TNF alpha and NGF antibodies for animals

Abstract

Provided are various embodiments relating to caninized TNFα and NGF antibodies. Such antibodies can be used in methods of treating dogs with inflammatory conditions, such as inflammatory bowel disease, and / or in methods of treating dogs with pain, such as osteoarthritis pain, back pain, cancer pain, and / or neuropathic pain.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 127994, filed December 18, 2020, and U.S. Provisional Application No. 63 / 128804, filed December 21, 2020, the contents of each of which are incorporated herein by reference in their entirety.

[0002] [Field] The present disclosure relates to caninized TNF alpha antibodies and / or caninized NGF antibodies for treating inflammatory conditions, such as, for example, inflammatory bowel disease in dogs, and / or for treating pain in dogs. [Background technology]

[0003] TNF is a cytokine involved in systemic inflammation and stimulates the acute phase response. TNF promotes the inflammatory response and, as a result, causes or contributes to many of the clinical problems associated with autoimmune diseases such as ankylosing spondylitis, asthma, cancer, Crohn's disease, inflammatory bowel disease (IBD), juvenile idiopathic arthritis, psoriasis including plaque psoriasis, psoriatic arthritis, rheumatoid arthritis, ulcerative colitis, and other chronic inflammatory diseases.

[0004] The present disclosure provides methods and compositions for treating diseases such as IBD in companion animals with TNFα antibodies, and thus relates to the fields of biology, molecular biology, and veterinary medicine.

[0005] Companion animals such as cats, dogs, and horses suffer from many diseases similar to human diseases, including autoimmune diseases, cancer, and chronic inflammatory diseases. However, it has not been demonstrated so far that adalimumab or infliximab can be used to treat such diseases in companion animals. Furthermore, proteins with significant amounts of human-derived amino acid sequences can be immunogenic in non-human animals, especially if those sequences are derived from antibodies. Also, human drugs that bind to human TNF may not bind to companion animal TNF in a manner that provides an equally beneficial therapeutic effect in companion animals as in humans, especially if the sequence of the companion animal TNF differs from that of human TNF. Similarly, human drugs may not be as effective if they induce an immune response in companion animals. See Mauldin et al., Aug. 2010, 21(4):373-382.

[0006] IBD is a collective term used to describe diseases of the gastrointestinal tract in dogs with associated histological evidence of inflammation. See Ettinger SJ and Feldman EC. Diseases of the Small Intestine. Textbook of Veterinary Internal Medicine: Diseases of the Dog and the Cat, Elsevier Saunders, 2010. The exact etiology of canine IBD remains unclear, but altered immune system tolerance to dietary antigens and enteric bacteria is thought to play a major role, as does genetic susceptibility. Canine IBD may share a similar etiology to diseases that occur in humans (also known as Crohn's disease and ulcerative colitis), but the clinical syndromes and histological changes associated with dogs and humans are different. See Ettinger and Feldman; Suchodolski JS et al. "The fecal microbiome in dogs with acute diarrhea and idiopathic inflammatory bowel disease." PLoS ONE. 2012; 7(12):e51907. doi:10.1371 / journal.pone.0051907; Suchodolski JS. "Companion-animals symposium: Microbes and gastrointestinal health of dogs and cats." J Anim Sci. 2010; 89(5):1520-1530. Clinically, canine IBD is characterized by persistent or recurrent symptoms such as vomiting, diarrhea, abdominal pain, weight loss, or appetite changes.

[0007] Accurate diagnosis of canine IBD can be difficult, and the term "idiopathic" is often used when the exact causative agent cannot be identified. Typically, a complete medical history and physical examination, followed by laboratory tests, imaging, and intestinal biopsy (to demonstrate the presence of inflammation) are recommended. Furthermore, dogs with lymphocytic-plasmacytic colitis (LPC), a common form of IBD, failed to express a predominant cytokine profile (including TNFα) in the inflamed colonic mucosa, in contrast to human IBD. See Tamura Y et al. "Evaluation of Selected Cytokine Gene Expression in Colonic Mucosa from Dogs with Idiopathic Lymphocytic-plasmacytic Colitis." J Vet Med Sci. 2014;76(10):1407-10. Therapeutic strategies for managing these patients include administration of antiparasitic agents (anthelmintics), antibiotics, dietary modifications, and immunosuppressants, with the goal of remission of clinical signs. See Ettinger and Feldman. In humans, anti-inflammatory monoclonal antibodies that block TNF are widely used to treat this disease. In our canine patients, the prognosis and response to traditional treatments are variable and can vary from excellent to poor. Therefore, treatment outcomes in dogs cannot be predicted based on human treatments.

[0008] Thus, there remains a need for methods and compounds that can be used in companion animals to bind companion animal TNF to treat TNF-related conditions in companion animals. Ideally, these compounds would bind specifically to companion animal TNF and have a plasma half-life long enough to be of therapeutic utility, but not be highly immunogenic in companion animals. The present invention meets this need.

[0009] The present disclosure also provides methods and compositions for treating pain, such as chronic or inflammatory pain, in companion animals using NGF antibodies, and thus relates to the fields of biology, molecular biology, and veterinary medicine.

[0010] Nerve growth factor (NGF) is a neurotrophic factor that exerts a wide range of effects on regulating the growth, maintenance, proliferation, and survival of certain neurons. NGF is also associated with chronic and inflammatory pain. NGF binds to two classes of receptors: tropomyosin receptor kinase A (TrkA) and low affinity NGF receptors. Binding of dimeric NGF to the TrkA extracellular domain leads to receptor dimerization and activation of downstream kinase activity. NGF antibodies may be useful to antagonize NGF activity, reduce free NGF, and / or alleviate clinical signs and symptoms associated with NGF-associated pain. Companion species of animals, such as cats, dogs, and horses, can suffer from chronic and inflammatory pain. There remains a need for methods and species-specific compounds that specifically bind companion animal NGF that can be used to treat NGF-induced conditions and reduce NGF signaling activity.

[0011] The present disclosure also provides bispecific antibodies capable of binding both TNF and NGF, and methods for using such molecules therapeutically. For example, the bispecific molecules of the present disclosure may be used to treat both inflammation and associated pain, such as treating osteoarthritis, chronic pain, back pain, cancer pain, and neuropathic pain. Summary of the Invention

[0012] Embodiment 1. An isolated antibody that binds to canine TNFα, the antibody being a caninized antibody comprising: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:1; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:2; (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3; and (iv) a variable light chain comprising an LC-FR2 comprising glutamine at position 3 and lysine at position 8. Embodiment 2. An isolated antibody of embodiment 1, wherein the antibody comprises a variable heavy chain comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5; and (iii) a CDR H3 comprising the amino acid sequence of SEQ ID NO:6. Embodiment 3. The isolated antibody of any one of the preceding embodiments, wherein LC-FR2 comprises the amino acid sequence of SEQ ID NO:45. Embodiment 4. The isolated antibody of any one of the preceding embodiments, wherein the antibody comprises (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:46, or a variant thereof in which one, two, three, four, five, or six amino acids of the variable light chain are replaced by different amino acids; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof in which one, two, three, four, five, or six amino acids of the variable heavy chain are replaced by different amino acids; or (iii) the variable light chain of (i) and the variable heavy chain of (ii). Embodiment 5. The isolated antibody of any one of the preceding embodiments, wherein the antibody comprises: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:46, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:46; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:22; or (iii) the variable light chain of (i) and the variable heavy chain of (ii). Embodiment 6. An isolated antibody that binds to canine TNFα, comprising: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, or SEQ ID NO:46; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22; or (iii) a variable light chain of (i) and a variable heavy chain of (ii). Embodiment 7. An isolated antibody that binds to canine TNFα, comprising a variable light chain comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, or SEQ ID NO:46, and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22. Embodiment 8. An isolated antibody that binds to canine TNFα, the antibody comprising a variable light chain comprising the amino acid sequence of SEQ ID NO:43 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22. Embodiment 9. An isolated antibody that binds to canine TNFα, the antibody comprising a variable light chain comprising the amino acid sequence of SEQ ID NO:46 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22. Embodiment 10. An isolated antibody that binds to canine TNFα, comprising: (a) (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50; (iii) a CDR L3 comprising the amino acid sequence of SEQ ID NO: 51; and (iv) a light chain comprising the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 56, or a variant thereof in which 1, 2, 3, 4, 5, or 6 amino acids of the variable light chain are replaced by different amino acids; (b) (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53; (iii) a CDR H3 comprising the amino acid sequence of SEQ ID NO: 54; (iv) a heavy chain comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58, or a variant thereof in which 1, 2, 3, 4, 5, or 6 amino acids of the variable heavy chain are replaced by different amino acids; or (c) The light chain of (a) and the heavy chain of (b) The antibody, which is a caninized antibody comprising the antibody. Embodiment 11. An isolated antibody that binds to canine TNFα, comprising: (a) (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:49; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:50; (iii) a CDR L3 comprising the amino acid sequence of SEQ ID NO:51; and (iv) a variable light chain comprising the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56; (b) (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53; (iii) a CDR H3 comprising the amino acid sequence of SEQ ID NO:54; and (iv) a heavy chain comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58; or (c) The light chain of (a) and the heavy chain of (b) The antibody, which is a caninized antibody comprising the antibody. Embodiment 12. An isolated antibody that binds to canine TNFα, comprising: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58; or (iii) a variable light chain of (i) and a variable heavy chain of (ii). Embodiment 13. The antibody of any one of the preceding embodiments, comprising a canine constant heavy chain region and / or a canine constant light chain region. Embodiment 14. The antibody of any one of the preceding embodiments, comprising a canine heavy chain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region. Embodiment 15. The antibody comprises: (i) a light chain comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, or SEQ ID NO:44, or a light chain having a sequence identical to or different from the amino acid sequence of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, or SEQ ID NO:44 at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, or SEQ ID NO:44. 15. The isolated antibody of any one of embodiments 1 to 9, 13, or 14, comprising: (i) a light chain of (i) and a heavy chain of (ii); (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:64, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:64; or (iii) a light chain of (i) and a heavy chain of (ii). Embodiment 16. An isolated antibody of any one of embodiments 1 to 9 or embodiments 13 to 15, wherein the antibody comprises (i) a light chain having the amino acid sequence of SEQ ID NO: 44, (ii) a heavy chain having the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 64, or (iii) a light chain of (i) and a heavy chain of (ii). Embodiment 17. An isolated antibody that binds to canine TNFα, comprising a light chain comprising the amino acid sequence of SEQ ID NO:44 and a heavy chain comprising the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:64. Embodiment 18. An isolated antibody that binds to canine TNFα, comprising a light chain comprising the amino acid sequence of SEQ ID NO:15 and a heavy chain comprising the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:64. Embodiment 19. An isolated antibody that binds to canine TNFα, comprising: (a) (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:49; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:50; (iii) a CDR L3 comprising the amino acid sequence of SEQ ID NO:51; and (iv) a light chain comprising the amino acid sequence of SEQ ID NO:59 or SEQ ID NO:60, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:59 or SEQ ID NO:60; (b) (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53; (iii) a CDR H3 comprising the amino acid sequence of SEQ ID NO:54; and (iv) a heavy chain comprising the amino acid sequence of SEQ ID NO:61 or SEQ ID NO:62, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:61 or SEQ ID NO:62; or (c) The light chain of (a) and the heavy chain of (b) The antibody, which is a caninized antibody comprising the antibody. Embodiment 20. An isolated antibody that binds to canine TNFα, comprising a light chain comprising the amino acid sequence of SEQ ID NO:59 or SEQ ID NO:60, and a heavy chain comprising the amino acid sequence of SEQ ID NO:61 or SEQ ID NO:62. Embodiment 21. An isolated antibody that binds to canine NGF, the antibody being a caninized antibody comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70. Embodiment 22. The isolated antibody of embodiment 21, wherein the antibody further comprises: (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 65; (iv) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 66; and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 67. Embodiment 23. The isolated antibody of embodiment 21 or embodiment 22, wherein the antibody comprises: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 73; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 74; or (iii) the variable heavy chain of (i) and the variable heavy chain of (ii). Embodiment 24. An isolated antibody that binds to canine NGF, the antibody comprising: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 73; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 74; or (iii) a caninized antibody comprising the variable heavy chain of (i) and the variable heavy chain of (ii). Embodiment 25. An isolated antibody of any one of embodiments 21 to 24, wherein the antibody comprises (i) a variable light chain sequence of SEQ ID NO: 73, or a variant thereof in which 1, 2, 3, 4, 5, or 6 amino acids in the variable light chain are replaced by different amino acids, (ii) a variable heavy chain sequence of SEQ ID NO: 74, or a variant thereof in which 1, 2, 3, 4, 5, or 6 amino acids in the variable heavy chain are replaced by different amino acids, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii). Embodiment 26. The antibody of any one of embodiments 21 to 25, wherein the antibody comprises a canine constant heavy chain region and / or a canine constant light chain region. Embodiment 27. The antibody of any one of embodiments 21 to 26, wherein the antibody comprises a canine heavy chain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region. Embodiment 28. The isolated antibody of any one of embodiments 21 to 27, wherein the antibody comprises (i) a light chain sequence of SEQ ID NO: 77, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 77; (ii) a heavy chain sequence of SEQ ID NO: 79, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 79, or (iii) the light chain sequence of (i) and the heavy chain sequence of (ii). Embodiment 29. An isolated antibody of any one of embodiments 21 to 28, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:77 and a heavy chain comprising the amino acid sequence of SEQ ID NO:79. Embodiment 30. An isolated antibody that binds to canine NGF, the antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:77 and a heavy chain comprising the amino acid sequence of SEQ ID NO:79. Embodiment 31. An antibody of any one of embodiments 1 to 30, wherein the antibody is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2, and Fab'-SH. Embodiment 32. An isolated antibody that binds to canine TNFα and canine NGF, comprising: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:55, or SEQ ID NO:56, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:55, or SEQ ID NO:56; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22, SEQ ID NO:57, or SEQ ID NO:58, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:22, SEQ ID NO:57, or SEQ ID NO:58; (iii) a variable light chain comprising an amino acid sequence of SEQ ID NO:73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:73; (iv) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:74; or (v) the variable light chain sequence of (i), the variable heavy chain sequence of (ii), the variable light chain sequence of (iii), and the variable heavy chain sequence of (iv). An antibody comprising: Embodiment 33. The antibody comprises: (i) a variable light chain comprising an amino acid sequence of SEQ ID NO:43, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:43; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:22; (iii) a variable light chain comprising an amino acid sequence of SEQ ID NO:73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:73; (iv) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:74; or (v) the variable light chain sequence of (i), the variable heavy chain sequence of (ii), the variable light chain sequence of (iii), and the variable heavy chain sequence of (iv). 33. The isolated antibody of embodiment 32, comprising: Embodiment 34. An isolated antibody that binds to canine TNFα and canine NGF, comprising: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:43; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22; (iii) a variable light chain comprising the amino acid sequence of SEQ ID NO:73; and (iv) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:74. Embodiment 35. An isolated antibody of embodiment 33, comprising the amino acid sequence of SEQ ID NO:77 and the amino acid sequence of SEQ ID NO:81. Embodiment 36. An isolated nucleic acid encoding an antibody of any one of embodiments 1 to 35. Embodiment 37. A host cell comprising the nucleic acid of embodiment 36. Embodiment 38. A method for producing an antibody, comprising culturing a host cell according to embodiment 37 and isolating the antibody. Embodiment 39. A pharmaceutical composition comprising the antibody of any one of embodiments 1 to 35 and a pharma- ceutically acceptable carrier. Embodiment 40. A method for treating a dog having a condition associated with TNFα, comprising administering to the dog a therapeutically effective amount of an antibody of any one of embodiments 1 to 20 and 31 to 35 or a pharmaceutical composition of embodiment 39. Embodiment 41. A method for maintaining remission of a condition associated with TNFα in a dog, comprising administering to the dog a therapeutically effective amount of an antibody of any one of embodiments 1 to 20 and 31 to 35 or a pharmaceutical composition of embodiment 37. Embodiment 42. The method of embodiment 40 or embodiment 41, wherein the condition associated with TNFα is an inflammatory disease. Embodiment 43. The method of any one of embodiments 40 to 42, wherein the condition associated with TNFα is a gastrointestinal inflammatory disease. Embodiment 44. The method of any one of embodiments 40 to 43, wherein the condition associated with TNFα is inflammatory bowel disease. Embodiment 45. The method of any one of embodiments 40 to 44, wherein the condition associated with TNFα is ankylosing spondylitis, asthma, cancer, Crohn's disease, idiopathic arthritis, psoriasis, plaque psoriasis, psoriatic arthritis, rheumatoid arthritis, or ulcerative colitis. Embodiment 46. A method for treating a dog having an NGF-related condition, comprising administering to the dog a therapeutically effective amount of an antibody of any one of embodiments 21 to 35 or a pharmaceutical composition of embodiment 39. Embodiment 47. A method for maintaining remission of an NGF-related condition in a dog, comprising administering to the dog a therapeutically effective amount of an antibody of any one of embodiments 21 to 35 or a pharmaceutical composition of embodiment 39. Embodiment 48. A method for treating pain in a dog, comprising administering to the dog a therapeutically effective amount of the antibody of any one of embodiments 21 to 35 or the pharmaceutical composition of embodiment 39. Embodiment 49. The method of any one of embodiments 46 to 48, wherein the NGF-associated condition or pain is chronic pain, acute pain, and / or inflammatory pain. Embodiment 50. The method of any one of embodiments 46 to 49, wherein the condition or pain associated with NGF is osteoarthritis pain, back pain, cancer pain, and / or neuropathic pain. Embodiment 51. The method of any one of embodiments 46 to 50, wherein the NGF-associated condition or pain is pain associated with surgery, a broken or fractured bone, dental work, burns, cuts, and / or labor. Embodiment 52. The method of any one of embodiments 40 to 51, wherein the antibody or pharmaceutical composition is administered parenterally. Embodiment 53. The method of any one of embodiments 40 to 52, wherein the antibody or pharmaceutical composition is administered by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal, or inhalation route. Embodiment 54. The method of any one of embodiments 40 to 53, further comprising administering an IL17 antibody, an IL-5 antibody, an IL-31 antibody, an IL4 antibody, an IL13 antibody, an IL23 antibody, an IgE antibody, a CD11α antibody, an IL6R antibody, an α4-intergrin antibody, an IL12 antibody, an IL1β antibody, or an anti-BlyS antibody. Embodiment 55. The method of any one of embodiments 40 to 54, further comprising administering an NGF kinase inhibitor, a PI3K inhibitor, a ras inhibitor, a CGRP inhibitor, a TNF inhibitor, an IL17 inhibitor, an EGFR inhibitor, and / or a phospholipase C pathway inhibitor. Embodiment 56. The method of any one of embodiments 40 to 55, further comprising administering one or more pain medications, such as a corticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), a cyclooxygenase inhibitor, an opioid, and / or a cannabinoid. Embodiment 57. The method of any one of embodiments 40 to 56, wherein the antibody is administered in an amount ranging from 0.01 mg / kg body weight to 100 mg / kg body weight per dose. Embodiment 58. The method of any one of embodiments 40 to 57, wherein the antibody is administered at a dose of 2 mg / kg body weight. Embodiment 59. A method for reducing TNFα and / or NGF signaling function in a cell, comprising exposing the cell to an antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of embodiment 39 under conditions that permit binding of the antibody to TNFα and / or NGF, thereby reducing binding to TNFα and / or NGF signaling function by the cell. Embodiment 60. The method of embodiment 59, wherein the cells are exposed to the antibody or pharmaceutical composition ex vivo. Embodiment 61. The method of embodiment 59, wherein the cell is exposed to the antibody or pharmaceutical composition in vivo. Embodiment 62. The method of any one of embodiments 59 to 61, wherein the cell is a dog cell, a cat cell, or an horse cell. Embodiment 63. A method for detecting TNFα and / or NGF in a sample derived from a companion animal species, comprising contacting the sample with an antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of embodiment 39 under conditions allowing binding of the antibody to TNFα and / or NGF, and detecting whether a complex is formed between the antibody and TNFα and / or NGF in the sample. Embodiment 64. The method of embodiment 63, wherein the sample is a biological sample obtained from a dog, cat, or horse. [Brief description of the drawings]

[0013] [Figure 1] Alignment of the variable light chain sequences of D2E7, KBA VL v1, KBA VL v2, KBA VL v3, KBA VL v4, KBA VL v5, KBA VL v6, and KBA VL v7, and alignment of the variable heavy chain sequences of D2E7 and KBA. Description of a given sequence

[0014] Table 1 provides a list of certain sequences referenced herein.

[0015] TIFF2024507318000002.tif250170TIFF2024507318000003.tif255170TIFF2024507318000004.tif25517 0TIFF2024507318000005.tif255170TIFF2024507318000006.tif255170TIFF2024507318000007.tif25517 0TIFF2024507318000008.tif255170TIFF2024507318000009.tif255170TIFF2024507318000010.tif25517 0TIFF2024507318000011.tif255170TIFF2024507318000012.tif255170TIFF2024507318000013.tif62170 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Antibodies that bind canine TNFα and / or canine NGF are provided. Antibody heavy and light chains capable of forming antibodies that bind canine TNFα and / or canine NGF are also provided. Additionally, antibodies, heavy and light chains comprising one or more particular complementarity determining regions (CDRs) are provided. Polynucleotides encoding antibodies to canine TNFα and / or canine NGF are provided. Methods of producing or purifying antibodies to canine TNFα and / or canine NGF are also provided. Methods of treatment using antibodies to canine TNFα and / or canine NGF are provided. Such methods include, but are not limited to, methods of conditions associated with TNFα and / or NGF in dogs. Methods of detecting TNFα and / or NGF in a sample from a companion animal species are provided.

[0017] For the convenience of the reader, the following definitions are provided for terms used herein.

[0018] As used herein, numerical terms such as Kd are calculated based on scientific measurements and, therefore, are subject to appropriate measurement errors. In some instances, the numerical terms may include values ​​that are rounded to the nearest significant figure.

[0019] As used herein, "a" or "an" means "at least one" or "one or more," unless otherwise defined. As used herein, the term "or" means "and / or," unless otherwise defined. In the context of a claim that is multiple dependent, the use of "or" when referring to another claim refers only to that alternative claim.

[0020] Exemplary TNFα and / or NGF Antibodies Novel antibodies against TNFα and / or NGF are provided, for example, antibodies that bind to canine TNFα and / or canine NGF. The TNFα and / or NGF antibodies provided herein include, but are not limited to, monoclonal antibodies, chimeric antibodies, and caninized antibodies. In some embodiments, the TNFα antibody is KIND-509.

[0021] Also provided herein are amino acid sequences of monoclonal antibodies. For example, variable heavy chain CDRs, variable light chain CDRs, variable region heavy chain framework sequences, and variable region light chain framework sequences for the monoclonal antibodies described herein are provided. For example, the amino acid sequences of the variable light chain and variable heavy chain of monoclonal antibody KIND-509 are provided (SEQ ID NOs: 14 and 22, respectively). In addition, the amino acid sequences of the CDRs, framework sequences, variable light chain sequences, and variable heavy chain sequences of different caninized light and heavy chains are provided in FIG. 1.

[0022] Also provided herein are chimeric antibodies derived from monoclonal antibody D2E7. In some embodiments, the amino acid sequences of caninized monoclonal antibody D2E7 are provided, such as SEQ ID NOs: 13 to 46, 55 to 64, and 80 to 83. In some embodiments, the amino acid sequences of chimeric antibodies derived from monoclonal antibody D2E7 are provided, such as SEQ ID NOs: 9 to 12.

[0023] Also provided herein are chimeric antibodies derived from the monoclonal antibody αD11. In some embodiments, the amino acid sequences of caninized monoclonal antibody αD11, such as SEQ ID NOs:73-83, are provided.

[0024] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific (e.g., bispecific T cell engagers) and trispecific antibodies), and antibody fragments (such as Fab, F(ab')2, ScFv, minibodies, diabodies, triabodies, and tetrabodies) so long as they exhibit the desired antigen-binding activity. Canine, feline, and equine species have different types (classes) of antibodies shared by many mammals.

[0025] The term antibody includes, but is not limited to, fragments capable of binding antigen, such as Fv, single chain Fv (scFv), Fab, Fab', di-scFv, sdAb (single domain antibody) and (Fab')2 (including chemically bound F(ab')2). Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, the name of which reflects its ability to crystallize easily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species, such as mouse, human, cynomolgus monkey, dog, cat, and horse. Furthermore, for all antibody constructs provided herein, variants with sequences derived from other organisms are also contemplated. Antibody fragments also include single chain scFvs in any orientation, tandem di-scFvs, diabodies, tandem tri-sdcFvs, minibodies, etc. Antibody fragments also include nanobodies (sdAbs, antibodies with a single monomer domain, such as a heavy chain variable domain pair without a light chain). Antibody fragments can be said to be species specific in some embodiments (e.g., mouse scFv or dog scFv). This indicates the sequence of at least a portion of the non-CDR regions, not the source of the construct. In some embodiments, the antibody comprises a label or is conjugated to a second moiety.

[0026] The terms "label" and "detectable label" refer to a moiety attached to an antibody or its analyte to render the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein into which a label has been introduced that provides for the identification of the binding protein. In some embodiments, the label is a detectable marker that can generate a signal that is detectable by visual or instrumental means, for example, by incorporation of a radiolabeled amino acid or attachment of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected optically or colorimetrically). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-producing moieties, e.g., acridinium compounds, and fluorescence-producing moieties, e.g., fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety.

[0027] The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, having been raised against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies against different determinants (epitopes), each monoclonal antibody is raised against a single determinant on the antigen. Thus, a sample of monoclonal antibody is capable of binding to the same epitope on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. The monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0028] In some embodiments, the monoclonal antibody is KIND-509.

[0029] "Amino acid sequence" refers to the sequence of amino acid residues in a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, including, but not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) relative to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host producing the protein or errors during PCR amplification.

[0030] As used herein, "TNFα" refers to any native TNFα resulting from expression and processing of TNFα in cells. The term includes TNFα from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and horses), unless otherwise defined. The term also includes naturally occurring variants of TNFα, such as splice variants or allelic variants.

[0031] As used herein, "NGF" or "nerve growth factor" refers to any NGF resulting from the expression and processing of NGF in cells. The term includes NGF from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and horses), unless otherwise specified. The term also includes naturally occurring variants of NGF, such as splice variants or allelic variants.

[0032] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen, e.g., a protein, nucleic acid, sugar, or lipid) to which an antigen-binding molecule (e.g., an antibody, antibody fragment, or scaffold protein containing an antibody binding region) binds. Epitopes often include chemically active surface groupings of molecules, such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be formed from both contiguous or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3 residues, at least 5 residues, or 8-10 residues (e.g., amino acids or nucleotides). In some instances, an epitope is less than 20 residues (e.g., amino acids or nucleotides) long, less than 15 residues, or less than 12 residues. Two antibodies may bind to the same epitope in an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope may be identified at a predetermined minimum distance to the CDR residues on the antigen-binding molecule. In some embodiments, an epitope may be identified by the above distances and further limited to those residues involved in binding (e.g., hydrogen bonds) between the antibody residues and the antigen residues. Epitopes may also be identified by a variety of scans, for example, an alanine or arginine scan may indicate one or more residues with which the antigen-binding molecule may interact. Unless explicitly indicated, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antibody. Rather, the existence of such a set represents a minimal series (or species set) of epitopes. Thus, in some embodiments, the set of residues identified as an epitope represents a minimal epitope associated with the antigen, rather than an exclusive list of epitope residues on the antigen.

[0033] The term "CDR" refers to a complementarity determining region defined by at least one method of identification by one of skill in the art. In some embodiments, the CDRs can be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, the contact definition, or a combination of the Kabat, Chothia, AbM, or contact definitions. The various CDRs within an antibody can be represented by their appropriate number and chain type, including but not limited to CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term "CDR" is used herein to also encompass "hypervariable regions" or HVRs, which include the hypervariable loops.

[0034] In some embodiments, the caninized TNFα antibody comprises a variable light chain comprising (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:1; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:2; and / or (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the caninized TNFα antibody comprises a variable light chain comprising an LC-FR2 comprising a glutamine at position 3 (corresponding to KABAT position 37) and a lysine at position 8 (corresponding to KABAT position 42). In some embodiments, the caninized TNFα antibody comprises a variable heavy chain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5; and / or (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:6.

[0035] In some embodiments, the caninized TNFα antibody comprises a variable light chain that comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:49; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:50; and / or (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:51. In some embodiments, the caninized TNFα antibody comprises a variable heavy chain that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53; and / or (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:54.

[0036] In some embodiments, the caninized NGF antibody comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69; and / or (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the caninized NGF antibody comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 65; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 66; and / or (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 67.

[0037] The term "variable region" as used herein refers to a region that comprises at least three CDRs. In some embodiments, a variable region comprises three CDRs and at least one framework region ("FR"). The terms "heavy chain variable region" or "variable heavy chain" are used interchangeably and refer to a region that comprises at least three heavy chain CDRs. The terms "light chain variable region" or "variable light chain" are used interchangeably and refer to a region that comprises at least three light chain CDRs. In some embodiments, a variable heavy chain or variable light chain comprises at least one framework region. In some embodiments, an antibody comprises at least one light chain framework region selected from LC-FR1, LC-FR2, LC-FR3, and LC-FR4. In some embodiments, an antibody comprises at least one heavy chain framework region selected from HC-FR1, HC-FR2, HC-FR3, and HC-FR4. The framework region may be juxtaposed between the light chain CDRs or between the heavy chain CDRs. For example, the antibody may comprise a variable light chain having the following structure: (LC-FR1)-(CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3)-(LC-FR4). The antibody may also comprise a variable light chain having the following structure: (CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3). The antibody may also comprise a variable heavy chain having the following structure: (HC-FR1)-(CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3)-(HC-FR4). The antibody may comprise a variable heavy chain having the following structure: (CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3).

[0038] In some embodiments, the TNFα antibody comprises the LC-FR2 sequence of SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, or SEQ ID NO:45.

[0039] In some embodiments, the TNFα antibody comprises (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:46, or a variant thereof in which 1, 2, 3, 4, 5, or 6 amino acids of the variable light chain are replaced by different amino acids; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof in which 1, 2, 3, 4, 5, or 6 amino acids of the variable heavy chain are replaced by different amino acids; or (iii) the variable light chain of (i) and the variable heavy chain of (ii).

[0040] In some embodiments, the TNFα antibody comprises (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:46, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:46; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:22; or (iii) the variable light chain of (i) and the variable heavy chain of (ii).

[0041] In some embodiments, the TNFα antibody comprises: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, or SEQ ID NO:46; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22; or (iii) the variable light chain of (i) and the variable heavy chain of (ii).

[0042] In some embodiments, the TNFα antibody comprises a variable light chain comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, or SEQ ID NO:46.

[0043] In some embodiments, the TNFα antibody comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22.

[0044] In some embodiments, the TNFα antibody comprises a variable light chain comprising the amino acid sequence of SEQ ID NO:43 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22.

[0045] In some embodiments, the TNFα antibody comprises a variable light chain comprising the amino acid sequence of SEQ ID NO:46 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22.

[0046] In some embodiments, the TNFα antibody comprises (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56, or a variant thereof in which one, two, three, four, five, or six amino acids of the variable light chain are replaced by different amino acids; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58, or a variant thereof in which one, two, three, four, five, or six amino acids of the variable heavy chain are replaced by different amino acids; or (iii) a light chain of (i) and a heavy chain of (ii).

[0047] In some embodiments, the TNFα antibody comprises (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56, (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58; or (iii) the variable light chain of (i) and the variable heavy chain of (ii).

[0048] In some embodiments, the TNFα antibody comprises: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58; or (iii) the variable light chain of (i) and the variable heavy chain of (ii).

[0049] In some embodiments, the TNFα antibody comprises a variable light chain comprising the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56.

[0050] In some embodiments, the TNFα antibody comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58.

[0051] In some embodiments, the caninized NGF antibody comprises: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:73; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:74; or (iii) the variable heavy chain of (i) and the variable heavy chain of (ii).

[0052] In some embodiments, the caninized NGF antibody comprises: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:73; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:74; or (iii) the variable heavy chain of (i) and the variable heavy chain of (ii).

[0053] In some embodiments, the caninized NGF antibody comprises (i) the variable light chain sequence of SEQ ID NO: 73, or a variant thereof in which 1, 2, 3, 4, 5, or 6 amino acids in the variable light chain are replaced by different amino acids; (ii) the variable heavy chain sequence of SEQ ID NO: 74, or a variant thereof in which 1, 2, 3, 4, 5, or 6 amino acids in the variable heavy chain are replaced by different amino acids; or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii).

[0054] As used herein, the term "constant region" means a region containing at least three constant domains.

[0055] The terms "heavy chain constant region" or "constant heavy chain" are used interchangeably and refer to a region that includes at least three heavy chain constant domains, CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include gamma, delta, alpha, epsilon, and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody that includes a gamma constant region is an IgG antibody, an antibody that includes a delta constant region is an IgD antibody, an antibody that includes an alpha constant region is an IgA antibody, an antibody that includes a mu constant region is an IgM antibody, and an antibody that includes an epsilon constant region is an IgE antibody. A given isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a gamma 1 constant region), IgG2 (containing a gamma 2 constant region), IgG3 (containing a gamma 3 constant region), and IgG4 (containing a gamma 4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an alpha 1 constant region) and IgA2 (containing an alpha 2 constant region) antibodies; IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0056] The terms "light chain constant region" or "constant light chain" are used interchangeably and refer to the region that includes the light chain constant domain CL. Non-limiting exemplary light chain constant regions include lambda and kappa. Deletions and changes within the domain that do not alter function are included within the scope of the term "constant region" unless otherwise specified. Dogs, cats, and horses have antibody classes such as IgG, IgA, IgD, IgE, and IgM. Among the dog IgG antibody classes are IgG-A, IgG-B, IgG-C, and IgG-D.

[0057] The term "chimeric antibody" or "chimera" refers to an antibody in which a portion of the heavy or light chain is derived from a particular source or species, while at least a portion of the remaining heavy or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody that comprises at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (e.g., human, dog, cat, horse, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one dog constant region. In some embodiments, all of the variable regions of a chimeric antibody are from a first species and all of the constant regions of a chimeric antibody are from a second species. In some embodiments, a chimeric antibody comprises a constant heavy chain region or a constant light chain region from a companion animal. In some embodiments, a chimeric antibody comprises a mouse variable heavy and light chain and a companion animal constant heavy and light chain. For example, a chimeric antibody can comprise a mouse variable heavy and light chain and a dog constant heavy and light chain.

[0058] In some embodiments, the TNFα antibody comprises (a) (i) the light chain amino acid sequence of SEQ ID NO:10; (ii) the heavy chain amino acid sequence of SEQ ID NO:12; or (iii) the light chain amino acid sequence of (i) and the heavy chain sequence of (ii); or (b) (i) the light chain amino acid sequence of SEQ ID NO:40; (ii) the heavy chain amino acid sequence of SEQ ID NO:12; or (iii) the light chain amino acid sequence of (i) and the heavy chain sequence of (ii).

[0059] "Canine chimera," "chimeric dog," or "canine chimeric antibody" refers to a chimeric antibody having at least a portion of a heavy chain or a portion of a light chain derived from a dog. In some embodiments, the canine chimeric antibody comprises mouse variable heavy and light chains and canine constant heavy and light chains. In some embodiments, the antibody is a chimeric antibody comprising a mouse variable heavy chain framework region or a mouse variable light chain framework region.

[0060] In some embodiments, the TNFα and / or NGF antibody comprises a canine heavy chain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region.

[0061] "Caninized antibody" refers to an antibody in which at least one amino acid in a portion of a non-canine variable region has been replaced with the corresponding amino acid from a canine variable region. In some embodiments, the caninized antibody comprises at least one canine constant region (e.g., gamma constant region, alpha constant region, delta constant region, epsilon constant region, mu constant region, etc.) or a fragment thereof. In some embodiments, the caninized antibody is an antibody fragment, e.g., Fab, scFv, (Fab')2, etc. The term "caninized" also refers to forms of non-canine (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that comprise a minimal sequence of the non-canine immunoglobulin (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of an antibody). Caninized antibodies can include a canine immunoglobulin (recipient antibody) in which residues from the recipient's CDRs have been replaced by residues from the CDRs of a non-canine species (donor antibody), such as mouse, rat, or rabbit, that has the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the canine immunoglobulin are replaced by corresponding non-canine residues. Furthermore, caninized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

[0062] In some embodiments, at least one amino acid residue in a portion of the mouse variable heavy chain or the mouse variable light chain is replaced with the corresponding amino acid from a canine variable region, hi some embodiments, the modified chain is fused to a canine constant heavy chain or a canine constant light chain.

[0063] In some embodiments, the TNFα antibody has (i) a light chain sequence of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, or SEQ ID NO:44, or a light chain sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or similar to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, or SEQ ID NO:44. %, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:64; (ii) a heavy chain sequence of SEQ ID NO:23 or SEQ ID NO:64, or a variant thereof having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:64; or (iii) a caninized antibody comprising the light chain sequence of (i) and the heavy chain sequence of (ii).

[0064] In some embodiments, the TNFα antibody is a caninized antibody comprising (i) the light chain sequence of SEQ ID NO:44, (ii) the heavy chain sequence of SEQ ID NO:23 or SEQ ID NO:64, or (iii) the light chain sequence of (i) and the heavy chain sequence of (ii).

[0065] In some embodiments, the TNFα antibody is a caninized antibody comprising: (i) a light chain sequence of SEQ ID NO:59 or SEQ ID NO:60, or a variant thereof having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:59 or SEQ ID NO:60; (ii) a heavy chain sequence of SEQ ID NO:61 or SEQ ID NO:62, or a variant thereof having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:61 or SEQ ID NO:62; or (iii) a caninized antibody comprising the light chain sequence of (i) and the heavy chain sequence of (ii).

[0066] In some embodiments, the TNFα antibody is a caninized antibody comprising (i) the light chain sequence of SEQ ID NO:59 or SEQ ID NO:60, (ii) the heavy chain sequence of SEQ ID NO:61 or SEQ ID NO:62, or (iii) the light chain sequence of (i) and the heavy chain sequence of (ii).

[0067] In some embodiments, the caninized NGF antibody comprises (i) a light chain sequence of SEQ ID NO:77, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:77; (ii) a heavy chain sequence of SEQ ID NO:79, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:79; or (iii) the light chain sequence of (i) and the heavy chain sequence of (ii).

[0068] In some embodiments, the caninized NGF antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:77 and a heavy chain comprising the amino acid sequence of SEQ ID NO:79.

[0069] In some embodiments, the antibody binds to canine TNFα and canine NGF and has (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:55, or SEQ ID NO:56, or a variable light chain comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:55, or SEQ ID NO:56. (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22, SEQ ID NO:57, or SEQ ID NO:58, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:22, SEQ ID NO:57, or SEQ ID NO:58; (iii) a variable light chain comprising an amino acid sequence of SEQ ID NO:73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:73. (iv) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:74; or (v) the variable light chain sequence of (i), the variable heavy chain sequence of (ii), the variable light chain sequence of (iii), and the variable heavy chain sequence of (iv).

[0070] In some embodiments, the antibody binds to canine TNFα and canine NGF and has (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:43, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:43; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:22; (iii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:73, (iv) a variable light chain comprising the amino acid sequence of SEQ ID NO:74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:74; or (v) a variable light chain comprising the variable light chain sequence of (i), the variable heavy chain sequence of (ii), the variable light chain sequence of (iii), and the variable heavy chain sequence of (iv).

[0071] In some embodiments, the antibody binds to canine TNFα and canine NGF and comprises (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:43; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22; (iii) a variable light chain comprising the amino acid sequence of SEQ ID NO:73; and (iv) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:74.

[0072] In some embodiments, the antibody binds to canine TNFα and canine NGF and comprises the amino acid sequence of SEQ ID NO:77 and the amino acid sequence of SEQ ID NO:81.

[0073] A "fragment crystallizable polypeptide" or "Fc polypeptide" is the portion of an antibody molecule that interacts with effector molecules and cells. It comprises the C-terminal portion of an immunoglobulin heavy chain. As used herein, an Fc polypeptide includes fragments of an Fc domain that have one or more biological activities of the entire Fc polypeptide. An "effector function" of an Fc polypeptide is an action or activity carried out in whole or in part by any antibody in response to a stimulus, and may include complement fixation and / or ADCC (antibody-dependent cellular cytotoxicity) induction and / or ADCP (antibody-dependent cellular phagocytosis).

[0074] In some embodiments, the biological activity of an Fc polypeptide is the ability to bind FcRn. In some embodiments, the biological activity of an Fc polypeptide is the ability to bind C1q. In some embodiments, the biological activity of an Fc polypeptide is the ability to bind CD16. In some embodiments, the biological activity of an Fc polypeptide is the ability to bind Protein A.

[0075] The term "IgX Fc" means that the Fc region is derived from a particular antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where "X" refers to the antibody isotype. Thus, "IgG Fc" means the Fc region of the gamma chain, "IgA Fc" means the Fc region of the alpha chain, "IgD Fc" means the Fc region of the delta chain, "IgE Fc" means the Fc region of the epsilon chain, "IgM Fc" means the Fc region of the mu chain, etc. In some embodiments, an IgG Fc region comprises CH1, hinge, CH2, CH3, and CL1. "IgX-N-Fc" means that the Fc region is derived from a particular subclass of an antibody isotype (e.g., canine IgG subclasses A, B, C, or D; or feline IgG subclasses 1, 2a, or 2b), where "N" refers to the subclass. In some embodiments, the IgX Fc or IgX-N-Fc region is derived from a companion animal, such as a dog. In some embodiments, the IgG Fc region is isolated from a canine gamma heavy chain, such as IgG-A, IgG-B, IgG-C, or IgG-D. Antibodies comprising an IgG-A, IgG-B, IgG-C, or IgG-D Fc region may provide higher expression levels in recombinant production systems.

[0076] The terms "IgX Fc" and "IgX Fc polypeptide", unless otherwise specified, include wild-type IgX Fc polypeptide and variant IgX Fc polypeptides.

[0077] In some embodiments, the variant IgG Fc polypeptide comprises a variant IgG Fc polypeptide of a companion animal species. In some embodiments, the variant IgG Fc polypeptide comprises a variant canine IgG Fc polypeptide. In some embodiments, the variant IgG Fc polypeptide (e.g., a variant canine IgG-A Fc polypeptide, a variant canine IgG-C Fc polypeptide, or a variant canine IgG-D Fc polypeptide, a variant feline IgG1a Fc polypeptide) has an activity that is substantially lacking in the reference (e.g., wild-type) polypeptide.

[0078] The antibody may be modified to increase or decrease its half-life. In some embodiments involving higher doses of the antibody, a shorter half-life may be desired for acute treatment. In some embodiments involving lower doses of the antibody, a longer half-life may be desired for chronic treatment. For example, IgG Fc mutations that affect FcRn interactions may be introduced, as discussed below.

[0079] In some embodiments, the TNFα and / or NGF antibody comprises a wild-type or variant IgG Fc that has complement fixation activity (or complement dependent cytotoxicity (CDC)). In some embodiments, the TNFα and / or NGF antibody comprises a wild-type or variant IgG Fc that has antibody-dependent cellular cytotoxicity (ADCC) activity. In some embodiments, the TNFα and / or NGF antibody comprises a wild-type or variant IgG Fc that has antibody-dependent cellular phagocytosis (ADCP) activity. In some embodiments, the TNFα and / or NGF antibody comprises a wild-type or variant IgG Fc that has complement fixation activity and / or ADCC activity and / or ADCP activity. The IgG Fc polypeptide may be modified to have effector function or to have enhanced effector function.

[0080] In some embodiments, the TNFα and / or NGF antibodies comprise a wild-type or variant IgG Fc that binds to canine FcRn at low pH.

[0081] In some embodiments, the variant IgG Fc (e.g., variant canine IgG Fc polypeptide) has an altered FcRn binding affinity compared to the reference polypeptide. In some embodiments, the variant IgG Fc has an increased FcRn binding affinity at an acidic pH (e.g., a pH ranging from about 5.0 to about 6.5, e.g., a pH of about 5.0, a pH of about 5.5, a pH of about 6.0, or a pH of about 6.5) compared to the reference polypeptide. Exemplary variant IgG Fc polypeptides with increased FcRn binding affinity are disclosed in WO2020 / 082048, which is incorporated herein by reference in its entirety.

[0082] In some embodiments, the variant IgG Fc (e.g., variant canine IgG Fc polypeptide) has altered Protein A binding affinity compared to the reference polypeptide. In some embodiments, the variant IgG Fc has increased Protein A binding affinity compared to the reference polypeptide. Exemplary variant IgG Fc polypeptides with increased Protein A binding affinity are disclosed in WO 2020 / 139984 (e.g., Example 2), which is incorporated herein by reference in its entirety.

[0083] The term "affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D The affinity can be measured by common methods known in the art, such as immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance.

[0084] "K D ", "K d The terms ", "Kd" or "Kd value" are used interchangeably to refer to the equilibrium dissociation constant of an antibody-antigen interaction. In some embodiments, the K d is measured by using a Biolayer Interference Assay using a biosensor such as the Octet® system (Pall ForteBio LLC, Fremont, Calif.) according to the supplier's instructions.

[0085] For example, biotinylated antigen is bound to the sensor tip, antibody binding is monitored for 90 seconds, and dissociation is monitored for 600 seconds. The buffer for the dilution and binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2. A buffer-only blank curve is subtracted to correct for drift. Data are fitted to a 2:1 binding model using ForteBio data analysis software to derive the binding rate constant (k on ), dissociation rate constant (k off ), and K d Determine the equilibrium dissociation constant (K d ) is k off / k on The term "kon" refers to the rate constant for an antibody to bind to an antigen, and the term "koff" refers to the rate constant for an antibody to dissociate from the antibody / antigen complex.

[0086] The term "binding" to an antigen or epitope is a term well understood in the art, and methods for determining such binding are also well known in the art. A molecule is said to exhibit "binding" if it reacts, associates, or has affinity for a particular cell or substance, and the reaction, association, or affinity is detectable, for example, by one or more methods known in the art, such as immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance, etc.

[0087] "Surface plasmon resonance" is, for example, TM The system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ) is used to detect changes in protein concentration within a biosensor matrix, an optical phenomenon that allows for the analysis of real-time biospecific interactions. For further description, see Jonsson et al. (1993) Ann. Biol. Clin. 51:19-26.

[0088] "Biolayer interferometry" refers to an optical analysis technique that analyzes the interference pattern of light reflected from a layer of immobilized proteins on a biosensor tip and an internal reference layer. Changes in the number of molecules bound to the biosensor tip cause a shift in the interference pattern that can be measured in real time. A non-limiting exemplary device for biolayer interferometry is the Octet® system (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal. Biochem. 377:209-277.

[0089] In some embodiments, the TNFα antibody has a titer of 5×10 as measured by biolayer interferometry. -6 Less than M, 1×10 -6 Less than M, 5×10 -7 Less than M, 1×10 -7 Less than M, 5×10 -8 Less than M, 1×10 -8 Less than M, 5×10 -9 Less than M, 1×10 -9 Less than M, 5×10 -10 Less than M, 1×10 -10 Less than M, 5×10 -11 Less than M, 1×10 -11 Less than M, 5×10 -12 Less than M or 1×10 -12 It binds to canine TNFα, human TNFα, feline TNFα, or equine TNFα with a dissociation constant (Kd) of less than M.

[0090] In some embodiments, the TNFα antibody has a titer of 5×10 as measured by biolayer interferometry. -6 M and 1×10 -6 Between M, 5×10 -6 M and 5×10 -7 Between M, 5×10 -6 M and 1×10 -7 Between M, 5×10 -6 M and 5×10 -8 Between M, 5×10 -6 M and 1×10 -8 Between M, 5×10 -6 M and 5×10 -9 Between M, 5×10 -6 M and 1×10-9 Between M, 5×10 -6 M and 5×10 -10 Between M, 5×10 -6 M and 1×10 -10 Between M, 5×10 -6 M and 5×10 -11 Between M, 5×10 -6 M and 1×10 -11 Between M, 5×10 -6 M and 5×10 -12 Between M, 5×10 -6 M and 1×10 -12 Between M, 1×10 -6 M and 5×10 -7 Between M, 1×10 -6 M and 1×10 -7 Between M, 1×10 -6 M and 5×10 -8 Between M, 1×10 -6 M and 1×10 -8 Between M, 1×10 -6 M and 5×10 -9 Between M, 1×10 -6 M and 1×10 -9 Between M, 1×10 -6 M and 5×10 -10 Between M, 1×10 -6 M and 1×10 -10 Between M, 1×10 -6 M and 5×10 -11 Between M, 1×10 -6 M and 1×10 -11 Between M, 1×10 -6 M and 5×10 -12 Between M, 1×10 -6 M and 1×10 -12 Between M, 5×10 -7 M and 1×10 -7 Between M, 5×10 -7 M and 5×10 -8 Between M, 5×10 -7 M and 1×10 -8 Between M, 5×10 -7 M and 5×10 -9 Between M, 5×10 -7 M and 1×10 -9 Between M, 5×10 -7 M and 5×10 -10 Between M, 5×10 -7 M and 1×10-10 Between M, 5×10 -7 M and 5×10 -11 Between M, 5×10 -7 M and 1×10 -11 Between M, 5×10 -7 M and 5×10 -12 Between M, 5×10 -7 M and 1×10 -12 Between M, 1×10 -7 M and 5×10 -8 Between M, 1×10 -7 M and 1×10 -8 Between M, 1×10 -7 M and 5×10 -9 Between M, 1×10 -7 M and 1×10 -9 Between M, 1×10 -7 M and 5×10 -10 Between M, 1×10 -7 M and 1×10 -10 Between M, 1×10 -7 M and 5×10 -11 Between M, 1×10 -7 M and 1×10 -11 Between M, 1×10 -7 M and 5×10 -12 Between M, 1×10 -7 M and 1×10 -12 Between M, 5×10 -8 M and 1×10 -8 Between M, 5×10 -8 M and 5×10 -9 Between M, 5×10 -8 M and 1×10 -9 Between M, 5×10 -8 M and 5×10 -10 Between M, 5×10 -8 M and 1×10 -10 Between M, 5×10 -8 M and 5×10 -11 Between M, 5×10 -8 M and 1×10 -11 Between M, 5×10 -8 M and 5×10 -12 Between M, 5×10 -8 M and 1×10 -12 Between M, 1×10 -8 M and 5×10 -9 Between M, 1×10 -8 M and 1×10-9 Between M, 1×10 -8 M and 5×10 -10 Between M, 1×10 -8 M and 1×10 -10 Between M, 1×10 -8 M and 5×10 -11 Between M, 1×10 -8 M and 1×10 -11 Between M, 1×10 -8 M and 5×10 -12 Between M, 1×10 -8 M and 1×10 -12 Between M, 5×10 -9 M and 1×10 -9 Between M, 5×10 -9 M and 5×10 -10 Between M, 5×10 -9 M and 1×10 -10 Between M, 5×10 -9 M and 5×10 -11 Between M, 5×10 -9 M and 1×10 -11 Between M, 5×10 -9 M and 5×10 -12 Between M, 5×10 -9 M and 1×10 -12 Between M, 1×10 -9 M and 5×10 -10 Between M, 1×10 -9 M and 1×10 -10 Between M, 1×10 -9 M and 5×10 -11 Between M, 1×10 -9 M and 1×10 -11 Between M, 1×10 -9 M and 5×10 -12 Between M, 1×10 -9 M and 1×10 -12 Between M, 5×10 -10 M and 1×10 -10 Between M, 5×10 -10 M and 5×10 -11 Between M, 1×10 -10 M and 5×10 -11 Between M, 1×10 -10 M and 1×10 -11 Between M, 1×10 -10 M and 5×10 -12 Between M, 1×10 -10 M and 1×10-12 Between M, 5×10 -11 M and 1×10 -12 Between M, 5×10 -11 M and 5×10 -12 Between M, 5×10 -11 M and 1×10 -12 Between M, 1×10 -11 M and 5×10 -12 Between M or 1×10 -11 M and 1×10 -12 It binds to canine TNFα, human TNFα, feline TNFα, or equine TNFα with a Kd between M

[0091] In some embodiments, the TNFα antibody binds to canine TNFα, human TNFα, feline TNFα, or equine TNFα as determined by immunoblot analysis.

[0092] In some embodiments, the NGF antibody has a titer of 5×10 as measured by biolayer interferometry. -6 Less than M, 1×10 -6 Less than M, 5×10 -7 Less than M, 1×10 -7 Less than M, 5×10 -8 Less than M, 1×10 -8 Less than M, 5×10 -9 Less than M, 1×10 -9 Less than M, 5×10 -10 Less than M, 1×10 -10 Less than M, 5×10 -11 Less than M, 1×10 -11 Less than M, 5×10 -12 Less than M or 1×10 -12 It binds to canine NGF, human NGF, feline NGF, or equine NGF with a dissociation constant (Kd) of less than M.

[0093] In some embodiments, the NGF antibody has a titer of 5×10 as measured by biolayer interferometry. -6 M and 1×10 -6 Between M, 5×10 -6 M and 5×10 -7 Between M, 5×10 -6 M and 1×10 -7 Between M, 5×10 -6 M and 5×10-8 Between M, 5×10 -6 M and 1×10 -8 Between M, 5×10 -6 M and 5×10 -9 Between M, 5×10 -6 M and 1×10 -9 Between M, 5×10 -6 M and 5×10 -10 Between M, 5×10 -6 M and 1×10 -10 Between M, 5×10 -6 M and 5×10 -11 Between M, 5×10 -6 M and 1×10 -11 Between M, 5×10 -6 M and 5×10 -12 Between M, 5×10 -6 M and 1×10 -12 Between M, 1×10 -6 M and 5×10 -7 Between M, 1×10 -6 M and 1×10 -7 Between M, 1×10 -6 M and 5×10 -8 Between M, 1×10 -6 M and 1×10 -8 Between M, 1×10 -6 M and 5×10 -9 Between M, 1×10 -6 M and 1×10 -9 Between M, 1×10 -6 M and 5×10 -10 Between M, 1×10 -6 M and 1×10 -10 Between M, 1×10 -6 M and 5×10 -11 Between M, 1×10 -6 M and 1×10 -11 Between M, 1×10 -6 M and 5×10 -12 Between M, 1×10 -6 M and 1×10 -12 Between M, 5×10 -7 M and 1×10 -7 Between M, 5×10 -7 M and 5×10 -8 Between M, 5×10 -7 M and 1×10 -8 Between M, 5×10 -7 M and 5×10-9 Between M, 5×10 -7 M and 1×10 -9 Between M, 5×10 -7 M and 5×10 -10 Between M, 5×10 -7 M and 1×10 -10 Between M, 5×10 -7 M and 5×10 -11 Between M, 5×10 -7 M and 1×10 -11 Between M, 5×10 -7 M and 5×10 -12 Between M, 5×10 -7 M and 1×10 -12 Between M, 1×10 -7 M and 5×10 -8 Between M, 1×10 -7 M and 1×10 -8 Between M, 1×10 -7 M and 5×10 -9 Between M, 1×10 -7 M and 1×10 -9 Between M, 1×10 -7 M and 5×10 -10 Between M, 1×10 -7 M and 1×10 -10 Between M, 1×10 -7 M and 5×10 -11 Between M, 1×10 -7 M and 1×10 -11 Between M, 1×10 -7 M and 5×10 -12 Between M, 1×10 -7 M and 1×10 -12 Between M, 5×10 -8 M and 1×10 -8 Between M, 5×10 -8 M and 5×10 -9 Between M, 5×10 -8 M and 1×10 -9 Between M, 5×10 -8 M and 5×10 -10 Between M, 5×10 -8 M and 1×10 -10 Between M, 5×10 -8 M and 5×10 -11 Between M, 5×10 -8 M and 1×10 -11 Between M, 5×10 -8 M and 5×10-12 Between M, 5×10 -8 M and 1×10 -12 Between M, 1×10 -8 M and 5×10 -9 Between M, 1×10 -8 M and 1×10 -9 Between M, 1×10 -8 M and 5×10 -10 Between M, 1×10 -8 M and 1×10 -10 Between M, 1×10 -8 M and 5×10 -11 Between M, 1×10 -8 M and 1×10 -11 Between M, 1×10 -8 M and 5×10 -12 Between M, 1×10 -8 M and 1×10 -12 Between M, 5×10 -9 M and 1×10 -9 Between M, 5×10 -9 M and 5×10 -10 Between M, 5×10 -9 M and 1×10 -10 Between M, 5×10 -9 M and 5×10 -11 Between M, 5×10 -9 M and 1×10 -11 Between M, 5×10 -9 M and 5×10 -12 Between M, 5×10 -9 M and 1×10 -12 Between M, 1×10 -9 M and 5×10 -10 Between M, 1×10 -9 M and 1×10 -10 Between M, 1×10 -9 M and 5×10 -11 Between M, 1×10 -9 M and 1×10 -11 Between M, 1×10 -9 M and 5×10 -12 Between M, 1×10 -9 M and 1×10 -12 Between M, 5×10 -10 M and 1×10 -10 Between M, 5×10 -10 M and 5×10 -11 Between M, 1×10 -10 M and 5×10-11 Between M, 1×10 -10 M and 1×10 -11 Between M, 1×10 -10 M and 5×10 -12 Between M, 1×10 -10 M and 1×10 -12 Between M, 5×10 -11 M and 1×10 -12 Between M, 5×10 -11 M and 5×10 -12 Between M, 5×10 -11 M and 1×10 -12 Between M, 1×10 -11 M and 5×10 -12 Between M or 1×10 -11 M and 1×10 -12 It binds to canine NGF, human NGF, feline NGF, or equine NGF with a Kd between

[0094] In some embodiments, the NGF antibody binds to canine NGF, human NGF, feline NGF, or equine NGF as determined by immunoblot analysis.

[0095] "Wild type" refers to a non-mutated form of a naturally occurring polypeptide, or a fragment thereof. A wild type polypeptide may be recombinantly produced.

[0096] By "variant" is meant a biologically active polypeptide having at least about 50% amino acid sequence identity with a native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues have been added or deleted at the N-terminus or C-terminus of the polypeptide.

[0097] In some embodiments, the variant has at least 1, 2, 3, 4, 5, or 6 amino acids replaced with different amino acids.

[0098] In some embodiments, a variant has at least about 50% sequence identity with a reference nucleic acid molecule or polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues have been added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, a variant has at least about 50% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity with a reference nucleic acid or polypeptide sequence.

[0099] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be performed using, for example, BLAST, BLAST-2, ALIGN, or MEGALINE. TM This can be accomplished in a variety of ways within the skill of the art using publicly available computer software, such as (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0100] "Amino acid substitution" refers to the replacement of one amino acid in a polypeptide with another amino acid. In some embodiments, the amino acid substitution is a conservative substitution. Non-limiting exemplary conservative amino acid substitutions are shown in Table 2. Amino acid substitutions can be introduced into a molecule of interest and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC or enhanced pharmacokinetics.

[0101] TIFF2024507318000014.tif167170

[0102] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0103] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0104] The term "vector" is used to describe a polynucleotide that can be engineered to contain a cloned polynucleotide or a polynucleotide that can be propagated in a host cell. A vector may contain one or more of the following elements: an origin of replication, one or more control sequences that regulate the expression of a polypeptide of interest (such as, for example, a promoter or enhancer), or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0105] "Host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NS0 cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, and their derivatives, such as 293-6E, DG44, CHO-S, and CHO-K cells. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. A host cell includes cells transfected in vivo with a polynucleotide encoding the amino acid sequences provided herein.

[0106] The term "isolated" as used herein refers to a molecule that is separated from at least some of the components in which it is typically found or produced in nature. For example, a polypeptide is referred to as "isolated" if it is separated from at least some of the components of the cell in which it is produced. If the polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell in which it was produced is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide in which it is typically found in nature (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) or, for example, in the case of an RNA polynucleotide, is separated from at least some of the components of the cell in which it was produced. Thus, a DNA polynucleotide contained in a vector within a host cell may be referred to as "isolated". In some embodiments, the TNFα and / or NGF antibodies are purified using chromatography, such as size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.

[0107] The term "companion animal species" refers to animals suitable to be companions to humans. In some embodiments, companion animal species are small mammals, such as canines, felines, dogs, cats, horses, rabbits, ferrets, guinea pigs, rodents, etc. In some embodiments, companion animal species are farm animals, such as horses, cows, pigs, etc.

[0108] "Reduce" or "inhibit" means to decrease, reduce, or stop an activity, function, or amount compared to a reference. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 20% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 50% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or more. In some embodiments, the amount is inhibited or reduced over a period of time compared to a control dose (such as a placebo) over the same period of time. As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. A reference can be obtained from a healthy or non-diseased sample. In some examples, a reference is obtained from a non-diseased or untreated sample of a companion animal. In some examples, a reference is obtained from one or more healthy animals of a particular species that are not the animal being tested or treated.

[0109] The term "substantially reduced" as used herein refers to a sufficiently high degree of reduction between a numerical value and a reference numerical value such that one of skill in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological characteristic measured by the values. In some embodiments, a substantially reduced numerical value is reduced by more than about any one of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.

[0110] In some embodiments, the TNFα antibody may reduce TNFα signaling function in a companion animal species by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to TNFα signaling function in the absence of the antibody.In some embodiments, the reduction in TNFα signaling function is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and 60%, between 15% and 70%, between 15% and 80%, between 15% and 90%, between 15% and 100%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and 80%, between 20% and 90%, between 20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 25% and 60%, between 25% and 70%, between 25% and 80%, between 25% and 90% between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80%, 40% between 40% and 100%, between 45% and 50%, between 45% and 60%, between 45% and 70%, between 45% and 80%, between 45% and 90%, between 45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and 100%, between 60% and 70%, between 60% and 80%, between 60% and 90%, between 60% and 100%, between 70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and 90%, between 80% and 100%, or between 90% and 100%.

[0111] In some embodiments, the NGF antibody may reduce NGF signaling function in a companion animal species by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to NGF signaling function in the absence of the antibody.In some embodiments, the decrease in NGF signaling function is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and 60%, between 15% and 70%, between 15% and 80%, between 15% and 90%, between 15% and 100%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and 80%, between 20% and 90%, between 20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 25% and 60%, between 25% and 70%, between 25% and 80%, between 25% and 90% between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80%, 40% between 40% and 100%, between 45% and 50%, between 45% and 60%, between 45% and 70%, between 45% and 80%, between 45% and 90%, between 45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and 100%, between 60% and 70%, between 60% and 80%, between 60% and 90%, between 60% and 100%, between 70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and 90%, between 80% and 100%, or between 90% and 100%.

[0112] Exemplary Pharmaceutical Compositions The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0113] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid excipient, diluent, encapsulating material, formulation aid, or carrier common in the art for use with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration employed and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation in which it is used. Examples of pharma- ceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin, canine or other animal albumin; buffers, such as phosphate, citrate, tromethamine or HEPES buffers; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, or magnesium trisilicate; polyvinylpyrrolidone, cellulose-based materials; polyethylene glycol; sucrose; mannitol; or amino acids, including, but not limited to, arginine.

[0114] The pharmaceutical composition can be stored in lyophilized form. Thus, in some embodiments, the preparation method includes a lyophilization step. The lyophilized composition can then be reformulated before administration to dogs, typically as an aqueous composition suitable for parenteral administration. In other embodiments, especially when the antibody is highly stable to thermal and oxidative denaturation, the pharmaceutical composition can be stored as a liquid, i.e., as an aqueous composition, that can be administered to dogs directly or with a suitable dilution. The lyophilized composition can be reconstituted with sterile water for injection (WFI). A bacteriostatic agent such as benzyl alcohol can be included. Thus, the present invention provides pharmaceutical compositions in solid or liquid form.

[0115] The pH of the pharmaceutical composition may range from about pH 5 to about pH 8 when administered. The compositions of the present invention are sterile when they are used for therapeutic purposes. Sterility can be achieved by any of several means known in the art, including filtration through a sterile filtration membrane (e.g., 0.2 micron membrane). Sterility can be maintained with or without antibacterial agents.

[0116] Exemplary Uses of Antibodies and Pharmaceutical Compositions The antibodies of the invention or pharmaceutical compositions comprising the antibodies may be useful for treating conditions associated with TNFα and / or NGF.

[0117] As used herein, "TNFα-associated condition" refers to a disease associated with, caused by, or characterized by elevated levels or gradients of TNFα concentration, including, but not limited to, autoimmune diseases such as ankylosing spondylitis, asthma, cancer, Crohn's disease, inflammatory bowel disease (IBD), juvenile idiopathic arthritis, psoriasis, including plaque psoriasis, psoriatic arthritis, rheumatoid arthritis, ulcerative colitis, and other chronic inflammatory diseases.

[0118] As used herein, "NGF-associated condition" refers to a disease associated with, caused by, or characterized by an elevated level or gradient of NGF concentration. Such conditions include, but are not limited to, pain, such as chronic pain, acute pain, and / or inflammatory pain. In some embodiments, the pain is osteoarthritis pain, back pain, cancer pain, and / or neuropathic pain. In some embodiments, the pain is associated with surgery, broken bone, dental work, burns, cuts, and / or labor.

[0119] Conditions associated with TNFα and / or NGF may be manifested in companion animals, including but not limited to dogs.

[0120] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" includes any administration or application of a therapeutic agent for a disease in a mammal, including a companion animal. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or slowing of disease spread, prevention or slowing of disease recurrence, slowing or slowing of disease progression, amelioration of pathology, inhibition of disease or disease progression, inhibition or slowing of disease or its progression, arrest of its occurrence, and remission (partial or complete). Also included in "treatment" is the reduction of pathological consequences of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. Consistent with the above, the term treatment does not require 100 percent elimination of all aspects of the disorder.

[0121] In some embodiments, TNFα and / or NGF antibodies or pharmaceutical compositions comprising same may be utilized in accordance with the methods herein to treat a condition associated with TNFα and / or NGF. In some embodiments, TNFα and / or NGF antibodies or pharmaceutical compositions are administered to a companion animal, such as a dog, to treat a condition associated with TNFα and / or NGF. In some embodiments, TNFα and / or NGF antibodies or pharmaceutical compositions are administered to a companion animal, such as a dog, to maintain remission of a condition associated with TNFα and / or NGF.

[0122] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist may vary depending on factors such as the type of disease being treated, the disease state, the severity and course of the disease, the type of therapeutic objective, previous treatments, clinical history, response to previous treatments, the discretion of the attending veterinarian, the age, sex, and weight of the animal, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the animal. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the substance / molecule, agonist or antagonist are outweighed by therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0123] In some embodiments, the TNFα and / or NGF antibody or pharmaceutical composition comprising the TNFα and / or NGF antibody is administered parenterally by subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, the TNFα and / or NGF antibody or pharmaceutical composition comprising the TNFα and / or NGF antibody is administered as a bolus injection or by continuous infusion over a period of time. In some embodiments, the TNFα and / or NGF antibody or pharmaceutical composition comprising the TNFα and / or NGF antibody is administered intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intraarterially, intrasynovially, intrathecally, or by inhalation routes.

[0124] The TNFα and / or NGF antibodies described herein may be administered in an amount ranging from 0.01 mg / kg to 100 mg / kg of body weight per dose. In some embodiments, the TNFα and / or NGF antibodies may be administered in an amount ranging from 0.5 mg / kg to 50 mg / kg of body weight per dose. In some embodiments, the TNFα and / or NGF antibodies may be administered in an amount ranging from 0.1 mg / kg to 10 mg / kg of body weight per dose. In some embodiments, the TNFα and / or NGF antibodies may be administered in an amount ranging from 0.1 mg / kg to 100 mg / kg of body weight per dose. In some embodiments, the TNFα and / or NGF antibodies may be administered in an amount ranging from 1 mg / kg to 10 mg / kg of body weight per dose. In some embodiments, the TNFα and / or NGF antibodies may be administered in an amount ranging from 0.5 mg / kg to 100 mg / kg body weight, 1 mg / kg to 100 mg / kg body weight, 5 mg / kg to 100 mg / kg body weight, 10 mg / kg to 100 mg / kg body weight, 20 mg / kg to 100 mg / kg body weight, 50 mg / kg to 100 mg / kg body weight, 1 mg / kg to 10 mg / kg body weight, 5 mg / kg to 10 mg / kg body weight, 0.5 mg / kg to 10 mg / kg body weight, 0.01 mg / kg to 0.5 mg / kg body weight, 0.01 mg / kg to 0.1 mg / kg body weight, or 5 mg / kg to 50 mg / kg body weight. In some embodiments, the TNFα and / or NGF antibodies may be administered in an amount of 0.5 mg / kg body weight. In some embodiments, the TNFα and / or NGF antibodies may be administered in an amount of 2 mg / kg body weight.

[0125] The TNFα and / or NGF antibody, or pharmaceutical composition comprising the TNFα and / or NGF antibody, may be administered to the companion animal at one time or over a series of treatments. For example, the TNFα and / or NGF antibody, or pharmaceutical composition comprising the TNFα and / or NGF antibody, may be administered at least once, more than once, at least two times, at least three times, at least four times, or at least five times.

[0126] In some embodiments, the dose is administered once per week for at least two or three consecutive weeks, and in some embodiments, this treatment cycle is repeated two or more times, optionally interspersed with one or more weeks of treatment-free periods. In other embodiments, the therapeutically effective dose is administered once per day for two to five consecutive days, and in some embodiments, this treatment cycle is repeated two or more times, optionally interspersed with one or more days or weeks of treatment-free periods.

[0127] Administration of one or more additional therapeutic agents "in combination with" includes simultaneous (concurrent) and sequential or sequential administration in any order. The term "concurrent" is used herein to refer to administration of two or more therapeutic agents, where at least a portion of the administration overlaps in time or where administration of one therapeutic agent is within a short time period relative to administration of another therapeutic agent. For example, two or more therapeutic agents are administered at a time interval of about a certain number of minutes or less. The term "sequential" is used herein to refer to administration of two or more therapeutic agents, where administration of one or more agents continues after administration of one or more other agents is stopped, or administration of one or more agents is initiated before administration of one or more other agents. For example, administration of two or more therapeutic agents is administered at a time interval of about more than a certain number of minutes. As used herein, "in conjunction with" refers to administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" refers to administration of one therapeutic modality before, during, or after administration of another therapeutic modality to an animal.

[0128] In some embodiments, the methods include administering an IL17 antibody, an IL-5 antibody, an IL-31 antibody, an IL4 antibody, an IL13 antibody, an IL23 antibody, an IgE antibody, a CD11α antibody, an IL6R antibody, an α4-intergrin antibody, an IL12 antibody, an IL1β antibody, or an anti-BlyS antibody in combination with a TNFα and / or NGF antibody, or a pharmaceutical composition comprising a TNFα and / or NGF antibody.

[0129] Provided herein are methods of exposing cells to TNFα and / or NGF antibodies, or pharmaceutical compositions comprising TNFα and / or NGF antibodies, under conditions that permit binding of the antibodies to TNFα and / or NGF. In some embodiments, the cells are exposed to the antibodies or pharmaceutical compositions ex vivo. In some embodiments, the cells are exposed to the antibodies or pharmaceutical compositions in vivo. In some embodiments, the cells are exposed to the TNFα and / or NGF antibodies or pharmaceutical compositions under conditions that permit binding of the antibodies to intracellular TNFα and / or NGF. In some embodiments, the cells are exposed to the TNFα and / or NGF antibodies or pharmaceutical compositions under conditions that permit binding of the antibodies to extracellular TNFα and / or NGF.

[0130] In some embodiments, cells may be exposed to the TNFα and / or NGF antibody or pharmaceutical composition in vivo by any one or more of the administration methods described herein, including, but not limited to, intraperitoneal, intramuscular, or intravenous injection into a subject. In some embodiments, cells may be exposed to the TNFα and / or NGF antibody or pharmaceutical composition ex vivo by exposing the cells to culture medium containing the antibody or pharmaceutical composition. In some embodiments, the permeability of the cell membrane may be affected by the use of any number of methods understood by one of skill in the art, such as electroporation of the cells or exposure of the cells to a solution containing calcium chloride, prior to exposing the cells to culture medium containing the antibody or pharmaceutical composition.

[0131] In some embodiments, binding results in a decrease in TNFα and / or NGF signaling function by the cell. In some embodiments, the TNFα and / or NGF antibody may decrease TNFα and / or NGF signaling function in a cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to TNFα and / or NGF signaling function in the absence of the antibody.In some embodiments, the reduction in TNFα and / or NGF signaling function is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and between 60%, between 15% and 70%, between 15% and 80%, between 15% and 90%, between 15% and 100%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and 80%, between 20% and 90%, between 20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 25% and 60%, between 25% and 70%, between 25% and 80%, 25% between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80%, 4 between 0% and 90%, between 40% and 100%, between 45% and 50%, between 45% and 60%, between 45% and 70%, between 45% and 80%, between 45% and 90%, between 45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and 100%, between 60% and 70%, between 60% and 80%, between 60% and 90%, between 60% and 100%, between 70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and 90%, between 80% and 100%, or between 90% and 100%.

[0132] Provided herein are methods of using TNFα and / or NGF antibodies, polypeptides, and polynucleotides for the detection, diagnosis, and monitoring of conditions associated with TNFα and / or NGF. Provided herein are methods of determining whether a companion animal will respond to TNFα and / or NGF antibody therapy. In some embodiments, the methods include using TNFα and / or NGF antibodies to detect whether the animal has cells that express TNFα and / or NGF. In some embodiments, the method of detection includes contacting a sample with an antibody, polypeptide, or polynucleotide and determining whether the level of binding differs from that of a reference or comparative sample (such as a control). In some embodiments, the methods may be useful for determining whether an antibody or polypeptide described herein is an appropriate therapeutic agent for a subject animal.

[0133] In some embodiments, the sample is a biological sample. The term "biological sample" refers to a quantity of material from a living organism or formerly living organism. In some embodiments, the biological sample is a cell or cell / tissue lysate. In some embodiments, biological samples include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, synovial fluid, and epithelial cells.

[0134] In some embodiments, the cells or cell / tissue lysates are contacted with a TNFα and / or NGF antibody and binding between the antibody and the cells is determined. If the test cells show binding activity compared to reference cells of the same tissue type, it may indicate that the subject would benefit from treatment with a TNFα and / or NGF antibody. In some embodiments, the test cells are from a tissue of a companion animal.

[0135] Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays that can be performed include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), turbidimetric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). Indicator moieties, or label groups, can be attached to the antibody of interest and are selected to meet the needs of the various methods used, which are often determined by the availability of assay equipment and compatible immunoassay procedures. Suitable labels include, but are not limited to, radionuclides (e.g., 125 I, 131 I, 35 S, 3 H, or 32 P), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or p-glucosidase), a fluorescent moiety or protein (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or a luminescent moiety (e.g., Qdot supplied by Quantum Dot Corporation, Palo Alto, Calif. TM General techniques to be used in performing the various immunoassays noted above are known to those of skill in the art.

[0136] For diagnostic purposes, polypeptides, including antibodies, can be labeled with detectable moieties, including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels known in the art. Methods for conjugating labels to antibodies are known in the art. In some embodiments, the TNFα and / or NGF antibodies need not be labeled, and their presence can be detected using a second, labeled antibody that binds to the first TNFα and / or NGF antibody. In some embodiments, the TNFα and / or NGF antibodies can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). TNFα and / or NGF antibodies and polypeptides can also be used in in vivo diagnostic assays, such as in vivo imaging. Generally, the antibodies or polypeptides are coupled to a radionuclide (e.g., a 5'-diaminobutyric acid ester) so that the location of cells or tissues of interest can be identified using immunoscintigraphy. 111 In, 99 Tc, 14 C. 131 I, 125 I, 3 3H, or any other radionuclide label including those outlined herein. The antibodies may be used as staining reagents in pathology using techniques well known in the art.

[0137] In some embodiments, the first antibody is used as a diagnostic and the second antibody is used as a therapeutic. In some embodiments, the first antibody and the second antibody are different. In some embodiments, the first antibody and the second antibody can both bind to the antigen simultaneously by binding to distinct epitopes.

[0138] The following examples illustrate certain aspects of the disclosure and are not intended to limit the disclosure in any way. EXAMPLES

[0139] Example 1 Synthesis and purification of caninized TNFα D2E7 antibody from CHO cells A DNA sequence encoding the caninized TNFα D2E7 antibody is chemically synthesized and inserted into an expression vector suitable for transfection into CHO host cells and secretion of the light or heavy chain proteins or both from the cells. The expression vector is transfected into CHO cells. CHO cells are selected for high yield and stability of expression of the TNFα antibody or components thereof, optionally using a DHFR gene on the expression vector and methotrexate-mediated gene amplification, as known in the art. The CHO cells are cultured until sufficient amounts of the TNFα antibody are produced. The TNFα antibody is purified by one or more of a variety of steps including Protein A column chromatography, Protein G column chromatography, Protein L column chromatography, or other chromatographic methods, such as ion exchange column chromatography, hydrophobic interaction column chromatography, mixed mode column chromatography such as CHT, and / or multimodal mode column chromatography such as CaptoMMC. Low pH or other viral inactivation and removal steps can be applied. The purified protein is mixed with additives and sterilized by filtration to prepare the pharmaceutical composition of the invention. The pharmaceutical composition is administered to a dog, cat, or horse having a condition associated with TNFα in a dosage sufficient to bind and inhibit TNFα.

[0140] More specifically, the variable light chain of the TNFα antibody D2E7 (SEQ ID NO:7) was caninized to SEQ ID NO:14 (KBA VL v1) and SEQ ID NO:18 (KBA VL v2), and the variable heavy chain of the TNFα antibody D2E7 (SEQ ID NO:8) was caninized to SEQ ID NO:22 (KBA VH). DNA was synthesized to provide the translated protein sequences and leader sequences shown as SEQ ID NO:15 (KBA VL v1 and canine kappa light chain constant region), SEQ ID NO:19 (KBA VL v2 and canine kappa light chain constant region), and SEQ ID NO:23 (caninized variable heavy chain of D2E7 and canine IgG-B constant region) and used to prepare the respective expression vectors. The light chain expression vectors had a different selection marker than that of the heavy chain. To facilitate the identification of clones efficiently expressing the corresponding TNFα antibody, the light and heavy chain vectors were co-transfected in various ratios.

[0141] Next, use Vector to create the FreestyleMax TM Pilot-scale transfections (1 L working volume) were performed in CHO-KS cells using transfection reagent (Life Technologies). Supernatants were collected by clarifying the conditioned medium when cell viability dropped below 80%. Proteins were purified with a single-pass Protein A chromatography step. From a 1 L pilot-scale transient production, 12.2 mg of purified KBA v1 was obtained, along with 6.0 mg of KBA v2 at a lower titer. Both caninized versions of KBA v1 and KBA v2 expressed low titers compared to the typical titers of about 200 to 500 mg / L for other antibodies obtained in the lab. Purified KBA v1 and KBA v2 were analyzed by SDS-PAGE and HPLC gel filtration and appeared suitable for use. KBA v1 was further selected to generate a stable cell line, but low productivity was observed.

[0142] Example 2 Demonstration of TNFα binding activity This example demonstrates that the TNFα antibodies described herein, designated as TNFα antibodies KBA v1 (SEQ ID NO:15 and SEQ ID NO:23) and KBA v2 (SEQ ID NO:19 and SEQ ID NO:23), also referred to herein as KIND-509, bind to TNFα with kinetics required for therapeutic activity.

[0143] Binding analysis was performed as follows: Briefly, canine and human TNFα were biotinylated. Free unreacted biotin was removed from the biotinylated TNFα by extensive dialysis. Biotinylated TNFα was captured on a streptavidin sensor tip. Binding of TNFα antibodies at five different concentrations (150, 50, 17, 5.6, and 1.9 nM) to TNFα (human and canine in different studies) was monitored for 90 seconds. Dissociation was monitored for 600 seconds. A buffer-only blank curve was subtracted to correct for drift. ForteBio TM Using data analysis software, the data were fitted to a 1:1 binding model to determine k (association rate constant), k (dissociation rate constant), and K (dissociation constant). Binding statistics fell within acceptable parameters (chi-squared ≤ 3.0; R-squared ≥ 0.9). The buffer for dilution and all binding steps was 20 mM phosphate, 150 mM NaCl, 0.02% Tween-20, 0.05% sodium azide, and 0.1 mM BSA, pH 7.4.

[0144] Canine TNFα was from Sino Biological, catalog #7003-DNAE, lot #LC05JU2002; human TNFα was from Sigma, catalog #T6674; EZ-Link NHS-LC-Biotin was from Thermo Scientific, catalog #21336, lot #PB194183; and StreptAvidin biosensor was from ForteBio, catalog #18-509, lot #1403251.

[0145] The binding kinetics were as follows: For the ligand canine TNFα, the KD(M) of KBA v1 was 9.88×10 -11and 1.54 × 10 for adalimumab. -10 kon(1 / Ms) is 8.39×10 5 and 7.22 × 10 for adalimumab. 5 koff(1 / s) is 8.28×10 -5 and 1.12 × 10 for adalimumab. -4 Rmax(nm) was 0.77 for KBA v1 and 0.76 for adalimumab; total chi-square was 0.14 for both KBA v1 and adalimumab; and total R-square was 1 for both KBA v1 and adalimumab. For KBA v2, these values ​​correspond to a KD of 3.03×10 -10 ;kon 7.13×10 5 ;koff 2.16×10 -4 ; Rmax 0.71; total chi-squared 0.38; and total R-squared 1. Thus, KBA v1 has a higher affinity for canine TNFα than KBA v2.

[0146] For the ligand human TNFα, the KD(M) of KBA v1 is 2.07×10 -11 and 3.06 × 10 for adalimumab. -11 kon(1 / Ms) is 7.41×10 5 and 6.74 × 10 for adalimumab. 5 koff(1 / s) is 1.53×10 -5 and 2.06 × 10 for adalimumab. -5 Rmax(nm) was 1.05 for KBA v1 and 1.06 for adalimumab; total chi-square was 0.30 for KBA v1 and 0.27 for adalimumab; and total R-square was 1 for both KBA v1 and adalimumab. For KBA v2, these values ​​were 0.43×10 for KD 2.69×10 -10 ;kon 5.75×10 5 ;koff 1.50×10 -4 ; Rmax 0.98; total chi-square 0.23; and total R-square 1.

[0147] TIFF2024507318000015.tif34170

[0148] Example 3 Identifying caninization issues that affect antibody production A chimeric D2E7 antibody comprising a chimeric variable light chain of D2E7 and canine kappa (SEQ ID NO: 10) and a chimeric variable heavy chain of D2E7 and canine IgG-B (SEQ ID NO: 12) was prepared according to the method of Example 1. Typical expression levels were observed from 1 L pilot-scale transient production. Thus, canine kappa and canine IgG-B did not appear to affect production.

[0149] To determine whether the caninized variable light chain or the caninized variable heavy chain affected antibody production, antibodies comprising a caninized light chain paired with a chimeric heavy chain and a chimeric light chain paired with a caninized heavy chain were prepared according to the method of Example 1. Specifically, a caninized variable heavy chain and a canine IgG-B (SEQ ID NO: 23) were paired with a chimeric variable light chain of D2E7 and a canine kappa (SEQ ID NO: 10). The 1L pilot scale production titers were typical, indicating that the caninization of the variable heavy chain did not affect antibody production. However, when KBA VL v1 and a canine kappa (SEQ ID NO: 15) were paired with a chimeric variable heavy chain of D2E7 and a canine IgG-B (SEQ ID NO: 12), the resulting 1L pilot scale antibody production titers were low. In addition, KBA VL v2 and canine kappa (SEQ ID NO:19) paired with D2E7 and canine IgG-B chimeric variable heavy chains (SEQ ID NO:12) had undetectable or very low 1 L pilot scale production titers. The low titers of these latter two antibodies suggest that a significant reduction in antibody production was observed upon caninization of the variable light chain. Despite the low production titers, the antibodies maintained TNFα binding activity.

[0150] Example 4 Caninization of variable light chains to restore antibody production and maintain TNFα binding activity The amino acids and positions of the variable light chain that affect antibody production were investigated by preparing a series of antibodies with different caninized variable light chains. Specifically, the following additional caninized variable light chains of D2E7 were designed: KBA VL v3 (SEQ ID NO: 25); KBA VL v4 (SEQ ID NO: 29); KBA VL v5 (SEQ ID NO: 33); KBA VL v6 (SEQ ID NO: 37); and KBA VL v7 (SEQ ID NO: 43). Each of KBA VL v3-v7 and canine kappa (SEQ ID NO: 26, 30, 34, 38, and 44, respectively) was paired with the chimeric variable heavy chain of D2E7 and canine IgG-B (SEQ ID NO: 12) or the caninized variable heavy chain and canine IgG-B (SEQ ID NO: 23), and the resulting 1L pilot-scale transient antibody production in CHO cells was accessed. Antibodies containing KBA VL v7 (SEQ ID NO:43) showed restored productivity and maintained binding activity to canine and human TNFα.

[0151] Furthermore, when the chimeric variable light chain of D2E7 and canine lambda (SEQ ID NO: 41) was paired with the chimeric variable heavy chain of D2E7 and canine IgG-B (SEQ ID NO: 12) or the caninized variable heavy chain and canine IgG-B (SEQ ID NO: 23), the resulting 1 L pilot scale antibody production titers showed no improvement.

[0152] Stable CHO cell lines were generated expressing a TNFα antibody containing KBA VL v7 and canine kappa (SEQ ID NO: 44) and a canine variable heavy chain and canine IgG-B (SEQ ID NO: 23). Antibody production titers in shake flasks exceeded 4g / L.

[0153] The light chain framework region 2 appears to affect antibody production. In particular, two residues, glutamine (Q) at position 3 of the light chain framework region 2 (LC-FR2) and lysine (K) at position 8 of LC-FR2, are important for the expression of the caninized D2E7 light chain in anti-TNFα antibody production.

[0154] Example 5 Additional Exemplary Caninized TNFα Antibodies Additional caninized variable light chains of D2E7 can be designed with a glutamine (Q) at position 3 and a lysine (K) at position 8 of LC-FR2 (e.g., SEQ ID NO: 45). For example, another exemplary caninized variable light chain of D2E7 can be represented by SEQ ID NO: 46 (KBA VL v8).

[0155] Example 6 Trials for the treatment of canine IBD using TNFα antibodies A multi-site, randomized, blinded, placebo-controlled pilot clinical field study will be conducted to evaluate the efficacy and safety of KIND-509 for the management of inflammatory bowel disease (IBD) in client-owned dogs.

[0156] The study includes dogs of any age, breed, weight, or sex that have been diagnosed with IBD and meet the inclusion and exclusion criteria set forth below. The first dose of caninized TNFα antibody (e.g., KIND-509 or any other caninized TNFα antibody described herein) administered is 2 mg / kg based on body weight (BW) determined at Visit 1 (Day 0) and rounded to the nearest tenth. All subsequent doses of caninized TNFα antibody are 1 mg / kg based on BW determined at Visits 2, 3, and 4 and rounded to the nearest tenth. Dose volumes of control product (CP, e.g., PBS or formulation buffer) are matched to those of the treatment groups. All dogs receive four doses of either caninized TNFα antibody or CP, with the first dose administered at Visit 1 (Day 0). Subsequent doses are administered at Visits 2, 3, and 4 every 7 (±2) days.

[0157] Histopathological diagnosis of endoscopic gastrointestinal biopsies will be used to determine the efficacy of caninized TNFα antibodies in the management of IBD.

[0158] [Containment and General Control]

[0159] Enrolled dogs live with their owners and spend at least 60% of their time in the home. Only one dog at a time from a multi-dog household will be enrolled in the study. Boarding should be avoided during the study, and if boarding is necessary, it should ideally occur at the study site.

[0160] Water: Water is provided at the owner's discretion on a schedule and in amounts habitual to the dog. No sampling or analysis of water is required.

[0161] Diet: A food trial of at least 14 consecutive days with a specific diet will be completed within 90 days prior to the screening visit to exclude food-responsive enteropathy. During the food trial, only the prescription diet will be fed. The investigator will determine whether the food trial is sufficient to exclude food-responsive enteropathy. Details of the food trial will be recorded at the screening visit. A food trial is not required if the dog has a Canine Inflammatory Bowel Disease Activity Index (CIBDAI) score of ≥ 6 and the investigator determines that the dog has unstable disease (e.g., fever, rapid weight loss, loss of appetite, etc.).

[0162] Ideally, when dogs are screened for the study they will be receiving the particular diet as their only diet, although this is not a requirement for study inclusion.

[0163] Diet brand and formulation will be recorded at the screening visit. There should be no significant changes in diet brand and formulation within 14 days prior to the screening visit or during the study. The investigator will determine whether there has been a significant diet change. At each subsequent visit, study site personnel will record whether the diet has changed significantly. The decision to terminate the study for dogs that have a significant diet change before Visit 5 will be made on a case-by-case basis in consultation with the sponsor.

[0164] [Inclusion criteria] Dogs: · Easy to manage and cooperative with testing procedures Meets the water specifications above A history of at least one symptom of IBD (weight loss, anorexia / anorexia, vomiting, or diarrhea) for at least 3 weeks prior to the screening visit and at least one symptom of IBD (weight loss, anorexia / anorexia, vomiting, or diarrhea) at the time of the screening visit Have a CIBDAI score of ≥4 (mild to severe disease) at the screening visit - Have been diagnosed with IBD based on histopathological evaluation of endoscopic gastrointestinal biopsies obtained at the screening visit - Treatment with fenbendazole (50 mg / kg) by oral administration (PO) for 3 consecutive days within 60 days prior to the screening visit Completed at least 2 weeks (14 consecutive days) of a dietary trial with a specific diet as deemed appropriate by the investigator within 90 days prior to the screening visit. This inclusion criterion is required if the dog has a CIBDAI score of ≥ 6 and the dog has an unstable illness (e.g., fever, rapid weight loss, loss of appetite, etc.) as determined by the investigator. · Have been treated with an anti-diarrheal antibiotic, such as metronidazole or tylosin, for at least 14 consecutive days within the 90 days prior to the screening visit; dogs receiving an anti-diarrheal antibiotic at the time of the screening visit must discontinue the antibiotic and wait 7 days prior to enrollment / randomization (Visit 1). This inclusion criterion is required if the dog has a CIBDAI score ≥ 6 and the investigator determines that the dog has an unstable illness (e.g., fever, rapid weight loss, loss of appetite, etc.). The investigator believes the patient has at least 6 months of life expectancy

[0165] [Exclusion Criteria] Dogs: - History of severe hypoalbuminemia (albumin < 2.0 mg / dL) or severe hypoalbuminemia (albumin < 2.0 mg / dL) at the time of the screening visit - History of pancreatitis diagnosed by ultrasound examination within 6 months prior to the screening visit - Having a history, suspicion, or diagnosis of exocrine pancreatic insufficiency - History of abdominal surgery within 6 months prior to screening visit Have clinically significant liver or kidney disease, neoplasms, diabetes, or other intercurrent illnesses that may interfere with study completion / study results. Have had clinical signs of or been diagnosed with an infection (other than gingivitis or periodontal disease) and / or been treated with an antibiotic for an infection (other than an antidiarrheal antibiotic) within 30 days prior to the screening visit - Suspected or diagnosed with hyperadrenocorticism, hypoadrenocorticism, or hypothyroidism - Have made significant dietary changes within 14 days prior to the screening visit (significance of dietary changes will be determined by the investigator) Have been treated with any of the following products: o Systemic nonsteroidal anti-inflammatory drugs within 14 days prior to the screening visit Pancreatic enzyme supplementation within 30 days prior to the screening visit o Use of systemic, topical, intraocular, or inhaled glucocorticoids or mineralocorticoids within 30 days prior to the screening visit Long-acting glucocorticoids within 90 days prior to screening visit o Cyclosporine, azathioprine, mycophenolate mofetil, or other immunosuppressants within 90 days prior to the screening visit Chlorambucil within 90 days prior to screening visit - Pregnant or lactating females or dogs intended for breeding - Be enrolled in another clinical trial within 30 days prior to the screening visit

[0166] Exclusion Criteria: The Investigator will complete the Study End eCRF for all screened dogs and record the termination date. Study end for each enrolled dog completing the study is defined as the conclusion of scheduled assessments and procedures and the last date on which data are collected from the dog or owner. Laboratory results will not be received until after the termination date. Visit 5 will be the study end visit for all randomized cases completing the study. If a dog is randomized and is removed before Visit 5, all procedures expected to be performed at Visit 5 will be performed to the extent possible. The reason for removal will be documented in the Study End eCRF.

[0167] [Criteria for Removal After Enrolment] Enrolled dogs may be removed early from further participation in the study for the following reasons: Significant changes in diet The dog requires treatment with a prohibited substance. Dogs experience adverse events that prevent continuation of the study The dog meets the removal criteria under the rescue clause below. Dogs no longer comply with testing procedures The dog was found to have been registered by mistake. Owners are not cooperative with the testing procedures Dogs miss one or more scheduled study visits, exceed required treatment intervals, or require monitoring procedures that prevent the response to treatment from being evaluated. Owners selectively withdrawing their dogs from the study The sponsor decides to stop the trial - Other reasons such as the owner being unable to contact them or moving.

[0168] When possible, the investigator and sponsor will consult before prematurely removing a dog from the study.

[0169] [Rescue clause] If the CIBDAI score at Visit 3 (Day 14 ± 2) is the same or higher than the CIBDAI score at the Screening Visit and, in the opinion of the Investigator, the IBD performance status has worsened, the dog will be removed from the study and treated with appropriate standard of care as directed by the Investigator. These dogs will be considered treatment failures.

[0170] Disposal and Withdrawal of Animals Dogs completing the study will be placed in the custody of their owners. After euthanasia or natural death (and post-mortem examination, if applicable), the remains will be returned to the owner, if desired, or disposed of in accordance with the study site's policies, if approved by the owner.

[0171] Study Schedule The screening phase of the study will be up to 10 days and the treatment phase of the study will be 28±2 days, resulting in a total study duration of 38±2 days. The schedule of events is outlined in Table 4 below. Unscheduled visits will be allowed whenever dogs are enrolled in the study for further evaluation or if needed due to adverse events.

[0172] TIFF2024507318000016.tif255170TIFF2024507318000017.tif255170

[0173] [Test procedure / evaluation]

[0174] Medical History: To distinguish adverse events from ongoing or pre-existing conditions, all significant conditions (including chronic or recurrent conditions) that occurred within the 12 months prior to the Screening Visit, and any significant events that occurred prior to that, will be recorded in the Medical History eCRF for each dog. This record will include a brief description of each condition, along with the dates of onset and resolution, if available. For conditions that are ongoing at the time of the Screening Visit, the date of resolution will be recorded during the study, if applicable, or they may be listed as ongoing.

[0175] As part of the medical history, the investigator will assess the dog's history of clinical signs associated with IBD, including but not limited to lethargy, dehydration, anorexia / anorexia, weight loss, vomiting, diarrhea, tenesmus, bloody stools, and / or mucus in the stool. The investigator will also classify the dog's diarrhea as small intestinal, large intestinal, or mixed intestinal diarrhea.

[0176] Physical Examination: The signalment of each dog screened for enrollment, including date of birth, breed, and sex (including whether intact or neutered), will be recorded on the Signalment eCRF.

[0177] Physical examinations (PE) will be performed at all scheduled study visits (Screening Visit and Visits 1-5) and at any unscheduled visits as needed. The examination will include general appearance and demeanor, hydration status, and subjective evaluation of the ear, ophthalm, oral cavity, mucous membranes, respiratory, cardiovascular, gastrointestinal, neurological, musculoskeletal, lymphatic, integumentary, and genitourinary systems. Physical examination results will be recorded on the Physical Examination eCRF. Abnormalities (excluding pre-existing conditions) and worsening abnormalities recorded after Visit 1 (Day 0) will be reviewed as described below and recorded as adverse events.

[0178] Vital Signs: Temperature (°F), heart rate (HR) (beats / min), and respiratory rate (RR) (breaths / min) will be assessed at each PE and recorded on the Physical Examination eCRF.

[0179] Body weight and scale: Body weight will be recorded in kilograms (kg) on ​​the Physical Examination eCRF during each PE. For all dogs, body weight should be measured in the fasting state, defined as BW taken at least 2 hours after a meal.

[0180] Body Condition Score: Body condition score (BCS) is measured as part of the PE. The Nestle PURINA Body Condition System (LaFlamme, DP, 1997) is utilized to assess BCS. BCS uses visualization and palpation to assess the dog's overall conformation along with the amount of fat coverage over the ribs, spine, and loins. The BCS is on a scale of 1 to 9; a score of 9 being an extremely overweight dog and a score of 1 being an extremely underweight dog. Each dog is preferably assessed by the same investigator at each visit.

[0181] Muscle Condition Score: Muscle condition score (MCS) will be measured as part of PE. The World Small Animal Veterinary Association (WSAVA) Muscle Condition Score System (WSAVA, 2014) will be utilized to assess MCS. MCS grades muscle mass as normal, mildly reduced, moderately reduced, or severely reduced using visualization and palpation of the spine, scapula, skull, and ileal wings. Each dog is preferably assessed by the same investigator at each visit.

[0182] Canine Inflammatory Bowel Disease Activity Index (CIBDAI) Score: The Canine Inflammatory Bowel Disease Activity Index (CIBDAI) is a scoring system developed to assess the activity / severity of canine IBD. Utilizing the CIBDAI, six gastrointestinal signs are scored from 0 to 3 in dogs with IBD compared to normal dogs. The scores from each of the six gastrointestinal signs are summed to create a cumulative CIBDAI score. Based on the CIBDAI score, IBD is clinically classified as treatment-negative, mild, moderate, or severe (Appendix 1).

[0183] At each visit, the investigator will complete the CIBDAI score eCRF. The investigator will complete the CIBDAI score taking into account the owner's report / assessment of clinical signs, PE, changes in BW, etc. The investigator will evaluate behavior / activity, appetite, vomiting, stool consistency, stool frequency, and weight loss compared to the dog's normal state at the screening visit. At all subsequent visits, the investigator will score behavior / activity, appetite, vomiting, stool consistency, stool frequency, and weight loss compared to baseline. Preferably, the same owner is interviewed and the same investigator will complete the CIBDAI score eCRF at each visit.

[0184] Owner Assessment: At the screening visit, the owner completes the Owner Assessment-Baseline CRF for the dog's clinical signs (activity, appetite, vomiting, stool consistency, defecation frequency) and overall quality of life (QoL) that best represent the dog's clinical signs and QoL over the two weeks prior to the screening visit. At each subsequent scheduled visit, the owner completes an Owner Assessment CRF to record changes in the dog's clinical signs since the screening visit. The Owner Assessment-Baseline CRF and Owner Assessment CRF are transcribed by study site personnel into the Owner Assessment-Baseline eCRF and Owner Assessment eCRF. Preferably, the same owner completes the assessments at each visit. If the dog is fed within the scheduled visit time, the Owner Assessment is not completed and an explanation is recorded in the File Note (NTF).

[0185] Stool consistency will be graded according to the Purina fecal scoring system (VET 1502B-06FSC / 15-6843, Societe des Produits Nestle SA, Vevey, Switzerland). At the screening visit, owners will be shown the Purina fecal score chart and will report the fecal score that best represents their dog's stool consistency over the two weeks prior to the screening visit. At each subsequent visit, owners will be shown the Purina fecal score chart and will report the fecal score that best represents their dog's stool consistency since the previous visit.

[0186] [Clinical pathology]

[0187] Clinical Pathology at Central Laboratory: Blood will be collected by venipuncture at multiple study visits specified in Table 3 and at unscheduled visits as needed. Blood and urine samples will be processed and sent according to central laboratory guidance. At the screening visit, blood samples will be collected after a >8 hour fast. At Visit 3 and Visit 5, blood samples will be collected after a >6 hour fast. All blood samples will be collected prior to administration of caninized TNFα antibody or CP and cyanocobalamin.

[0188] The following tests will be performed in the central laboratory:

[0189] Hematology (completed at Screening Visit and Visits 3 and 5): Total white blood cell count (WBC), differential white blood cell count, red blood cell count (RBC), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin content (MCHC), hemoglobin (Hgb), and platelet count.

[0190] Biochemistry (completed at the Screening Visit and Visits 3 and 5): Amylase, alanine aminotransferase (ALT), lipase, alkaline phosphatase (ALP), cholesterol, serum aspartate aminotransferase (AST), creatinine, total protein, glucose, albumin, calcium, globulin, sodium, total bilirubin, chloride, potassium, blood urea nitrogen (BUN), phosphorus, creatine phosphokinase (CPK), magnesium, gamma-glutamyltransferase (GGT), and triglycerides.

[0191] Cortisol (completed at screening visit): Blood will be collected by venipuncture at the screening visit for assessment of cortisol concentrations. Blood samples will be processed and sent according to central laboratory guidance.

[0192] Trypsin-Like Immunoreactivity (TLI), Cobalamin, and Folate (Completed at Screening Visit): Blood will be collected by venipuncture at the Screening Visit for assessment of TLI, cobalamin, and folate concentrations. Blood samples will be processed and sent per central laboratory guidance. Dogs will receive a cyanocobalamin injection following blood collection at the Screening Visit. Dogs with low cobalamin concentrations based on results at the Screening Visit will be treated with cyanocobalamin supplementation as further described below.

[0193] Urinalysis (UA) and urine culture ± sensitivity (completed at Screening Visit and Visit 5 (if urinalysis) and completed at Screening Visit (urine culture ± sensitivity)): Complete UA (including urine specific gravity, dip strip and urine sediment evaluation) and urine culture ± sensitivity will be assessed. Urine is ideally obtained by cystocentesis, but if cystocentesis is not possible, urine can be obtained by catheterization or voiding.

[0194] Additional laboratory evaluations may be completed at the discretion of the investigator.

[0195] Histoplasma Antigen Enzyme Immunoassay: At the screening visit, a minimum of 1 mL of urine will be collected for Histoplasma antigen enzyme immunoassay (EIA).

[0196] Abdominal Ultrasound: An abdominal ultrasound will be performed at the screening visit prior to colonoscopy, anesthesia, and preparation for gastroduodenoscopy and colonoscopy.

[0197] Gastroduodenoscopy and colonoscopy and endoscopic gastrointestinal biopsies: Gastroduodenoscopy and colonoscopy are performed at the screening visit. At least 6 endoscopic biopsies are taken from the stomach, duodenum, and colon. If possible, the ileum is evaluated endoscopically and biopsies are taken under endoscopic guidance or blinded endoscopic examination. The screening visit may take place over 2 to 4 days (not necessarily consecutive) to allow for the completion and review of diagnostic testing required before proceeding with colonoscopy, anesthesia, and preparation for gastroduodenoscopy and colonoscopy, and to allow for preparation for colonoscopy. Pending diagnostic testing, owners may take their dog home and return 1 to 2 days later to begin preparation for colonoscopy.

[0198] Prior to preparation for colonoscopy, a screening visit signalment, medical history, PE, CIBDAI score, owner evaluation, and abdominal ultrasound are completed. In addition to the above, hematology, biochemistry, and cortisol are also completed and reviewed prior to performing anesthesia, gastroduodenoscopy, and colonoscopy.

[0199] All requirements for the Screening visit will be completed within a time frame that allows for receipt and review of histopathology by Visit 1 (Day 0).

[0200] Histopathology of endoscopic gastrointestinal biopsies: Endoscopic gastrointestinal biopsies will be submitted to a pathologist of the investigator's choice for histopathological evaluation. A histopathological diagnosis of idiopathic IBD (e.g., lymphoplasmacytic enteritis, eosinophilic enteritis, etc.) is required for dogs to be eligible for enrollment in the study.

[0201] Concomitant medications and therapies: Any medications knowingly prescribed or administered from 1 month prior to the screening visit through completion will be considered concomitant medications. Medications administered prior to study initiation will be identified to ensure that dogs meet the study inclusion criteria.

[0202] The Clinical Development Manager or designated study monitor will be contacted prior to administration of any concomitant medication with unclear acceptable use.

[0203] Medications Allowed: New medications that are not expected to interfere with treatment will be allowed during the study. This may include, but is not limited to, the following: Fluid therapy (lactated Ringer's, 0.9% NaCl, etc.) Antibiotics not intended to treat diarrhea Antihistamines (e.g. diphenhydramine) Preventive treatments (e.g. flea treatments, heartworm prevention, etc.)

[0204] Conditionally Allowed Medications: Existing medications will be allowed if there are no prescription changes in the two weeks prior to the screening visit and during the study. This includes, but is limited to, the following: Antiemetics (e.g., metoclopramide, ondansetron, maropitant, mirtazapine, etc.) Antacids (e.g. omeprazole, famotidine, ranitidine, cimetidine, etc.) Prokinetic drugs (e.g. cisapride, erythromycin, metoclopramide, ranitidine, etc.) Probiotics (e.g., Fortiflora, Proviable, etc.) Prescription opioids for pain relief (e.g. tramadol, buprenorphine, etc.)

[0205] Antiemetics administered during a screening visit for endoscopy are an exception to the requirements for existing medications. Antiemetic administration is permitted at the time of endoscopy during the screening visit.

[0206] Cyanocobalamin Supplementation: All dogs will receive a subcutaneous (SQ) injection of cyanocobalamin 25 μg / kg after a blood sample is taken at the screening visit. Dogs determined to be cobalamin deficient based on cobalamin concentrations at the screening visit will receive cyanocobalamin 25 μg / kg SQ at Visits 1, 2, 3, and 4. The dose of cyanocobalamin at any visit should not exceed a total dose of 1500 μg. Cyanocobalamin will be provided to the study site by the sponsor.

[0207] Prohibited Medication: Medications that are expected to interfere with the treatment and / or clinical signs of IBD will not be permitted during the study. This may include, but may not be limited to, the following medications: · vaccination Glucocorticoids (e.g., prednisone, prednisolone, dexamethasone, triamcinolone, betamethasone, etc.) Mineralocorticoids (e.g. desoxycorticosterone pivalate, fludrocortisone, etc.) Immunosuppressants (e.g. cyclosporine, azathioprine, mycophenolate mofetil, etc.) Alkylating agents (e.g., chlorambucil, etc.) Systemic nonsteroidal anti-inflammatory drugs (e.g., carprofen, deracoxib, etc.) Antidiarrheal antibiotics (e.g., metronidazole, tylosin, sulfasalazine, etc.) Antidiarrheal medications (e.g. loperamide, lomotil, etc.) Pancreatic enzyme supplements

[0208] Owner Diary: Owners will record any untoward or unexpected events observed throughout the study from Screening Visit through Visit 5 / End of Study on the Owner Diary: Observational Events CRF. The Owner Diary: Observational Events CRF will be reviewed by the Investigator at each visit. Observations recorded by owners prior to Visit 1 will not be recorded as adverse events. For each observation recorded by owners after Visit 1, the Investigator will indicate whether the observation constitutes an adverse event as defined below.

[0209] Adverse Event: An adverse event (or AE) is any finding in a dog that is untoward, unintended, and occurs following the use of a veterinary drug, whether or not it is considered IVP related. An event that fits this description is considered an AE if it occurs at or between the time of the first treatment administration at Visit 1 (Day 0) and the end of the study.

[0210] New clinically significant (CS) laboratory results (or corresponding diseases / conditions) determined from samples taken after the dog received study treatment will be recorded as AEs on the Adverse Events eCRF for each case. Non-clinically significant (NCS) laboratory results will not be recorded as AEs.

[0211] Using the Adverse Event eCRF, the investigator will document and describe the AE, including clinical signs / abnormalities, start / end dates or ongoing, effect of study drug administered, assessment of the relationship of the AE to the study drug, severity of the event, reason for being considered an SAE (if applicable), and outcome of the event. The sponsor will code each AE using the Veterinary Dictionary for Drug Regulatory Authorities (VeDDRA) terminology. The sponsor will evaluate AEs on a case-by-case basis at the end of the study and perform a final classification as serious or non-serious.

[0212] The investigator will contact the monitor about any SAEs and / or if an unusually high frequency of non-serious AEs is observed.

[0213] Serious Adverse Event (SAE): An SAE is any AE that is deemed by the Investigator to be fatal or life-threatening or requiring specialist intervention, clinically serious, or that causes miscarriage, stillbirth, infertility, birth defects, or long-term or permanent disability or disfigurement. Owners will contact the Investigator as soon as reasonably possible after the occurrence of an SAE. The Investigator will evaluate the case and determine the need for treatment. A description of the SAE will be provided in the Adverse Event eCRF.

[0214] Causality Assessment: Investigators will complete a causality assessment for each AE using the following criteria:

[0215] Category A - Likely: All of the following apply: · There is a reasonable temporal relationship between the administration of IVP and the onset and duration of the reported event; · The description of the clinical phenomenon should be consistent with, or at least plausible to, the known pharmacology and toxicology of a given product; There should be no equally plausible explanation for the case. In particular, the concurrent use (and possible interactions) of other products or intercurrent illnesses should be taken into account in the evaluation.

[0216] Category B - Possible: The causal relationship is one of the (other) possible, plausible causes of the described AE, but the data do not meet the criteria for inclusion in Category A.

[0217] Category O – Unclassifiable / Not evaluable: Reliable data regarding the AE are not available or are insufficient to allow an assessment of causality.

[0218] Category O1 - Inconclusive: The association with IVP cannot be discounted, but other factors make it impossible to draw a conclusion.

[0219] Category N - Unlikely: Sufficient information exists to establish beyond a reasonable doubt that an alternative explanation exists for the AE that is not related to IVP.

[0220] [Autopsy and histopathology]

[0221] If necessary, necropsies may be performed on enrolled dogs that die before study completion to determine the cause of death or morbidity leading to euthanasia, but only after obtaining voluntary written consent from the owners. Necropsies may include a complete gross examination for pathological changes and tissue sampling for histopathology.

[0222] Tissues taken at necropsy for histopathology may include the heart chambers, lungs, liver, gallbladder, stomach, duodenum, jejunum, ileum, colon / rectum, spleen, left and right kidneys, adrenal glands, bladder, and any other tissues deemed necessary.

[0223] [Analysis population]

[0224] Efficacy Population: The efficacy population consisted of all enrolled dogs that were eligible for efficacy analyses. Eligibility criteria included study completion including Visit 5. Ineligibility criteria included discontinuation for reasons other than safety and efficacy, and protocol violations affecting efficacy assessments. All efficacy analyses were based on the efficacy population. The inclusion or exclusion of efficacy data for each dog that terminated from the study prior to the primary efficacy endpoint was determined on a case-by-case basis.

[0225] Safety Population: The safety population consists of all enrolled dogs that received at least one dose of caninized TNFα antibody or CP. Concomitant medications, AEs, and IVP exposures will be summarized based on the safety population.

[0226] [Efficacy outcomes]

[0227] Primary Variable: Reduction in CIBDAI score was the primary efficacy variable. Disease remission and response were defined as: Complete remission: 75% or more decrease in CIBDAI score or a CIBDAI score of less than 4 Partial remission: A decrease in CIBDAI score of 25% or more but less than 75% No response: <25% decrease in CIBDAI score, no change in CIBDAI score, or increase in CIBDAI score

[0228] Response or remission will be assessed at Visit 5 compared to the CIBDAI score obtained at the Screening Visit. Additional exploratory statistical analyses of components of the CIBDAI score will be performed at the sponsor's discretion.

[0229] The effect of caninized TNFα antibody on the percentage of dogs with disease response and the percentage of dogs in remission will be assessed using Fisher's exact test (FREQ procedure of SAS, SAS Institute, Cary NC; version 9.4 or higher). In addition, CIBDAI scores collected over time will be subjected to a repeated measures analysis of covariance (RMANCOVA) with treatment group, time, and treatment-by-time interaction as fixed effects, and baseline values ​​included as covariates. A compound symmetry structure will be assumed for the covariance matrix. Site will be ignored in this analysis due to the small sample size. If the interaction is significant, the within-time treatment effect will be assessed. If the interaction is not significant, the main effect of treatment will be assessed.

[0230] Secondary Variables: Additional variables will be assessed such as improvement / increase in BW, improvement / increase in BCS, improvement / increase in MCS, increase in appetite, decrease in vomiting, decrease in stool frequency, improvement in fecal score, etc. These results will be summarized as appropriate and may be subjected to statistical analysis at the sponsor's discretion.

[0231] [Safety outcomes]

[0232] Adverse Events and Concomitant Medications: Adverse events and concomitant medication data will be summarized and tabulated for final reporting.

[0233] Clinical Pathology Laboratory Data: Each clinical pathology laboratory variable will be summarized as appropriate. Urinalysis results will be summarized.

[0234] Vital signs, body weight data and categorical findings: Vital signs and body weight data will be assessed as above for clinical pathology outcomes. Outcomes that are categorical in nature, including AEs, will be summarized by frequency and number.

[0235] Example 7 Preliminary analysis of the treatment of canine IBD with TNFα antibodies Ten dogs diagnosed with IBD completed the study described in Example 6 through Visit 5. The primary efficacy variable was reduction in Canine Inflammatory Bowel Disease Activity Index (CIBDAI) scores, which were assessed at screening and at days 0, 7, 14, 21, and 28. Complete remission, defined as a 75% or greater reduction in mean post-dose CIBDAI score from baseline, was achieved in 75% of the TNFα antibody group (KIND-509 at a dose of 2 mg / kg) compared to 17% of the placebo group. Treatment efficacy was rapid and durable. At day 7, the first post-dose visit, 75% of the KIND-509-treated dogs showed a 75% or greater reduction in CIBDAI score from baseline compared to 17% of the placebo group. Additionally, 50% of dogs treated with KIND-509 achieved and maintained a 100% reduction in CIBDAI scores from baseline across all post-dosing visits, while no dogs in the placebo group achieved the same outcome.

[0236] Example 8 Design of caninized NGFαD11 antibody The variable light chain of the αD11 rat anti-NGF monoclonal antibody (SEQ ID NO: 71) was caninized to SEQ ID NO: 73, and the variable heavy chain of the αD11 rat anti-NGF monoclonal antibody (SEQ ID NO: 72) was caninized to SEQ ID NO: 74. This resulted in two amino acid changes in the variable heavy chain CDR-H1 (see SEQ ID NO: 75). Exemplary light and heavy chains comprising these caninized variable light and heavy chain sequences were also designed (SEQ ID NOs: 76-79).

[0237] Example 9 Expression and purification of a bispecific anti-canine TNF and anti-canine NGF molecule from CHO cells. Bispecific molecules were designed to simultaneously target TNF and NGF to reduce inflammation and pain associated with conditions such as osteoarthritis, chronic pain, back pain, cancer pain, and neuropathic pain.

[0238] An exemplary bispecific molecule designed comprises (1) a caninized anti-NGF variable light chain with a canine kappa light constant region (SEQ ID NO:77); and (2) a caninized anti-NGF variable heavy chain (SEQ ID NO:77), a variant canine IgG-B Fc engineered for reduced C1q and CD16 binding and long-acting, and SEQ ID NO:81, which comprises a single chain variable fragment (ScFv) of the caninized anti-TNF variable heavy chain (SEQ ID NO:22) and light chain (SEQ ID NO:43).

[0239] The molecules (SEQ ID NO: 77 and 81) were expressed from mammalian cells and purified by single-step Protein A column chromatography. Binding analysis was performed using the biosensor Octet. The Kd of the bispecific molecule containing SEQ ID NO: 77 and SEQ ID NO: 81 was 8.38 x 10 for canine TNF-α. -10 , which was within the single pM range for canine NGF.

[0240] A second exemplary bispecific molecule was reverse designed comprising (1) a caninized anti-TNF variable light chain with a canine kappa light constant region (SEQ ID NO: 44) and (2) a caninized anti-TNF variable heavy chain (SEQ ID NO: 22), a variant canine IgG-B Fc engineered for reduced C1q and CD16 binding and long-acting, and SEQ ID NO: 83, which comprises a single chain variable fragment (ScFv) of the caninized anti-NGF variable heavy chain (SEQ ID NO: 74) and light chain (SEQ ID NO: 73).

[0241] Surprisingly, no expression was observed with the reverse bispecific molecule (ie, comprising SEQ ID NOs: 44 and 83).

Claims

1. An isolated antibody that binds to canine TNFα, the antibody being a caninized antibody comprising: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:1; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:2; (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3; and (iv) a variable light chain comprising an LC-FR2 comprising glutamine at position 3 and lysine at position 8.

2. 2. The isolated antibody of claim 1, comprising a variable heavy chain comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:

6.

3. The isolated antibody of claim 1 or 2, wherein LC-FR2 comprises the amino acid sequence of SEQ ID NO:

45.

4. 4. The isolated antibody of claim 1 , comprising: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 46, or a variant thereof in which one, two, three, four, five, or six amino acids of the variable light chain are replaced by different amino acids; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof in which one, two, three, four, five, or six amino acids of the variable heavy chain are replaced by different amino acids; or (iii) the variable light chain of (i) and the variable heavy chain of (ii).

5. 5. The isolated antibody of claim 1 , comprising: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 46, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 46; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 22; or (iii) the variable light chain of (i) and the variable heavy chain of (ii).

6. An isolated antibody that binds to canine TNFα, comprising: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, or SEQ ID NO:46; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22; or (iii) a variable light chain of (i) and a variable heavy chain of (ii).

7. 1. An isolated antibody that binds to canine TNFα, (a) (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:49; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:50; (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:51; and (iv) a light chain comprising the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56, or a variant thereof in which 1, 2, 3, 4, 5, or 6 amino acids of the variable light chain are replaced by different amino acids; (b) (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:54; and (iv) a heavy chain comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58, or a variant thereof in which 1, 2, 3, 4, 5, or 6 amino acids of the variable heavy chain are replaced by different amino acids; or (c) the light chain of (a) and the heavy chain of (b).

2. An isolated antibody, which is a caninized antibody comprising:

8. 1. An isolated antibody that binds to canine TNFα, (a) a light chain comprising: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:49; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:50; (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:51; and (iv) a variable light chain comprising the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56; (b) (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:54; and (iv) a heavy chain comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58; or (c) the light chain of (a) and the heavy chain of (b).

2. An isolated antibody, which is a caninized antibody comprising:

9. 9. The isolated antibody of claim 1 , comprising a canine constant heavy chain region and / or a canine constant light chain region.

10. 10. The isolated antibody of any one of claims 1 to 9, comprising a canine heavy chain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region.

11. (i) a light chain comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, or SEQ ID NO:44, or a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, or SEQ ID NO:

44.

11. The isolated antibody of any one of claims 1 to 6, claims 9 or 10, comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:64, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:64; or (iii) a light chain of (i) and a heavy chain of (ii).

12. 12. An isolated antibody described in any one of claims 1 to 6 or claims 9 to 11, comprising: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 44; (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 64; or (iii) a light chain of (i) and a heavy chain of (ii).

13. 1. An isolated antibody that binds to canine TNFα, (a) (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:49; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:50; (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:51; and (iv) a light chain comprising the amino acid sequence of SEQ ID NO:59 or SEQ ID NO:60, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:59 or SEQ ID NO:60; (b) (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:54; and (iv) a heavy chain comprising the amino acid sequence of SEQ ID NO:61 or SEQ ID NO:62, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:61 or SEQ ID NO:62; or (c) the light chain of (a) and the heavy chain of (b).

2. An isolated antibody, which is a caninized antibody comprising:

14. An isolated antibody that binds to canine NGF, the antibody being a caninized antibody comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:

70.

15. 15. The isolated antibody of claim 14, further comprising: (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 65; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 66; and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

67.

16. 16. The isolated antibody of claim 14 or claim 15, comprising: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 73; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 74; or (iii) a variable light chain of (i) and a variable heavy chain of (ii).

17. 1. An isolated antibody that binds to canine NGF, the antibody being: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:73; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:74; or (iii) a caninized antibody comprising the variable light chain of (i) and the variable heavy chain of (ii).

18. 18. The isolated antibody of any one of claims 15 to 17, comprising: (i) a variable light chain sequence of SEQ ID NO: 73, or a variant thereof in which one, two, three, four, five, or six amino acids in the variable light chain are replaced by different amino acids; (ii) a variable heavy chain sequence of SEQ ID NO: 74, or a variant thereof in which one, two, three, four, five, or six amino acids in the variable heavy chain are replaced by different amino acids; or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii).

19. 19. The isolated antibody of any one of claims 15 to 18, comprising a canine constant heavy chain region and / or a canine constant light chain region.

20. 20. The isolated antibody of any one of claims 15 to 19, comprising a canine heavy chain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region.

21. 21. The isolated antibody of any one of claims 15 to 20, comprising: (i) a light chain sequence of SEQ ID NO: 77, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 77; (ii) a heavy chain sequence of SEQ ID NO: 79, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 79; or (iii) the light chain sequence of (i) and the heavy chain sequence of (ii).

22. 22. An isolated antibody described in any one of claims 15 to 21, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 77 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

79.

23. 23. The isolated antibody of any one of claims 1 to 22, which is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2, and Fab'-SH.

24. 1. An isolated antibody that binds to canine TNFα and canine NGF, comprising: (i) a variable light chain comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:55, or SEQ ID NO:56, or a variable light chain comprising at least the amino acid sequence of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:55, or SEQ ID NO:

56. (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:22, SEQ ID NO:57, or SEQ ID NO:58, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:22, SEQ ID NO:57, or SEQ ID NO:58; (iii) a variable light chain comprising the amino acid sequence of SEQ ID NO:73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:73; (iv) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:74; or (v) an isolated antibody comprising the variable light chain sequence of (i), the variable heavy chain sequence of (ii), the variable light chain sequence of (iii), and the variable heavy chain sequence of (iv).

25. (i) a variable light chain comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 43; (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 22; (iii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:

73.

25. The isolated antibody of claim 24, comprising: (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 74; (iv) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 74; or (v) the variable light chain sequence of (i), the variable heavy chain sequence of (ii), the variable light chain sequence of (iii), and the variable heavy chain sequence of (iv).

26. 26. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 25.

27. 27. A host cell comprising the nucleic acid of claim 26.

28. 30. A method for producing an antibody comprising culturing the host cell of claim 27 and isolating the antibody.

29. 26. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 25 and a pharma- ceutically acceptable carrier.

30. A method for treating a dog having a condition associated with TNFα, comprising administering to the dog a therapeutically effective amount of an antibody described in any one of claims 1 to 13 and claims 23 to 25 or a pharmaceutical composition described in claim 29.

31. A method for maintaining remission of a TNFα-associated condition in a dog, the method comprising administering to the dog a therapeutically effective amount of an antibody described in any one of claims 1 to 13 and claims 23 to 25 or a pharmaceutical composition described in claim 29; optionally, the TNFα-associated condition is an inflammatory disease, a gastrointestinal inflammatory disease, an inflammatory bowel disease, ankylosing spondylitis, asthma, cancer, Crohn's disease, idiopathic arthritis, psoriasis, plaque psoriasis, psoriatic arthritis, rheumatoid arthritis, or ulcerative colitis.

32. A method for treating a dog having an NGF-related condition, comprising administering to the dog a therapeutically effective amount of an antibody described in any one of claims 14 to 25 or a pharmaceutical composition described in claim 29.

33. A method for maintaining remission of an NGF-related condition in a dog, comprising administering to the dog a therapeutically effective amount of an antibody described in any one of claims 14 to 25 or a pharmaceutical composition described in claim 29.

34. A method for treating pain in a dog, comprising administering to the dog a therapeutically effective amount of an antibody of any one of claims 14 to 25 or a pharmaceutical composition of claim 29; optionally, the NGF-associated condition or pain is chronic pain, acute pain, inflammatory pain, osteoarthritic pain, back pain, cancer pain, neuropathic pain, pain associated with surgery, pain associated with a fracture or broken bone, pain associated with dental work, pain associated with a burn, pain associated with a cut, or pain associated with labour.

35. 35. The method of any one of claims 30 to 34, wherein the antibody or pharmaceutical composition is administered parenterally, intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intraarterially, intrasynovially, intrathecally, or by inhalation.

36. 36. The method of any one of claims 30 to 35, further comprising administering one or more pain medications, such as an IL17 antibody, an IL-5 antibody, an IL-31 antibody, an IL4 antibody, an IL13 antibody, an IL23 antibody, an IgE antibody, a CD11α antibody, an IL6R antibody, an α4-intergrin antibody, an IL12 antibody, an IL1β antibody, or an anti-BlyS antibody, an NGF kinase inhibitor, a PI3K inhibitor, a ras inhibitor, a CGRP inhibitor, a TNF inhibitor, an IL17 inhibitor, an EGFR inhibitor, a phospholipase C pathway inhibitor, and / or a corticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), a cyclooxygenase inhibitor, an opioid, and / or a cannabinoid.

37. 37. The method of any one of claims 30 to 36, wherein the antibody is administered in an amount ranging from 0.01 mg / kg body weight to 100 mg / kg body weight per dose, or is administered at a dose of 2 mg / kg body weight.

38. A method for reducing TNFα and / or NGF signaling function in a cell, comprising exposing the cell to an antibody described in any one of claims 1 to 25 or a pharmaceutical composition described in claim 29 under conditions that allow binding of the antibody to TNFα and / or NGF, thereby reducing binding to TNFα and / or NGF signaling function by the cell, optionally wherein the cell is exposed to the antibody or pharmaceutical composition ex vivo or wherein the cell is exposed to the antibody or pharmaceutical composition in vivo, and optionally wherein the cell is a dog cell, a feline cell, or an horse cell.

39. A method for detecting TNFα and / or NGF in a sample from a companion animal species, comprising contacting the sample with an antibody described in any one of claims 1 to 25 or a pharmaceutical composition described in claim 29 under conditions that allow binding of the antibody to TNFα and / or NGF, and detecting whether a complex is formed between the antibody and TNFα and / or NGF in the sample, optionally wherein the sample is a biological sample obtained from a dog, cat, or horse.