2012 / 153122, WO 2013 / 034900, WO 2012 / 153121, and WO 2012 / 153123, the contents of which are incorporated herein by reference in their entireties.

[0061] The phrases "specifically bind" or "specific binding" refer to a binding reaction between two molecules that is at least twice background, and more typically exceeds 10-100 times background molecular association, under physiological conditions. When using one or more detectable binding agents that are proteins, specific binding is determinant of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under specified immunoassay conditions, a particular antigen-binding molecule binds to a particular antigenic determinant, thereby identifying its presence. Specific binding to an antigenic determinant under such conditions requires an antigen-binding molecule that is selected for its specificity for that determinant. This selection can be achieved by subtracting out antigen-binding molecules that cross-react with other molecules. Using a variety of immunoassay formats, antigen-binding molecules (e.g., immunoglobulins) can be selected such that they are specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Methods for determining binding affinity and specificity are also well known in the art (see, e.g., Harlow and Lane, supra; Friefelder, "Physical Biochemistry: Applications to biochemistry and molecular biology" (WH Freeman and Co. 1976)).

[0062] "Affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair, e.g., an antigen-binding molecule. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (k, respectively). off and k on ). Thus, equivalent affinities may include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

[0063] The terms "polypeptide," "peptide," or "protein" are used interchangeably herein to refer to a linear series of amino acid residues connected to one another by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acid residues are normally in the naturally occurring "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide.

[0064] As used herein, the term "modified antibodies" includes synthetic forms of antibodies that have been altered so that they do not occur in nature, such as antibodies that contain at least two heavy chain portions but do not contain two complete heavy chains (such as domain deleted antibodies or minibodies), multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that have been altered to bind to two or more different antigens or different epitopes on a single antigen, heavy chain molecules linked to scFv molecules, etc. ScFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. In addition, the term "modified antibodies" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen).

[0065] As described elsewhere herein, the anti-NGF binding molecules described herein may suitably comprise further modifications within their framework and / or constant regions, such as for compatibility with a target species. As an illustrative example, one or more of the framework regions of a modified anti-NGF binding molecule suitably comprises an amino acid sequence that is native (not foreign) to the target species to which the modified anti-NGF binding molecule is administered.

[0066] In embodiments, the antigen binding molecule has the amino acid sequence QVQLX 1 ESGX 2 X 3 LVQPX 4 X 5 SLRLTCX 6 VH framework region 1 (FR1) comprising AS (SEQ ID NO:74), (a)X 1 is selected from the group consisting of valine, methionine, and a conservative amino acid substitution of any of the foregoing; (b)X 2 is selected from the group consisting of glycine, alanine, and a conservative amino acid substitution of any of the foregoing; (c)X 3 is selected from the group consisting of aspartic acid, glutamic acid, and a conservative amino acid substitution of any of the foregoing; (d)X 4is selected from the group consisting of glycine, serine, and a conservative amino acid substitution of any of the foregoing; (e)X 5 is selected from the group consisting of glycine, glutamic acid, and a conservative amino acid substitution of any of the foregoing; (f)X 6 is selected from the group consisting of valine, alanine, and a conservative amino acid substitution of any of the foregoing.

[0067] In embodiments, the antigen binding molecule comprises: (a) QVQLVESGADLVQPSESLRLTCVAS (SEQ ID NO: 75), (b) QVQLVESGGDLVQPSESLRLTCVAS (SEQ ID NO: 76), (c) QVQLVESGADLVQPGESLRLTCVAS (SEQ ID NO: 77), (d) QVQLVESGADLVQPSGSLRLTCVAS (SEQ ID NO: 78), (e) QVQLVESGGDLVQPGESLRLTCVAS (SEQ ID NO: 79), (f) QVQLVESGGDLVQPSGSLRLTCVAS (SEQ ID NO: 80), (g) QVQLVESGGDLVQPGGSLRLTCVAS (SEQ ID NO: 72), (h) QVQLMESGADLVQPSESLRLTCVAS (SEQ ID NO: 13), (i) QVQLMESGGDLVQPSESLRLTCVAS (SEQ ID NO: 81), (j) QVQLMESGADLVQPGESLRLTCVAS (SEQ ID NO: 82), (k) QVQLMESGADLVQPSGSLRLTCVAS (SEQ ID NO: 83), (l) QVQLMESGGDLVQPGESLRLTCVAS (SEQ ID NO: 84), (m) QVQLMESGGDLVQPSGSLRLTCVAS (SEQ ID NO: 85), (n) QVQLMESGGDLVQPGGSLRLTCVAS (SEQ ID NO: 86), (o) QVQLVESGADLVQPSESLRLTCAAS (SEQ ID NO: 87), (p) QVQLVESGGDLVQPSESLRLTCAAS (SEQ ID NO: 88), (q) QVQLVESGADLVQPGESLRLTCAAS (sequence number 89), (r) QVQLVESGADLVQPSGSLRLTCAAS (SEQ ID NO: 90), (s) QVQLVESGGDLVQPGESLRLTCAAS (sequence number 91), (t) QVQLVESGGDLVQPSGSLRLTCAAS (SEQ ID NO: 92), (u) QVQLVESGGDLVQPGGSLRLTCAAS (SEQ ID NO: 93), (v) QVQLMESGADLVQPSESLRLTCAAS (SEQ ID NO: 94), (w) QVQLMESGGDLVQPSESLRLTCAAS (SEQ ID NO: 95), (x) QVQLMESGADLVQPGESLRLTCAAS (sequence number 96), (y) QVQLMESGADLVQPSGSLRLTCAAS (sequence number 97), (z) QVQLMESGGDLVQPGESLRLTCAAS (SEQ ID NO: 98), (aa) QVQLMESGGDLVQPSGSLRLTCAAS (SEQ ID NO: 99), (bb) QVQLMESGGDLVQPGGSLRLTCAAS (SEQ ID NO: 100), (cc) QVQLVESGAELVQPSESLRLTCVAS (SEQ ID NO: 101), (dd) QVQLVESGGELVQPSESLRLTCVAS (SEQ ID NO: 102), (ee) QVQLVESGAELVQPGESLRLTCVAS (SEQ ID NO: 103), (ff) QVQLVESGAELVQPSGSLRLTCVAS (SEQ ID NO: 104), (gg) QVQLVESGGELVQPGESLRLTCVAS (SEQ ID NO: 105), (hh) QVQLVESGGELVQPSGSLRLTCVAS (SEQ ID NO: 106), (ii) QVQLVESGGELVQPGGSLRLTCVAS (SEQ ID NO: 107), (jj) QVQLMESGAELVQPSESLRLTCVAS (SEQ ID NO: 108), (kk) QVQLMESGGELVQPSESLRLTCVAS (SEQ ID NO: 109), (ll) QVQLMESGAELVQPGESLRLTCVAS (SEQ ID NO: 110), (mm) QVQLMESGAELVQPSGSLRLTCVAS (SEQ ID NO: 111), (nn) QVQLMESGGELVQPGESLRLTCVAS (SEQ ID NO: 112), (oo) QVQLMESGGELVQPSGSLRLTCVAS (SEQ ID NO: 113), (pp) QVQLMESGGELVQPGGSLRLTCVAS (SEQ ID NO: 114), (qq) QVQLVESGAELVQPSESLRLTCAAS (SEQ ID NO: 115), (rr) QVQLVESGGELVQPSESLRLTCAAS (SEQ ID NO: 116), (ss) QVQLVESGAELVQPGESLRLTCAAS (SEQ ID NO: 73), (tt) QVQLVESGAELVQPSGSLRLTCAAS (SEQ ID NO: 117), (uu) QVQLVESGGELVQPGESLRLTCAAS (SEQ ID NO: 118), (vv) QVQLVESGGELVQPSGSLRLTCAAS (SEQ ID NO: 119), (ww)QVQLVESGGELVQPGGSLRLTCAAS (SEQ ID NO: 120), (xx) QVQLMESGAELVQPSESLRLTCAAS (SEQ ID NO: 121), (yy) QVQLMESGGELVQPSESLRLTCAAS (SEQ ID NO: 122), (zz) QVQLMESGAELVQPGESLRLTCAAS (SEQ ID NO: 123), (aaa) QVQLMESGAELVQPSGSLRLTCAAS (SEQ ID NO: 124), (bbb) QVQLMESGGELVQPGESLRLTCAAS (SEQ ID NO: 125), (ccc)QVQLMESGGELVQPSGSLRLTCAAS (SEQ ID NO: 126), and (ddd)QVQLMESGGELVQPGGSLRLTCAAS (SEQ ID NO: 127), and

[0068] In embodiments, the antigen binding molecule comprises: (a) a VH framework region 1 (FR1) comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 36, 40, 44, 48, 52, and 72 to 127; (b) a VH FR2 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 37, 41, 45, 49, and 53; (c) a VH FR3 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 38, 42, 46, 50, and 54; (d) a VH FR4 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 39, 43, 47, 51, and 55; (e) a VL FR1 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 56, 60, 64, 68, and 129; (f) a VL FR2 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 57, 61, 65, and 69; (g) a VL FR3 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 58, 62, 66, and 70; and (h) a VL FR4 comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 59, 63, 67, and 71.

[0069] In embodiments, the antigen binding molecule comprises: (a) a VH framework region 1 (FR1) comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 72; and (b) a VL FR1 comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 129.

[0070] In an embodiment, (a) VH comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NOs: 27 to 31, and (b) VL comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 128, and SEQ ID NOs: 32-35.

[0071] In another embodiment, the antigen binding molecule comprises: (a) a VH FR1 comprising an amino acid sequence having at least 80% sequence identity to a VH FR1 amino acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NOs: 72 to 127; (b) a VH FR2 comprising an amino acid sequence having at least 80% sequence identity to the VH FR2 amino acid sequence of SEQ ID NO: 14; (c) a VH FR3 comprising an amino acid sequence having at least 80% sequence identity to the VHFR3 amino acid sequence of SEQ ID NO: 15; (d) a VH FR4 comprising an amino acid sequence having at least 80% sequence identity to the VHFR4 amino acid sequence of SEQ ID NO: 16; (e) a VLFR1 comprising an amino acid sequence having at least 80% sequence identity to the VLFR1 amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 129; (f) a VLFR2 comprising an amino acid sequence having at least 80% sequence identity to the VLFR2 amino acid sequence of SEQ ID NO: 18; (g) a VL FR3 comprising an amino acid sequence having at least 80% sequence identity to the VLFR3 amino acid sequence of SEQ ID NO: 19; and (h) a VL FR4 comprising an amino acid sequence having at least 80% sequence identity to the VHFR4 amino acid sequence of SEQ ID NO:20.

[0072] In another embodiment, the antigen binding molecule comprises: (a) the VH comprises an amino acid sequence having at least 80% sequence identity to the VH amino acid sequence of SEQ ID NO: 12; and (b) the VL comprises an amino acid sequence having at least 80% sequence identity to the VL amino acid sequence of SEQ ID NO:128.

[0073] In an embodiment, (a) VH comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 31, and (b) VL comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-35.

[0074] In embodiments, the antigen binding molecule comprises: (a) a VH framework region 1 (FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 36, 40, 44, 48, 52, and 72 to 127; (b) a VH FR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 37, 41, 45, 49, and 53; (c) a VH FR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 38, 42, 46, 50, and 54; (d) a VH FR4 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 39, 43, 47, 51, and 55; (e) a VL FR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 56, 60, 64, 68, and 129; (f) a VL FR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 57, 61, 65, and 69; (g) a VL FR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 58, 62, 66, and 70; and (h) comprises, consists of, or consists essentially of a VL FR4 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 59, 63, 67, and 71.

[0075] In an embodiment, (a) VH comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NOs: 27-31; and (b) VL comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 128, and SEQ ID NOs: 32-35.

[0076] In yet another embodiment, the antigen binding molecule comprises: (a) VH FR1 comprising the amino acid sequence of SEQ ID NO: 72; (b) VH FR2 comprising the amino acid sequence of SEQ ID NO: 14; (c) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 15; (d) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 16; (e) VL FR1 comprising the amino acid sequence of SEQ ID NO: 17; (f) VL FR2 comprising the amino acid sequence of SEQ ID NO: 18; (g) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 19; and (h) comprises, consists of, or consists essentially of a VL FR4 comprising the amino acid sequence of SEQ ID NO:20.

[0077] In another embodiment, the antigen binding molecule comprises: (a) the VH comprises the amino acid sequence of SEQ ID NO: 12; and (b) the VL comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 128.

[0078] In an embodiment, (a) VH comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-31; and (b) The VL comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-35.

[0079] References herein to "at least 80%" include 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the listed sequences after optimal alignment or best-fit analysis.

[0080] Optimal alignment of sequences to align a comparison window can be performed by computer implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and best alignment (i.e., resulting in the highest homology percentage over the comparison window) generated by any suitable method known to those skilled in the art. Also, reference can be made to the BLAST family of programs, for example, as disclosed by Altschul et al. (1997) Nucl. Acids. Res. 25:3389. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al. (1994-1998) in Current Protocols in Molecular Biology, John Wiley & Sons Inc.

[0081] The term "sequence identity" as used herein refers to the degree to which sequences are identical or structurally similar nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Two or more peptide sequences can be compared by determining their "percent identity." The percent identity of two sequences can be described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be extended for use with peptide sequences using a score matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov, Nucl. Acids Res. 14(6): 6745-6763 (1986). Suitable methods and computer programs for aligning two or more amino acid sequences and determining their sequence identity or homology are well known to those skilled in the art. For example, the percentage of identity or similarity of two amino acid sequences can be easily calculated using algorithms such as BLAST, FASTA, or Smith-Waterman algorithms.Thus, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. For example, the "sequence identity" is the "percentage of match" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

[0082] Sequence identity includes exact identity between compared sequences at the nucleotide or amino acid level. Sequence identity typically relates to the percentage of amino acid residues in a candidate sequence that are identical to the residues of the corresponding peptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of homology, as described herein, and does not consider any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions should be construed as reducing sequence identity or homology.

[0083] In another embodiment, the antigen-binding molecule is an antibody or antigen-binding fragment thereof as described elsewhere herein, In embodiments, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a scFab, a Fab', a single-chain variable fragment (scFv), and a single-arm antibody.

[0084] Also disclosed herein are chimeric molecules comprising the NGF binding molecules described herein and a heterologous moiety. In some embodiments, the heterologous moiety can be a detectable moiety, a half-life extending moiety, or a therapeutic moiety. Thus, as used herein, a "chimeric" molecule is a molecule that comprises one or more unrelated types of components or that comprises two or more chemically distinct regions that can be conjugated to each other, fused, linked, translated, linked via a linker, chemically synthesized, expressed from a nucleic acid sequence, etc. For example, a peptide and a nucleic acid sequence, a peptide and a detectable label, an unrelated peptide sequence, etc. In embodiments in which the chimeric molecule comprises amino acid sequences of different origins, the chimeric molecule comprises (1) polypeptide sequences that are not found together in nature (i.e., at least one of the amino acid sequences is heterologous to at least one of the other amino acid sequences), or (2) amino acid sequences that are not naturally adjacent. For example, a "chimeric" antibody, as used herein, refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0085] The present disclosure also extends to an isolated polynucleotide comprising a nucleic acid sequence encoding an NGF binding molecule described herein.

[0086] In embodiments, a nucleic acid molecule encoding an immunoglobulin heavy chain of an NGF binding molecule disclosed herein has at least 80% sequence identity to SEQ ID NO: 25. In embodiments, a nucleic acid molecule encoding an immunoglobulin heavy chain of an NGF binding molecule disclosed herein comprises, consists of, or consists essentially of the nucleic acid sequence of SEQ ID NO:25.

[0087] In embodiments, a nucleic acid molecule encoding an immunoglobulin light chain of an NGF binding molecule disclosed herein has at least 80% sequence identity to SEQ ID NO: 26. In embodiments, a nucleic acid molecule encoding an immunoglobulin light chain of an NGF binding molecule disclosed herein comprises, consists of, or consists essentially of the nucleic acid sequence of SEQ ID NO:26.

[0088] The terms "polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides, which may be mRNA, RNA, cRNA, cDNA, or DNA. The term refers to a polymeric form of nucleotides, typically at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The term includes single- and double-stranded forms of DNA.

[0089] Also disclosed herein are vectors that contain a nucleic acid encoding the NGF binding molecules described herein.

[0090] By "vector" is meant a nucleic acid molecule, preferably a DNA molecule, for example from a plasmid, bacteriophage, or virus, into which a nucleic acid sequence can be inserted or cloned. The vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell, including the target cell or tissue, or a precursor cell or tissue thereof, or may be capable of integrating into the genome of the defined host so that the cloned sequence is reproducible. Thus, the vector may be a self-replicating vector, i.e. a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. linear or closed circular plasmids, extrachromosomal elements, minichromosomes, or artificial chromosomes. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates together with the chromosome into which it is integrated. The vector system may comprise a single vector or plasmid, two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of vector typically depends on the compatibility of the vector with the host cell into which it is introduced. The vector may also contain a selection marker, such as an antibiotic resistance gene, that can be used for the selection of suitable transformants. Examples of such resistance genes are well known to those skilled in the art.

[0091] In one embodiment, the vector is an adeno-associated virus (AAV) vector that allows the NGF binding molecules described herein to be safely administered to a subject and provide sustained expression of the NGF binding molecule in the subject.

[0092] Adeno-associated viruses are members of the Parvoviridae family and contain a linear, single-stranded DNA genome of less than about 5,000 nucleotides. AAV requires co-infection with a helper virus (i.e., adenovirus or herpesvirus) or expression of helper genes for efficient replication. AAV vectors used for administration of therapeutic nucleic acids typically have about 96% of the parent genome deleted, leaving only the terminal repeats (ITRs) that contain recognition signals for DNA replication and packaging. This eliminates immunological or toxic side effects due to expression of viral genes. In addition, delivery of specific AAV proteins to producer cells allows integration of AAV vectors containing AAV ITRs into specific regions of the cellular genome, if desired (see, e.g., U.S. Pat. Nos. 6,342,390 and 6,821,511). Host cells containing an integrated AAV genome do not exhibit changes in cell growth or morphology (see, e.g., U.S. Pat. No. 4,797,368).

[0093] AAV vectors typically contain an AAV protein capsid in which a nucleic acid sequence is packaged for delivery to a target cell. The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, which are arranged in an icosahedral symmetry in a ratio of about 1:1:10 to 1:1:20, depending on the AAV selected. AAV serotypes can be selected as the source of capsids for AAV viral vectors (DNase-resistant viral particles), including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhl0, AAVrh64Rl, AAVrh64R2, AAVrh8, AAVrh10, variants of any of the known or mentioned AAVs, or AAVs yet to be discovered. Illustrative examples are described in US Patent Application Publication No. 2007 / 0036760(A1), US Patent Application Publication No. 2009 / 0197338(A1), EP 1310571, WO 2003 / 042397, U.S. Patent No. 7,790,449, U.S. Patent No. 7,282,199, WO 2005 / 033321, U.S. Patent No. 7,906,111, WO 2006 / 110689, and WO 2003 / 042397, the entire contents of which are incorporated herein by reference. Alternatively, recombinant AAV can be used as a source of AAV capsid. In some embodiments, AAV capsid can be generated by mutagenesis (e.g., by one or more insertions, deletions, or substitutions) of one of the aforementioned AAV capsids or its encoding nucleic acid. In some embodiments, the AAV capsid is chimeric and contains domains from two or more AAV capsid proteins. In some embodiments, the AAV capsid is a mosaic of Vpl, Vp2, and Vp3 monomers from two or more different AAV or recombinant AAV. In some embodiments, the AAV capsid shares about 90% sequence identity to about 99.9% identity, preferably about 95% to about 99% sequence identity, or more preferably about 97% to about 98% sequence identity to an AAV capsid provided herein and / or known in the art.In embodiments, the AAV capsid shares at least 95% sequence identity with the AAV capsid.

[0094] In embodiments, the AAV vector is selected from the group consisting of AAV1, AAV5, AAV6, AAV8, AAVrh64Rl, AAV9, AAVrh91, AAVhu.37, AAV3b, AAV3b.AR2.12, and AAVrh10 vectors. In embodiments, the AAV is AAV1. In embodiments, the AAV vector is an AAVrh91 vector.

[0095] Methods for generating and isolating AAV vectors suitable for delivery to a subject are known to those of skill in the art (see, e.g., U.S. Pat. No. 7,790,449, U.S. Pat. No. 7,282,199, WO 2003 / 042397, WO 2005 / 033321, WO 2006 / 110689, U.S. Pat. No. 7,588,772, and WO 2017 / 040524, the entire contents of which are incorporated herein by reference). In an embodiment, a producer cell line is transiently transfected with a construct encoding a transgene flanked by inverted terminal repeats (ITRs) and a construct encoding rep and cap. In another embodiment, a packaging cell line stably supplying rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus, necessitating separation of rAAV from contaminating viruses. More recently, systems have been developed that do not require infection with a helper virus to recover AAV, and the necessary helper functions (e.g., adenovirus El, E2a, VA, and E4, or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied in trans by the system. In these newer systems, the helper functions can be provided by transient transfection of cells with constructs encoding the necessary helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In yet another system, a transgene flanked by ITRs and rep / cap genes is introduced into insect cells by infection with a baculovirus-based vector (see, e.g., Zhang et al., 2009, Human Gene Therapy 20:922-929, the entire contents of which are incorporated herein by reference).Illustrative examples of suitable methods for making and using these and other AAV production systems can be found in U.S. Pat. Nos. 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065, Grieger & Samulski (2005, Adv. Biochem. Engin / Biotechnol. 99:119-145), and Buning et al. (2008, J. Genet. Med. 10:717-733), the entire contents of which are incorporated herein by reference. Other illustrative examples of genetic engineering, recombinant engineering, and synthetic techniques are also described in Green and Sambrook et al, (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference. Suitable methods for producing rAAV virions are also well known to those of skill in the art (see, e.g., Fisher et al. (1993) J. Virol., 70:520-532 and U.S. Patent No. 5,478,745, the entire contents of which are incorporated herein by reference). The viral vectors described herein can be delivered in a single composition or in multiple compositions. Optionally, two or more different AAVs, or multiple viruses, can be delivered (see, e.g., WO 2011 / 126808 and WO 2013 / 049493, the entire contents of which are incorporated herein by reference).

[0096] The AAV ITRs are flanked by unique coding nucleotide sequences for the nonstructural replication (Rep) proteins and the structural capsid (Cap) proteins (also known as virion proteins (VPs)). The terminal 145 nucleotides are self-complementary and organize such that energetically stable intramolecular duplexes forming T-shaped hairpins can form. These hairpin structures serve as origins of viral DNA replication by serving as primers for cellular DNA polymerase complexes. The Rep genes encode the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. The Rep78 and Rep68 proteins are multifunctional DNA-binding proteins that perform helicase and nickase functions during productive replication to allow for degradation of AAV termini (see, e.g., Im et al., Cell, 61:447-57 (1990)). These proteins also regulate transcription from the endogenous AAV promoter and promoters in the helper virus (see, e.g., Pereira et al., J. Virol., 71:1079-1088 (1997)). Other Rep proteins modify the function of Rep78 and Rep68. The cap gene encodes the capsid proteins VP1, VP2, and VP3. The cap gene is transcribed from the p40 promoter.

[0097] Also disclosed herein are expression constructs that include a nucleic acid sequence encoding an NGF binding molecule described herein operably linked to one or more regulatory sequences.

[0098] The term "construct" refers to a recombinant genetic molecule that contains one or more isolated nucleic acid sequences from different sources. Thus, a construct is a chimeric molecule in which two or more nucleic acid sequences of different origins are assembled into a single nucleic acid molecule, and includes any construct that contains (1) a nucleic acid sequence that contains regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous to at least one of the other nucleotide sequences), or (2) a sequence that encodes a portion of a functional RNA molecule or protein that is not naturally adjacent, or (3) a portion of a promoter that is not naturally adjacent. Representative constructs include any recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source and capable of genomic integration or autonomous replication, and that contains a nucleic acid molecule to which one or more nucleic acid molecules are operably linked. Constructs of the invention generally contain elements necessary to direct the expression of a nucleic acid sequence of interest that is also included in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence. Such elements may include control elements or regulatory sequences, such as a promoter operably linked to the nucleic acid sequence of interest (to direct its transcription), and often also include a polyadenylation sequence. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, e.g., prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the genome of the host cell. Two or more constructs may be contained within a single nucleic acid molecule, e.g., a single vector, or may be contained within two or more separate nucleic acid molecules, e.g., two or more separate vectors. An "expression construct" generally includes at least a control sequence operably linked to the nucleotide sequence of interest. In this manner, for example, a promoter operably connected to the nucleotide sequence to be expressed is provided in the expression construct for expression in an organism or part thereof, including a host cell.Conventional compositions and methods for preparing and using the constructs and host cells for the practice of the present invention are well known to those of skill in the art; see, e.g., Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3. JF Sambrook, DW Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0099] As used herein, "control element", "control sequence", "regulatory sequence" and the like refer to nucleic acid sequences (e.g., DNA) necessary for the expression of an operably linked coding sequence in a particular host cell. Control sequences suitable for prokaryotic cells include, for example, promoters and, optionally, cis-acting sequences such as operator sequences and ribosome binding sites. Control sequences suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that regulate mRNA stability, and targeting sequences that target the product encoded by the transcribed polynucleotide to an intracellular compartment within the cell or to the extracellular environment. Suitable control elements are well known to those of skill in the art, and illustrative examples include liver-specific promoters and non-tissue specific promoters (e.g., CB7). Illustrative examples of suitable liver-specific promoters include alpha 1 antitrypsin (e.g., The Liver Specific Gene Promoter Database, Cold Spring Harbor, http: / / rulai.schl.edu / LSPD), human albumin (humAlb) (see, e.g., Miyatake et al., J. Virol., 71:512432 (1997)), and the Hepatitis B virus core promoter (see, e.g., Sandig et al., Gene Ther., 3:10029 (1996)). In embodiments, the liver-specific promoter thyroxin binding globulin (TBG) is used. Other suitable promoters, such as viral promoters, constitutive promoters, regulatable promoters (see, e.g., WO 2011 / 126808 and WO 2013 / 04943), or promoters responsive to physiological cues, can also be utilized in the vectors described herein. In embodiments, expression of a polynucleotide encoding an antigen binding protein or NGF binding fragment thereof described herein is under the control of a liver-specific promoter (TBG).In another embodiment, expression of a polynucleotide encoding an antigen binding protein or NGF binding fragment thereof described herein is under the control of a non-tissue specific promoter (CB7).

[0100] Also disclosed herein are host cells containing the constructs defined herein.

[0101] The terms "host", "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", including the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell. A host cell is any type of cell line that can be used to produce the antigen-binding molecules of the present invention. Host cells include cultured cells, such as cultured mammalian cells, e.g., CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, but also include transgenic animals, transgenic plants, or cells contained within cultured plants or animal tissues, to name just a few. In one embodiment, the host cell is a CHO or HEK293 cell line.

[0102] Methods are provided for producing the modified NGF binding molecules described herein, such methods comprising culturing a host cell disclosed herein and recovering the NGF binding molecule from the host cell or culture medium.

[0103] Also disclosed herein are pharmaceutical compositions comprising an NGF binding molecule or vector described herein and a pharma- ceutically acceptable carrier.

[0104] By "pharmaceutically acceptable carrier" is meant a pharmaceutical vehicle composed of materials that are not biologically or otherwise undesirable, i.e., the materials may be administered to a subject together with a selected active agent without causing any or substantial adverse reactions. Carriers may include excipients and other additives, such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

[0105] Representative pharma-ceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and similar materials, and combinations thereof, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference).Except in the case that any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical composition is contemplated.

[0106] The pharmaceutical compositions may be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Suitable pharmaceutical compositions may be administered intravenously, subcutaneously, or intramuscularly. In some embodiments, the compositions are in the form of injectable or infusible solutions. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In certain embodiments, the pharmaceutical compositions are administered by intravenous infusion or injection. In other embodiments, the pharmaceutical compositions are administered by intramuscular or subcutaneous injection.

[0107] The phrases "parenteral administration" and "administered parenterally", as used herein, mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0108] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. In the present invention, pharma-ceutically acceptable carriers include, but are not limited to, 0.01-0.1M, preferably 0.05M, phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0109] More specifically, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage, and is preferably preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, and / or by maintaining the required particle size. In certain embodiments, the agents of the present disclosure can be conjugated to a vehicle for cell delivery. In these embodiments, the agents can be encapsulated in a suitable vehicle to either aid in the delivery of the agent to the target cell, to increase the stability of the agent, or to minimize the potential toxicity of the agent. As will be appreciated by those skilled in the art, a variety of vehicles are suitable for delivering the agents of the present disclosure. Non-limiting examples of suitable structured fluid delivery systems include nanoparticles, liposomes, microemulsions, micelles, dendrimers, and other phospholipid-containing systems. Methods for incorporating the agents of the present disclosure into delivery vehicles are known in the art. Although various embodiments are presented below, it will be understood that other methods known in the art for incorporating the antigen-binding molecules described herein into delivery vehicles are contemplated.

[0110] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. The antigen-binding molecules of the present disclosure may be administered multiple times. The intervals between single doses may be daily, weekly, monthly, or yearly. The intervals may be irregular as indicated by measuring the blood levels of the modified polypeptide or antigen in the patient. Alternatively, the antigen-binding molecules may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0111] For ease of administration and uniformity of dosage, it may be advantageous to formulate the composition in unit dosage form.As used herein, unit dosage form refers to a physically separate unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the necessary pharmacologic carrier.The specification for the unit dosage form of the present invention is determined by and directly depends on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the art of compounding such active compound for the treatment of susceptibility in an individual.

[0112] The dosage and treatment regimen of the antigen-binding molecule can be determined by one of skill in the art. In a specific embodiment, the antigen-binding molecule is administered by injection (e.g., subcutaneously, intramuscularly, or intravenously) at a dose of about 0.01 to 40 mg / kg, for example, about 0.01 to about 0.1 mg / kg, for example, about 0.1 to about 1 mg / kg, about 1 to about 5 mg / kg, about 5 to about 25 mg / kg, about 10 to about 40 mg / kg, or about 0.4 mg / kg. In an embodiment, the antigen-binding molecule is administered to a subject (e.g., subcutaneously, intramuscularly, or intravenously) at a dose of about 0.01 mg / kg to about 40 mg / kg, preferably about 0.1 mg / kg to about 20 mg / kg, preferably about 0.2 mg / kg to about 20 mg / kg, preferably about 0.2 mg / kg to about 10 mg / kg, preferably about 0.2 mg / kg to about 5 mg / kg, or more preferably about 0.2 mg / kg to about 2 mg / kg body weight. In an embodiment, the antigen-binding molecule is administered to a subject (e.g., subcutaneously, intramuscularly, or intravenously) at a dose of about 0.2 mg / kg to about 0.5 mg / kg body weight (e.g., about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.35 mg / kg, about 0.4 mg / kg, about 0.45 mg / kg, or about 0.5 mg / kg body weight). In embodiments, the antigen-binding molecule is administered to a subject (e.g., subcutaneously, intramuscularly, or intravenously) at a dose of about 1 mg / kg to about 40 mg / kg, more preferably about 2 mg / kg to about 20 mg / kg body weight. In an embodiment, the antigen-binding molecule is administered to a subject subcutaneously (e.g., subcutaneously, intramuscularly, or intravenously) at a dose of about 1 mg / kg to about 40 mg / kg, more preferably about 2 mg / kg to about 20 mg / kg body weight (e.g., about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg body weight). In an embodiment, the antigen-binding molecule is administered to a subject subcutaneously (e.g., subcutaneously, intramuscularly, or intravenously) at a dose of about 1 mg / kg body weight. In embodiments, the antigen binding molecule is administered subcutaneously to a subject at a dose of about 1 mg / kg body weight.In an embodiment, the antigen-binding molecule is administered to a subject (e.g., subcutaneously, intramuscularly, or intravenously) at a dose of about 2 mg / kg body weight. In an embodiment, the antigen-binding molecule is administered to a subject subcutaneously at a dose of about 2 mg / kg body weight. Without being bound by theory or a particular application mode, the dose administered to a subject may be lower (e.g., about 0.2 to 0.5 mg / kg body weight) when administered over a shorter dosing interval (e.g., about once every 1, 2, 3, or 4 weeks), or may be higher (e.g., about 1 to 20 mg / kg body weight) when administered over a longer dosing interval (e.g., about once every 5, 6, 7, or 8 weeks, 2, 3 months, or more).

[0113] Dosing schedules can vary, for example, from about once per week to once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks. In embodiments, an antigen binding molecule or NGF binding fragment thereof described herein is administered to a subject at a dosing frequency of about once per week or less, preferably once every 2 weeks, preferably once every 3 weeks, preferably once every 4 weeks, preferably once every 5 weeks, preferably once every 6 weeks, preferably once every 7 weeks, preferably once every 8 weeks, preferably once every 9 weeks, preferably once every 10 weeks, preferably once every 11 weeks, or more preferably once every 12 weeks. In embodiments, an antigen binding molecule or NGF binding fragment thereof described herein is administered to a subject at a frequency of about once every two months or more (e.g., about once every two months, about once every 2 1 / 2 months, about once every three months, about once every 3 1 / 2 months, about once every four months, about once every 4 1 / 2 months, about once every five months, about once every 5 1 / 2 months, about once every six months, etc.).

[0114] In an embodiment, the antigen-binding molecule is administered every other week at a dose of about 10 to about 20 mg / kg.In an embodiment, the antigen-binding molecule is administered every other week at a dose of about 10 to about 20 mg / kg.

[0115] As described elsewhere herein, the inventors have also unexpectedly demonstrated that NGF-binding antibodies such as those described herein are clinically effective (e.g., in pain relief) for at least 60 days (about 2 months) after administration. This therapeutic benefit was independent of any modifications to the Fc region of the anti-NGF antibody that would otherwise extend its serum half-life. Thus, in embodiments, the antigen-binding molecule, vector, or pharmaceutical composition described herein is administered to a subject once every about 2 months or more.

[0116] In an embodiment, the antigen-binding molecule is administered at a dose of about 0.01 to about 40 mg / kg every other week, preferably about 0.01 to about 20 mg / kg every other week, preferably about 0.10 to about 10 mg / kg every other week, preferably about 0.5 to about 10 mg / kg every other week, or more preferably about 0.5 to about 50 mg / kg every other week. In one embodiment, the antigen-binding molecule is administered at a dose of about 1 mg / kg body weight every other week. An exemplary, non-limiting range of an effective amount of an antigen-binding molecule of the present disclosure is 0.01 to 5 mg / kg, more preferably 0.03 to 2 mg / kg.

[0117] In view of the inventors' unexpected finding that anti-NGF antibodies are therapeutically effective for at least 60 days (about 2 months) after administration to a subject, as described elsewhere herein, the present disclosure also extends to a method of treating or preventing a condition associated with increased expression and / or activity of NGF, comprising administering to a subject in need thereof an NGF binding molecule or an NGF binding fragment thereof at a dose of about 0.5 to about 10 mg / kg body weight, preferably at a dose of about 1 mg / kg body weight, and at an administration frequency of about once every two months or more. In an embodiment, the dose administered to the subject is about 0.5 to about 5 mg / kg body weight, preferably about 0.5 to about 2 mg / kg body weight, or more preferably about 1 mg / kg body weight. In an embodiment, the subject is a cat or a dog. In an embodiment, the NGF binding molecule or an NGF binding fragment thereof is administered subcutaneously to the subject. In an embodiment, the condition associated with increased expression and / or activity of NGF is pain, as described elsewhere herein. In an embodiment, the condition associated with increased expression and / or activity of NGF is pain associated with arthritis. In one embodiment, the condition associated with increased expression and / or activity of NGF is pain associated with osteoarthritis.

[0118] In another aspect, there is provided a method for treating or preventing a condition associated with increased NGF expression and / or activity, comprising administering to a subject in need of said treatment or prevention, wherein the condition associated with increased NGF expression and / or activity is pain, preferably pain associated with osteoarthritis, and the NGF-binding molecule or NGF-binding fragment thereof is administered subcutaneously at a dose of about 0.5 to about 2 mg / kg body weight, preferably about 1 mg / kg body weight, with an administration frequency of about 2 months or more.

[0119] The present disclosure also extends to a therapeutic anti-NGF antibody capable of delivering pain relief in a subject in need thereof for at least 60 days after administration, characterized in that the therapeutic anti-NGF antibody does not require modifications to the amino acid sequence of the Fc region to achieve pain relief in the subject.

[0120] Dosages and treatment regimens for polynucleotides or vectors comprising nucleic acid sequences encoding the NGF-binding molecules or NGF-binding fragments thereof described herein can also be determined by those skilled in the art. In certain embodiments, the polynucleotides or vectors are administered by injection (e.g., subcutaneously, intramuscularly, or intravenously) at doses of, for example, about 1×10 6 Gene copies (gc) / kg body weight ~ approx. 1 x 10 16 gc / kg body weight (e.g., 1×10 6 gc / kg, 1×10 7 gc / kg, 1×10 8 gc / kg, 1×10 9 gc / kg, 1×10 10 gc / kg, 1×10 11 gc / kg1×10 12 gc / kg, 1×10 13 gc / kg, 1×10 14 gc / kg, 1×10 15 gc / kg, or 1 x 10 16 In embodiments, the polynucleotide or vector described herein is administered at a dose of about 1×10 10 gc / kg ~ approx. 1×10 14 gc / kg body weight, preferably about 1×10 11 gc / kg ~ approx. 1×10 13 gc / kg body weight, more preferably about 1×10 12 gc / kg ~ approx. 5×10 12 In embodiments, the polynucleotide or vector described herein is administered to a subject at a dose of about 1×10 12The subject is administered a dose of gc / kg body weight. The administration schedule of the polynucleotide or vector described herein can vary, for example, from once a week to once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks. In an embodiment, the antigen-binding molecule or NGF-binding fragment thereof described herein is administered to the subject about once a week, preferably once every 2 weeks, preferably once every 3 weeks, preferably once every 4 weeks, preferably once every 5 weeks, preferably once every 6 weeks, preferably once every 7 weeks, preferably once every 8 weeks, preferably once every 9 weeks, preferably once every 10 weeks, preferably once every 11 weeks, or more preferably once every 12 weeks.

[0121] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0122] The pharmaceutical composition of the present invention may comprise an effective amount of the agent (i.e., NGF binding molecule) disclosed herein. The effective amount may be a "therapeutically effective amount" or a "prophylactically effective amount". A "therapeutically effective amount" refers to an amount effective at the dosage and for the period of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of an agent may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to induce a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. Alternatively, this property of the composition can be evaluated by examining the ability of the compound to inhibit in vitro, for example, by assays known to the practitioner.

[0123] In contrast, a "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0124] Also disclosed herein is a method of treating, inhibiting, or ameliorating pain in a subject, comprising administering to a subject in need thereof an NGF binding molecule or vector described herein.

[0125] The term "treating," as used herein, may refer to: (1) slowing the progression of one or more symptoms of a condition; (2) inhibiting the development of a condition or one or more symptoms of a condition; (3) alleviating the condition, i.e., causing a regression of the condition or at least one or more symptoms of the condition; and / or (4) causing a decrease in the severity of the condition or one or more symptoms of the condition.

[0126] The terms "treating," "treatment," and the like are used interchangeably herein to mean alleviating, reducing, alleviating, ameliorating, or otherwise inhibiting a condition, including one or more symptoms of the condition. The terms "prevent," "preventing," "prophylaxis," "prophylactic," "preventative," and the like are used interchangeably herein to mean preventing or delaying the onset of a condition or the risk of developing a condition.

[0127] The terms "treating," "treatment," and the like also include alleviating, reducing, alleviating, ameliorating, or otherwise suppressing the effects of a condition for at least a period of time. It should also be understood that the terms "treating," "treatment," and the like do not mean that a condition or its symptoms are permanently alleviated, reduced, alleviated, ameliorated, or otherwise suppressed, and thus also encompass temporary alleviation, reduction, alleviation, amelioration, or otherwise suppression of a condition or its symptoms.

[0128] The terms "subject", "patient", "host" or "individual", as used interchangeably herein, refer to any subject for which therapy or prevention is desired, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrates falling within the scope of the present invention include primates (e.g., humans, monkeys, and apes, including monkey species from the genus Macaca (e.g., cynomolgus monkeys, such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatta)) and baboons (Papio ursinus), as well as ape species such as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri), and tamarins (species from the genus Saguinus), and chimpanzees (Pan troglodytes)), rodents (e.g., mice, The subject may be any member of the subphylum Chordata, including, but not limited to, any animal, including rats, guinea pigs, lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), birds (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), as well as fish. In one embodiment, the subject is a human subject. In another embodiment, the subject is a canine subject. In another embodiment, the subject is a feline subject. In another embodiment, the subject is an equine subject.

[0129] Conditions associated with abnormal (e.g., increased) levels and / or abnormal (e.g., increased) activity of NGF are well known to those skilled in the art. In embodiments disclosed herein, the condition is pain. In embodiments, the pain is selected from the group consisting of neuropathic pain, inflammatory pain, pruritic pain, perioperative pain, postoperative pain, and postoperative pain.

[0130] As defined herein, the term "pain" means an unpleasant sensory and emotional experience typically associated with or described in terms of actual or potential tissue damage.

[0131] Regarding surgical or postoperative pain, the US Animal Welfare Act (Animal Welfare Act 2002. AWA regulations, CFR, Title 9 (Animals and Animal Products), Chapter 1 (Animal and Plant Health Inspection Service, Department of Agriculture). Subchapter A (Animal Welfare), Parts 1-4) defines a painful procedure as any procedure that is reasonably expected to cause more than slight or momentary pain or distress in the subject to whom the procedure is administered, i.e., pain that exceeds the pain caused by an injection or other minor procedure. Therefore, if an animal (e.g., a canine, feline, or porcine subject) undergoes a painful surgical procedure, the animal should receive postoperative analgesia.

[0132] The subject may be experiencing significant or chronic pain as a result of rheumatoid arthritis, osteoarthritis, inflammation, or an associated medical condition, such as a cancerous or malignant condition.

[0133] Also provided herein is an antigen-binding molecule or vector described herein for use in treating, suppressing, or ameliorating pain in a subject.

[0134] Also provided herein is the use of an NGF binding molecule or vector described herein in the manufacture of a medicament for treating, inhibiting, or ameliorating a condition associated with abnormal (e.g., increased) levels of NGF and / or abnormal (e.g., increased) NGF activity in a subject in need thereof. In an embodiment, the condition is pain. In another embodiment, the condition is pain associated with arthritis. In another embodiment, the condition is arthritis. Thus, also disclosed herein is a method of treating or preventing arthritis or an arthritic condition in a subject, comprising administering to a subject in need thereof an NGF binding molecule, or vector, or pharmaceutical composition described herein.

[0135] In embodiments, the arthritis or arthritic condition is selected from the group consisting of immune-mediated polyarthritis, rheumatoid arthritis, and osteoarthritis.

[0136] Also provided herein is an NGF binding molecule or vector described herein for use in treating or preventing arthritis or an arthritic condition in a subject.

[0137] Also provided herein is the use of an NGF binding molecule or vector described herein in the manufacture of a medicament for the treatment or prevention of arthritis or an arthritic condition in a subject.

[0138] Also disclosed herein is a method of treating or preventing a condition caused by, associated with, or resulting from increased expression of or sensitivity to NGF in a subject in need thereof, comprising administering to a subject in need thereof an NGF binding molecule or vector described herein.

[0139] Also disclosed herein is an NGF binding molecule, or vector, or pharmaceutical composition as described herein for use in treating a condition caused by, associated with, or resulting from increased expression of or increased sensitivity to NGF in a subject.

[0140] The disclosure also extends to the use of an NGF binding molecule or vector described herein in the manufacture of a medicament for the treatment of a condition caused by, associated with, or resulting from increased expression of or increased sensitivity to NGF in a subject.

[0141] The present disclosure also extends to a method for the treatment or prevention of tumors whose growth is induced by NGF and conditions associated therewith, comprising administering to a subject in need thereof an NGF binding molecule, or vector, or pharmaceutical composition as described herein.

[0142] In one embodiment, the tumor is osteosarcoma.

[0143] Also provided herein is an NGF binding molecule, or vector, or pharmaceutical composition as described herein for use in treating or preventing NGF-induced growth of tumors and conditions associated therewith in a subject in need thereof.

[0144] The present disclosure also extends to the use of an NGF binding molecule or vector as described herein in the manufacture of a medicament for the treatment or prevention of NGF-induced growth tumours and conditions associated therewith in a subject in need thereof.

[0145] The present disclosure also extends to a kit comprising an NGF binding molecule, or a vector, or a pharmaceutical composition described herein.

[0146] Also disclosed herein is the use of an NGF binding molecule or vector described herein for detecting NGF in a sample.

[0147] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives (or).

[0148] As used in this application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0149] "About" means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0150] Throughout this specification and the following description, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a stated integer or step, or group of integers or steps, but not to the exclusion of any other integers or steps, or group of integers or steps.

[0151] Reference in this specification to any prior publication (or information derived therefrom), or to any known matter, is not, and should not be construed as, an acknowledgment or admission, or any form of suggestion, that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification pertains.

[0152] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the invention includes all such variations and modifications that fall within its spirit and scope. The invention also includes all of the steps, features, compositions, and compounds referred to or shown herein, individually or collectively, and any and all combinations of any two or more of such steps or features.

[0153] Certain embodiments of the invention will now be described with reference to the following examples, which are for illustrative purposes only and are not intended to limit the scope of the generality described above. EXAMPLES

[0154] Example 1: Fetinized anti-NGF antibody Two felineized anti-NGF monoclonal antibodies, -fe_αD11_HCCDR1(F / L) and fe_αD11_HCCDR2(L / V), were engineered. As shown in Tables 5 and 6, fe_αD11_HCCDR1(F / L) contains an F to L substitution at the position corresponding to position 2 of the heavy chain CDR1 sequence of rat αD11 (previously described in WO 2006 / 131951), and fe_αD11+HCCDR2(L / V) contains an L to V substitution at the position corresponding to position 14 of the heavy chain CDR2 sequence of rat αD11. The amino acid sequences of the heavy and light chain variable regions of fe_αD11_HCCDR1(F / L) are shown in Table 8. The amino acid sequences of the heavy and light chain framework regions of fe_αD11_HCCDR1(F / L) are shown in Table 12. The amino acid sequences of the heavy and light chain variable regions of fe_αD11_HCCDR2(L / V) are shown in Table 9. The amino acid sequences of the heavy and light chain framework regions of fe_αD11_HCCDR2(L / V) are shown in Table 13. The nucleic acid sequences encoding the heavy and light chain variable regions of fe_αD11_HCCDR2(L / V) are shown in Table 14.

[0155] For comparison, a felinized anti-NGF monoclonal antibody (fe_αD11) was engineered using CDR sequences from rat anti-NGF monoclonal antibody αD11. Felinized anti-NGF monoclonal antibody fe_αD11 has heavy chain CDR sequences of SEQ ID NOs: 1, 8, and 3, and light chain CDR sequences of SEQ ID NOs: 4 to 6. The amino acid sequences of the heavy and light chain variable regions of fe_αD11 are shown in Table 7. The amino acid sequences of the heavy and light chain framework regions of fe_αD11 are shown in Table 11.

[0156] The engineered feline anti-NGF monoclonal antibody was expressed in Chinese Hamster Ovary (CHO) cells.

[0157] Example 2: In vivo pharmacokinetics A pharmacokinetic (PK) study was performed in healthy cats. Four animals were administered subcutaneously (sc) with felineized anti-NGF antibodies (fe_αD11, fe_αD11+HCCDR1(F / L), and fe_αD11+HCCDR2(L / V)) at 2.0 mg / kg each on days 0, 21, 42, and 63. Serum concentrations of the antibodies were assessed over a 91-day period. The concentration of each antibody in serum was determined using an NGF-binding ELISA. Briefly, ELISA plates were coated with 0.1 μg / mL murine NGF (muNGF) and blocked with PBS / 0.05% Tween 20 / 1% BSA. The muNGF-coated wells were then incubated with antibody preparations diluted in PBS / 0.05% Tween 20 / 1% BSA (100 μL / well) for 1 h at room temperature. Binding curves were established using antibody concentrations ranging from 100ng / mL to 1.56ng / mL. After washing, plates were incubated with a 1 / 5000 dilution of goat anti-cat IgG-HRP in PBS / 0.05% Tween 20 / 1% BSA. Plates were washed with PBS / 0.05% Tween 20 and developed by the addition of TMB substrate. 2M H 2 SO 4 Color development was stopped by the addition of 0.01% EDTA, absorbance was read at 450 nm, and background values ​​were subtracted from absorbance readings. Non-compartmental pharmacokinetic parameters of the three felinized anti-NGF antibodies after doses 1 and 4 were calculated using PKsolver. software. Data for each animal is shown in Figure 1 and further summarized in Table 1 below.

[0158] Table 1: Pharmacokinetic parameters of felineized anti-NGF antibodies in serum after subcutaneous administration in cats.

[0159] [Table 3]

[0160] [Table 4]

[0161] Note that one animal from each of the fe_αD11 and fe_αD11_HCCDR1(F / L) cohorts was excluded from the post-dose 4 analysis due to rapid loss of detectable circulating antibodies associated with anti-drug antibody (ADA) formation in those animals (see also Example 3 below).

[0162] The three fetinized anti-NGF antibodies exhibited a pharmacokinetic (PK) profile typical of a subcutaneously administered antibody. After absorption from the injection site, peak plasma levels (Cmax) were achieved in approximately 3-4 days (Tmax). The mean elimination half-life (T1 / 2) after the first dose was calculated to be approximately 9 days (range approximately 8-11 days). The PK profile of the second dose was similar to that of the first, with a T1 / 2 estimated to be approximately 7 days (range approximately 4-10 days).

[0163] Example 3: Immunogenicity profile of felineized anti-NGF antibodies in serum after subcutaneous administration in cats. Three feline anti-NGF antibodies were administered subcutaneously to four cats at 2 mg / kg each on days 0, 21, 42, and 63. The presence of anti-drug antibodies (ADA) at various time points was determined using a bridging immunoassay. Data for each animal is shown in Figure 2. A summary of the data is also shown below in Table 2.

[0164] [Table 5]

[0165] These data show that the majority of animals administered fe_αD11 developed a strong ADA response, which was associated with a significant reduction in the detectable expression of anti-NGF antibodies in the serum in at least one animal and a reduction in T1 / 2 in the other animals. In animals administered fe_αD11_HCCDR1(F / L), one animal developed a significant ADA response associated with a loss of detection of anti-NGF antibodies in the serum. Advantageously, no significant ADA response was observed in cats administered fe_αD11_HCCDR2(L / V). These data demonstrate that the V to L substitution at the position corresponding to position 14 of the rat αD11 heavy chain CDR2 sequence (SEQ ID NO:2) unexpectedly and advantageously removed an epitope in the rat αD11 heavy chain CDR2 sequence that is otherwise immunogenic in species other than rat.

[0166] Example 4: Efficacy of fe_αD11_HCCDR2(L / V) in cats A multicentre, placebo-controlled, randomised, blinded study was conducted to evaluate the efficacy and field safety of fe_αD11_HCCDR2(L / V) administered as monthly subcutaneous (SQ) injections compared with placebo for the control of pain associated with osteoarthritis (OA) in cats.

[0167] Thirty-two cats under an established Veterinary Client Patient Relationship (VCPR) diagnosed with OA by physical examination and radiography were enrolled in this study.

[0168] Day 0 was defined as the first day of dosing. Cats were randomized in a 1:1 ratio and stratified by site of enrollment into the following groups: Group 1 (placebo) - received one SQ injection of placebo on day 0 and one SQ injection of placebo on day 30. Group 2 (mAb) - received one SQ injection of anti-NGF monoclonal antibody (mAb), fe_αD11_HCCDR2(L / V), at 2.0 mg / kg body weight on day 0 and a second dose on day 30.

[0169] The primary outcome measure was successful improvement in an owner-reported client-specific outcome measure (CSOM) for pain. Cats were assessed at days 0, 30, and 90. Statistical significance was determined using an alpha of 0.1.

[0170] [Table 6] [a]CSOM success was defined as a reduction in total CSOM score of at least 2 compared to day 0, with no increase in any individual activity. [b] P values, LS means, and LS mean differences generated by generalized linear mixed models assuming a binomial distribution and logit link. Group, day, and group-day were included as fixed effects, and site and group-site interaction were included as random effects.

[0171] As shown in Table 3 above, at day 30, a statistically significant difference was observed between the mAb-treated (n=17) and placebo (n=15) groups (mAb: 82.5% vs. placebo: 40.1%, p=0.0357, success defined as a reduction in total CSOM score of at least 2 compared to day 0).

[0172] At day 90, not all animals completed the study through their visit, reducing the total number of cats that were evaluable to 23. In any case, when success was defined by a reduction in total CSOM score of at least 2 compared to day 0, there was a notable difference between the mAb-treated (n=12) and placebo (n=11) groups at day 90 (mAb: 75.8% vs. placebo: 38.7%, p=0.1161). This trend was also observed when a higher success bar was applied, as defined by a reduction in total CSOM score of at least 3 compared to day 0 (mAb: 60.1% vs. placebo: 9.9%, p=0.0608).

[0173] No treatment-related adverse effects were identified in this study.

[0174] These data clearly demonstrate that fe_αD11_HCCDR2(L / V) is safe and clinically effective in reducing pain associated with OA in cats. Unexpectedly, this reduction in pain was evident up to 60 days (i.e., day 90) after administration, which advantageously allows the possibility of administering the agents described herein as infrequently as every two months.

[0175] Example 5: Pharmacokinetics of caninized anti-NGF antibody in dogs Pharmacokinetic (PK) studies were performed in healthy dogs. Four animals were administered purified ca_αD11_HCCDR2(L / V) subcutaneously (SC) at 1.0 mg / kg body weight on days 0 and 28 of the study. The ca_αD11_HCCDR2(L / V) antibody has the same CDR sequences as those described in Tables 3 and 4 below. Serum concentrations of ca_αD11_HCCDR2(L / V) were assessed over a 56-day period using an NGF-binding ELISA, as detailed below. Pharmacokinetic parameters were determined using PKSolver software (Zhang Y et al. Computer Methods and Programs in Biomedicine. 2010;99(3):306-314). The emergence of anti-drug antibodies was assessed on days 28 and 56 using a bridging electrochemiluminescence (ECL) assay, as detailed below.

[0176] A. Quantification of canine anti-NGF antibodies in canine serum The concentration of ca_αDD11_HCCDR2(L / V) in dog serum was determined using an NGF-binding ELISA. Briefly, ELISA plates were coated with 0.1 μg / mL murine NGF (muNGF) and blocked with PBS / 0.05% Tween 20 / 1% BSA. muNGF-coated wells were incubated for 1 h at room temperature with serum diluted in PBS / 0.05% Tween 20 / 1% BSA (100 μL / well). A standard curve was established using antibody concentrations ranging from 100 ng / mL to 1.56 ng / mL. After washing, plates were incubated with a 1 / 10,000 dilution of goat anti-dog IgG-HRP in PBS / 0.05% Tween 20 / 1% BSA. Plates were then washed with PBS / 0.05% Tween 20 and developed by the addition of 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Development was stopped by the addition of 2M H2SO4 and absorbance was read at 450 nm and background subtracted.

[0177] B. Evaluation of anti-drug antibodies (ADA) in dog serum A bridging electrochemiluminescence (ECL) assay with acid dissociation was used to assess the presence of ca_αD11_HCCDR2(L / V) in dog serum at the time points when circulating levels of drug were lowest (day 28 pre-dose and day 56 pre-dose). Prior to assessment in the bridging assay, samples were pretreated with acid to disrupt any pre-existing ADA-caNGF mAb complexes. Briefly, samples were diluted to 2.5% with 300 mM acetic acid to dissociate ADA-drug complexes prior to analysis. Acidified samples were incubated for 40 minutes with shaking at ambient temperature. 25 μL of acidified samples were transferred to wells of a 96-well plate containing 90 μL of master mix reagent (0.125 μg / mL biotinylated caNGF mAb and 0.125 μg / mL ruthenylated caNGF mAb with 1% Meso Scale Discovery (MSD) Blocker A in PBS-T) and 11 μL of 1.2 M Tris solution (pH 9.5). The acidified samples + master mix reagent were incubated at ambient temperature in the dark with shaking for 60 minutes. Concurrently, the streptavidin-coated MSD plate was blocked with 200 μL / well of PBS-T buffer with 3% (MSD) Blocker A for 60 minutes at ambient temperature.

[0178] The streptavidin-coated MSD plate was then washed and 25 μL of acidified sample + master mix reagent was transferred to the plate, which was then incubated at ambient temperature in the dark for 60-90 minutes with shaking. After washing the MSD plate and adding 150 μL of 2× MSD Read T-Buffer per well, the plate was read on an MSD MESO QuickPlex SQ 120 instrument. The resulting response was recorded in relative light units (RLU).

[0179] C. Results As shown in Figure 3, the canine anti-NGF antibody ca_αD11_HCCDR2(L / V) showed a typical pharmacokinetic (PK) profile for an antibody administered SC. After absorption from the injection, peak plasma levels (Cmax) were achieved at approximately 4 days (Tmax). The mean elimination half-life (T1 / 2) after the first dose was calculated to be 6.6 days (range approximately 5.2-7.3 days). The PK profile of the second dose was similar to the first, with a T1 / 2 estimated to be 10 days (range approximately 5.7-8 days). After the second dose of ca_αD11_HCCDR2(L / V) on day 28, there was no change in the PK profile, indicating that no neutralizing anti-drug antibodies had developed. Furthermore, no anti-drug antibodies were detected in a bridging electrochemiluminescence (ECL) assay on days 0, 28, and 56, demonstrating that ca_αD11_HCCDR2(L / V) is non-immunogenic after repeated dosing.

[0180] Example 6: AAV construct encoding fe_αD11_HCCDR2(L / V) as a therapeutic agent in cats Cats were administered a single intramuscular (IM) injection of an adeno-associated virus (AAV) vector construct containing a nucleic acid sequence encoding a fetinized anti-NGF monoclonal antibody, fe_αD11_HCCDR2(L / V), under the control of the CB7 promoter at 1 × 10 12 gc / kg body weight and administered on study day 14 (AAV group, n=18). Placebo controls received a single IM injection of saline (n=15).

[0181] The AAV used in this study was AAVrh91, previously described in WO 2021 / 176362, the entire contents of which are incorporated herein by reference.

[0182] The concentration of FeNGF mAb in feline serum was determined using an NGF binding ELISA. ELISA plates were coated with 0.1 pg / mL muNGF and blocked with PBS / 0.05% Tween 20 / 1% BSA. muNGF-coated wells were incubated with serum diluted in PBS / 0.05% Tween 20 / 1% BSA in a volume of 100 pl / well for 1 h at room temperature. Binding curves were established using FeNGF antibody concentrations ranging from 1.56 ng / mL to 100 ng / mL. Well plates were washed after incubation and then incubated with a 1 / 10,000 dilution of goat anti-cat IgG-HRP in PBS / 0.05% Tween 20 / 1% BSA. Plates were washed with PBS / 0.05% Tween 20 and developed by addition of 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Color development was stopped by the addition of 2M H2SO4, absorbance was read at 450 nm, and background subtracted from the resulting readings. As shown in Figure 4, fe_αD11_HCCDR2(L / V) was detected in the serum of cats on days 30 and 90 of the study after IM administration of the AAV construct.

[0183] As in Example 4 above, the primary outcome measure was successful improvement in owner-reported Client Specific Outcome Measures (CSOM). Success was defined as a reduction in CSOM score of at least 2 or at least 3 on days 30 and 90 of the study compared to day 0. Statistical significance was determined using an alpha of 0.1.

[0184] As shown in Table 4 below and in Figure 5, there was a notable difference between the groups at day 90, with cats treated with the AAV constructs showing a higher success rate at day 90 of the study when compared to placebo (see Figure 5A: CSOM score > 2; AAV: 84.1%; placebo: 39.2%; p = 0.0637, and Figure 5B: CSOM score > 3; AAV: 84.0%; placebo: 9.9%; p = 0.0215). The success rate of cats treated with AAV was comparable to that observed at day 30 of the study in a separate cohort of cats with osteoarthritis-related pain treated with a monoclonal anti-NGF antibody, fe_αD11_HCCDR2(L / V) (CSOM score > 2, mAb: 82.4% vs. placebo: 40.0%, p = 0.0357).

[0185] [Table 7] CSOM success is defined as a reduction in the total CSOM score of at least 2 compared to day 0, with no increase in any individual activity. P values ​​(compared to placebo), LS means, and LS mean differences generated by generalized linear mixed models assuming a binomial distribution and logit link. Group, day, and group-day were included as fixed effects, and site and group-site interaction were included as random effects.

[0186] These data demonstrate that treatment with a single dose of a nucleic acid construct encoding the fetinized anti-NGF antibody fe_αD11_HCCDR2(L / V) was effective in reducing pain associated with osteoarthritis in cats for at least 76 days.

[0187] [Table 8]

[0188] [Table 9]

[0189] [Table 10]

[0190] [Table 11]

[0191] [Table 12]

[0192] [Table 13] * Alternative FR1 sequences

[0193] [Table 14]

[0194] [Table 15]

[0195] [Table 16]

[0196] [Table 17-1]

[0197] [Table 17-2]

[0198] [Table 18]

[0199] [Table 19]

[0200]

Table 20-1

[0201]

Table 20-2

[0202]

Table 21

[0203]

Table 22-1

[0204]

Table 22-2

[0205]

Table 23-1

[0206]

Table 23-2

[0207]

Table 24-1

[0208]

Table 24-2

Claims

1. 1. An antigen-binding molecule that specifically binds to nerve growth factor (NGF), the antigen-binding molecule comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and the VL comprises a complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

2. (a) the VH comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NOs: 27-31; and (b) the VL comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NOs: 32 to 35. The antigen-binding molecule of claim 1. (a) the VH comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12; and (b) the VL comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 128; Preferably, the VH comprises the amino acid sequence of SEQ ID NO: 12 and the VL comprises the amino acid sequence of SEQ ID NO:

128. The antigen-binding molecule of claim 1.

4. The antigen-binding molecule (a) a VH FR1 comprising an amino acid sequence having at least 95% sequence identity to the VH FR1 amino acid sequence of SEQ ID NO: 72; (b) a VH FR2 comprising an amino acid sequence having at least 95% sequence identity to the VH FR2 amino acid sequence of SEQ ID NO: 14; (c) a VH FR3 comprising an amino acid sequence having at least 95% sequence identity to the VH FR3 amino acid sequence of SEQ ID NO: 15; (d) a VH FR4 comprising an amino acid sequence having at least 95% sequence identity to the VH FR4 amino acid sequence of SEQ ID NO: 16; (e) a VL FR1 comprising an amino acid sequence having at least 95% sequence identity to the VL FR1 amino acid sequence of SEQ ID NO: 129; (f) a VL FR2 comprising an amino acid sequence having at least 95% sequence identity to the VL FR2 amino acid sequence of SEQ ID NO: 18; (g) a VL FR3 comprising an amino acid sequence having at least 95% sequence identity to the VL FR3 amino acid sequence of SEQ ID NO: 19; and (h) a VL FR4 comprising an amino acid sequence having at least 95% sequence identity to the VH FR4 amino acid sequence of SEQ ID NO:

20. (a) the VH comprises an amino acid sequence having at least 95% sequence identity to the VH amino acid sequence of SEQ ID NO: 31; and (b) the VL comprises an amino acid sequence having at least 95% sequence identity to the VH amino acid sequence of SEQ ID NO: 35; Preferably, the VH comprises the amino acid sequence of SEQ ID NO: 31, and the VL comprises the amino acid sequence of SEQ ID NO:

35. The antigen-binding molecule of claim 1.

6. The antigen-binding molecule (a) a VH FR1 comprising an amino acid sequence having at least 95% sequence identity to the VH FR1 amino acid sequence of SEQ ID NO: 52; (b) a VH FR2 comprising an amino acid sequence having at least 95% sequence identity to the VH FR2 amino acid sequence of SEQ ID NO: 53; (c) a VH FR3 comprising an amino acid sequence having at least 95% sequence identity to the VH FR3 amino acid sequence of SEQ ID NO: 54; (d) a VH FR4 comprising an amino acid sequence having at least 95% sequence identity to the VH FR4 amino acid sequence of SEQ ID NO: 55; (e) a VL FR1 comprising an amino acid sequence having at least 95% sequence identity to the VL FR1 amino acid sequence of SEQ ID NO: 68; (f) a VL FR2 comprising an amino acid sequence having at least 95% sequence identity to the VL FR2 amino acid sequence of SEQ ID NO: 69; (g) a VL FR3 comprising an amino acid sequence having at least 95% sequence identity to the VL FR3 amino acid sequence of SEQ ID NO: 70; and (h) a VL FR4 comprising an amino acid sequence having at least 95% sequence identity to the VL FR4 amino acid sequence of SEQ ID NO:

71.

7. The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule is an antibody or an NGF-binding fragment thereof, and preferably the NGF-binding fragment is selected from the group consisting of a Fab fragment, an scFab, an Fab', a single-chain variable fragment (scFv), and a single-arm antibody.

8. The antigen-binding molecule of claim 7, wherein the molecule is a humanized, canine, feline, or equine antibody, or an NGF-binding fragment thereof.

9. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antigen-binding molecule of any one of claims 1 to 6, wherein preferably the nucleic acid sequence encoding the immunoglobulin heavy chain has at least 95% sequence identity to SEQ ID NO: 25, or the nucleic acid sequence encoding the immunoglobulin light chain has at least 95% sequence identity to SEQ ID NO:

26.

10. An expression construct comprising a nucleic acid sequence encoding the antigen-binding molecule of any one of claims 1 to 6, operably linked to one or more regulatory sequences.

11. A host cell comprising the expression construct of claim 10.

12. A vector comprising a nucleic acid sequence encoding the antigen-binding molecule of any one of claims 1 to 6, wherein the vector is preferably an AAV vector.

13. A pharmaceutical composition comprising the antigen-binding molecule of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition for treating or preventing a condition associated with increased expression and / or activity of NGF in a subject in need thereof, comprising: The pharmaceutical composition comprises an antigen-binding molecule according to any one of claims 1 to 6, preferably The condition associated with increased expression and / or activity of NGF is (a) pain, preferably selected from the group consisting of neuropathic pain, inflammatory pain, pruritic pain, perioperative pain, postoperative pain, and post-surgical pain; or (b) arthritis, preferably wherein the arthritis is selected from the group consisting of immune-mediated polyarthritis, rheumatoid arthritis, and osteoarthritis, more preferably; The pharmaceutical composition, wherein the antigen-binding molecule is administered to the subject once every two months or more.

15. A kit comprising the antigen-binding molecule of any one of claims 1 to 6.

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